嵌合抗原受体修饰的NK-92MI细胞对HER2阳性乳腺癌细胞的杀伤

Killing effect of NK-92MI cells modified with chimeric antigen receptor on HER2^+ breast carcinoma cell

目的:探讨用特异性靶向HER2抗原的嵌合抗原受体(HER2-CAR)修饰的NK-92MI细胞对HER2阳性乳腺癌细胞的杀伤效率。方法:通过PCR获得HER2-CAR片段,运用常规分子克隆技术,将其构建到慢病毒载体上,利用包装的慢病毒颗粒对NK-92MI细胞进行转染;应用流式细胞术检测细胞转染效率以及转染前后NK-92MI的IFN-γ和颗粒酶分泌差异;使用7-AAD和流式细胞术体外检测HER2-CAR-NK92MI细胞对乳腺癌细胞的杀伤效率;构建小鼠乳腺癌原位肿瘤模型进行体内细胞毒性的检测。结果:用HER2-CAR修饰的NK-92MI细胞对HER2阳性乳腺癌肿瘤细胞MCF-7和T47D的杀伤效率明显高于未被基因修饰的NK-92MI细胞[分别为(62.4±1.3)%vs(22.1±1.2)%和(50.0±1.9)%vs(16.9±0.6)%,P<0.01];而两者对HER2阴性的乳腺癌细胞MDA-MB-468的杀伤效率没有明显差异[(13.6±1.4)%vs(12.7±0.8)%,P>0.05]。同时,经HER2-CAR修饰的NK-92MI细胞相比未被基因修饰的NK-92MI细胞,IFN-γ的分泌明显升高(P<0.01)。结论:经HER2-CAR修饰的NK-92MI细胞对HER2阳性乳腺癌细胞的杀伤效率明显提高,且具有特异性靶向,此为治疗乳腺癌等恶性肿瘤提供了临床依据。

Objective：To explore killing efficiency of NK-92MI cells modified with chimeric antigen receptor of specific targeting HER2 antigen （HER2-CAR） on HER2＋ breast carcinoma cells. Methods： HER2-CAR fragments were obtained by PCR, then built into lentiviral vectors via molecular cloning technology and transfected into NK-92MI cells using the lentiviral particles with HER2-CAR. Flow cytometry assay was used to detect efficiency of the cell transfection and, secretion differences between IFN-γ and granzyme in NK-92MI cells before and after the transfection. Killing efficiency of HER2-CAR-NK92MI cells on breast carcinoma cells in vitro was tested with 7-AAD and flow cytometry assays. Mouse model with breast tumor in situ was constructed and used to detect killing efficiency of the HER2-CAR-NK92MI cells in vivo. Results：Killing efficiencies of the NK-92MI cell modified with HER2-CAR on HER2＋ breast carcinoma MCF-7 and T47T cell lines were significantly higher than those of the NK-92MI cell that did not modified with HER2-CAR gene （[62.4±1.3]% vs[22.1±1.2／]%. [50.0±1.9]% vs [16.9±0.6]%, respectively, all P〈0.01）. However, there was no obvious difference in killing efficiency of both the modified and un-modified NK-92MI cells on HER2- breast carcinoma MDA-MB-468 cell （[13.6±1.4]% vs [12.7±0.8]%,P〉0.05）. At the same time, amount of IFN-γ secreted by the NK-92MI cell modified with HER2-CAR was significantly higher than that secreted by the NK-92MI cell which did not modified with HER2-CAR gene （P〈0.01）.Conclusion： Killing efficiency of the NK-92MI cell modified with HER2-CAR on HER2＋ breast carcinoma cell significantly increased, and could have specific targeting property, which might provide a clinical evidence for the treatment of malignant tumor, such as breast carcinoma and so on.