RNAi技术沉默HSP70基因治疗喉癌的实验研究

发布时间：2018-07-11 16:37

本文选题：RNAi + HSP70　；参考：《吉林大学》2012年硕士论文

【摘要】：目的：探讨RNA干扰技术沉默HSP70基因表达对人喉癌细胞株Hep-2生物活性的影响。方法：采用免疫组化SP染色方法检测6例声带白斑(声带白斑组)、6例喉乳头状瘤（喉乳头状瘤组）和7例声带息肉（对照组）的组织标本中HSP70和HSF1蛋白表达。利用免疫荧光染色检测HSP70在Hep-2中的表达，构建针对HSP70基因的siRNA真核表达载体，以携带HSP70基因的人喉癌Hep-2细胞系为靶细胞，通过阳离子脂质体法将重组质粒pHSP70-shRNA转入Hep-2喉癌细胞。采用流式细胞术检测Hep-2细胞的细胞周期分布情况，实时定量RT-PCR、免疫荧光及其它技术研究siRNA沉默HSP70表达后对其下游基因CyclinD1、C-myc、Gadd45和P21表达的影响。结果:1.免疫组化结果显示，，HSP70阳性表达为棕黄色颗粒，定位于细胞浆和细胞核。在声带息肉中低水平表达，HSP70主要定位于细胞核表达。在喉乳头状瘤、声带白斑表达较强。H-score法半定量结果表明：喉乳头状瘤组HSP70表达（4.25±0.53）与声带息肉组（3.63±0.58）比较明显增高（P0.05），声带白斑组HSP70表达最强（5.43±0.40），与声带息肉组比较差异有统计学意义（P0.01）。HSF1阳性表达大部分定位于细胞浆，少量细胞核。其表达趋势与HSP70表达趋势相似。对声带息肉、喉乳头状瘤和声带白斑组织中HSP70蛋白表达量与HSF1表达的关系进行直线回归分析，结果显示：HSP70蛋白表达量与HSF1的变化呈现正的直线相关（r=0.867，P0.01）。 2．免疫荧光结果显示，HSP70在Hep-2细胞中呈高表达，明显高于鼻咽上皮永生细胞系NP-69。 3.流式细胞术检测结果显示，转染重组质粒pHSP70-shRNA后, Hep-2细胞增殖明显受到抑制，细胞凋亡增加, G1期比例上升, G2期比例明显减少； 4.免疫荧光结果显示:实验组(pHSP70-shRNA质粒)较对照组（pRNAT-U6.1/Neo对照质粒）HSP70的表达减弱，同时伴有下游基因C-myc、CyclinD1、Gadd45基因表达的下降。 5.实时定量RT-PCR结果显示，转染组HSP70和CyclinD1的mRNA表达明显低于对照组，而P21mRNA的表达明显高于对照组。转染组中CyclinD1mRNA表达的下调和P21mRNA表达的上调可能与HSP70的基因沉默相关。结论：1. HSP70在Hep-2细胞中高表达，可能在喉癌的发生发展中发挥着重要的作用。2.重组质粒pHSP70-shRNA明显下调HSP70蛋白在Hep-2喉癌细胞中的表达,并抑制肿瘤细胞的增殖,促进其凋亡,导致细胞中与增殖和凋亡相关基因的表达改变，推测HSP70基因沉默可能与促进喉癌细胞的凋亡相关。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.65

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