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.

国家自然科学基金资助项目（No.31471283）