本文選題：COX-2 + shRNA�。� 參考：《南華大學(xué)》2014年碩士論文

【摘要】：目的 研究COX-2對HSC-T6細(xì)胞中Acsl家族相關(guān)基因表達(dá)的影響，明確COX-2調(diào)節(jié)HSC-T6細(xì)胞脂類代謝的作用環(huán)節(jié)。 方法 1、實(shí)驗(yàn)分組：HSC-T6細(xì)胞組（空白組），pYr-1.1-COX-2shRNA組（COX-2shRNA組）；pYr-1.1-NC組（空載體組）。 2、將構(gòu)建好的pYr-1.1-COX-2shRNA及pYr-1.1-NC分別轉(zhuǎn)染至HSC-T6細(xì)胞中，并于轉(zhuǎn)染后48小時用熒光倒置顯微鏡觀察轉(zhuǎn)染效率。 3、利用Real-time PCR技術(shù)檢測轉(zhuǎn)染后各組細(xì)胞內(nèi)COX-2、Acsl1、Acsl3、Acsl4、Acsl5、Acsl6基因表達(dá)情況。 4、根據(jù)Real-time PCR技術(shù)檢測結(jié)果，選取Acsl家族中有意義的基因，再利用Western Blot技術(shù)檢測HSC-T6細(xì)胞中Acsl家族相關(guān)基因的蛋白表達(dá)。 結(jié)果 1、轉(zhuǎn)染HSC-T6細(xì)胞48小時后，在熒光倒置顯微鏡下可見細(xì)胞質(zhì)中分布著綠色熒光，且轉(zhuǎn)染效率達(dá)75%以上。 2、HSC-T6細(xì)胞不同組別中COX-2mRNA表達(dá)情況：COX-2mRNA在COX-2shRNA組中表達(dá)低于空白組和空載體組，統(tǒng)計(jì)學(xué)處理有顯著意義(p＜0.05)。 3、HSC-T6細(xì)胞不同組別中Acsl家族相關(guān)基因mRNA表達(dá)情況：COX-2shRNA組中Acsl6mRNA表達(dá)較空白組及空載體組明顯增加，統(tǒng)計(jì)學(xué)處理有顯著意義(p＜0.05)，，但Acsl1、Acsl3、Acsl4、Acsl5mRNA表達(dá)變化不大，沒有統(tǒng)計(jì)學(xué)意義（p＞0.05）。 4、HSC-T6細(xì)胞不同組別中Acsl6蛋白質(zhì)表達(dá)情況：COX-2shRNA組中Acsl6蛋白質(zhì)表達(dá)較空白組及空載體組明顯增加，統(tǒng)計(jì)學(xué)處理有顯著意義(p＜0.05)。 結(jié)論 1、靶向COX-2的shRNA可以明顯沉默HSC-T6細(xì)胞中COX-2的表達(dá)。 2、在大鼠HSC-T6細(xì)胞中，Acsl1、Acsl3、Acsl4、Acsl5、Acsl6五種亞型均有表達(dá)；但沉默HSC-T6細(xì)胞COX-2表達(dá)后，僅Acsl6mRNA表達(dá)明顯增強(qiáng)，Acsl1、Acsl3、Acsl4、Acsl5mRNA的表達(dá)無顯著作用。 3、沉默HSC-T6細(xì)胞COX-2表達(dá)后，Acsl6蛋白質(zhì)表達(dá)增加。 4、COX-2可能通過下調(diào)Acsl6的表達(dá)來調(diào)控HSC-T6細(xì)胞的脂類代謝。
[Abstract]:Purpose To study the effect of COX-2 on the expression of Acsl family related genes in HSC-T6 cells and to clarify the role of COX-2 in regulating lipid metabolism in HSC-T6 cells. Method 1. HSC-T6 cells were divided into two groups: pYr-1.1-COX-2shRNA group (pYr-1.1-COX-2shRNA group) and pYr-1.1-NC group (empty vector group). 2. The constructed pYr-1.1-COX-2shRNA and pYr-1.1-NC were transfected into HSC-T6 cells respectively, and the transfection efficiency was observed by fluorescence inverted microscope 48 hours after transfection. 3. Real-time PCR technique was used to detect the expression of Acsl6 gene in the transfected cells. 4According to the results of Real-time PCR technique, the significant genes of Acsl family were selected and the protein expression of Acsl family related genes in HSC-T6 cells was detected by Western Blot technique. Result 1. Green fluorescence was observed in the cytoplasm of HSC-T6 cells 48 hours after transfection, and the transfection efficiency was over 75%. 2The expression of COX-2mRNA in different groups of HSC-T6 cells was significantly lower in COX-2shRNA group than that in blank group and empty vector group (P < 0.05). 3The expression of Acsl family related gene mRNA in different groups of HSC-T6 cells was significantly higher in the group of Acsl family associated with mRNA than in the control group and empty vector group (P < 0.05), but there was no significant difference in the expression of Acsl6mRNA mRNA between the two groups (p > 0.05), but there was no significant difference in the expression of Acsl6mRNA gene between the two groups (p > 0.05). The expression of Acsl6mRNA family related gene in different groups of HSC-T6 cells was significantly higher than that in the control group and empty vector group. (4) the expression of Acsl6 protein in different groups of HSC-T6 cells was significantly higher than that in control group and empty vector group, and there was significant difference in Acsl6 protein expression between two groups (P < 0.05). Conclusion 1. ShRNA targeting COX-2 could significantly inhibit the expression of COX-2 in HSC-T6 cells. 2. All the five subtypes of Acsl1, Acsl3, Acsl4, Acsl5 and Acsl6 were expressed in rat HSC-T6 cells, but after silencing the expression of COX-2 in HSC-T6 cells, only the expression of Acsl6mRNA increased significantly. 3. After silencing the expression of COX-2 in HSC-T6 cells, the expression of Acsl6 protein increased. 4 COX-2 may regulate lipid metabolism of HSC-T6 cells by down-regulating the expression of Acsl6.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.5

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