| Objective:(1)Build the knockout SLC5A8 gene knockout model based on CRISPR/Cas9 technology;(2)To observe the changes of tumor microenvironment in C57 negative tumor mice with SLC5A8 gene knockout model;(3)To observe the changes of T lymphocyte subsets in peripheral blood and lymph node organs of SLC5A8 knockout model mice;(4)To observe the changes of organ structure in SLC5A8 knockout model mice;(5)To clarify the antitumor mechanism of SLC5A8 gene at the organ level.Method:(1)The gene knockout model of SLC5A8 was constructed and identified using CRISPR/Cas9 technology;(2)Experimental group:C57 mice were divided into three groups:negative tumor mice with SLC5A8 gene knockout were the experimental group(KO group),normal negative tumor mice were the reference group(WT group),and the blank control group(Control group).All negative tumor mouse models were constructed by subcutaneous implantation of malignant melanoma B16 cells in the abdomen of C57 mice.(3)Observe and record the tumor growth status of the experimental group and the reference group,including the change of tumor volume,tumor weight of negative tumor mice,etc.;The tumor volume inhibition rate and tumor reinhibition rate were calculated and the time-growth curve was drawn.(4)Flow cytometry was used to detect the levels of t-lymphocyte subsets in the tumor microenvironment of the experimental group and the reference group.The levels of T lymphocyte subsets in peripheral blood and lymph node organs of the experimental group,reference group and blank control group were detected.The effect of SLC5A8 gene on tumor microenvironment was analyzed.(5)HE staining was used to observe the changes of tumor tissue structure in the experimental group and reference group.The changes of organ structure in each group mice were observed and analyzed.Result:(1)The SLC5A8 gene knockout model was successfully constructed by usingCRISPR/Cas9 technology.(2)The growth of melanoma:Compared with the reference group,the experimental group had faster tumor growth,larger volume and increased tumor weight(P<0.01).(3)CD4+ proportion:in the tumor microenvironment,CD4+ in the experimental group decreased significantly compared with that in the reference group(P<0.01).In peripheral blood,tumor draining lymph nodes,spleen and thymus,the experimental group was significantly lower than the blank control group,with significant differences(P<0.01).Except thymus,the reference group was all lower than the blank control group(P<0.05).Except the peripheral blood,the experimental group was all lower than the reference group(P<0.05).(4)CD8+ proportion:in the tumor microenvironment,CD8+ in the experimental group was significantly lower than that in the reference group(P<0.01).In the lymph nodes of tumor drainage,the experimental group was significantly lower than the other two groups(P<0.01).In the spleen,the experimental group and the reference group were lower than the blank group(P<0.05).In the thymus gland,both the experimental group and the reference group increased compared with the blank control group(P<0.05).(5)CD4+/CD8+ ratio level:In the peripheral blood,tumor draining lymph nodes,spleen and thymus,the experimental group and the reference group were lower than the blank control group(P<0.05),among which the experimental group was the most significant(P<0.01).In the tumor drainage lymph nodes,spleen and thymus,the experimental group was lower than the reference group(P<0.05).(6)CD4+&CD8+ ratio level:in the thymus gland of C57 mice,both the experimental group and the reference group were significantly lower than the control group(P<0.01),and the experimental group was significantly lower than the Control group(P<0.05).(7)HE staining:tumor:compared with the reference group,tumor cells in the experimental group showed a deeper degree of atypia,with uneven cell density,complicated tumor cell structure,disordered arrangement,diverse cell morphology,and a large number of inflammatory cells infiltration.Colon:in the experimental group,the structural integrity of the colon mucosa was damaged,the arrangement was disordered,part of the fossa disappeared,and a large number of inflammatory cells were seen to infiltrate,submucosal edema,the colon muscle layer thickened significantly,mucosal inflammation.The colonic mucosa of the reference group was slight mucosal inflammation.(3)stomach:in the experimental group,gastric mucosa was slightly edema,cells were not arranged neatly,and a small numberof inflammatory cells were infiltrated under the mucosa.(4)lungs:in the experimental group,the alveolar cavity was enlarged,the alveolar wall was widened,individual alveolar fusion was observed,some alveolar compensatory dilation was observed,and alveolar interstitium was infiltrated with a small number of inflammatory cells.(5)Liver and kidney:no significant difference was found in each group.Conclusion:(1)Knockout of SLC5A8 gene can reduce the cellular immunity(T lymphocyte subsets)function in peripheral blood and tumor draining lymph nodes,spleen and thymus of C57 negative tumor mice.(2)The knockout of SLC5A8 gene affected the changes of the colon and other organ structures in C57 mice,which may have damaged the immune barrier of tissues and organs by affecting the microenvironment,further leading to inflammatory lesions of organs.(3)SLC5A8 may inhibit the growth and proliferation of tumor cells in C57 negative tumor mice by affecting the cellular immunity(T lymphocyte subgroup)function in the tumor microenvironment of C57 negative tumor mice. |
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