CTLA4.FasL促进异种肝卵圆细胞在大鼠脾脏的定植  

CTLA4.FasL promotes the engraftment of xenogeneic hepatic oval cells in rat spleen

作　　者：万真[1] 王旭珍 张晓刚[3] Wan Zhen;Wang Xuzhen;Zhang Xiaogang(Department of General Surgery,the First Affiliated Hospital of Nanchang University,Nanchang 330006,Jiangxi Province,China;Department of Critical Care Medicine,the First Affiliated Hospital of Nanchang University,Nanchang 330006,Jiangxi Province,China;Department of Hepatobiliary Surgery,the First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,Shaanxi Province,China)

机构地区：[1]南昌大学第一附属医院普外科,江西南昌330006 [2]南昌大学第一附属医院重症医学科,江西南昌330006 [3]西安交通大学第一附属医院肝胆外科,陕西西安710061

出　　处：《中国组织工程研究》2022年第23期3728-3732,共5页Chinese Journal of Tissue Engineering Research

基　　金：江西省重点研发计划(20203BBGL73142),项目负责人:万真。

摘　　要：背景:CTLA4.FasL可有效抑制同种反应性T淋巴细胞及自身反应性T淋巴细胞,但CTLA4.FasL在异种肝卵圆细胞经脾移植中对异种反应性T淋巴细胞的抑制作用还不明确。目的:研究CTLA4.FasL对异种反应性淋巴细胞增殖的作用,监测CTLA4.FasL修饰的小鼠肝卵圆细胞(CTLA4.FasL-肝卵圆细胞)在大鼠脾脏内的存活与定植。方法:①于小鼠肝卵圆细胞悬液中,加入携带CTLA4.FasL融合基因的重组慢病毒载体Lv/CTLA4.FasL和聚凝胺进行感染,以未感染慢病毒的肝卵圆细胞和感染空白慢病毒-肝卵圆细胞为对照;在荧光显微镜下观察肝卵圆细胞的活力和红色荧光蛋白慢病毒载体的表达;②对行2/3肝切除的SD大鼠分别经脾移植CTLA4.FasL-肝卵圆细胞、空白慢病毒-肝卵圆细胞、肝卵圆细胞和PBS(不含肝卵圆细胞),分别于肝切除术后1,5,14和21 d,采用ELISA法测定大鼠血清CTLA4.FasL浓度,取受体大鼠脾脏进行CK-19的免疫组化检测;③以C57BL/6小鼠淋巴细胞作为刺激细胞,以SD大鼠淋巴细胞作为反应细胞;在混合淋巴细胞反应中,分别接种CTLA4.FasL-肝卵圆细胞,空白慢病毒-肝卵圆细胞及肝卵圆细胞,或分别加入CTLA4.FasL-肝卵圆细胞(质量浓度分为10,50和100μg/L)、空白慢病毒-肝卵圆细胞及肝卵圆细胞培养上清液,培养96 h后,用5-Brdu法测定各组大鼠淋巴细胞的增殖情况。结果与结论:①在异种混合淋巴细胞培养体系中,CTLA4.FasL-肝卵圆细胞及培养上清均能有效抑制大鼠淋巴细胞的增殖;②CTLA4.FasL-肝卵圆细胞移植受体大鼠的脾脏淋巴细胞对小鼠淋巴细胞低免疫应答,而空白慢病毒-肝卵圆细胞和肝卵圆细胞移植受体大鼠来源的脾细胞则表现出强应答;③CTLA4.FasL-肝卵圆细胞组移植后21 d大鼠脾脏实质内可见CK-19阳性细胞,而在PBS组、肝卵圆细胞组和空白慢病毒-肝卵圆细胞组术后21 d未见CK-19阳性细胞;④结果说明,CTLA4.FasL可有效抑制异种BACKGROUND:Cytotoxicn lymphocyte antigen 4.fasrymndrit ligand(CTLA4.FasL)can effectively inhibit alloreactive and autoreactive T lymphocytes.However,the inhibitory effect of CTLA4.FasL on xenogeneic T lymphocytes in hepatic oval cell transplantation via the spleen remains unclear.OBJECTIVE:To investigate the effect of CTLA4.FasL on the proliferation of xenogeneic lymphocytes,and to monitor the survival and engraftment of CTLA4.FasLhepatic oval cells in rat spleen.METHODS:The recombinant lentiviral vectors carrying CTLA4.FasL fusion gene(Lv/CTLA4.FasL)and polybrene were added to the rat hepatic oval cell suspension.We used hepatic oval cells that were not infected with lentivirus and infected blank lentivirus-hepatic oval cells as controls,and then observed the viability of hepatic oval cells and the expression of red fluorescent protein lentiviral vector under a fluorescence microscope.Sprague-Dawley rats undergoing 2/3 hepatectomy were transplanted with CTLA4.FasL-hepatic oval cells,blank lentivirus-hepatic oval cells,hepatic oval cells,or phosphate buffered saline(without hepatic oval cells)through the spleen.At 1,5,14,and 21 days after hepatectomy,the concentration of CTLA4.FasL in rat serum was determined by enzyme linked immunosorbent assay,and the spleen of the recipient rats was taken for CK-19 immunohistochemical staining.C57BL/6 mouse lymphocytes were used as stimulator cells,and Sprague-Dawley rat lymphocytes as response cells.In the mixed lymphocyte reaction,CTLA4.FasL-hepatic oval cells,blank lentivirus-hepatic oval cells,and hepatic oval cells were inoculated,or CTLA4.FasL-hepatic oval cells(a mass concentration of 10,50,and 100μg/L),blank lentivirus-hepatic oval cells,and hepatic oval cell supernatant.After 96 hours of culture,5-bromodeoxyuridine method was used to detect the proliferation of lymphocytes in each group.RESULTS AND CONCLUSION:Both CTLA4.FasL-hepatic oval cells and the culture supernatant could effectively inhibit the proliferation of rat lymphocytes in the xenogeneic mixed lymphocyte

关 键 词：CTLA4.FasL 异种 肝卵圆细胞 脾脏 定植 

分 类 号：R446[医药卫生—诊断学] R496[医药卫生—临床医学]