Study Of Mingjing Granula On The Bone-marrowderived Cells To Inhibit Choroidal Neovascularization

ObjectiveTo observe the effect of Mingjing Granula on the mobilization, recruitment, adhesion and differentiation of bone-marrow derived cells due to induced choroidal neovascularization (CNV) by laser, and investigate the possible mechanism of Mingjing granula in preventing CNV.Methods1. C57BL/6mice were induced CNV by krypton laser(697nm,180mw,50μ m,0.1s), and randomly divided into2groups:control group and treatment group.10normal mice were used as the normal control without any treatment. The amount of CD34+cells in peripheral blood were detected by flow cytometer on day7,14and28after treatment. The level of GM-CSF, VEGF, G-CSF, MMP-9, TSP-1, VE-cadherin were determined respectively by ELISA on day7,14and28after treatment.2. C57BL/6mice were received krypton laser photocoagulation (697nm,180mw,50μ m,0.1s)to induce CNV, then injected through tail vein with BMCs from GFP-transgenic mice. The BMC-treated mice were randomly divided into control group and treatment group.10normal mice were used as the normal control without any treatment. On day7,14,28after laser surgery, histopathologic study, FFA, and choroidal flatmount were performed to measure the CNV severity and BMCs recruitment and adhesion. Stromal cell derived factor-1α (SDF-1α)and CXCR4levels in peripheral blood were measured by ELISA. SDF-1α, vascular cell adhesion molecule-1(VCAM-1), inter-cellular adhesion molecule1(ICAM-1) were detected by immunofluorescent staining.3. C57BL/6mice were induced CNV by krypton laser(697nm,180mw,50μm,0.1s), then injected through tail vein with BMCs from GFP-transgenic mice. The BMC-treated mice were randomly divided into control group and treatment group.10normal mice were used as the normal control without any treatment. On day 7,14,28after laser surgery, the expression of GFP, CD31, α-SMA, F4/80, RPE65in CNV area were detected by immunofluorescence double labeling.Results1. The amount of CD34+cells in control group were significantly higher than that of the treatment group and normal group on day7and14(P<0.05), and there was no significant difference between the treatment group and the normal group (P>0.05). The GM-CSF and VEGF levels in peripheral blood in the treatment group were significantly lower than that of the control group and normal group on day7and14(P<0.05). TSP-1level in peripheral blood in the treatment group were significantly higher than the control group on day7(P<0.05).2. On day7and day14, CNV lesions in the treatment group showed less incorporation of GFP-cells and smaller in size compared with the control group. Histologic sections also showed less severe lesions in the treatment group. The CNV leakage area of FFA in the treatment group was significantly smaller than that of the control group on day28after treatment (P<0.05). SDF-1and CXCR4levels in peripheral blood in the treatment group was less than that of control group on day7(P<0.05). The expression of SDF-1α, VCAM-1, ICAM-1detected by immunofluorescence staining in the treatment group were lower compared with the control group on day7,day14.3. Immunofluorescence double label staining showed that BMCs in the CNV area can differectiate into RPE cells, vascular endothelial cells, macrophage, and vascular smooth muscle cells. The amount of GFP+cells in CNV area in treatment groups were less than that of control group on day7and14(P<0.05).The amount of CD31+cells, RPE65+cells,F4/80+cells which differentiated from GFP+labled cells were singnificantly less in treatment group than in control group on day7(P<0.05).But the differentiation ratio of BMCs to these four kinds of cells was no significantly deviation (P>0.05)ConclusionThis experiment initially indicated that Mingjing Granula could inhibit the mobilization of bone-marrow derived cells in CNV mice at the early stage, and reduced chemokines levels in peripheral blood and expression of adhesion molecules around CNV, thus could inbiht the recruitment and adhersion of bone marrow derived cells in CNV. The research also showed that Mingjing Granula can reduce the amount of BMCs in CNV area, and reduce the amount of RPE cells, vascular endothelial cells, macrophages in CNV area at the early stage. But Mingjing Granula couldn’t reduce the differentiation ratio of BMCs into RPE cells, vascular endothelial cells, macrophages.