發(fā)布時(shí)間：2018-04-15 22:21

  本文選題：慢病毒載體 + PLCE1基因�。� 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：目的:探討PLCE1(Phospholipase C Epsilon-1)基因RNA干擾(RNAi)的重組慢病毒載體對(duì)食管癌Eca109細(xì)胞系裸鼠移植瘤生長(zhǎng)的影響及機(jī)制。方法:前期研究已明確了具有下調(diào)PLCE1基因的si RNA干擾序列,設(shè)計(jì)并合成sh RNA oligo,將其構(gòu)建入干擾慢病毒載體p SIH1-H1-cop GFP,用構(gòu)建的慢病毒載體p SIH1-H1-cop GFP/sh R-PLCE1轉(zhuǎn)染293T細(xì)胞來(lái)包裝慢病毒,用包裝的p L/sh RNA/PLCE1慢病毒轉(zhuǎn)染Eca109細(xì)胞,觀察轉(zhuǎn)染效率。用實(shí)時(shí)定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(q RT-PCR)、蛋白印跡技術(shù)(Western Blot)分別檢測(cè)慢病毒轉(zhuǎn)染后Eca109細(xì)胞株中PLCE1 mRNA及蛋白水平表達(dá)的變化,明確處理后PLCE1沉默的效果。將處于對(duì)數(shù)生長(zhǎng)期的食管癌Eca109細(xì)胞常規(guī)消化,將20只裸鼠隨機(jī)分成實(shí)驗(yàn)組、陰性對(duì)照組,分別向裸鼠其右腋皮下分別注射p SIH1-H1-cop GFP/sh R-PLCE1或p SIH1-H1-cop GFP的食管癌Eca109細(xì)胞200微升(3×107個(gè)細(xì)胞),從肉眼可見(jiàn)腫瘤形成時(shí)開(kāi)始,每三天測(cè)量一次各組腫瘤體積的大小及裸鼠體重,繪制腫瘤生長(zhǎng)曲線,35天后處死裸鼠,取瘤測(cè)量體積和重量,分別計(jì)算體積抑瘤率和重量抑瘤率。免疫組化法檢測(cè)PLCE1、Ki-67、Bcl-2蛋白在兩組裸鼠移植瘤中的表達(dá)變化;免疫熒光法檢測(cè)兩組裸鼠移植瘤中PLCE1、Ki-67、Bcl-2和Beclin-1蛋白的表達(dá);原位末端標(biāo)記(TUNEL)染色法檢測(cè)實(shí)驗(yàn)組和陰性對(duì)照組裸鼠移植瘤中的細(xì)胞凋亡變化情況。結(jié)果:1.qRT-PCR及Western bolt檢測(cè)結(jié)果表明:PLCE1沉默組與陰性對(duì)照組相比,PLCE1的mRNA及蛋白表達(dá)水平明顯下降,干擾效率為(73.00±7.95)%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。2.繪制裸鼠移植瘤生長(zhǎng)曲線顯示,實(shí)驗(yàn)組比陰性對(duì)照組的腫瘤生長(zhǎng)速度明顯減慢(P0.001);實(shí)驗(yàn)組和陰性對(duì)照組剝脫的瘤體體積分別為(61.65±6.44)mm3和(0.88±0.30)mm3,體積抑瘤率達(dá)(98.48±0.47)%,差異具有統(tǒng)計(jì)學(xué)意義(P=0.0020)。實(shí)驗(yàn)組和陰性對(duì)照組剝脫的瘤體重量分別為(4.29±1.39)mg和(410±80.97)mg,重量抑瘤率(98.90±0.46)%,差異具有統(tǒng)計(jì)學(xué)意義(P0.0001)。3.免疫組化結(jié)果顯示PLCE1沉默組的PLCE1、Ki-67和Bcl-2陽(yáng)性率分別為(17.67±3.06)%、(7.33±1.53)%、(8.67±2.08)%,均低于各自陰性對(duì)照組(71.33±3.21)%、(20.67±4.01)%和(46.67±5.86)%,差異具有統(tǒng)計(jì)學(xué)意義(P=0.0030,P=0.0117,P=0.0064)。4.免疫熒光結(jié)果顯示PLCE1沉默組PLCE1、Ki-67熒光強(qiáng)度降低,而B(niǎo)eclin-1蛋白熒光強(qiáng)度增強(qiáng);PLCE1沉默組組移植瘤組織中凋亡細(xì)胞的數(shù)量(19.00±2.64)%顯著高于對(duì)照組(10.67±2.08)%,P=0.0462。結(jié)論:沉默PLCE1可能是通過(guò)促進(jìn)食管癌細(xì)胞Eca109的自噬和凋亡,從而抑制裸鼠移植瘤生長(zhǎng)的;因此靶向作用PLCE1基因抑制腫瘤生長(zhǎng),有望成為食管癌癌治療的新途徑。
[Abstract]:Aim: to investigate the effect and mechanism of recombinant lentivirus vector containing PLCE1(Phospholipase C Epsilon-1 gene RNA interference RNAi on the growth of esophageal carcinoma Eca109 cell line in nude mice.Methods: si RNA interference sequence with down-regulation of PLCE1 gene was identified in previous studies. Sh RNA oligo was designed and synthesized, and constructed into interfering lentivirus vector p SIH1-H1-cop GFP. The constructed lentivirus vector p SIH1-H1-cop GFP/sh R-PLCE1 was transfected into 293T cells to package lentivirus.Eca109 cells were transfected with packaged p L/sh RNA/PLCE1 lentivirus and the transfection efficiency was observed.The expression of PLCE1 mRNA and protein in lentivirus-transfected Eca109 cell lines were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting technique. The effect of PLCE1 silencing was determined.Eca109 cells of esophageal carcinoma in logarithmic growth phase were routinely digested and 20 nude mice were randomly divided into experimental group and negative control group.#number0# SIH1-H1-cop GFP/sh R-PLCE1 or p SIH1-H1-cop GFP Eca109 cells of esophageal carcinoma were injected subcutaneously into the right axilla of nude mice respectively. The tumor volume and body weight of each group were measured every three days from the time of tumor formation.The tumor growth curve was drawn 35 days later, the nude mice were killed, the tumor volume and weight were measured, and the volume inhibition rate and the weight inhibition rate were calculated respectively.Immunohistochemical method was used to detect the expression of Bcl-2 protein in transplanted tumor of two groups of nude mice, and the expression of Bcl-2 and Beclin-1 protein in transplanted tumor of two groups of nude mice was detected by immunofluorescence method.Apoptosis was detected by Tunel staining in nude mice in both experimental group and negative control group.Results 1. The results of Western bolt and RT-PCR showed that the expression of mRNA and protein in the control group was significantly lower than that in the control group, and the interference efficiency was 73.00 鹵7.95g. The difference was statistically significant (P 0.05).The growth curve of transplanted tumor in nude mice showed that the tumor growth rate in the experimental group was significantly slower than that in the negative control group (P 0.001), the volume of tumor was 61.65 鹵6.44)mm3 and 0.88 鹵0.30mm3 in the experimental group and the negative control group, respectively, and the tumor inhibition rate was 98.48 鹵0.47mm. the difference was statistically significant.The tumor weight of the experimental group and the negative control group was 4.29 鹵1.39)mg and 410 鹵80.97 mg, respectively. The inhibition rate of tumor weight was 98.90 鹵0.46 mg, and the difference was statistically significant (P 0.0001 路3).Immunohistochemical results showed that the positive rates of PLCE1 Ki-67 and Bcl-2 in the PLCE1 silencing group were 7.33 鹵1.53 and 7.33 鹵1.53, respectively, which were lower than those in the negative control group (71.33 鹵3.21 鹵20.67 鹵4.01% and 46.67 鹵5.86l%, respectively). The difference was statistically significant.The results of immunofluorescence showed that the fluorescence intensity of PLCE1 + Ki-67 was decreased in PLCE1 silencing group, while the number of apoptotic cells in transplanted tumor tissue of PLCE1 silencing group was 19.00 鹵2.64% higher than that of control group (10.67 鹵2.08).Conclusion: silencing PLCE1 may inhibit the growth of transplanted tumor in nude mice by promoting the autophagy and apoptosis of Eca109 cells, therefore, targeting PLCE1 gene to inhibit tumor growth may be a new approach for the treatment of esophageal carcinoma.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

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