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

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

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。

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 293 T 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 293 T 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.