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目的研究腺病毒介导CTLA4Ig基因转染正常人肝细胞株L02后,CTLA4Ig在L02细胞内的表达、生物学活性以及对L02细胞生物学特性的影响。方法重组腺病毒载体Ad-CTLA4Ig-EGFP转染体外培养的正常人肝细胞株L02,观察绿色荧光在细胞内的表达情况。以免疫细胞化学、Western blot、ELISA等方法检测CTLA4Ig在L02细胞内、外的表达情况;通过绘制细胞生长曲线、检测尿素合成能力观察转染前后L02细胞的生物学特性;转染后的L02细胞与大鼠脾脏细胞共培养,通过检测脾脏细胞增殖情况评价表达CTLA4Ig的L02细胞在体外的免疫耐受活性;收集共培养体系中的大鼠脾脏细胞,再分别与正常L02细胞、HeLa细胞共培养,检测脾细胞增殖情况以评价转染后L02细胞免疫耐受的特异性。结果转染后的L02细胞可在细胞质内大量表达CTLA4Ig蛋白,并分泌至培养上清中;转染后L02细胞生长速度无明显改变(P>0.05),单个细胞尿素合成能明显升高[(0.56±0.01)pmol/d,P<0.01];转染后的L02细胞在体外可显著抑制大鼠脾脏细胞的增殖(抑制率约为36.8%,P<0.05),被抑制后的大鼠脾脏细胞在正常L02细胞刺激下增殖较低,但在HeLa细胞刺激下则可显著增殖(P<0.01)。结论Ad-CTLA4Ig-EGFP转染后的L02细胞可表达具有生物学活性的CTLA4Ig,并在体外表现出显著的具有抗原特异性的免疫抑制活性。
Objective To study the expression of CTLA4Ig in L02 cells induced by adenovirus mediated CTLA4Ig gene and its effect on the biological characteristics of L02 cells. Methods Recombinant adenovirus vector Ad-CTLA4Ig-EGFP was transfected into human hepatoma cell line L02 cultured in vitro to observe the expression of green fluorescence in cells. The expression of CTLA4Ig in L02 cells was detected by immunocytochemistry, Western blot and ELISA. The biological characteristics of L02 cells before and after transfection were observed by measuring the cell growth curve. The L02 cells were transfected Co-cultured with rat spleen cells, the immune tolerance of L02 cells expressing CTLA4Ig was evaluated by measuring the proliferation of spleen cells in vitro. Spleen cells of co-cultured rats were collected and co-cultured with normal L02 cells and HeLa cells , Detect the proliferation of spleen cells to evaluate the specificity of L02 cells immune tolerance after transfection. Results The transfected L02 cells could express CTLA4Ig protein abundantly in the cytoplasm and secreted into the culture supernatant. The growth rate of L02 cells did not change significantly after transfection (P> 0.05), while the single cell urea synthesis increased significantly 0.56 ± 0.01) pmol / d, P <0.01]. The L02 cells transfected could significantly inhibit the proliferation of rat spleen cells in vitro (the inhibition rate was about 36.8%, P <0.05) The proliferation of cells was lower under stimulation of normal L02 cells but significantly on the stimulation of HeLa cells (P <0.01). Conclusion L02 cells transfected with Ad-CTLA4Ig-EGFP can express CTLA4Ig with biological activity and exhibit significant antigen-specific immunosuppressive activity in vitro.