Table of Content

05 July 2017, Volume 37 Issue 7

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Down-regulation of FTO in human gastric cancer and its effects on cell line MGC-803 function

2017, 37(7):  907-911. 

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Objective To investigate the expression of FTO in gastric cancer tissues and the functional significance of FTO in MGC-803 cell line. Methods The FTO mRNA was detected by RT-qPCR in 54 cases of gastric cancer samples and their paired adjacent normal control tissues. The effect of FTO overexpression in MGC-803 on cell proliferation, cell migration and invasion were detected by CCK-8, wounding heal and transwell assays, respectively. Results The expression of FTO mRNA was significantly lower than that of adjacent tissues (P<0.05). Furthermore,overexpression of FTO in MGC-803 cells inhibited cell proliferation, cell migration and invasion.Conclusions FTO was low expressionin gastric cancer tissues and inhibits gastric cancer cell line MGC-803 proliferation, migration and invasion.FTO is closely associated with the development of gastric cancer.

Murine pancreatic injury induced by D-galactose

2017, 37(7):  912-917. 

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Objective To explore the effect of D-galactose(D-gal)on murine pancreatic injury and its mechanism.Methods C57BL/6J mice were randomly divided into Control group and D-gal model group(D-gal 120mg/kg/d for 42 days).On the 2nd day after drug injection completed,the peripheral blood were taken for measuring the level of fasting blood glucose(FBG) and fasting insulin(FINS);and then the organ index of pancreas were calculated by the ratio of pancreatic wet weight(mg) and mouse body weight(g);HE stain were routinely prepared to observe the histologic structure of pancreatic tissue;the transmission electron microscopy was used to analyze ultrastructural changes of pancreatic cells;the pancreatic frozen sections were prepared to test senescence-associated β-galactosidase(SA-β-Gal) and its relative absorbance(RA) of positively stained cells in the pancreatic islets; mmunohistochemistry assays to study advanced glycation end products(AGEs) and its RA; pancreas tissue homogenate was made to detect the content of superoxide dismutase(SOD), malonaldehyde(MDA) and total antioxidant capacity(T-AOC). Results In D-gal group mice, the FBG increased markedly(P<0.05) and FINS reduced; pancreas wet weight and organ index raised obviously(P<0.01); light microscopic structure of the pancreas presented without typical pathologic change, however the single nucleated cell’s area within the islet was increased significantly(P<0.05); the pancreas endocrine and exocrine cells were showed the ultrastructure damaged and lipofuscin formation increased; the RA of positive pancreas cells in SA-β-Gal staining increased obviously(P<0.05); the RA of AGEs positive regional expression markedly increased(P<0.01); the content of SOD and T-AOC decreased obviously(P<0.05), the content of MDA increased markedly(P<0.01). Conclusion Aging mice model replicated by D-gal can cause the pancreatic injury, its mechanisms may be closely related to oxidative injury of pancreatic cells caused by D-gal.

Immunogenic cell death of human osteosarcoma MG-63 cells induced by capsaicin

2017, 37(7):  918-922. 

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Objective To investigate the effect of capsaicin and cisplatin on the proliferation and immunogenic cell death of human osteosarcoma cells. Methods MTT assay was used to examine the growth inhibiting effects of capsacin and cisplation on MG-63 cells; Mitochondrial membrane potential (MMP) was used to investigate the apoptosis; flow cytometry was used to detect the expression of calreticulin(CRT) on the cell membrane, fluorescein enzymatic method was used to detect the release of ATP and ELISA was used to detect the secretion of high mobility group B1 (HMGB1). Results Capsaicin and cisplatin can inhibit MG-63 cells proliferation in the dose-dependent manner and induce MG-63 apoptosis（P＜0.01）. Only capsaicin can induce translocation of CRT from endoplasmic reticulum to the cell surface and release of extracellular ATP and HMGB1（P＜0.01）. Conclusions Capsaicin can induce human osteosarcoma cells apoptosis and immunogenic cell death.

Hyperthermic intraperitoneal perfusion associated with cisplatin promote apoptosis in human ovarian cancer cell lines

2017, 37(7):  923-928. 

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Objective To investigate the effect of hyperthermic intraperitoneal perfusion and cisplatin on apoptosis in human ovarian cancer cells. Metheds Two human ovarian cancer cells (OVCAR-3, A2780) were divided into control group, cisplatin group, hyperthermia group and thermo-chemotherapy group; microscope was used to observe the morphological changes of the four groups; AO/GV stain and flow cytometry(FCM) was used to analyse cell apoptosis; Detection of apoptosis related genes CASPASE7、 CASPASE8 and BAX in ovarian cancer cells by fluorescence quantitative PCR. Results Inverted microscope observeed that the ovarian cancer cells were retracted and suspended partially in the cisplatin group and hyperthermia group, especially in the thermo-chemotherapy group. After AO/GV staining, the apoptotic cells were increased in the cisplatin group and hyperthermia group compared with the control group, and the thermo-chemotherapy group is more than cisplatin group and hyperthermia group. FCM results indicated that the proportion of cells apoptosis were higher in the cisplatin group and hyperthermia group, the thermo-chemotherapy group is the highest than the other groups(p<0.05). q-PCR results showed that in the thermo-treatment group the expression of pro-apoptotic genes, including CASPASE3、CASPASE6、CASPASE7、CASPASE8、CASPASE9，BAX、BAK and BID, were significantly higher than other groups, apoptosis inhibitory gennes, such as BCL-2、BCL-XL、MCL-1、c-FLIP, were significantly decreased than the others. Conclusion Cisplatin and hyperthermia can promote the apoptosis in ovarian cancer cells, especially by the thermo-chemotherapy.

Construction of lung cancer cell model overexpressing human MutS Homologue 2（hMSH2）

2017, 37(7):  929-934. 

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Objective To construct human lung cancer cell model with human MutS homologous protein 2 (hMSH2) overexpression, for exploring the effect of hMSH2 molecule in the cytotoxicity of γδ T cell against lung cancer cells. Methods hMSH2 coding sequence was cloned by PCR for construction of recombinant vector which over expressed hMSH2-EGFP fusion protein using homologous recombination. The recombinant vector was transfected to lung cancer cell line NCI-H520 to construct human lung cancer cell model overexpressing hMSH2 molecule. The expression of hMSH2 molecule in NCI-H520 was detected by Western blot. The expression of hMSH2 on the cell membrane was measured by flow cytometry. Cytotoxicity of expanded γδ T cells from peripheral blood mononuclear cells against NCI-H520 cells was detected by LDH release assay in vitro. Results hMSH2 coding sequence (2805 bp) was cloned and the result of restriction endonuclease digestion of Fugw-hMSH2 recombinant vector was accordance with the anticipated objective strip size. Exogenous hMSH2-EGFP fusion protein was expressed in NCI-H520 cells. The level of hMSH2 molecule on the surface of NCI-H520 cells with overexpression of hMSH2 was significantly increased (P<0.001). Cytotoxicity of γδ T cells against NCI-H520 cells with overexpression of hMSH2 was significantly increased compared to the wild type NCI-H520 cells (P<0.05). Conclusions Lung cancer cell model that overexpressed hMSH2 molecule is successfully constructed, hMSH2 molecule on the cell membrane is increased and the cytotoxicity of γδ T cells against lung cancer cells is enhanced.

Identification of a novel DNA aptamer against albumin

2017, 37(7):  935-938. 

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Objective To develop an albumin aptamer that may potentially serve as a selective ligand for albumin removal from experimental samples. Methods A single-stranded 59nt DNA library that contains 21 random oligonucleotides was synthesized in vitro．An albumin aptamer A6 was developed by SELEX technique using bovine serum albumin (BSA) as target．The enrichment of aptamer and evaluation of its binding properties were monitored by flow cytometry. The secondary structure of A6 was predicted by MFord software. Results The aptamer A6 bound to BSA strongly with a Kd of 77.4 nmol/L, and had minimal cross reactivity with control proteins including ovalbumin, IgG, and trypsin. Conclusions Aptamer A6 may have potential in albumin removal applications.

Establish HPLC method to detect urocanic acid in serum and investigate its significance for childhood leukemia

2017, 37(7):  939-944. 

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Objective To establish a method for detection of serum urocanic acid (UCA) by high performance liquid chromatography (HPLC), and explore the clinical significance of serum UCA concentration for children acute leukemia. Methods The chromatographic conditions of HPLC were set up and optimized, and the linearity of standard curve, precision, accuracy and stability were validated.Then the serum from ninety acute leukemia children and ninety non-tumor blood disease children was collected,the concentration of serum UCA was detected with HPLC,and the differences of two groups were compared to study the clinical significance of UCA in children acute leukemia. Results The HPLC method for detecting serum UCA was successfully optimized and established.The standard curves of trans-UCA and cis-UCA both showed good linearities (R2=0.9996 and 0.9999) at the condition of the mobile phase of acetonitrile-20 mmol/L KH2PO4,pH 3.7(5:95,V/V),flow rate of 1.2 ml/min,detection wavelength of 264 nm in HPLC. The relative standard deviation RSD% of intra-assay and inter-assay were lower than 5%. Compared with non-tumor blood disease, the serum concentration of cis urocanic acid (cis-UCA) and trans urocanic acid (trans-UCA) of children acute leukemia were significantly increased (P<0.001). Compared with cis-UCA, trans-UCA was more valuable for risk classification of acute lymphoblastic leukemia (ALL). Conclusions HPLC is a good way to determine the concentration of UCA in serum. The concentration of serum UCA in children acute leukemia may provide the clues for diagnosis and prognosis, with important clinical significance.

Construction and identification of shRNA Lentiviral vector targeting TPX2 gene

2017, 37(7):  945-952. 

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Objective To construct the Lentiviral RNA interference vector targeting TPX2 and obtain the human cervical cancer hela cell line stably infected by TPX2-shRNA ,which laid research foundation for studying the relationship between human cervical carcinoma and TPX2 gene. Methods By targeting TPX2 gene, four double-stranded DNA hairpin structures corresponding to shRNA were designed, synthesized and connected with Pglv2-U6-Puro to construct the recombinant plasmids.Then these recombinant plasmids were transformed into DH5α competent cells. The positive clone was extracted and transfected into 293T cells for virus packages after sequenced correctly.Human cervical carcinoma HeLa cell line infected by these recombinant Lentiviral was screened by Puromycin, then stable cell lines was obtained.The silencing effect of TPX2 in HeLa cell was detected by Real-time fluorescent quantitative PCR and Western blot. Cell cycle and cell apoptosis wer detected by Flow cytometry.Results Sequencing results confirmed that 5 Lentiviral is packaged successfully. The steady cell lines transfered TPX2-shRNA was screened with 0.4ug/ml puromycin. HeLa cells infected by recombinant lentivirus all play the gene silencing effect especially in the group of TPX2-shRNA-1.In the group of TPX2-shRNA-1,TPX2mRNA (0.21 ± 0.07) and protein(0.19 ± 0.28) relative expression levels are lower than those in the control group (1.08 ± 0.07) (p<0.01)and(0.64 ± 0.03)(p<0.01)respectively;G2 and S-phase cells are higher than those in the control group (p<0.05)and the apoptosis rate was significantly more than those in the control group (p<0.05).Conclusions.The effective TPX2 genetic interference sequence was Obtained, Lentiviral vectors carrying TPX2shRNA was successfully constructed,and the HeLa cell line with TPX2 Silenced were successfully screened,which lay the research foundation for the role of TPX2 in cervical cancer.

Cytotoxic role of γδ T cells to latency cells in patients with early human immunodeficiency virus-1 infection

2017, 37(7):  953-958. 

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Objective To investigate the cytotoxicity of γδ T cells to HIV-1 latency cell in patients with early HIV-1 infection. Methods Sixteen early HIV-1-infected patients were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) of patients were isolated and γδ T cells were expanded using zoledronate (5 μmol/L) and interleukin (IL)-2 (1 000 IU/mL) ex vivo. Lactic dehydrogenase (LDH) was used to detect the cytotoxic role of γδ T cells to HIV-1 latency cell (J-Lat Full Length Clone10.6). The phenotype of γδ T cells before and after expansion and the intensity of GFP in HIV-1 latency cells were detected by flow cytometry. Results Zoledronate plus IL-2 stimulate rapid and large γδ T cells proliferation ex vivo (P<0.001). γδ T cells showed high cytotoxicity to latency cells, and the intensity of GFP in latency cells were decreased significantly (P<0.05). Moreover, expanded γδ T cells displayed cytotoxic NK-like phenotype, the frequency of CD56+Vδ2 T cells in patients with early HIV-1 infection was significant higher than that in healthy controls. Conclusions γδ T cell has an ability to eradicate HIV-1 latency, and γδ T cell-based autologous or xenogenous adoptive immunotherapy will be promising prospects to cure HIV-1 infection.

miR-130b accelerates the neuronal migration in the developing embryonic mice cortex

2017, 37(7):  959-962. 

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Objective Using In Utero Electroporation technique to study the effects of miR-130b on the neuronal migration in the developing embryonic mice cortex. Methods Pregnancy 15.5 KM mice were selected and plasmid of miR-130b was injected into the lateral ventricle of the embryonic brain. In 2 days after eletroporation, mouse embryos were collected and cut into frozon coronal slices, then surveyed the neuronal migration under the fluorescence microscope. Results The neuronal migration rates were higher in miR-130b overexpression embryonic cortex, of which 75.1% eletroporated neurons migrated into MZ, compared with the 22.1% in control group. On the contrary, only 7.9% eletroporated neurons stayed in the VZ/SVZ in the experimental group, compared with the 69.3% in control group. Conclusions miR-130b accelerates the neuronal migration in the developing embryonic mice cortex, its regulational role is worth studying.

Interferonγ promotes terminal erythroid differentiation

2017, 37(7):  963-969. 

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Objective To study the effects of interferonγ(IFN-γ) on in vitro terminal erythroid differentiation. Methods Real-time PCR was used to detect the expression of IFN-γ receptors during erythroid differentiation of K562 induced by hemin. Both hemin-induced K562 cells and human umbilical cord CD34+ cell derived primary erythroid cells were treated with IFN-γ. Erythroid differentiation of the cells was evaluated using real-time PCR to detect the mRNA level of erythroid specific surface markers CD71 and CD235a, and benzidine staining assay was applied to explore the change of hemoglobin expression. Results The expression of IFN-γ receptors in K562 cells was first decreased and climbed up again after reaching the lowest point at 48 h of hemin induction. IFN-γ treatment increased CD71 and CD235a expression in both hemin-induced K562 cells and the later stage (E15D) primary erythroid cells. Benzidine staining showing increased globin protein expression in hemin-induced K562 cells after IFN-γstimulation. Furthermore, our results indicate that IFN-γ promotes hemin-induced K562 erythroid differentiation in a time dependent manner. Mechanistically, the results showed that IFN-γ treatment stimulates the expression of erythroid transcription factors NFE2, which is critically for erythroid maturation. Conclusions IFN-γ accelerates terminal erythroid differentiation in hemin-induced K562 cells and human umbilical cord CD34+ derived primary erythroid cells.

UrotensinⅡ promoting collagen expression in rats cardiac tissues and proliferation of cardiac myofibroblasts in new-born rats

2017, 37(7):  970-974. 

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Objective To investigate the effect of urotensin II on myocardial fibrosis in rats. Methods The pressure overload animal model was established in rats by abdominal aorta coarctation. The rats were divided into sham operation group, modeled for 4, 8 and 12 weeks group. The expression changes of UⅡ, GPR14, col-Ⅰ, col-Ⅲ, and PKA in cardiac tissues were detected by Western blot. Isolated and cultured cardiac myofibroblasts (CFs) from new-born SD rats were treated with UⅡ、KT5720 or SB-611812, and then the proliferation of CFs were observed by microscope and CKK-8. Results The expression of UⅡ,GPR14, col-Ⅰ,col-Ⅲand PKA increased markedly in cardiac tissues of model rat, which were time-dependent. UⅡpromoted the proliferation of CFs (P<0.05), which could be inhibited by KT5720 or SB-611812. Conclusion UⅡ/UT system promoted the occurring and development of myocardial fibrosis.

Isolating culture of adipose mesenchymal stem cells in psoriasis vulgaris patients and the differentiation into immune regulation function

2017, 37(7):  975-981. 

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Objective To study the differences of the biological characteristics and immune regulation function of adipose mesenchymal stem cells (AMSCs)from psoriasis patients and healthy people. Methods AMSCs were isolated and cultured from human psoriatic and healthy adipose tissue, the phenotypes and cell cycle of AMSCs taken from three generation were detected by flow eytometry. Alkaline phosphate enzyme staining and oil red o staining were used respectively to identify their adipogenic and osteogenic capacity . Next，the levels of inflammation antimicrobial proinflammatory factor were detected by PCR and ELISA.Then gene expression profile of AMSCs were screen by gene expression profile chip，as so to bolting the the gene array related with immunology gene. Results We found that there were no significant change in cell morphology, and cell surface markers were expressed high for CD29、CD44、CD73, while lower for CD31、CD45 and HLA-DR. AMSCs of psoriasis patients and healthy people both had the ability of adipogenic and osteogenic differentiation. But the cell cycle showed the third generation AMSCs proliferation rates were slower than of normal control , as compared with healthy controls，adipogenic differentiation ability was stronger. What’more, the levels of inflammatory cytokines in psoriasis group was lower than that in controls such asIL-10、IDO、TGF-β, on the contrary the levels of proinflammatory factor in psoriasis group were higher than that in controls, such as TNF-α，IFN-Y. In addition, gene chip results suggested that psoriasis group AMSCs had obvious expression differences on JAK - STAT pathway with healthy controls. Conclusion Compared with the control, there are significant differences in patients AMSCs proliferation and adipogenic differentiation ability, immune inflammation suppression control ability is weaken,we speculate that this phenomenon may be associated with JAK - STAT immune pathways related to downgrade.

Effects of estrogen replacement therapy on the myelin sheath of cerebral white matter and hippocampus and Lingo-1 expression in middle-aged ovariectomized rats

2017, 37(7):  982-987. 

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Objective To detect the expressions of Lingo-1 and myelin associated protein in the white matter and hippocampus of ovariectomized rats after short-term estrogen replacement therapy in order to explore the possible mechanisms for the effects of estrogen on the brain myelin sheaths and cognitive function. Methods 24 middle-aged (9-12 months) female Sprague-Dawley (SD) rats were bilaterally ovariectomized (OVX) and randomly divided into vehicle replacement (OVX+Veh) group and estrogen replacement (OVX+E) group. After one month ERT, The spatial learning and memory ability of all rats were assessed with Morris water maze. Then, 10 rats were randomly selected from each group. The ultrastucture of myelin sheaths in the cerebral white matter and hippocampus were observed using electron microscope transmission, and the protein expression levels of MBP and Lingo-1 were investigated with western blot and immunohistochemical staining. Results The escape latencies of OVX+E rats in navigation test were significantly shorter than that of OVX+Veh rats(P < 0.05). The myelin sheaths in the white matter and hippocampus of OVX+Veh rats showed obviously degeneration. In the OVX+E group, the expression of MBP in the white matter and hippocampus were significantly higher than that of OVX+Veh group(P < 0.05), however, the expression of Lingo-1 were significantly lower than that of OVX+Veh group(P < 0.05). Conclusions 1-month ERT had significant beneficial effects on the spatial learning capacity and myelin sheaths in the white matter and hippocampus. The protective effects may be related to estrogen-induced downregulation of the Lingo-1 expression in the white matter and hippocampus of rats.

NS4A protein of Zika virus influences the neuronal migration of mouse cortex

2017, 37(7):  988-993. 

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Objective To determine the effect of NS3 and NS4A proteins of Zika virus on the neuronal migration in vivo. Methods To identify the coding sequence of NS3 and NS4A, the genome of Zika SZ01 was sequenced by rapid amplification of cDNA ends (RACE) and reverse-transcription PCR, then NS3 and NS4A was constructed in pCIG vector fused with Flag-tag to express these proteins. And then these plasmids was transfected into the embryo brain of E13.5 mice by in utero electroporation, the distribution of the cells which express these proteins in the cortex was detected by Flag, GFP and TBR1 fluorescence in E18.5 mice through immunohistochemistry so as to assess the influence of viral proteins on the neuronal migration of mouse cortex. Results 1) Sequence results showed that the amino acid sequence of NS4A is consistent with NCBI data, while NS3 has an amino acid mutant. 2) As the fluorescence of Flag and eGFP can co-localization, the eGFP fluorescence signal can indicate the cells that have expressed these virus proteins in cortex. 3) TBR1 fluorescence shows the distribution of the cells that express NS4A in vivo are significantly different from pCIG control and NS3 (p<0.001). Conclusions The NS4A protein of Zika virus may affect the neuronal migration in vivo.

Up-regulation of Keap-1 inhibits the nuclear import of Nrf-2 and decreases cisplatin resistance in ovarian cancer cells Skov3/DDP

2017, 37(7):  994-999. 

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Objective To test the status of Keap-1/Nrf-2 pathway in cisplatin-resistant Skov3/DDP cells, and the contribution of these molecular in cisplatin (cis-pt) resistance. Methods Skov3/DDP was divided into four groups：blank, cis-pt, Keap-1 up-regulated and Keap-1 plus cis-pt. Keap-1 was up-regulated in Skov3/DDP cells by trans-faction with pFlag-CMV-Keap-1 plasmid which was validated by expression of flag and increase of Keap-1 level using Western blot. The effect of Keap-1 up-regulation on Nrf-2 re-distribution was analyzed by Western blot. FCM was employed to detect apoptosis. ROS level was measured by DHE staining. Results The expression of Keap-1 was decreased obviously in Skov3/DDP cells compared with Skov3 cells (P<0.05), Nrf-2 in Skov3/DDP cells detected mainly in nucleus. Then, the Keap-1 was successfully up-regulated (3.5 fold) by exogenous expression of pFlag-CMV-Keap-1 plasmid, as a result, nucleus portion of Nrf-2 was significantly reduced (0.2 fold). Next, the FCM results showed significant increase of apoptosis in Keap-1 up-regulated Skov3/DDP cells after cis-pt administration (P<0.05). Finally, ROS level was raised significantly in Skov3/DDP cells treated with cis-pt plus Keap-1 up-regulation. Conclusions Up-regulation of Keap-1 reduced the nucleus portion of Nrf-2 and reverses cisplatin resistance in Skov3/DDP cells.

Value of neutrophil-lymphocyte count ratio in diagnosing bloodstream infection

2017, 37(7):  1000-1003. 

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Objective To explore the diagnostic value of five infection markers in bloodstream infection. Methods Randomly selected 110 bloodstream infection patients with positive blood cultures and 30 bacterial infection patients with negative blood cultures. Blood were drawn simultaneously with blood cultures; the complete blood count and C-reactive protein (CRP) levels were measured. The white blood cell count(WBC), neutrophil count (NEU), lymphocyte count (LMY), CRP level and neutrophil-lymphocyte count ratio (NLCR) were compared between the two groups. Results The levels of WBC, NEU, NLCR and CRP in bloodstream infection group were significantly higher than those in control group (P <0.05), while LYM was significantly lower than that in control group (P <0.05).Among these five infection markers, the area under the receiver operating characteristic curve (ROC-AUC) was the highest for NLCR (0.808) and LMY (0.756); when the cutoff value for NLCR was >9.33, sensitivity was 63.6%, specificity was 93.3%; and the cutoff value for LYM was ≤0.97, sensitivity was 58.2%, specificity was 86.7%. Furthermore, the NLCR of patients with gram-negative bloodstream infection was higher than those in patients with gram-positive bloodstream infection. NLCR showed important clinical significance in distinguishing strains of different bloodstream infections. Conclusion NLCR is the better predictors than routine parameters in diagnosing bloodstream infection.

Quantification of the nonpolar amino acids in amniotic fluid of congenital malformation by gas chromatography mass spectrometry (GC-MS) derivatization method

2017, 37(7):  1004-1009. 

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Objective To develop a gas chromatography–mass spectrometry (GC–MS) detection method for quantification of the nonpolar amino acids in amniotic fluid of congenital malformation including alanine (Ala), leucine (Leu), isoleucine (Ile), proline (Pro) and phenylalanine (Phe). To compare the different amino acids in amniotic fluid between pregnant women with congenital malformation and normal control and anayze the corresponding pathogenesis. Methods After precipitated protein of the 100 μL supernatant of amniotic fluid by methyl alcohol, the supernatant was dried by nitrogen. The extractions were treated with MSTFA for derivatization. Then gas chromatography-mass spectrometry was used to detect and analyze the amino acids. Results This method was proved to be good sensitivity, precision, accuracy and recoveries. Under the optimum testing conditions, the limit of detection (LOD) was 0.12~0.38 mg/L. The calibration curves showed good linearity over the investigated concentration range between 0.5 and 10 mg/L. The recoveries were 91.12 % to 104.41 %. The relative standard deviation of intra and inter-day were 0.84 % to 9.33 %. The developed method was applied to the quantification of 5 nonpolar amino acids in amniotic fluid of 17 pregnant women with congenital malformation and 13 normal control pregnant woman. The contents of leucine and isoleucine decreased in disease group compared to controls. The difference of the other three amino acids between the two groups had no statistical significance. Conclusions The validation results showed that the method was suitable for detection of the amino acids in amniotic fluid and having broad prospect of clinical application. Leucine may participate in the pathogenesis of congenital malformation.

Culture and characterization of human pseudomyxoma peritonei cells

2017, 37(7):  1010-1014. 

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Objective Culture, expansion and characterization of pseudomyxoma peritonei cells, which laid the foundation for the study of pseudomyxoma peritonei and drug screening. Methods Tumors from 5 cases of human pseudomyxoma peritonei were cultured by collagenase digestion. The cultured cells were then identified as tumor cells by chromosome karyotyping. The secretion of neutral mucopolysaccharide was detected by PAS staining. To simulate the tumor growth condition in vivo, 3D culture was applied and the morphology of the cultured organoid was observed with HE staining, xenografts were used to test the tumorigenicity of PMP primary cells. Results The primary culture of pseudomyxoma peritonei cells was successful in all of the 5 cases. 2D cultured cells were adherent, polygonal pebble-like arranged, and passed on up to 18 generations. Chromosome karyotype analysis showed that most of the cells were subdiploid karyotype tumor cells. Positive PAS staining suggests that these cells secrete mucus. The morphology of 3D cultured organoid was similar to that of tumor tissues. The tumor formation rate of xenografts was low, and the tumoris not similar to patient’s PMP. Conclusion Pseudomyxoma peritonei tumor cells can be cultured and expanded in vitro.

miR-27a is negatively regulated by mTOR and inhibits liver cancer cell invasion via targeting GP73

2017, 37(7):  1015-1020. 

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Objective This study aims to explore the mechanism of mTOR-mediated liver cancer cell invasion. Methods q-PCR was used to check the expression of miR-27a and GP73; miR-27a mimics were transfected into GP73-high expressing M97H cells and miR-27a inhibitors were transfected into GP73-low expressing HepG2 cells, q-PCR and Western blot were performed to observe the expression of GP73; Dual-luciferase assay was also performed to verify the binding sites of miR-27a in GP73 3’UTR; miR-27a mimics were transfected into M97H cells and miR-27a inhibitors were transfected into HepG2 cells, Transwell assay was used to measure cell invasion. Results mTOR downregulated miR-27a and upregulated GP73; GP73 was downregulated by miR-27a and upregulated by miR-27a suppression; GP73 was a target gene of miR-27a; miR-27a inhibited the invasion of M97H cells rather than HepG2 cells. Conclusions miR-27a is negatively regulated by mTOR and inhibits liver cancer cell invasion via targeting GP73.

Effects of rapamycin on expression of miR-144 in ovarian cancer cell and regulation of Beclin1 gene expression about cell autophagy

2017, 37(7):  1021-1025. 

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Objective To detect the influence of rapamycin on the expression of 4 kinds of miRNAs about cell autophagy.To study the the relationship of miR-144 and Beclin-1 gene. Methods SKOV-3 cells were treated 50ng/mL rapamycin 2 hours and 10nmol/L 3-methyl adenine 12 hours,the expression of miR-17,miR-20a,miR-144 and miR-155 was detected by RT-qPCR in SKOV-3 cell of different groups，the protein expression of Beclin-1 was detected by Western blot.The targeting effect of miR-144 on Beclin-1 gene was verified by the dual-luciferase reporter assay,Western blot and RT-qPCR.Results The expression of miR-17,miR-144 and miR-155 were increased compared with NC groups in rapamycin group (P<0.05); miR-17,miR-20a and miR-144 were down regulated compared with NC group in 3-MA group(P<0.05)；the protein of Beclin-1 was down expression compared with NC group in rapamycin group.miR-144 could suppress Beclin-1 expression by targeting the specific 3’ untranslated region sequence of Beclin-1 gene. Conclusions miR-144 can directly inhibit the autophagy-related gene Beclin-1 expression and participate in the regulation of autophagy process in SKOV-3 cells.

Periostin siRNA transfection inhibits ox-LDL-induced human aortic endothelial cell line injury

2017, 37(7):  1026-1030. 

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Objective To investigate the effects of periostin (Postn) on oxidized low density lipoprotein (ox-LDL) -induced injury in human artery endothelial cells (HAECs) and its underlying mechanisms. Methods The HAECs were randomly divided into 4 groups: control group, ox-LDL group, Postn siRNA group and negative siRNA group. The mRNA and protein expression levels were analyzed by RT-qPCR and Western blot respectively. Cell proliferation was tested by MTT. Cell apoptosis was determined by flow cytometry. NF-κB DNA binding ability was measured by EMSA. Results Compared with control group, the mRNA and protein levels of Postn were increased significantly (P < 0.05); the ability of cell proliferation was reduced (P < 0.05); the cell apoptosis rate was increased (P < 0.05); the protein expression levels of VCAM1, ICAM1, E-selectin, IL-1β, IL-6, TNF-α, p65 and p-IκB-α were significantly up-regulated (P < 0.05), and the NF-κB DNA binding ability was markedly increased (P < 0.05) in ox-LDL group, which were all reversed in Postn siRNA group. Conclusion Postn si-RNA transfection could reduce ox-LDL-induced endothelial cell injury, which may be related with the inhibition of NF-κB signaling pathway.

Down-regulating HIPK2 promotes apoptosis of human kidney tubular epithelial cells induced by cisplatin

2017, 37(7):  1031-1036. 

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Objective To explore the effect of HIPK2 on apoptosis of human kidney tubular epithelial cells (HKC) induced by cisplatin. Methods Apoptosis of HKC cells was induced by cisplatin and the expression of HIPK2 was detected by real-time PCR and western blot. Two HIPK2 siRNAs were designed according to gene sequence of HIPK2 and cell lines with HIPK2 knockdownwere established through transfecting the HIPK2 siRNAs into HKC cells by liposome. The expression of HIPK2 mRNA and protein were detected by real-time PCR and western blot after induced by cisplatin. Then cell apoptosis was detected by Annexin V/PI after the HIPK2-knockdown cells were treated with cisplatin. Moreover, the expression of pro-apoptotic protein bax was detected by western blot after HIPK2 was knockdown. Results The expression level of HIPK2 mRNA and protein were down-regulated obviously on the process ofHKC apoptosis whichinduced by dose-dependent cisplatin (p<0.05). The transfection of siRNA can significantly reduce the expression of HIPK2 mRNA and protein in HKC (p<0.05), which will promote the HKC cells apoptosis induced by cisplatin. Conclusions HIPK2 can suppress the HKC cells apoptosis induced by cisplatin.

Expression level of lncRNA-ENST00000460164 in luminal A breast cancer and its effect on cell cycle

2017, 37(7):  1037-1041. 

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Objective To validate the expression level and the role of ENST00000460164 in luminal A breast cancer. Methods The expression level of ENST00000460164 in breast cancer tissues was detected by RT-qPCR. pll3.7-ENST00000460164-shRNA and empty vector，pll3.7，were transfected into MCF-7 cells. Cell cycle was measured by flow cytometry. Western blot was used to detect the expression of p16INK4A and cyclinD1. Results ENST00000460164 was highly expressed in luminal A breast cancer tissues compared to the adjacent non-cancer tissues. The knockdown of ENST00000460164 results in the G1 cell-cycle arrested, cyclin D1 downregulated and P16INK4A upregulated in MCF-7 cells. Conclusions ENST00000460164 is overexpressed in luminal A breast cancer. ENST00000460164 may control G1/S transition by regulating p16INK4A or cyclin D1 expression.

Identification of genes associated with gastric cancer prognosis from TCGA datasets

Xiao-Yi ZHANG Xiao-Yue WANG

2017, 37(7):  1042-1046. 

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Objective To identify genes associated with prognosis or differentiated type in gastric cancer from frequently mutated genes or highly-expressed genes, using large-scale genomic data from The Cancer Genome Atlas. Methods The somatic mutation data, RNAseqV2 data and clinical information were downloaded from the TCGA website. The frequency of deleterious somatic mutations for each gene was counted to select the frequently mutated genes. DESeq2 was used to analyze the gene expression data. Then survival analysis was performed on genes highly-expressed in the tumor tissue. Kaplan-Meier curves were generated by R-survival package, and significance was evaluated by log-rank test. Results The frequency for pathogenic mutations in PIK3CA and APC was significantly discordant between different grades of gastric cancer. 2 040 genes were up-regulated in tumor tissue, while 2 357 genes were down -regulated. Among the up-regulated genes, 7 genes were associated with poor prognosis of gastric cancer and one was associated with better prognosis. Conclusion In this study, by analyzing TCGA gastric cancer dataset, we have identified several genes associated with differentiation types or prognosis in gastric cancer. Our work will provide clues for future research on potential prognostic markers in clinical treatment.

Naringin attenuates myocardial ischemia reperfusion injury in rats

2017, 37(7):  1047-1048. 

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Preliminary analysis of the causes of CK-MB inversion in children

2017, 37(7):  1049-1050. 

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Necroptosis and tumor

Lian-zi LI Tao

2017, 37(7):  1051-1054. 

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Necroptosis, it is activated by the formation of necrosome, is a way of programmed cell death that independent of the activation of caspase. Necroptosis is regulated by many factors, for example, RIPK1 can initiate necroptosis, but also inhibit necroptosis; caspase-8 is the key negative regulation factor of necroptosis; CHIP is a new regulation protein of necroptosis. Triggering necroptosis is a promising strategy to overcome apoptosis resistance in cancer.

Progress in the research of tumor adoptive cellular immunotherapy

2017, 37(7):  1055-1058. 

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Adoptive immune cells can regulate and increase immune function of cancer patients, while effectively overcoming tumor escape mechanism. Cytokine induced killer cells (CIK), natural killer cells (NK), tumor infiltrating lymphocytes (TIL), dendritic cells (DC), T cell receptor-modified T cells (TCR-T) and chimeric antigen receptor-modified T cells (CAR-T) eliminate tumor by killing tumor cells directly or stimulating the immune response against tumor cells through different mechanisms.?

Role of uric acid in fructose-induced metabolic syndrome

Ke-Yue ZHANG Xue-jun ZENG

2017, 37(7):  1059-1063. 

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Excessive fructose intake can induce metabolic syndrome and increase serum uric acid levels. Uric acid levels are high in types of fructose –induced metabolic syndrome and may play a pathogenic role in the progression of these diseases. In this review, we focus on the role of uric acid in fructose-induced metabolic syndromes.

Improving humanistic knowledge for a new medical model

Ren-zhi WANG,

2017, 37(7):  1064-1066. 

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With the development of productive forces and the improvement of science and technology, medical model, as the total understanding of health and disease at a certain historical period, has undergone several different phases: spirtualism medical model, mechanistic medical model, Biological Medical Model and biological-psychological-social medical model. Under the background of biologic and psychogenic society medicine, the professional quality of contemporary doctor faces new requirements and challenges. This article will discuss the necessity, importance and approaches of intensifying studying medical humanistic knowledge and adjusting the transformation of medical model.