本文選題：慢病毒 + GRP78�。� 參考：《廣西醫(yī)科大學(xué)》2016年碩士論文

【摘要】：腎細胞癌(renal cell carcinoma, RCC)又簡稱腎癌,起源于腎實質(zhì)腎小管上皮細胞,是泌尿生殖系統(tǒng)中最為常見惡性腫瘤之一,在腎癌中最常見病理類型為腎透明細胞癌(CRCC)。CRCC治療以根治性切除術(shù)(radicalnephrectomy, RN)為主,由于早期臨床癥狀不明顯,臨床上以晚期患者為多。并且部分腎癌患者存在術(shù)后腫瘤復(fù)發(fā)或轉(zhuǎn)移以及多耐藥性,對放、化療治療不敏感,療效不佳。免疫治療由于長期療效尚不能確定和總緩解率不高限制其廣泛推廣。因此深入研究腎細胞癌的增殖和侵襲的分子機制對提高早期診斷率和后續(xù)治療療效十分關(guān)鍵。葡萄糖調(diào)節(jié)蛋白78 (glucose regulated protein78, GRP78) 是 GRP家族中的重要成員,與熱休克蛋白(heat shock protein70, HSP70)具有較高的同源性,屬于HSP70家族成員之一。 GRP78在腫瘤細胞株和腫瘤組織如肝癌、結(jié)腸癌及肺癌、神經(jīng)膠質(zhì)細胞瘤等高表達,并可能與腫瘤的凋亡、耐藥密切相關(guān)。但GRP78在腎細胞癌中的癌細胞增殖和侵襲等方面所起的作用尚未清楚。本研究在證實GRP78基因在腎癌細胞中呈明顯高表達的基礎(chǔ)上,初步提示了GRP78基因可能與腎細胞癌的發(fā)生發(fā)展存在相關(guān)。本實驗首先成功構(gòu)建好的小干擾RNA(small interference RNA, siRNA),然后轉(zhuǎn)染進入到表達GRP78基因的腎癌786-0細胞中。確認siRNA-GRP78在基因和蛋白水平可以有效抑制GRP78的表達。接著研究干擾GRP78基因后對于腎癌細胞增殖和細胞侵襲力的影響。通過RNA干擾法探討GRP78基因后對腎癌786-0細胞的細胞增殖和侵襲力的影響,為今后腎癌的基因治療提供一定的理論和實驗基礎(chǔ)。第一部分：慢病毒RNA干擾GRP78基因載體的構(gòu)建及篩選目的探討慢病毒RNA干擾GPR78基因載體的構(gòu)建及篩選。方法構(gòu)建慢病毒靶向siRNA-GPR78干擾載體并轉(zhuǎn)染進入腎癌786-0細胞株。轉(zhuǎn)染48h后利用Realtime-PCR和Western blot分別檢沏G RP78mRNA和蛋白表達。篩選出一條抑制率最高的siRNA-GPR78進入下一步研究。結(jié)果Realtime-PCR結(jié)果顯示與空白對照組(1.85±0.08)和陰：性對照組(1.79±0.15)比較,GRP78mRNA的表達水平在siRNAl(0.32±0.09)、siRNA2(0.83±0.07)以及siRNA3組(0.79±0.11)均降低,差異有統(tǒng)計學(xué)意義(P0.01)；Western blot結(jié)果顯示與空白對照組(1±0.03)或陰性對照組(1±0.07)比較,GRP78蛋白表達水平在siRNAI組(0.21±0.03)、siRNA2組(0.79±0.03)以及siRNA3組(0.86±0.04)均降低,差異有統(tǒng)計學(xué)意義(P0.0 1)；空白對照組與陰性對照組的GRP78mRNA和蛋白的表達水平差異無統(tǒng)計學(xué)意義(P0.05)。siRNA1抑制GRP78蛋白效率最佳。結(jié)論構(gòu)建成功的siRNA-GRP78慢病毒載體可有效抑制腎癌786-0細胞中的GRP78基因及蛋白表達,以siRNAl抑制GRP78蛋白效率最佳,選擇siRNA1進下一步研究。第二部分：RNA干擾GRP78基因?qū)δI癌細胞增殖和侵襲力的影響目的探討RNA干擾GPR78基因后對腎癌細胞的增殖和侵襲能力的影響。方法將siRNAl-GRP78干擾載體轉(zhuǎn)染進入腎癌786-0細胞中。利用MTT法和Transwell法分別檢測出腎癌細胞的增殖和侵襲力。結(jié)果MTT法檢測結(jié)果顯示與空白對照組和陰性對照組相比,siRNAl組腎癌細胞生長抑制明顯,差異有統(tǒng)計學(xué)意義(P0.01)。Transwell法檢測結(jié)果顯示與空白對照組和陰性對照組相比,siRNAl組腎癌細胞生長抑制明顯,差異有統(tǒng)計學(xué)意義(P0.01)。而陰性對照組與空白對照組比較,細胞的生長抑制不明顯,差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論RNA干擾GRP78基因后,下調(diào)GRP78的表達,同時影響腎癌細胞的增殖和降低細胞的侵襲力。GRP78在促進腎癌細胞發(fā)生發(fā)展、細胞的增殖,以及維持細胞侵襲力等方面中可能起到重要作用。本研究也為以GRP78作為靶點的腫瘤基因治療提供可靠的理論和實驗基礎(chǔ)。
[Abstract]:Renal cell carcinoma (RCC), also referred to as renal carcinoma, is derived from renal tubular epithelial cells. It is one of the most common malignant tumors in the genitourinary system. The most common pathological type of renal cell carcinoma is renal clear cell carcinoma (CRCC).CRCC treatment with radical resection (radicalnephrectomy, RN), due to early clinical symptoms. It is not obvious that the patients with advanced stage are more clinically, and some of the patients with renal cancer have recurrence or metastasis of postoperative tumor and multidrug-resistant. Chemotherapy is not sensitive to chemotherapy, and the curative effect is poor. Molecular mechanism is crucial to improving early diagnostic rate and follow-up treatment. Glucose regulatory protein 78 (glucose regulated protein78, GRP78) is an important member of the GRP family. It has a high homology with the heat shock protein (heat shock protein70, HSP70), one of the members of the HSP70 family. GRP78 in tumor cell lines and tumors. Tissues such as liver cancer, colon cancer, lung cancer, glioma and so on are highly expressed, and may be closely related to the apoptosis and resistance of the tumor. But the role of GRP78 in the proliferation and invasion of the cancer cells in renal cell carcinoma is not clear. This study has suggested that the GRP78 gene is highly expressed in the cell cell of renal cell carcinoma. GRP78 gene may be associated with the development of renal cell carcinoma. First, a successful small interference RNA (small interference RNA, siRNA) was successfully constructed and then transfected into 786-0 cells expressing the GRP78 gene. It was confirmed that siRNA-GRP78 at the gene and protein level could effectively inhibit the expression of GRP78. Then, the interference of GRP78 base was studied. The effect of RNA interference on the proliferation and invasiveness of 786-0 cells in renal carcinoma after GRP78 gene interference, and to provide a theoretical and experimental basis for the gene therapy of renal cancer in the future. Part 1: Construction and screening of GRP78 gene vector of lentivirus RNA interference The construction and screening of the lentivirus RNA interference GPR78 gene carrier. Methods the lentivirus targeted siRNA-GPR78 interference carrier was constructed and transfected into 786-0 cell lines of renal carcinoma. 48h and Western blot were used to detect G RP78mRNA and protein expression respectively after transfection of Realtime-PCR and Western blot. A new siRNA-GPR78 with the highest inhibition rate was selected for the next step of study. Ealtime-PCR results showed that the expression level of GRP78mRNA was in siRNAl (0.32 + 0.09), siRNA2 (0.83 + 0.07) and siRNA3 group (0.79 + 0.11) in the blank control group (1.85 + 0.08) and the sex control group (1.79 + 0.15), and the difference was statistically significant (P0.01). The Western blot results showed that the control group was (1 + 0.03) or negative control group (1). The expression level of GRP78 protein in siRNAI group was (0.21 + 0.03), siRNA2 group (0.79 + 0.03) and siRNA3 group (0.86 + 0.04) decreased, the difference was statistically significant (P0.0 1), and there was no statistical difference between the blank control group and the negative control group in the expression level of GRP78mRNA and protein (P0.05) and the conclusion that the efficiency of the inhibition of GRP78 protein was the best. The construction of a successful siRNA-GRP78 lentivirus vector can effectively inhibit the GRP78 gene and protein expression in 786-0 cells of renal cell carcinoma, with siRNAl to inhibit the efficiency of GRP78 protein, and select siRNA1 for the next step. The second part: RNA interference of GRP78 gene to the proliferation and invasiveness of renal cell carcinoma cells to investigate the effect of RNA interfering with GPR78 gene on renal cancer The effect of cell proliferation and invasion. Methods the siRNAl-GRP78 interference carrier was transfected into 786-0 cells of renal carcinoma. The proliferation and invasiveness of renal cell carcinoma cells were detected by MTT and Transwell. Results the results of MTT assay showed that the growth inhibition of renal cell carcinoma cells in siRNAl group was significantly lower than that in the blank control group and the negative control group. The results of different statistical significance (P0.01).Transwell assay showed that the growth inhibition of renal cell carcinoma cells in siRNAl group was significantly higher than that in the blank control group and the negative control group, and the difference was statistically significant (P0.01). Compared with the blank control group, the cell growth inhibition was not obvious, and the difference was not statistically significant (P0.05). Conclusion RNA interfered with GRP7. After the 8 gene, the expression of GRP78, the proliferation of renal cell carcinoma cells and the reduction of cell invasiveness.GRP78 may play an important role in promoting the development of renal cell carcinoma cells, cell proliferation, and maintaining cell invasiveness. This study also provides a reliable theory and experiment for the tumor gene therapy with GRP78 as a target. Basics.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R737.11

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