Effects Of Bushen Antai Granule On Proliferation And Migration And Differentiation Of EPCs From Bone Marrow In Rats And Clinical Observation On Treatment Of Kidney Deficiency Type Early Threatened Abortion By Bushen Antai Granule

Fart Ⅰ experimental researchObjective:To observe the effects of different concentrations of serum of Bushen Anfeng Granule on the proliferation activity,migration ability and differentiation of rat bone marrow endothelial progenitor cells(EPCs)to VECs(vascular endothelial cells).To explore the effects of traditional Chinese medicine on angiogenesis from the level of cell differentiation,and to reveal the scientific connotation of traditional Chinese medicine theory of "kidney-dominant reproduction" and "kidney-dominant bone marrow generation.Methods:Identffication of separation of cultured cells of rat bone marrow-derived EPCs:first of all take 3-4 weeks SD rats(female)5,only using density gradient centrifugatioin separation mononuclear cells,and then collect training 7 d cells,EPCs specific surface antigen by immunofluprescence method to 34 people leukocyte differentiation antigen(CD34),vascular endothelial growth factor receptor 2(VEGFR-2,also called KDR+),human leukocyte differentiation antigen 133(CD133)to flow cytometry instrument testing,line Dil markers of acetylated LDL(Dil-AC-LDC)and fluorescein isothiocyanate)Whin lectin-1(FITC-UEA-1)double intake experiment,the identification of EPCs.In accordance with 23.4 g/kg.D preparation of traditional Chinese medicine dose kidney drug serum fetus infusion of traditional Chinese medicine.Using tetramethyl azo salt azole trace enzyme reaction colorimetry(determined by MTT method)to detect different concentrations(5%,10%,15%,20%)and in different time(24 h,48 h,72 h)under the intervention of EPCs cell proliferation activity.Through the cell migration experiment technology(Transwell method)to detect the EPCs migration ability,timing,quantitative PCR(Real-time PCR)The differentiation of EPCs to VECs was detected.Results:1 The dentification of EPCs1.1 Cell immunofluorescence detectionDAPI stain out of the nucleus under the excitation of ultraviolet to blue,cell surface antigen CD34 specificity,VEGFR-2,the expression of CD133 positive for the corresponding fluorescence labeled red or green Most visible cultivate EPCs specificity of surface antigen CD34,VEGFR-2,both CD133 positive expression,7 days positive rate were significantly increased,the difference was statistically significant(P<0.05)1.2 Flow cytometryAfter EPCs were cultured for 7 days,the expression of cell surface markers CD34,CD133 and VEGFR on the 7th day of culture was analyzed by flow cytometry,as shown in Figure 2.The results showed that CD34 positive cells accounted for 94.7%and CD133 positive cells accounted for 93.8%.The number of VEGFR-positive cells was 98.8%,and the difference was statistically significant(P<0.05),suggesting that the cells were EPCs.2.Proliferative activity of EPCs was detectedThe results showed that the absorbance values of blank control group at 24h,48h and 72h were(0.034±0.003),(0.026±0.002)and(0.029±0.005),respectively.The absorbance values of the negative control group at 24h,48h and 72h were(0.404±0.017),(0.569±0.033)and(0.546±0.025),respectively.The OD(490)values of the experimental group containing 5%drug serum at 24h,48h and 72h were(0.526±0.018),(0.768±0.015)and(0.675±0.051),respectively.The OD(490)values at 24h,48h and 72h of the 10%drug senum group were(0.532±0.014),(0.843±0.023)and(0.770±0.034),respectively,and the OD(490)values at 24h,48h and 72h of the 15%drug serum group were(0.482±0.039),(0.073±0.025)and(0.705±0.013),respectively.TheOD(490)values of 20%serum in the experimental group at 24h,48h and 72h were(0.381±0.025),(0.458±0.034)and(0.454±0.095),respectively.The absorbance value of the negative control group and each concentration of drug-containing serum group was significantly higher than that of the blank group,and the difference was statistically significant(P<0.01).The proliferation activity of bone marrow derived EPCs of rats with different concentrations of Bushen Anfei Granule in vitro was statistically significant.3.Migration ability test of EPCsTranswell assay showed that the average number of migrated cells per high-power field was(51.0±7.94)in the control group and(105.5±15.22)in the drug serum group,respectively.Compared with the blank control group,the migration ability of EPCs in the drug serum group was significantly increased(P<0.01).4.Differentiation of EPCs to VECsRNA extraction of endothelial progenitor cells,Real-timePCR primer design,through the Real-timePCR detection,and compared with the blank serum,serum drug group of vascular endothelial growth factor(VEGF)and vascular pseudo willebrand factor(vWF)the relative expression quantity(2-delta delta CT)were significantly increased,the drug-containing serum biomarkers VEGF treatment can significantly induce angiogenesis and the expression of vWF.(P<0.01,significant statistical difference.)drug serum vWF set of relative expression is higher,the more significant difference(P<0.01).Conclusion:1.The cultured cells were identified as rat bone marrow endothelial progenitor cells by immunofluorescence and flow cytometry.2.Bushen Antai Granule can enbance the proliferation activity and migration ability of EPCs,and the influence degree may be related to the concentration.The proliferation rate of serum containing 10%drug reached the highest value at 48h.3.Bushen Antai Granule can significantly promote the differentiation function of EPC to VECs,and play a role by stimulating the expression of VEGF and VWF.Part Ⅱ clinical researchObjective:With oral kidney fetus infusion of joint flexion progesterone and individual oral flexor progesterone therapy in kidney type of the early threatened abortion randomized controlled clinical research,evaluation of kidney fetus granule in improving the TCM syndrome type kidney early threatened abortion patients,serum progesterone(P),estradiol(E2)value,VEGF expression level of clinical curative effect,provide data support for spuc advantages of Chinese medicine.Methods:Collected between March 2019 and March 2020 in The First Affiliated Hospital of Anhui University of Chinese Medicine outpatient service of department of gynaecology of kidney patients with 60 cases of early threatened abortion,according to the proportion of 1:1 randomly divided into control group(30 cases)and observation group(30 cases)in the two groups.The control group with oral flexor progesterone,observation group with oral kidney fetus granule+flexor progesterone combined treatment,treatment for two weeks(14 days).Using the software IBMSPSS25.0 to deal with the data analysis,by comparing the two groups of patients before and after medication in TCM syndrome integral,serum P and E2 value changes,the level of VEGF expression,evaluating kidney fetus granule Clinical effect of combination with dydrogesterone on early threatened abortion of kidney deficiency type.Results:1.Comparison of clinical comprehensive efficacy:the total effective rates of the observation group(Bushen Anfeng granule combined with dydrogesterone)and the control group(dydrogesterone)were 96.67%and 90%,respectively,P<0.05,indicating that both groups had good therapeutic effects,and the total efficacy of the observation group was better than that of the control group.2.TCM syndrome score:Compared with before treatment,TCM syndrome score of the observation group and the control group decreased significantly after treatment,P<0.01,the difference was statistically significant;The comparison of TCM syndrome scores between the two groups after treatment was statistically significant,P<0.01,indicating that both the observation group(Bushen Anfeng granule combined with dydrogesterone)and the control group(dydrogesterone)could effectively improve the clinical syndromes in the treatment of threatened abortion,and the observation group was better than the control group.3.Efficacy of TCM syndromes:Comparison of TCM syndromes efficacy between the two groups after treatment(P=0.035,P<0.05)showed statistically significant differences.The total effective rate of the observation group was 93.33%,while the total effective rate of the control group was 86.67%,indicating that the observation group had better efficacy of TCM syndromes than the control group.4.Serum levels of E2 and E2:Serum levels of E2 and E2 in the observation group and the control group were significantly increased after treatment,compared with before treatment(P＜0.05),the difference was statistically significant;After treatment,P was less than 0.01,the difference was statistically significant,indicating that both groups could effectively improve serum P and E2 levels,and the observation group was better than the control group.5.Peripheral blood VEGF level:After treatment,the VEGF level in peripheral blood of both the observation group and the control group was significantly increased,compared with that before treatment,P<0.05,the difference was statistically significant.After treatment,the comparison between the two groups was P<0.01,the difference was statistically significant,indicating that both groups could effectively improve the level of VEGF in peripheral blood,and the observation group was better than the control group.Conclusion:1.Bushen Antai Granule combined with ddrogesterone has a clear clinical comprehensive effect on the treatment of early threatened abortion,and is better than ddrogesterone alone.2.Bushen Antai Granule combined with dydrogesterone can significantly improve the syndrome of kidney deficiency in patients.3.Bushen Antai Granule combined with dydrogesterone can increase serum P and E2 levels,which is obviously better than that of single use The treatment of dydrogesterone is worth popularizing.4.Bushen Anfetal Granule combined with dydrogesterone can up-regulate VEGF level,contribute to the angiogenesis of blood vessels,thereby correcting the maternal-fetal interface angiogenesis disorder and changing the outcome of pregnancy.