【作者】 柳洋；

【导师】 文继舫；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2003， 硕士

【摘要】 目的：研究DPC4基因转染对结肠癌细胞生长的抑制作用及其作用机制。 方法：利用脂质体转染技术将PcDNA3.1-DPC4质粒和空载体PcDNA3.1质粒分别导入结肠癌细胞SW620；经G418筛选后获得稳定转染细胞克隆，采用Western blot和免疫组化S-P法检测细胞中DPC4的表达；把三组细胞分别接种到裸鼠皮下，观察其出瘤时间，并每三天测量一次肿瘤结节的最长径和最短径，30天后统一处死裸鼠，测量瘤重与瘤体积，观察各脏器有无转移；RT-PCR检测DPC4基因转染前后细胞内p21^WAF1mRNA的表达；免疫组化S-P法检测三组裸鼠瘤纠织内p21^WAF1蛋白的表达。 结果： 1、成功构建DPC4基因稳定高表达的结肠癌细胞株SW620。 2、成功构建结肠癌细胞SW620裸鼠移植瘤模型。 3、DPC4基因转染后导致出瘤时间延迟（P＜0.05）。处死裸鼠分离瘤组织后，PcDNA3.1-DPC4-SW620组肿瘤的体积、重量均小于SW620组、PcDNA3.1-SW620组（P＜0.05），抑瘤率为66％。 4、PcDNA3.1-DPC4-SW620细胞的p21^WAF1mRNA含量高于SW620细胞、PcDNA3.1-SW620细胞（P＜0.05）。 5、PcDNA3.1-DPC4-SW620组裸鼠瘤组织p21^WAF1蛋白表达阳性强度高于SW620组、PcDNA3.1-SW620组（P＜0.01）。 结论： 1、DPC4基因能够抑制结肠癌细胞SW620的裸鼠成瘤性。 2、DPC4基因通过诱导细胞周期负性调节因子p21^WAF1的mRNA、蛋白质表达从而抑制细胞周期，实现抑制结肠癌细胞生长的功能。 3、DPC4基因具有抑制结肠癌细胞生长的作用，为结肠癌的侯选肿瘤抑制基因。更多还原

【Abstract】 Objective: To explore the effect of DPC4 gene on the growth of colorectal carcinoma cell and its associated mechanism.Methods: Human colorectal carcinoma cell line SW620 was transfected with plasmid PcDNA3.1-DPC4 using lipofectamine transfecting technique and stable G418-resistant clones were isolated. The expression of DPC4 protein was detected by immunohistochemistry and Western blot respectively. Then the SW620 cells, PcDNA3.1-SW620 cells and PcDNA3.1-DPC4-SW620 cells were injected into the flanks of the nude mice subcutaneously. The time of xenografts appearing was observed. The longest and shortest diameters of the xenografts were measured every two days. p21 WAF1 mRNA of the cells was detected by RT-PCR and p21 WAF1 protein of the xenografts in the nude mice was analyzed by immunohistochemistry.Results: The PcDNA3.1-DPC4-SW620 cells steadily overexpressing the DPC4 protein were obtained. The expression of DPC4 protein in SW620 cells transfected with the plasmid PcDNA3.1-DPC4 was stronger than that in SW620 cells and PcDNA3.1-SW620 cells. In contrast with the nude mice in group of SW620 cells and PcDNA3.1 -SW620 cells, the time of xenografts appearing of the nude mice in the group of PcDNA3.1-DPC4-SW620 cells was delayed (P<0.05). The volume and the weight of the xenografts in the group of PcDNA3.1- DPC4-SW620 cells were reduced markedly (P<0.05) . The expression of p21WAF1 mRNA of PcDNA3.1-DPC4-SW620 cells was enhanced (P<0.05) . The expression of p21WAF1 protein in the xenografts of the nude mice incubated with PcDNA3.1-DPC4-SW620 cells was increased (P<0.01) .Conclusion: DPC4 gene may play an important role in suppressing the growth of colorectal carcinoma cell SW620. DPC4 gene may inhibit the growth of colorectal carcinoma cell SW620 by positively regulating the expression of p21WAFI mRNA and protein.更多还原