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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 prepare chimeric antigen receptor T cell (CAR-T) targeting human trophoblast cell surface antigen 2 (Trop-2) Effect of T cells on the proliferation of ovarian cancer cells in. METHODS: Trop-2 CAR was constructed by molecular cloning and gene recombination technique. The expression of Trop-2 CAR in 293T cells was detected by Western blot. The effect of Trop-2 CAR-T cells on the proliferation of ovarian cancer cells was detected by CCK-8 assay. ELISA to detect changes in cytokine secretion. Results: The results of restriction endonuclease digestion and sequencing showed that the Trop-2 CAR gene fragments were ligated correctly. The result of Western blot showed that the plasmid could express efficiently in 293T cells. The results of CCK-8 showed that Trop-2 CAR- T cells could significantly inhibit the proliferation of ovarian cancer cells expressing Trop-2 in vitro (P <0.05). ELISA results showed that Trop-2 CAR-T cells co-cultured with ovarian cancer cells expressing Trop- interferon-γ, IFN-γ) and interleukin-2 (IL-2) increased (P <0.01). Conclusion: Trop-2 CAR-T cells were successfully prepared and could effectively inhibit the proliferation of Trop-2-positive ovarian cancer cells.