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

【摘要】：目的:觀察食管癌中Skp2的表達特征,并探討Ad-shSkp2轉(zhuǎn)染食管癌細胞Eca109后,對其生物學特性的影響以及對食管癌體內(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,用免疫組織化學方法檢測Skp2的表達情況。(2)利用Ad-shSkp2轉(zhuǎn)染293細胞,擴增后,CsCl密度梯度純化、離心以獲得滿足實驗需要的Ad-shSkp2。(3)用Ad-shSkp2轉(zhuǎn)染Eca109細胞后,通過熒光顯微鏡和免疫印跡試驗(Western blot)來檢測Skp2的表達,用CCK-8檢測Ad-shSkp2對食管癌細胞增殖的影響,流式細胞術(shù)檢測對細胞凋亡和細胞周期的影響,Western blot檢測Bcl-2、Bax、PARP以及cleaved-PARP蛋白的表達。(4)利用腺病毒Ad-GFP-RFP-LC3轉(zhuǎn)染Eca109細胞后,在熒光顯微鏡下觀察并記錄自噬的發(fā)生;以及通過Western blot檢測LC3Ⅱ/Ⅰ的比值以及Beclin1的表達。(5)將Eca109細胞接種到裸鼠皮下,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,繪制裸鼠生長曲線,計算腫瘤生長抑制率。剝離瘤體,利用免疫組織化學方法檢測各組瘤體中Skp2、PCNA的表達情況。結(jié)果:(1)Skp2在癌組織和癌旁組織中均有表達,且在癌組織中的表達率明顯高于正常組織。(2)成功獲得滿足實驗需要的Ad-shSkp2與Ad-NC。(3)AdshSkp2轉(zhuǎn)染Eca109細胞后,顯著降低了Skp2的表達,Ad-shSkp2轉(zhuǎn)染Eca109細胞后,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)食管癌細胞中Skp2的表達后,降低了Bcl-2的表達,升高了Bax以及cleaved-PARP的表達。(4)熒光顯微鏡下觀察到Ad-shSkp2作用于食管癌細胞后,誘導了了自噬的發(fā)生,LC3Ⅱ/Ⅰ的比值以及Beclin1的表達均增加。(5)蘇木精-伊紅染色法(HE染色)證實了裸鼠瘤體確實為食管癌,成功構(gòu)建人食管癌裸鼠移植瘤,Ad-shSkp2組腫瘤的生長明顯受限,與對照組比較,差異有統(tǒng)計學意義(P0.05)。實驗組中的Skp2以及PCNA的表達明顯低于對照組,差異有統(tǒng)計學意義(P0.05)。結(jié)論:Skp2在人類食管癌中高表達,沉默Skp2誘導了食管癌細胞Eca109凋亡與自噬的發(fā)生;不管在體內(nèi)還是體外,沉默Skp2都對食管癌的生長有一定的抑制作用。
[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：1672530