本文選題：FLIP + 前列腺癌　； 參考：《南昌大學》2014年碩士論文

【摘要】：背景與目的： Fas相關死亡域樣白介素1β轉換酶抑制蛋白(cellular-FLICE inhibitoryprotein,c-FLIP)，簡稱FLIP，研究發(fā)現(xiàn)其過量表達于各類腫瘤細胞中，通過競爭性抑制Fas, TNFR-1, DR4及TRAILR等死亡受體介導的細胞凋亡途徑中的caspase-8(半胱氨酸的天冬氨酸蛋白水解酶8)凋亡蛋白而阻止細胞凋亡程序，促使腫瘤細胞過量生長。FLIP過量表達于PCa細胞，抑制細胞凋亡，維持細胞過量生長，可能是PCa基因治療的有效靶點。本研究旨在通過siRNA靶向沉默PC3細胞中FLIP基因的表達，探討其作為PCa基因治療靶點的可行性。 方法： 將體外培養(yǎng)的PC3細胞分為空白對照組(僅加培養(yǎng)基)，陰性對照組（NC-siRNA，脂質體）實驗組(FLIPL-siRNA，脂質體)。QPCR檢測PC3細胞FLIPLmRNA，caspase-8mRNA表達水平；western blot檢測PC3細胞FLIPL蛋白表達水平；MTT法檢測PC3細胞生長抑制率；流式細胞術檢測PC3細胞凋亡率及Transwell模型檢測PC3細胞侵襲性。 結果： 1）FLIPL-siRNA轉染PC3細胞后48h，QPCR檢測FLIPLmRNA表達水平。相對于空白PC3組與陰性對照PC3組，實驗組PC3細胞的FLIPLmRNA表達水平顯著下降（P0.001）；siRNA-1，siRNA-2，siRNA-3和siRNA-4的抑制率分別為(69.9士2.2)%、(65.7士2.3)%、(75.4士1.6)%和(70.3士3.2)%，其中siRNA-3的抑制作用最強(P0.05）；而空白PC3組和陰性對照PC3組之間FLIPLmRNA的表達水平無顯著差異(P0.05)。siRNA-3組的caspase-8mRNA表達水平較空白組和陰性對照組顯著升高(P0.001)，且空白組與陰性對照組之間的mRNA表達水平差異無統(tǒng)計學意義(P0.05)。 2）FLIPL-siRNA轉染PC3細胞后48h，Western blot法檢測FLIPL蛋白表達水平。相對于空白PC3組，siRNA-3PC3組的FLIPL蛋白表達水平顯著降低(P0.01)。 3）MTT，流式細胞術，Transwell模型檢測發(fā)現(xiàn)siRNA-3對PC3細胞生長抑制率，凋亡率，侵襲性等生物學特征的影響有統(tǒng)計學意義（P0.01）。 結論： 1）實驗發(fā)現(xiàn)FLIP靶向特異性siRNA能在轉錄和翻譯環(huán)節(jié)有效抑制PC3細胞中FLIP基因表達并誘導caspase8表達上調。 2）實驗結果表明FLIPL-siRNA靶向沉默F(xiàn)LIPL基因可抑制PC3細胞生長，促進其凋亡并降低侵襲性。 3）實驗結果提示FLIP基因可能是PCa基因治療的有效靶點，，以FLIP基因為靶點的基因治療可以成為現(xiàn)行PCa治療方法的有效輔助治療措施并提高PCa（尤其是AIPC）患者的生存率。
[Abstract]:Background and purpose:
Fas related death domain like interleukin 1 beta converting enzyme inhibitory protein (cellular-FLICE inhibitoryprotein, c-FLIP), referred to as FLIP, has been found to be overexpressed in various tumor cells by competitive inhibition of caspase-8 (cysteine aspartate protein water) in cell withering pathways mediated by Fas, TNFR-1, DR4 and TRAILR. Deapoptotic protein 8) apoptosis protein, which prevents cell apoptosis, excessively overgrowth of.FLIP in PCa cells, inhibits apoptosis and maintains cell growth, may be an effective target for PCa gene therapy. The aim of this study is to explore the expression of FLIP gene in the silent PC3 cells by targeting siRNA and explore the target as the target of PCa gene therapy. The feasibility of it.
Method:
The PC3 cells in vitro were divided into blank control group (only adding medium), and negative control group (NC-siRNA, liposome) test group (FLIPL-siRNA, liposome).QPCR was used to detect FLIPLmRNA, caspase-8mRNA expression level, Western blot detection of PC3 cell FLIPL protein expression level; MTT method was used to detect the inhibition rate of cell growth; flow cytometry was used. The apoptosis rate of PC3 cells was detected and Transwell cell model was used to detect the invasiveness of PC3 cells.
Result:
1) the expression level of FLIPLmRNA was detected by 48h and QPCR after transfection of FLIPL-siRNA to PC3 cells. The FLIPLmRNA expression level of PC3 cells in the experimental group was significantly lower than that in the blank PC3 group and the negative control group PC3 group (P0.001). SiRNA-1, siRNA-2, (65.7 and 2.3)%, (75.4 1.6)% and (70.3 3.2)%, respectively, (75.4) and (70.3 and 3.2)%, respectively. The inhibitory effect of 3 was the strongest (P0.05), while there was no significant difference in the expression level of FLIPLmRNA between the blank PC3 group and the negative control group (P0.05), the caspase-8mRNA expression level in the group.SiRNA-3 group was significantly higher than that in the blank group and the negative control group (P0.001), and there was no significant difference in the mRNA expression level between the blank group and the negative control group (P0.05).
2) after transfection of FLIPL-siRNA to PC3 cells, the expression level of FLIPL protein was detected by 48h and Western blot. The level of FLIPL protein expression in siRNA-3PC3 group was significantly lower than that in the blank PC3 group (P0.01).
3) MTT, flow cytometry, and Transwell model detection found that siRNA-3 had significant effects on the growth inhibition rate, apoptosis rate and invasiveness of PC3 cells (P0.01).
Conclusion:
1) it was found that FLIP targeted specific siRNA could effectively inhibit FLIP gene expression and induce the up regulation of caspase8 expression in PC3 cells during transcription and translation.
2) the experimental results showed that silencing of FLIPL gene by FLIPL-siRNA could inhibit the growth of PC3 cells, promote apoptosis and reduce invasiveness.
3) the experimental results suggest that FLIP gene may be an effective target for PCa gene therapy, and gene therapy targeting FLIP gene can be an effective adjuvant therapy for current PCa therapy and improve the survival rate of PCa (especially AIPC) patients.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.25

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