Research Of Survivin Regulation Of ROCK-1 Promotes Hpasmcs Proliferation In Normoxia And Hypoxia

Objecive :1)To detect whether Rho associated coiled coil forming protein kinase(ROCK-1)was expressed in human pulmonary artery smooth cell under hypoxia.2) To survey whether Survivin promoted proliferation of human pulmonary artery smooth cell via ROCK-1 signal pathway.Mehtods :1) In vitro the normal tissues surrounding human lung was separated and cultured them by tissue explant. Cells at passage 3 to 8 were used for the experiment. The cells are preconditioned by hypoxia for 0h, 0.5h, 1h, 2h, 4h, 8h, 12 h. At every time the expression of protein ROCK-1 was detected in human pulmonary artery smooth cells(HPASMCs) by western blotting and immunofluorescence staining, in the meantime expression of ROCK-1 mRNA was explored by realtime-PCR. In addition,the ability of cell proliferation was observed by CCK8.2) Subsequent to primary HPASMCs culturing, three sequences Survivin siRNA in vitro was costructed and synthesised. The target sequence for the highest inhibition rate(at least 70%) to inhibit expression of Survivin and ROCK-1 mRNA of HPSMCs was Screening respectedly. And then they was transfected respectively into the HPASMCs cultured in vitro with the liposome 2000.Then cells are randomly grouped into 6 groups. normoxic blank control group, normoxic Survivin siRNA transfecting group, normoxic ROCK-1 siRNA transfecting group, hypoxic blank control group, hypoxic Survivin siRNA transfecting group, hypoxic ROCK-1 siRNA transfecting group.Successively the change of expression of protein Survivin and ROCK-1was watched by western blotting, meanwhile the change of expression of Survivin mRNA and ROCK-1 mRNA was examined by realtime-PCR, Complementary, the ability of cell proliferation was surveied with CCK8 and EDU immunofluorescence.Results :1) Western blotting and immunofluorescence staining showed that the expression of protein ROCK-1 is significantly increased by hypoxic preconditioning, and it peaked in hypoxia for 4h. the same as realtime-PCR mRNA. CCK8 test shows that the ability of proliferation is significantly enhancing in hypoxa for 24 h, 48 h, 72 h.2) Silencing Survivin gene can not only reduce the expression of ROCK-1 mRNA but also decress protein of HPSMCs, but Silencing ROCK-1 gene fail to cut down the level of Survivin mRNA and protein.Conclusion :1) Hypoxia conditions can promote the proliferation of HPASMCs.2)Hypoxia conditions can lead to expression of ROCK-1 increases in HPASMCs. Which suggested ROCK-1 siganal maybe participate the proliferation of HPSMCs.3)Silencing Survivin gene can reduce the expression protein and mRNA of ROCK-1 in HPSMCs, but Silencing ROCK-1 gene cannot block the expression of Survivin protein and mRNA. Which reals Survivin siganal locating in the upstream of Rock-1, Both Survivin and ROCK- 1 participate the proliferation of HPASMCs. As the signaling pathways they may promote HPASMCs proliferation.