【摘要】 目的探讨体外培养对成年小鼠肝组织及肝癌组织理化特性的影响。方法采取9月龄H-ras12V转基因雄性小鼠的肝癌组织和癌旁组织及C57/BL6J正常雄性小鼠的正常肝组织进行体外培养,苏木精-伊红染色法检测肝组织在12 d内的生长状态;考马斯亮蓝染色法检测组织中总体蛋白在48 h内的变化趋势;Western blot法检测48 h内培养组织中p-ERK、p-AKT、p-mTOR、GAPDH和Cyclin D1的蛋白表达水平;RT-qPCR法检测48 h内培养组织中18s RNA、Cyclin D1、GAPDH、UQCRB的mRNA表达水平。结果体外培养6 d内,正常雄性小鼠肝组织、H-ras12V转基因雄性小鼠肝癌及癌旁组织的细胞形态变化相似,存活状态良好。体外培养6 d后,正常肝细胞和癌旁细胞逐渐减少,相比之下,肝癌细胞开始出现大量死亡,但出现少数克隆样细胞团。考马斯亮蓝染色检测结果显示,正常肝组织、肝癌组织和癌旁组织的总体蛋白变化趋势相似,随培养时间的延长,在130~170 k D之间的蛋白条带丰度先升高再降低,40~130 k D之间的蛋白条带丰度逐渐增加。Western Blot检测结果显示,随着体外培养时间的延长,组织中p-ERK、p-AKT、p-mTOR、GAPDH和Cyclin D1的蛋白表达水平呈逐渐下降趋势。RT-qPCR检测结果显示,Cyclin D1在正常肝细胞、肝癌细胞及癌旁细胞和GAPDH在正常肝细胞、癌旁细胞中随培养时间的延长,呈现逐级升高趋势;而GAPDH在肝癌细胞和UQCRB在正常肝细胞、肝癌细胞及癌旁细胞中随培养时间的延长,呈现先下降后又逐渐升高的趋势;18s RNA的mRNA表达水平在正常肝细胞、肝癌细胞及癌旁细胞中没有规律性趋势变化。结论成年小鼠肝组织及肝癌组织在体外培养期间,虽然肝细胞的形态学变化不明显,存活良好,但短时间内,众多基因在mRNA和蛋白的表达水平上发生了显著的变化,对实验结果将有较大的影响。更多还原

【Abstract】 To investigate the influence of primary culture on physiochemical properties of normal hepatic tissues and hepatic tumor tissues of adult mice. The hepatic tumor tissues( T) and tumor adjacent tissues( P) of 9 month-aged H-ras12 V transgenic mice and normal hepatic tissues( Wt) of 9 month-aged C57/BL6 J mice were collected to perform the in vitro primary culture. The morphological changes of cultured hepatocytes were detected by hematoxylin-eosin staining during 12 days. The whole-protein variation tendency of cultured hepatic tissues were detected by bradford staining during 48 h. The protein expression levels of p-ERK,p-AKT,p-mTOR,Cyclin D1,and GAPDH of cultured hepatic tissues were detected by western blot during 48 h. The mRNA expression levels of Cyclin D1,GAPDH,UQCRB,and 18 s RNA of cultured hepatic tissues were detected by RT-qPCR during 48 h.The survival condition of primary cultured hepatocytes of Wt, P, and T were fine during 6 days and the morphological changes were resemble. After 6 days,the cell number of hepatocytes of Wt and P decreased gradually. However,hepatocytes of T dead quickly and appeared a few clone-like cell masses. The whole-protein variation tendencies in Wt,P,and T were resemble. The abundance of protein bands in 130 ~ 170 kμ increased first and then decreased during 48 h. The abundance of protein bands in 40 ~ 130 kμ aincreased during 48 h. The protein expression levels of p-ERK,p-AKT,p-m TOR,Cyclin D1,and GAPDH in primary cultured hepatic tissues decreased gradually during 48 h. The mRNA expression levels of Cyclin D1 in hepatic tissues of Wt,P,T and GAPDH in Wt,P increased during 48 h. The mRNA expression levels of GAPDH in hepatic tissues of T and UQCRB in Wt,P,T decreased first and then increased during 48 h. There is no regularity variation tendency for the expression levels of 18 s RNA. During the short-term primary culture for hepatic tissues and hepatic tumor tissues of adult mice,although morphology changes of hepatocytes are not obviously different,the mRNA and protein expression levels of many geneschange significantly. These changes will give great influences on the result of primary culture related experiments.更多还原