| Background:Multiple myeloma (Myeloma Multiple, MM) is a malignant clonal disease of plasma cell system, which is mainly characterized by the clonal growth of plasma cells and the emergence of a large number of immunoglobulin M protein in the blood and urine. At present the main treatment is according to the risk stratification of patients for individualized treatment, although new drugs such as the use of immune modulators and proteasome inhibitor prolonged the survival time of the patients, but most of the patients with the disease will relapse, so we need to update the drug treatment and method. The chimeric antigen receptor is a new method for the treatment of malignant tumor. We found that the chimeric antigen receptor modified NK cells have an anti-myeloma effect, which provides a theoretical basis for the clinical use of the technology.Objective:This study focused on the effect of CAR on NK cell killing myeloma cells.Materials and methods:This study first extracted the total RNA of Jurkat cells and cDNA were synthesized by RT-PCR method. The CD28 and CD3 zeta fragment were synthesized by PCR and were cut under UV. By the methods of double enzyme digestion and ligation, each fragment was connected to pcDNA3.1 plasmid. Anti CD138scFv segment was obstained by department of biological treatment in our hospital, and the segment was connected to the plasmid pcDNA3.1 by gene synthesized. Three fragments are connected to each other as a fusion fragment after sequencing by the methods of double enzyme digestion and ligation. The fusion fragment was connected to pHBLV-CMVIE-Luc-T2A-puro plasmid by double digestion fragments after sequencing. Then together with three plasmid lentiviral packaging system (pSPAX2, pMD2G, pHBLV-CMVIE-Luc-T2A-puro) they were transfected into 293T cells to collect the virus, and the virus titer was determined. Furthermore, in order to get the stably transfected cell lines, the NK92MI cell line were infected by the virus after using puromycin screening positive cells Strains. The expression of the CAR (anti CD138-CD28-CD3 zeta) NK cells were verified by PCR and Western blot. At last the ability of secreting cytokine CD 107a was detected by flow cytometry, and the statistical analysis was carried out to verify the anti-myeloma effect of CAR-NK cells.Results:①Gene fragment of the CAR (antiCD138scFv-CD28-CD3ζ) was constructed by gene engineering technique in vitro, and the gene sequence was verified by sequencing.② After virus packaging technology, we obtained the virus expressing the protein of the CAR.③ we obtained the stable CAR NK cell line expressing the fusion protein after virus transfection.④ we further verified the expression of the fusion protein of the CAR on the sufurce of NK cells by PCR and Western Blot.⑤ NK cells containing the fusion protein of CAR had obvious killing effect on multiple myeloma by cell killing experiments, the ability of CAR NK cells to secrete cytokine CD 107a was significantly better than that of the control group.Conclusion:①The fusion protein of the CAR (antiCD138scFv-CD28-CD3ζ) can be constructed in vitro and expressed on the surface of NK92MI cells.② The CAR (antiCD138scFv-CD28-CD3ζ) modified NK cells plays an important role in the anti-myeloma effect, and the killing effect on multiple myeloma tumor cell lines which express CD138 antigens was enhanced. |
