| 张 灿,庄清慧,白 玥,王 芳,钟晓松.一种新的人源化抗CD19嵌合抗原受体NK细胞的制备和对体外肿瘤细胞杀伤作用[J].,2023,(3):412-416 |
| 一种新的人源化抗CD19嵌合抗原受体NK细胞的制备和对体外肿瘤细胞杀伤作用 |
| Construction and Antitumor Activity of a New Humanized anti-CD19 Chimeric Antigen Receptor NK Cells in vitro |
| 投稿时间：2022-07-26 修订日期：2022-08-21 |
| DOI：10.13241/j.cnki.pmb.2023.03.003 |
| 中文关键词: 嵌合抗原受体NK细胞 CD19 人源化scFv 淋巴瘤 |
| 英文关键词: Chimeric antigen receptor NK cells CD19 Humanized scFv Lymphoma |
| 基金项目:北京市科技计划脑科学专项基金项目（Z161100000216136） |
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| 中文摘要: |
| 摘要 目的：构建人源化抗CD19嵌合抗原受体NK细胞（hCAR19-NK），并且在体外证明其对CD19 阳性血液病肿瘤细胞杀伤作用。方法：构建人源化的第二代CD19 CAR的逆转录病毒载体，使用辐照的K562-4-1BBL-mIL21细胞刺激外周血来源的NK细胞，通过逆转录病毒转导NK细胞获得hCAR19-NK细胞；采用流式细胞术和Western blot检测转导效率；采用4 h荧光杀伤实验和ELISA法检测hCAR19-NK细胞对淋巴瘤细胞的杀伤能力和IFN-γ释放水平；采用CD107a脱颗粒实验评估淋巴瘤细胞对hCAR19-NK细胞的特异性激活；比较对照组（Mock）和hCAR19-NK组细胞扩增倍数。结果：流式细胞术和Western blot 证明构建的CAR可以成功转导外周血来源的NK细胞；4 h荧光杀伤实验证明随着效靶比例升高，hCAR19-NK对Raji-GL杀伤率增加，明显高于Mock组；ELISA法检测显示Raji和K562-CD19作为靶细胞时，hCAR19-NK细胞的IFN-γ释放明显高于Mock组（P<0.01）；CD19+细胞（Raji和K562-CD19）可以特异性刺激hCAR19-NK细胞表达CD107a，具有统计学意义（P<0.05）；Mock组和hCAR19-NK组细胞扩增倍数无显著差异。结论：成功构建了可以杀伤CD19+ 肿瘤的人源化scFv的第二代hCAR19-NK细胞。 |
| 英文摘要: |
| ABSTRACT Objective: Humanized anti-CD19 chimeric antigen receptor NK cells (hCAR19-NK) were constructed successfully and proved to kill CD19 positive hematological tumor cells in vitro. Methods: The retroviral vector of humanized second-generation CD19 chimeric antigen receptor was constructed. Irradiated K562-4-1BBL-mIL21 as feeder cells were used to stimulate peripheral blood derived NK cells, and hCAR19-NK cells were obtained by retroviral transduction of NK cells; The transduction efficiency was detected by flow cytometry and Western blot; The killing ability and IFN-γ release level of hCAR19-NK cells on lymphoma cells were detected by 4 h fluorescence killing test and ELISA; CD107a degranulation test was used to evaluate the specific activation of lymphoma cells to hCAR19-NK cells; The cell expansion times of control group (Mock) and hCAR19-NK group were compared. Results: Flow cytometry and Western blot showed that the constructed CAR could successfully transduce NK cells from peripheral blood; The hCAR19-NK cells exhibited good anti-tumor activity in vitro via killing Raji-GL cells after 4 hours co-culture; After co-culture of hCAR19-NK and different CD19 positive cell lines, the IFN-γ release was significantly higher than those in the Mock group(P<0.01); CD19+ cells (Raji and K562-CD19) could specifically stimulate the expression of CD107a in hCAR19-NK cells and CD107a degranulation was significantly higher than those in the Mock group(P<0.05); And the fold expansion of hCAR19-NK cells in vitro was similar to Mock cells. Conclusion: The second-generation hCAR19-NK cells using humanized scFv have been successfully constructed for the effectively treatment of CD19 positive cell lines. |
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