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

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可有效抑制异种反应性淋巴细胞的增殖,促进小鼠肝卵圆细胞在大鼠脾实质内的存活及定植。

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 culture system.Spleen lymphocytes from the recipient rats transplanted with CTLA4.FasL-hepatic oval cells had a low immune response to mouse lymphocytes,while the spleen cells derived from the recipient rats transplanted with blank lentivirus-hepatic oval cells and hepatic oval cells showed strong responses.CK-19 positive cells were detected in rat spleen parenchyma of the CTLA4.FasL-hepatic oval cell group but not in the phosphate buffered saline group,hepatic oval cell group,and blank lentivirus-hepatic oval cell group at 21 days after transplantation.The results show that CTLA4.FasL can promote the survival and engraftment of hepatic oval cells in the rat spleen parenchyma through inhibiting the proliferation of xenogeneic lymphocytes.