mGITRL转染Lewis细胞株的建立及生物学功能的初步研究

发布时间：2018-03-22 19:11

本文选题：GITRL　切入点：调节性T细胞　出处：《江苏大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的: 构建携带小鼠mGITRL基因的真核表达质粒pIRES2-eGFP/mGITRL,建立稳定表达mGITRL的小鼠Lewis细胞株,并通过体内外功能实验,探讨mGITRL蛋白在肿瘤生物治疗中的作用。方法: (1)利用PCR方法,以mGITRL TA克隆为模板扩增mGITRL基因,将mGITRL基因克隆至pIRES2-eGFP载体,酶切筛选阳性克隆并进行序列测定。 (2)将测序正确的pIRES2-eGFP/mGITRL载体通过电穿孔法(电压为0.22KV、电容为960μf)转染4.0×10~6Lewis细胞,转染后的细胞用800μg/ml G418筛选,并经过克隆化生长,获得稳定表达mGITRL的Lewis细胞株。 (3)在抗CD3 mAb存在下,5.0×10~4丝裂霉素C处理过的Lewis、eGFP-Lewis、GITRL-Lewis细胞分别和5.0×10~4 CD4~+CD25~-T细胞,或CD4~+CD25~+T细胞加CD4~+CD25~-T细胞共培养,观察CD4~+CD25~-T细胞增殖能力和CD4~+CD25~-T细胞抑制功能。 (4)2.0×10~6Lewis、eGFP-Lewis、GITRL-Lewis细胞分别致瘤,观察小鼠肿瘤的生长情况及小鼠生存率,并检测荷瘤小鼠CD4~+CD25~+T细胞比例,以及CD4~+CD25~-T细胞增殖能力和CD4~+CD25~+T细胞抑制功能。结果: (1)经PCR扩增获得目的基因,经酶切、连接,构建了真核表达质粒pIRES2-eGFP/mGITRL,酶切鉴定为阳性质粒,基因测序与GenBank中发表的序列(AY359852.1)完全一致。 (2)测序正确的pIRES2-eGFP/mGITRL载体体外转染Lewis细胞,经G418筛选,克隆化生长,获得稳定表达细胞株,该细胞株经RT-PCR检测表达mGITRL,流式细胞仪检测表达EGFP和mGITRL。 (3)体外共培养试验显示,在抗CD3 mAb刺激下,丝裂霉素C处理过的GITRL-Lewis能增加CD4~+CD25~-T细胞的增殖,并能部分解除CD4~+CD25~+T细胞的免疫抑制功能。 (4)GITRL-Lewis组小鼠较eGFP-Lewis组及Lewis组小鼠肿瘤生长明显缓慢,生存率显著提高。 (5)荷瘤小鼠的脾细胞中CD4~+CD25~+T细胞占CD4~+T细胞的比例无明显差异但高于正常小鼠。荷瘤小鼠的TIL细胞中CD4~+CD25~+T细胞占CD4~+T细胞的比例明显高于脾细胞,而GITRL-Lewis荷瘤小鼠的TIL细胞中CD4~+CD25~+T细胞的比例低于Lewis及eGFP-Lewis组小鼠。 (6)荷瘤小鼠的免疫功能低下,其CD4~+CD25~-T细胞的增殖能力明显低于正常小鼠;但GITRL-Lewis组小鼠CD4~+CD25~-T细胞的增殖能力高于eGFP-Lewis组小鼠。荷瘤小鼠CD4~+CD25~+T细胞均能抑制正常小鼠CD4~+CD25~-T细胞增殖,但GITRL-Lewis荷瘤小鼠CD4~+CD25~+T细胞抑制正常小鼠CD4~+CD25~-T细胞增殖的作用低于eGFP-Lewis组。结论: (1)成功构建了真核表达质粒pIRES2-eGFP/mGITRL。 (2)建立了稳定表达mGITRL的Lewis细胞株。 (3)GITRL-Lewis细胞能增强CD4~+CD25~-T细胞的增殖,部分解除Treg细胞的免疫抑制功能。 (4)GITRL-Lewis细胞的致瘤能力明显低于野生型细胞株。
[Abstract]:Objective:. The eukaryotic expression plasmid pIRES2-eGFP / mGITRL carrying mouse mGITRL gene was constructed, and the mouse Lewis cell line expressing mGITRL stably was established. The role of mGITRL protein in tumor biotherapy was investigated by in vivo and in vitro functional experiments. Methods:. 1) the mGITRL gene was amplified by PCR and mGITRL TA clone was used as template. The mGITRL gene was cloned into pIRES2-eGFP vector. The positive clones were screened by enzyme digestion and sequenced. The correct pIRES2-eGFP/mGITRL vector was transfected into 4.0 脳 10~6Lewis cells by electroporation (voltage 0.22 KV, capacitance 960 渭 f). The transfected cells were screened by 800 渭 g/ml G418 and cloned to obtain Lewis cell lines expressing mGITRL stably. In the presence of anti CD3 mAb, 5. 0 脳 10 ~ 4 mitomycin C treated LewiseGFP-LewisGITRL-Lewis cells and 5. 0 脳 10 ~ (4) CD4 ~ CD25~-T cells, or CD4 ~ CD25 ~ T cells and CD4 ~ CD25~-T cells were co-cultured to observe the proliferation ability of CD4 ~ CD25~-T cells and the inhibitory function of CD4 ~ CD25~-T cells. The tumor was induced by 2.0 脳 10 ~ (6) LewiseGFP-LewisGITRL-Lewis cells respectively. The tumor growth and survival of mice were observed. The percentage of CD4 ~ CD25 ~ T cells, the proliferation ability of CD4 ~ CD25~-T cells and the inhibitory function of CD4 ~ CD25 ~ T cells in tumor-bearing mice were measured. Results:. The eukaryotic expression plasmid pIRES2-eGFP / mGITRL was constructed by PCR amplification. The plasmid was identified as positive by restriction endonuclease digestion. The sequence of the gene was identical to that of AY359852.1 published in GenBank. 2) the pIRES2-eGFP/mGITRL vector was transfected into Lewis cells in vitro. After G418 selection, stable expression cell line was obtained. The cell line expressed mGITRL by RT-PCR, EGFP and mGITRL were detected by flow cytometry. Co culture test in vitro showed that GITRL-Lewis treated with mitomycin C could increase the proliferation of CD4 ~ CD25~-T cells and partially relieve the immunosuppressive function of CD4 ~ CD25 ~ T cells stimulated by anti CD3 mAb. The tumor growth and survival rate of GITRL-Lewis group were significantly slower than those of eGFP-Lewis group and Lewis group. (5) the percentage of CD4- CD25T cells in spleen cells of tumor-bearing mice was higher than that of normal mice, but the percentage of CD4- CD25T cells in TIL cells of tumor-bearing mice was significantly higher than that of spleen cells. The percentage of CD4 ~ CD25 ~ T cells in TIL cells of GITRL-Lewis bearing mice was lower than that of Lewis and eGFP-Lewis groups. (6) the immune function of tumor-bearing mice was low, and the proliferation ability of CD4 ~ CD25~-T cells was lower than that of normal mice, but the proliferative ability of CD4 ~ CD25~-T cells in GITRL-Lewis group was higher than that in eGFP-Lewis group. CD4 ~ CD25 ~ T cells in tumor-bearing mice could inhibit the proliferation of CD4 ~ CD25~-T cells in normal mice. However, CD4 ~ CD25 ~ T cells inhibited the proliferation of normal CD4 ~ CD25~-T cells in GITRL-Lewis tumor bearing mice compared with eGFP-Lewis group. Conclusion:. 1) the eukaryotic expression plasmid pIRES2-eGFP / mGITRLwas successfully constructed. A stable Lewis cell line expressing mGITRL was established. GITRL-Lewis cells could enhance the proliferation of CD4 ~ CD25~-T cells and partially relieve the immunosuppressive function of Treg cells. The tumorigenicity of GITRL-Lewis cells was significantly lower than that of wild-type cell lines.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.11

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