| Objective:1. Observe the Astragalus Polysaccharides intervention in vitro culture of rat bone marrow derived Endothelial Progenitor Cells quantity, migration, adhesion, proliferation and differentiation, and the influence of cycle distribution.2.Observe the astragalus polysaccharides intervention in rat bone marrow derived endothelial progenitor cells in vitro co-culture on proliferation of human umbilical vein endothelial cells.Methods:1. The male Wistar rats after intraperitoneal injection of chloral hydrate anesthesia, are taken off their necks and put to death. Remove the femur, tibia, sternum, humerus, the marrow cavity is washed with PBS buffer, using density gradient centrifugation mononuclear cells isolated from rat bone marrow cells, and cultivate adherent cells after seven days of collection. The cells are detected by multiple wavelength laser confocal microscopy identification and flow cytometry. Collect the rat bone marrow derived endothelial progenitor cells, and it will be randomly divided into 6 groups:Set up the control group;Other groups for various concentrations of astragalus polysaccharides.Be DMEM(low concentration of sugar)in the culture medium to join the final concentration of 0.05 mg/ml,0.1 mg/ml,0.2 mg/ml,0.4 mg/ml,0.8 mg/ml Astragalus polysaccharides training for 24 hours. The Astragalus polysaccharides concentration with 0.4 mg/ml group to be timed differently (Oh,6h,12h,24h,48h)were sent to cultivate. Inverted microscope to observe rat bone marrow derived endothelial progenitor cells in each group and to count;Transwell Chambers to cultivate rat bone marrow origin EPCs quantity detection;Adhesion ability experiment test of rat bone marrow derived endothelial progenitor cell adhesion number;Proliferation experiment test of rat bone marrow derived endothelial progenitor cell proliferation; Flow cytometry to detect the rat bone marrow derived endothelial progenitor cell differentiation and cell cycle distribution.2. Separation of rat bone marrow derived endothelial progenitor cells after 7 days is to collect adherent cells, and sampling for dyeing and fluid identification. After being cell expression conforms to the standard and Establish Transwell co-culture system, the control group is to set up and Rat bone marrow derived endothelial progenitor cells and human umbilical vein endothelial cells together to be DMEM(low concentration of sugar) medium cultivation,And astragalus polysaccharides in various concentration(0.05mg/ml,0.1mg/ml,0.2mg/ml,0.4mg/ml,0.8mg/ml)intervent ion training is to be detected after 24h. Proliferation test detected the increase of astragalus polysaccharides and concentration intervention trained posterity umbilical vein endothelial cell proliferation. Different time(Oh,6h,12h,24h,48h)is set for the intervetion group training of astragalus polysaccharide concentration of 0.4mg/ml. Proliferation experiment testing trained each time point of human umbilical vein endothelial progenitor cells proliferation. Results:(1)the influence of the amount of endothelial progenitor cells of rat bone marrow derived based on the intervention cultivation of astragalus polysaccharides in vitro:Astragalus polysaccharide cells can obviously increase the number of rat bone marrow derived endothelial progenitor cells, and can display a certain concentration effect and time efficiency relations. When the astragalus polysaccharide concentration is 0. 01mg/ml, the number was significantly increased(P< 0.05), when astragalus polysaccharide concentration is 0.4mg/ml it achieved the best effect (P<0.01);when astragalus polysaccharide concentration is 0.4mg/ml after 12h intervention training, the number increased significantly(P<0.01),24h to achieve the best effect(P<0.01),48 h a slight decline, but still better than that of control group (P< 0.01). (2)The influence of migration ability astragalus of rat bone marrow derived endothelial progenitor cells based on the polysaccharides intervention in vitro:Astragalus polysaccharide can obviously increase the migration ability of the rat bone marrow derived endothelial progenitor cell, and displayed a certain concentration and time efficiency. When astragalus polysaccharide concentration is 0.1mg/ml, the number of its migration increased(P<0.05), when astragalus polysaccharide concentration is 0.4 mg/ml, it achieves the best effect (P <0.01);when astragalus polysaccharide concentration is 0.4 mg/ml with intervention training after 6 h, its migration quantity increased(P< 0.05),24h to achieve the best effect(P<0.01),48h a slight decline, but still better than that of control group(P<0.01). (3)The influence of proliferation ability of rat bone marrow derived endothelial progenitor cell based on astragalus polysaccharides intervention in vitro:the astragalus polysaccharide can obviously increase the proliferation ability of the rat bone marrow derived endothelial progenitor cell, and a certain concentration and time efficiency, when astragalus polysaccharide concentration is 0.05mg/ml,the proliferation ability increased(P<0.05),when astragalus polysaccharide concentration is 0.4mg/ml, it achieved the best effect(P<0.01);when astragalus polysaccharide concentration is 0.4mg/ml with intervention training after 6h, its proliferation ability increased(P<0.05),24h to achieve the best effect (P<0.01),48h a slight decline, but still better than that of control group (P<0.01). (4) The influence of cycle distribution of rat bone marrow derived endothelial progenitor cell baasd on astragalus polysaccharides intervention in vitro:when astragalus polysaccharide concentration is 0.1 mg/ml, the Go/G, phase in proportion to the endothelial cells of rat bone marrow progenitor cells began to decrease (P<0.05), and the S phase and G2 phase proportion of rat bone marrow derived endothelial progenitor cells began to increase(P<0.05), when astragalus polysaccharide concentration is 0.4mg/ml, it is of best effect(P< 0.01). (5)The influence of differentiation of rat bone marrow derived endothelial progenitor cell based on astragalus polysaccharides intervention in vitro:when astragalus polysaccharide concentration is 0.1 mg/ml, it began to promote the line direction of endothelial progenitor cells in the rat bone marrow cells, the expression of monocyte/macrophage surface sign(CD14+ and CD64+) percentage reduced compared with control subjects(P<0.05 or P<0.01), while the endothelial cells specificity mark(vWF+)is of higher percentage compared with control group(P< 0.05), when astragalus polysaccharide concentration is 0.4mg/ml, it is of best effect(P<0.01). (6)The influence of proliferation ability of rat bone marrow derived endothelial progenitor cells in human umbilical vein endothelial cell in transwell indirect common culture under the condition of astragalus polysaccharides intervention:the influence of the different concentrations of astragalus polysaccharide in vitro, human umbilical vein endothelial cells cultivation in a certain concentration and time efficiency. Astragalus polysaccharides intervention to cultivate human umbilical vein endothelial cells in vitro compared with control group human umbilical vein endothelial cell proliferation ability increased(P<0.01), endothelial progenitor cells in the rat bone marrow cells and human umbilical vein endothelial cells compared with the control group human umbilical vein endothelial cell proliferation ability increased(P< 0.01), astragalus polysaccharides intervention in vitro endothelial progenitor cells and human umbilical vein endothelial cells were cultivated and astragalus polysaccharides intervention in vitro human umbilical vein endothelial cells than human umbilical vein endothelial cell proliferation ability increased(P<0.01).Astragalus polysaccharides in different time (0.4mg/ml) intervention in the rat bone marrow derived endothelial progenitor cell co-cultivated compared with control group human umbilical vein endothelial cell proliferation ability increased(P<0.05or P<0.01), and displayed a certain time dependence,24 h to reach the best effect (P<0.01).Conclusion:(1)The APS group intervention in endothelial cells of rat bone marrow cells in vitro can increase the quantity, promote the proliferation, migration, adhesion, a certain concentration and time dependence. (2)The APS intervention in vitro endothelial progenitor cells in the rat bone marrow cells can make its Go/G, phase ratio decrease, and the S phase and G2 cell percentage increase. (3) The APS rat bone marrow derived endothelial progenitor cells in vitro intervention can make the percentage of monocyte/macrophage surface marks (CD14+, CD64+) cells decrease, while the percentage of endothelial cells specificity mark(vWF*) cells increase significantly. (4)The APS intervention in rat bone marrow derived endothelial progenitor cells in vitro culture with human umbilical vein endothelial cells can significantly promote the proliferation of human umbilical vein endothelial cells, and display certain concentration and changes of time dependence. |
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