融合蛋白Tandab（CD3/CD19/4-1BBL）介导的 T细胞对B细胞淋巴瘤细胞杀伤作用观察.

Objective To investigate the killing effect of Tandab（CD3/CD19/4-1BBL) mediated T cells on human B-cell lymphoma Raji cells. Methods The methods of restriction enzyme cleavage and linkage were employed to con‐ struct the lentiviral expression vector pLentiR. SV40-Tandab（CD3/CD19/4-1BBL)-06633，which was then transiently transfected into 293T cells. The Tandab（CD3/CD19/4-1BBL）was extracted and purified from the culture medium. The binding abilities of Tandab（CD3/CD19/4-1BBL）to CD19+ (Raji cells), CD3+ （human acute T-lymphoblastic leukemia cell line Jurkat cells)，and 4-1BB+ Jurkat cells）cells were determined by flow cytometry，respectively. T cells were co-cultured with Raji cells（E∶T=20∶1）in the presence of Tandab（CD3/CD19/4-1BBL)，Tandab（CD3/CD19)，and 4-1BBL with the indicated concentrations of 0. 08，0. 8，and 8 pmol/mL. Lactic dehydrogenase（LDH）release assay was applied to detect the killing efficiency of T cells mediated by Tandab（CD3/CD19/4-1BBL）on Raji cells. Expression of IL-2 in the cul‐ ture medium treated with 8 pmol/mL Tandab（CD3/CD19/4-1BBL）was determined by enzyme-linked immunosorbent as‐ say（ELISA). Flow cytometry was used to measure the percentages of CD3+ CD69+ and CD3++CD25+ cells. Results The pLentiR. SV40-Tandab（CD3/CD19/4-1BBL）was successfully constructed. Tandab（CD3/CD19/4-1BBL）could specifi‐ cally bind to CD19+ ，CD3+ ，and 4-1BB+ cells. The Tandab（CD3/CD19/4-1BBL）group and Tandab（CD3/CD19）group had significantly higher percentages of cell lysis than the 4-1BBL group（both P<0. 01）. With the increased concentrations of Tandab，the percentages of cell lysis increased in a dose-dependent manner at the same E/T ratio（both P<0. 01). The expression of IL-2 in the Tandab（CD3/CD19/4-1BBL）group，Tandab（CD3/CD19）group，and 4-1BBL group was higher than that of the PBS group（all P<0. 05）. The percentages of CD3+ CD69+ cells and CD3+ CD25+ cells in the Tandab （CD3/CD19/4-1BBL）group and Tandab（CD3/CD19）group were higher than those in the 4-1BBL group and PBS group （all P<0. 01). Conclusion Tandab（CD3/CD19/4-1BBL）could efficiently result in cell lysis of B-cell lymphoma cells induced by T cells. [ABSTRACT FROM AUTHOR]

目的 观察融合蛋白Tandab（CD3/CD19/4-1BBL）介导的T细胞对人B细胞淋巴瘤细胞Raji的杀伤作用。 方法 采用常规酶切、连接等分子克隆方法构建慢病毒表达载体pLentiR.SV40-Tandab（CD3/CD19/4-1BBL），将其 瞬时转染至293T细胞，收集培养上清并纯化融合蛋白Tandab（CD3/CD19/4-1BBL）。采用流式细胞术分析Tandab （CD3/CD19/4-1BBL）与CD19+细胞（Raji细胞）、CD3+细胞（人急性T淋巴细胞白血病细胞Jurkat）、 4-1BB+细胞（Jurkat 细胞）的结合活性。收集Raji细胞，按照效靶比20∶1加入T细胞，分别加入0.08、 0.8、 8pmol/mL的Tandab（CD3/ CD19/4-1BBL）、Tandab（CD3/CD19）、 4-1BBL，采用LDH释放实验测算T细胞对Raji细胞的杀伤效率，采用ELISA法 检测8pmol/mL融合蛋白作用后细胞培养液上清中的IL-2、采用流式细胞术检测CD3+CD69+细胞、CD3+CD25+细胞比 例。结果 pLentiR.SV40-Tandab（CD3/CD19/4-1BBL）构建成功，表达的融合蛋白Tandab（CD3/CD19/4-1BBL）可 与CD19+、CD3+、 4-1BB+细胞结合。Tandab（CD3/CD19/4-1BBL）组、Tandab（CD3/CD19）组T细胞对Raji细胞杀伤百 分比均高于4-1BBL组（P均<0.01）。在效靶比一定时，随着融合蛋白Tandab浓度增高， T细胞对Raji细胞的杀伤百 分比逐渐增高（P均<0.01）。Tandab（CD3/CD19/4-1BBL）组、Tandab（CD3/CD19）组、 4-1BBL组细胞培养液上清中 的IL-2水平均高于PBS组（P均<0.05）。Tandab（CD3/CD19/4-1BBL）组、Tandab（CD3/CD19）组CD3+CD69+细胞、 CD3+CD25+细胞比例高于4-1BBL组、PBS组（P均<0.01）。结论 融合蛋白Tandab（CD3/CD19/4-1BBL）可有效介导 T细胞对B细胞淋巴瘤细胞的杀伤作用。 [ABSTRACT FROM AUTHOR]