摘要
目的了解神经生长因子(nerve growth factor,NGF)受体p75NTR在肝癌组织和肝癌细胞株HepG2中的表达,探讨p75NTR对肝癌细胞凋亡的影响及其关键功能域。方法收集2006年9月至2008年4月于华中科技大学同济医学院附属同济医院行手术切除患者的肝癌组织标本及对应的癌旁组织标本共26对,应用免疫组织化学法检测trkA和p75NTR在肝癌组织和癌旁组织中的表达。应用Western blot检测NGF在肝癌细胞株HepG2和人胚胎肝细胞株L02中的表达。p75NTR组通过脂质体将p75NTR质粒转染入HepG2细胞中,空载体组将pcDNA3.1空载体转染HepG2细胞,空白对照组为未处理的HepG2细胞。采用Western blot和免疫荧光法检测各组p75NTR的表达。应用聚合酶链式反应(polymerase chain reaction,PCR)构建p75NTR肿瘤坏死因子2受体相关因子结构域(tumor necrosis factor 2 receptor associated factor domain,TRAF)、突触后密度蛋白结合结构域(postsynaptic density protein domain,PSD)和Ⅱ型死亡结构域(typeⅡdeathdomain,T2DD)突变质粒,转染入HepG2细胞中,应用流式细胞术检测细胞凋亡,采用方差分析和LSD-t检验比较不同功能域点突变对HepG2细胞凋亡的影响。结果免疫组织化学检测表明TrkA和p75NTR在26对肝癌组织和癌旁组织中均未见明显表达。Westernblot结果表明,NGF在HepG2细胞中的相对表达量为0.749±0.302,显著高于L02细胞的0.452±0.290,差异有统计学意义(t=7.421,P=0.037)。p75NTR质粒转染入HepG2细胞株后,应用Western blot和免疫荧光进行验证,均可见p75NTR高表达,而空载体组和空白对照组未见明显表达。流式细胞术检测表明,p75NTR组、空载体组和空白对照组细胞凋亡率分别为(25.3±3.6)%、(3.2±0.7)%、(3.0±0.8)%,差异有统计学意义(F=21.740,P <0.001),其中p75NTR组显著高于空载体组和空白对照组(t=25.230、23.156,P均<0.001),空载体组和空白对照组差异无统计学意义(t=0.417,P=0.692)。进行p75NTR质粒功能域突变后,空白对照组、空载体组、p75NTR组、TRAF组、T2DD组和PSD组的HepG2细胞凋亡率分别为(3.1%±0.8)%、(3.5%±0.6)%、(25.8%±3.3)%、(24.8%±4.1)%、(4.4±0.9)%、(23.4%±3.3)%,差异有统计学意义(F=47.794,P <0.001),其中T2DD组Hep G2细胞凋亡率降至与空载体组一致(t=-0.386,P=0.708);而TRAF组和PSD组HepG2细胞凋亡率与p75NTR组接近(t=0.429、0.987,P=0.677、0.347)。结论 p75NTR在肝癌组织及HepG2细胞中无明显表达,过表达p75NTR可促进肝癌细胞的凋亡,提示p75NTR在肝癌细胞中发挥肿瘤抑制因子的作用,T2DD是其关键功能域。
Objective To investigate the expression of nerve growth factor(NGF)receptor p75NTR in liver cancer tissues and HepG2 cell line,as well as the effects of p75NTR on the apoptosis of HepG2 cells and its key functional domains.Methods A total of 26 pairs of HCC tissues and para-carcerous liver tissues in Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology from September 2006 to April 2008 were collected.The expression of p75NTR in HCC tissues and para-carcerous tissues were examined by immunohistochemistry.The expression of NGF in HepG2 cell line and human embryonic hepatocyte lines L02 were detected by Western blot.The p75NTR plasmid was transfected into HepG2 cells by liposome as p75NTR group,pcDNA3.1 empty vector was transfected into HepG2 cells as empty vector group and untreated HepG2 cells were taken as control group.The expression of p75NTR was detected by Western blot and immunofluorescence assay.The p75NTR point mutation plasmids of tumor necrosis factor 2 receptor associated factor domain(TRAF),postsynaptic density protein domain(PSD),typeⅡdeath domain(T2DD)were constructed by polymerase chain reaction(PCR)and transfected into HepG2 cells,respectively.The apoptosis of HepG2 cells were determined by flow cytometry and the effects of mutations in different functional areas on the apoptosis of HepG2 cells were compared by ANOVA and LSD-t test.Results Immunohistochemistry showed that there were no obvious expression of p75NTR in 26 pairs of HCC tissues and para-carcerous tissues.Western blot showed that the relative expression of NGF in HepG2 cells was 0.749±0.302,which was significantly higher than that in L02 cells(0.452±0.290),the difference was statistically significant(t=7.421,P=0.037).p75NTR protein was overexpressed in HepG2 cells which were transfected with p75NTR plasmid and detected by Western blot and immunofluorescence method while there was no obvious expression in empty vector group and blank control group.Flow cytometry showed that the apoptosis rates of HepG2 cells in p75NTR group,empty vector group and blank control group were(25.3±3.6)%,(3.2±0.7)%and(3.0±0.8)%,respectively,the difference was statistically significant(F=21.740,P<0.001).The apoptosis rate of HepG2 cells in p75NTR group was significantly higher than those of empty vector control group and blank control group(t=25.230,23.156;all P<0.001)and there were no significant difference between empty vector group and blank control group(t=0.417,P=0.692).After the mutation of p75NTR plasmid functional domain,the apoptosis rates of HepG2 cells in blank control group,empty vector group,p75NTR group,TRAF group,T2DD group and PSD group were(3.1%±0.8)%,(3.5%±0.6)%,(25.8%±3.3)%,(24.8%±4.1)%,(4.4±0.9)%and(23.4%±3.3)%,respectively,the difference was statistically significant(F=47.794,P<0.001).The apoptosis rate of HepG2 cells in T2DD group was close to the empty vector group(t=-0.386,P=0.708)and the apoptosis rate of HepG2 cells in TARF and PSD group were close to p75NTR group(t=0.429,0.987;P=0.677,0.347).Conclusions There are no obvious expression of p75NTR in HCC tissues and HepG2 cells.Overexpression of p75NTR could promote the apoptosis of HCC cells.It is suggested that p75NTR may be a tumor suppressor in HCC cells and T2DD domain maybe the key functional domain.
作者
柯小丽
朱倩
田德安
黎培员
Ke Xiaoli;Zhu Qian;Tian De’an;Li Peiyuan(Division of Gastroenterology,Puren Hospital,Beijing 100062,China;Division of Gastroenterology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)
出处
《中国肝脏病杂志（电子版）》
CAS
2021年第2期6-12,共7页
Chinese Journal of Liver Diseases：Electronic Version
基金
国家自然科学基金(81372663)
肿瘤生物学国家重点实验室开放基金(CBSKL201720)
湖北省自然科学基金(2017CFB457)。
关键词
肝细胞癌
神经生长因子受体
P75NTR
凋亡
功能域
Hepatocellular carcinoma
Nerve growth factor receptor
p75NTR
Apoptosis
Functional domain
