Study On The Biological Function Of CD7 Molecule

CD7 protein is expressed on T cells and NK cells,and has higher expression on T-ALL and T-LBL.At present,CD7-targeted immune adoptive therapy is increasingly used in clinic.However,the biological function of CD7 and the influence of CD7 protein on hematopoietic system are still unclear.Objective:This paper aims to explore the biological function of CD7 and the influence of CD7 deletion on hematopoietic system.Methods:1.CD7 knockout mice were constructed by CRISPR/Cas9 technology,and CD7+/mice were hybridized to obtain wild mice and CD7-/-mice.2.The immune organs of mice were sectioned with H&E,and the organ weight and cell number were detected.Thymus cells of mice and NK cells in bone marrow obtained by flow sorting were selected for RNA-sequence analysis.In vitro,the proliferation ability of mouse spleen and thymocytes,as well as the migration and adhesion ability of mouse thymocytes were detected.3.Flow Cytometry(FCM)was used to detect the proportion of various types of cells in hematopoietic system,T cells in thymus and immune cells in spleen.The proportion of peripheral blood cells in mice was detected by five-category blood cell analyzer.4.B16-F10 cells were subcutaneously inoculated,the percentage of immune cells in tumor and spleen of mice inoculated with tumor was detected by flow cytometry,RNA from thymus and spleen cells was extracted,and the relative expression contents of cytokines TNF-α,IFN-γ,IL-6 and IL-2 were detected by Q-PCR.CD8+T cells of spleen were sorted by flow sorting method,and their killing ability,migration,invasion,adhesion and relative expression of cytokines were detected in vitro.5.The normal donor peripheral blood T cells of CD7-were constructed by the method of Protein Expression Blocker(PEBL).The common maker on the surface of T cells,cell apoptosis and cell cycle were detected by flow cytometry,and the relative expression contents of migration,chemotaxis,adhesion and adhesion related genes ITGB1 and ITGB3 were detected in vitro.And build CAR-T cells that block CD7 protein,and insist on testing its killing ability to tumor.6.The tumor cell line of CD7-was constructed by intracellular blocking.The common maker,apoptosis and cell cycle on the surface of the tumor cell line were detected by flow cytometry,and the relative expression contents of migration,chemotaxis,adhesion and adhesion related genes ITGB1 and ITGB3 were detected in vitro.Tumor cells were injected into NCG mice through tail vein,and the survival time,body weight and tumor load in peripheral blood were observed.Finally,the mice were killed to detect the tumor infiltration of mouse organs.Results:1.We successfully obtained CD7-/-mice,and RNA-sequence results showed that the deletion of CD7 resulted down-regulation genes of the interaction between mouse thymocytes and extracellular matrix and the focal adhesion.2.Under normal physiological conditions,The proportion of all kinds of cells in hematopoietic system,thymus and spleen of mice is normal.3.After B16-F10 cells were inoculated,the tumor volume of CD7-/-mice increased faster,and the proportion of CD8+T cells in spleen and tumor decreased.4.After B16-F10 cells were inoculated,the invasion and adhesion ability of CD8+T in spleen of CD7-/-mice was weakened.5.After blocking CD7,there is no difference in the detection indexes of T cells in peripheral blood of normal donors.6.After blocking CD7,the invasion and adhesion of tumor cells are weakened.After blocking CD7 in NCG mice,the tumor cells were more concentrated in the liver.Conclusions:It was found that the deletion of CD7 gene did not affect the development of hematopoietic system and the growth of mice.In this paper,the biological function of CD7 and the conditions of its action were expounded,which provided a theoretical basis for clinical evaluation of the possible side effects caused by CD7 deletion after targeted therapy.Finally,this paper found that the invasion ability of tumor cells weakened after blocking CD7,which is of great significance for the follow-up treatment of patients with negative recurrence after targeted CD7 therapy.