Liu Ling-bo, Li Wei-ming, He Wei, Zhang Min, Xiao Juan, Zhong Zhao-dong, Li Lei, Zou Ping
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zhonghua Yi Xue Za Zhi. 2005 Dec 28;85(49):3469-74.
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.
A hammerhead ribozyme gene targeting the Fas mRNA was synthesized and its expression vector pEGFP-RZ596 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-RZ596 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 annexin V-FITC. The proliferation and killing ability in vitro against WEHI-3 cells of the T cells were detected by MTT colorimetry.
The luminance of Fas Western bloting results from the mouse spleen T cells negative control, transfected with pEGFPC1 and transfected with pEGFP-RZ596 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-RZ596 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-RZ596, was further promoted to 82% (P < 0.01)
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.
研究抗Fas核酶对细胞毒性T淋巴细胞(CTL)Fas表达的调控作用、CD80-IgG融合蛋白对急性粒单核细胞白血病细胞表面CD80表位的增加作用及其对CTL凋亡和杀伤急性粒单核细胞白血病细胞能力的影响。
合成针对Fas mRNA的锤头状核酶基因,构建其表达载体pEGFP-RZ596,经电穿孔转染入小鼠脾T细胞。采用RT-PCR和Western印迹法检测T细胞上Fas的表达。同时,用基因重组技术构建真核表达载体pcDNA/CD80-IgG,转染入CHO系仓鼠卵巢细胞。用蛋白G亲和层析法从经G418筛选的CHO细胞培养上清中纯化CD80-IgG融合蛋白。然后进行pEGFP-RZ596转染的小鼠脾T细胞与经CD80-IgG融合蛋白孵育的WEHI-3细胞(小鼠急性粒单核细胞白血病细胞系)之间的同种异体混合淋巴细胞培养。用膜联蛋白V-异硫氰酸荧光素检测T细胞的凋亡率。用MTT比色法检测T细胞体外对WEHI-3细胞的增殖和杀伤能力。
小鼠脾T细胞阴性对照、转染pEGFPC1和转染pEGFP-RZ596的Fas Western印迹结果灰度值分别为1、0.98和0.45(P<0.01)。与Fas配体表达较高(64%±3%)的WEHI-3细胞共培养后,转染pEGFP-RZ596的小鼠脾T细胞的凋亡率和对WEHI-3细胞的杀伤能力分别为37%和67%。而小鼠脾T细胞阴性对照和转染pEGFPC1的凋亡率分别为88%、84%(P<0.01),杀伤能力分别为32%、31%(P<0.01)。WEHI-3细胞经CD80-IgG融合蛋白预孵育后,CD80阳性表达率从5.1%±0.4%上调至27.4%±2.2%(P<0.01)。小鼠脾T细胞对经CD80-IgG融合蛋白预孵育和未预孵育的WEHI-3细胞的杀伤能力分别为64%和49%(P<0.01),但转染pEGFP-RZ596的小鼠脾T细胞的杀伤能力进一步提高到82%(P<0.01)。
联合应用抗Fas核酶和CD80-IgG融合蛋白可避免FasL-Fas途径诱导的小鼠CTL凋亡,同时显著提高小鼠CTL对WEHI-3细胞的杀伤能力。