Down Regulation Of ROCK-Ⅰ Gene Expression And Its Inhibition To Vascular Smooth Muscle Cell Migration

Objective: According to the relevant data, there were 15.3 million people die of cardiovascular diseases and cerebrovascular diseases, which account 1/4 of the total number of death.So the cardiovascular diseases and cerebrovascular diseases have been become one of the most fatal diseases. According to some animal experiment research, it was confirmed that Rho-kinase participated in the pathogenesy of the diseases such as angios- clerosis, angiospasm, ischemical reperfusion injury, pulmonary artery hy- pertension, palsy, hypertension and congestive heart failure. The recent research has found that Rho/Rho-kinase signal transduction pathway participates the development of the cardiovascular diseases and cerebrovas- cular diseases, so the inhibitor of the signal transduction pathway will prevent and treat these diseases. Rho-kinase(Rho-associated coiled-coil kinase, ROCK)is the key enzyme in Rho/ROCK signal pathway, therefore, ROCK will become one of the important target of drug treatment of the diseases.Rho-kinase generally exists in every kind of tissues, and it contains two isomers: ROCK-Ⅰand ROCK-Ⅱ, and Rho-kinase plays an important part in smooth muscle of many kind of tissues.Rho/ROCK signal transduction pathway participates in numerous physiological functional regulations, such as cytoskeleton recombination, smooth muscle contraction, cellular proliferation and stick, migration and other inflammatory reactions. And these cell functions are close correlated with many disease of blood vessel. In recent years, Rho/ROCK signal transduction pathway has been concerned wide attention. ROCK was activated by Rho protein, then phosphorylated 697-threonine of myosin subunit downstream, and raised myosin light chain phosphorylation in endochylema, and increased actin-myosin cross linkage, then actin micro- filament framework get together, finally it results in cells migration.For this reason, Rho/ROCK was hoped to become the important treated target of artherosclerosis, restenosis, hypertension, brain or coronary artery vasospasm and so on.To sum up, if we can forbid the vascular smooth muscle cells to migrate, we will provide significant theory for study and treat the above diseases. So, ROCK is considered to become an important treated target. After literature retrieval, we have not found the relevant news reports, so this essay will be studied and researched.Method : We designed and synthesized siRNA according to rat ROCK-ⅠcDNA sequence, then screen the effective siRNA . Bring siRNA to A7r5 cells through Lipofectamine? 2000 Reagent, and then do the following experiments:1. In order to check the down regulation of ROCK-Ⅰgene expression from mRNA level, the total RNA was extracted from A7r5 cell by TaKaRa RNAiso Reagent, and then followed by reverse transcription and PCR.2. To confirm the down regulation of ROCK-Ⅰgene expression from protein level, the total protein was extracted and examined by Western Blotting.3. The migration of smooth muscle cell (A7r5) was determined by Boyden Chamber. The variation of cell migration mobility was analyzed with down regulation of ROCK-Ⅰgene expression.Result: After repeating and considerable experiments many times, we get the following results:The efficiency of transfection: We used siRNA which contain fluorescent labeling to do the experiment of interference. The cells were transfected respectively by 5h, 24h, 48h and 72h, and then observed under the fluorescence microscope. From the pictures we obtained, it showed that the best efficiency of transfection is transfection after 48h or 72h.Assay of Lipofectamine toxicity: In order to find out the best concentration of Lipofectamine, we use different concentration of Lipo- fectamine to transfect the cells. The cells mortality rate was calculate after different transfection time. The results suggest that the best concentration of Lipofectamine is 0.56ng/μl ~ 0.83ng/μl.Assay from gene: To confirm the down regulation of ROCK-Ⅰgene expression from mRNA level, the total RNA was extracted from A7r5 cell after transfection 48h or 72h and then followed by reverse transcription and PCR. Through analysis by SPSS software, the data showed that the expression was greatly decreased after RNA interference.Assay from protein: In order to detect the down regulation of ROCK-Ⅰgene expression from protein, the total protein was extracted form A7r5 cell after transfection 72h and then followed by Western blot . Then we detected the protein by ECL and analyzed by SPSS software . The data showed that that the expression was greatly decreased after RNA interference.Selection of the optimal concentration of cell migration inductor (PDGF-BB): Because of the dose-dependent relationship between cell migration and PDGF-BB, we should choose the optimal concentration to do the experiment. From the experiment of cell migration, we found that the optimal concentration of PDGF-BB is 5ng/ml.Experiment of cell migration: We used different dose of siRNA to do experiment of cell migration using the optimal PDGF-BB through the method of Boyden chamber. Then we observed under the microscope and analyzed by the software of SPSS, and obtained the coincident results: The numbers of cell migration were greatly decreased after RNA interference.Conclusion: Through the method of RNA interference, we can successfully down regulate the expression of ROCK-Ⅰgene. So it made great sense of further research of the molecular mechanism of ROCK-Ⅰand cell migration.