RESUMO
Objective To establish method of constructing lentiviral vectors to express microRNA (miRNA) ''tough decoy''(TuD)and to detect the effects of the TuD on cellular endogenous miRNA level and cellular phenotypes. Methods Two-step cloning strategy was utilized to first generate a universal miRNA TuD frame vector,followed by con-structing the TuD expression vector specially targeting miR-203. The package of the recombinant lentivirus was per-formed in 293T cells. Then the rat bone marrow mesenchymal stem cells(BM-MSCs)were infected by the miR-203 TuD expression lentivirus. The pSIH1-H1-copGFP vector was also packaged and the BM-MSCs infected by this lentivirus were served as control. Endogenous miR-203 level in BM-MSCs was measured by quantitative RT-PCR,and cellular vi-ability and apoptosis were detected by CCK-8 test and Annexin V-PI staining respectively. Results The miR-203 TuD expression vector was successfully constructed and inserted sequence was validated. At the 3rd,6th and 9th days after in-fected by the miR-203 TuD expression lentivirus,rat BM-MSCs exhibited a repressed endogenous miR-203 level. The miR-203 TuD also promoted viability and inhibited apoptosis of BM-MSCs. All these differences between miR-203 TuD group and control group were statistically significant. Conclusion The two-step cloning method for the construction of miRNA TuD expression vector is simple and efficient. The miRNA TuD can effectively suppress the level of the target miRNA and affect cellular phenotypes.
RESUMO
Objective To investigate the immunomodulatory effects of heme oxygenase-1 ( HO-1) modified rat bone marrow mesenchymal stem cells ( BM-MSCs) on lymphocytes .Methods Rat BM-MSCs were cultured and identified in vitro.HO-1 gene was transduced into BM-MSCs via recombinant adenovirus (rAdV) to construct HO-1/BM MSCs, which were then co-cultured with rat spleen lymphocytes .The cellu-lar activity of lymphocytes was detected by using MTT method .The cell cycle distributions were analyzed by propidium iodide staining and flow cytometry .ELISA was used to detect the level of cytokines in the super-natant of cultured cells .The expression of lymphocyte activation markers , CD25 and CD71, were measured by using fluorescence labeled antibodies and flow cytometry .Results Compared with the control group , the lymphocytes co-cultured with HO-1/BM MSCs showed a suppressed proliferation and cell cycle progression . HO-1/BM MSCs inhibited the secretion of IL-2, TNF-αand IFN-γ, but enhanced the secretion of IL-10 in lymphocytes.Furthermore, the expression of CD25 and CD71 were also down-regulated by HO-1/BM MSCs. Conclusion Compared with BM MSCs , HO-1/BM MSCs show higher immunosuppressive effects on lym-phocytes .