| Fertilized egg implantation outside the uterine cavity called ectopic pregnancy(EP),commonly known as extrauterine pregnancy.EP is a common gynecologic acute abdomen,the incidence rate of about 1 to 2%.It is still one of the main causes of morbidity and mortality in the early pregnant women.The tubal pregnancy is the most common type,which about 98%occur in the fallopian tube.In recent years,with the booming development of assisted reproductive technology(ART),the common complications of ectopic pregnancy also increased.It lead to a serious life and health threaten to women of childbearing age.The development of modern medical technology makes ectopic pregnancy to be able to detect at an early stage.The opportunity of non-surgical treatment has increased.More patients with fertility needs have opportunities to preserve reproductive function.Ancient Chinese medicine book didn' t have the name of ectopic pregnancy records.According to the clinical manifestations,it can be seen in the name of disease such as" Abdominal pain during pregnancy ","colporrhagia","colporrhagia during pregnancy","Lower abdominal congestion ","Zheng Jia"and etc.Modern Chinese medicine using the disease name of ectopic pregnancy or extrauterine pregnancy in the book of "Chinese Medical Encyclopedia ·Chinese Gynecology" TCM theory holds that the pathogenesis of ectopic pregnancy are connected with following causes:lower abdominal stasis,Chong and Ren Meridian stagnant,uterine meridian and collateral stagnant,or congenital kidney deficiency,acquired spleen impairment.The essential pathogenesis is the lower abdominal blood stasis.Using blood stasis elimination and embryos killing formula for treatment of early tubal pregnancy is effective.The blood stasis elimination formula is composed of salvia miltiorrhiza,radix paeoniae rubra,peach kernel,Radix trichosanthis and lithospermum according to traditional Chinese medicine theory.Among them,salvia miltiorrhiza,radix paeoniae rubra and peach kernel have the main effect of promoting blood circulation and dispersing blood stasis.Radix trichosanthis and lithospermum have the auxiliary effect of promoting blood circulation,dispersing blood stasis and killing embryos.This formula has definite clinical effect with simplification ingredients,and Maximum saved the patient's fertility.1.Clinical researchObjectiveBased on the previous research of tubal pregnancy Integrative Medicine program,using the retrospective survey study on the efficacy and clinical advantages of tubal pregnancy Integrative Medicine program from clinical applications to further optimize and improve this treatment program.MethodsRetrospectively collected tubal pregnancy cases with drug treatment in January 2012 to December 2015.All cases were grouped in accordance with the scope of Western medicine conservative treatment and divided into the conventional group and beyond group.Through the analysis of ?-HCG value in each group on the effects of treatment outcome and the contrast between the two groups of efficient,to discuss the TCM clinical advantages of tubal pregnancy Integrative Medicine program and optimize the treatment program.Results1)Included in cases:According to the diagnostic criteria,inclusion criteria and exclusion criteria,from January 2012 to December 2015 were collected 664 cases in line with tubal pregnancy Integrative Medicine program,wherein the conventional group 528 cases,beyond group 136 cases.2)The total effective rate of beyond group was 62.5%;while the conventional group was 77.8%.The efficient differences between two groups were statistically significant(P<0.05).In which all conservative treatment ineffective patients with surgical treatment were successful,and clinical cured.Thus,scope of indications by tubal pregnancy Integrative Medicine program is wider than scope of indications by Western medicine conservative treatment.This treatment program can reduce the surgical probability of more than 60%,and provides patients with more opportunities to give birth again.3)In the beyond group,according to serum ?-HCG value comparison,HCG value<2000 effective rate was 85.9%;HCG value s from 2000 to 5000 effective rate was 45.5%;HCG value s from 5000 to 8000 effective rate was 44.4%%;HCG value>8000 a total of 8 cases,only 1 case of effective,efficient was 12.5%.When HCG values over 8000,ruptured risk of tubal pregnancy is extremely high,and the present study only 1 case was valid.Based on clinical safety and efficacy,it is recommended to optimize the current Integrative therapy program,that is,when the first ?-HCG value is greater than 8000IU/L,no matter how much the disease impact factor scores are recommended surgery.2.Experimental researchObjectiveThis study is intended to using Blood stasis elimination formula as an object,aiming at the important pathological link of early tubal pregnancy,using trophoblast cells cultured in vitro for the study of carriers,using the orthogonal design table L18(35)for compatibility study,combining the orthogonal design and multiple objective index analysis,integrated analyse the Chinese materia medica,pharmacology,molecular biology techniques and other means,systematically study the efficacy of Blood stasis elimination formula,elaborate its compatibility law of drug composition,and find the optimal combination dose(optimize the prescription).Methods1)Human gestational trophoblastic cell line HTR-8/SVneo cells were cultured.Cells were identified by immunofluorescence staining method.Using CCK8 reagent and the flow cytometry to assay the proliferation and apoptosis of Human gestational trophoblastic cell line HTR-8/SVneo cells after cultured 48h with different extraction of Blood stasis elimination formula(group A-water extraction,group B-alcohol extraction,group C-alcohol extraction after water extracted,group D-alcohol extraction and water extraction).Setting methotrexate group as the positive control group,without medication cells as blank control group.2)Using the orthogonal table L18(35)for compatibility study.Respectively using flow cytometry and RT-qPCR method to assay the effect of gestational trophoblastic cell line HTR-8/SVneo cell apoptosis after cultured with different compatibilities of Blood stasis elimination formula.Then analyze the experimental results through the SPSS software by orthogonal variance analysis.3)Using Western Blot and RT-qPCR method to assay the protein and gene expression of Human gestational trophoblastic cell line HTR-8/SVneo cells with Bax,Fasl,Caspase-3,Caspase-8,Caspase-9,by different concentrations of optimized Blood stasis elimination formula.Results1)Successfully recovered and cultured Human gestational trophoblastic cell line HTR-8/SVneo cells.Identification results of Immunofluorescence staining showed that the cytoplasm had CK-18,ErbB 2 positive signal,no vimentin signal.Functional Identification of chemiluminescence showed secretion of HCG.2)CCK8 method showed that compared with negative control group,on the HTR-8/SVneo cell function with different extraction of this formula on proliferation was inhibited(Figure 1).Group A inhibition greater than B,C,D group,the difference was statistically significant(P<0.05),MTX group than the A,B,C,D group,the difference was statistically significant(P<0.05).3)Hoechst staining was observed in addition to the negative control group in each group are different degrees of cell volume shrink,cell shrinkage,nuclear condensation,nuclear fragmentation and apoptotic bodies,the A group and MTX group the most obvious.4)Flow cytometry showed:after each group of drugs acting on the HTR-8/SVneo cell,the apoptosis rate was(34.067 ± 1.401)%(group A),(20.933± 5.900)%(group B),(22.233 ± 4.869)%(group C),(27.5 ± 5.047)%(group D),(36.133 ± 0.493)%(group MTX),which was respectively and significantly increased as compared to blank control group(3.6 ±0.529)%(group K).The difference was statistically significant(P<0.05).The apoptosis rate of group A was higher than group B and group C,group MTX higher than group D,the difference was statistically significant(P<0.05).There was no statistically significant between Group A and group D,group MTX and group A(P>0.05).5)variance analysis results of Orthogonal design:according to apoptosis rate of HTR-8/Svneo cells,the impact by salvia miltiorrhiza,radix paeoniae rubra,peach kernel,Radix trichosanthis other than lithospermum was statistically significant(P<0.01),their impact degree of the order:radix paeoniae rubra>peach kernel>salvia miltiorrhiza>Radix trichosanthis>lithospermum.According to Caspase-3 gene expression of HTR-8/Svneo cells,other than peach kernel,all herbs' impact was statistically significant(P<0.05),their impact degree of the order:Radix trichosanthis>Salvia miltiorrhiza>radix paeoniae rubra>lithospermum>peach kernel.All five herbs effect on Caspase-9 gene expression were statistically significant P<0.01),their impact degree of the order:Radix trichosanthis>Salvia miltiorrhiza>lithospermum>radix paeoniae rubra>peach kernel.Differences between groups of various dosage:effects on apoptosis rate,A3>A2>A1(P<0.05),B3>B2>B1(P<0.05),C3>C2>C1(P<0.05),D3>D2&D1,(P<0.05),no statistically significant between D2 and D1(P>0.05);E2>E1)E3,the difference was not significant(P>0.05);the expression of Caspase-3 gene impact,A3&A2>A,(P<0.05),no statistically significant between A3 and A2(P>0.05),B2&B1)B3(P<0.01),no statistically significant between B2 and B,(P>0.05),C2>C3>C,but no significant difference(P>0.05),D2>D3>,(P<0.01),E1>E2(P<0.01),no statistically significant between E1 and E3 or E1 and E3(P>0.05);on Caspase-9 gene expression,A2>A3>A1(P<0.01),B2>B3&B1(P<0.01),no statistically significant between B3 and B1(P>0.05),C2&C1>C3(P<0.05),no statistically significant between C2 and C1(P>0.05),D3>D2&D1(P<0.01),no statistically significant between D2 and D,(P>0.05),E2>E1>E3(P<0.01).6)Western-blot and RT-qPCR Detection:Compared with the control group,Low,medium and high dose group and methotrexate group could significantly increase protein expression of Caspase-3,Caspase-8,Caspase-9,Bax and Fasl on HTR-8/SVneo cells.Medium and high dose group and methotrexate group could significantly increase mRNA expression of Bax,Caspase-3,Caspase-8,Caspase-9 and Fasl on HTR-8/SVneo cells;while low dose group could significantly increase mRNA expression of Caspase-3,Caspase-8,Caspase-9 and Fasl.3.Conclusion1)Integrative treatment program of tubal pregnancy is effective,the success rate of cases which meet the Integrative treatment indications meanwhile beyond the western medicine conservative treatment indications was relatively high.2)When the first ?-HCG value is greater than 8000IU/L,no matter how much the disease impact factor scores are recommended surgery.3)Choose water extraction as the best extraction method for Blood stasis elimination formula.4)By orthogonal design analysis of variance screened the best compatibility of Blood stasis elimination formula as follows:Salvia miltiorrhiza 15g,radix paeoniae rubra 15g,Peach kernel 15g,Radix Trichosanthis 30g,Lithospermum 15g.5)Optimal Blood stasis elimination formula can increase apoptosis pathway related protein expression of Fas/FasL&Bcl-2/Bax,suggesting that the formula may through the induction of Caspase-mediated death receptors and mitochondrial apoptotic pathways to promote trophocyte cell apoptosis. |
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