肝癌边缘群细胞抑癌基因Tg737杂合性缺失的检测及其意义

目的 检测肝癌边缘群(SP)细胞中Tg737基因的杂合性缺失(LOH),探讨其与肝癌临床特征的关系及意义.方法 胶原酶结合机械消化法分别制备95例肝癌细胞及癌旁正常肝细胞悬液,Hoeehst33342染色并结合流式细胞仪分别分选肝癌及癌旁的边缘群细胞,分别提取DNA后以同一患者的外周血DNA为对照,采用PCR-SSCP-银染方法 检测肝癌和癌旁中SP细胞Tg737基因5个微卫星位点的LOH,然后进一步分析微卫星LOH情况与原发性肝癌临床特征的关系及意义.结果 95例肝癌边缘群细胞中,Tg737基因5个位点LOH发生的平均频率是71.24%,频率最高点为G64212(78.75%),其中24例(25.30%)标本中检出3个以上微卫星标记位点存在LOH.其中SHGC-57879和G64212的LOH与肝癌的转移和TNM分期密切相关(P<0.01).结论 肝癌边缘群细胞中Tg737基因的高频率LOH与原发性肝癌的临床分期和远处转移相关,提示肝癌干细胞中的基因组不稳定性是影响肝癌病理进程的重要因素.

Detection and significance of loss of heterozygosity of the tumor suppressor gene Tg737 in the side population cells of hepatocellular carcinomas

Objective To detect the loss of heterozygosity (LOH) of the Tg737 gene in side population (SP) cells of bepatocellular carcinoma (HCC) and investigate their association with the clinicopathological characteristics of HCC. Methods HCC cell and normal liver cell suspensions of 95 patients were prepared by collagenase and mechanical digestion method. SP cells were sorted by flow cytometry from suspensions of hepatocarcinoma or normal liver cells dyed by Hoechst33342. DNA was extracted from the two groups of SP cells and peripheral blood specimens. LOH of 5 microsatellite markers on the Tg737 gene was detected in the two groups of SP cells. Then the correlation between LOH status and clinicopathological characteristics was analyzed. Results The mean frequency of LOH in 5 markers was 71.24%. The highest frequency of LOH was observed with the G64212 marker (78.75%). Twenty-four (25.30%) tumor samples had a large deletion in mote than three microsatellite markers. LOH of SHGC-57879 and G64212 were closely associated with metastasis and TNM stage (P<0.01). Conclnsion The high frequency LOH of Tg737 is associated with TNM stage and metastasis of HCC, which indicates that genomic instability of liver cancer stem cells may play an important role during pathologic development of HCC.