慢病毒介导的PLCE1基因沉默对食管癌细胞Eca109裸鼠移植瘤生长影响及机制的研究

发布时间：2018-04-15 22:21

本文选题：慢病毒载体 + PLCE1基因　；参考：《石河子大学》2017年硕士论文

【摘要】：目的:探讨PLCE1(Phospholipase C Epsilon-1)基因RNA干扰(RNAi)的重组慢病毒载体对食管癌Eca109细胞系裸鼠移植瘤生长的影响及机制。方法:前期研究已明确了具有下调PLCE1基因的si RNA干扰序列,设计并合成sh RNA oligo,将其构建入干扰慢病毒载体p SIH1-H1-cop GFP,用构建的慢病毒载体p SIH1-H1-cop GFP/sh R-PLCE1转染293T细胞来包装慢病毒,用包装的p L/sh RNA/PLCE1慢病毒转染Eca109细胞,观察转染效率。用实时定量逆转录聚合酶链反应(q RT-PCR)、蛋白印迹技术(Western Blot)分别检测慢病毒转染后Eca109细胞株中PLCE1 mRNA及蛋白水平表达的变化,明确处理后PLCE1沉默的效果。将处于对数生长期的食管癌Eca109细胞常规消化,将20只裸鼠随机分成实验组、阴性对照组,分别向裸鼠其右腋皮下分别注射p SIH1-H1-cop GFP/sh R-PLCE1或p SIH1-H1-cop GFP的食管癌Eca109细胞200微升(3×107个细胞),从肉眼可见肿瘤形成时开始,每三天测量一次各组肿瘤体积的大小及裸鼠体重,绘制肿瘤生长曲线,35天后处死裸鼠,取瘤测量体积和重量,分别计算体积抑瘤率和重量抑瘤率。免疫组化法检测PLCE1、Ki-67、Bcl-2蛋白在两组裸鼠移植瘤中的表达变化;免疫荧光法检测两组裸鼠移植瘤中PLCE1、Ki-67、Bcl-2和Beclin-1蛋白的表达;原位末端标记(TUNEL)染色法检测实验组和阴性对照组裸鼠移植瘤中的细胞凋亡变化情况。结果:1.qRT-PCR及Western bolt检测结果表明:PLCE1沉默组与阴性对照组相比,PLCE1的mRNA及蛋白表达水平明显下降,干扰效率为(73.00±7.95)%,差异具有统计学意义(P0.05)。2.绘制裸鼠移植瘤生长曲线显示,实验组比阴性对照组的肿瘤生长速度明显减慢(P0.001);实验组和阴性对照组剥脱的瘤体体积分别为(61.65±6.44)mm3和(0.88±0.30)mm3,体积抑瘤率达(98.48±0.47)%,差异具有统计学意义(P=0.0020)。实验组和阴性对照组剥脱的瘤体重量分别为(4.29±1.39)mg和(410±80.97)mg,重量抑瘤率(98.90±0.46)%,差异具有统计学意义(P0.0001)。3.免疫组化结果显示PLCE1沉默组的PLCE1、Ki-67和Bcl-2阳性率分别为(17.67±3.06)%、(7.33±1.53)%、(8.67±2.08)%,均低于各自阴性对照组(71.33±3.21)%、(20.67±4.01)%和(46.67±5.86)%,差异具有统计学意义(P=0.0030,P=0.0117,P=0.0064)。4.免疫荧光结果显示PLCE1沉默组PLCE1、Ki-67荧光强度降低,而Beclin-1蛋白荧光强度增强;PLCE1沉默组组移植瘤组织中凋亡细胞的数量(19.00±2.64)%显著高于对照组(10.67±2.08)%,P=0.0462。结论:沉默PLCE1可能是通过促进食管癌细胞Eca109的自噬和凋亡,从而抑制裸鼠移植瘤生长的;因此靶向作用PLCE1基因抑制肿瘤生长,有望成为食管癌癌治疗的新途径。
[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 卤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 卤6.44)mm3 and 0.88 卤0.30mm3 in the experimental group and the negative control group, respectively, and the tumor inhibition rate was 98.48 卤0.47mm. the difference was statistically significant.The tumor weight of the experimental group and the negative control group was 4.29 卤1.39)mg and 410 卤80.97 mg, respectively. The inhibition rate of tumor weight was 98.90 卤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 卤1.53 and 7.33 卤1.53, respectively, which were lower than those in the negative control group (71.33 卤3.21 卤20.67 卤4.01% and 46.67 卤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 卤2.64% higher than that of control group (10.67 卤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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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