| Objective1.Establish rat model of combination of disease and syndrome based on theory of traditional Chinese medicine,which caused by endometriosis with Cold coagulation and blood stasis,using it to observe the change of appearance of ectopic endometrium and hemorheology and coag ul at ionin,pat ho morphism features of natral and ectopicand endometrium in different periods and determing the time of medication.2.To discuss the TCM pathogenesis of Endometriosis-associated Infertility that "lurking cold injures kidney and results in blood stasis damaging collaterals,that injures uterus meridians and results in infertility",detecting the different expressions of HOXA10?MLL1?Emx2 and av?3 in each group to discusse the relationship among the four indexs in Endometriosis-associated infertility.3.comparring the different expressions of HOXA10.MLL1 Emx2 and av?3 in each group to observe the effect of preventive intervention and therapeutic intervention of TONG YUSHU for endometrial receptivityof rat model caused by endometriosis.And then discuss the impact and mechanism of action of TONGYUSHU in treatment of Endometriosis-associated Infertility,and provide new ideas for clinical treatment.Methods1.PART 1:45 female SD rats were randomly divided into 3 groups:model group I haves 8 rats,model groupII haves 30 rats and blank control group haves 7 rats.The model groups were treated by autologous endometrial transplants and cold water immersion.The model groupI were separated in the model three days and five days after the surgery,4 rats were randomly selected to observe the rat appearance characterization,lesion appearance and ectopic endometrial pathology observation.30 rats in group II were separated in the model of four,six and eight weeks after the surgery,9 rats were randomly selected to observe the rat appearance characterization,lesion appearance and ectopic endometrial pathology observation.The rats in the comparison group were directly observe the pathology of right segment of uterine.Except for group I,all the rats were taken for blood rheology and blood coagulation function test by drawing blood from Abdominal aorta.2.PART 2:150 virgin female rats were randomly divided into two groups:18 rats in blank control group and the others are model group.4 weeks after the surgery,the model group are opened abdomen,was performed out that 108 successful model rats were randomly divided into model groups:4 weeks and 8 weeks model group,Low amount of drug group,normal amount of drug group,high amount of drug group and medicine group.There were 18 rats in each group.Model group in 4 weeks are mating in accordance with the ratio of male and female rats2:1.Mark them and kill them in the fourth days after pregnant.Each TCM group is treated according to the corresponding amount of drug,mark them after they mated,kill them in the fourth days after pregnant;medicine group were treated four weeks,stop medication for a week,then mark them after they mated,kill them in the fourth days after pregnant;model group in 8 weeks and the blank control group,after saline gavage for four weeks,mark them after they mated,kill them in the fourth days after pregnant.all the rats were taken for blood rheology and blood coagulation function test by drawing blood from Abdominal aorta.Observr pathology of the endometrium from two model groups and the treatment groups;the blank control group were directly observe the pathology of right segment of uterine.observed pinopode under scanning electron microscope,from RT-PCR and Western Blot observe the expression of MLLlmRNA and protein,HOXA10mRNA and protein;detection the expression of MLL1,HOXA10,Emx2 and av?3 by immunohistochemistry,determining the optimum treatment dosage of Tongyushu.3.PART 3:80 virgin female rats were randomly divided into two groups:18 rats in blank control group and the others are model group.Rats in model group were opened abdomen randomly in fifth days after laparotomy,1 8 successful model rats are preventive medication group.Using optimal dose of Tongyushu orally,one week after the operation began to soak in ice water,for 4 weeks,then mark them after they mated,kill them in the fourth days after pregnant;model group in 8 weeks and the blank control group,after saline gavage for four weeks,mark them after they mated,kill them in the fourth days after pregnant.All the rats had the same test items with test two.Results1.The results of PART 1:the successful rate of rat model was 76.7%.The pathology shows that lesions appear serious edema in the third days,the interstitial cells reduced,in fifth days edema ease and interstitial cells increaced,structure became loose;four weeks later typical endometrial morphology has been presented;sixth weeks and eight weeks made pathological model around as four weeks.The model groups showed the appearence caused by cold and unnomarl performance in blood,but there was no significant difference in blood between the model groups.The fifth days after surgry is determined for preventive medication.2.The results of PART2:the successful rate of rat model was87.9%.After two weeks of treatment,the appearance and mental state caused by cold coagulation and blood stasis were improved,and the state of the rats in the middle and high amount of drug were significantly improved.The change was not obvious in the medical group and the weight of the rats was clearly increased.The treatment group reduced more lesion volume than the four and eight weeks,endometrium pathological observation showed different changes.The endometrial structure gradually became continuous and complete,and the number of abnormal uterine glands decreased,and the structure of the submucosal layer of the uterus was restored.The changes of high and middel amount group of TCM are more significant.Compare the model group and the treatment group with the blank control group,RT-PCR and WB results shows MLL1mRNA and protein significantly decreased,the expression of MLL1 in model 8 weeks were significantly lower than that in model group 4 weeks,expression in each treatment group were significantly higher than the model,in the expression of high group increased the most obviously,the expression of MLLlmRNA protein and its expression trend is consistent with the.HOXA10mRNA and protein of the same with MLL1.Immunohistochemistry results showed that compared with the control group,MLL1 model group and treatment group,the expression of HOXA10 and integrin alpha av?3 was significantly decreased,Emx2 expression was significantly increased;the expression of the first three in the 8 weeks model group Below the model 4 weeks group,Emx2 expression was higher than the model group in the 4 week.The expression of in the treatment group was significantly higher than that in the model group,the expression of Emx2 was lower than that of the model group,and the expression of the middle and high group was the most significant.Spearman analysis showed that the expression of MLL1,HOXA10 and integrin av?3 in eutopic endometrium of endometriosis rats were positively correlated,and the expression was negatively correlated with the expression of Emx2.3.The results of PART 3:the successful rate of rat model was88.7%.There was not a typical apparence caused by cold in the model group.Prevention group and high amount group reduced significantly more lesion volume than model 4 weeks,and the adhesion relieved.prevention group were more remarkable.Two treatment group endometrium pathological changes appeared in different degrees.compared with high group,the preventive medication group was more obvious healing effect on endometrium.Blood rheology and coagulation the function test result sshows no significant difference between Two treatment group.RT-PCR and WB results showed that compared with the blank control group,the expression of MLLlmRNA and protein in the model group and two treatment groups significantly decreased,but the expression in the prevention group was higher than that in the high group.HOXA10mRNA and its protein is consistent with MLL1.Immunohistochemistry results showed that compared with the blank control group,in model group and treatment group,the expression of MLL1.HOXA10 and integrin av?3 was significantly decreased,Emx2 expression was significantly increased;the expression of the first three in the prevention group was significantly higher than that in the high group.Spearman analysis showed that the expression of Emx2,MLL1,HOXA10 and integrin av?3 in rats with endometriosis eutopic endometrium,the expression of he former three is negatively related to and Emx2.Conclusion1.The rat model of endometriosis caused by cold coagulation and blood stasis can be established successfully by the combination of autologous endometrial transplantation and cold water immersion.The method is Stable and reliable.2.The TCM pathogenesis of endometriosis-associated infertility is“lurking cold injures kidney and results in blood stasis damaging Research from" endometrial receptivity ".We can combine the two parts in order to discuss and seek medical treatment targets.3.MLL1mRNA and protein in rat model of endometriosis eutopic endometrium was significantly lower than that in normal uterine endometrium.The expression of MLL1 in eutopic endometrium was positively correlated with HOXA10 and av?3,negatively with Emx2.Abnormal expression of MLL1 may be involved in the pathogenesis ofendometriosis-associated infertility.We can speculated that MLL1 may affect the development of endometriosis-associated infertility in this method,and the molecular markers can be used as an index to predict the development of disease and the therapeutic effect.4.Tongyushu can significantly increase the expression of MLL1?HOXA10and integrin av?3 in eutopic endometrium of endometriosis,decrease the expression of Emx2,can improve the blood rheology,coagulation function and apparence caused by cold,no obvious side effects,and can save time of pregnant.Compared with danazuo,it has obvious advantages.5.Preventive administration is better than treatment to improve uterine receptivity inendometriosis-associated infertility.It is significant to improve the pregnancy rate after Laparoscope. |
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