本文選題：GITRL　切入點(diǎn)：調(diào)節(jié)性T細(xì)胞　出處：《江蘇大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 構(gòu)建攜帶小鼠mGITRL基因的真核表達(dá)質(zhì)粒pIRES2-eGFP/mGITRL,建立穩(wěn)定表達(dá)mGITRL的小鼠Lewis細(xì)胞株,并通過(guò)體內(nèi)外功能實(shí)驗(yàn),探討mGITRL蛋白在腫瘤生物治療中的作用。 方法: (1)利用PCR方法,以mGITRL TA克隆為模板擴(kuò)增mGITRL基因,將mGITRL基因克隆至pIRES2-eGFP載體,酶切篩選陽(yáng)性克隆并進(jìn)行序列測(cè)定。 (2)將測(cè)序正確的pIRES2-eGFP/mGITRL載體通過(guò)電穿孔法(電壓為0.22KV、電容為960μf)轉(zhuǎn)染4.0×10~6Lewis細(xì)胞,轉(zhuǎn)染后的細(xì)胞用800μg/ml G418篩選,并經(jīng)過(guò)克隆化生長(zhǎng),獲得穩(wěn)定表達(dá)mGITRL的Lewis細(xì)胞株。 (3)在抗CD3 mAb存在下,5.0×10~4絲裂霉素C處理過(guò)的Lewis、eGFP-Lewis、GITRL-Lewis細(xì)胞分別和5.0×10~4 CD4~+CD25~-T細(xì)胞,或CD4~+CD25~+T細(xì)胞加CD4~+CD25~-T細(xì)胞共培養(yǎng),觀察CD4~+CD25~-T細(xì)胞增殖能力和CD4~+CD25~-T細(xì)胞抑制功能。 (4)2.0×10~6Lewis、eGFP-Lewis、GITRL-Lewis細(xì)胞分別致瘤,觀察小鼠腫瘤的生長(zhǎng)情況及小鼠生存率,并檢測(cè)荷瘤小鼠CD4~+CD25~+T細(xì)胞比例,以及CD4~+CD25~-T細(xì)胞增殖能力和CD4~+CD25~+T細(xì)胞抑制功能。 結(jié)果: (1)經(jīng)PCR擴(kuò)增獲得目的基因,經(jīng)酶切、連接,構(gòu)建了真核表達(dá)質(zhì)粒pIRES2-eGFP/mGITRL,酶切鑒定為陽(yáng)性質(zhì)粒,基因測(cè)序與GenBank中發(fā)表的序列(AY359852.1)完全一致。 (2)測(cè)序正確的pIRES2-eGFP/mGITRL載體體外轉(zhuǎn)染Lewis細(xì)胞,經(jīng)G418篩選,克隆化生長(zhǎng),獲得穩(wěn)定表達(dá)細(xì)胞株,該細(xì)胞株經(jīng)RT-PCR檢測(cè)表達(dá)mGITRL,流式細(xì)胞儀檢測(cè)表達(dá)EGFP和mGITRL。 (3)體外共培養(yǎng)試驗(yàn)顯示,在抗CD3 mAb刺激下,絲裂霉素C處理過(guò)的GITRL-Lewis能增加CD4~+CD25~-T細(xì)胞的增殖,并能部分解除CD4~+CD25~+T細(xì)胞的免疫抑制功能。 (4)GITRL-Lewis組小鼠較eGFP-Lewis組及Lewis組小鼠腫瘤生長(zhǎng)明顯緩慢,生存率顯著提高。 (5)荷瘤小鼠的脾細(xì)胞中CD4~+CD25~+T細(xì)胞占CD4~+T細(xì)胞的比例無(wú)明顯差異但高于正常小鼠。荷瘤小鼠的TIL細(xì)胞中CD4~+CD25~+T細(xì)胞占CD4~+T細(xì)胞的比例明顯高于脾細(xì)胞,而GITRL-Lewis荷瘤小鼠的TIL細(xì)胞中CD4~+CD25~+T細(xì)胞的比例低于Lewis及eGFP-Lewis組小鼠。 (6)荷瘤小鼠的免疫功能低下,其CD4~+CD25~-T細(xì)胞的增殖能力明顯低于正常小鼠;但GITRL-Lewis組小鼠CD4~+CD25~-T細(xì)胞的增殖能力高于eGFP-Lewis組小鼠。荷瘤小鼠CD4~+CD25~+T細(xì)胞均能抑制正常小鼠CD4~+CD25~-T細(xì)胞增殖,但GITRL-Lewis荷瘤小鼠CD4~+CD25~+T細(xì)胞抑制正常小鼠CD4~+CD25~-T細(xì)胞增殖的作用低于eGFP-Lewis組。 結(jié)論: (1)成功構(gòu)建了真核表達(dá)質(zhì)粒pIRES2-eGFP/mGITRL。 (2)建立了穩(wěn)定表達(dá)mGITRL的Lewis細(xì)胞株。 (3)GITRL-Lewis細(xì)胞能增強(qiáng)CD4~+CD25~-T細(xì)胞的增殖,部分解除Treg細(xì)胞的免疫抑制功能。 (4)GITRL-Lewis細(xì)胞的致瘤能力明顯低于野生型細(xì)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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王勝軍,馬斌,仝佳,許化溪,楊勝利;小鼠GITRL基因的克隆和序列分析[J];江蘇大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2004年02期

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本文編號(hào)：1649993