本文選題：RNAi + HSP70　； 參考：《吉林大學》2012年碩士論文

【摘要】：目的：探討RNA干擾技術沉默HSP70基因表達對人喉癌細胞株Hep-2生物活性的影響。 方法：采用免疫組化SP染色方法檢測6例聲帶白斑(聲帶白斑組)、6例喉乳頭狀瘤（喉乳頭狀瘤組）和7例聲帶息肉（對照組）的組織標本中HSP70和HSF1蛋白表達。利用免疫熒光染色檢測HSP70在Hep-2中的表達，構建針對HSP70基因的siRNA真核表達載體，以攜帶HSP70基因的人喉癌Hep-2細胞系為靶細胞，通過陽離子脂質體法將重組質粒pHSP70-shRNA轉入Hep-2喉癌細胞。采用流式細胞術檢測Hep-2細胞的細胞周期分布情況，實時定量RT-PCR、免疫熒光及其它技術研究siRNA沉默HSP70表達后對其下游基因CyclinD1、C-myc、Gadd45和P21表達的影響。 結果:1.免疫組化結果顯示，，HSP70陽性表達為棕黃色顆粒，定位于細胞漿和細胞核。在聲帶息肉中低水平表達，HSP70主要定位于細胞核表達。在喉乳頭狀瘤、聲帶白斑表達較強。H-score法半定量結果表明：喉乳頭狀瘤組HSP70表達（4.25±0.53）與聲帶息肉組（3.63±0.58）比較明顯增高（P0.05），聲帶白斑組HSP70表達最強（5.43±0.40），與聲帶息肉組比較差異有統(tǒng)計學意義（P0.01）。HSF1陽性表達大部分定位于細胞漿，少量細胞核。其表達趨勢與HSP70表達趨勢相似。對聲帶息肉、喉乳頭狀瘤和聲帶白斑組織中HSP70蛋白表達量與HSF1表達的關系進行直線回歸分析，結果顯示：HSP70蛋白表達量與HSF1的變化呈現(xiàn)正的直線相關（r=0.867，P0.01）。 2．免疫熒光結果顯示，HSP70在Hep-2細胞中呈高表達，明顯高于鼻咽上皮永生細胞系NP-69。 3.流式細胞術檢測結果顯示，轉染重組質粒pHSP70-shRNA后, Hep-2細胞增殖明顯受到抑制，細胞凋亡增加, G1期比例上升, G2期比例明顯減少； 4.免疫熒光結果顯示:實驗組(pHSP70-shRNA質粒)較對照組（pRNAT-U6.1/Neo對照質粒）HSP70的表達減弱，同時伴有下游基因C-myc、CyclinD1、Gadd45基因表達的下降。 5.實時定量RT-PCR結果顯示，轉染組HSP70和CyclinD1的mRNA表達明顯低于對照組，而P21mRNA的表達明顯高于對照組。轉染組中CyclinD1mRNA表達的下調和P21mRNA表達的上調可能與HSP70的基因沉默相關。 結論：1. HSP70在Hep-2細胞中高表達，可能在喉癌的發(fā)生發(fā)展中發(fā)揮著重要的作用。2.重組質粒pHSP70-shRNA明顯下調HSP70蛋白在Hep-2喉癌細胞中的表達,并抑制腫瘤細胞的增殖,促進其凋亡,導致細胞中與增殖和凋亡相關基因的表達改變，推測HSP70基因沉默可能與促進喉癌細胞的凋亡相關。
[Abstract]:Objective: To investigate the effect of silencing HSP70 gene by RNA interference on the biological activity of human laryngeal carcinoma cell line Hep-2.
Methods: the expression of HSP70 and HSF1 protein in 6 cases of vocal cords leukoplakia (vocal cords leukoplakia group), 6 cases of laryngeal papilloma (laryngeal papilloma group) and 7 cases of vocal polyps (control group) were detected by immunohistochemical method. The expression of HSP70 in Hep- 2 was detected by immunofluorescence staining and siRNA eukaryotic expression vector for HSP70 gene was constructed. The human laryngeal carcinoma Hep-2 cell line with HSP70 gene was used as the target cell, and the recombinant plasmid pHSP70-shRNA was transferred into the Hep-2 larynx cell by the cationic liposome method. The cell cycle distribution of Hep-2 cells was detected by flow cytometry. The real-time quantitative RT-PCR, immunofluorescence and other techniques were used to study the downstream gene C after the siRNA silencing HSP70 expression. The effects of yclinD1, C-myc, Gadd45 and P21 expression.
Results: 1. the immunohistochemical results showed that the positive expression of HSP70 was brown yellow granules, located in the cytoplasm and nucleus. In the low level of the vocal polyps, the expression of HSP70 was mainly located in the nucleus expression. In the laryngeal papilloma, the expression of the leukoplakia in the vocal cords was strongly expressed by the semi quantitative.H-score method. The expression of HSP70 in the laryngeal papilloma group was (4.25 + 0.53) and the vocal cord. The polyp group (3.63 + 0.58) was significantly higher (P0.05), and the expression of HSP70 in the vocal cords leukoplakia group was the strongest (5.43 + 0.40). There was a significant difference between the vocal polyp group and the vocal polyp group (P0.01). The positive expression of.HSF1 was mostly located in the cytoplasm and a small number of nuclei. The expression trend was similar to that of the HSP70 expression. The relationship between the expression of HSP70 protein and the expression of HSF1 in the fabric was analyzed by linear regression. The results showed that the expression of HSP70 protein showed a positive linear correlation with the changes of HSF1 (r=0.867, P0.01).
2. immunofluorescence results showed that HSP70 was highly expressed in Hep-2 cells, which was significantly higher than that in the nasopharyngeal epithelial cell line NP-69..
The results of 3. flow cytometry showed that after transfection of recombinant plasmid pHSP70-shRNA, the proliferation of Hep-2 cells was obviously inhibited, cell apoptosis increased, the proportion of G1 phase increased, and the proportion of G2 phase decreased significantly.
4. the results of immunofluorescence showed that the expression of HSP70 in the experimental group (pHSP70-shRNA plasmid) was weaker than that of the control group (pRNAT-U6.1/Neo control plasmid), while the downstream gene C-myc, CyclinD1, Gadd45 gene expression decreased.
5. the results of real-time quantitative RT-PCR showed that the mRNA expression of HSP70 and CyclinD1 in the transfected group was significantly lower than that of the control group, while the expression of P21mRNA was significantly higher than that of the control group. The down regulation of CyclinD1mRNA expression and the up regulation of P21mRNA expression in the transfected group may be related to the gene silencing of HSP70.
Conclusion: 1. HSP70 is highly expressed in Hep-2 cells and may play an important role in the development and development of larynx cancer..2. recombinant plasmid pHSP70-shRNA obviously downplays the expression of HSP70 protein in Hep-2 larynx cancer cells, and inhibits the proliferation of tumor cells and promotes its apoptosis, which leads to the expression of the genes associated with proliferation and apoptosis in the cell and conjectured H. SP70 gene silencing may be associated with apoptosis in laryngeal carcinoma cells.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R739.65

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