| Ectopic pregnancy refers to development at the site of implantation outside the uterine cavity, where more than 95% of patients with tubal pregnancy . Tubal pregnancy is the most common gynecological acute abdomen, gynecology emergency accounted for more than 80% rate, first diagnosis delays of up to 40%, which is one of the main reasons the first trimester of death. In recent years, with the increase in pelvic inflammatory disease, abortion, assisted reproductive technology and other risk factors, the incidence of tubal pregnancy showed an increasing trend, a serious threat to women of childbearing age health and quality. With the development of technology to make an early diagnosis of ectopic pregnancy can be found, so that conservative medical treatment possible for more young patients provides opportunities for preservation of fertility.The etiopathogenesis of pregnancy is considered to be syndrome of blood stasis in the lower abdomen by the traditional Chinese medicine , so the Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo has been used to treat tubal pregnancy. The Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo is based on traditional Chinese medicine and traditional Chinese medicine theory.which has been considered to be useful composed by Ectopic Pregnancy I Recipe(salvia miltiorriza Bunge, Paeonia veithii Lynch, Peach kernel, Radices trichosanthis, Lithospermum erythrorhizon), Maximize the preservation of fertility patients and improve the quality of life. 一. The clinical researchObjective:Combined with previous tubal pregnancy Integrative Medicine Clinic study program, adopt prospective, non-blinded, randomized, controlled study, ranging from clinical medicine discusses the effectiveness of traditional Chinese medicine+MTX and traditional Chinese medicine+Mifepristone treatment of tubal pregnancy, which provide the basis for clinical choice of drugs.Methods:The patients of tubal pregnancy who were treated during 2012.1 to 2013.12 were collected., who were identified can be "Integrative Medicine" conservative medical treatment cases, the patients were randomly divided into Chinese medicine+MTX group and Chinese medicine+mifepristone group. By comparing the two groups the effective rate, effective cases of abdominal pain time, stop time vaginal bleeding, blood β-HCG values fall time, reduced mass, in order to explore the advantages and feasibility of traditional Chinese medicine+MTX and traditional Chinese medicinemi+fepristone treatment of tubal pregnancy.Results:(一)The cases for research:There were 103 cases from January 2012 to December 2013, who were fit to be treated by Chinese medicine+MTX group 54 case, Chinese medicine+mifepristone group of 49 cases(二)The condition factor case103 cases of cases aged 25-29 the highest proportion, accounting for 38.8%, and no history of production accounted for 43.00%;stopped after a few days for the highest proportion of 43-56 days, accounting for 64.28%;abdominal symptoms pain who accounted for the largest proportion, accounting for 53%; The diameter 3 to 5cm of the mass by B-ultrasound examination took up the largest proportion, reach maximum 54.29%. The diameter<3cm of the internal hemorrhage through B-ultrasound examination took up the largest proportion; The serumβ-HCG 1000 to 2000IU/L took up the largest proportion; The total score of illness impact factors were 8.648±1.456 and 8.833±1.209 separately, condition factor total score.Using t-test and rank-sum test, we can know that there is no difference between Chinese medicine+MTX group and Chinese medicine+mifepristone group in the illness impact factors (P>0.05), the two groups were comparable.(三)The results of the clinical curative effects:The effective rate was 88.89% in the traditional Chinese medicine+MTX group, and 89.80% in the traditional Chinese medicinemi+fepristone group, and there is difference between observative group and control group in clinical effect (P<0.05); the traditional Chinese medicine+MTX group abdominal pain duration was 4.23+ 2.52 days, the traditional Chinese medicine+mifepristone group was 4.40+ 1.45 days, the difference was not statistically significant (P> 0.05); serum β-HCG values to 50% of the traditional Chinese medicine+MTX group was 5.704 ±1.550 days, the traditional Chinese medicine+mifepristone group was 7.021 ± 1.670 days, the traditional Chinese medicine+MTX group better value down HCG levels than the traditional Chinese medicine+mifepristone group (P<0.05); decreased to 10% of the traditional Chinese medicine+MTX group was 20.34 ±6.22 days, the traditional Chinese medicine+mifepristone group was 21.47 ± 8.92 days, the two aspects of the overall effect of HCG levels drop considerably (P> 0.05); the traditional Chinese medicine+MTX group was 85.19% reduced mass, the traditional Chinese medicine+mifepristone group was 73.47%, the traditional Chinese medicine+MTX group was superior to the traditional Chinese medicine mifepristone group in terms of reduced mass(P<0.05).二 The experimental sectionObjective:Discussion the effect of Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo on morphology of tubal pregnancy trophoblast apoptosis, apoptosis and apoptotic pathways, comparative trophoblastic tubal pregnancy and early intrauterine gestational trophoblastic morphology, growth curve and circulation similarities and differences between apoptosis rate, apoptotic morphology and apoptosis pathway after the action of Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo.Method:(一)Tubal pregnancy and intrauterine first trimester trophoblast cell culture, cells were identified by immunofluorescence staining method, cell growth curve.(二)The use of CCK-8、Hoechst staining、TEM and Annexin V-FITC/PI assay effect of the Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo on two cells about cell growth inhibition、apoptotic morphology、ultrastucture、apoptosis rate.(三) Western Blot and qRT-PCR assay the effect of Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo on two cells about the protein and genes of Bax, Fasl Caspase-3, Caspase-8, Caspase-9.Results:(一)Successfully cultured trophoblast tubal pregnancy and normal intrauterine pregnancy trophoblast cells, identification results of immunofluorescence staining method showed cytoplasm have CK-18, cerb-2 positive signal, no signal vimentin. Roughly the same in both cell growth cycle, both in the 3-4 Telsda logarithmic growth cycle 5-6 days due to inhibition of cell growth and density into the plateau.(二)CCK-8 assay found that after a final concentration of 5,10,20,30,40 mg /ml in the two cells treated the Chinese medicine which activating blood, dissoving stasis, eliminating mass and killing embryo, the growth inhibitory effect of different time of its each is different, according to its line graph can be seen in a time-, concentration-dependent manner. The Chinese medicine which activating blood, dissoving stasis, eliminating mass and killing embryo role of tubal gestational trophoblastic 48h half maximum inhibitory concentration:IC50= 20.12mg/ml; role of intrauterine gestational trophoblastic 48h half maximum inhibitory concentration:IC50 = 23.98mg/ml. On this basis, the following experimental medicine concentration of high-dose group 20mg/ml, middle-dose group 10mg/ml, low-dose group 5mg/ml.(三) Hoechest staining was observed under an inverted fluorescence microscope shows that different concentrations of traditional Chinese medicine group, there are different degrees nuclear condensation, nuclear fragmentation and apoptotic bodies, to 20mg/ml concentration and Medicines+MTX group was most obvious. Observed under TEM circulation xiaozheng role traditional Chinese medicine to kill the embryo after 48h, nourish the cell surface microvilli smaller size, varying degrees of shrinkage, chromatin less often qualitative, increased heterochromatin, concentrated in the nucleolus edge, or nucleolus shrinking, cracking, individual formation of apoptotic bodies, the endoplasmic reticulum was bubbly, fusion with the plasma membrane, organelles extravasation, swelling, cytoplasmic vacuoles of lipid droplets increased significantly. Two nourish apoptotic cell morphology and ultrastructure no significant difference.(E3)Annexin V-FITC/PI assay each drug group acting on tubal gestational trophoblastic 48h, compared with the negative control group, with the increase of drug concentration, a corresponding increase in the rate of apoptosis, which high-dose group was significantly increased apoptosis rate (P<0.01); compared with the methotrexate group, the apoptosis rate of Chinese high-dose group increased (P<0.05), while Chinese medicine+MTX group and high-dose group, no significant difference in the rate of apoptosis. Each drug group acting on intrauterine trophoblast cells after 48h, compared with the negative control group, with the increase of drug concentration, a corresponding increase in the rate of apoptosis, including high-dose group was significantly increased apoptosis rate (P<0.01); methotrexate group、methotrexate+Chinese medicine group compared with Chinese medicine high、middle-dose group showed no significant difference in apoptosis (P> 0.05). Each concentrations of two cell apoptosis rate compared to no significant difference (P> 0.05).(五)Tubal pregnancy trophoblast cells, compared with the negative control group, Medicine and Medicines+MTX group can increase the Bax, Fasl, Caspase-3, Caspase-9 protein expression, and the high-dose group of Chinese medicine, Chinese medicine+MTX group also increase Fasl, Caspase-3 gene expression (P<0.05), whereas no significant increase in protein Caspase-8 (P>0.05), but the gene expression was significantly increased. Methotrexate group increased only Caspase-9 protein expression. In intrauterine gestational trophoblastic cells, compared with the negative control group, Medicine and Medicines+ methotrexate group can increase the Bax, Fasl, Caspase-3, Caspase-8, Caspase-9 protein expression, (P<0.05), and the high-dose group of Chinese medicine also increase Bax, Fasl, Caspase-3, Caspase-8, Caspase-9 gene expression (P<0.05). Methotrexate group can increase the Fasl, Caspase-3, Caspase-8, Caspase-9 protein expression (P<0.05), Fasl gene expression also increased (P<0.05).concluded:(一) Integrative curative treatment of tubal pregnancy, medicine+MTX reduced mass superior efficacy in medicine+mifepristone group, HCG drop in value, vaginal bleeding, abdominal pain, etc.considerable effect.(二) Tubal pregnancy trophoblastic and intrauterine gestational trophoblastic in morphology and proliferation were no significant difference, those has similar growth characteristics.(三) The Chinese medicine which activating blood, dissoving stasis, eliminating mass and killing embryo inhibit proliferation and promote tubal pregnancy trophoblast cells apoptosis, which may be one of the mechanisms to reduce HCG, reduced mass treatment of tubal pregnancy. The Chinese medicine which activating blood, dissoving stasis, eliminating mass and killing embryo induced intrauterine gestational trophoblastic cells apoptosis and apoptosis morphology with no significant difference in tubal pregnancy trophoblast cells.(四)The Chinese medicine which activating blood, dissoving stasis, eliminating mass and killing embryo effect trophoblast tubal pregnancy and intrauterine gestational trophoblastic cells, may regulate the expression of Fas/FasL, Bcl-2/Bax apoptosis pathway-related proteins, suggesting that The Chinese medicine which activating blood, dissoving stasis, eliminating mass and killing embryo can induce Caspase-mediated death receptor and mitochondrial apoptotic pathway and promote trophoblast apoptosis. |
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