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

【摘要】：研究目的:探討CLDN1基因在食管鱗癌細(xì)胞失巢凋亡中的作用及分子機(jī)制。方法:通過體外細(xì)胞培養(yǎng)正常食管上皮細(xì)胞(HEEC)、Eca109、Eca9706、TE1、TE10、TE11、Kyse150細(xì)胞系,使用q RT-PCR篩選出低表達(dá)及高表達(dá)CLDN1 m RNA的食管鱗癌細(xì)胞株作為沉默及過表達(dá)CLDN1基因的實(shí)驗(yàn)對(duì)象(TE11及TE10細(xì)胞);體外構(gòu)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(zhuǎn)染TE11及TE10細(xì)胞。轉(zhuǎn)染72小時(shí)后,使用嘌呤霉素篩選轉(zhuǎn)染的細(xì)胞,獲得穩(wěn)定低表達(dá)(sh-CLDN1組)及高表達(dá)CLDN1(CLDN1組)細(xì)胞株。Western blot技術(shù)檢測(cè)CLDN1沉默及過表達(dá)效果;通過流式細(xì)胞儀檢測(cè)篩選細(xì)胞轉(zhuǎn)染率。懸浮培養(yǎng)篩選后的細(xì)胞株,使用流式細(xì)胞儀檢測(cè)失巢凋亡率,觀察CLDN1的表達(dá)與細(xì)胞失巢凋亡的關(guān)系。利用Lv-GFP-RFP-LC3B腺病毒轉(zhuǎn)染篩選的食管鱗癌細(xì)胞,在激光共聚焦顯微鏡觀察下觀察CLDN1表達(dá)對(duì)細(xì)胞自噬影響,并通過蛋白質(zhì)免疫印跡技術(shù)檢測(cè)LC3B、p62蛋白及自噬相關(guān)通路p-AMPKα、ULK1相關(guān)蛋白的表達(dá)情況。使用AMPK通路激動(dòng)劑二甲雙胍激活p-AMPKα后使用蛋白質(zhì)免疫印跡技術(shù)檢測(cè)(sh-CLDN1+MET組)CLDN1、AMPKα、ULK1、LC3B蛋白質(zhì)的表達(dá)變化情況。使用自噬抑制劑三甲基腺嘌呤(3-methyladenine)及激動(dòng)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(xì)胞獲得(CLDN1+3MA組,sh-CLDN1+RAPA組)細(xì)胞并與處理前的細(xì)胞比較失巢凋亡率變化,觀察自噬與失巢凋亡的調(diào)控關(guān)系。動(dòng)物水平上,通過裸鼠皮下荷瘤實(shí)驗(yàn)驗(yàn)證CLDN1對(duì)食管鱗癌細(xì)胞失巢凋亡影響。結(jié)果:體外成功篩選出穩(wěn)定干擾CLDN1及過表達(dá)CLDN1的細(xì)胞。Western bolt檢測(cè)發(fā)現(xiàn):沉默組(sh-CLDN1組)CLDN1的表達(dá)與NC組相比下調(diào)(0.275±0.046比0.691±0.031,P0.05),過表達(dá)組(CLDN1組)CLDN1的表達(dá)與NC組相比上調(diào)(0.706±0.086比0.191±0.051,P0.05)。干擾組失巢凋亡率較對(duì)照組明顯升高(38.901±2.541%比19.882±3.568%,P0.05)。過表達(dá)組失巢凋亡率較對(duì)照組下降(17.170±2.122%比39.121±3.281%,P0.05)。結(jié)果表明干擾CLDN1的表達(dá)可促進(jìn)食管鱗癌細(xì)胞失巢凋亡,過表達(dá)CLDN1的表達(dá)可抑制食管鱗癌細(xì)胞失巢凋亡。激光共聚焦顯微鏡觀察統(tǒng)計(jì)自噬小體顯示:下調(diào)CLDN1能夠抑制細(xì)胞自噬(5.671±1.267比11.50±1.50,P0.05),上調(diào)CLDN1能夠促進(jìn)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(dá)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(dòng)劑雷帕霉素促進(jìn)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(xì)胞及動(dòng)物組織中Western blot顯示干擾CLDN1引起p-AMPKα和ULK1表達(dá)下調(diào),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A(yù)MPKα亞基磷酸化及CLDN1過表達(dá)后,ULK1表達(dá)上調(diào),LC3BII/LC3BI比值增加。結(jié)論:在食管鱗癌細(xì)胞中,CLDN1基因可以通過p-AMPKα/ULK1信號(hào)通路促進(jìn)自噬作用來抑制細(xì)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(xué)位授予單位】：西南醫(yī)科大學(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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