目的：构建新型靶向CD138的嵌合抗原受体T（CAR-T）细胞并研究其对骨髓瘤细胞的细胞毒性。方法：通过单克隆抗体筛选技术制备并获得能稳定分泌CD138单克隆抗体（mAb）的杂交瘤细胞株。将杂交瘤株细胞腹腔注射至小鼠体内，产生含有单克隆抗体的腹水，然后收集并纯化以获得纯CD138 mAb。进行了进一步检查以评估 CD138 mAb 的生物学特性。该抗体的可变区序列通过逆转录聚合酶链式反应扩增，作为CD138 CAR的抗原识别结构域，随后通过慢病毒感染表达在T细胞表面。采用流式细胞术评估 CD138 CAR-T 细胞的表型。进行体外细胞毒性和脱颗粒测定以评估其抗肿瘤作用。结果：①成功制备抗人CD138抗体杂交瘤细胞系，并筛选出能够持续稳定分泌抗CD138抗体的杂交瘤细胞株5G2。 ②纯化的CD138(5G2) mAb能特异性识别CD138( )细胞，结合亲和常数(K(D))为6.011×10(-9) mol/L，与CD138(-)细胞无明显结合活性。 ③利用分子克隆技术获得CD138（5G2）抗体可变区序列，并成功构建CD138（5G2）CAR，通过慢病毒感染在T细胞上表达，同时与重组人CD138蛋白有效结合。用 CD138 (5G2) CAR 转导的 T 细胞增殖效率很高。表型分析显示，CD138（5G2）CAR-T细胞表现出更大的分化为中央记忆T细胞和记忆干T细胞的倾向，而终末分化的效应记忆亚群的比例减少。 ⑤CD138（5G2）CAR-T细胞对CD138（）骨髓瘤细胞系H929表现出特异性细胞毒性，而CD138（-）细胞系K562不受影响。与CD138（5G2）CAR-T细胞共培养后，H929细胞残留率为（12.92±8.02）%，而Vector-T组残留（54.25±15.79）%（E∶T=1∶2） P<0.001）。 ⑥脱粒实验结果显示，CD138（5G2）CAR-T细胞与H929细胞系共培养后明显激活，而Vector-T细胞未观察到明显激活[（25.78±3.35）% vs（6.13±1.30）] )%，P<0.001]。 ⑦与CD138( )细胞共培养后，CD138(5G2) CAR-T细胞较Vector-T组细胞因子分泌显着增加[interleukin-2：(1 697.52±599.05) pg/ml vs (5.07± 1.17) 皮克/毫升，P<0.001； γ干扰素：(3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml，P<0.001；肿瘤坏死因子-α：（1 837.43±640.49）pg/ml vs（8.75±1.65）pg/ml，P<0.001]。然而，与CD138(-)细胞共培养后，两组细胞因子分泌水平没有观察到显着差异。结论：本研究成功制备了一种新型抗CD138单克隆抗体，并且用该5G2单克隆抗体衍生的抗原识别结构域构建的CAR-T细胞表现出对骨髓瘤细胞的有效抗肿瘤活性。这可以作为检测CD138抗原的新选择，并为多发性骨髓瘤免疫治疗提出新策略。

Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: ① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P<0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. ⑦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.