靶向Trop-2 CAR-T细胞的制备及其体外对卵巢癌细胞杀伤作用的研究
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国家自然科学基金(81602119);江苏省科学技术厅重点研发专项基金(BE2016799)


Construction of Trop-2-targeted chimeric antigen receptor-modified T cells and their effects on the proliferation of ovarian cancer cells in vitro
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    摘要:

    目的:制备靶向人滋养层细胞表面抗原2(human trophoblast cell surface antigen 2 , Trop-2)的嵌合抗原受体T细胞(chimeric antigen receptor T cell , CAR-T),观察Trop-2 CAR-T细胞在体外对卵巢癌细胞增殖的影响。方法:运用分子克隆及基因重组技术构建Trop-2 CAR;采用Western blot检测Trop-2 CAR在293T细胞中的表达;CCK-8法检测Trop-2 CAR-T细胞对卵巢癌细胞增殖的影响;ELISA检测细胞因子分泌的变化。结果:酶切鉴定及测序分析结果表明,Trop-2 CAR各基因片段连接正确;Western blot检测结果显示,该质粒能够在293T细胞中有效表达;CCK-8结果显示,制备的Trop-2 CAR-T细胞在体外能明显抑制表达Trop-2的卵巢癌细胞的增殖(P<0.05);ELISA检测结果表明Trop-2 CAR-T细胞与表达Trop-2的卵巢癌细胞共培养后,干扰素(interferon-γ,IFN-γ)、白介素(interleukin-2,IL-2)细胞因子分泌增加(P<0.01)。结论:该研究成功制备了Trop-2 CAR-T细胞,可有效抑制Trop-2表达阳性的卵巢癌细胞的增殖。

    Abstract:

    Objective: To produce Trop-2-targeted chimeric antigen receptor T cells(CAR-T cells) of human trophoblast cell surface antigen 2(Trop-2) and detect their effects on the proliferation of ovarian cancer cells in vitro. Methods: Molecular cloning and gene recombination technology were introduced to construct Trop-2 CAR vector. The expression of Trop-2 CAR on 293T cells was tested by Western blot. The effect of Trop-2 CAR-T cells on the proliferation of ovarian cancer cells was evaluated by CCK-8 assay. The secreting changes of cytokines by Trop-2 CAR-T cells were detected by ELISA. Results: The results of enzyme digestion and sequencing demonstrated that the Trop-2 CAR vector was constructed correctly. The results of Western blot showed that Trop-2 CAR was expressed on 293T cells. The results of CCK-8 verified that Trop-2 CAR-T cells prominently inhibited the growth of ovarian cancer cells with Trop-2 antigen expression(P<0.05), moreover, higher levels of IFN-γ and IL-2 were detected in supernatant by ELISA(P<0.01). Conclusion: Trop-2 CAR-T cells were prepared successfully and could inhibit the proliferation of ovarian cancer cells expressing Trop-2 antigen.

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徐亚如,周 荧,唐 奇,刘振云,黄骁辰,杨婷婷,张慧林,赵 薇,蒯兴旺,仇镇宁,朱 进,冯振卿.靶向Trop-2 CAR-T细胞的制备及其体外对卵巢癌细胞杀伤作用的研究[J].南京医科大学学报(自然科学版),2017,(6):653-658

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  • 收稿日期:2017-01-12
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  • 在线发布日期: 2017-06-30
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