The Study Of Specific Inhibition Of Osteopontin Gene By RNA Interference In Vascular Smooth Muscle Cell Of Rats

Part 1 Vascular smooth muscle cell of rats culture in vitro and identificationObjective To culture vascular smooth muscle cells isolated from the thoracic aortas of Sprague-Dawley rats in vitro.Methods Vascular smooth muscle cells were under isolated from the thoracic aortas of male Sprague-Dawley rats aseptic condition. The surrounding tissue was removed from the arterial segment. The outer tissue layer of the vessel was stripped off with watchmaker forceps and a scalpel. VSMC were cultured by tissue-sticking method. The VSMC were passaged after 2 weeks, andα-SM-actin was used to identify by immunohistochemistry.Results VSMC were Fusiform shape when observed under light microscope or contrast phase microscope. After reaching confluency, VSMC culture showed typical morphology and "hill and valley" growth pattern. Immunologic staining with anti-α-SM-actin antibody stained cells showed above 96% purity.Conclusion It is simple and cheap that culture VSMC of rats in vitro by tissue-sticking method. It can supply stable cells for following experiments.Part 2 Design and synthesis of osteopontin-specific small hairpin loop RNAObjective To design siRNA targeting osteopontin on Dharmacon siDESIGN Center and gain small hairpin loop RNA in vitro transcription.Methods Osteopontin mRNA sequence of rat was retrieved in nucleotide library of NCBI. Osteopontin-specific shRNA (shRNA1 and shRNA2) was designed and chosed on Dharmacon siDESIGN Center. DNA oligonucleotide was transformed to small hairpin loop RNA by annealing, fill-in, in vitro transcription reaction and purity. In addition, lcu-shRNA targeting luciferase gene was synthesized to negative control. These products were identified with 12% denaturing polyacrylamide gel. The yield of product was calculated according to optical density( OD260) .Results The specificity of shRNA1 and luc-shRNA was fine and bands were distinct located on 52bp, but the band of shRNA2 was dim. The yield of shRNA1, shRNA2 and luc-shRNA was 3.56μg, 0.91μg and 3.22μg, respectivly. Their optical density ratio of OD260/OD280 was between 1.8 and 2.0.Conclusion One osteopontin-specific shRNA and negative control shRNA were successful synthesized in vitro transcription through MessageMuter? shRNAi Production Kit.Part3 Study on the expression of osteopontin on vascular smooth muscle cell of rats inhibited by special shRNAObjective To detect the expression of osteopontin mRNA and protein after transfection by special shRNA on vascular smooth muscle cell. Methods VSMCs were divided into three groups: blank control group, negative control group and RNAi group. The cells were at 60% confluence when transfection. Forty-eight hours after transfection, all RNA and protein were extracted respectivly by Trizol. The inhibition effects on osteopontin gene were determined by semi-quantitative reverse transcription PCR (RT-PCR) and Western blot analysis.Results Semi-quantitative RT-PCR showed that mRNA transcription of osteopontin gene was reduced by 76.3%±3.4%(p<0.01). The introduction of RNAi group was showed to efficiently and specifically inhibit the expression of osteopontin according to results of Western blot, with inhibition rate at 68.7%±4.6% 48h(p<0.01). On the contrast, the blank control and negative control did exhibit no inhibitory effect on the protein expression and mRNA transcription of osteopontin (p>0.05).Conclusion Small hairpin RNA targeting osteopontin efficiently and specifically inhibits the expression of osteopontin.Part4 Effect on vascular smooth muscle cell of rats proliferation,adhesion and migration by special shRNAObjective To study the inhibition effects of shRNA targeting osteopontin on the proliferation, adhesion and migration of vascular smooth muscle cell of rats.Methods Cell proliferative activities were measured by MTT. VSMCs were seeded in 96-well plates coated by 1% laminin solution. Optical density (A490) was determined at 4h, 6h and 8h after transfection. Cell migration assay in vitro was assayed with Boyden chamber with an 8-μm pore size. The lower side of the filter membrane was fixed and stained for cell counting under a microscope at a magnification of×400.Results Compared with the blank control, VSMC proliferation was inhibited obviously when osteopontin expression was knocked down by RNAi, with a 52.2%±5.48%,46.2%±4.69% and 33.2%±4.87% reduction at 24h, 48h, 72h after transfection respectively( p< 0.01). The results of cell adhesion assay showed that VSMC adherent activities were significantly suppressed by 58.5%±5.6% , 65.2%±7.4% and 64.4%±6.7% at 4h, 6h, 8h after transfection respectively( p< 0.01) . Cell migratory capability demonstrated the inhibitory results in Boyden chamber study by RNAi, with a 23.4%±3.5%, 31.9%±4.9% and 30.9%±4.1% reduction at 4h, 6h, 8h after transfection respectively( p<0.05).Conclusion Following inhibition the expression of osteopontin by RNAi, vascular smooth muscle cell migration, adhesion and proliferation were inhibited at various extents. Part5 Effect on vascular smooth muscle cell of rats synthesis of collagen by special shRNAObjective To detect synthesis typeⅠcollagen and typeⅢcollagen after transfection by special shRNA on vascular smooth muscle cell. Methods Forty-eight hours after transfection, all RNA and protein were extracted respectivly by Trizol. The inhibition effects on osteopontin gene were determined by semi-quantitative RT-PCR. The expression of TypeⅠ,Ⅲcollagen in the supernatant was detected by ELISA.Results The content of typeⅠ,Ⅲcollagen in the supernatant was knocked down by RNAi, with a 24.2%±4.6% and 26.7%±5.2% reduction at 48h after transfection respectively( p< 0.05). The results of RT-PCR showed that three groups did exhibit no inhibitory effect on mRNA of typeⅠ,Ⅲcollagen, respectively (p>0.05). Conclusion The number of VSMCs in RNAi group was less than blank control group and negative control group. Thus, typeⅠ,Ⅲcollagen secreted from VSMC into the supernatant were different.