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Objective To investigate the effects of trefoil factor 3 (TFF3) on the proliferation and apoptosis of human thyroid papillary carcinoma cell line (TPC-1) and its molecular mechanism. Methods The lentiviral expression vector of overexpression and knockdown of TFF3 gene were constructed, 293T cell was packaged to produce lentiviral particles, virus solution was collected and transfected into TPC-1 cells, enhanced cell TFF3-TPC-1 and enhanced control group ConTFF3-TPC-1; silencing cell shRNA-TFF3-TPC-1 and silencing control cells shCon -TPC-1. Western blotting, and Real-time PCR were used to detect the expression of TFF3 protein and mRNA of four groups. Growth curve and colony formation assay were used to detect the proliferation. Flow cytometry was used to analyze the apoptosis level of the four groups; Western blotting and immunocytochemistry were used to detect apoptosis-related protein and pathway protein phosphatidylinositol 3-kinase/ protein kinase B (PI3K/ Akt), nuclear factor-κB (NF-κB) expression. Results 1. Overexpression and inhibition of expression of TFF3 stable cell TFF3-TPC-1 and shRNA-TFF3-TPC-1 were constructed suscessfully. 2. The proliferation and cloning ability of TFF3-TPC-1 cells were significantly higher than those of ConTFF3-TPC-1 cells(P<0. 05 or P< 0. 01), the proliferation and cloning ability of shRNA-TFF3-TPC-1 cells were significantly lower than those of shCon-TPC-1 cells(P<0. 01); 3. The apoptosis rate of TFF3-TPC-1 cells was lower than that of ConTFF3-TPC-1 (0. 75%±0. 08% vs 5. 62%±0. 3%, P<0. 01),and the apoptosis rate of shRNA-TFF3-TPC-1 was higher than that of shConTPC-1 (22. 2% ± 1. 2% vs 5. 34% ± 0. 4%, P<0. 01); 4. After silencing TFF3 gene, the expressions of Bax, cytochrome C (Cyt-C), cleaved-Caspase-9, cleaved-Caspase-3 were up-regulated, and the expressions of Bcl-2, Akt, p-Akt and NF-κB-P65 were down-regulated (P<0. 05 or P<0. 01). Conclusion TFF3 may regulate the proliferation and apoptosis of TPC-1 cells by affecting the PI3K/ Akt/ NF-κB signaling pathway.
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Objective To investigate the effects of VCAM-1 on migration and invasion of glioma cell lines . Methods The techniques of lentivirus pSGU6/GFP/Neo-based VCAM-1 shRNA and EF1 a-GFP/puro-based VCAM-1 expression vector, the scratch wound healing migration and transwell invasion assays , and the Western blot and cell staining were applied to observe the effects of VCAM-1 expression levels on migration and invasion of glioma cell line cells.There are four groups in T98G cells including control, vector, scramble and shRNA-VCAM-1 groups and three groups in U251 cells covering control, vector and VCAM-1 overexpressed groups ( n=6 per group) .Results The stabled glioma cell lines of T98 G cells with down-regulated VCAM-1 and U251 cells with VCAM-1 overexpression were established by using lentivirus-based VCAM-1 shRNA and expression vector.The ability of scratch wound healing (migration activity) decreased significantly (P<0.01) in T98G cells with lower VCAM-1 expression levels, while the migration activity was obviously improved in U251 cells with overexpressed VCAM-1 ( P <0.05 ) .Similarly, the invasion ability was significantly inhibited ( P <0.05) in T98G cells with silenced VCAM-1, as well as VCAM-1 overexpression could enhance the invasion ability of U251 cells ( P<0.01 ) .Conclusions VCAM-1 improves the migration activity and invasion ability of human glioma cell line cells.
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Objective To explore the effect of mouse proliferation-associated protein 2G4 (p38-2G4) high-expression on the proliferation and erythriod differentiation of murine erythroleukemia ( MEL ) cells.Methods To establish the recombinant lentivirus vector p 38-2G4-pLJM1, the p38-2G4-pLJM1 was cotransfected into HEK293T cells to obtain lentivirus with pCMV-VSV-G and pCMV-dR8.2.Lentivirus were infected into MEL cells to establish the stably p 38-2G4 high-expressed MEL cells.Western blotting was used to analyse the high-expression efficiency.MTT assay and benzidine staining were applied to detect the cell viability and hemoglobin synthesis of the stable cell line in presence /absence of inducers.Results Western blotting showed that the p38-2G4 high-expression stable cell strain had a higher expression of p38-2G4 as compared to the control group ( MEL) ( P 0.05), but the hemoglobin synthesis had been reduced as compared to the control group (P<0.05).Conclusion p38-2G4 high-expression does not affect the cell viability of MEL cells , but inhibits the erythriod differentiation of MEL cells in three independent experiments .
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Objective To study the bone marrow-derived mesenchymal stem cell(MSC) potential as seed cells for treatment of multiple organ dysfunction syndrome(MODS) in rabbit model, lay fundamental for clin-ical utilization of the MSC for treatment of MODS. Methods MODS rabbit model was established by hemor-rhagic shock combined with endotoxin, isolation and characterization of rabbit bone marrow mesenchymal stem cells labeled MSC with GFP by lentivirus transfection, administration of MSC by ear margin vein injec-tion, MSC integration determined by PCR and pathological section, the MSCs effect on MODS rabbits evalu-ated by physical observation. Results Compared with controls, ransplanted MSCs were found in liver, lung, kidney, MODS rabbits were physically improved obviously. Conclusions MSCs are able to integrate into host without any rejection reaction, and capable of ameliorating MODS evidently.