發(fā)布時間：2018-02-13 10:01

  本文關鍵詞： CLDN1 食管鱗癌 自噬 失巢凋亡　出處：《西南醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：研究目的:探討CLDN1基因在食管鱗癌細胞失巢凋亡中的作用及分子機制。方法:通過體外細胞培養(yǎng)正常食管上皮細胞(HEEC)、Eca109、Eca9706、TE1、TE10、TE11、Kyse150細胞系,使用q RT-PCR篩選出低表達及高表達CLDN1 m RNA的食管鱗癌細胞株作為沉默及過表達CLDN1基因的實驗對象(TE11及TE10細胞);體外構建含有GFP-Puro-sh-CLDN1序列的慢病毒及含有GFP-Puro-CLDN1序列慢病毒載體轉染TE11及TE10細胞。轉染72小時后,使用嘌呤霉素篩選轉染的細胞,獲得穩(wěn)定低表達(sh-CLDN1組)及高表達CLDN1(CLDN1組)細胞株。Western blot技術檢測CLDN1沉默及過表達效果;通過流式細胞儀檢測篩選細胞轉染率。懸浮培養(yǎng)篩選后的細胞株,使用流式細胞儀檢測失巢凋亡率,觀察CLDN1的表達與細胞失巢凋亡的關系。利用Lv-GFP-RFP-LC3B腺病毒轉染篩選的食管鱗癌細胞,在激光共聚焦顯微鏡觀察下觀察CLDN1表達對細胞自噬影響,并通過蛋白質免疫印跡技術檢測LC3B、p62蛋白及自噬相關通路p-AMPKα、ULK1相關蛋白的表達情況。使用AMPK通路激動劑二甲雙胍激活p-AMPKα后使用蛋白質免疫印跡技術檢測(sh-CLDN1+MET組)CLDN1、AMPKα、ULK1、LC3B蛋白質的表達變化情況。使用自噬抑制劑三甲基腺嘌呤(3-methyladenine)及激動劑雷帕霉素(Rapamycin)分別處理的篩選的CLDN1組及sh-CLDN1組食管鱗癌細胞獲得(CLDN1+3MA組,sh-CLDN1+RAPA組)細胞并與處理前的細胞比較失巢凋亡率變化,觀察自噬與失巢凋亡的調控關系。動物水平上,通過裸鼠皮下荷瘤實驗驗證CLDN1對食管鱗癌細胞失巢凋亡影響。結果:體外成功篩選出穩(wěn)定干擾CLDN1及過表達CLDN1的細胞。Western bolt檢測發(fā)現(xiàn):沉默組(sh-CLDN1組)CLDN1的表達與NC組相比下調(0.275±0.046比0.691±0.031,P0.05),過表達組(CLDN1組)CLDN1的表達與NC組相比上調(0.706±0.086比0.191±0.051,P0.05)。干擾組失巢凋亡率較對照組明顯升高(38.901±2.541%比19.882±3.568%,P0.05)。過表達組失巢凋亡率較對照組下降(17.170±2.122%比39.121±3.281%,P0.05)。結果表明干擾CLDN1的表達可促進食管鱗癌細胞失巢凋亡,過表達CLDN1的表達可抑制食管鱗癌細胞失巢凋亡。激光共聚焦顯微鏡觀察統(tǒng)計自噬小體顯示:下調CLDN1能夠抑制細胞自噬(5.671±1.267比11.50±1.50,P0.05),上調CLDN1能夠促進自噬(22.670±2.510比5.671±0.580,P0.05)。自噬抑制劑3-MA抑制自噬后可增加過表達組失巢凋亡率(CLDN1組:6.505±2.104%比CLDN1+3MA組:16.684±2.337%,P0.05),自噬激動劑雷帕霉素促進自噬后降低干擾組(sh-CLDN1組)的失巢凋亡率(sh-CLDN1組:42.035±3.092%比sh-CLDN1+RAPA組:25.984±3.356%,P0.05)。細胞及動物組織中Western blot顯示干擾CLDN1引起p-AMPKα和ULK1表達下調,LC3BII/LC3BI比值降低(0.590±0.036、0.59±0.05,0.627±0.150,P0.05);反之則升高。使用二甲雙胍激活AMPKα亞基磷酸化及CLDN1過表達后,ULK1表達上調,LC3BII/LC3BI比值增加。結論:在食管鱗癌細胞中,CLDN1基因可以通過p-AMPKα/ULK1信號通路促進自噬作用來抑制細胞的失巢凋亡。
[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 偽 -UULK1-associated protein in autophagy related pathway was detected by Western blotting technique. The expression of p-AMPK 偽 ULK1LC3B protein was detected by Western blotting after activation of p-AMPK 偽 by metformin, a AMPK pathway agonist, and Western blotting technique was used to detect AMPK 偽 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 鹵0.046 vs 0.691 鹵0.031 P0.05A, and the expression of CLDN1 in the overexpression group was increased by 0.706 鹵0.086 vs 0.191 鹵0.051 P0.050.The apoptosis rate of the interference group was significantly higher than that of the control group (38.901 鹵2.541% vs 19.882 鹵3.568 P0.05N). The apoptotic rate of the overexpression group was 17.170 鹵2.122% vs 39.121 鹵3.281 鹵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 鹵1.267 vs 11.50 鹵1.50P0.05, and upregulation of CLDN1 could promote autophagy 22.670 鹵2.510 vs 5.671 鹵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 鹵2.104% in CLDN1 group and 16.684 鹵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 鹵3.092% vs sh-CLDN1 RAPA group 25.984 鹵3.356N). Western blot showed that the down-regulation of p-AMPK 偽 and ULK1 expression induced by CLDN1 decreased the ratio of LC3BIIR / LC3BI by 0.590 鹵0.036 鹵0.036 鹵0.59 鹵0.05 + 0.627 鹵0.150P 0.05, whereas increased. The expression of ULK1 up-regulated the ratio of LC3BIILC3BI after the activation of AMPK 偽 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 偽 subunit phosphorylation by metformin. CLDN1 gene can promote autophagy through p-AMPK 偽 -ULK1 signaling pathway to inhibit cell apoptosis.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1

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