【摘要】： 第一部分 大鼠VSMC的體外培養(yǎng)及鑒定 目的體外分離Sprague-Dawley大鼠胸主動脈平滑肌細胞,建立血管平滑肌細胞體外培養(yǎng)模型。 方法無菌下取雄性Sprague-Dawley(SD)大鼠胸主動脈,清除外膜組織和內(nèi)膜,采用組織塊翻轉(zhuǎn)干涸培養(yǎng)法進行培養(yǎng)。2周后進行傳代,細胞爬片行免疫組織化學(xué)鑒定。 結(jié)果光鏡以及相差顯微鏡下觀察細胞呈梭形,生長至融合狀態(tài)時呈現(xiàn)特有的“峰”與“谷”特點。同時,運用免疫組化特異抗α-肌動蛋白(α-actin)單抗免疫組織化學(xué)染色陽性,純度達96%以上。結(jié)論采用組織塊培養(yǎng)法培養(yǎng)大鼠血管平滑肌細胞簡單易行,且又經(jīng)濟,可為后續(xù)實驗提供細胞來源。 第二部分骨橋蛋白特異性短發(fā)夾RNA的設(shè)計與合成 目的在線設(shè)計針對骨橋蛋白siRNA的序列,采用體外轉(zhuǎn)錄的方法,獲得發(fā)夾狀RNA,為下一步的細胞轉(zhuǎn)染奠定基礎(chǔ)。 方法在NCBI(http://www.ncbi.nlm.nih.gov/)的Nucleotide庫中檢索有關(guān)大鼠骨橋蛋白的mRNA序列。Dharmacon siDESIGN Center按照網(wǎng)頁要求和提示設(shè)計siRNA ,選擇其中的兩條siRNA序列(shRNA1和shRNA2)。按照MessageMuter? shRNAi Production Kit的說明設(shè)計DNA寡核苷酸,進行退火反應(yīng)、延伸反應(yīng)、體外轉(zhuǎn)錄反應(yīng)和RNA純化。此外,還合成了針對熒光素酶基因的陰性對照產(chǎn)物luc-shRNA。將合成產(chǎn)物在12%變性聚丙烯酰胺凝膠電泳鑒定產(chǎn)物。在紫外分光光度計上OD260和OD280讀取吸光度值,計算shRNA的產(chǎn)量(1OD260=40μgRNA)。 結(jié)果合成產(chǎn)物在12%變性聚丙烯酰胺凝膠電泳, shRNA1、luc-shRNA特異性好,在52bp Mark處呈現(xiàn)兩條平行清晰條帶,而shRNA2條帶較淡。shRNA1和shRNA2的產(chǎn)量分別為3.56μg和0.91μg, luc-shRNA的量為3.22μg。三種產(chǎn)物的吸光度OD260/OD280在1.8-2.0之間。 結(jié)論成功合成一條針對大鼠骨橋蛋白基因的發(fā)夾狀RNA和一條作為陰性對照實驗的發(fā)夾狀RNA。 第三部分短發(fā)夾RNA抑制大鼠VSMC骨橋蛋白基因表達的研究 目的將骨橋蛋白特異性shRNA轉(zhuǎn)染大鼠VSMC,檢測骨橋蛋白基因在mRNA和蛋白質(zhì)水平的表達情況。 方法實驗分為三組:(1)空白對照組,僅加入RNAiFect TransfectionReagent(2)陰性對照組,加入RNAiFect TransfectionReagent+luc-shRNA(3)RNAi組,RNAiFect TransfectionReagent+OPN-shRNA。在VSMC融合60%時轉(zhuǎn)染細胞轉(zhuǎn)染。48h后,利用Trizol試劑分別提取各組細胞總RNA和總蛋白。通過RT-PCR檢測骨橋蛋白基因mRNA的表達;Western blot方法檢測其蛋白質(zhì)的表達。RT-PCR擴增產(chǎn)物在1.5%瓊脂糖凝膠上進行電泳并進行半定量分析。 結(jié)果RNAi組的OPN基因mRNA的表達強度與空白對照組比較下調(diào)76.3%±3.4%(P0.01),顯著低于空白對照組。陰性對照組是空白對照組的98.1%±1.5%,兩者之間OPN基因mRNA的表達強度沒有明顯差異性(P0.05)。Westernblot印跡結(jié)果顯示:轉(zhuǎn)染OPN-shRNA組灰度值比明顯降低,OPN蛋白質(zhì)表達量較空白對照組下調(diào)68.7%±4.6%(P0.01)。陰性對照組OPN蛋白表達量是空白對照組間差異無顯著性(P0.05)。 結(jié)論通過體外轉(zhuǎn)錄合成的骨橋蛋白特異性短發(fā)夾RNA成功地轉(zhuǎn)染到大鼠VSMC中,并高效、特異性抑制了骨橋蛋白基因的表達,達到基因沉默的目的。 第四部分短發(fā)夾RNA對大鼠VSMC增殖、黏附及遷移能力的影響 目的在利用RNAi技術(shù)抑制血管平滑肌細胞OPN基因表達的基礎(chǔ)上,研究血管平滑肌細胞增殖、遷移和黏附特性的改變。 方法采用MTT法檢測細胞增殖,各組于24h和、48h和72h在490nm波長測量吸光度(A490)。1%層黏連蛋白包被96孔培養(yǎng)板,分別在轉(zhuǎn)染4h、6h、8h后檢測吸光度(A490)。采用Boyden’s小室檢測遷移能力的變化。轉(zhuǎn)染48h后,將遷移至下室面的VSMC用4%多聚甲醛固定,PBS洗滌,蘇木素、伊紅染色。在顯微鏡下計數(shù),每張濾膜隨機取5個高倍視野(×400)取均值。 結(jié)果轉(zhuǎn)染特異性shRNA后VSMC的增殖受到顯著的抑制,RNAi組的吸光度在24h,48h和72h為空白對照組的52.2%±5.48%,46.2%±4.69%和33.2%±4.87%(P0.01)。陰性對照組的吸光度在3個時間點則均與空白對照組比較,缺乏顯著性差異(P0.05)。這表明抑制OPN表達的shRNA對體外培養(yǎng)的VSMC的增殖有明顯抑制作用。在細胞黏附實驗中,RNAi組中VSMC數(shù)量明顯下降,在4h,6h和8h分別為空白對照組的58.5%±5.6%,65.2%±7.4%和64.4%±6.7%(P0.01);陰性對照組和空白對照組兩者間在相同的時間點均無顯著性差異(P0.05)。在細胞遷移中,RNAi組中VSMC遷移數(shù)量與空白對照組相比,在4h,6h和8h分別降低23.4%±3.5%,31.9%±4.9%和30.9%±4.1%(P0.05)。 結(jié)論OPN特異性shRNA在有效抑制OPN基因后,體外培養(yǎng)VSMC的遷移、黏附和增殖都受到不同程度的抑制。本實驗將為骨橋蛋白介導(dǎo)的血管成形術(shù)后再狹窄的基因沉默療法提供實驗基礎(chǔ)。 第五部分短發(fā)夾RNA對大鼠VSMCⅠ、Ⅲ型膠原合成的影響 目的檢測VSMC在轉(zhuǎn)染OPN特異性shRNA后,合成Ⅰ、Ⅲ型膠原的情況。 方法細胞轉(zhuǎn)染48h,提取細胞總RNA,RT-PCR檢測Ⅰ、Ⅲ型膠原mRNA表達。吸取培養(yǎng)上清液,利用ELISA法檢測OPN特異性shRNA對VSMCⅠ、Ⅲ型膠原合成的影響。 結(jié)果轉(zhuǎn)染OPN特異性shRNA組(RNAi組)48h后,培養(yǎng)上清液Ⅰ型、Ⅲ型膠原含量分別較空白對照組下調(diào)24.2±4.6%和26.7±5.2%,差異具有顯著性意義(P0.05)。RT-PCR結(jié)果經(jīng)半定量分析顯示:在各組之間Ⅰ型、Ⅲ型膠原基因mRNA灰度值均無顯著性差異(P0.05)。 結(jié)論出現(xiàn)上述結(jié)果的原因,可能是在RNAi組VSMC的數(shù)量在轉(zhuǎn)染后低于兩個對照組,導(dǎo)致VSMC分泌到上清液中的Ⅰ、Ⅲ型膠原有所不同。
[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緇，

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