| Background and AimsAlthouth in patients with ischemic heart disease,using conventional drugs, percutaneous coronary intervention,coronary artery bypass graft can significantly reduced mortality and morbility,there are still 5%-10%of patients should not be part of the selection of these methods.Explore new ways and means of treatment is particularly important.In past 10 years,animal experiments and clinical trials have indicated that therapeutic angiogenesis treatment is a more promising new method of cardiovascular diseases for therapeutic angiogenesis can increase collateral perfusion of ischemic myocardium.Angiogenesis is the biological process of growing new blood vessels baseing on the original structure of the blood vessels.Except for vascular endothelial growth factor,fibroblast growth factor and many other factors,animal experiments have already seen that estrogen may also accelerate endothelial cell migration and proliferation,and thus strengthen the effect of angiogenesis.However,the effect of estrogen on endothelial cell was investigated on the cells of large vessel such as human umbilical vascular or arteriae aorta.It is reported that cells originated from different location showed somewhat alterations in function eventhough they are in same category.In this paper,we investigate the effect of estrogen on rat cardiac microvascular endothelial cell in vitro through observing the 17β-E2 on proliferation,migration and tube-like structure formation,and we furtherly elucidate the mechanism that involved in these phenomenon,MethodsPartâ… :CMEC culture and biological surface marker identification.1.Left ventricular of SD female rats,was soaked in 75%ethanol 20s to remove endocardial endothelial cells of Endocardium and Epicardial.Using density gradient centrifugation and cell adhesion method,rat cardiac microvascular endothelial cells were isolated and cultured at 15%fetal calf serum DMEM medium.2.Identify cell phenotypes through immunofluorescence double staining.Partâ…¡:Investigating whether estrogen receptor expresses on CMEC by Immunofluorescence method.Partâ…¢:Study the effects of 17β-E2 on CMEC.1.After gaven different concentrations of 17β-estradiol,cell proliferation capacity was detected by MTT and the best concentration was selected.2.With the best concentration of 17β-estradiol and 17β-estradiol antagonist tamoxifen, following experiments was carried out:2.1.Using cell transfer experiments,the ability of 17β-E2 on cell migration was detected;2.2.Cell invasion assay detected the invasive capacity of 17β-E2 on cells.2.3.Tube-like structure formation assay detected the impact of 17β-E2 on cell differentiation;2.4.ELISA test observed the effects of 17β-E2 on VEGF secretion of myocardial microvascular endothelial cells.Statistical analysis was performed with SPSS version 12.0.One-way analysis of variation and post hoct(LSD-t) test were employed.Results1.Immunofluorescence cell staining results showed that:VW factor stained positive,and DAL phagocytosis experiments showed positive results.The resultes confirmed that the obtained cells are cardiac microvascular endothelial cells.2.Immunofluorescence analysis:negative control group is not fluorescent, while the positive control group was observed a positive fluorescent marker. Fluorescence microscopy showed that ER protein was expressed in CMECs.3.Compared with control group,17β-estradiol significantly promoted proliferation of rat cardiac microvascular endothelial cell at the concentration from 0.001 to 1μmol/L(P＜0.05),and in the concentration of 0.01μmol/L the promotion is at highest;But in 17β-estradiol + tamoxifen treatment group, there are no significant difference with control group(P＞0.05),so no effect on the proliferation of CMEC.4.Scratch assay studied the effect of 17β-E2 on cell migration.Compared with control group,the relative scratch area significantly reduced after cultured with 17β-E2(0.01μmol/ L)(cell migration rate was 53%P＜0.05).However, the relative scratch area showed no significant difference in 17β-estradiol + tamoxifen intervention group(P＞0.05).5.The effect of 17β-estradiol on cell invasive capacity was detected by Millicell chamber assay.17β-estradiol induced a large extent CMECs penetrating through cicroporous membrane from upper to lower.Compared with control group(P＜0.05,compared with control group),and 17β-estradiol antagonist tamoxifen blocked this effect.6.Compared with control group,the number of tube-like structure increased in the 17β-E2 group,and tubule spacing and weave mesh was reduced significantly.Resultes quantified by image analysis were as follows:at the control group,the average number of tubular branching points were23/vision at the 17β-E2 group(P＜0.05 compared with control group,12/vision);and, 12/vision at the 17β-E2 + tamoxifen group(P＞0.05,compared with control group).7.17β-estradiol(0.01μmol/L) significantly promoted VEGF secretion of rat cardiac microvascular endothelial cells(P＜0.05 vs.control group),very low concentrations of VEGF were detected in groups of control and 17β-estradiol + tamoxifen,and there were no significant difference between two groups.Conclusion1.Obtained cells were identified as cardiac microvascular endothelial cells.2.ER protein was expressed in CMECs.3.MTT assay show that 17β-E2 significantly promoted the proliferation of CMECs.And at the concentration of 0.01μmol/L,17β-E2 has the greatest effect.However,17β-estradiol antagonist tamoxifen blocked this effect.4.Cultured with 17β-E2(0.01μmol/L),the abilities of migration and tube-like structure formation of CMECs were significantly elevated.17β-estradiol antagonist tamoxifen blocked these effects.5.At the concentration of 0.01μmol/L,17β-E2 significantly promoted VEGF secretion of rat cardiac microvascular endothelial cells.
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