| Objectives:As one of the diseases that seriously threaten human health,cancer not only causes harm to the patient’s body and mind,but also brings a huge economic burden to the family,society and the country.At present,common clinical treatments for malignant tumors include surgery,radiotherapy and chemotherapy,and targeted therapy.However,this type of treatment cannot effectively control the progression and recurrence of some malignant tumors,nor can it effectively extend the survival period and improve the survival of such patients Quality of life.Therefore,there is an urgent need to seek new treatments.In recent years,immunotherapy has become an important tumor treatment method,which brings hope to overcome malignant tumors.Studies have shown that most tumor cells can evade the recognition of the immune system,thereby limiting the anti-cancer effect of immunotherapy.Chimeric antigen receptor T(CAR-T)cell therapy technology has good targeting,killing,proliferation and durability,and has become a hot spot for tumor immunotherapy.Although a large number of clinical studies have confirmed that this technology has achieved great success in improving the quality of life of patients with hematological malignancies and prolonging the survival of patients.However,the research of this technology in solid tumors is not mature enough to achieve the expected therapeutic effect.It still faces many challenges,including:the special pathological characteristics of solid tumor tissues,the immunosuppressive microenvironment around the tumor,the optimized combination of costimulatory molecules,Suicide gene design,adverse reactions after reinfusion,etc.The purpose of this study is to explore the targeted killing ability of MSLN-CAR-T cells targeting mesothelin(MSLN)to solid tumors through the piggyBac transposon system,and the MSLN-CAR-T cells under this culture system The anti-tumor activity and safety of the drug provide a research basis for the clinical transformation of this treatment method.Methods:The animal experiments involved in this study were approved by the Animal Ethics Committee of Yan’an Hospital Affiliated to Kunming Medical University.1.Functional verification of mesothelin chimeric antigen receptor modified T cells targeting malignant tumors in vitroThe tumor cells with high expression of mesothelin were screened by Western blot,and the tumor cells with low expression of mesothelin were artificially overexpressed by lentivirus transfection.The piggyBac transposon system was used to transfect T cells to obtain the third generation MSLN-CAR-T cells,and the empty plasmid was used as a control T cell.Flow cytometry was used to detect the expression of CAR in T cells and detect its transfection effectiveness.The killing effect of MSLN-CAR-T cells on cell lines with high expression of mesothelin and cell lines with low expression of mesothelin was tested in different ways.Furthermore,ELISA was used to detect the secretion of cytokines after MSLN-CAR-T cells and tumor cells were co-cultured.2.Functional verification of mesothelin chimeric antigen receptor-modified T cells targeting malignant tumors in vivoThe BGC-823 cell line and SPC-A-1 cell line that naturally express the mesothelin antigen,the MDA-MB-231 cell line that does not express the mesothelin antigen,and the MDA-MB-231MSLN(OE)cell line that artificially overexpresses the mesothelin antigen are selected as The tumor cells used in the mesothelin nude mouse tumor-bearing model were constructed,and Nude nude mice were used as the nude mouse strain for the nude mouse model to construct the hman mesothelin tumor-bearing nude mouse model.After successfully constructing a mesothelin tumor-bearing nude mouse model,inject newly cultured control T or MSLN-CAR-T cells into the model nude mouse,and compare control T or MSLN by measuring the size of the tumor and the change in tumor weight-The tumoricidal activity of CAR-T cells in nude mice.3.Study on Invasiveness and Safety of Mesothelin Chimeric Antigen Receptor Modified T Cells Targeting Malignant TumorsObserve the survival status and weight changes of tumor-bearing nude mice injected with control T or MSLN-CAR-T cells,and observe whether the pathological sections of each organ of the nude mice have organic lesions after the nude mice are killed at an appropriate time to judge the nude mice Whether adverse reactions occurred and after the nude mice were sacrificed,the infiltration of MSLN-CAR-T cells into tumor tissues,inhibition of tumor cell proliferation,and induction of tumor cell apoptosis were detected by immunofluorescence to reflect the infiltration of MSLN-CAR-T on tumor tissues.,The ability to inhibit proliferation and induce apoptosis.Results:1.Functional verification of mesothelin chimeric antigen receptor modified T cells targeting malignant tumors in vitro:We screened tumor cells with high and low expression of mesothelin by Western blot.Among them,mesothelin was highly expressed in gastric adenocarcinoma cells BGC-823 and lung adenocarcinoma cells SPC-A-1,while in triple-negative breast cancer cells MDA-MB-231 and prostate cancer cells LNCaP low expression;At the same time,the low-expressing tumor cells MDA-MB-231 and LNCaP were successfully overexpressed by lentiviral transfection,and detected by piggyBac transposon system and flow cytometry.Successfully constructed MSLN-CAR-T cells with a positive rate of about 20%.By co-cultivating MSLN-CAR-T cells with tumor cells,the results showed that control T cells had no killing effect on tumor cell lines.The killing effect of MSLN-CAR-T cells on cell lines with high expression of mesothelin is significantly stronger than that on cell lines with low expression,and the tumor-killing ability becomes more obvious with the increase of the effective target ratio;MSLN-CAR-T cells and tumors were detected by ELISA In the supernatant of cell co-culture,it was found that after MSLN-CAR-T cells and mesothelin over-expressing cell lines were co-cultured,IL-2,IL-6,IL-10,TNF-α,IFN-γ,Perforin and Granzyme B The secretion amount is significantly higher than that of cell lines that do not express mesothelin and increases with the increase in the effective target ratio;the tumor cells after co-culture were collected,and Western blot detection revealed that the tumors were co-cultured with MSLN-CAR-T cells The expression of apoptosis-related proteins Bax and Bad and the ratio of Cleaved-Caspase-3 to Caspase-3 were higher than those of the control group.2.Functional verification of mesothelin chimeric antigen receptor-modified T cells targeting malignant tumors in vivo:After successfully constructing a nude mouse tumor model one week later,MSLN-CAR-T cells were injected into the tail vein of nude mice.The study found that the tumor volume and weight of the MSLN-CAR-T cell treatment group were smaller than those of the blank control group and the control T group,indicating that MSLN-CAR-T cells can inhibit tumor growth to a certain extent in nude mice,which makes the weight change of nude mice more stable,and at the same time significantly prolongs the survival time of nude mice.3.Study on Invasiveness and Safety of Mesothelin Chimeric Antigen Receptor Modified T Cells Targeting Malignant Tumors:Nude mice injected with MSLN-CAR-T cells were not found to have obvious adverse reactions,and there were no obvious organic lesions in the important tissues and organs in the pathological sections.The MSLN-CAR-T immunofluorescence labeling detection of tumor-related proliferation,apoptosis and tumor infiltration found that MSLN-CAR-T cells in nude mice treated with MSLN-CAR-T cells have better infiltration and tumor cell proliferation is inhibited,Tumor cell apoptosis increased significantly,indicating that MSLN-CAR-T cells have good anti-tumor properties.Conclusions:1.In this experiment,piggyBac transposon can successfully construct MSLN-CAR-T cells.2.The MSLN-CAR-T constructed in this experiment has stronger killing activity and targeting ability on malignant tumor cell lines with high expression of mesothelin.3.MSLN-CAR-T cells can better inhibit the progression of solid tumors in animals,prolong the survival time of animals,and have good in vivo treatment safety. |
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