腺病毒介导CTLA4Ig转染正常人肝细胞株的研究

Normal human liver cell line L02 appears immunological inhibition after by CTLA4Ig gene modification

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 observe the expression of CTLA4Ig in normal human liver cell line L02 that transducted with Ad-CTLA4Ig-EGFP,and to explore their biological activity and changes in biological characteristics.Methods Normal liver cells L02 were transducted with Ad-CTLA4Ig-EGFP.The expression of green fluorescence in the cells was observed.Immunocytochemistry,Western blotting and ELISA were used to detect the expression of CTLA4Ig in L02 cells.Changes of biological characteristics in CTLA4Ig transducted L02 cells（CTLA4-L02） were observed through cell proliferation curve and urea synthesis.After the transducted cells were co-cultured with rat splenocytes for 72 h,the proliferation of splenocytes were detected by MTT assay.The co-cultured splenocytes were then co-cultured with HeLa cells and normal L02 cells respectively for another 72 h,then the proliferation of splenocytes was examined again.Results CTLA4Ig were highly expressed in the cytoplasm of CTLA4-L02 cells,and were secreted to the culture supernatant.No obvious change in growth speed was observed after transduction（P〉0.05）,but the synthesis of urea was enhanced after transduction [（0.56±0.01） pmol/d,P〈0.01].The proliferation of rat splenocytes was inhibited by 36.8%（P〈0.05） when co-cultured with CTLA4-L02.The inhibited splenocytes mentioned previously kept a low proliferative rate when co-cultured with normal L02 cells,but showed a high proliferative rate when co-cultured with HeLa cells（P〈0.01）.Conclusion After transfected with Ad-CTLA4Ig-EGFP,L02 cells express active CTLA4Ig and show immunological inhibition,but no obvious negative change in biological characteristics is observed.