CLDN1基因对食管鳞癌细胞失巢凋亡的影响及机制探讨

发布时间：2018-02-13 10:01

本文关键词： CLDN1 食管鳞癌自噬失巢凋亡　出处：《西南医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:探讨CLDN1基因在食管鳞癌细胞失巢凋亡中的作用及分子机制。方法:通过体外细胞培养正常食管上皮细胞(HEEC)、Eca109、Eca9706、TE1、TE10、TE11、Kyse150细胞系,使用q RT-PCR筛选出低表达及高表达CLDN1 m RNA的食管鳞癌细胞株作为沉默及过表达CLDN1基因的实验对象(TE11及TE10细胞);体外构建含有GFP-Puro-sh-CLDN1序列的慢病毒及含有GFP-Puro-CLDN1序列慢病毒载体转染TE11及TE10细胞。转染72小时后,使用嘌呤霉素筛选转染的细胞,获得稳定低表达(sh-CLDN1组)及高表达CLDN1(CLDN1组)细胞株。Western blot技术检测CLDN1沉默及过表达效果;通过流式细胞仪检测筛选细胞转染率。悬浮培养筛选后的细胞株,使用流式细胞仪检测失巢凋亡率,观察CLDN1的表达与细胞失巢凋亡的关系。利用Lv-GFP-RFP-LC3B腺病毒转染筛选的食管鳞癌细胞,在激光共聚焦显微镜观察下观察CLDN1表达对细胞自噬影响,并通过蛋白质免疫印迹技术检测LC3B、p62蛋白及自噬相关通路p-AMPKα、ULK1相关蛋白的表达情况。使用AMPK通路激动剂二甲双胍激活p-AMPKα后使用蛋白质免疫印迹技术检测(sh-CLDN1+MET组)CLDN1、AMPKα、ULK1、LC3B蛋白质的表达变化情况。使用自噬抑制剂三甲基腺嘌呤(3-methyladenine)及激动剂雷帕霉素(Rapamycin)分别处理的筛选的CLDN1组及sh-CLDN1组食管鳞癌细胞获得(CLDN1+3MA组,sh-CLDN1+RAPA组)细胞并与处理前的细胞比较失巢凋亡率变化,观察自噬与失巢凋亡的调控关系。动物水平上,通过裸鼠皮下荷瘤实验验证CLDN1对食管鳞癌细胞失巢凋亡影响。结果:体外成功筛选出稳定干扰CLDN1及过表达CLDN1的细胞。Western bolt检测发现:沉默组(sh-CLDN1组)CLDN1的表达与NC组相比下调(0.275±0.046比0.691±0.031,P0.05),过表达组(CLDN1组)CLDN1的表达与NC组相比上调(0.706±0.086比0.191±0.051,P0.05)。干扰组失巢凋亡率较对照组明显升高(38.901±2.541%比19.882±3.568%,P0.05)。过表达组失巢凋亡率较对照组下降(17.170±2.122%比39.121±3.281%,P0.05)。结果表明干扰CLDN1的表达可促进食管鳞癌细胞失巢凋亡,过表达CLDN1的表达可抑制食管鳞癌细胞失巢凋亡。激光共聚焦显微镜观察统计自噬小体显示:下调CLDN1能够抑制细胞自噬(5.671±1.267比11.50±1.50,P0.05),上调CLDN1能够促进自噬(22.670±2.510比5.671±0.580,P0.05)。自噬抑制剂3-MA抑制自噬后可增加过表达组失巢凋亡率(CLDN1组:6.505±2.104%比CLDN1+3MA组:16.684±2.337%,P0.05),自噬激动剂雷帕霉素促进自噬后降低干扰组(sh-CLDN1组)的失巢凋亡率(sh-CLDN1组:42.035±3.092%比sh-CLDN1+RAPA组:25.984±3.356%,P0.05)。细胞及动物组织中Western blot显示干扰CLDN1引起p-AMPKα和ULK1表达下调,LC3BII/LC3BI比值降低(0.590±0.036、0.59±0.05,0.627±0.150,P0.05);反之则升高。使用二甲双胍激活AMPKα亚基磷酸化及CLDN1过表达后,ULK1表达上调,LC3BII/LC3BI比值增加。结论:在食管鳞癌细胞中,CLDN1基因可以通过p-AMPKα/ULK1信号通路促进自噬作用来抑制细胞的失巢凋亡。
[Abstract]:Objective: to investigate the role and molecular mechanism of CLDN1 gene in the apoptosis of esophageal squamous carcinoma cells. Methods: normal esophageal epithelial cells were cultured in vitro. Using Q RT-PCR to screen esophageal squamous cell carcinoma cell lines with low expression and high expression of CLDN1 m RNA as silencing and overexpression of CLDN1 gene, and constructing lentivirus containing GFP-Puro-sh-CLDN1 sequence and lentivirus containing GFP-Puro-CLDN1 sequence in vitro. TE11 and TE10 cells were transfected in vivo. 72 hours after transfection, Purine mycin was used to screen the transfected cells, and the stable and low expression CLDN1(CLDN1 cells were obtained. Western blot technique was used to detect the silencing and overexpression of CLDN1. The transfection rate of selected cells was detected by flow cytometry, the cell lines were screened by suspension culture, and the apoptotic rate of cell loss was detected by flow cytometry. To observe the relationship between the expression of CLDN1 and apoptosis. The effect of CLDN1 expression on autophagy of esophageal squamous cell carcinoma cells transfected with Lv-GFP-RFP-LC3B adenovirus was observed under confocal laser microscope. The expression of LC3BP62 protein and p-AMPK 伪 -UULK1-associated protein in autophagy related pathway was detected by Western blotting technique. The expression of p-AMPK 伪 ULK1LC3B protein was detected by Western blotting after activation of p-AMPK 伪 by metformin, a AMPK pathway agonist, and Western blotting technique was used to detect AMPK 伪 ULK1LC3B protein in AMPK pathway agonist metformin. Changes of cytoplasm expression. Selected CLDN1 and sh-CLDN1 esophageal squamous carcinoma cells treated with autophagy inhibitor trimethyladenine 3-methyladenine and agonist rapamycinin were treated with CLDN1 group and sh-CLDN1 group, respectively. To compare the change of apoptosis rate of loss of nest, To observe the relationship between autophagy and apoptosis. The effect of CLDN1 on apoptosis of esophageal squamous carcinoma cells was verified by subcutaneous tumor-bearing experiment in nude mice. Results: the stable interfering CLDN1 and overexpression of CLDN1 were successfully screened out in vitro. Western bolt detection showed that the expression of CLDN1 in silencing group was similar to that in NC group. The down-regulation was 0.275 卤0.046 vs 0.691 卤0.031 P0.05A, and the expression of CLDN1 in the overexpression group was increased by 0.706 卤0.086 vs 0.191 卤0.051 P0.050.The apoptosis rate of the interference group was significantly higher than that of the control group (38.901 卤2.541% vs 19.882 卤3.568 P0.05N). The apoptotic rate of the overexpression group was 17.170 卤2.122% vs 39.121 卤3.281 卤3.281P 0.05N. The results showed that the apoptotic rate of the overexpression group was significantly higher than that of the control group. The results showed that the apoptosis rate of the overexpression group was significantly higher than that of the control group. Can promote apoptosis of esophageal squamous carcinoma cells. Overexpression of CLDN1 could inhibit the apoptosis of esophageal squamous carcinoma cells. The results of laser confocal microscopy showed that down-regulation of CLDN1 could inhibit autophagy 5.671 卤1.267 vs 11.50 卤1.50P0.05, and upregulation of CLDN1 could promote autophagy 22.670 卤2.510 vs 5.671 卤0.580P0.05.The inhibition of autophagy was observed by laser confocal microscopy. After inhibiting autophagy, 3-MA increased the apoptotic rate of loss of nests in the overexpression group: 6.505 卤2.104% in CLDN1 group and 16.684 卤2.337 in CLDN1 3MA group, respectively. Autophagy agonist rapamycin promoted the apoptosis rate of sh-CLDN1 group after autophagy and decreased the apoptosis rate of sh-CLDN1 group (42.035 卤3.092% vs sh-CLDN1 RAPA group 25.984 卤3.356N). Western blot showed that the down-regulation of p-AMPK 伪 and ULK1 expression induced by CLDN1 decreased the ratio of LC3BIIR / LC3BI by 0.590 卤0.036 卤0.036 卤0.59 卤0.05 + 0.627 卤0.150P 0.05, whereas increased. The expression of ULK1 up-regulated the ratio of LC3BIILC3BI after the activation of AMPK 伪 subunit phosphorylation by metformin and the overexpression of CLDN1. Conclusion: the ratio of LC3BIILC3BI in esophageal squamous cell carcinoma cells is increased after activation of AMPK 伪 subunit phosphorylation by metformin. CLDN1 gene can promote autophagy through p-AMPK 伪 -ULK1 signaling pathway to inhibit cell apoptosis.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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