【作者】 索涛莉；

【导师】 徐酉华；

【作者基本信息】 重庆医科大学 ， 儿科学， 2010， 硕士

【摘要】 目的:探讨RNA干扰技术沉默Hsp90β（Heat shock protein 90 beta）基因表达对Jurkat细胞生长的抑制作用及化疗敏感性的影响。方法:1.采用Real-Time PCR检测Jurkat、Raji、K562、HL-60细胞株Hsp90βmRNA表达的情况,筛选出高表达Hsp90β的细胞株。2.针对Hsp90β基因全长cDNA序列Hsp90B1（NM₀03299.1）,设计并构建3对Hsp90β干扰序列和一对随机对照序列,分别克隆获得质粒pSOS-Hsp90βi1, pSOS-Hsp90βi2, pSOS-Hsp90βi3及pSOS-Hsp90βicontrol;应用Lipofectamine? LTX将质粒分别转染入高表达Hsp90β的细胞中, Real-Time PCR检测Hsp90βmRNA水平的变化,筛选出沉默效果最好的Hsp90βsiRNA干扰片段;Western印迹法检测Hsp90β蛋白表达水平。3.MTT法和流式细胞仪检测Hsp90βsiRNA对Jurkat细胞生长抑制作用。4.检测Hsp90βsiRNA对Jurkat细胞化疗敏感性的影响,选不同浓度的长春新碱、阿霉素和依托泊苷,通过MTT法抗癌药物敏感试验检测干扰前后对Jurakt细胞化疗敏感性的变化。结果:1.Real-Time PCR结果显示Hsp90β在Jurkat细胞中的表达明显高于其它三株白血病细胞株（Raji,K562,HL-60）,表达差异有统计学意义（P<0.05）。2.应用RNA干扰技术成功构建Hsp90β基因三条特异性真核载体pSOS-Hsp90βi1、2、3并分别转染Jurkat细胞,发现pSOS-Hsp90βi2的沉默效果最明显,Jurkat细胞Hsp90βmRNA及蛋白表达均明显降低（P<0.05）。3.Hsp90β基因表达沉默后,Jurkat细胞增殖明显受抑,细胞凋亡率比空白对照组及转染pSOS-Hsp90βicontrol组明显增加,差异具有显著意义（P<0.05）。4.转染了pSOS-Hsp90βi2的Jurkat细胞对VCR、ADM和VP16药物的IC50显著低于其余两组（P<0.05）。结论:不同的白血病细胞株Hsp90β的表达存在差异,在Jurkat、Raji、K562、HL-60中Jurkat细胞表达Hsp90β最高;成功构建了3种真核表达载体pSOS-Hsp90βsiRNA,并筛选出沉默效果最好的pSOS-Hsp90βi2;靶向pSOS-Hsp90βi2可下调Hsp90β表达,对人白血病Jurkat细胞有明显的生长抑制作用,显著增加了Jurkat细胞对长春新碱、阿霉素和依托泊苷的敏感性,为白血病基因治疗的靶向性奠定理论依据。更多还原

【Abstract】 Objective To investigate the effect of Hsp90β（Heat shock protein 90 beta） gene silencing by small interfering RNA （siRNA） on the growth inhibition and chemosensitivity of Jurkat cells.Methods 1.The cell line overexpressing Hsp90βgene was distinguished by means of Real-Time PCR from the cells of Jurkat, Raji, K562 and HL-60. 2.According to Hsp90βgene cDNA sequence Hsp90B1（NM₀03299.1）, three specific interference sequences and a random controlled sequence were inserted into the pSOS-HUS, respectively. The recombinant eukaryotic expression plasmids pSOS-Hsp90βi1, pSOS-Hsp90βi2, pSOS-Hsp90βi3 and pSOS-Hsp90βicontrol were constructed and transfected into Jurkat cells by Lipofectamine ? LTX with PLUS ? Reagent. The gene silencing efficiency of recombinant plasmid pSOS-Hsp90βi was monitored by real-time PCR and effective Hsp90β-specific RNAi sequences were screened as well. Western blotting was used to detect the protein expression of Hsp90β. 3.The changes in Hsp90βsiRNA, the cell growth and apoptotic rate were determined by MTT assays and flow cytometry. 4. The gradient concentrations of Vincristine, Adriamycin and Etopodide were applied respectively in the cell culture medium of Jurkat before and after the Hsp90βsiRNA transfection. The cell sensitivity to chemotherapy drugs was measure by MTT.Results 1. Real-Time PCR results revealed that Jurkat cell had a highest expression level of Hsp90βof four leukemia cell lines （Raji, K562, HL-60）, the expression difference was statistically significant （P<0.05）; 2. Recombinant plasmid pSOS-Hsp90βi1, 2, 3, eukaryotic vector targeting Hsp90βwas successfully constructed. The expression of Hsp90βin the cells transfected with pSOS-Hsp90βi2 were inhibited significantly at both mRNA and protein levels （P<0.05）. 3. MTT assays and flow cytometry showed that Hsp90βsiRNA inhibited the proliferation of Jurkat cells and induced apoptosis of leukemia cells. 4. pSOS-Hsp90βi2 transfection can increase the sensibility of Jurkat cells to anticancer drugs （for example, VCR, ADM and VP16）. The IC50 of drugs were lower than that of two control groups （p<0.05）.Conclusion Hsp90βis expressed higher in Jurkat cells than that in other leukemia cell lines（Raji, K562, HL-60）; The successfully constructed eukaryotic expression vector pSOS-Hsp90βsiRNA. Highly interference pSOS-Hsp90βi2 was screen; The chemically synthesized specific siRNA targeting Hsp90βcould effectively reduce the expression of Hsp90βgene, inhibit growth and increase the sensitivity of Jurkat cells to VCR, ADM and VP16. In conclusion, it laid a good foundation for further prepare a condition for targeted gene therapy in leukemia.更多还原