Construction Of A Recombinant Lentiviral Vector For TSG-6 And TSG-6 ShRNA And Its Effect And Apoptosis Of Human Keloid Fibroblast

Background: Current research has shown that tumor necrosis factor α stimulated gene6(TSG-6) has anti-inflammatory effect, and the scar formation can be inhibited by local injection of TSG-6 protein at the early stage of trauma. However, the mechanism of this effect is still unclear.Objective: To construct the lentiviral expression vector and sh RNA vector for human TSG-6, and explore the effect of TSG-6 on proliferation and apoptosis of human keloid fibroblast cell lines.Methods: Human keloid fibroblast cells were isolated from the keloid’s tissue by enzyme digestion and identified by immunocytochemistry assay. Lentiviral vectors p LVX-puro-TSG-6 and p LVX-sh RNA1-TSG-6 were constructed and transfected into human keloid fibroblast, exclusively. Expression levels of TSG-6 m RNA and protein were detected by RT-PCR and Western blotting. Then the MTT and flow cytometry were used to estimate the effect of TSG-6 on the proliferation and apoptosis in each group. In addition, expression of bcl-2 、 p53 and active-caspase-3 were detected by Western blotting respectively.Results and Conclusion: Human keloid fibroblast cells were separated and purified.After transfection, RT-PCR and Western blotting results showed that TSG-6 gene expression was altered as expected. In the group of TSG-6 over-expression,proliferation of HKFs was inhibited and apoptosis was increased significantly compared with the control group(P < 0.05). On the contrary, proliferation of HKFs was promoted and apoptosis was decreased significantly in the interference group(P < 0.05). And the result of Western blotting assay shown that the alteration in proliferation and apoptosis were related to the variation in expression of bcl-2、p53 and activity of active-caspase-3.