RNA干扰技术特异性抑制大鼠VSMC骨桥蛋白基因表达的研究

发布时间：2019-05-28 16:30

【摘要】： 第一部分 大鼠VSMC的体外培养及鉴定 目的体外分离Sprague-Dawley大鼠胸主动脉平滑肌细胞,建立血管平滑肌细胞体外培养模型。 方法无菌下取雄性Sprague-Dawley(SD)大鼠胸主动脉,清除外膜组织和内膜,采用组织块翻转干涸培养法进行培养。2周后进行传代,细胞爬片行免疫组织化学鉴定。 结果光镜以及相差显微镜下观察细胞呈梭形,生长至融合状态时呈现特有的“峰”与“谷”特点。同时,运用免疫组化特异抗α-肌动蛋白(α-actin)单抗免疫组织化学染色阳性,纯度达96%以上。结论采用组织块培养法培养大鼠血管平滑肌细胞简单易行,且又经济,可为后续实验提供细胞来源。 第二部分骨桥蛋白特异性短发夹RNA的设计与合成 目的在线设计针对骨桥蛋白siRNA的序列,采用体外转录的方法,获得发夹状RNA,为下一步的细胞转染奠定基础。 方法在NCBI(http://www.ncbi.nlm.nih.gov/)的Nucleotide库中检索有关大鼠骨桥蛋白的mRNA序列。Dharmacon siDESIGN Center按照网页要求和提示设计siRNA ,选择其中的两条siRNA序列(shRNA1和shRNA2)。按照MessageMuter? shRNAi Production Kit的说明设计DNA寡核苷酸,进行退火反应、延伸反应、体外转录反应和RNA纯化。此外,还合成了针对荧光素酶基因的阴性对照产物luc-shRNA。将合成产物在12%变性聚丙烯酰胺凝胶电泳鉴定产物。在紫外分光光度计上OD260和OD280读取吸光度值,计算shRNA的产量(1OD260=40μgRNA)。 结果合成产物在12%变性聚丙烯酰胺凝胶电泳, shRNA1、luc-shRNA特异性好,在52bp Mark处呈现两条平行清晰条带,而shRNA2条带较淡。shRNA1和shRNA2的产量分别为3.56μg和0.91μg, luc-shRNA的量为3.22μg。三种产物的吸光度OD260/OD280在1.8-2.0之间。 结论成功合成一条针对大鼠骨桥蛋白基因的发夹状RNA和一条作为阴性对照实验的发夹状RNA。 第三部分短发夹RNA抑制大鼠VSMC骨桥蛋白基因表达的研究 目的将骨桥蛋白特异性shRNA转染大鼠VSMC,检测骨桥蛋白基因在mRNA和蛋白质水平的表达情况。 方法实验分为三组:(1)空白对照组,仅加入RNAiFect TransfectionReagent(2)阴性对照组,加入RNAiFect TransfectionReagent+luc-shRNA(3)RNAi组,RNAiFect TransfectionReagent+OPN-shRNA。在VSMC融合60%时转染细胞转染。48h后,利用Trizol试剂分别提取各组细胞总RNA和总蛋白。通过RT-PCR检测骨桥蛋白基因mRNA的表达;Western blot方法检测其蛋白质的表达。RT-PCR扩增产物在1.5%琼脂糖凝胶上进行电泳并进行半定量分析。 结果RNAi组的OPN基因mRNA的表达强度与空白对照组比较下调76.3%±3.4%(P0.01),显著低于空白对照组。阴性对照组是空白对照组的98.1%±1.5%,两者之间OPN基因mRNA的表达强度没有明显差异性(P0.05)。Westernblot印迹结果显示:转染OPN-shRNA组灰度值比明显降低,OPN蛋白质表达量较空白对照组下调68.7%±4.6%(P0.01)。阴性对照组OPN蛋白表达量是空白对照组间差异无显著性(P0.05)。 结论通过体外转录合成的骨桥蛋白特异性短发夹RNA成功地转染到大鼠VSMC中,并高效、特异性抑制了骨桥蛋白基因的表达,达到基因沉默的目的。 第四部分短发夹RNA对大鼠VSMC增殖、黏附及迁移能力的影响 目的在利用RNAi技术抑制血管平滑肌细胞OPN基因表达的基础上,研究血管平滑肌细胞增殖、迁移和黏附特性的改变。 方法采用MTT法检测细胞增殖,各组于24h和、48h和72h在490nm波长测量吸光度(A490)。1%层黏连蛋白包被96孔培养板,分别在转染4h、6h、8h后检测吸光度(A490)。采用Boyden’s小室检测迁移能力的变化。转染48h后,将迁移至下室面的VSMC用4%多聚甲醛固定,PBS洗涤,苏木素、伊红染色。在显微镜下计数,每张滤膜随机取5个高倍视野(×400)取均值。 结果转染特异性shRNA后VSMC的增殖受到显著的抑制,RNAi组的吸光度在24h,48h和72h为空白对照组的52.2%±5.48%,46.2%±4.69%和33.2%±4.87%(P0.01)。阴性对照组的吸光度在3个时间点则均与空白对照组比较,缺乏显著性差异(P0.05)。这表明抑制OPN表达的shRNA对体外培养的VSMC的增殖有明显抑制作用。在细胞黏附实验中,RNAi组中VSMC数量明显下降,在4h,6h和8h分别为空白对照组的58.5%±5.6%,65.2%±7.4%和64.4%±6.7%(P0.01);阴性对照组和空白对照组两者间在相同的时间点均无显著性差异(P0.05)。在细胞迁移中,RNAi组中VSMC迁移数量与空白对照组相比,在4h,6h和8h分别降低23.4%±3.5%,31.9%±4.9%和30.9%±4.1%(P0.05)。 结论OPN特异性shRNA在有效抑制OPN基因后,体外培养VSMC的迁移、黏附和增殖都受到不同程度的抑制。本实验将为骨桥蛋白介导的血管成形术后再狭窄的基因沉默疗法提供实验基础。 第五部分短发夹RNA对大鼠VSMCⅠ、Ⅲ型胶原合成的影响 目的检测VSMC在转染OPN特异性shRNA后,合成Ⅰ、Ⅲ型胶原的情况。 方法细胞转染48h,提取细胞总RNA,RT-PCR检测Ⅰ、Ⅲ型胶原mRNA表达。吸取培养上清液,利用ELISA法检测OPN特异性shRNA对VSMCⅠ、Ⅲ型胶原合成的影响。 结果转染OPN特异性shRNA组(RNAi组)48h后,培养上清液Ⅰ型、Ⅲ型胶原含量分别较空白对照组下调24.2±4.6%和26.7±5.2%,差异具有显著性意义(P0.05)。RT-PCR结果经半定量分析显示:在各组之间Ⅰ型、Ⅲ型胶原基因mRNA灰度值均无显著性差异(P0.05)。 结论出现上述结果的原因,可能是在RNAi组VSMC的数量在转染后低于两个对照组,导致VSMC分泌到上清液中的Ⅰ、Ⅲ型胶原有所不同。
[Abstract]:the first part VSMC in rats In vitro culture and identification purposes, Sprague-Dawley rat thoracic aortic smooth muscle cells were isolated and established in vitro In vitro culture model of vascular smooth muscle cells, male Sprague-Dawley (SD) rat thoracic aorta was removed aseptically, adventitia tissue and inner membrane were removed, tissue mass was used to reverse dry culture, and the culture was performed. After a week of passage, the cells were stained by immunohistochemistry. The results showed that under the light microscope and under the phase-contrast microscope, the cells were found to be in the form of a shuttle, and the characteristic of "trunk>" peak "was present when the cells were grown to the fused state. / unk> and "valley" characteristics. At the same time, the immunohistochemistry-specific anti-interference-actin (E-actin) was used. The immunohistochemical staining of actin was positive and the purity was over 96%. Conclusion The rat's blood vessel was cultured by tissue culture method. smooth muscle cells are simple and easy to operate, and are economical and can be provided for subsequent experiments Cell origin. The design and synthesis of the second part of the osteopontin-specific short hairpin RNA is designed on-line for osteopontin The sequence of siRNA uses in vitro transcription to obtain hairpin RNA, laying the foundation for next-step cell transfection. The method is in NCBI (

http://www.ncbi.n The mRNA sequence of the rat bone bridge protein was retrieved from the Nucleotide library of lm.nih.gov/ ). er to design si in accordance with the web page requirements and prompts RNA, select two of the siRNA sequences (shRNA 1 and shRNA 2), according to the MessageMuter? shRNAi Pr The description of the Ducky Kit is designed to design a DNA oligonucleic acid to perform an annealing reaction, in addition, a needle is also synthesize, The negative control product luc-shRNA of the luciferase gene was identified. The product was identified by gel electrophoresis of the 12% denatured polytetramine amine. The absorbance values were read from the OD260 and OD280 on the photometer to calculate the yield of shRNA (1 OD260 = 40. mu.gRNA). The resultant product was electroformed at 12% of the denatured polytetramine. The specificity of the shRNA 1 and the luc-shRNA was good, two parallel clear bands were presented at the position of 52 bp, while the shRNA 2 was weak. The yield of shRNA 1 and shRNA 2 was 3.56. m 0.91. mu.g, the amount of luc-shRNA was 3.22. m u.g. The absorbance of the three products OD260/ OD28 0 is between 1.8 and 2.0. Conclusion One is successfully synthesized Hairpin RNA of the rat bone bridge protein gene and a hairpin RNA as a negative control experiment. The third part of the short hairpin R The purpose of this study was to study the expression of the osteopontin-specific shRNA in the rat VSMC, and to detect the expression of the osteopontin gene in the mRNA and protein level. RNAiFect TransfectionReagent+luc-shRNA(3)RNAi缁，

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