Establishment Of Mouse Stably Knockout Of-MYSM1 In MSC Cell Line C3H10T1/2 And Its Effect On The Immune Regulation In Vitro

The problem of malignant tumor has not yet been breached,nor does malignant diseases of blood system.However,Hematopoietic stem cell transplant?HSCT?brings a new hope.After several years of development,the application of HSCT is more wide and the transplant technology is also more mature.As a result,it provides the chance to survival to more patients.But HSCT also brings many side effects and GVHD is one of them.Clinically,the patient will appear more or less GVHD,which will affect the quality of life or even cause transplant failure or death.So a lot of researchers devoted themselves to finding a better solution to this problem.The pathogenesis of aGVHD and cGVHD demonstrates they both are immune related diseases.So clinical treatment is the hormone and other immunosuppressive agents.But it is invalid to many patients,and it also brings some adverse effects like infection.MSCs has become a promising therapy for aGVHD,which comes lessen adverse effects.So it will come more clinical significances for us to probe into its immunoregulation capacity.Mesenchymal stem cells?MSC?was originally due to the multi-directional differentiation capacity and was applied in tissue repair.It also has the ability of immune inhibition.But we donnot clearly understand the mechanism,which limits its clinical application.In order to know more about the mechanism and provide theoretical basis to clinical researches,whether can we deeply go to explore the mechanism of MSCs from epigenetics?Gene expression is a complicated process controlled by various factors,which makes the control of gene expression very complicated.The histones is constituted with two parts,one is histone octamer and the other is an DNA about 147 bp,and it can be methylated,deubiquited and so on.Histone H₂A is an important part of the nucleosome and its deubiquited can effectively regulate the gene expression.Our research aimed to explore how deubiquitinase MYSM1 changed the immunomodulatory function of MSC.In order to achieve this goal,we proposed to knockout the MYSM1 gene in MSCs of the mice by the gene editing techniques.Then co-cultured the supernatant with the spleen lymphocytes of mice and detect its effects on the lymphocyte proliferation and various factors secreted by lymphocyte subgroup.So how to knockout MYSM1 gene from the MSC of mice? We use the CRISPR-Cas9 technology.We first use CRISPR/Cas9 to incorporate MYSM1 gene into CRISPR,then use the corresponding CRISPR RNSs to guide the degradation of MYSM1.We use for reference of the new technology in “Sicience”.We let the company accomplish the setting-up of the virus.Then we finish the other jobs.Moreover,we insert the puro resistence genes into the virus,only to make it easier for us to screen the cells which have already been infected by the virus.After the virus been generated by the company,we finished the following parts.First of all,use lentivirus,coupled with GFP sign,infect the mice MSCs-C3H10 T 1/2.After using the technology of CRISPR-Cas9 to knock out the MYSM1 gene from mice MSCs,we check the efficiency with FCM.After successful transfection,we detect whether MYSM1 gene has already been knocked out from both transcription and translation by using qPCR and western-blotting.After successfully knocking out,we first verify specific markers on the surface of MSC,then try to verify its immunomodulatory effects via co-cultured the cell supernatant with Spleen lymphocyte of mice.We can conclude the following results through the experiment:1.By FCM to detect lentivirus after transfection with GFP,MSCs expression of GFP efficiency is high as 99%.The results of qPCR and Western-blotting showed that we succeed in knocking out the MYSM1 gene.2.Gene knockout MYSM1 measured by the antibody marker streaming MSC specific markers on the surface unchanged and the immune inhibition ability is strengthened.So we may draw the conclusion: We successfully knock out MYSM1 gene from MSCs via virus transfection technique and CRISPR-Cas9 technology.And it then preliminarily convicted that it can play a stronger immune inhibition in vitro.