化瘀消癥杀胚法对输卵管妊娠影响的临床与实验研究
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摘要
引言：
孕卵在子宫腔以外部位种植、发育称为异位妊娠。近年来,随着人工流产、盆腔炎患者的增多,发病率有上升趋势,已经成为妇科临床中的常见病和多发病之一。据资料统计,异位妊娠发病率占所有已知怀孕总数的1%以上,其中大部分为输卵管妊娠(95%)。
中医目前认为其发病机理是少腹血瘀证,故多采用活血化瘀、消癥杀胚方药对早期输卵管妊娠进行治疗。应用由宫外孕Ⅰ号方(丹参、赤芍、桃仁)加紫草、天花粉等组成的化瘀消癥杀胚的复方对符合适应症的输卵管妊娠患者治疗,同时配合口服中成药、中药散剂外敷及中药针剂静滴,在临床治疗上已取得较好的效果,使这部分患者避免了因手术而承受的痛苦,减少了术后并发症,同时最大限度保全了患者的生育功能。
1.临床部分
目的：
通过前瞻性的随机对照研究,从临床有效性方面论证中医药在治疗输卵管妊娠的疗效及优势,评价原制定的“中西医结合输卵管妊娠治疗方案”的正确性和可行性,为建立规范、量化、易推广、能够被国内妇科同行广泛认可的输卵管妊娠治疗方案提供可参考的临床依据。
研究方法：
拟选择2009年1月至2010年12月间,临床上相同辨病分期、辨证分型,按病影响因子评分模型实施评分后属于相同用药范围内的,适合药物保守治疗的,β-HCG为阳性的输卵管妊娠病例,按单盲法、不等随机对照研究(2：1),将患者分为观察组和对照组。观察组按照“输卵管妊娠中西医结合治疗方案”治疗,对照组用西药米非司酮治疗。通过对比两组间的有效率、有效病例的血β-HCG值下降时间及疗程,探讨中西医结合治疗输卵管妊娠的优势及可行性。
研究结果：
(1)纳入试验病例情况：根据诊断标准、纳入标准及排除标准,2009年1月至2010年12月共收集符合药物保守治疗的输卵管妊娠病例184例,其中符合纯中药保守治疗119例,观察组(中-观察)82例,对照组(中-对照)37例；符合中西医结合保守治疗65例,观察组(中西-观察)45例,对照组(中西-对照)20例。
(2)病情影响因子情况：184例符合保守治疗的输卵管妊娠病例中,年龄在25-29岁年龄段所占比例最大,达37.5%；停经时间在43-56天之间所占比例最大,达52.72%；腹痛情况表现为隐痛者所占比例最大,达49.46%；B超下包块最大径<3cm者所占比例最大,达73.37%；B超下出血最大径<3cm者所占比例最大,达76.63%；初始血β-HCG值<1000IU/L者所占比例最大,达71.2%。病情影响因子总积分,中-观察组为6.96±1.13分,中-对照组为7.19±1.15分；中西-观察组为8.49±0.97分,中西-对照组为8.40±1.05分。
使用t检验,符合药物保守治疗的输卵管妊娠,观察组和对照组在年龄、停经时间、B超下包块最大径、B超下出血最大径、初始血β-HCG值和病情影响因子比较,差异无统计学意义(P>0.05),两组具有可比性。
(3)临床疗效研究结果：中-观察组有效率为81.71%,中-治疗组有效率为64.86%,两组比较,差异具有统计学意义(P<0.05)；中西-观察组有效率为68.89%,中西-对照组有效率为60%,两组比较,差异无统计学意义(P>0.05)。
中-观察组有效病例实际用药时间为10.76±5.92天；中-对照组有效病例实际用药时间为11.25±4.14天,两组比较,差异无统计学意义(P>0.05)；中西-观察组有效病例实际用药时间为16.03±5.60天,中西-对照组有效病例实际用药时间为13.17±4.73天,两组比较,差异无统计学意义(P>0.05)。
中-观察组有效病例血β-HCG值下降至50%的时间为4.66±2.07天,下降至10%的时间为9.28±3.40天；中-对照组有效病例下降至50%的时间为5.58±3.19天,下降至10%的时间为11.17±4.99天,两组血β-HCG值下降至50%时间比较,差异无统计学意义(P>0.05),下降至10%的时间比较,差异具有统计学意义(P<0.05)。中西-观察组有效病例血β-HCG值下降至50%的时间为5.87±2.88天,下降至10%的时间为12.52±4.19天；中西-对照组血β-HCG值下降至50%的时间为6.00±2.66天,下降至10%的时间为12.50±4.52天,两组血β-HCG值下降至50%时间比较,差异无统计学意义(P>0.05),下降至10%的时间比较,差异无统计学意义(P>0.05)。
2.实验部分
目的：
本研究拟从细胞凋亡率及凋亡相关蛋白等方面探讨化瘀消癥杀胚复方对输卵管妊娠滋养细胞靶部位的作用机制。这对丰富中医妇科学的基础理论,找寻中医治疗理论依据有着重大的意义,并为探索化瘀消癥杀胚复方的药效物质奠定研究基础。
研究方法：
(1)滋养细胞体外培养：拟选择符合药物治疗的早期输卵管妊娠患者的输卵管妊娠绒毛组织,采用以体外培养的人输卵管妊娠滋养细胞作为研究载体。
(2)含药血清的制备
①化瘀消癥杀胚复方水煎剂的制备
中药浓缩成剂量为1.5g/ml的药液,-20℃保存备用。
②含药血清的制备
将大鼠分为化瘀消癥杀胚复方高、中、低剂量组,分别灌胃7天,日两次；甲氨喋呤组,末日采血前肌注甲氨喋呤；生理盐水组,一直予灌胃生理盐水。
末次给药1小时后在麻醉下行腹主动脉采血,将同组大鼠血清混合,灭活、除菌后,-20℃保存,以备体外培养绒毛滋养层用。
③指标检测
一般情况观察：每周测量3次体重、饮食量、饮水量,每日观察小鼠精神状态、活动力、反应,大便形态,腹泻等。
生殖器官湿重：解剖当日,取大鼠的子宫、卵巢进行称重。
(3)三组含药血清与体外培养的滋养细胞作用72h后,通过①采用流式细胞法(FCM)检测细胞凋亡率；②蛋白质印迹法(Western Blot)检测凋亡相关蛋白Bcl-2和Bax的表达。
研究结果：
(1)输卵管妊娠滋养细胞培养
①细胞形态学：大部分细胞伸展呈圆形,1h后可见部分细胞贴壁,24h后大部分细胞贴壁,5、6天细胞数量明显增多,呈三角形,连接成片,部分呈长梭形。可见细胞为上皮样细胞形态,呈片状铺展生长。
②免疫荧光染色：细胞胞浆中有CK18阳性信号,未见波形蛋白信号。
③滋养细胞分泌功能：传代培养过程中的细胞培养液,通过化学发光法可检测出β-HCG表达。
④细胞生长曲线：6天为滋养细胞的对数生长期,12天后即进入平台期。
(2)化瘀消癥杀胚复方含药灌胃对大鼠的影响
与同一时间生理盐水组比较,化瘀消癥杀胚复方高、中、低剂量组大鼠体重从开始饲养/用药第4天/用药第7天差异无统计学意义(P>0.05)；与同一时间生理盐水组比较,化瘀消瘤杀胚复方高、中、低剂量组大鼠饮水量的差异无统计学意义(P>0.05)；在饲养的第7天,中药高剂量组的大鼠进食量下降,与同一时间的生理盐水组比较,差异具有统计学意义(P<005)。
与生理盐水组比较,化瘀消癥杀胚复方高、中、低剂量组,甲氨喋呤组大鼠在按照实验方案用药后,其子宫湿重及卵巢湿重的差异无统计学意义(P>0.05)。
(3)化瘀消癥杀胚复方含药血清对输卵管妊娠滋养细胞的细胞凋亡的影响
①滋养细胞与5%含药血清共同作用72h后,可提高滋养细胞的凋亡率,且中药的影响作用更显著。甲氨喋呤组与生理盐水组比较,细胞凋亡率上升,差异具有统计学意义(P<0.05)；化瘀消癥杀胚复方组与生理盐水组凋亡率比较,细胞凋亡率上升,差异具有显著统计学意义(P<0.01)；与甲氨喋呤组比较,差异无统计学意义(P>0.05)。
②滋养细胞与5%含药血清共同作用72h后,可降低Bcl-2蛋白表达量。甲氨喋呤组与生理盐水组比较,Bcl-2蛋白表达量降低,差异具有统计学意义(P<0.05)；化瘀消癥杀胚复方组与生理盐水组比较,Bcl-2蛋白表达量降低,差异具有显著统计学意义(P<0.01)；与甲氨喋呤组比较,差异无统计学意义(P>0.05)。
③含药血清与滋养细胞共同培养72h后,Bax蛋白表达量未见有明显变化。两组含药血清与生理盐水组比较,差异均无统计学意义(P>0.05)。
④化瘀消癥杀胚杀胚中药能使Bcl-2/Bax比例降低,从而起到促进凋亡的作用。
研究结论：
1.通过与纯西药组相比较,证实在符合中西医保守治疗输卵管妊娠范围内患者,依照“输卵管妊娠中西医结合治疗方案”进行治疗,治疗效果切实,副作用少。
2.“输卵管妊娠中西医结合治疗方案”仍有需要优化和完善的部分,如设立保守治疗的血β-HCG值上限,或动态血β-HCG值波动上限。
3.本研究成功地建立了输卵管妊娠滋养细胞体外培养模型,证实其可为其他的实验研究提供足够量的研究载体。
4.本研究探讨了化瘀消瘤杀胚复方对输卵管妊娠滋养细胞细胞凋亡的影响。结论：化瘀消癥杀胚复方提高输卵管妊娠滋养细胞细胞凋亡率,诱导滋养细胞凋亡可能是其发挥杀胚效应的机制之一。
5.本研究探讨了化瘀消癥杀胚复方对输卵管妊娠滋养细胞凋亡相关蛋白表达的影响。结论：下调Bcl-2的表达,使Bcl-2/Bax比例下降,诱导凋亡的发生可能是其发挥杀胚效应的机制之一。
Introduction
In the recent years, with the increase of abortion and infections in the department of gynecology and obstetrics, the rate of ectopic pregnancy is rising. According to the statistics, the incidence of ectopic pregnancies among the all is above 1%[1], and the overwhelming majority of them are tubal pregnancy(95%).
The etiopathogenesis of tubal 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 for a long time. The treatment program is put into practice to treat the patients of tubal pregnancy who are fit the indication. The treatment program has been considered to be useful which is composed by Ectopic Pregnancy I Recipe (Salvia miltiorrhiza Bunge, Paeonia. veitchii Lynch, Peach kernel) plus Radices trichosanthis, Lithospermum erythrorhizon, Chinese formulated products for oral use, the powder of Chinese medicine for external use and the Chinese medicine parenteral solution for intervenous drop infusion. The purpose is to decrease the passions and postoperative complications because of the operations, and to assure the safety of reproductive activity.
1. The clinical research
Objective:
Adopt prospective disparation case-control study method to observe the clinical curative effects of the traditional Chinese medicine for treating tubal pregnancy. To evaluate the correctness and the feasibility of the "Chinese and western treatment program of tubal pregnancy"。Through the results of the statistics to prove the traditional Chinese medicine is an effective treatment to treat tubal pregnancy, and to promote and provide better clinical evidence for the further verification of rationality and utility of the Chinese and western treatment program for tubal pregnancy.
Research Contents and Research Methods
The patients of tubal pregnancy who were treated in the First Affiliated Hospital of Guangzhou University of Chinese Medicine during 2009.1-2010.12 were collected. By adopting prospective disparation case-control study method (2:1)and single blind method, the patients those who were considered to be in the same condition were divided into observative group and control group randomly, after had been distinguished the disease stages, syndrome differentiation and calculated the total score of illness impact factors. The observative group were treated by "Chinese and western treatment program of tubal pregnancy", while the control group were treated by pure western medicine-Mifepristone. The effective rate, the change trend and rules of serumβ-HCG, the treatment course of the valid cases of the two groups were collected severally in order to investigate the clinical rationality of the Chinese and western treatment program of tubal pregnancy.
Results
(1)The cases for test:There were 184 cases from January 2009 to December 2010, who were fit to be treated by expectant treatment by drug.119 cases were fit to be treated by pure Chinese medicine treatment among the all, which were divided into two groups, the observative group 82 cases while the control group 37 cases.65 cases were fit to be treated by conbined treatment of traditional Chinese medicine and western medicine among the all, which were divided into two groups, the observative group 45 cases while the control group 20 cases.
(2)Illness impact factors:Among the 184 cases, age 25 to 29 years took up the largest proportion, reach maximum 37.5%. The onset time were 43-56 days from the last menstrual time took up the largest proportion, reach maximum 52.72%. The syndrome of vague pain took up the largest proportion, reach maximum 49.46%. The diameter<3cm of the mass through B-ultrasound examination took up the largest proportion, reach maximum 73.37%. The diameter<3cm of the internal hemorrhage through B-ultrasound examination took up the largest proportion, reach maximum 76.63%. The serumβ-HCG<1000 IU/L took up the largest proportion, reach maximum 71.2%. The total score of illness impact factors were 6.96±1.13 and 7.19±1.15 of those fit to be treated by pure Chinese medicine. The score of illness impact factors were 8.49±0.97 and 8.40±1.05 of those fit to be treated by conbined treatment of traditional Chinese medicine and western medicine.
Using T-test, we can know that the there is no difference between observative group and control group in the illness impact factors(P>0.05), the two groups were comparable.
(3)The results of the clinical curative effects:There is difference between observative group and control group in effective rate (P<.0.05) of the two groups which were fit to be treated by pure Chinese medicine. There is no difference between observative group and control group in effective rate (P >0.05) of the two groups which were fit to be treated by conbined treatment of traditional Chinese medicine and western medicine.
There is no difference between observative groups and control groups in treatment course of different therapeutic method (P>0.05).
There is difference between the two groups which were fit to be treated by pure Chinese medicine, in the time that the serumβ-HCG dropping down to 10%(P<0.05). While there is no difference between the same two groups in the time that the serumβ-HCG dropping down to 50%(P>0.05).
There is no difference between the two groups which were fit to be treated by conbined treatment of traditional Chinese medicine and western medicine, in the time that the serumβ-HCG dropping down to 50% and to 10%(P>0.05).
2. The experimental research
Objective
In the experimental research, the apoptosis and the apoptosis related proteins of the trophocytes in the fallopian tube were checked to observe the functional mechanism of the traditional Chinese medicine (Ectopic I, Decoction), which activating blood, dissolving stasis, eliminating mass and killing embryo for treating tubal pregnancy. Such study has positive influence in enriching the theory of Chinese gynecology, setting basis for clinical dialectics medication, developing the traditional Chinese medicine and providing the scientific theoretical basis for treating tubal pregnancy.
Research Contents and Research Methods
(1)The trophocytes cultured in vitro:The villus in the fallopian tube of those whom were fit to be treated by drugs but chose to take operation were collected. The trophocytes were cultured in vitro to be treated as research carrier.
(2) The preparation of medicated serum
①The medicinal broth of traditional Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo:The traditional Chinese medicine was thickened to be 1.5g/ml and conserved in-20℃.
②The preparation of medicated serum
The SD rats were divided into five groups, three of them were feeded by traditional Chinese medicine with different dose- high dose group, moderate dose group and low dose group. The western medicine- Methotrexate had been intramuscular injected 1 hour before the serum was collected to prepare another group. The rats of the last group were feeded by normal saline.
1 hour after the last time that given medicine, the serum of the SD rats were collected. The serum of the same group was mixed together, inactivated, degermed and conserved in-20℃.
③Same index
The observation of general state of health:the weights, the diet quantity and the water quantity of the rats were recorded 3 times every week. The mental state, the motoricity, the defecate situation of the rats were observed every day.
The weights of the generative organs:The weights of the uteruses and ovaries of the rats were weighted in the day when they were killed.
(3) Three groups of medicated serum were respectively added into cultured trophocytes cell in vitro. Then the cells were incubated for 72 hours.
①The apoptotic rate of different groups were checked by flow cytometry.
②The expression of Bcl-2 and Bax were checked by the western blot technique.
Results
(1)The trophcyotes were cultured in vitro
①Cell morphology:Most of the cells extended to be round, the number of the cells raised after 5 to 6 days. Some of them connected together and presented to be long spindle. The cell morphology presented to be epithelioid cell.
②mmune fluorescence staining:The CK18 signals existed in the cells while the vimentin signals didn't.
③The secretory function of the trophocytes:The expression ofβ-HCG can be checked in the culture solution of passage cell.
④The growth curve of the cells:The exponential phase of growth of the trophocytes was 6 days, the Platform period of the trophocytes was 12 days.
(2)The impact of the traditional Chinese medicine to rats
There are no difference among the groups of traditional Chinese medicine and the group of the normal saline in the same time in the weights- the first day/the forth day/the seventh day after had been feeded the traditional Chinese medicine (P>0.05). There are no difference among the groups of traditional Chinese medicine and the group of the normal saline in the same time in the water quantity- the first day/the forth day/the seventh day-after had been feeded the traditional Chinese medicine (P>0.05).In the seventh day after had been feeded the traditional Chinese medicine, the diet quantity of the high dose group descended, there is significant differences between such group and the group of the normal saline(P<0.05)
There are no difference among the groups of traditional Chinese medicine, the group of the normal saline and the group of western medicine in the weights of the uteruses and ovaries (P>0.05).
(3)The impact of the apoptosis after the trophocytes were cultured by medicated serum that contained Chinese medicine
①After had been treated with different serum that contained different medicine in vitro for 72 hours, the apoptotic rate of the trophocytes raises, the group of Chinese medicine raised most obviously. There is difference between the group of normal saline and group of Methotrexate (P<0.05). There is significant difference between the group of normal saline and group of Chinese medicine (P<0.01). There is no difference between the group of Methotrexate and group of Chinese medicine (P>0.05)
②After had been treated with different serum that contained different medicine in vitro for 72 hours, the expressions of Bcl-2 protein of the trophocytes descended, the group of Chinese medicine descended most obviously. There is difference between the group of normal saline and group of Methotrexate (P<0.05). There is significant difference between the group of normal saline and group of Chinese medicine(P0.05)
③fter had been treated with different serum that contained different medicine in vitro for 72 hours, the expressions of Bax protein of the trophocytes didn't change obviously. There is no difference among the three groups (P>0.05).
④he rate of Bcl-2/Bax down-regulated when treated with the serum contained Chinese medicine, and the apoptosis rate raised as a result.
Research Conclusion
1. After compared with the western medicine, the "Chinese and western treatment program of tubal pregnancy" was good stability in drug treatment of tubal pregnancy. Moreover, the treatment is effective and reasonable.
2. Some points of the "Chinese and western treatment program of tubal pregnancy" should'be optimized through more perspective clinical research.
3. The trophocytes in the fallopian tube were successfully cultivated in vitro, and proved that it can provide enough vector for further research.
4. The traditional Chinese medicine that activating blood, dissolving stasis, eliminating mass and killing embryo induced the apoptosis. Inducing apoptosis may be one of the mechanisms of the effect of killing embryo.
5. The traditional Chinese medicine that activating blood, dissolving stasis, eliminating mass and killing embryo deceased protein expression of Bcl-2 and deceased the rate of Bcl-2/Bax. Down-regulating of Bcl-2 may play an important role in the apoptosis in tubal pregnancy.
孕卵在子宫腔以外部位种植、发育称为异位妊娠。近年来,随着人工流产、盆腔炎患者的增多,发病率有上升趋势,已经成为妇科临床中的常见病和多发病之一。据资料统计,异位妊娠发病率占所有已知怀孕总数的1%以上,其中大部分为输卵管妊娠(95%)。
中医目前认为其发病机理是少腹血瘀证,故多采用活血化瘀、消癥杀胚方药对早期输卵管妊娠进行治疗。应用由宫外孕Ⅰ号方(丹参、赤芍、桃仁)加紫草、天花粉等组成的化瘀消癥杀胚的复方对符合适应症的输卵管妊娠患者治疗,同时配合口服中成药、中药散剂外敷及中药针剂静滴,在临床治疗上已取得较好的效果,使这部分患者避免了因手术而承受的痛苦,减少了术后并发症,同时最大限度保全了患者的生育功能。
1.临床部分
目的：
通过前瞻性的随机对照研究,从临床有效性方面论证中医药在治疗输卵管妊娠的疗效及优势,评价原制定的“中西医结合输卵管妊娠治疗方案”的正确性和可行性,为建立规范、量化、易推广、能够被国内妇科同行广泛认可的输卵管妊娠治疗方案提供可参考的临床依据。
研究方法：
拟选择2009年1月至2010年12月间,临床上相同辨病分期、辨证分型,按病影响因子评分模型实施评分后属于相同用药范围内的,适合药物保守治疗的,β-HCG为阳性的输卵管妊娠病例,按单盲法、不等随机对照研究(2：1),将患者分为观察组和对照组。观察组按照“输卵管妊娠中西医结合治疗方案”治疗,对照组用西药米非司酮治疗。通过对比两组间的有效率、有效病例的血β-HCG值下降时间及疗程,探讨中西医结合治疗输卵管妊娠的优势及可行性。
研究结果：
(1)纳入试验病例情况：根据诊断标准、纳入标准及排除标准,2009年1月至2010年12月共收集符合药物保守治疗的输卵管妊娠病例184例,其中符合纯中药保守治疗119例,观察组(中-观察)82例,对照组(中-对照)37例；符合中西医结合保守治疗65例,观察组(中西-观察)45例,对照组(中西-对照)20例。
(2)病情影响因子情况：184例符合保守治疗的输卵管妊娠病例中,年龄在25-29岁年龄段所占比例最大,达37.5%；停经时间在43-56天之间所占比例最大,达52.72%；腹痛情况表现为隐痛者所占比例最大,达49.46%；B超下包块最大径<3cm者所占比例最大,达73.37%；B超下出血最大径<3cm者所占比例最大,达76.63%；初始血β-HCG值<1000IU/L者所占比例最大,达71.2%。病情影响因子总积分,中-观察组为6.96±1.13分,中-对照组为7.19±1.15分；中西-观察组为8.49±0.97分,中西-对照组为8.40±1.05分。
使用t检验,符合药物保守治疗的输卵管妊娠,观察组和对照组在年龄、停经时间、B超下包块最大径、B超下出血最大径、初始血β-HCG值和病情影响因子比较,差异无统计学意义(P>0.05),两组具有可比性。
(3)临床疗效研究结果：中-观察组有效率为81.71%,中-治疗组有效率为64.86%,两组比较,差异具有统计学意义(P<0.05)；中西-观察组有效率为68.89%,中西-对照组有效率为60%,两组比较,差异无统计学意义(P>0.05)。
中-观察组有效病例实际用药时间为10.76±5.92天；中-对照组有效病例实际用药时间为11.25±4.14天,两组比较,差异无统计学意义(P>0.05)；中西-观察组有效病例实际用药时间为16.03±5.60天,中西-对照组有效病例实际用药时间为13.17±4.73天,两组比较,差异无统计学意义(P>0.05)。
中-观察组有效病例血β-HCG值下降至50%的时间为4.66±2.07天,下降至10%的时间为9.28±3.40天；中-对照组有效病例下降至50%的时间为5.58±3.19天,下降至10%的时间为11.17±4.99天,两组血β-HCG值下降至50%时间比较,差异无统计学意义(P>0.05),下降至10%的时间比较,差异具有统计学意义(P<0.05)。中西-观察组有效病例血β-HCG值下降至50%的时间为5.87±2.88天,下降至10%的时间为12.52±4.19天；中西-对照组血β-HCG值下降至50%的时间为6.00±2.66天,下降至10%的时间为12.50±4.52天,两组血β-HCG值下降至50%时间比较,差异无统计学意义(P>0.05),下降至10%的时间比较,差异无统计学意义(P>0.05)。
2.实验部分
目的：
本研究拟从细胞凋亡率及凋亡相关蛋白等方面探讨化瘀消癥杀胚复方对输卵管妊娠滋养细胞靶部位的作用机制。这对丰富中医妇科学的基础理论,找寻中医治疗理论依据有着重大的意义,并为探索化瘀消癥杀胚复方的药效物质奠定研究基础。
研究方法：
(1)滋养细胞体外培养：拟选择符合药物治疗的早期输卵管妊娠患者的输卵管妊娠绒毛组织,采用以体外培养的人输卵管妊娠滋养细胞作为研究载体。
(2)含药血清的制备
①化瘀消癥杀胚复方水煎剂的制备
中药浓缩成剂量为1.5g/ml的药液,-20℃保存备用。
②含药血清的制备
将大鼠分为化瘀消癥杀胚复方高、中、低剂量组,分别灌胃7天,日两次；甲氨喋呤组,末日采血前肌注甲氨喋呤；生理盐水组,一直予灌胃生理盐水。
末次给药1小时后在麻醉下行腹主动脉采血,将同组大鼠血清混合,灭活、除菌后,-20℃保存,以备体外培养绒毛滋养层用。
③指标检测
一般情况观察：每周测量3次体重、饮食量、饮水量,每日观察小鼠精神状态、活动力、反应,大便形态,腹泻等。
生殖器官湿重：解剖当日,取大鼠的子宫、卵巢进行称重。
(3)三组含药血清与体外培养的滋养细胞作用72h后,通过①采用流式细胞法(FCM)检测细胞凋亡率；②蛋白质印迹法(Western Blot)检测凋亡相关蛋白Bcl-2和Bax的表达。
研究结果：
(1)输卵管妊娠滋养细胞培养
①细胞形态学：大部分细胞伸展呈圆形,1h后可见部分细胞贴壁,24h后大部分细胞贴壁,5、6天细胞数量明显增多,呈三角形,连接成片,部分呈长梭形。可见细胞为上皮样细胞形态,呈片状铺展生长。
②免疫荧光染色：细胞胞浆中有CK18阳性信号,未见波形蛋白信号。
③滋养细胞分泌功能：传代培养过程中的细胞培养液,通过化学发光法可检测出β-HCG表达。
④细胞生长曲线：6天为滋养细胞的对数生长期,12天后即进入平台期。
(2)化瘀消癥杀胚复方含药灌胃对大鼠的影响
与同一时间生理盐水组比较,化瘀消癥杀胚复方高、中、低剂量组大鼠体重从开始饲养/用药第4天/用药第7天差异无统计学意义(P>0.05)；与同一时间生理盐水组比较,化瘀消瘤杀胚复方高、中、低剂量组大鼠饮水量的差异无统计学意义(P>0.05)；在饲养的第7天,中药高剂量组的大鼠进食量下降,与同一时间的生理盐水组比较,差异具有统计学意义(P<005)。
与生理盐水组比较,化瘀消癥杀胚复方高、中、低剂量组,甲氨喋呤组大鼠在按照实验方案用药后,其子宫湿重及卵巢湿重的差异无统计学意义(P>0.05)。
(3)化瘀消癥杀胚复方含药血清对输卵管妊娠滋养细胞的细胞凋亡的影响
①滋养细胞与5%含药血清共同作用72h后,可提高滋养细胞的凋亡率,且中药的影响作用更显著。甲氨喋呤组与生理盐水组比较,细胞凋亡率上升,差异具有统计学意义(P<0.05)；化瘀消癥杀胚复方组与生理盐水组凋亡率比较,细胞凋亡率上升,差异具有显著统计学意义(P<0.01)；与甲氨喋呤组比较,差异无统计学意义(P>0.05)。
②滋养细胞与5%含药血清共同作用72h后,可降低Bcl-2蛋白表达量。甲氨喋呤组与生理盐水组比较,Bcl-2蛋白表达量降低,差异具有统计学意义(P<0.05)；化瘀消癥杀胚复方组与生理盐水组比较,Bcl-2蛋白表达量降低,差异具有显著统计学意义(P<0.01)；与甲氨喋呤组比较,差异无统计学意义(P>0.05)。
③含药血清与滋养细胞共同培养72h后,Bax蛋白表达量未见有明显变化。两组含药血清与生理盐水组比较,差异均无统计学意义(P>0.05)。
④化瘀消癥杀胚杀胚中药能使Bcl-2/Bax比例降低,从而起到促进凋亡的作用。
研究结论：
1.通过与纯西药组相比较,证实在符合中西医保守治疗输卵管妊娠范围内患者,依照“输卵管妊娠中西医结合治疗方案”进行治疗,治疗效果切实,副作用少。
2.“输卵管妊娠中西医结合治疗方案”仍有需要优化和完善的部分,如设立保守治疗的血β-HCG值上限,或动态血β-HCG值波动上限。
3.本研究成功地建立了输卵管妊娠滋养细胞体外培养模型,证实其可为其他的实验研究提供足够量的研究载体。
4.本研究探讨了化瘀消瘤杀胚复方对输卵管妊娠滋养细胞细胞凋亡的影响。结论：化瘀消癥杀胚复方提高输卵管妊娠滋养细胞细胞凋亡率,诱导滋养细胞凋亡可能是其发挥杀胚效应的机制之一。
5.本研究探讨了化瘀消癥杀胚复方对输卵管妊娠滋养细胞凋亡相关蛋白表达的影响。结论：下调Bcl-2的表达,使Bcl-2/Bax比例下降,诱导凋亡的发生可能是其发挥杀胚效应的机制之一。
Introduction
In the recent years, with the increase of abortion and infections in the department of gynecology and obstetrics, the rate of ectopic pregnancy is rising. According to the statistics, the incidence of ectopic pregnancies among the all is above 1%[1], and the overwhelming majority of them are tubal pregnancy(95%).
The etiopathogenesis of tubal 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 for a long time. The treatment program is put into practice to treat the patients of tubal pregnancy who are fit the indication. The treatment program has been considered to be useful which is composed by Ectopic Pregnancy I Recipe (Salvia miltiorrhiza Bunge, Paeonia. veitchii Lynch, Peach kernel) plus Radices trichosanthis, Lithospermum erythrorhizon, Chinese formulated products for oral use, the powder of Chinese medicine for external use and the Chinese medicine parenteral solution for intervenous drop infusion. The purpose is to decrease the passions and postoperative complications because of the operations, and to assure the safety of reproductive activity.
1. The clinical research
Objective:
Adopt prospective disparation case-control study method to observe the clinical curative effects of the traditional Chinese medicine for treating tubal pregnancy. To evaluate the correctness and the feasibility of the "Chinese and western treatment program of tubal pregnancy"。Through the results of the statistics to prove the traditional Chinese medicine is an effective treatment to treat tubal pregnancy, and to promote and provide better clinical evidence for the further verification of rationality and utility of the Chinese and western treatment program for tubal pregnancy.
Research Contents and Research Methods
The patients of tubal pregnancy who were treated in the First Affiliated Hospital of Guangzhou University of Chinese Medicine during 2009.1-2010.12 were collected. By adopting prospective disparation case-control study method (2:1)and single blind method, the patients those who were considered to be in the same condition were divided into observative group and control group randomly, after had been distinguished the disease stages, syndrome differentiation and calculated the total score of illness impact factors. The observative group were treated by "Chinese and western treatment program of tubal pregnancy", while the control group were treated by pure western medicine-Mifepristone. The effective rate, the change trend and rules of serumβ-HCG, the treatment course of the valid cases of the two groups were collected severally in order to investigate the clinical rationality of the Chinese and western treatment program of tubal pregnancy.
Results
(1)The cases for test:There were 184 cases from January 2009 to December 2010, who were fit to be treated by expectant treatment by drug.119 cases were fit to be treated by pure Chinese medicine treatment among the all, which were divided into two groups, the observative group 82 cases while the control group 37 cases.65 cases were fit to be treated by conbined treatment of traditional Chinese medicine and western medicine among the all, which were divided into two groups, the observative group 45 cases while the control group 20 cases.
(2)Illness impact factors:Among the 184 cases, age 25 to 29 years took up the largest proportion, reach maximum 37.5%. The onset time were 43-56 days from the last menstrual time took up the largest proportion, reach maximum 52.72%. The syndrome of vague pain took up the largest proportion, reach maximum 49.46%. The diameter<3cm of the mass through B-ultrasound examination took up the largest proportion, reach maximum 73.37%. The diameter<3cm of the internal hemorrhage through B-ultrasound examination took up the largest proportion, reach maximum 76.63%. The serumβ-HCG<1000 IU/L took up the largest proportion, reach maximum 71.2%. The total score of illness impact factors were 6.96±1.13 and 7.19±1.15 of those fit to be treated by pure Chinese medicine. The score of illness impact factors were 8.49±0.97 and 8.40±1.05 of those fit to be treated by conbined treatment of traditional Chinese medicine and western medicine.
Using T-test, we can know that the there is no difference between observative group and control group in the illness impact factors(P>0.05), the two groups were comparable.
(3)The results of the clinical curative effects:There is difference between observative group and control group in effective rate (P<.0.05) of the two groups which were fit to be treated by pure Chinese medicine. There is no difference between observative group and control group in effective rate (P >0.05) of the two groups which were fit to be treated by conbined treatment of traditional Chinese medicine and western medicine.
There is no difference between observative groups and control groups in treatment course of different therapeutic method (P>0.05).
There is difference between the two groups which were fit to be treated by pure Chinese medicine, in the time that the serumβ-HCG dropping down to 10%(P<0.05). While there is no difference between the same two groups in the time that the serumβ-HCG dropping down to 50%(P>0.05).
There is no difference between the two groups which were fit to be treated by conbined treatment of traditional Chinese medicine and western medicine, in the time that the serumβ-HCG dropping down to 50% and to 10%(P>0.05).
2. The experimental research
Objective
In the experimental research, the apoptosis and the apoptosis related proteins of the trophocytes in the fallopian tube were checked to observe the functional mechanism of the traditional Chinese medicine (Ectopic I, Decoction), which activating blood, dissolving stasis, eliminating mass and killing embryo for treating tubal pregnancy. Such study has positive influence in enriching the theory of Chinese gynecology, setting basis for clinical dialectics medication, developing the traditional Chinese medicine and providing the scientific theoretical basis for treating tubal pregnancy.
Research Contents and Research Methods
(1)The trophocytes cultured in vitro:The villus in the fallopian tube of those whom were fit to be treated by drugs but chose to take operation were collected. The trophocytes were cultured in vitro to be treated as research carrier.
(2) The preparation of medicated serum
①The medicinal broth of traditional Chinese medicine which activating blood, dissolving stasis, eliminating mass and killing embryo:The traditional Chinese medicine was thickened to be 1.5g/ml and conserved in-20℃.
②The preparation of medicated serum
The SD rats were divided into five groups, three of them were feeded by traditional Chinese medicine with different dose- high dose group, moderate dose group and low dose group. The western medicine- Methotrexate had been intramuscular injected 1 hour before the serum was collected to prepare another group. The rats of the last group were feeded by normal saline.
1 hour after the last time that given medicine, the serum of the SD rats were collected. The serum of the same group was mixed together, inactivated, degermed and conserved in-20℃.
③Same index
The observation of general state of health:the weights, the diet quantity and the water quantity of the rats were recorded 3 times every week. The mental state, the motoricity, the defecate situation of the rats were observed every day.
The weights of the generative organs:The weights of the uteruses and ovaries of the rats were weighted in the day when they were killed.
(3) Three groups of medicated serum were respectively added into cultured trophocytes cell in vitro. Then the cells were incubated for 72 hours.
①The apoptotic rate of different groups were checked by flow cytometry.
②The expression of Bcl-2 and Bax were checked by the western blot technique.
Results
(1)The trophcyotes were cultured in vitro
①Cell morphology:Most of the cells extended to be round, the number of the cells raised after 5 to 6 days. Some of them connected together and presented to be long spindle. The cell morphology presented to be epithelioid cell.
②mmune fluorescence staining:The CK18 signals existed in the cells while the vimentin signals didn't.
③The secretory function of the trophocytes:The expression ofβ-HCG can be checked in the culture solution of passage cell.
④The growth curve of the cells:The exponential phase of growth of the trophocytes was 6 days, the Platform period of the trophocytes was 12 days.
(2)The impact of the traditional Chinese medicine to rats
There are no difference among the groups of traditional Chinese medicine and the group of the normal saline in the same time in the weights- the first day/the forth day/the seventh day after had been feeded the traditional Chinese medicine (P>0.05). There are no difference among the groups of traditional Chinese medicine and the group of the normal saline in the same time in the water quantity- the first day/the forth day/the seventh day-after had been feeded the traditional Chinese medicine (P>0.05).In the seventh day after had been feeded the traditional Chinese medicine, the diet quantity of the high dose group descended, there is significant differences between such group and the group of the normal saline(P<0.05)
There are no difference among the groups of traditional Chinese medicine, the group of the normal saline and the group of western medicine in the weights of the uteruses and ovaries (P>0.05).
(3)The impact of the apoptosis after the trophocytes were cultured by medicated serum that contained Chinese medicine
①After had been treated with different serum that contained different medicine in vitro for 72 hours, the apoptotic rate of the trophocytes raises, the group of Chinese medicine raised most obviously. There is difference between the group of normal saline and group of Methotrexate (P<0.05). There is significant difference between the group of normal saline and group of Chinese medicine (P<0.01). There is no difference between the group of Methotrexate and group of Chinese medicine (P>0.05)
②After had been treated with different serum that contained different medicine in vitro for 72 hours, the expressions of Bcl-2 protein of the trophocytes descended, the group of Chinese medicine descended most obviously. There is difference between the group of normal saline and group of Methotrexate (P<0.05). There is significant difference between the group of normal saline and group of Chinese medicine(P
③fter had been treated with different serum that contained different medicine in vitro for 72 hours, the expressions of Bax protein of the trophocytes didn't change obviously. There is no difference among the three groups (P>0.05).
④he rate of Bcl-2/Bax down-regulated when treated with the serum contained Chinese medicine, and the apoptosis rate raised as a result.
Research Conclusion
1. After compared with the western medicine, the "Chinese and western treatment program of tubal pregnancy" was good stability in drug treatment of tubal pregnancy. Moreover, the treatment is effective and reasonable.
2. Some points of the "Chinese and western treatment program of tubal pregnancy" should'be optimized through more perspective clinical research.
3. The trophocytes in the fallopian tube were successfully cultivated in vitro, and proved that it can provide enough vector for further research.
4. The traditional Chinese medicine that activating blood, dissolving stasis, eliminating mass and killing embryo induced the apoptosis. Inducing apoptosis may be one of the mechanisms of the effect of killing embryo.
5. The traditional Chinese medicine that activating blood, dissolving stasis, eliminating mass and killing embryo deceased protein expression of Bcl-2 and deceased the rate of Bcl-2/Bax. Down-regulating of Bcl-2 may play an important role in the apoptosis in tubal pregnancy.
引文
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