本文選題：轉(zhuǎn)化生長因子β1 + RNA干擾��； 參考：《暨南大學(xué)》2007年碩士論文

【摘要】： 目的：探討TGFβ1siRNA對(duì)TGFβ1基因表達(dá)的干擾作用，為進(jìn)一步深入研究TGFβ1與高氧肺損傷發(fā)生發(fā)展機(jī)理提供一種新的工具；為研究早產(chǎn)兒CLD的基因治療方法提供依據(jù)。 方法：針對(duì)大鼠TGFβ1基因mRNA序列，，設(shè)計(jì)、合成攜帶3條TGFβ1siRNA和TGFβ1基因的綠色熒光蛋白融合表達(dá)質(zhì)粒載體，并設(shè)陰性質(zhì)粒組和空質(zhì)粒組為對(duì)照，通過脂質(zhì)體包裹分別轉(zhuǎn)染293細(xì)胞。轉(zhuǎn)染后24h、48h和72h收集細(xì)胞，在熒光顯微鏡下觀察干擾效果，采用熒光定量PCR檢測TGFβ1基因表達(dá)情況，并計(jì)算干擾效率。 結(jié)果：(1)成功設(shè)計(jì)并構(gòu)建出攜帶大鼠TGFβ1siRNA和TGFβ1的綠色熒光蛋白融合表達(dá)質(zhì)粒載體。(2)脂質(zhì)體包裹質(zhì)粒轉(zhuǎn)染293細(xì)胞后在熒光顯微鏡下觀察，可見轉(zhuǎn)染后24h、48h和72h TGFβ1siRNA質(zhì)粒組細(xì)胞綠色熒光強(qiáng)度均明顯弱于陰性質(zhì)粒組細(xì)胞，空質(zhì)粒載體組未產(chǎn)生綠色熒光。(3)熒光定量PCR檢測轉(zhuǎn)染后293細(xì)胞TGFβ1mRNA表達(dá)量，其中轉(zhuǎn)染后48h陰性質(zhì)粒組表達(dá)量最高(P＜0.05)，轉(zhuǎn)染后24hTGFβ1siRNA質(zhì)粒組表達(dá)量最低(P＜0.05)，空質(zhì)粒組未見TGFβ1mRNA表達(dá)；轉(zhuǎn)染后24h、48h和72h TGFβ1siRNA質(zhì)粒組TGFβ1mRNA表達(dá)量均顯著低于陰性質(zhì)粒組(P＜0.01)，其基因干擾效率則依次遞減，分別為97.2％、97.1％和67.7％。 結(jié)論：研究證明自行設(shè)計(jì)的TGFβ1siRNA轉(zhuǎn)染293細(xì)胞后24h、48h和72hTGFβ1siRNA均能夠高效干擾TGFβ1基因的表達(dá)，其基因干擾效率隨轉(zhuǎn)染后時(shí)間的增加而遞減。
[Abstract]:Objective: to investigate the interfering effect of TGF 尾 1siRNA on the expression of TGF 尾 1 gene, to provide a new tool for further study on the mechanism of TGF 尾 1 and hyperoxia lung injury, and to provide the basis for studying the gene therapy of CLD in premature infants.Methods: according to the mRNA sequence of rat TGF 尾 1 gene, three green fluorescent protein expression plasmids carrying TGF 尾 1siRNA and TGF 尾 1 genes were designed and synthesized. The negative plasmid group and the empty plasmid group were used as control group and transfected into 293 cells by liposome.The cells were collected at 24 h and 72 h after transfection. The interference effect was observed under fluorescence microscope. The expression of TGF 尾 1 gene was detected by fluorescence quantitative PCR, and the interference efficiency was calculated.Results the fusion expression plasmid of green fluorescent protein carrying rat TGF 尾 1siRNA and TGF 尾 1 was successfully designed and constructed. The liposome encapsulated plasmid was transfected into 293 cells and observed under fluorescence microscope.The green fluorescence intensity of the TGF 尾 1siRNA plasmid group was significantly lower than that of the negative plasmid group at 24 h or 72 h after transfection. No green fluorescence was produced in the empty plasmid vector group. The expression of TGF 尾 1mRNA in 293 cells was detected by fluorescence quantitative PCR.The expression of TGF 尾 1mRNA was the highest in the negative plasmid group 48 hours after transfection and the lowest in the 24hTGF 尾 1siRNA plasmid group after transfection (P < 0.05). No TGF 尾 1mRNA expression was found in the empty plasmid group.The expression of TGF 尾 1mRNA in 24 h and 72 h TGF 尾 1siRNA plasmid group was significantly lower than that in negative plasmid group (P < 0.01), and the gene interference efficiency was significantly decreased to 97.2% and 67.7%, respectively.Conclusion: the results showed that both the self-designed TGF 尾 1siRNA and 72hTGF 尾 1siRNA could interfere with the expression of TGF 尾 1 gene in 293 cells 24 h after transfection, and the gene interference efficiency decreased with the increase of transfection time.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R722;R346

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