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Objective To study the isolation method and the culture method of primary human endometrial cells and to iden‐tify the purity and biological activity .Methods We digested human endometrial tissue by collagenase at first ,and then to separate , purify and culture the human glandular stromal cells(ESC) and endometrial epithelial cells(EEC)by differential centrifugation ,dab‐ble screen filtration and adhesion purification technology .Ultimately ,we identified the isolated cells with cytokeratin and vimentin immunocytochemical staining and immunofluorescence method .Results Stromal cells showed a parallel growth .The cells were spindle or polygonal ,and the vimentin antibody showed a positive immunohistochemical staining ,the purity was more than 95% .At the same time ,glandular epithelial cells grew in whorls .The cells were polygonal or tadpole shaped ,and the cytokeratin antibody immunohistochemical staining ,the purity were up to 90% .Conclusion The successful isolation and culture of high quantity ,viabili‐ty and purity by collagenase digestion and dabble screen filter method of endometriall cells make a strong operability .The laboratory which has the basic cell culture conditions can develop the experiment .
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Background and purpose:It has beenreported that miR-1284 is associated with gastric cancer lymph node metastasis in the research of microRNA microarray in human gastric cancer tissues. But the specific role of miR-1284 in gastric cancer has not been reported. The aim of this study was to investigate the effect of miR-1284 over-expression on the gene expression profiling and invasion/metastasis of human gastric cancer SGC-7901 cells. Methods:Gastric cancer SGC-7901 cells of LV-miR-1284 group were transfected with lentiviral vectors of miR-1284, cells of LV-NC-GFP group were transfected with lentiviral vectors without miR-1284, and cells of control group were not transfected with lentiviral vectors. The expression of miR-1284 was detected by the real-time fluorescent quantitative PCR. Differential expression genes were detected by the microRNA chip. Target genes of miR-1284 were predicted by the bioinformatics. Invasive ability was detected by the Transwell invasion assay. Metastasis ability was detected by subcutane-ously transplanted tumor model of nude mice.Results:Compared with LV-NC-GFP and control groups, the expressions of miR-1284 and 20 genes were up-regulated, and the expression of 17 genes was down-regulated in LV-miR-1284 group. One hundred and thirty-eight target genes of miR-1284 were predicted by the bioinformatics website. Compared with invasive cell number of LV-NC-GFP group (168.67±4.55) and control group (170.33±3.08), the ability of invasion ofcells was weakened in LV-miR-1284 group (70.00±2.37). Compared with the liver metastasis rate of LV-NC-GFP group (85.71%) and control group (85.71%), the ability of metastasis of cells was weakened in LV-miR-1284 group (14.29%). Conclusion:The ability of invasion and metastasis of SGC-7901 cells is suppressed by over-expression of miR-1284. The mechanism may be related to regulating the expression ofSUMO1 andJUNgenes.
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AIM:To evaluate the correlation between microRNA-1284 (miR-1284) and gastric cancer, and to investigate the underlying mechanism.METHODS: The expression of miR-1284 was examined by real-time PCR in 63 gastric cancer ( GC) tissue samples and 63 non-malignant adjacent tissue samples.The correlation between miR-1284 and the clinicopathological feature of GC was analyzed.Lentiviral vector containing miR-1284 was constructed and transfected into GC SGC-7901 cells.After transfection, the expression of miR-1284 was examined by real-time PCR.The cell activity was evaluated by CCK-8 assay.The cell cycle and apoptosis were determined by flow cytometry.The ability of cell migra-tion was measured by wound-healing assay.The potential target gene of miR-1284 was predicted by online bioinformatic softwares.The expression of JAG1 mRNA was examined by real-time PCR.The protein levels of JAG1, Notch1 and NF-κB were analyzed by Western blotting.RESULTS:Compared with non-malignant adjacent tissue samples, the results of real-time PCR showed significant downregulation of miR-1284 in 42 GC tissue samples ( P0.05), but significantly associated with histologic grading (P<0.05).Compared with LV-NC-GFP group and control group, after transfection of miR-1284 in LV-miR-1284 group, the expression of miR-1284 was significantly in-creased (P<0.05), the percentages of apoptotic cells and the cells in G0/G1 phase were significantly increased (P<0.05), the cells activity and ability of migration were significantly decreased (P<0.05), and the expression of JAG1, Notch1 and NF-κB was significantly decreased (P<0.05).CONCLUSION:The inhibitory effect of miR-1284 on gastric cancer may be associated with the regulation of its targeting gene JAG1.
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AIM:To study the effect and the molecular mechanism of CDX 2 over-expression on the prolifera-tion, growth and cell cycle of human gastric cancer cell line SGC-7901.METHODS:The SGC-7901 cells in LV-CDX2-GFP group were transfected with the recombinant lentivirus vector LV-CDX2-GFP, the cells in LV-GFP group were trans-fected with the negative control lentiviral vector for the negative control , and the cells in blank control group were without any treatment.The cell proliferation was detected by CCK-8 assay.The cell cycle distribution was analyzed by flow cytome-try.The expression of CDX2, Bax, Bcl-2, cyclin D1 and survivin was determined by semi-quantitative RT-PCR and Wes-tern blotting .RESULTS:Compared with LV-GFP group and blank control group , the proliferation activity of the SGC-7901 cells was significantly lower (P0.05) between LV-GFP group and blank control group .CONCLUSION:Over-expression of CDX2 mediated by lentivirus inhibits the proliferation and growth of human gastric cancer SGC-7901 cells and arrestes the cell cycle at G 0/G1 phase, which may be related to down-regulation of Bcl-2, cyclin D1 and survivin and up-regulation of Bax .