本文選題：食管癌　切入點(diǎn)：凋亡　出處：《湖北醫(yī)藥學(xué)院》2017年碩士論文

【摘要】：目的:觀察食管癌中Skp2的表達(dá)特征,并探討Ad-shSkp2轉(zhuǎn)染食管癌細(xì)胞Eca109后,對(duì)其生物學(xué)特性的影響以及對(duì)食管癌體內(nèi)及體外的抑制作用。方法:(1)24例食管癌切除手術(shù)患者的癌組織以及相應(yīng)的癌旁正常組織,用免疫組織化學(xué)方法檢測(cè)Skp2的表達(dá)情況。(2)利用Ad-shSkp2轉(zhuǎn)染293細(xì)胞,擴(kuò)增后,CsCl密度梯度純化、離心以獲得滿足實(shí)驗(yàn)需要的Ad-shSkp2。(3)用Ad-shSkp2轉(zhuǎn)染Eca109細(xì)胞后,通過(guò)熒光顯微鏡和免疫印跡試驗(yàn)(Western blot)來(lái)檢測(cè)Skp2的表達(dá),用CCK-8檢測(cè)Ad-shSkp2對(duì)食管癌細(xì)胞增殖的影響,流式細(xì)胞術(shù)檢測(cè)對(duì)細(xì)胞凋亡和細(xì)胞周期的影響,Western blot檢測(cè)Bcl-2、Bax、PARP以及cleaved-PARP蛋白的表達(dá)。(4)利用腺病毒Ad-GFP-RFP-LC3轉(zhuǎn)染Eca109細(xì)胞后,在熒光顯微鏡下觀察并記錄自噬的發(fā)生;以及通過(guò)Western blot檢測(cè)LC3Ⅱ/Ⅰ的比值以及Beclin1的表達(dá)。(5)將Eca109細(xì)胞接種到裸鼠皮下,構(gòu)建裸鼠移植瘤模型,分別將Ad-shSkp2、Ad-NC、PBS注射到裸鼠食管癌瘤體中,繪制裸鼠生長(zhǎng)曲線,計(jì)算腫瘤生長(zhǎng)抑制率。剝離瘤體,利用免疫組織化學(xué)方法檢測(cè)各組瘤體中Skp2、PCNA的表達(dá)情況。結(jié)果:(1)Skp2在癌組織和癌旁組織中均有表達(dá),且在癌組織中的表達(dá)率明顯高于正常組織。(2)成功獲得滿足實(shí)驗(yàn)需要的Ad-shSkp2與Ad-NC。(3)AdshSkp2轉(zhuǎn)染Eca109細(xì)胞后,顯著降低了Skp2的表達(dá),Ad-shSkp2轉(zhuǎn)染Eca109細(xì)胞后,Eca109的增殖受到了抑制,PI(碘化丙啶)染色流式分析顯示G2/M期阻滯以及G0/G1期出現(xiàn)一個(gè)明顯的亞二倍體峰即凋亡峰。Western blot結(jié)果顯示下調(diào)食管癌細(xì)胞中Skp2的表達(dá)后,降低了Bcl-2的表達(dá),升高了Bax以及cleaved-PARP的表達(dá)。(4)熒光顯微鏡下觀察到Ad-shSkp2作用于食管癌細(xì)胞后,誘導(dǎo)了了自噬的發(fā)生,LC3Ⅱ/Ⅰ的比值以及Beclin1的表達(dá)均增加。(5)蘇木精-伊紅染色法(HE染色)證實(shí)了裸鼠瘤體確實(shí)為食管癌,成功構(gòu)建人食管癌裸鼠移植瘤,Ad-shSkp2組腫瘤的生長(zhǎng)明顯受限,與對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)組中的Skp2以及PCNA的表達(dá)明顯低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:Skp2在人類(lèi)食管癌中高表達(dá),沉默Skp2誘導(dǎo)了食管癌細(xì)胞Eca109凋亡與自噬的發(fā)生;不管在體內(nèi)還是體外,沉默Skp2都對(duì)食管癌的生長(zhǎng)有一定的抑制作用。
[Abstract]:Objective: to observe the expression of Skp2 in esophageal carcinoma, and to investigate the expression of Eca109 in esophageal carcinoma cells transfected with Ad-shSkp2. Methods the tumor tissues and adjacent normal tissues of 24 patients with esophageal carcinoma undergoing resection of esophageal carcinoma were studied. The expression of Skp2 was detected by immunohistochemical method. (2) Ad-shSkp2 was transfected into 293 cells, then purified by CSCL density gradient, centrifuged to obtain Ad-shSkp2. 3) Eca109 cells were transfected with Ad-shSkp2. The expression of Skp2 was detected by fluorescence microscope and Western blotting, and the effect of Ad-shSkp2 on the proliferation of esophageal carcinoma cells was detected by CCK-8. The effect of flow cytometry on cell apoptosis and cell cycle. Western blot was used to detect the expression of Bcl-2Pax-PARP and cleaved-PARP protein. After transfection of Eca109 cells with adenovirus Ad-GFP-RFP-LC3, autophagy was observed and recorded under fluorescence microscope. Western blot was used to detect the ratio of LC3 鈪，

本文編號(hào)：1672530