Table of Content

05 March 2021, Volume 41 Issue 3

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Original Articles

Anti-tumor drug FTY720 regulates p53-dependent transcription

YAO Han, XU Wen-bin, WANG Dong-lai

2021, 41(3):  311-317. 

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Objective To explore whether anti-tumor drug FTY720 participates in regulation of p53-dependent gene transcription and to evaluate the potential roles of FTY720-p53 axis in the modulation of cellular biological processes. Methods CRISPR-Cas9 technique was used to generate a p53-knockout (p53-KO) cell strain with human osteosarcoma cell line U2OS that expressed the wildtype p53. Control cells or p53-KO cells were treated with or without FTY720 and the transcriptional was then assessed by RNA-sequencing (RNA-seq) analysis. The differentially expressed genes as well as their potential functions were annotated through bioinformatic analysis. Results The U2OS p53-KO cell strain was successfully established. 220 p53-dependent downstream genes were identified in response to FTY720 treatment. These genes were functionally enriched in the regulation of multiple biological processes including negative regulation of platelet activation, cellular response to chemokine, positive regulation of Rho protein signal transduction, multicellular organismal homeostasis, mucin type O-glycan biosynthesis, ECM-receptor interaction, viral protein interaction with cytokine and cytokine receptor. Conclusions p53 activation is involved in FTY720-mediated tumor suppressive functions.

Construction of B16 cell strain expressing CSDE1-EGFP using CRISPR-PITCH system

LIU Meng-yu, ZHOU Ya-bo, HUANG Bo, LYU Jia-di

2021, 41(3):  318-324. 

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Objective To construct a melanoma cell strain expressing endotopic CSDE1-EGFP using new CRISPR-PITCH system, then to select the cell strain of low expression CSDE1 and high expression CSDE1. Methods The EGFP coding sequence was inserted into the last exon of the CSDE1 gene using a micro-homology-mediated end joining(MMEJ) method; Using flow cytometry sorting, PCR, immunofluorescence and live cell imaging to detect whether CSDE1-EGFP was inserted successfully, and whether the fluorescence intensity of EGFP was consistent with the expression level of CSDE1; The Transwell experiment and mouse tumor-bearing experiment were used to observe the functional differences between the B16 cell strain with low and high expression of CSDE1. Results The B16 cell strains of low expression CSDE1 and high expression CSDE1 were selected, and the strength of EGFP expression was found to be consistent with CSDE1 expression. The melanoma cells with different expression level of CSDE1 protein had different functions. Conclusions The new CRISPR-PITCH system provides a fast and effective intuitive method for studying the dynamic positioning of CSDE1 in other tumor cells and the functional differences are resulted from different expressions.

Establishing a CRISPR-Cas9 system targeting at mouse Krt14 that simulates pathogenic site mutations of human

YUE Peng-peng, LI Cheng-qin, NONG Yue-juan, CHEN Ling, FU Can, YU Hong-hao

2021, 41(3):  325-332. 

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Objective To establish an effective CRISPR-Cas9 system targeting at mouse Krt14 that may simulate the pathogenic site mutation of human KRT14. Methods Databases such as OMIM, ExAC and ClinVar were retrieved to identify the strong pathogenic sites of human KRT14, the causative gene of human epidermolysis bullosa simplex. Then, the human and mouse KRT14 protein sequence alignment analysis was carried out to identify the strong pathogenic sites on the mouse genome. Based on the gene targeting principle of the CRISPR-Cas9 system, 4 sgRNAs targeting the mouse Krt14 and 4 expression plasmids were constructed. Then, the sgRNA expression plasmids and Cas9 expression plasmid were co-transfected into mouse NIH 3T3 cells simultaneously. PCR product sequencing, TA clone sequencing of the positive cells screened by drugs were performed to verify the target efficiency of the 4 sgRNAs. Results The p.Arg125 site of human KRT14 protein was identified to be a strong pathogenic site, which corresponded to the p.Arg131 site of mouse KRT14 protein. According to the DNA sequence of the mouse p.Arg131 site, 4 sgRNA expression plasmids targeting at this site was successfully constructed. Sequencing of PCR amplification products of the target site from drug screen positive cells showed that 4 sites were all mutated. The results of TA cloning sequencing showed that the mutation efficiency of the four sites was 70%, 90%, 65% and 100% respectively. Conclusions The above results indicates that efficient CRISPR-Cas9 system targeting at the mouse Krt14 based on the highly pathogenic sites of the human KRT14 has successfully constructed. This result lays a foundation for the in-depth study of Krt14 function and for the establishment of Krt14 gene editing mouse model in order to explore the mechanism and treatment strategy of disease.

miR-29b inhibits LPS-induced apoptosis in human bronchial epithelial cell line 16HBE

YUAN Dong, JIANG Yao-na, LI Ya-qing

2021, 41(3):  333-339. 

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Objective To study the effect of miR-29b on lipopolysaccharide(LPS)-induced apoptosis of human bronchial epithelial cell line 16HBE. Methods 16HBE cells were divided into control group, LPS group (LPS treatment), transfection mimics control group, transfection miR-29b mimics group. MTT assay for proliferation, flow cytometry was used for apoptosis; Western blot was used for checking the expression of cleaved cysteinyl aspartate specific proteins 3(c-caspase-3), cleaved cysteinyl aspartate specific proteinase 12(c-caspase-12), glucose-regulated protein 78(GRP78) and CCAAT/enhancer-binding protein C/EBP (CHOP) protein. Human bronchial epithelial cells 16HBE were co-transfected with miR-29b mimics and pcDNA-CHOP, the proliferation and apoptosis were also determined using the above method. Results Compared with the control group, the cell proliferation- activity and apoptosis rate of the LPS group decreased, and the protein expression level of c-caspase-3, c-caspase-12, GRP78, and CHOP all increased. Compared with the LPS+NC group, the LPS+miR-29b group showed an increased cell proliferation, decreased apoptosis and decreased expression of c-caspase-3, c-caspase-12, GRP78, and CHOP protein. pcDNA-CHOP reversed the effects of miR-29b mimics on the proliferation and apoptosis of bronchial epithelial cells under LPS conditions. Conclusions miR-29b inhibits 16HBE apoptosis induced by LPS; the mechanism might be related to the inhibition of endoplasmic reticulum stress.

Macrophage polarization imbalance and metabolic changes in autoimmune thyroiditis mice

WANG Shi-qi, ZHAO Lei, SHI Xiao-guang

2021, 41(3):  340-345. 

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Objective To investigate the significance of macrophage polarization imbalance and metabolic pathway changes in the occurrence and development of autoimmune thyroiditis (AIT). Methods Four weeks old NOD.H-2^h4 female mice were randomly divided into control group and AIT group(0.05% sodium iodide water). Hematoxylin-eosin(HE) staining was used to observe the lymphocyte infiltration in thyroid tissue. TgAb was measured by ELISA technology. The ratio of M1 type and M2 type macrophages in thyroid tissue was analyzed by immunofluorescence. Seahorse was used to analyze the energy metabolism of cells, and the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured. Results The thyroid tissue of mice in AIT group had obvious lymphocyte infiltration, and the incidence of AIT was significantly higher than the control group(P＜0.05). Compared with the control group, the ratio of M1 and M2 macrophages in the thyroid tissue of mice in the AIT group increased, while the proportion of M2 type macrophages decreased (P＜0.05); the ECAR and OCR values of mice in the high iodine drinking water group were significantly higher than those of the control group (P＜0.05). Conclusions The changes of macrophage metabolism and polarization imbalance affect the occurrence and development of AIT.

Analyses of factors influencing myocardial fibrosis in non-ischemic heart failure patients

WANG Fang, DONG Zhe, SUN Yi-hong, REN Jing-yi, LI Jing, ZHOU Yi-feng, ZHENG Jin-gang

2021, 41(3):  346-351. 

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Objective To investigate the potential factors influencing myocardial fibrosis (MF) in non-ischemic heart failure(HF) patients with an emphasis on the role of right heart catheter (RHC) parameters. Methods This retrospective research collected clinical data from 33 non-ischemic heart failure patients admitted to China-Japan Friendship Hospital who received endomyocardial biopsy (EMB) and right heart catheter (RHC) examination. All the patients were divided into two groups: mild MF (n=17) and severe MF (n=16), by means of K-means cluster analysis. MF was quantitatively evaluated by Masson staining. Potential factors influencing MF were analyzed. Results Compared with that in mild MF group, patients in severe MF group had aggravated cardiac function (cardiac index, 2.15±0.76 vs. 2.94±1.10,P＜0.05), accompanied by higher systolic right ventricle pressure (RVP), systolic pulmonary arterial pressure (PAP), pulmonary arterial wedge pressure (PAWP) and total pulmonary resistance (TPR) (P＜0.05). Hypertension and TPR were independently correlated with the degree of MF in multiple linear regression analysis (Adjusted R²=0.454, P＜0.001). Conclusions Hypertension and TPR, reflecting afterload of left and right ventricle, are found to be independently correlated with degree of MF in non-isch- emic heart failure patients. Cardiologists should pay more attention to high-risk patients in order to retard MF progression through timely intervention.

Whole-exome sequencing from a case of pediatric aldosterone- and cortisol-coproducing adrenal adenoma

WANG Hui-ping, WEN Jin, CUI Yun-ying, MA Xiao-sen, REN Wei-dong, TONG An-li

2021, 41(3):  352-357. 

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Objective To identify the genetic mutations of aldosterone- and cortisol-coproducing adrenal adenoma in a young boy by whole-exome sequencing. Methods The clinical data of a boy with aldosterone- and cortisol-coproducing adrenal adenoma were collected. Whole-exome sequencing was performed with DNA extracted from the blood and tumor tissue to identify germline and somatic mutations. Results A 13-year-old boy with hypertension and hypokalemia was diagnosed as primary aldosteronism complicated with sub-clinical Cushing's syndrome diagnosed by clinical manifestations and adrenal hormone testing. CT scan found a 4.5 cm×3.6 cm mass in right adrenal gland. The mass was surgically removed and adenoma was proved by pathologic microscopy. A somatic CTNNB1 c.133T＞C (p.S45P) mutation was detected in the adenoma tissue. Somatic mutations, such as KCNJ5, ATP1A1, ATP2B3 and CACNA1D were not detected. Conclusions Somatic CTNNB1 mutation is probably the main cause of this rare disease characterized by aldosterone- and cortisol-coproducing adrenal adenoma. But the profound mechanism needs further study.

miR-205 inhibits cell proliferation of papillary thyroid carcinoma by regulating Wnt-5a

LIU Ya-qi, ZHENG Cheng-hong

2021, 41(3):  358-363. 

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Objective To investigate whether miR-205 can regulate wingless-type MMTV integration site family member 5a (Wnt-5a) to inhibit the papillary thyroid carcinoma (PTC) cell proliferation. Methods RT-qPCR was used to detect the expression of miR-205 and Wnt-5a in PTC tissues and adjacent normal tissues.Further correlation analysis between miR-205 and Wnt-5a was carried out. The low expression of miR-205 related to the stage of PTC TMN and lymph node metastasis was also discussed. Meanwhile, RT-qPCR was also used to detect NTHY-OR-3-1 and PTC cell lines (K1 and BCPAP). In addition, the proliferation of PTC cells was detected by CCK-8 after over expression and inhibition of miR-205. Xenograft tumor formation test was used to observe whether inhibition of miR-205 acceler- ates the growth of tumor. The online target gene prediction software and dual luciferase reporter gene test were used to predict and verify whether Wnt-5a is the target gene of miR-205. Results The expression of miR-205 was significantly lower than that of surrounding normal tissues, and the expression of Wnt-5a was significantly higher. TNM stage and lymph node metastasis severity of PTC patients were significantly higher in patients with low miR-205 expression than in patients with high miR-205 expression. Low expression of miR-205 showed a poor survival rate. Expression of miR-205 in NTHY-OR-3-1 was significantly lower than that of K1 and BCPAP. miR-205 inhibited the proliferation of PTC cells. Over-expression of miR-205 also inhibited the growth of PTC. It was confirmed that Wnt-5a was the target gene of miR-205 by luciferase reporter assay. Conclusions miR-205 plays an anti-cancer role in PTC, which may be a potential therapeutic target.

Over-expression of c-SKI inhibits isoprenaline-induced myocardial fibrosis in mice

ZHANG Ying, XIANG Yan, ZHANG Yue, WANG Juan

2021, 41(3):  364-369. 

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Objective To investigate the effect of c-SKI on isoprenaline(ISO)-induced cardiac fibrosis in mice. Methods Mice were divided into 4 groups: the control group,the model group,the c-SKI gene infection group (LV-SKI group) and the normal control virus infection group (LV-NC group), with 10 in each. After two weeks, in the model group, isoprenaline(ISO) was injected subcutaneously for 30 d, with the first dose of 5 mg/kg followed by 2.5 mg/kg. In the LV-SKI group and the LV-NC group, c-SKI lentivirus and control virus were injected into the tail vein on days 18, 21, 24, 27 and 30 at a dose of 100 μL, titer was 1×10⁸ TU.HE staining and Masson staining was used to observe the pathological changes of myocardial tissue. ELISA was used to detect type Ⅰ and type Ⅲ collagen in myocardial tissue. Western blot was used to detect the expression of c-SKI, interstitial cell marker vimentin, α SMA,endothelial cell marker CD31 and the expression levels of transcription factors Snail, Twist and Slug. Results Compared with the control group, the expression of c-ski in the model group was time-dependent (P＜0.01). In the model group, the myocardial cells were disordered, the interstitium was significantly increased, and the contents of type Ⅰ and type Ⅲ collagen were increased (P＜0.01). Expressions of vimentin, α-SMA, Snail, Twist and Slug were all up regulated, while CD31 protein expression was down-regulated(P＜0.05). After over-expressing c-SKI, compared with the LV-NC group, the myocardial structure of the mice tended to be neat, the interstitial collagen fibers decreased, and the type Ⅰ and Ⅲ collagen in the myocardial tissue decreased (P＜0.01). The expression of vimentin and belt-sma was down-regulated (P＜0.05), and the expression of CD31 was up-regulated (P＜0.05). Meanwhile, the expression of transcription factors Snail, Twist and Slug was also down-regulated (P＜0.01). Conclusions c-SKI expression is down-regulated during myocardial fibrosis in mice, and over-expression of c-SKI can improve ISO-induced myocardial fibrosis by inhibiting End-MT.

Effects of administration of omeprazole on the esophageal microbiota composition in rat model with reflux esophagitis

GAO Feng, JIA Meng-zhen, SUN Yue

2021, 41(3):  370-375. 

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Objective To investigate the effect of proton pump inhibitor (PPI) omeprazole on the composition of esophageal microbiota in rats with reflux esophagitis. Methods The rat model of reflux esophagitis was established by the method of pyloric partial ligation and complete anterior gastric ligation. Rats were randomly divided into sham operation control group, reflux esophagitis control group and PPI treatment group (omeprazole). One week after the operation, distilled water/omeprazole tablets were intragastric administration once a day and two pieces of lower esophageal mucosa were obtained 2 weeks later, one for bacteria 16S rDNA gene sequencing, and another for pathological examination. Results Six surviving rats in each group were selected. The abundance values of sham operation control, reflux esophagitis control and PPI treatment groups at the genus level were as follows:Peptostreptococcus 2.5%±0.5%, 1.7%±0.7% and 1.9%±0.5%; Helicobacter 1.5%±0.1%, 1.0%±0.5% and 1.2%±0.3%;Turicibacter 1.0%±0.2%, 0.5%±0.2% and 1.0%±0.4% (P＜0.05). The abundances of Peptostreptococcus, Helicobacter, and Turicibacterin the reflux esophagitis control group were signifi-cantly reduced than those in the sham operation control group. The pathological grades of reflux esophagitis (mild/moderate/severe) in reflux esophagitis control and PPI treatment groups were 0/4/2 and 5/1/0(P＜0.05). Conclusions There are moderate changes in the composition of esophageal microbiota in rats with reflux esophagitis,the proportion of beneficial bacteria reduced. After the treatment of PPI omeprazole, the esophageal mucosal injury is alleviated, the proportion of beneficial bacteria increased, and the composition of esophageal microbiota returns to normal.

Salvianolic acid B alleviates LPS-induced acute lung injury in mice

LI Yu-ting, LUO Le, YIN Su-juan, ZHU Xiao-lan

2021, 41(3):  376-381. 

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Objective To study whether salvianolic acid B(SAB) can reduce lipopolysaccharide(LPS)-induced acute lung injury. Methods Fourty-eight mice were randomly divided into control group, model group (intratracheal instillation of LPS), SAB low/medium/high dose intervention group, and sulforaphane positive control group. The lung wet / dry weight ratio (W/D) was recorded; The protein concentration of alveolar lavage fluid (BALF) was detected; Lung tissue pathological damage was evaluated by HE staining and microoscopy; TNF-α,IL-1β and IL-6 in BALF were detected by ELISA.Western blot was used to detect the protein expression of Nrf2, NQO1, HO1, Keap1,NF-κB and p-NF-κB in lung tissue. Results Compared with the control group, pathological changes were observed in the lung tissue of the model group, W/D, protein concentration in BALF and inflammatory cytokine content were significantly increased (P＜0.05). Compared with the model group, SAB intervention group had significant changes (P＜0.05) that alleviated the oxidative stress induced by LPS, increased the expression of Keap1(P＜0.01), increased the expression of Nrf2, NQO1 and HO1 (P＜0.01), and inhibited the activation of NF-κB and p-NF-κB induced by LPS. Conclusions SAB can alleviate LPS-induced acute lung injury of mice.

Sophoridine alleviates hypoxia-induced injury of rat myocardial cell line H9c2

LU Hou-xin, YANG Qing-quan, GUO Shi

2021, 41(3):  382-387. 

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Objective To investigate the inhibitory effect and mechanism of sophoridine(Sop) on cell injury caused by hypoxia in rat myocardial cell line H9c2. Methods H9c2 cells were exposed to 1% O₂ and cultured with different concentrations (1, 4 and 16 mmol/L) of Sop for 48 h. Cell viability was measured by MTT assay. The release level of LDH and the content of MDA were determined by the kit methods. Cell apoptosis was detected by flow cytometry. The protein expression of cleaved-caspase-3, p-PI3K and p-AKT was detected by Western blot. Results Sop alleviated the cell viability, LDH release, MDA content and hypoxia-induced apoptosis of H9c2 cells in a concentration-dependent manner (P＜0.05, P＜0.01, P＜0.001). Sop decreased the cleaved-caspase-3 expression and increased the ratio of p-PI3K/PI3K and p-AKT/AKT in a concentration-dependent manner (P＜0.05, P＜0.01, P＜0.001). Conclusions Sop reduces hypoxia-induced cell injury and has a protective effect in vitro model of H9c2 cells, the potential mechanism is enhancing the stimulation activity of PI3K/AKT signal.

Diagnostic value of traditional sweat chloride test for Chinese cystic fibrosis patients

CHEN Ke-qi, LI Xue, XU Wen-shuai, GAO Jun, XIA Liang-yu, XU Kai-feng, TIAN Xin-lun

2021, 41(3):  388-392. 

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Objective To explore the accuracy of traditional sweat chloride test and its diagnostic value for Chinese patients with cystic fibrosis (CF). Methods The sweat chloride level in 118 patients who were suspected to have CF in Peking Union Medical College Hospital from Jan 2012 to Oct 2019 was measured,and the results were compared with the confirmed diagnosis. Results The patients' ages were (25.8±13.6)years old (15 months-67 years old). One hundred and two out of 118 patients obtained valid chloride level from their sweat chloride tests with median concentration of 43.3 mmol/L(IQR:23.8-120.1 mmol/L). Eighty were consistent with their diagnosis. Thirty-nine patients had sweat chloride values higher than 60 mmol/L, while 31(79.49%) of them were finally confirmed CF diagnosis. Sweat chloride levels of fourty cases were lower than 30 mmol/L, of which 32(80%) cases were excluded from CF. Twenty-three patients had sweat chloride level between 30 and 59 mmol/L, while 17(73.91%) of them were excluded from CF diagnosis. 66(64.71%) of our patients had reproducible results of the sweat chloride level. Conclusions The positive predictive value of the sweat chloride test is 79.49% and the negative predictive value is 80% using traditional sweat chloride test measurement. The traditional sweat chloride test has high diagnostic value for Chinese CF patients.

Beneficial effect of skin micro-needling in the treatment of alopecia areata and its clinical assessment

PENG Ya-wen, LEI Tie-chi

2021, 41(3):  393-397. 

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Objective To investigate the effects of skin micro-needling in the treatment of alopecia areata (AA) using a dermaroller of 1.5 mm sized needles and in the stimulation of mouse dorsal hair growth using a dermaroller of 0.5 mm sized needles. Methods Fifty-two patients with AA were recruited from the outpatient department of Renmin Hospital of Wuhan University (Mar 2018 to Aug 2019) and further divided into two group, 5% minoxidil spray and 0.05% halomethasone cream were applied topically to the control group. The micro-needling group received micro-needling treatment combined with 5% minoxidil spray and 0.05% halomethasone cream. Moreover, the dorsal hair of C57BL/6 mouse was removed and then divided into model group, minoxidil group and micro-needling group. From the next day, the corresponding treatment was given to the mice for 14 consecutive days. Anagen-associated pigmentation changes and hair growth were recorded using a digital camera. The morphology and number of anagenic hair follicles were observed using HE staining, the expression of K15 in the hair follicle was detected by an immunohistochemistry staining. Results The effective rate of the micro-needling group was superior to that of the control group (P＜0.05), after 12 weeks of treatment. As compared with the model group, the skin darkening appeared earlier and the hair length longer in the micro-needling group (P＜0.05), the number of hair follicles and the K15 expression level were greatly increased in the micro-needling group compared with those in the model group (P＜0.05). Conclusions The hair follicle epithelial stem cells may be activated by micro-needling and thus stimulates hair growth and treats AA.

Increased expression of G protein-coupled receptor 84 after cerebral ischemia-reperfusion in rats

ZHAO Chong-chong, GUO Wen-ting, CAI Hong-bin, WANG Huan

2021, 41(3):  398-403. 

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Objective To determine the expression and function of G protein-coupled receptor 84 (GPR84) in rat cerebral ischemia-reperfusion(I/R). Methods The rat middle cerebral artery occlusion (MCAO) models were performed, and rats were then divided into sham group, cerebral ischemia for 2 h and reperfusion for 12 h group (I/R 12 h), 24 h group (I/R 24 h), 48 h group (I/R 48 h), 72 h group (I/R 72 h); rat primary cortical neurons were isolated and cultured in vitro, neurons were then divided into control group (control), oxygen-glucose deprivation (OGD) for 1 h and recovery for 24 h group (OGD/R 1 h), OGD for 2 h and recovery for 24 h group (OGD/R 2 h); neurons were transfected with GPR84 small interfering RNA (siRNA), cells were divided into control group, OGD/R 1 h group, neurons pretreated with negative control and exposed to OGD for 1 h and recovery for 24 h group (NC+OGD/R 1 h), neurons pretreated with GPR84 siRNA and exposed to OGD for 1 h and recovery for 24 h group (GPR84 siRNA+OGD/R 1 h). Western blot was used to determine the expression of GPR84 protein. Immunofluorescence assay was used to analyze the localization of the immunofluorescence signals of GPR84 in cerebral ischemic tissues after MCAO. TdT-mediated dUTP nick end labeling (TUNEL) assay was used to detect the percentage of apoptotic neurons. Results Compared with the sham group, the expression of GPR84 protein was significantly up-regulated at each time point after cerebral I/R(P＜ 0.01). In addition, GPR84 was mainly localized in neurons and microglia, while little GPR84 was found in astrocytes. Neurons in the MCAO group showed a higher level of GPR84 expression than those in the sham group. GPR84 knockdown significantly inhibited the increase of GPR84 expression induced by OGD/R. Compared with the control group, neurons in the OGD/R 1 h group exhibited higher apoptotic cells percentage. Compared with the NC+OGD/R 1 h group, the percentage of apoptotic neurons in GPR84 siRNA +OGD/R 1 h group was significantly decreased. Conclusions These results suggest that the elevated GPR84 expression induced by cerebral I/R may play an important role in cerebral ischemia injury.

Lnc-MALAT1 attenuates miR-217 inhibiting LPS-induced inflammatory response in rat alveolar macrophages

LUO Chuan-ling, SUN Li-ping, QI Cheng-dong

2021, 41(3):  404-408. 

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Objective To investigate the regulatory mechanism of long-chain noncoding RNA metastasis-associated lung adenocarcinoma 1 (lnc-MALAT1) in the inflammatory response of lipopolysaccharide (LPS)-induced alveolar macrophages (AMOs). Methods AMOs cells were treated with LPS for 12 hours to create an inflammation model, and liposome method was used to transfect pcDNA, pcDNA-MALAT1, miR-NC, miR-217 mimics, pcDNA-MALAT1 and miR-NC, pcDNA-MALAT1 and miR-217 mimics to AMOs. The level of IL-1β and TNF-α was detected by ELISA. RT-qPCR was used to detect the expression of MALAT1 and miR-217 in cells. The dual luciferase report experiment and RIP experiment were used to detect potential target-attacking relationship between MALAT1 and miR-217. Results Compared with the AMOs group, the expression of IL-1β and TNFα, MALAT1 in the cells of the LPS+AMOs group were significantly increased (P＜0.05) and the expression level of miR-217 was significantly reduced(P＜0.05). The dual luciferase report experiment and RIP experiment confirmed that MALAT1 targeted at miR-217. Over-expression of MALAT1 could obviously promote the secretion of IL-1β and TNF-α induced by LPS in AMOs, while over-expression of miR-217 inhibited the secretion of IL-1β and TNF-α induced by LPS in AMOs. Furthermore, over-expression of miR-217 reduced the secretion of IL-1β and TNF-α induced by MALAT1 in LPS-induced AMOs. Conclusions Lnc-MALAT1, a long non-coding RNA, can promote the inflammatory response of AMOs induced by LPS, and its mechanism is related to its targeting at miR-217.

Constructing a risk predictive model for recurrence following nephron-sparing surgery in the treatment of clear cell renal cell carcinoma

CHEN Zhi-gang, HONG Peng, YANG Bin, TIAN Xiao-jun, WANG Guo-liang, ZHANG Shu-dong, MA Lu-lin

2021, 41(3):  409-414. 

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Objective To detect factors associated with recurrence after nephron-sparing surgery (NSS) in patients with localized clear cell renal cell carcinoma (ccRCC), and to construct a model for predicting the recurrence risk in five years following NSS. Methods Data of the patients who underwent NSS for renal occupied lesions between January 2006 and July 2019 at Peking University Third Hospital were retrospectively analyzed. Univariate and multivariate logistic regression analyses were used to assess the factors associated with recurrence of ccRCC after NSS, and a nomogram was established to predict recurrence risk in 5 years following NSS. Results Totally 795 patients who were pathologically diagnosed with ccRCC were included in the study. Recurrence occurred in 17 cases, with the recurrence rate 2.1%. The median time to relapse was 35.7 months. In the univariable analysis, BMI (HR=4.102, P＜ 0.05), the artery blocking time (HR=0.194, P＜ 0.01) and tumor necrosis or sarcomatoid change (HR=5.160, P＜ 0.01) were associated with recurrence-free survival of the ccRCC patients after NSS. The multivariate analysis showed that these three factors were independent factors of recurrence. The accuracy of our nomogram for recurrence risk was verified using internal validation, and the value of c-index was 0.843. Conclusions BMI, artery blocking time and tumor necrosis or sarcomatoid change were independent factors for recurrence following NSS in the ccRCC patients. An accurate model to predict the recurrence risk is constructed, which may help to provide personalized managements for the aforementioned patients after NSS.

Clinical Sciences

Changes of peripheral blood lymphocyte subsets in patients with psoriasis

LIU Yan-min, ZHAO Xin-cheng, LI Jun-qin, ZHANG Kai-ming

2021, 41(3):  415-418. 

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Objective To investigate the changes and immune mechanisms of peripheral blood lymphocyte subsets in psoriasis patients with progressive, stationary and regression stages. Methods The percentage of lymphocytes in each group of peripheral blood mononuclear cells (PBMCs) in 77 patients with psoriasis vulgaris including 28 patients during progression, 23 patients during stationary, and 26 patients during regression. Results The percentages of CD3⁺, CD4⁺ and CD8⁺ lymphocytes were significantly increased in both the progression and stationary stages as compared to the regression stage (P＜0.01). CD4^- and CD8^- subsets in the peripheral blood lymphocyte subsets of the patients showed a decreasing trend according to progression, stationary and regression stages(P＜0.05). The result showed that the proportion of CD3^-CD16⁺CD56⁺ increased with the stability of the disease, the proportion of CD45⁺CD14⁺ gradually decreased, and the proportion of CD3^-CD19⁺ was negatively correlated with the disease (P＜0.05). Conclusions There are significant differences in lymphocyte subsets in the three groups of patients with psoriasis, which further supported the immune pathogenesis of psoriasis.

Analysis of 2 cases of glycogen storage disease typeⅠb with Crohn's disease in children

TANG Xiao-yan, ZHANG Yu, LI Zheng-hong, QIU Zheng-qing

2021, 41(3):  419-422. 

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Objective To investigate the clinical characteristics of glycogen storage disease typeⅠb(GSDⅠb) with Crohn's Disease(CD) and to improve the recognition of the disease. Methods Retrospectively analyze the clinical characteristics of 2 cases diagnosed with the GSDⅠb and CD in the pediatric department of Peking Union Medical College Hospital. Results A boy and a girl were included in the study. They were genetically diagnosed as GSDⅠb at 2 years age. Gastrointestinal symptoms including abdominal pain and diarrhea were observed at 4, 5 years old respectively. They were diagnosed as CD with endoscopic characteristics and pathology results. Besides corn starch, granulocyte colony-stimulating factor(G-CSF) was used to elevate the neutrophil to above 1.0×10⁹/L. The gastrointestinal symptoms got alleviated. 5-aminosalicylic acid agents were used as supportive treatment. Conclusions Evaluation of the gastrointestinal system is necessary as the abdominal pain or diarrhea were observed in GSDⅠb patients. G-CSF with a relatively low dose is the mainstay to elevate neutrophil with few side effects. Splenomegaly is the most common side effect. Monitor renal function is necessary in the use of 5-ASA.

Mini Reviews

Research progress of blood hypercoagulable state in liver cirrhosis

QI Wan-rong, MAO Xiao-rong, LI Jun-feng

2021, 41(3):  423-427. 

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The patients with liver cirrhosis are often complicated with coagulation dysfunction, and blood hypercoagulable state is one of the manifestations. Blood hypercoagulable state is not only involved in thrombosis, the latest evidence confirms that it is also involved in liver injury, liver regeneration and liver fibrosis. This review attempts to discuss the mechanism of blood hypercoagulable state in liver cirrhosis, the role of intestinal microecological environment in it and the latest influence mechanism of blood hypercoagulable state in liver cirrhosis.To provide a new concept for the diagnosis and treatment of patients with liver cirrhosis.

Research progress on metabolism-related biomarkers of frailty in the elderly

PAN Yi-ming, LI Yun, MA Li-na

2021, 41(3):  428-432. 

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Frailty is a common syndrome in the elderly, identified by the decline of physiological functions in multiple organ systems, which may lead to adverse outcomes such as falls, cognitive impairment, hospitalization or death. Early diagnosis of frailty is of great value in promoting healthy aging. At present, it can only be diagnosed by assessment tools in clinical without specific biomarkers. Frailty is often accompanied by metabolic disorders in many aspects, such as changes in metabolism of energy, muscle, bone, and in endocrine system. The study of metabolism-related biomarkers can help early diagnosis and intervention of frailty.

Progress in molecular mechanism and targeted drugs of bone metastases in breast cancer

ZHENG Ya-nan, LI Mei, ZHAO Ji-yuan

2021, 41(3):  433-437. 

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Breast cancer is the most common malignant tumor that threatens the health of women around the world, and bone is the site most prone to metastasis. The molecular mechanism concerned and targeted therapy against bone metastasis of breast cancer have become the current research hotspots. This article summarizes the key molecules involved in regulating bone metastasis of breast cancer. The important targeted therapy drugs are introduced and their efficacy is compared in two categories of those targeting at osteoclasts and those targeting at osteoblasts.

Research progress of lipoedema

WANG Li-quan, LONG Xiao

2021, 41(3):  438-441. 

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Lipoedema is an uncommon disease disorder characterized by bilateral enlargement of the legs due to abnormal depositions of subcutaneous fat and essentially affects females. The disease has been reported with genetic tendency and can also be affected by hormone level of the host. Lipoedema has characteristic clinical manifestations, like pain, easy bruising and “cuff sign”. In clinical, main diseases considered for differential diagnosis are lymphedema, obesity, Dercum's disease and chronic venous disease. Management of lipoedema includes psychological health education, weight loss, conservative treatment and operative treatment. There are increasing reports supporting liposuction using super tumescent local anesthesia and vibrating cannulas as the preferred surgical option.

Progress of ferroptosis mechanism and application in lung cancer therapy

XU Fei, CHEN Wei-da, GUO Ming-hao, CHEN Ze-tao

2021, 41(3):  442-447. 

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Ferroptosis is a newly discovered mechanism of cell death characterized by accumulation of iron dependent lipid peroxidation and reactive oxygen species, which is different from apoptosis, necrosis and autophagy in morphology and biochemical findings. It is involved in the occurrence and development of lung cancer, so inducing ferroptosis seems to become a new strategy for anti-lung cancer therapy. This review includes the followings: mechanism of ferroptosis from the aspects of iron metabolism, amino acid and glutathione metabolism and lipid metabolism; its effects on lung cancer occurrence and progression, chemotherapy resistance, radiation resistance and immunotherapy respectively, in order to update information on ferroptosis and thus provides a new direction and new strategy for lung cancer treatment.

Advances on the roles of tumor microenvironment in bone cancer pain

ZHOU Jia-hong, LI Min

2021, 41(3):  448-451. 

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Tumor microenvironment (TME) is the in vitro microenvironment in which tumors live. Cancer-induced bone pain(CIBP) is one of the most common symptoms of tumor bone metastasis. The mechanisms of bone cancer pain(BCP) are complex, and the tumor acidic microenvironment, the related ion channels and inflammatory mediators released by cancer, play a role in BCP.

Medical Education

Analysis of the needs and feedbacks of diagnostic ultrasound teaching based on questionnaire at Peking Union Medical College

WANG Xin, WANG Ming, ZHAO Jun, WANG Ya-hong, LYU Ke, ZHU Qing-li, LI Jian-chu, JIANG Yu-xin, YANG Meng

2021, 41(3):  452-455. 

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Objective This study aims to investigate the current situation of ultrasonographic teaching and learning needs from eight-year program undergraduates in Peking Union Medical College. Methods Questionnaires of the current situation about ultrasonographic teaching and learning needs from students were collected and compared with feedbacks from students from 4+4 pilot project curriculum who received a new pattern of ultrasonographic teaching. Results The students of eight-year program showed weakness in understanding of definition and development of ultrasound discipline, basic knowledge and ultrasonographic technology. They were not satisfied with the current ultrasonographic teaching and hoped to improve the curriculum content and teaching pathway. Conclusions Based on these results, the ultrasonographic curriculum design and teaching patterns should be adjusted according to students' demands and emphasize essential and basic knowledge such as ultrasonographic manifestations of normal human anatomy and that found in common diseases.

Application of virtual simulation experiments in physiological experimental teaching

KANG Ji-hong, HAN Li-li, PANG Wei, ZHU Shi-gong

2021, 41(3):  456-458. 

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Objective To evaluate the teaching effects of physiological virtual simulation experiments. Methods In spring semester of 2020, virtual simulation experiments were applied in the physiological experimental course in Peking University Health Science Center. The teaching effect was evaluated thereafter by a questionnaire based on the network tool “SOJUMP”. Results The results from the analysis of the 278 questionnaires showed that virtual simulation experiments based on the internet platform improved the students'interest in learning, promoted the integration of theoretical and experimental teaching, and helped training the students'independent learning capacity. Virtual simulation experiments were recognized by the vast majority of the students. Conclusions The teaching platform applied in physiological virtual simulation experiment is a new teaching model to supplement to the traditional physiology experimental course. The combined application of virtual simulation experiments and animal experiments may support the integration of virtual and actual experiments so as to promote the quality of teaching and the capacity building of students for independent learning.

Application and evaluation of inquiry teaching mode for graduated students in large-scale biomedical equipment courses

SUN Hai-dan, GUO Zheng-guang, ZHOU Jun, PIAN Hong-ru, YANG Ming-zhu, WANG Xin, ZHENG Zhi

2021, 41(3):  459-462. 

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Objective To test and evaluate the inquiry teaching (IT) mode for graduates students in course of Theory and Application of Large-scale Biomedical Equipments and provide a basis to support its routine application in the future. Methods Design the experiments basing on the IT mode. Summarize the strategy, performance and results. Evaluate the training outcomes with the education monitoring theory and the statistic approaches. Analyze the questionnaire data quantitatively according to reliability, correlation coefficient and discrimination. Results Experiments derived from inquiry teaching (IT) mode were employed in grade 2017 and 2018 graduats . The primary results are satisfied. There are 23 students involving these tests. The reliability of questionnaire is good with α value as 0.852. Twenty-three questions in 27 ones show the significant differences. Conclusions The results of questionnaire show satisfied reliability, correlation coefficient, discrimination and coverage. This indicates that this mode may be employed in evaluation of teaching. The design, difficulty and schedule are major influencing factors of IT experiments.

Effect evaluation and feedback of ultrasound medicine online teaching during the COVID-19 pandemic

SUN Yan, JIANG Ling, WANG Shu-min, JIANG Jie, CHEN Wen

2021, 41(3):  463-466. 

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Objective To explore the application of online teaching of ultrasonic theory during the COVID-19 pandemic. Methods The standardized training of undergraduates, graduates and refresher doctors who have participated in the online theoretical teaching of ultrasound in the Peking University Third Hospital. Compared with the assessment results in previous years as the Objective index, the questionnaire was used as the subjective evaluation index. Results There was no significant difference between the experimental group and the control group. It is shown that 78.3% of learners belives that the online teaching method got better than the conventional offline teaching method. Conclusions The quality of online teaching is similar to that of conventional teaching mode but both trainers and trainees prefer the online teaching.