| Objective:To study the influence of Qi-tonifying and Blood-activating Chinese herb (YQHX) serum on the proliferous ability of bone mesenchymal stem cells (BMSCs) and angiogenesis mechanism of ischemic vessel on diabetic rats with hind limbs ischemia by injection BMSCs and administering YQHX herb, thereby providing new understandings and methods for preventing and treating lower limb arterial ischemic diseases.Methods:(1) In vitro, BMSCs was purified by all cells adherent culture method, which identified by flow cytometry. The serum was extracted from the rats in control group and the rats were given Qi-tonifying (YQ) herbs and Blood-activating (HX) herbs by intragastric administration. MTT assay was used to detect the influence of10%concentration of4different drug serums on the proliferous ability of the third generation of BMSCs and to detect the influence of5%,10%and20%concentration of control and Yiqihuoxue serum on the proliferous ability of the third generation of BMSCs.(2) In vivo,80Sprague Dawley (SD) rats were randomly divided into8groups:normal blood sugar control (NMC) group, diabetes control (DMC) group, diabetes ischemic (DMI) group, Yiqihuoxue (YQHX) group, local injection BMSCs (L-BMSCs) group, L-BMSCs+YQHX group, acupoint injection BMSCS (Acu-BMSCs) group, and Acu-BMSCs+YQHX group,10rats per group. Establishing animal model of diabetic mellitus:50mg/kg of Streptozotocin (STZ) per rat was injected intraperitoneally. Ischemic model:arteria femoralis as well as all of collateral artery in the left hind limb of the rats were excised. The general state, level of blood sugar and weight were observed at the2nd,7th,14th and21th day after modeling. The change of blood flow in hind limb of the rats was detected by Laser Doppler Perfusion Imager (PeriScan PIM). Immunohisto-chemistry was used to detect the vascular density which signed by a-actin, platelet endothelial cell adhesion molecule-1(PECAM-1/CD31), and von Willebrand factor (vWF), and the expression of vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) in muscular tissue. Elisa method was used to detect the serum level of VEGF, G-CSF and SDF-1.Results:Most of the BMSCs obtained by all cells adherent culture method was elliptical and long-fusiform shape, and grow spirally in vitro. The membranes of third generation of BMSCs were positive for CD44and CD106and negative for CD45and CD34detected by flow cytometry. BMSCs cultured by various medicated serum increased significantly on the2nd,4th and5th day compared with the previous day. On the6th day, BMSCs proliferation by HX serum and YQHX serum began to decrease, on the other hand, the other three groups reached a peak proliferation, and decreased on the7th day. There was no significant difference among each group on the2nd day, but BMSCs cultured by HX serum increased the most. On the4th day, BMSCs cultured by YQHX serum was highest of all groups, and HX serum group was the second. On the6th day, BMSCs YQHX group was second then followed by HX group which was still higher than the control group.(2) Among different concentration of medicated serum, only10%YQHX serum cultured BMSCs began to increased significantly on the2nd day. BMSCs cultured by20%control and YQHX serum were higher than the10%FBS group. In vivo, there was87.14%rats induced diabetes. The DM rat model rat of DM was high and stable. The survival rate of ischemic rat model established by excising arterial femoral is as well as all of collateral artery in the left hind limb was88.52%in DM rats.(1) Ethological results:the rat models exhibited cachexia, pallid and pale limb, even turning dark purple, claudication and ulcer in some. The PUI of the DM rats was lower than the control group.(2) The PUI of ischemic hind limb of the rats in the Acu-BMSCs+YQHX group increased most obviously, second were Acu-BMSCs and L-BMSCs+YQHX group, and lastly the YQHX and Acu-BMSCs group.(3) The expression of a-actin and CD31in the ischemic limb were higher than the healthy limb in each group. The expression of VEGF and bFGF mRNA, a-actin and CD31increased, and VWF decreased in the Acu-BMSCs+YQHX group compared with other groups. The levels of VEGF, SDF-1and G-CSF in rat serum tested by ELISA of Acu-BMSCs and Acu-BMSCs+group were higher than other groups.Conclusion:The rat BMSCs cultured by all cells adherent culture method has features of BMSCs in cellular morphology and phenotypic expression.(1) The influence of HX serum on proliferation capacity of BMSCs appeared earliest of all. The influence of YQ serum on proliferation activation of BMSCs continued longest of all. The influence of YQHX serum on proliferous capacity of BMSCs was biggest of all. In vitro, the proliferous capacity of BMSCs was markedly increased obviously by10%concentration of YQHXsemn.(2) The blood flow of the ischemic hind limb of the DM rats improved efficiency and the expression of VEGF and bFGF increased by injecting BMSCs and YQHX medicine at acupuncture points. It also enhances the hyperplasia of vascular endothelial cells and vascular smooth muscle cells and prevents atherosclerosis.(3)The expression of VEGF, SDF-1and G-SGF in serum was increased in injection BMSCs group and Acu-BMSCs+YQHX medicine group. |
PDF Full Text Request