| Objective:This study verified the repair effect of BSJDTL on the bone marrow microenvironment of leukemia,and further explored whether the bone marrow microenvironment restored the hematopoietic support function of BSJDTL,providing a new method for the treatment of leukemia.Methods:The effect of BSJDTL formula on animal level was preliminarily verified by establishing leukemia mouse model.Then,mouse mesenchymal stem cells were extracted and cultured,and the repair effect of BSJDTL Formula on bone marrow microenvironment was verified by testing the cell proliferation of mesenchymal stem cells.After that,the restoration of hematopoietic function of bone marrow microenvironment was verified by studying the activity of hematopoietic stem cells in bone marrow microenvironment.Finally,the restoration of restored bone marrow microenvironment function was verified by detecting the cytokines of mesenchymal stem cells to hematopoietic stem cells.Study 1:Forty-eight healthy BALB/C male mice were selected and randomly divided into four groups:Normal control group,Model control group,CVPtern medicine treatment group,BSJDTL group,with 12 mice in each group.The L1210 cell line was established byγ-ray irradiation and tail vein injection.The growth status and bone marrow smear of mice were evaluated,and the levels of CD4~+and CD8~+subsets in peripheral blood routine and serum were detected.The proportion of CD34~+cells and CD3~-CD19~+cells in mouse bone marrow cells was detected by flow cytometry to verify the repair effect of Bukiden Jiedu Tongluo recipe on mouse bone marrow.Study 2:Study one femoral bone marrow cells in mice,the whole bone marrow adherent method is adopted to select training room of mesenchymal stem cells by flow cytometry identification phenotypic mesenchymal stem cells,cell colony experimental tests between the proliferation of mesenchymal stem cells,the number of flow cytometry detection cycle of mesenchymal stem cells,apoptosis,CCK8 method to detect the cell activity,q RT-PCR was used to detect the proliferation,differentiation and tumor-related genes of Mesenchymal stem cells,and to verify the repair effect of BSJDTL recipe on core cell-mesenchymal stem cells in bone marrow microenvironment.Study 3:In study one mouse bone marrow cells,with the method of magnetic bead separation extracting hematopoietic stem cell culture,choose ectomesenchymal stem cell culture at the same time,through ectomesenchymal stem cells and hematopoietic stem cells will be trained after inspection CCK8 of hematopoietic stem cell proliferation activity and streaming apoptosis of hematopoietic stem cells and the colony of hematopoietic stem cells,Finally,CDK1 gene of hematopoietic stem cells was detected by q RT-PCR to verify the supporting effect of bone marrow microenvironment repaired by BSJDTL recipe on hematopoietic stem cells.Study 4:The mesenchymal stem cells and hematopoietic stem cells co-cultured in study2 were collected separately.The supernatant of cell culture medium co-cultured in study 2was detected by ELISA,and cytokines CXCL12,SCF and ICAM1 secreted by mesenchymal stem cells were detected.The expression of cytokines secreted by mesenchymal stem cells at gene level was detected by q RT-PCR.It was proved that BSJDTL prescription could restore the function of repaired mesenchymal stem cells and support the mechanism of hematopoietic action.Results:Study 1:Compared with model control group and CVPtern medicine treatment group,the number of white blood cells in peripheral blood increased,the ratio of CD4~+/CD8~+in peripheral blood showed an upward trend,and the proportion of CD34~+cells in bone marrow cells increased significantly(P<0.05).The proportion of CD3~-CD19~+cells in bone marrow also showed that the proportion of mice in the kidney-tonifying,detoxifying and collection-clearing group was higher than that in the CVPtern medicine group(P<0.05).Study 2:The MSCs cell cloning ability of mice treated with BSJDTL formula was enhanced,and the cell colony analysis experiment showed that the number of cell colonies in BSJDTL group was significantly increased compared with model control group and CVPtern medicine treatment group(P<0.05).Compared with the model control group and CVPtern medicine treatment group,the ratio of G0/G1 phase in the kidney-tonifying,detoxifying and collection-clearing group was significantly lower,and the ratio of S+G2 phase was significantly higher(P<0.05).Apoptosis detection also showed that the MSCs apoptosis rate was the lo CVPt in tonifying kidney-jiedu tongluo group.The expression of MSCs gene in each group showed no significant difference in CDK1 gene expression after bushun Jiedu Tongluo prescription.The expression of CREB1 and BIRC5 genes in MSCs of leukemia mouse model control group was significantly increased,while the expression of CREB1 and BIRC5 genes in MSCs of CVPtern medicine treatment group was lower than that in model control group,while the expression of CREB1 and BIRC5 genes in MSCs of Bushen Jiudu Tongluo formula was significantly decreased(P<0.05).Study 3:The number of BFU-E colonies of hematopoietic stem cells co-cultured with MSCs treated with Bukiden Jiedu Tongluo Formula increased significantly,and the difference was statistically significant(P<0.05).The number of CFU-GM colonies also increased.The proliferation ability of HSC was tested by CCK8 experiment,and the results also showed that the proliferation ability of hematopoietic stem cells was significantly improved after co-culture with MSCs treated with Bukiden Jiudu Tongluo formula compared with CVPtern medicine group(p<0.05),but there was no statistical difference in apoptosis.q RT-PCR detection of CDK1 gene expression of hematopoietic stem cells showed that the CDK1 gene expression in model control group was significantly higher than that in normal control group,and the expression of CDK1 gene in CVPtern medicine treatment group was higher than that in model control group.After co-culture with MSCs treated with Bukidney-jiudu Tongluo formula,the expression of CDK1 gene was inhibited(P<0.05).Study 4:ELISA results showed that compared with the normal control group,the secretion of CXCL12 and SCF in the model control group and the CVPtern medicine treatment group decreased,but the secretion of CXCL12 in the Kidney-tonifying jiudu Tongluo group increased,and the increase of kidney-tonifying Jiudu Tongluo prescription was more significant than that in the CVPtern medicine treatment group(P<0.05).For ICAM1secretion,Compared with the normal control group,both the model control group and the CVPtern medicine treatment group showed an upward trend,but the kidney tonifying,detoxification and collating-clearing group showed a downward trend,with no statistical difference.The results of q RT-PCR at the gene level showed that the expression of CXCL12and SCF showed a consistent trend in all groups.Compared with the normal control group,the model control group and the CVPtern medicine treatment group were down-regulated,and there were significant statistical differences in CXCL12 secretion(P<0.05).Compared with the model control group,CXCL12 expression was up-regulated in both the CVPtern medicine treatment group and the kidney-tonifying,detoxifying and collating-clearing group,and the up-regulation was more obvious in the kidney-tonifying,detoxifying and collating-clearing group,with statistically significant difference(P<0.05).Compared with the model control group,SCF expression was significantly up-regulated in the kidney-tonifying,detoxifying and collating-clearing group,with statistically significant difference(P<0.05).Compared with the normal control group,the expression of ICAM1 in the model control group and the CVPtern medicine treatment group was significantly up-regulated(P<0.05),while the expression of ICAM1 in the kidney-tonifying,detoxifying and collasing-clearing group was not significantly different from the normal control group,and there was a statistical difference between the model control group and the CVPtern medicine treatment group(P<0.05).Compared with the model control group,the secretion of ICAM1 in the CVPtern medicine treatment group was also significantly up-regulated,with statistical significance(P<0.05).Conclusion:Proved by assessment model of leukemia mice BSJDTL effect on mouse bone marrow repair,and then by mesenchymal stem cells in the BSJDTL between side effect after cell proliferation ability enhancement,the number of settlement increased,cell activity,proliferation gene expression and differentiation,tumor gene is proof BSJDTL inhibition of ectomesenchymal stem cells to repair,At the same time,the detection results of bone marrow hematopoietic stem cells also showed that the proliferation and self-renewal ability of hematopoietic stem cells were restored,which further indicated that the bone marrow microenvironment was repaired.Finally,the high expression of CXCL12 and SCF in mesenchymal stem cells and the low expression of ICAM1 indicated that the restored mesenchymal stem cells restored the function of hematopoietic stem cells. |
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