Table of Content

    05 October 2021, Volume 41 Issue 10
    Chaperone Rer1 involves in trafficking of hERG potassium channel in cell line HEK293T
    2021, 41(10):  1403-1409. 
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    Objective To investigate the role of Rer1 in hERG potassium channel transport,and to study the medicine Baf A1 that may restore abnormal hERG transport.Methods HEK293T cells were transiently transfected with constructed A561V mutant and WT plasmids,and the wild-type,mutant and mixed transfection cell models were established.The expressions of hERG and Rer1 were detected by immunofluorescence and Western blot.SiRNA was used to knockdown the expression of Rer1 and detect the expression of hERG;Bafilomycin A1,a V-ATPase inhibitor was incubated by 1 mmol/L for 6 h.Western blotting was used to detect the changes of hERG protein before and after the incubation.Patch clamp was used to measure the current of potential functional in mixed transfection cells.Results Compared with the WT group,A561V-mutation could cause trafficking deficient of hERG protein.The PM expression of hERG in mutant group was significantly reduced(P<0.01).The expression of Rer1 in mutation and mixed transfection groups decreased significantly(P<0.01).Further knockdown of Rer1 can promote the forward transport of mature hERG.Besides,after incubation with Baf A1,the mature hERG protein in WT and mutant groups increased significantly(P<0.05),while the immature hERG in the mixed transformation group was significantly increased(P<0.05).Relative density of tail current was elevated following incubation with Baf A1 compared with control.Conclusions Rer1 is involved in the transport of hERG potassium channel and can inhibit the forward transport of some abnormal hERG.Baf A1 can significantly enhance the tail current density of mixed transfection cells.
    RNA-seq analysis on cisplatin down-regulating expression of DRG neurons’ axon guidance related genes
    2021, 41(10):  1410-1416. 
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    Objective To explore the effect of cisplatin on the expression of axon guidance related gene expression in mouse dorsal root ganglion (DRG) neurons using transcriptome sequencing technique, and further explore the neurotoxic of cisplatin. Methods The primary cultures of DRG neurons was isolated from Kunming mice, and divided into control group and injury group. Neurons in the control group were cultured for 24 hours, and the neurons in the injury group were pre-cultured for 4 hours and then treated with 20 μmol/L cisplatin for 20 hours. To observe the neurites and the growth cones in two groups by the NF200 immunofluorescence staining. Total RNA was extracted from the cells, and transcriptome sequencing was performed using RNA-seq technology. The axon guidance related genes were searched through literature and database (total 231), and analyzed the differential expression of related genes. Real-time fluorescence quantitative PCR (Q-PCR) was used to verifies the expressions of related genes (Cxcl12,Ext1,Gdnf,Plxna4,Nrp2,Apbb2,Sema6a,Nrp1,Nrcam,Slit1,Ank3,Ncam1,Sema7a,Dag1,Klf7,Ntn1). Rusults Compared with the control group, the neurites of the injury group were significantly shortened and reduced, and the growth cones showed barb retraction. A large number of genes were down-regulated in the injury group, compared with the control group, among them, 49 genes related to axon guidance pathway were down-regulated. The proteins interact closely with each other, mainly focus on Ntn1,Nrp1,Nrp2,Sema6a,plxna2,plxna4,Slit2,Robo1, etc.The Q-PCR verification of related genes expression was consistent with the trend of RNA-seq results. Conclusions The neurotoxicity mechanism of cisplatin may be related to the down-regulation of many axon guidance related genes.
    Decreased integrinβ1 in lipid raft inhibits proliferation and migration of HPS rat serum-induced PMVECs
    qi jiazhou
    2021, 41(10):  1417-1422. 
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    Objective To investigate the effects of integrinβ1 within lipid raft on PMVECs. Method PMVECs were divided into four groups: control group: normal rat serum stimulated cells; HPS group: HPS rat serum stimulated cells; MβCD group: the cells were pretreated with 0.01mol/L MβCD for 1 hour, and then cultured with HPS rat serum; si-integrinβ1 group: construction of integrinβ 1 low expression cells using siRNA technology, and then cultured with HPS rat serum. The expression of integrin β1,FAK and pY397FAK withinin lipid rafts of PMVECs in each group was detected by Western blot; Cell proliferation, migration were observed by CCK-8 and wound healing assay; The secretion of angiogenic factors (VEGF) was detected by ELISA. Results Compared with the control group , the expression of integrin β1 and the level of activated FAK in the lipid rafts of HPS group was higher than that of the control group(P<0.05), the ability of PMVECs proliferation, migration and the level of secreted VEGF were significantly increased(P<0.05). Compared with the HPS group, the expression of integrin β1 and the level of activated FAK in the lipid rafts was significantly reduced in the MβCD group or siRNA integrinβ1 group(P<0.05), the ability of PMVECs proliferation, migration and the level of secreted VEGF significantly were also significantly reduced(P<0.05). Conclusions The up-regulation of integrinβ1 within lipid raft activates integrinβ1 / FAK pathway thereby promotes cell proliferation, migration and secretion of VEGF, may mediates pulmonary vascular remodeling of HPS.
    Hydroxysafflor yellow A inhibits epithelial-mesenchymal transition in hepatocellular carcinoma cell line Huh7
    2021, 41(10):  1423-1427. 
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    Objective To investigate the effect of hydroxysafflor yellow A (HSYA) on the migration and invasion in Huh7 cells, and to explore whether HSYA affects the epithelial-mesenchymal transition (EMT) of liver cancer in vitro and in vivo. Methods Human Huh7 cells were cultured in vitro and divided into control group, 80 and 160 μmol/L HSYA experimental groups and treated for 24h respectively. Wound healing and transwell assay were used to detect the migration and invasion ability of HCC cells. Western blot analysis was used to detect the expression of vimentin and E-cadherin. To establish a model of Huh7 nude mice with subcutaneous xenograft, the morphology of transplanted tumor and liver tissues in nude mice were observed by HE staining. The expression of TGF-β1, vimentin and E-cadherin in tumor tissues were detected by immunohistochemistry. Results Compared with the control group, cell migration and invasion of HSYA experimental group were significantly decreased, E-cadherin expression was increased, and vimentin expression was decreased(P<0.05). Compared with the transplantation group, there was no tumor cell metastasis and tumor cell necrosis in the HSYA experimental group (2.25mg/kg), increased expression of E-cadherin and decreased vimentin expression as well as decreased TGF-β1 expression(P<0.05). Conclusions HSYA can inhibit the migration and invasion of HCC cells by inhibiting the occurrence of EMT.
    Effects of splenectomy on liver homing and cirrhosis therapy after transplantation with adipose tissue derived mesenchymal stem cells in rats
    2021, 41(10):  1428-1433. 
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    Objective To explore the influence of splenectomy on liver homing and treatment of cirrhosis after adipose tissue derived mesenchymal stem cells (ADSCs) infusion. Methods The Rats liver cirrhosis model was established and randomly divided into four groups, sham operation group (sham), sham operation + ADSCs infusion group (ADSCs), splenectomy group (Sp), splenectomy + ADSCs infusion group (Sp+ADSCs). The cell infusion was performed one week after sham or splenectomy. Then, liver function, pathological and homing changes were detected one week later. Results The isolated ADSCs were CD29 positive and have adipogenic and chondrogenic differentiation ability, the hepatic fibrosis area in ADSCs group was significantly smaller than that in sham operation group (p<0.05), and the fibrotic area in Sp+ADSCs group reduced as compared with that in ADSCs group (p<0.05). The expression level of collagen I mRNA in ADSCs group were significantly decreased as compared with that in sham group (P<0.05), and the collagen I mRNA level in Sp+ADSCs group was further decreased as compared with that in ADSCs group. As compared with ADSCs group[ALT: (280±56)U/L], the Sp+ADSCs group [ALT: (213±89)U/L] had a significant difference in inhibiting liver injury (P<0.05); Ex vitro fluorescence imaging showed that the fluorescence signal intensity in liver tissue of the Sp+ADSCs was significantly higher than that in ADSCs group (P<0.05). Conclusions Splenectomy can significantly improve the live homing of ADSCs and improve their therapeutic effect on liver cirrhosis.
    Over-expression of ERP29 inhibits invasion and migration of human ovarian cancer cell line OVCAR3
    2021, 41(10):  1434-1439. 
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    Objective To explore the effect of over-expression of endoplasmic reticulum protein 29 (ERP29) on the invasion and migration ability of human ovarian cancer OVCAR3 cells, and to explore related mechanisms. Methods The OVCAR3 cell line in the logarithmic proliferation stage was divided into over-expression group, empty vector group, and control group. The over-expression group was transfected with ERP29 over-expression RNA recombinant eukaryotic vector pcDNA 3.1-ERP29 plasmid by liposome transfection method. The empty vector group was transfected with the negative control vector pcDNA 3.1 empty plasmid, and the control group was not treated. Observe the transfection efficiency with an inverted fluorescence microscope, and use real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) to detect the expression of ERP29 mRNA after transfection. Transwell chamber experiment was used to detect the invasion. The scratch experiment was used to detect the migration. Western blot was used to detect the expression of AKT, p-AKT, mTOR, p-mTOR, 4EBP1, p-4EBP1 protein. Results The plasmid transfection efficiency of the overexpression group and the empty vector group both exceeded 80%. Compared with the control group and the empty vector group, the relative expression of ERP29 mRNA in the over-expression group was increased (P<0.05), which of the number of transmembrane cells was decreased (P<0.05), which of the migration rate was decreased (P<0.05), which of the p-AKT/AKT , P-mTOR/mTOR, p-4EBP1/4EBP1 were all decreased (P<0.05). Conclusions Over-expression of ERP29 can significantly inhibit the invasion and migration ability of ovarian cancer OVCAR3 cells, which may be inhibited by down-regulating phosphorylation of AKT, mTOR, and 4EBP1.
    Role of autophagy in hypoxia-reoxygenation-induced injury of rat alveolar epithelial cell type II
    2021, 41(10):  1440-1445. 
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    Objective To investigate the role of autophagy in the hypoxia-reoxygenation (H/R) model of rat alveolar epithelial cell type II (AEC II). Methods Cells were randomly divided into control group (C) 、hypoxia-reoxygenation group (H/R)、solvent group (HD)、autophagy agonist group(Rap) and autophagy inhibitor group (3-MA): pretreat cell with 3-mA 1h before hypoxia. The cell viability was determined with CCK-8, and use RT-PCR, Western blot to detect the expression of autophagy related mRNA transmission and its proteins, and ultrastructure of cell was examined by electron microscope. Results Compared to the C group, the cell were all viability decreased, the expression level of autophagy related proteins and gene transcription increased, and the cell ultrastructure was damaged greatly (P<0.05 ). Compared to the H/R group, the cell viability of 3-MA group was higher, the autophagy related proteins and their gene transcription level were decreased ((P<0.05 ), the cell structure was damaged slightly, and autophagy bodies were not found. Conclusions Hypoxia-reoxygenation can activate autophagy and induce alveolar epithelial cell type II damage.
    Nrf2 activator relieves pulmonary vascular remodeling in rats with hypoxic pulmonary arterial hypertension
    2021, 41(10):  1446-1450. 
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    Objective To investigate the effect of activation of nuclear factor E2-related factor 2 (Nrf2) on vascular remodeling in hypoxic-induced pulmonary hypertension (PAH) rats and its mechanism. Methods The rats were divided into groups: control group, hypoxic group (hypoxic treatment of rats), Nrf2 activated group (gavage 5mg/kg Nrf2 agonist sulforaphane every day for 4 weeks before hypoxic treatment). The cardiopulmonary hemodynamics and right ventricular hypertrophy index mean pulmonary artery pressure (mPAP) were measured, the right ventricular hypertrophy index = right ventricle (RV)/(left ventricle + interventricular septum) (LV + S) was calculated; HE staining assay the related indexes of pulmonary vascular remodeling were measured, and the wall area (WA)/total area (TA) of pulmonary arterioles and the thickness of vascular mesothelium (MT)/external diameter (ED) of pulmonary arterioles were calculated; malondialdehyde (MDA) and superoxide dismutase (SOD) were measured by thiobarbituric acid method and nitrogen blue tetrazole method; the mRNA levels of matrix metalloproteinase (MMP) and MMP-9 in pulmonary artery were detected by qRT-PCR; and the protein levels of Nrf2, antioxidant response element (ARE), NADPH quinineoxidoreductase-1 (NQO-1) and heme oxygenase1 (HO1) were detected by WB. Results Compared with the control group, the levels of mPAP, RV/(LV + S), levels of WA/TA and MT/ED, level of MDA and mRNA levels of MMP-2 and MMP-9 in pulmonary artery tissue, the levels of Nrf2, ARE, NQO1 and HO1 proteins in the nucleus of the hypoxic group were increased (P < 0.05); and SOD activity in pulmonary artery tissue and level of Nrf2 protein in plasma were decreased (P < 0.05). Compared with hypoxia group, the levels of mPAP, RV/(LV + S), levels of WA/TA and MT/ED, level of MDA and mRNA levels of MMP-2 and MMP-9 in pulmonary artery tissue, the level of Nrf2 protein in cytoplasm of the Nrf2 activation group were decreased (P < 0.05); and SOD activity in pulmonary artery tissue and levels of Nrf2, ARE, NQO1 and HO1 proteins in the nucleus were increased (P < 0.05). Conclusions Nrf2 activation can delay the vascular remodeling induced by PAH, and may be achieved by activating Nrf2/ARE pathway.
    Dl-3-n-butylphthalide attenuates H2O2-induced oxidative damage of human umbilical vein and umbilical artery endothelial cells
    2021, 41(10):  1451-1456. 
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    Objective To investigate the protective effect and mechanism of dl-3-n-butylphthalide (NBP) on hydrogen peroxide (H2O2) - induced oxidative damage in vascular endothelial cells(VECs). Methods Human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs) were cultured in vitro. MTT assay was used to find the appropriate dosage of H2O2 to establish oxidative stress injury model. Then the cells were pre-incubated with two dosages of NBP (5 and 10 μmol/L) 2 h before exposure to H2O2. The cell viability was detected by MTT assay, mitochondrial membrane potential was measured by rhodamine 123, cell apoptosis was detected by Annexin-V/ PI double staining, and intracellular free Ca2+ was measured by Fura-4/AM. Results H2O2-induced oxidative injury reduced the cell viability of HUVECs and HUAECs, increased the intracellular free Ca2+ concentration, leading to mitochondrial membrane potential decline and cell apoptosis(P<0.05). However, NBP treatment could improve the above changes induced by H2O2。Conclusions NBP protects vascular endothelial cells by reducing the oxidative damage induced by H2O2.
    Edaravone reduces oxidative stress and inflammation in COPD rats
    2021, 41(10):  1457-1462. 
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    Objective To explore the mechanism of edaravone (EDA) on oxidative stress and inflammation in chronic obstructive pulmonary disease (COPD) rats. Methods The rats were randomly divided into control group, model group, EDA high dose group, EDA medium dose group and EDA low dose group, with 10 rats in each group. The COPD model was prepared by intratracheal infusion of lipopolysaccharide (LPS) combined with smoking, and rats were injected intraperitoneally with 40, 20, and 10 mg/kg EDA. 28 days after modeling, the pulmonary function and the wet-to-dry weight ratio (W/D) of the lung tissues were detected. ELISA method was used to detect the levels of SOD, MDA, TNF-α, IL-1β and IL-6 in lung tissues. The pathological changes of lung tissues were observed by HE staining. Western blot was used to detect the protein expression levels of Caspase-3, MMP-9, TLR4, MyD88, NF-κB p65 protein expression and IκBα phosphorylation. Results Compared with control group, the resting breathing frequency and airway resistance (RI) of model group increased, the values of peak inhale flow (PIF) and dynamic lung compliance (Cdyn) decreased, the lung injury was obvious, the SOD activity of the lung tissues decreased, the contents of MDA, TNF-α, IL-1β, and IL-6 increased, and Caspase-3. MMP-9, TLR4, MyD88 expression and IκBα phosphorylation increased, and NF-κB p65 intranuclear metastasis was obvious (P<0.05). Compared with model group, EDA reduced resting respiratory frequency, RI, increased PIF and Cdyn, reduced lung injury, increased SOD activity of lung tissues, reduced MDA, TNF-α, IL-1β, IL-6 contents, and reduced Caspase-3, MMP-9, TLR4, MyD88 expression and IκBα phosphorylation levels, NF-κB p65 nuclear metastasis was restricted (P<0.05). Conclusions EDA can improve the lung function of COPD rats, reduce lung tissues damage, inflammation and oxidative stress.
    miR-133a-3p targeted regulating CD2AP gene reduces RSV-infected damage of human bronchial epithelial cell line 16-HBE
    2021, 41(10):  1463-1469. 
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    Objective To explore the effect of miR-133a-3p on the apoptosis and inflammatory response of bronchial epithelial cells infected by respiratory syncytial virus (RSV) and its regulatory effect on CD2-associated protein (CD2AP). Methods The bronchial mucosal tissue specimens of 59 children with bronchial asthma were collected as the AsP group, and 59 bronchial mucosal tissue specimens of non-asthmatic children with bronchiectasis were selected as the NC group. The RT-qPCR method was used to detect the expression of miR-133a-3p and CD2AP in the tissues. RSV infected bronchial epithelial cells (16-HBE) to establish a cell injury model (RSV group). miR-NC, miR-133a-3p mimics, si-NC, si-CD2AP, pcDNA-NC, pcDNA-CD2AP, miR-133a-3p mimics and pcDNA-NC, miR-133a-3p mimics and pcDNA-CD2AP were transferred respectively 16-HBE cells infected with RSV. RT-qPCR and Western blot were used to detect the expression of miR-133a-3p and CD2AP in the cells. ELISA was used to detect the levels of TNF-α, IL-6, and IL-1α. Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporter experiment was used to detect the targeting relationship between miR-133a-3p and CD2AP. Western blot method was used to detect the protein expression of cleaved-caspase-3. Results Compared with the NC group, the expression level of miR-133a-3p in the bronchial mucosa tissue of the AsP group was decreased (P<0.05), and the expression level of CD2AP mRNA was increased (P<0.05). The expression level of miR-133a-3p in RSV-infected 16-HBE cells was decreased (P<0.05), and the expression level of CD2AP and the levels of TNF-α, IL-6 and IL-1α were increased (P<0.05), the apoptosis rate and the protein level of cleaved-caspase-3 were increased (P<0.05). Transfection of miR-133a-3p mimics or transfection of si-CD2AP into RSV-infected 16-HBE cells could significantly reduce the levels of TNF-α, IL-6, IL-1α, the apoptosis rate and the protein level of cleaved-caspase-3 (P<0.05). The dual luciferase report experiment confirmed that CD2AP was the target gene of miR-133a-3p. Co-transfection of miR-133a-3p mimics and pcDNA-CD2AP could significantly reverse the effects of miR-133a-3p mimics transfection on 16-HBE cell apoptosis and inflammation. Conclusions The overexpression of miR-133a-3p could inhibit the inflammatory response and apoptosis of RSV-infected bronchial epithelial cells by negatively regulating the expression of CD2AP, thereby reducing cell damage.
    BMP9 promotes bone differentiation of bone marrow mesenchymal stem cells via Orai1 pathway
    2021, 41(10):  1470-1475. 
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    Objective To explore the role of calcium release activated calcium channel protein 1 (Orai1) in bone marrow mesenchymal stem cells (BMSCs) osteogenic induced by bone morphogenetic protein 9 (BMP9). Methods BMSCs were isolated by adherent method, and the third generation BMSCs were added with 10-5mol / L of BMP9, and the mRNA expression of Orai1 was observed at different time points. q-PCR and Western blot were used to value the transfection efficiency after Orai1knockdown. The osteogenic markers of Runx2, OPN and OCN were detected by q-PCR. Hydroxyapatite / PLA / chitosan, a scaffold material for bone tissue engineering, was implanted into the subcutaneous of nude mice. After 4 weeks, the osteogenic effect was observed by HE staining and Masson staining. Results The mRNA expression of ORAI1 increased gradually at the 5th day after BMP9 induction (P<0.05). Our data suggested that the gene and protein expressions of ORAI1 decreased significantly after Orai1knockdown (P<0.05). Besides, the osteogenic effect of Orai1knockdown decreased significantly, and the expression of Runx2, OPN and OCN gene decreased significantly (P<0.05). Moreover, BMP9 could significantly improve the ectopic osteogenic ability of BMSCs, and Orai1 knockdown significantly inhibited this effect (P<0.05). Conclusions BMP9 plays an important role in osteogenic differentiation of BMSCs probably by regulating ORAI1.
    Correlations of MGMT promoter methylation and IDH1 mutations with the clinical prognosis of malignant glioma patients
    2021, 41(10):  1476-1480. 
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    Objective To analyse retrospectively the IDH mutation and MGMT promoter methylation of malignant glioma patients and identify the association with their clinical relevance. Method Twenty-five patients with glioma who were received treatment from October 2017 to October 2019 were selected, the survival rate was calculated by Kaplan-Meier method and analyzed by Log-Rank test. Result Univariate analysis showed that negative status of MGMT gene promoter methylation(χ2=4.22,P=0.04) was an important factor affecting the survival rates of patients, and patients with both negative status of MGMT gene promoter methylation and mutation of IDH1 had a significantly prolonged survival(P<0.05). Conclusion Our study demonstrates that IDH mutation and MGMT promoter methylation status are significantly associated with clinical prognosis of malignant glioma patients.
    Pathological classification and surgical prognosis of idiopathic hyperaldosteronism
    2021, 41(10):  1481-1485. 
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    Objective Few cases with idiopathic hyperaldosteronism (IHA) underwent surgical treatment. Their pathological characteristics and surgical prognosis were rarely reported. The clinical and pathological characteristics and surgical prognosis of 23 patients with IHA were retrospectively analyzed to improve the level of clinical diagnosis and treatment. Methods Clinical characteristics of 23 patients with IHA were analyzed retrospectively. HE staining and CYP11B2 immunostaining were performed on the pathological sections. Pathological features and surgical prognosis were analyzed. Results All samples were categorized as nonclassic pathology, including 12 cases with multiple aldosterone-producing nodules,10 cases with multiple or single aldosterone-producing micronodules. Only one case had aldosterone-producing diffuse hyperplasia. Unilateral adrenal resection was performed in all patients. Postoperatively, blood pressure and serum potassium were recovered to normal range in 6 patients with a cure rate 26%.Blood pressure decreased but did not return to normal in 17 patients (improve rate 74%). Compared with the improved group, patients in the cured group had a lower preoperative blood pressure (P<0.05). Conclusion Multiple functional nodules and micronodules are the main changes in adrenal pathology of IHA. Unilateral adrenal resections probably bring clinical benefits in these patients.
    Effect of combined treatment of the first metatarsophalangeal joint fusion with the second to fifth metatarsophalangeal arthroplasty on rheumatoid forefoot deformity
    2021, 41(10):  1486-1490. 
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    Objective To analyze the effect of the first metatarsophalangeal joint fusion combined with 2-5 metatarsophalangeal joint arthroplasty in the treatment of rheumatoid foot. Methods From January 2015 to December 2019, 21 patients (36 feet) who have rheumatoid anterior foot deformity, with an average age of 63.6 ± 6.0 years, were performed first metatarsophalangeal joint fusion and 2-5 metatarsophalangeal joint arthroplasty. The results of the operation were evaluated by the American Orthopaedic Foot and Ankle Society (AOFAS) scores and visual analog scale (VAS). The hallux valgus angle (HVA) and the intermetatarsal angle (IMA) were measured on the X-ray preoperative and postoperative films. Results The follow-up period of 21 cases (36 feet) was 39.2 ± 5.1 months, without any complications such as nonunion, incision infection and necrosis of toe. The preoperative and postoperative scores of AOFAS, VAS, HVA and IMA were 81.6 ± 9.1, 8.4 + 1.1, 37.2 ° ± 7.5 °, 16.9 ° ± 2.7 °, and 84.25 ± 2.87, 1.9 ± 0.9, 14.4 ° ± 2.0 °, 11.1 ° ± 1.7 ° respectively. The improvement was statistically significant (P < 0.05). Conclusion The first metatarsophalangeal joint fusion combined with 2-5 metatarsophalangeal joint arthroplasty was effective on rheumatoid forefoot deformity, which can correct the deformity, relieve pain and improve the function.
    Construction and optimization of detection technology platform for EGFR gene exon 19 del18bp mutation
    2021, 41(10):  1491-1496. 
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    Objective To construct and optimize a high-fidelity polymerase-mediated mutation sensitive molecular switch technique platform for detecting exon 19 del18bp of EGFR gene. Methods Using the genome DNA from a healthy volunteer as PCR template, the wild-type and mutant-type DNA fragment containing mutation site were amplified by common PCR and overlap PCR respectively, which then were connected into the vector pMD19-T. The constructed wild-type and mutant-type recombinant plasmid were transformed into competent cells E. coli DH5α further appraised by bacteria solution PCR and genome sequencing. The mutation-specific upstream detection primer was designed with 3' phosphorothioate modification, and two-directional primers extension was performed using high-fidelity polymerase. The annealing temperature, primer concentration, template concentration, cycle number, and other high-fidelity polymerase-mediated PCR conditions were all optimized through an orthogonal test protocol to establish a molecular switch detection platform for exon 19 del18bp of EGFR gene. Results The molecular switch detection platform for exon 19 del18bp of EGFR gene was successfully established. The optimal PCR condition of detecting EGFR gene exon 19 del18bp using mutation-sensitive molecular switch technique was as follows: 57℃optimal annealing temperature, 0.16μmol/L primer concentration, 1.48×10-2μmol/L template concentration and 25 PCR cycles. And the allele-specific primer for del18bp can detect the perfectly matched template at a sensitivity of about 10-3μmol/L. Conclusions Mutation-sensitive molecular switch technology composed of high-fidelity polymerase with 3' phosphorothioate-modified prime is expected to provide a new technical means which can implement EGFR mutation test for personalized targeted meditation for lung cancer.
    Progress of study on inflammatory factors regulating function of vascular endothelial cells from type 1 diabetes mellitus
    2021, 41(10):  1497-1501. 
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    Inflammatory factors are widely involved not only in the physiopathological process of type 1 diabetes mellitus (T1DM), but also in the regulation of vascular endothelial dysfunction. A variety of inflammatory factors affect the insulin signaling pathway through the activation of oxidative stress or regulation of related receptors, leading to apoptosis of pancreatic β-cells and vascular endothelial dysfunction. This article reviews the relationship between type 1 diabetes and the inflammatory factors interleukin family, tumor necrosis factor-α, interferon-γ, transforming growth factor β1, and forkhead transcription factor p3. Furthermore, the studies of inflammatory factors like lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), soluble vascular cell adhesion molecule (sVCAM), and nuclear factor of activated T cells (NFAT) regulating type 1 diabetes complicated with vascular endothelial dysfunction are reviewed and the possibility of cytokine immunotherapy modulating vascular endothelial dysfunction is envisioned.
    Recent advances in rosacea comorbidities
    2021, 41(10):  1502-1506. 
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    Rosacea is a chronic inflammatory skin disease mainly affecting the middle of the face with a variety of systemic comorbidities. This article reviews comorbidities of rosacea, including cardiovascular diseases, metabolic diseases, digestive diseases, malignancies, and neuropsychiatric diseases, and provides suggestions on how to deal with comorbidities of rosacea. If rosacea patients have symptoms or signs of other diseases, or abnormal results of laboratory examinations or imaging examinations, they should be referred to the corresponding department for further treatment. It is necessary to further investigate the relationship between rosacea and its comorbidities, especially the relationship between different subtypes of rosacea and their comorbidities, to facilitate the treatment and management of rosacea patients.
    Research progress of spinophilin in nervous system diseases
    2021, 41(10):  1507-1509. 
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    Spinophilin (SPN) was first isolated from the dendritic spines of neurons as a binding protein of protein phosphatase PP1, thus it is also known as PPP1R9B. SPN can not only bind to PP1, but also interacts with more than 30 proteins, including cytoskeleton proteins and adhesion molecules. SPN participates in biological processes such as synaptic plasticity, neuronal migration, intracellular signal transduction, and plays an important role in the development of neurodegenerative diseases, psychiatric disorders and neuronal tumors. Therefore, understanding the role and mechanism of SPN in the physiological and pathological processes abovementioned is helpful to advance the basic research and clinical transformation of SPN.
    Research progress on the role of glomerular mesangial cell proliferation in glomerulosclerosis of diabetic nephropathy
    2021, 41(10):  1510-1513. 
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    Diabetic nephropathy is a common chronic microvascular complication in diabetic patients. Mesangial cells are the main cell components in glomeruli. High glucose stimulation can promote mesangial cell proliferation by reducing endogenous hydrogen sulfide concentration, activating transforming growth factor-β superfamily and influencing long non-coding RNA level. Abnormal activation of mesangial cells plays an important role in the process of diabetic glomerulosclerosis.
    Progress in research on autophagy and hepatocellular carcinoma
    2021, 41(10):  1514-1517. 
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    Autophagy is a protective mechanism for eukaryotes to maintain homeostasis. Meanwhile, it plays an important role in maintaining the survival and energy metabolism of tumor cells, and also impacts on the therapeutic effect of liver cancer through different mechanisms, such as autophagy maintains liver cells homeostasis, autophagy causes liver cancer cells develop resistance, and autophagy inhibitors or natural extracts play a role of resistance to liver cancer through autophagy pathways. It is of great significance to explore the regulation of autophagy in the prevention and treatment of hepatocellular carcinoma.
    Research progress on the effects of gestational metabolic syndrome on maternal health and offspring growth and development
    2021, 41(10):  1518-1522. 
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    In order to meet the needs of the mother and fetus during pregnancy, the various systems in the body have changed in varying degrees. However, as a result of individual differences, part of pregnant women will appear metabolic syndrome such as type 2 diabetes(T2D) mellitus, lipid disorder, and hypertension, which will affect the health of mothers and the development of nervous and cardiovascular systems of their offspring. Therefore, controlling various metabolic abnormalities is beneficial to improve pregnancy outcomes.
    Research progress on the mitochondrial translocation and degradation
    2021, 41(10):  1523-1528. 
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    The mitochondrial proteins translocation and degradation coordinate to maintain the proteostasis network of mitochondria. Their dysfunction is one of the reasons of metabolic diseases. The dysfunction of transportation and degradation prevents the nuclear-encoded mitochondrial protein from importing and cells from clearing abnormal proteins in time, which ultimately leads to mitochondrial damage. In the diabetic cardiomyocyte, decreased activity of electron transport chain (ETC), increased production of reactive oxygen species (ROS) and decreased energy productivity caused by disorders of mitochondrial transportation and degradation are main reasons for the development of diabetic cardiomyopathy (DCM).
    Curriculum design and outcomes analysis of cochlear implant training course
    2021, 41(10):  1529-1532. 
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    Objective To analyze the course design and training outcomes of cochlear implantation special training course, and to explore the training methods for otology microsurgeons to master cochlear implant technology. Methods A total of 24 students in the two sessions of cochlear implant training courses in 2014 and 2017 were collected through questionnaires made by ourselves, and their basic information, cognition of cochlear implant technology and evaluation and harvest of the training courses were investigated and analyzed. Results Among the 24 students, 33% are senior (8/24), 17% are deputy senior (4/24), 50% are intermediate (12/24), 58% are PhD, 25% are master and 17% are undergraduate. 92% (22/24) of the students thought the training was very helpful for surgery, and 8% (2/24) of the students thought it was helpful to some extent for surgery. The most helpful part of participating in the course is the facial nerve recess approach training. The highest score for participating in the class was the training method of cochlear implant surgery, which could be improved constantly in accordance with the training method in their own hospital. As for the organization and course setting of the training course, the teaching method of step by step scored the highest for the most beneficial part, followed by the course setting including the facial nerve recess approach, subtotal petrosectomy and transotic approach. Conclusions The teaching methods of step by step, centralized comments and discussion helps students to understand the cochlear implant technology course quickly.
    Application and experience of Moodle for biochemistry teaching in 4 + 4 clinical medicine pilot class
    杨啸林 Xiao-LinYang Yan-Li ZHANG
    2021, 41(10):  1533-1537. 
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    Peking Union Medical College sets up a review course of Biochemistry in the general introduction of medicine for the 4 + 4 clinical medicine pilot class. The mixed classroom design was carried out with Moodle platform to help students of different majors to review, consolidate and improve their knowledge of Biochemistry, a compulsory course for premedical students. Under the support of Moodle, we built an autonomous learning environment, integrating Macmillan sapling e-learning resources, and establishing a chapter-based pre-class test to improve students' mastery of basic knowledge. Moodle's "Quiz" and "Workshop" were used in case-based learning to help students establish the relationship between biochemical changes and diseases. The positive exploration of the teaching mode of Biochemistry by Moodle targets to accommodate the characteristics of 4 + 4 students.
    Application of CBL in cardiovascular system integration teaching for students from 4+4 pilot class at Peking Union Medical College
    2021, 41(10):  1538-1541. 
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    Objective To explore the value of case-based learning (CBL) teaching method in the basic medicine module of cardiovascular system integration course in the 4 + 4 medical teaching pilot class of Peking Union Medical College (PUMC), and optimize future improvements based on teaching feedbacks. Method The students of Grade 2020 4 + 4 medical teaching pilot class in PUMC were selected as the research objects, CBL of 6 class hours was carried out according to the content of basic medicine module in cardiovascular system integration teaching. Online examinations, questionnaire survey and teacher-student discussion were used to evaluate the teaching effect. Results The examination scores were improved in 87.5% (28 / 32) of the students and the average score of all the students was significantly higher after the CBL teaching compared to those before the CBL teaching. Questionnaire survey and teacher-student discussion showed that the vast majority of the students believed that CBL teaching method was helpful in improving the understanding and learning of basic knowledge of cardiovascular system, inspiring the learning interest, and promoting the ability of clinical thinking and analysis. Conclusions The application of CBL teaching medthod in cardiovascular system integration courses will help to improve students' understanding and acquisition of basic medical knowledge, and enhance the teaching quality and effect of the integration courses of basic medical module teaching. Meanwhile, the students should pay more attention to improving their knowledge framework and basic medical knowledge reserve.
    Application of social hotspots on case-based teaching method during the teaching of endocrinology
    2021, 41(10):  1542-1545. 
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    In order to improve the quality of clinical endocrinology courses, social hotspots were used in professional instructions of endocrinology, and aligned effectively with curriculum knowledge. Social hot topics on case-teaching can enrich teaching materials and broaden students’ scope of knowledge; enliven classroom dynamics and make instructions more efficient; in addition precise selection of hotspots can make the study more interesting. This paper, by means of illustrating the application cases of hotspots in endocrinology teaching, analyzed the advantages of such a method over conventional methods. In addition, the paper made practical suggestions on such a teaching model, for the purposes of ideal teaching outcomes and nurturing excellent clinical medical students in the end.
    Reflections on the training of internal medicine residents in the post COVID-19 pandemic period
    2021, 41(10):  1546-1549. 
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    The COVID-19 pandemic has exerted huge impact on medical education. Having experienced the outbreak of the pandemic, we are more aware of the importance of well-trained internal residents with medical professionalism. Therefore, we should keep the good tradition of resident training in the Department of Medicine, Peking Union Medical College Hospital. At the same time, as the pandemic shows it could be a trend of normalization, we need to pay more attention to the mental health of residents and enhance the professionalism training of residents in response to the change of teaching environment, so as to improve the competence of residents in the post-pandemic period.
    Application and exploration of comprehensive experiment in biochemistry experiment teaching
    2021, 41(10):  1550-1553. 
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    Biochemistry experiment courses are important practical courses for medical specialties. Traditional teaching has the problems of single teaching method, insufficient student participation, weak learning enthusiasm, and no assessment method. Comprehensive experiment is a teaching method that takes students as the main body and uses relevant theoretical knowledge and practical operation skills to solve specific experimental problems. In the teaching process, the assessment is used as an experiment course test method, which not only urges students to complete the experimental content seriously, but also Can reduce students' slack in the experiment course. The introduction of comprehensive experiments into biochemistry experiment teaching is expected to improve the quality of teaching and enhance students' ability to apply theoretical knowledge, practical skills and independent thinking.
    Application of medical record quality control system in blood transfusion records
    2021, 41(10):  1554-1558. 
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    Objective In order to improve the level of blood transfusion records by evaluating the transfusion cases in the whole hospital through the transfusion case evaluation system and monitoring the transfusion cases through the medical record quality control system. Methods Blood transfusion medical records were evaluated according to the Implementation Rules of Beijing Regional Hospital Management Assessment and Evaluation Standards (2009 Edition), Medical Records Writing Standards, Technical Standards for Blood Transfusion and other relevant requirements.A total of 21,790 clinical blood transfusion records from 2015 to 2020 were collected through the medical record quality control system, among which 5515 were from non-surgical departments and 16275 were from surgical departments (4134 were from actual blood transfusion records).Using blood transfusion evaluation system for each classification and summary of problem to register, by withholding statement that is associated with the performance, and real-time feedback and publish monthly bulletin of blood transfusion of the public, the common problems of blood transfusion duration omissions, evaluation of unfinished after blood transfusion, informed consent problem such as lack of item, through the electronic medical record quality control system to or mandatory restrictions, not banned again to apply for a phased and prohibited discharge settlement methods such as blood, thoroughly to the whole process of blood transfusion medical supervision. Results From 2015 to 2020, the qualified medical records showed an increasing trend year by year, each year is compared to the previous year (P< 0.05).The qualified rate of blood transfusion medical records in non-surgical departments (96.33%) was higher than that in surgical departments (88.90%). After the intervention of medical record quality control system in 2020, there was a significant improvement compared with the previous five years (P < 0.01). Conclusions The quality of clinical blood transfusion records can be improved effectively by monitoring the whole process of blood transfusion records through the information system.