Skp2基因沉默对食管癌细胞Eca109生物学特性的影响

发布时间：2018-03-27 17:26

  本文选题：食管癌　切入点：凋亡　出处：《湖北医药学院》2017年硕士论文

【摘要】：目的:观察食管癌中Skp2的表达特征,并探讨Ad-shSkp2转染食管癌细胞Eca109后,对其生物学特性的影响以及对食管癌体内及体外的抑制作用。方法:(1)24例食管癌切除手术患者的癌组织以及相应的癌旁正常组织,用免疫组织化学方法检测Skp2的表达情况。(2)利用Ad-shSkp2转染293细胞,扩增后,CsCl密度梯度纯化、离心以获得满足实验需要的Ad-shSkp2。(3)用Ad-shSkp2转染Eca109细胞后,通过荧光显微镜和免疫印迹试验(Western blot)来检测Skp2的表达,用CCK-8检测Ad-shSkp2对食管癌细胞增殖的影响,流式细胞术检测对细胞凋亡和细胞周期的影响,Western blot检测Bcl-2、Bax、PARP以及cleaved-PARP蛋白的表达。(4)利用腺病毒Ad-GFP-RFP-LC3转染Eca109细胞后,在荧光显微镜下观察并记录自噬的发生;以及通过Western blot检测LC3Ⅱ/Ⅰ的比值以及Beclin1的表达。(5)将Eca109细胞接种到裸鼠皮下,构建裸鼠移植瘤模型,分别将Ad-shSkp2、Ad-NC、PBS注射到裸鼠食管癌瘤体中,绘制裸鼠生长曲线,计算肿瘤生长抑制率。剥离瘤体,利用免疫组织化学方法检测各组瘤体中Skp2、PCNA的表达情况。结果:(1)Skp2在癌组织和癌旁组织中均有表达,且在癌组织中的表达率明显高于正常组织。(2)成功获得满足实验需要的Ad-shSkp2与Ad-NC。(3)AdshSkp2转染Eca109细胞后,显著降低了Skp2的表达,Ad-shSkp2转染Eca109细胞后,Eca109的增殖受到了抑制,PI(碘化丙啶)染色流式分析显示G2/M期阻滞以及G0/G1期出现一个明显的亚二倍体峰即凋亡峰。Western blot结果显示下调食管癌细胞中Skp2的表达后,降低了Bcl-2的表达,升高了Bax以及cleaved-PARP的表达。(4)荧光显微镜下观察到Ad-shSkp2作用于食管癌细胞后,诱导了了自噬的发生,LC3Ⅱ/Ⅰ的比值以及Beclin1的表达均增加。(5)苏木精-伊红染色法(HE染色)证实了裸鼠瘤体确实为食管癌,成功构建人食管癌裸鼠移植瘤,Ad-shSkp2组肿瘤的生长明显受限,与对照组比较,差异有统计学意义(P0.05)。实验组中的Skp2以及PCNA的表达明显低于对照组,差异有统计学意义(P0.05)。结论:Skp2在人类食管癌中高表达,沉默Skp2诱导了食管癌细胞Eca109凋亡与自噬的发生;不管在体内还是体外,沉默Skp2都对食管癌的生长有一定的抑制作用。
[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，

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