摘要
目的 :检测Glypican 3(GPC3 )在肝细胞癌中的表达 ,探讨GPC3 在肝细胞癌中的作用。方法 :( 1 )定向克隆GPC3 基因C末端 1 80bp的片段 ,IPTG诱导原核表达载体 pGEX 5X 1、鉴定并利用谷胱苷肽转移酶纯化GPC3 末端融合蛋白 ;( 2 )免疫家兔 ,制备抗体 ;( 3)应用免疫组化 (SP法 )检测了肝细胞癌及其癌旁组织中GPC3 的表达。结果 :( 1 )纯化了GPC3 末端融合蛋白 ;( 2 )制备了抗人GPC3 多克隆抗体 ;( 3)免疫组化结果表明 2 5例低分化肝细胞癌中 1 8例为阳性 ,表达率为 72 % ;阳性结果为棕色颗粒均匀分布于肝细胞癌的细胞膜上和胞浆内 ;正常肝细胞、胆管细胞、血管内皮细胞、Ito细胞及成纤维细胞均呈阴性 ;癌旁组织不表达。GPC3 的表达和肿瘤大小及门脉癌栓形成有密切关系。结论 :GPC3 在肝细胞癌中高表达 ,且具有细胞特异性 ;GPC3 在低分化的肝细胞癌中有重要作用。
Objective:To investigate the expression of GPC 3 in hepatocelular carcinoma (HCC) and its clinical significance.Methods:(1) Fragment of the last 180 bp length of the ORF was subcloned into prokaryotic expression plasmid pGES 5X 1;the glutathione S transferase fusion protein was induced by IPTG and purified by Glutathione Sepharose 4B.(2) The polyclonal antibody against human GPC 3 was generated by injecting white rabbit with the fusion protein.The polyclonal antibody was salted out from the rabbit serum,purified througth dialysis,and detected by double agargel immune diffusion test.(3) Immunohistochemistry assay was conducted in 25 poorly differentiated HCC tissues.Results:(1) The glutathione S transferase fusion protein was purified.(2) The polyclonal antibody against human GPC 3 was generated;(3) Immunohistochemistry showed that among 25 poorly differentiated HCCs,18 (72%) were positive for GPC 3 protein.In cancer cells,GPC 3 was expressed both on cell membrane and in cytosol in a yellow brown granular pattern.The non tumor portion was not stained.Expression of GPC 3 was closely related to tumor size and portal venous embolus ( P <0.05).Conclusion:GPC 3 is over expressed in HCC,and its expressionis is closely correlated with the tumor size and portal venous embolus.GPC 3 may play important roles in poorly differentiated hepatocellular carcinoma.
出处
《解剖学杂志》
CAS
CSCD
北大核心
2001年第6期499-503,共5页
Chinese Journal of Anatomy
基金
国家重大基础研究项目 (批准号 3982 5 114)
国家自然科学基金 (批准号 39770 379)
