| Objective:This project constructed the lentivirus over-expression vector of mice C-X-C chemokine receptor type4(Cxcr4) gene based on previous research, conducted in vitro transfection of mice mesenchymal stem cell (MSCs), co-transplanted with hematopoietic stem cell (HSCs) acquired through magnetic activated cell sorting, and reconstructed the mice bone marrow chimeric models to explore the effects of over-expressed CXCR4 on MSCs homing, together with the influence on the recovery of the entire mice bone marrow hematopoietic system. The chimeric mice Lewis lung cancer models were constructed after successful modeling, and the formation of tumor tissue and peritumoral lymphatic vessel as well as tumor growth situation were explored after combined orall administration of Jinfukang to mice, to determine whether Jinfukang achieve anti-tumor effects on the basis of lymphatic vessel formation regulated by over-expressed CXCR4.Methods:(1) The growth of mice MSCs under different culture conditions in vitrowas observed, its growth curve according to daily cell countwas drew, its OD value was measured with MTT method, and its purity was detectedwith flow cytometry.(2) The mice bone marrow HSCs was selectedby CD117 and magnetic activated cell sorting, and its sorting efficiency was determinedwith flow cytometry.(3) The optimal MOI value for transfecting MSCswas explored with lentivirus over-expression CXCR4, and its transfection efficiencywas determined with flow cytometry.(4)The C57BL/6 male mice were treated as the donors and female mice as the receptors, and homogenic mice bone marrow chimeric modelwas constructed. The recipient mice received TBI (total dose of 8Gy,2Gy/min, with the field of view of 30*30cm), and then were divided into 5 groups:the over-expressed CXCR4-MSCs combined with Jinfukang group, the over-expressed CXCR4-MSCs combined with cyclophosphamide group, the over-expressed CXCR4-MSCs combined with normal saline group, the over-expressed empty-MSCs combined with Jinfukang group, and the over-expressed empty-MSCs combined with normal saline group. The expression of red fluorescence in peripheral blood and bone marrow smears, as well as the influence of over-expressed CXCR4 on MSCs homing was observed under laser scanning confocal microscope after successful modeling; meanwhile, the recoveries of while blood cells, red blood cells, and platelets in peripheral blood were detected to observe their effects on the recovery of mice bone marrow hematopoietic system.(5)Right axillary subcutaneous injection of Lewis lung cancer cells (1×107/ml/mouse) was conducted 35 days after successful modeling of mice bone marrow chimeric models to construct chimeric mice Lewis lung cancer model; andoral administration of Jinfukang (0.4ml/mouse)and normal saline (0.4ml/mouse), cyclophosphamide intraperitoneal injection (200mg/kg/mouse) were conducted 1 week later. All the mice were sacrificed at 28 days after to get the tumor tissue for weighing, froze the section, and performed immunofluorescence staining; the lymphatic endothelium was labeled in green through LYVE-1 antibody, and the cell nucleus was labeled in blue with DAPI; the red and green fluorescence in newly formed lymphatic vesselwere observed after over-expression of CXCR4, together with the expression of fluorescence after merging; and the growth and changes of tumor tissue after applying Jinfukangwere analyzed.Results:(1)Mice bone marrow MSCs with high purity were successfully obtained through whole bone marrow culture, and Flow identification of generation P3 MSCs showed,the positive rates of CD90.2=42.7% and CD105= 98.2%, respectively,which demonstrated that the culture condition of complete medium (10%FBS combined with DMEM/F12) was suitable for in vitro culture of bone marrow MSCs, and was superior to those of 10%FBS combined with DMEM group (CD90.2=19.8% and CD 105=95%), and 15%FBS combined with DMEM group (CD90.2=21.3% and CD105=95.7%).(2) CD117 combined with magnetic activated cell sorting successfully sorted out mice bone marrow HSCs with high purity; and the results of flow identification demonstrated that compared with those before sorting (positive rate of 1.1%), the sorting efficiency was outstandingly improved after MACS (positive rate of 54.5%). (3)Transfection of generation P3 MSCs was conducted successfully with lentivirus over-expressed CXCR4, and was determined that the best MOI for transfection was 40. The content of CXCR4 on MSCs was detected with flow identification; when compared with the lentivirus NC-MSCs group (positive rate of 12.5%), high transfection efficiency was obtained after lentivirus LV-CXCR4-MSCs (positive rate of 71.5%).(4)Mice bone marrow chimeric model was successfully constructed, and red fluorecytes obtained through differentiation and migration of bone marrow MSCs were observed in peripheral blood and bone marrow smears under laser scanning confocal microscope; when compared with those in non-over-expression group, the content of red fluorescence was notably increased in the over-expression group, with statistical significance (P<0.05).(5)The recoveries of white blood cells, red blood cells and platelets in mice peripheral bloodwere significantly enhanced rapidly after lentivirus over-expressed CXCR4, compared with that of non-over-expression group (P<0.05). (6) The tumor was taken for weighing after successfully constructing Lewis lung cancer modelin the chimeric mices.The tumor tissue was apparently enlarged after lentivirus over-expressed CXCR4 compared with the other groups (P<0.01); while the tumor tissue was distinctly smaller after oral administration of Jinfukang compared with that ofuntreatedgroup (P<0.05). (7) The expression of red fluorescence in the newly formed lymphatic vessel was remarkably increased after lentivirus over-expressed CXCR4, among which some of the newly formed lymphatic vessel tissues might showed RFP/LYVE-1(GFP) double positivewere observed. The average conversion rate in all over-expression groups was markedly higher than that in the other groups (P<0.05); and after administration of Jinfukang, theaverage conversion rate was outstandingly lower than that in the over-expression group (P<0.05).Conclusion:(1)Mice bone marrow MSCs with high purity were successfully cultured in vitro, and were used to co-transplant with HSCs obtained through magnetic activated cell sorting, and the mice bone marrow chimeirc model was constructed successfully.(2)The lentivirus over-expressed CXCR4-MSCs promoted MSCs homing as well as the recovery of bone marrow hemopoietic system in chimeric mice.(3)The lentivirus over-expressed CXCR4-MSCs promoted MSCs to migrate to lung tumor, participated in the lymphatic vessel formation in tumor tissue, and accelerated the tumor growth.(4)Jinfukang oral liquid displayed a distinct inhibition of on the growth of Lewis lung tumor tissue, the mechanism might be through inhibiting the conversion of bone marrow MSCs into lymphatic endothelial cells, and SDF-1/CXCR4 might be one of the pathways. |
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