Experimental Study Of Rhodiola On High Glucose In Rats MMVECs And VEGF

[Objective]1. To cuture high purity MMVECs in vitro by digestion method.2. To observe Rhodiola containing medicated serum on high glucose in rats MMVECs and VEGF content and expression,in contrast with Perindopril containing medicated serum.[Methods]To open the newborn 5-7 days Wistar rats breast and draw matierial,repeatedly wash them 3 times in cold D-Hanks solution,use twice digestion method(digest in 0.25% trysin and type II collagenase) to separate and culture MMVECs,purify cells in differential attachment and sub-cell cloning method,identify cells by factorâ…§and CD31 related antigen.The 3rd passage MMVECs were cultured respectively in following conditions including DMEM, high glucose(50mmol/L) media supplemented with 5% rat serum, 50mmol/L glucose media containing medicated serum of NDK and PD. DMEM served as negative control. After 72h, MTT colorimetric assay was adopted to measure MMVECs proliferation. Furthermore,VEGF secretory volume of MMVECs' supernate fluid, VEGF-mRNA expression level were detected by ELISA,QT-PCR.[Results]1. Cultured high purified MMVECs successfully and it can be used in the experiment study.2. MTT colorimetric assay:MMVECs isolated from newborn rats primary cultured in different medium. The OD values of 72h group increased significantly compared with those of 48h group (P<0.05).The OD values of high glucose group diminished significantly compared with those of normal group at 72h group,especially 50mmol/L and 75mmol/L group(P<0.01). The OD values of NDK and PD group increased greatly compared with 50mMGlu group (P<0.05) at 72h and there were no significant differences between these two treated groups(P>0.05).3. Transmission electron microscopy observation:MMVECs emerges digitationes, increased cytolysosome, engorged chondriosome, broaden rough endoplasmic reticulum in high glucose.NDK low dose group and PD group can improve the damage.4. ELISA detection:The VEGF secretory volume in the supernate fluid of high glucose group diminished significantly compared with those of control group (P<0.01). The VEGF secretory volume in the supernate fluid of NDK middle and low dose group and PD group increased greatly compared with high Glu group (P<0.05, P<0.01),there were significant differences between NDK low dose group and PD group(P<0.05).5. QT-PCR detection:The VEGFmRNA expression level in MMVECs of high glucose group,NDK high dose group,NDK middle dose group, NDK low dose group and PD group were diminished significantly compared with those of control group (P<0.01). The VEGFmRNA expression level of NDK low dose group increased greatly compared with 50mmol/L Glu group (P<0.01),and PD group also increased,but no significant differences.There were significant differences between NDK low dose group and PD group.[Conclusion]1. The primary culture methods of MMVECs, after twice digestion and two purify methods,and two identification, are proved to be successful. It can be used in the formal experiment.2. High glucose inhibited the proliferation of MMVECs.NDK low dose group can increase the proliferation,similar to PD group.3. Transmission electron microscopy can observe the injury of MMVECs.NDK low group can improve the damage, precede to PD group. 4. High glucose depressed the secretory volume of VEGF in the supernate fluid of MMVECs,its mRNA expression level in MMVECs obviously.5. NDK low dose group can increase the VEGF secretory volume of MMVECs'supernate fluid and VEGF-mRNA protein expression level of MMVECs,partly precede to PD group.[Innovation]To study the effects of medicated serum of NDK on the role of proliferation and elaborating VEGF of MMVECs cultured in high glucose from the aspects of integral level, cellular level and molecular level, which hasn't been reported home and abroad,compare with PD medicated serum. This research can provide the basic theory and experimental foundation for the application of NDK to clinical treatment of diabetic cardiomyopathy.