CD138-CAR-T对多发性骨髓瘤株RPMI8226及耐药株MM1R杀伤作用的研究

发布时间：2018-04-05 13:05

本文选题：多发性骨髓瘤　切入点：耐药株　出处：《青岛大学》2017年硕士论文

【摘要】：目的:多发性骨髓瘤是一种恶性血液肿瘤近几年其发病率逐渐增高,而随着疾病的进展给患者带来极大的痛苦,当前治疗方法有限。由于化疗药的联合应用,极易产生多发耐药。多发耐药一旦产生,患者大多预后不良。因此,我们不断寻求新的治疗方法,细胞免疫疗法CAR-T技术其实就是将目的基因植入病毒载体内然后将病毒转入患者的T细胞使其表达特定的抗体。在前期研究中发现单链可变片断(SCFV)取代了细胞外的抗原结合域使得TCR发生了改变,这样就使CAR-T具有MHC非限制性识别的优点,让其更好地发挥识别和抗肿瘤细胞的作用。特别是一些研究发现CD19-CAR-T在CD19阳性B细胞恶性肿瘤上取得了很好的效果。而CD138是一个表面蛋白,它的作用是当作粘附分子结合细胞外基质分子的胶原蛋白和纤维连接蛋白。CD138在骨髓瘤细胞表面高表达,但是几乎不表达于其他的造血细胞,包括T细胞和B细胞。因此,CD138成为了一个用CAR-T疗法治疗多发性骨髓瘤很好的靶点。基于以上设计了本实验。我们研究的主要目的是探讨细胞免疫疗法CD138-CAR-T对于多发性骨髓瘤细胞普通株RPMI8226的杀伤作用,以及对耐药株MM1R的杀伤作用。评估CD138分子作为细胞疗法CAR-T靶点的效果。方法:构建携带有CD138抗体的慢病毒以及未携带目的基因的慢病毒,然后分离提取人的T淋巴细胞,将慢病毒转入T淋巴细胞构建成CD138-CAR-T和携带空病毒的T细胞。然后用流式细胞术检测CD138-CAR-T的转染效率,检测多发性骨髓瘤普通株RPMI8226、耐药株MM1R及阴性对照细胞293T细胞表面CD138分子的表达水平,流式细胞术检测CD138-CAR-T和携带空病毒的T细胞在效靶比为10:1和1:1时分别对多发性骨髓瘤普通株RPMI8226,耐药株MM1R以及CD138阴性对照细胞株293T细胞的杀伤作用。结果:CD138-CAR转染至T细胞的效率为46.25%,RPMI8226细胞表面CD138表达量较高而MM1R细胞表面表达量较低,因此为探究CAR-T细胞对于多发性骨髓瘤耐药株CD138高表达与低表达的作用我们又将MM1R CD138阳性细胞分选出来培养提高其阳性率。293T细胞表面CD138基本不表达。对于RPMI8226和分选后的MM1R来说,CD138-CAR-T组比携带空病毒的T细胞组杀伤作用强。而对于分选前的MM1R及293T细胞来说,CD138-CAR-T组与携带空病毒的T细胞组杀伤作用无明显差别。同时分选后的MM1R细胞组CD138-CAR-T杀伤作用显著强于未分选MM1R细胞组。并且在效靶比为10:1时杀伤作用要好于效靶比1:1时。对于293T细胞组来说CD138-CAR-T组与对照T细胞组无显著统计学差异。结论:CD138-CAR-T对于肿瘤细胞表面CD138阳性的多发性骨髓瘤普通株RPMI8226和耐药株MM1R均有显著的杀伤作用,而对于CD138阴性细胞株293T则没有显著的杀伤作用,因此不论耐药株还是普通株其杀伤作用的好坏依赖于细胞表面CD138分泌水平的高低,分泌水平高杀伤作用好。
[Abstract]:Objective: multiple myeloma is a malignant hematologic tumor whose incidence has been increasing in recent years, but with the progress of the disease to bring great pain to patients, the current treatment is limited.Because of the combination of chemotherapeutic agents, it is easy to produce multiple drug resistance.Multiple drug resistance, once produced, the majority of patients with poor prognosis.Therefore, we are constantly looking for new treatment methods. Cellular immunotherapy (CAR-T) technology is to insert the target gene into the virus vector and then transfer the virus into the T cells of patients to express specific antibodies.In previous studies, it was found that SCFV) replaced the extracellular antigen-binding domain (TCR), which made CAR-T have the advantage of MHC non-restrictive recognition and make it play a better role in recognition and anti-tumor cells.In particular, some studies have found that CD19-CAR-T has a good effect on CD19-positive B-cell malignant tumors.CD138 is a surface protein that acts as an adhesion molecule to collagen and fibronectin. CD138, which binds to extracellular matrix molecules, is highly expressed on the surface of myeloma cells, but hardly expressed in other hematopoietic cells.Including T cells and B cells.CD138 has therefore become a good target for CAR-T therapy in the treatment of multiple myeloma.Based on the above, the experiment is designed.The main purpose of our study was to investigate the cytotoxicity of cellular immunotherapy (CD138-CAR-T) on the RPMI8226 of multiple myeloma cell line and the killing effect on the drug-resistant cell line MM1R.To evaluate the effect of CD138 molecule as a target of CAR-T for cell therapy.Methods: lentivirus carrying CD138 antibody and lentivirus without target gene were constructed. Human T lymphocytes were isolated and transformed into CD138-CAR-T and empty T cells.Then flow cytometry was used to detect the transfection efficiency of CD138-CAR-T, the expression of CD138 molecules on the surface of multiple myeloma cell line RPMI8226, drug-resistant cell line MM1R and negative control cell line 293T.Flow cytometry was used to detect the killing effect of CD138-CAR-T and T cells carrying empty virus on RPMI8226, MM1R and 293T cells at 10:1 and 1:1, respectively.Results the efficiency of CD138 transfection to T cells was 46.25% and that of RPMI8226 cells was higher than that of MM1R cells.Therefore, in order to investigate the effect of CAR-T cells on the high and low expression of CD138 in multiple myeloma resistant cell lines, we isolated and cultured MM1R CD138 positive cells to increase the positive rate of CD138 on the surface of the cell line .293T.The cytotoxicity of CD138-CAR-T group was stronger than that of empty-carrying T cell group for RPMI8226 and MM1R.However, there was no significant difference between CD138-CAR-T group and empty virus carrying T cell group for MM1R and 293T cells before sorting.At the same time, the cytotoxicity of CD138-CAR-T in MM1R cells group was significantly stronger than that in MM1R cell group.The killing effect is better at 10:1 than at 1:1.There was no significant difference between CD138-CAR-T group and control T cell group for 293 T cell group.Conclusion the cell surface CD138 positive CD138 positive multiple myeloma strain RPMI8226 and drug-resistant cell line MM1R have significant killing effect on the tumor cell line: CD138-CAR-T, but not on the CD138 negative cell line 293T, but not on the CD138 negative cell line 293T, but not on the CD138 negative cell line 293T, but not on the CD138 negative cell line 293T.Therefore, the cytotoxicity of drug resistant strain and common strain depends on the level of CD138 secretion on the cell surface, and the high level of secretion is good.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.3

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