Studies Of Inhibition Of Hyperplasia Of VSMCs In Rat By The Recombinate Plasmid Containing RNA Interference Of Cysteine Rich 61

PurposeThe formation of stenosis after artery injury is a complicated pathology and physiology process. It has already been confirmed that activation, proliferation and migration of vascular smooth muscle cells(VSMCs) were centro-link for hyperplasia. Inhibition of proliferation of VSMCs have significant for atherosclerosis and restenosis after percutaneous transluminal coronary angioplasty (PTCA).1998, Fire and his colleagues found a phenomenon called RNA interference (RNAi). This phenomenon refers to the gene target specificity post transcriptional gene silencing (PTGS) induced by the double-stranded RNA(dsRNA) which is injected into the Caenorhabditis elegans. During the past a few years, substantial improvements about RNAi were gained worldwide. RNAi became a new gene inhib- iting technique, and can successfully interfere the express of the specific genes using RNAi in vitro-cultured mammalian cells such as from human or rat. This provides a new possibility for human geng therapy.Cysteine-rich61(Cyr61) is a heparin-binding, extracellular, matrix associated protein of the cysteine-rich 61/nephroblastoma family, which also includes connective tissue growth factor, nephroblastoma overexpressed, Wnt-induced secreted protein-1 (WISP-1), WISP-2, and WISP-3. Cyr61 induces angiogenesis in vivo and supports cell adhesion, promotes cell migration, and enhances growth factor stimulated mitogenesis in fibroblasts and endothelial cells, s strongly expressed in smooth muscle cells of arterial walls during embryonic develooment. Immunoblot and histological analyses showed that Cyr61 accumulates at high levels in the vessel media and neointima from d7 to d14 after balloon angioplasty, corresponding to the period of migratory and proliferative activity of smooth muscle cells in restenosis. Cyr61 supported VSMCs migration, proliferation and adhesion, most likely because of saturation of the Cyr61 heparin-binding sites. In the present study we constructed plasmids with rat Cyr61 short haitpin RNA (shRNA) and transfect the plasmids into rat VSMCs to investigate the changes of the expression of Cyr61 mRNA and protein respectively and the effect on hyperplasia of VSMCs in rat.Methods1. Finding the sequence of rat Cyr61 mRNA in the GeneBank, putting into the corresponding software and designing the primer rank. The fragments amplificated by PCR were inserted to the pGenesil-1 vector, and then selected and identified.2. The plasmids containing the shRNA of Cyr61 were constructed, and the change of rnRNA and protein were detected by RT-PCR and Western blot after transfecting vascular smooth musle cells.3. The plasmids containing the shRNA of Cyr61 were constructed and transfe cted vascular smooth musle cells, observe the shape by the inverted phasecon trast microscope and fluorescence microscope, and detect the hyperplasia of VSMCs by trypan blues taining and MTT. Detected DNA content by incorporating ³H-TDR. Through FCM method, we detected VSMCs cycleResults1. The result of confirmation test is consistent with the prediction, and the pl asmid was certified to be in the right rank.2. After transfecting Cells, there was significant different(P＜0.01)in the expression of Cyr61 mRNA between the gene transfected group (pCyr61 -shRNA₁ 0.114±0.012; pCyr61-shRNA₂0.105±0.010)and the control group (Control group 1.256±0.138; p-Hk group 1.342±0.147); and there was significant different (P＜0.01) in the expression of Cyr61 protein between the geng transfected group (pCyr 61-shRNA₁ 0.012±0.004; pCyr61-shRNA₂ 0.009±0.001)and the control group (Control group 0.984±0.082; p-Hk group 0.898±0.076). It is shown that pCyr61- shRNA can decrease the expression of Cyr61 mRNA and protein. 3. After transfecting cells, there was significant different(P＜0.01)in the Cell number between the gene transfected group (pCyr61-shRNA₁2.031±0.096; pCyr 61-shRN A₂ 2.431±0.765)and the control group(Control group 8.234±0.428; p-Hk group 9.642±0.465); there was significant different (P＜0.01) in the Ratio of light density by MTT between the gene transfected group (pCyr61 -shRNA₁0.145±0.008; pCyr61-shRNA₂ 0.175±0.013)and the control group (Control group 0.856±0.046; p-HKgroup0.876±0.033); there was significant different (P＜0.01) in the DNA content by incorporating ³H-TDR between the gene transfected group (pCyr61-shRNA₁ 158.333±11.480; pCyr61- shRNA₂ 162.000±12.156)and the control group (Control group543.000±27.622; p-HK group 536.667±36.866).4. Through ELISA method, we detected VSMCs substance Cyr61 protein, there was significant different(P＜0.01) between Angâ…¡group and Losartan group. (204.179±7.131 vs 115.423±5.782).5. Through FCM method, we detected VSMCs cell cycle. We found there was significant different(P＜0.01)in the Cell cycle between the gene transfected group (pCyr61-shRNA₁ G0/Glphase 84.3%,S phase7.69%,G2/M phase8.1%; pCyr61shRNA₂ 0/G1 phase 82.5%,S phase6.5%,G2/M phase11%)and the control group (Control group G0/G1 phase52.7%,S phase16.9%,G2/M phase 30.4%; p-HK group G0/G1 phase53.8%,S phase16.6%,G2/M phase29.6%9.642±0.465).Conclusion1. The successful construction of Cyr61 shRNA plamids have the RNA interference activity.2. The plasmids containing the shRNA of Cyr61 can inhibit the expression of Cyr61 mRNA and protein in VSMCs.3. VSMCs can excreted Cyr61 protein by stimuluing of Angâ…¡.4. After transfecting cells, The plasmids containing the shRNA of Cyr61 inhibitted Cyr61 protein and inhibited VSMCs proliferation.