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

【摘要】：目的 研究COX-2對HSC-T6細胞中Acsl家族相關基因表達的影響，明確COX-2調節(jié)HSC-T6細胞脂類代謝的作用環(huán)節(jié)。 方法 1、實驗分組：HSC-T6細胞組（空白組），pYr-1.1-COX-2shRNA組（COX-2shRNA組）；pYr-1.1-NC組（空載體組）。 2、將構建好的pYr-1.1-COX-2shRNA及pYr-1.1-NC分別轉染至HSC-T6細胞中，并于轉染后48小時用熒光倒置顯微鏡觀察轉染效率。 3、利用Real-time PCR技術檢測轉染后各組細胞內COX-2、Acsl1、Acsl3、Acsl4、Acsl5、Acsl6基因表達情況。 4、根據(jù)Real-time PCR技術檢測結果，選取Acsl家族中有意義的基因，再利用Western Blot技術檢測HSC-T6細胞中Acsl家族相關基因的蛋白表達。 結果 1、轉染HSC-T6細胞48小時后，在熒光倒置顯微鏡下可見細胞質中分布著綠色熒光，且轉染效率達75%以上。 2、HSC-T6細胞不同組別中COX-2mRNA表達情況：COX-2mRNA在COX-2shRNA組中表達低于空白組和空載體組，統(tǒng)計學處理有顯著意義(p＜0.05)。 3、HSC-T6細胞不同組別中Acsl家族相關基因mRNA表達情況：COX-2shRNA組中Acsl6mRNA表達較空白組及空載體組明顯增加，統(tǒng)計學處理有顯著意義(p＜0.05)，，但Acsl1、Acsl3、Acsl4、Acsl5mRNA表達變化不大，沒有統(tǒng)計學意義（p＞0.05）。 4、HSC-T6細胞不同組別中Acsl6蛋白質表達情況：COX-2shRNA組中Acsl6蛋白質表達較空白組及空載體組明顯增加，統(tǒng)計學處理有顯著意義(p＜0.05)。 結論 1、靶向COX-2的shRNA可以明顯沉默HSC-T6細胞中COX-2的表達。 2、在大鼠HSC-T6細胞中，Acsl1、Acsl3、Acsl4、Acsl5、Acsl6五種亞型均有表達；但沉默HSC-T6細胞COX-2表達后，僅Acsl6mRNA表達明顯增強，Acsl1、Acsl3、Acsl4、Acsl5mRNA的表達無顯著作用。 3、沉默HSC-T6細胞COX-2表達后，Acsl6蛋白質表達增加。 4、COX-2可能通過下調Acsl6的表達來調控HSC-T6細胞的脂類代謝。
[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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575.5

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