| Object1.To observe the clinical efficacy of traditional Chinese medicine for tonifying kidney and activating blood in the treatment of patients with thin endometrium infertility,so as to provide an effective method for the treatment of thin endometrium.2.In vitro experiments were conducted to observe the changes in cell proliferation and apoptosis rate of endometrial stem cells(EDSCs)treated with traditional Chinese medicine for tonifying kidney and promoting blood circulation.The regulatory effect of Wnt/?-catenin signal pathway in thin endometrium and the effect of traditional Chinese medicine for tonifying kidney and activating blood on EDSCs markers after blocking Wnt/?-cateninsignal pathway were analyzed to further analyze the mechanism of traditional Chinese medicine for tonifying kidney and activating blood in the treatment of thin endometrium.3.After establishing the thin endometrium model of kidney deficiency and blood stasis in rats through in vivo experiments,the effects of traditional Chinese medicine for tonifying kidney and activating blood on endometrium receptivity,Wnt/?-catenin signal pathway key factor ?-catenin protein and endometrium stem cell markers in rats were observed,providing a scientific basis for the clinical application of traditional Chinese medicine for tonifying kidney and activating blood.Methods1.A total of 70 patients with thin endometriosis admitted to the traditional Chinese medicine department of Beijing maternity hospital were selected and divided into the treatment group(bushenhuoxue&prognova)and the control group(prognova)by random number table method.Control group:started to take bprognova on the 5th day of the menstrual cycle,once a day,each time 2 mg,for 21 consecutive days.Treatment group:on the basis of treatment measures in the control group at the same time with traditional Chinese medicine tonifying kidney and activating blood,the dominant follicle side out when the drug.The menstrual volume,TCM syndrome integral,endometrial thickness,endometrial type,uterine artery pulse index,uterine artery resistance index and clinical pregnancy of the two groups were observed before and after treatment.2.Selected regions in Beijing maternity hospital thin endometrium of patients,after taking the endometrium EDSCs,were isolated and cultured in serum with the kidney invigorate the circulation of Chinese traditional medicine drug intervention,the EDSCs proliferation force determined by MTT method and flow cytometry instrument method detecting EDSCs apoptosis rate,the method of the immunized group Wnt/?-catenin signaling pathways critical factor beta catenin expression in EDSCs and TCF dominant negative mutant(dnTCF)expression plasmid transfection EDSCs blocking Wnt/?-cateninsignaltransduction,The changes of CD34/CD117,oct-4 and ABCG2 were detected by immunofluorescence assay,q-pcr and Westernblot.3.The rat endometrium model with kidney deficiency and blood stasis was established.After the effect of traditional Chinese medicine of tonifying kidney and promoting blood circulation,HE staining was used to observe the effects of endometrial thickness,endometrial stromal thickness,endometrial gland number,endometrial blood vessel number,epithelial cells and mesenchymal cells.Immunohistochemical method was used to detect the effect of traditional Chinese medicine for tonifying kidney and activating blood on the expression of Wnt/?-cateninprotein,a key factor in the Wnt/?-catenin signal pathway,as well as the effect of q-pcr and Westernblot on the expression of oct-4 and ABCG2,markers of endometrial stem cells.Results1.There was no significant difference between the treatment group and the control group(P>0.05)in general data such as age,education level and number of qing dynasty visits.Changes in menstrual volume:there were significant differences between the treatment group and the control group before and after treatment(P<0.01).Compared with the control group after treatment,the improvement of menstrual blood loss graph score in the treatment group was significantly better than that in the control group(P<0.05).TCM syndrome points:there was a significant difference between the treatment group and the control group before and after treatment(P<0.05),and the improvement of TCM syndrome points in the treatment group was significantly better than that in the control group(P<0.05).Improvement of endometrial thickness:there was a significant difference between the treatment group and the control group before and after treatment(P<0.05),and the improvement of endometrial thickness in the treatment group was significantly better than that in the control group(P<0.05).Proportion of type A endometrium:there was A significant difference between the treatment group and the control group before and after treatment(P<0.05),and the improvement of the proportion of type A endometrium in the treatment group was significantly better than that in the control group(P<0.05).Uterine arterial pulse index PI:there was a significant difference between the treatment group and the control group(P<0.05).Uterine arterial resistance index(RI):there was a significant difference between the treatment group and the control group(P<0.05),and the reduction degree of PI and RI in the treatment group was significantly higher than that in the control group(P<0.05).Clinical pregnancy:after 3 menstrual cycles of treatment,the clinical pregnancy rate of the treatment group was higher than that of the control group(P<0.05).2.In vitro experiment:EDSCs proliferation:the results showed that there were significant differences between the administration group and the control group(P<0.01).Cell proliferation under inverted microscope and fluorescence microscope showed that the serum of traditional Chinese medicine for tonifying kidney and activating blood could increase the proliferation of EDSCs.Apoptosis of EDSCs:transmission electron microscopy and flow cytometry showed that the serum of traditional Chinese medicine can reduce the apoptosis of EDSCs.Expression of ?-catenin in EDSCs:there were significant differences between the high-dose and bujialue groups compared with the control group(P<0.01).Transcriptional activity OF ?-catenin/TCF in EDSCs:transfected with different plasmids,there were significant differences between transfected with OF and transfected with OT in the same group(P<0.05).After OT transfection,there were significant differences between the medium and high dose groups and the bujialue group and the control group(P<0.05).Expression of CD34/CD117 protein in EDSCs:CD34 protein:there was a significant difference between the medium dose and the control group(P<0.05),while there was a significant difference between the high dose and prognova group(P<0.01).CD117 protein:medium dose and high dose showed significant difference compared with the control group(P<0.05),and prognovagroup showed significant difference compared with the control group(P<.01).Oct-4 gene expression in EDSCs:there was a significant difference between the administration group and the control group(P<0.05).ABCG2 protein expression in EDSCs:there were significant differences between the administration group and the control group(P<0.05).CD34/CD117,oct-4 and ABCG2 were all decreased after blocking Wnt/?-catenin signal pathway,with significant difference(P<0.05).3.In vivo experiment:endometrial thickness:the endometrial thickness of the model group was lower than that of the control group,with a very significant difference(P<0.001);Endometrial thickness of rats in low dose group,medium dose group,high dose group and prognova group was higher than that in model group,with significant difference(P<0.05).Uterine interstitial thickness:the model group was significantly lower than the control group(P<0.001).The interstitial thickness of rats in the low dose group,medium dose group,high dose group and prognova group was higher than that in the model group,with significant difference(P<0.001).The number of uterine glands in the model group was less than that in the control group(P<0.001).The number of uterine glands in rats in the medium dose,high dose and bujialue group was higher than that in the model group,with significant difference(P<0.001).Number of uterine blood vessels:the number of uterine blood vessels in the model group was less than that in the control group,showing a significant difference(P<0.001).The number of uterine vessels in the medium dose,high dose and bujialue group was higher than that in the model group(P<0.05).Endometrial epithelial cell area:the area of endometrial epithelial cells in the model group was smaller than that in the control group,with a significant difference(P<0.001).The area of rat endometrial epithelial cells in the medium dose,high dose and bujialue group was larger than that in the model group,with significant difference(P<0.05).Endometrial stromal cell area:the area of endometrial stromal cells in the model group was smaller than that in the control group,with a significant difference(P<0.001).The values of low dose group,medium dose group,high dose group and prognova group were significantly higher than those of model group(P<0.001).?-catenin protein:the model group was significantly lower than the control group(P<0.001);?-catenin increased in different degrees in each group after administration.There were significant differences between the low-dose group and the model group(P<0.05).There were significant differences between medium dose group,high dose group,bujialue group and model group(P<0.001).Oct gene:the model group was significantly lower than the control group(P<0.001).OCT4 expression was increased in each group after administration.There was no significant difference between low dose,medium dose and model group.There were significant differences between the high dose group and the model group(P<0.001).There were significant differences between prognova group and model group(P<0.05).ABCG2 protein:the model group was significantly lower than the control group(P<0.001).The expression level of ABCG2 increased in each group after administration.There was no significant difference between the low dose group and the model group.There were significant differences between the medium dose group and the model group(P<0.05).At high dose,there was significant difference between prognova group and model group(P<0.001).Conclution1.Combined treatment with kidney-tonifying and blood-activating traditional Chinese medicine can increase the menstrual volume of patients with thin endometriosis,improve the symptoms of kidney deficiency and blood stasis,increase the thickness of endometrium,increase the proportion of type A endometrium,reduce the uterine artery beat and resistance index,and improve the clinical pregnancy rate.2.The serum of traditional Chinese medicine for tonifying kidney and activating blood can increase the proliferation of EDSCs,reduce apoptosis,promote the nuclear transcription mediated by ?-catenin/TCF in EDSCs,increase the expression of CD34/CD117,oct-4 and ABCG2 in EDSCs markers,and reduce the expression of CD34/CD117,oct-4 and ABCG2 in different degrees after blocking the Wnt/?-catenin signal pathway.3.Kidney-tonifying and blood-activating herbs can increase the endometrial thickness,endometrial stromal thickness,endometrial gland number,endometrial blood vessel number,endometrial epithelial cell and interstitial cell area,the expression of-catenin protein,Oct4 gene and ABCG2 protein in rat endometrial model with kidney deficiency and blood stasis. |
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