可溶性CD40基因修饰树突状细胞对实验动物淋巴细胞增殖及胞毒活性的影响

目的探讨可溶性CD40-增强绿色荧光蛋白C(sCD40-EGFP)修饰树突状细胞(DC)对实验动物免疫功能的影响,为利用DC诱导供体特异性移植免疫耐受提供实验依据。方法应用基因工程技术构建CD40胞外区与EGFP重组载体质粒pEGFP-N1/sCD40,采用脂质体转染法,将其导入DC2.4细胞株。采用荧光分光光度计和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定转染质粒pEGFP-N1/sCD40的DC培养上清;将经未修饰DC细胞及sCD40-EGFP及空载体修饰DC经腹腔注射致敏Balb/c小鼠,取单个核细胞作为反应细胞,分别以不同组DC为刺激细胞,行混合淋巴细胞培养,采用MTS比色法检测细胞增殖,乳酸脱氢酶法测定细胞毒活性。结果sCD40分子与EGFP融合基因载体构建成功,在树突状细胞中获得表达,并分泌至上清;sCD40-EGFP融合蛋白对不同组DC致敏或未致敏小鼠的同种细胞刺激的增殖反应均有明显的抑制作用,sCD40-EGFP基因修饰DC诱导不同DC致敏组小鼠淋巴细胞增殖反应均明显降低,sCD40-EGFP融合蛋白对各实验组淋巴细胞胞毒活性有显著的抑制作用,sCD40-EGFP基因修饰DC致敏组小鼠淋巴细胞胞毒活性较其余实验组明显降低。结论稳定表达sCD40-EGFP融合蛋自的DC致敏可显著降低同种小鼠淋巴细胞的增殖反应,并能诱导淋巴细胞对靶细胞的胞毒活性降低。

The effect of sCD40 modified dendritic cells on proliferation and cytotoxicity of lymphocytes in experimental animals

Objective To study the effects of sCD40-EGFP modified dendritic ceils （DC） on immunological function of experimental animals and to provide experimental basis for DC-induced dornor-specific immune torlerance. Methods Standard cloning techniques were employed to construct sCD40-EGFP fusion gene which was identified with double enzyme digestion and DNA sequencing. Then the recombinant plasmid was transfected into mouse dendritic cells with lipofectamine, sCD40-EGFP fusion protein secreted by DC in the culture supernatant was confirmed by fluorescence spectrophotometer and SDS-PAGE. Balb/c mice were sensitized with intraperitoneal injection of unmodified or modified dendritic cells. The mouse peripheral blood mononuclear cells （PBMC） as reaction cells were incubated with different groups of dendrite cells. MTS assay was used to detect lymphocytes proliferation. Lactate dehydrogenase release method was used to examine the cytotoxic activity. Results The fusion gene was observed in dendritic cells and the fusion protein was detected in supernatant, sCD40-EGFP fusion protein was shown to have an evidenced inhibition on lymphocyte proliferation in response to unsensitized or sensitized Balb/c mice. Lymphocyte proliferation in different groups of mice induced by sCD40-EGFP modified dendritic cells were significant decreased, sCD40-EGFP fusion protein obviously inhibited the cytotoxic activity of lymphocytes, and the cytotoxic activity of lymphocytes of mice induced by sCD40-EGFP modified dendritic cells were significantly reduced. Conclusion Dendritic cells with stable expression of sCD40-EGFP fusion protein can obviously reduce lymphocyte proliferation in response to allogenic mice and reduce the cytotoxic activity of lymphocytes.