摘要
目的研究抗Fas锤头状核酶下调细胞毒T细胞(CTL)的Fas表达,以及CD80-免疫球蛋白G(CD80-IgG)融合蛋白增加急性粒-单核白血病细胞表面的CD80分子表位对CTL杀伤急性粒-单核白血病细胞能力的影响。方法构建可有效切割Fas mRNA的锤头状核酶真核质粒,通过电穿孔法导入小鼠脾脏T细胞,逆转录-聚合酶链反应(RT-PCR)和Western印迹检测T细胞Fas的表达;同时构建pcDNA/CD80-IgG融合基因真核表达载体,采用脂质体技术导入中国地仓鼠卵巢细胞株(CHO)细胞中,并采用免疫亲和层析法纯化CHO细胞上清中的CD80-IgG融合蛋白,用其孵育急性粒-单核白血病细胞株(WEHI-3)后,与上述转染Fas mRNA锤头状核酶的T细胞进行同种异体白血病细胞和淋巴细胞混合培养;膜联蛋白V-异硫氰酸荧光素(FITC)检测T细胞凋亡率,四甲基偶氮唑蓝微量反应比色法(MTT法)检测T细胞增殖和CTL体外杀伤WEHI-3细胞的能力。结果凝胶图像分析小鼠脾脏T细胞Fas蛋白电泳条带的灰度比值显示以对照组为1,空载体(pEGFPC1)和Fas核酶RZ596重组真核表达载体(pEGFP-RZ596)转染组分别为0.98和0.45(P<0.01);当转染pEGFP-RZ596的小鼠脾脏T细胞与高表达Fas配体的(64%±3%)WEHI-3细胞混合培养后,T细胞凋亡率为37%,对WEHI-3细胞的杀伤活性为67%,而对照及转染pEGFPC1小鼠脾脏T细胞组的凋亡率和对WEHI-3细胞的杀伤活性分别为88%、84%(P<0.01)和32%、31%(P<0.01)。WEHI-3细胞表面CD80表达率仅为5.1%±0.4%,经CD80-IgG融合蛋白预孵育后增至27.4%±2.2%(P<0.01);小鼠脾脏T细胞对CD80-IgG融合蛋白预孵育和未孵育的WEHI-3细胞杀伤率分别为64%和49%(P<0.01)。转染Fas核酶的小鼠脾脏T细胞对CD80-IgG融合蛋白预孵育的WEHI-3细胞杀伤率增至82%(P<0.01)。结论联合抗Fas核酶和CD80-IgG融合蛋白能使小鼠CTL免于FasL-Fas途径的凋亡;并显著增强其杀伤急性粒单核白血病细胞的效应。
Objective To study Fas expression regulation of cytotoxic T lymphocyte（CTL） via anti-Fas ribozyme, increasing of CD80 epitope on the surface of acute myelomonocytic leukemia cells by CD80-IgG fusion protein and their effects on the apoptosis and killing ability against acute myelomonocytic leukemia cells of CTL. Methods A hammerhead ribozyme gene targeting the Fas mRNA was synthesized and its expression vector pEGFP-RT596 was constructed and transfected into the mouse spleen T cells via electroporation. The Fas expression on T cells was detected by RT-PCR and Western bloting. In the meantime the eukaryotic expression vector pcDNA/CD80-IgG was constructed by gene recombinant technique and transfected into ovarian cells of hamster of the line CHO. The CD80-IgG fusion protein was purified from the supernatant of G418-selected CHO cells by Protein G affinity chromatography method. Then allogeneic mixed lymphocytes culture between the mouse spleen T cells transfected with pEGFP-RZ96 and WEHI-3 cells（ mouse acute myelomonocyte leukemia cell line ） incubated with CD80-IgG fusion protein was performed. The apoptosis rate of the T cells was detected with annexinV-FITC. The proliferation and killing ability in vitro against WEHI-3 cells of the T cells were detected by MrlT colorimetry. Results The luminance of Fas Western bloting results from the mouse spleen T cells negative control, transfected with pEGFPC1 and transfected with pEGFP-RZ96 were separately 1,0.98 and 0.45 （ P 〈 0.01 ）. After being cocultured with WEHI-3 cells, which has higher expression of Fas ligand （64% ± 3% ） , the apoptosis rate and the killing ability against WEHI-3 cells of the mouse spleen T cells transfected with pEGFP-RZ96 were separately 37% and 67%. Whereas that of the mouse spleen T cells negative control and transfected with pEGFPC1 were separately 88% ,84% （ P 〈 0.01 ） and 32% ,31% （ P 〈 0.01 ）. The CD80 positive expression rate of WEHI-3 cells was upregulated from 5. 1% ± 0.4% to 27.4% ± 2. 2% after these cells were preincubated with CD80-IgG fusion protein （ P 〈 0.01 ）. The killing ability of the mouse spleen T cells against WEHI-3 cells preincubated and not preincubated with CD80-IgG fusion protein were separately 64% and 49% （ P 〈 0.01 ）, but that of the mouse spleen T cells, which were transfected with pEGFP-RT^96, was further promoted to 82% （ P 〈 0.01 ） Conclusion The apoptosis of mouse CTL inducing by FasL-Fas pathway could be avoided and the killing ability of mouse CTL against WEHI-3 cells can be significantly promoted at the same time by combining anti-Fas ribozyme and CD80-IgG fusion protein.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2005年第49期3469-3474,共6页
National Medical Journal of China
基金
国家自然科学基金资助项目(30240022)
