本文選題：RNA干擾 + 慢病毒載體　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：目的:構(gòu)建靶向特異尿激酶型纖溶酶原激活物(uPA)-siRNA慢病毒表達(dá)載體后并篩選出高效靶向序列,轉(zhuǎn)染兔軟骨細(xì)胞,觀察其對軟骨細(xì)胞增殖、u PA、MMP-3基因及蛋白表達(dá)水平的影響。方法:取原代生長良好的兔膝關(guān)節(jié)軟骨細(xì)胞傳代接種培養(yǎng),根據(jù)Gen Bank中兔u PA基因序列,參照si RNA靶點設(shè)計原則,設(shè)計、合成構(gòu)建靶向兔u PA-si RNA序列,利用RT-PCR篩選高效靶向序列,按照不同的感染復(fù)數(shù)(MOI)值加入慢病毒顆粒液,共同培養(yǎng)后確定最佳感染復(fù)數(shù)(MOI)值,并測定分析病毒感染效率,高效靶向序列經(jīng)慢病毒包裝后,通過Lipofectamine 2000轉(zhuǎn)染兔軟骨細(xì)胞,并按實驗進(jìn)行隨機(jī)分組,分為實驗組(轉(zhuǎn)染u PA-si RNA慢病毒載體組)、空載體組(轉(zhuǎn)染空慢病毒載體組)、空白對照組(未予任何處理組)、藥物對照組(加載MMP特異性抑制劑TIMP)。細(xì)胞離體培養(yǎng)96h后,應(yīng)用實時定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)及蛋白免疫印跡法(Western blot)分別檢測軟骨細(xì)胞u PA-si RNA對細(xì)胞內(nèi)u PA、MMP-3 m RNA和蛋白的表達(dá)水平,并通過CCK-8法檢測u PA-si RNA對軟骨細(xì)胞增殖的影響。結(jié)果:慢病毒載體成功轉(zhuǎn)染原代軟骨細(xì)胞,通過si RNA載體質(zhì)粒測序與預(yù)期si RNA序列一致,DNA測序序列正確無突變,從中篩選出高效靶向序列(即沉默效果最佳)。轉(zhuǎn)染后軟骨細(xì)胞培養(yǎng)可見,早期軟骨細(xì)胞呈多角形,細(xì)胞貼壁生長;傳2代時,細(xì)胞呈現(xiàn)梭形,并聚集生長;傳5代時,細(xì)胞呈現(xiàn)典型纖維細(xì)胞樣;符合軟骨細(xì)胞生長特點,可進(jìn)行后續(xù)實驗。隨著感染復(fù)數(shù)(MOI)的增加,慢病毒的感染效率也隨之增加,當(dāng)感染復(fù)數(shù)(MOI)為100時感染率超過85%,符合后續(xù)實驗要求。通過CCK-8檢測si RNA對軟骨細(xì)胞增殖能力的影響發(fā)現(xiàn),給予轉(zhuǎn)染u PA-si RNA慢病毒載體后的軟骨細(xì)胞增殖并未受到抑制。實驗組u PA m RNA、MMP-3 m RNA及u PA、MMP-3蛋白的表達(dá)水平顯著低于空載體租、空白對照組和藥物對照組(P0.05),而空載體組、空白對照組及藥物對照組u PA m RNA、MMP-3 m RNA及u PA、MMP-3蛋白的表達(dá)水平差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:成功構(gòu)建高效靶向u PA-si RNA慢病毒載體,其可穩(wěn)定轉(zhuǎn)染兔軟骨細(xì)胞并能高效抑制u PA基因及蛋白表達(dá),也可對MMP-3基因及蛋白表達(dá)呈抑制作用,對軟骨細(xì)胞增殖起到促進(jìn)作用。
[Abstract]:Aim: to construct a plasminogen activator targeting urokinase type plasminogen activator (UPA) -siRNA lentivirus expression vector and screen out a highly efficient target sequence for transfection into rabbit chondrocytes and observe its effect on the expression of MMP-3 gene and protein in chondrocytes. Methods: rabbit knee articular chondrocytes were cultured and cultured. According to the gene sequence of rabbit u PA gene in GenBank, according to the principle of si RNA target design, the target rabbit u PA-si RNA sequence was designed and synthesized. The highly efficient target sequences were screened by RT-PCR, and the lentivirus particles were added to different infected plural moi values. After co-culture, the optimal infection complex moi values were determined, and the efficiency of virus infection was analyzed, and the highly efficient target sequences were packaged by lentivirus. Rabbit chondrocytes were transfected with Lipofectamine 2000. They were divided into experimental group (transfection of UPA-si RNA lentivirus vector group), empty vector group (transfection group of empty lentivirus vector group, blank control group (no treatment group), drug control group (loaded with TIMP, a specific inhibitor of MMP). After cultured in vitro for 96 h, the expression of u PA-si mRNA in chondrocytes was detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotanalysis, respectively, and the expression of uPA-MMP-3 mRNA and protein in chondrocytes were detected. The effect of u PA-si RNA on the proliferation of chondrocytes was detected by CCK-8 method. Results: the lentivirus vector was successfully transfected into the primary chondrocytes. The siRNA plasmid sequencing was consistent with the expected si RNA sequence. After transfection, the chondrocytes were found to be polygonal and adherent to the wall; at passage 2, the cells were fusiform and aggregated; at passage 5, the cells were typical fibrocyte-like, which was consistent with the characteristics of chondrocyte growth. Further experiments can be carried out. The infection efficiency of lentivirus increased with the increase of the number of infected moi. The infection rate of lentivirus was more than 85 when the infection number of moi was 100, which met the requirements of subsequent experiments. The effect of si RNA on the proliferation of chondrocytes was detected by CCK-8. It was found that the proliferation of chondrocytes transfected with lentivirus vector of uPA-si RNA was not inhibited. The expression levels of MMP-3 mRNA and UPA-MMP-3 protein in the experimental group were significantly lower than those in the empty vector group, while in the blank control group and the drug control group, the expression levels of MMP-3 mRNA and uPA-MMP-3 protein were significantly lower than those in the empty vector group. There was no significant difference in the expression level of UPA m RNA-MMP-3 mRNA and uPA-MMP-3 protein between blank control group and drug control group (P 0.05). Conclusion: the highly efficient targeting uPA-si RNA lentivirus vector can be successfully constructed, which can stably transfect rabbit chondrocytes and inhibit the expression of UPA gene and protein, and also inhibit the expression of MMP-3 gene and protein. Promote the proliferation of chondrocytes.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R684.3

【共引文獻(xiàn)】

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