妇科Ⅰ号方治疗盆腔炎性疾病病理与免疫机制初探

【作者】 刘弘；

【导师】 金哲；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 目的：分析盆腔炎性疾病及其后遗症住院患者普通细菌、沙眼衣原体、解脲支原体、人型支原体的阳性构成比及药敏情况；探索我科治疗盆腔炎性疾病的协议处方妇科Ⅰ号方(四逆散加味)分别对于大肠杆菌造模和解脲支原体造模的两种小鼠盆腔炎性疾病模型炎性病理改善情况及其对于小鼠免疫机制的影响；探索盆腔炎性疾病动物模型不同建立方法的特点。方法：临床研究：收集我科住院患者资料,按照诊断标准入选盆腔炎性病例124人输卵管阻塞性不孕病例92人,排除检查资料不完整者16人,进行宫颈分泌物普通细菌、沙眼衣原体、解脲支原体、人型支原体加药敏检测结果的数据分析,分析各种病原体阳性构成比以及抗生素的耐药情况。实验研究分为两个部分。第一部分采用大肠杆菌菌液致小鼠盆腔炎性疾病模型,随机分为空白组、模型组、左氧氟沙星组、妇科Ⅰ号方四组,中药灌胃21天,阳性对照药给予14天,剩余天数给予同等剂量的蒸馏水,空白对照组和模型组给予同等剂量同等天数的蒸馏水。观察妇科Ⅰ号方对于阴道、宫颈、子宫、输卵管四个部位病理改善情况,同时采用小鼠血清细胞因子IL-2、IL-6、IL-10、TNF-α浓度和胸腺指数、脾指数作为免疫观测指标。第二部分采用雌激素预处理下,解脲支原体血清8型阴道接种感染致小鼠盆腔炎性疾病模型,随机分为空白对照组、雌激素对照组、模型组、阿齐霉素组、妇科Ⅰ号方五组,给药干预方法同上,观察阴道、宫颈、子宫、输卵管四个部位病理炎症状况,同时采用小鼠血清细胞因子IL-2、IL-6、IL-10、TNF-α浓度(Elisa法)和胸腺指数、脾指数以及T-bet/GATA3 mRNA(real-time PCR法)作为免疫观测指标,多组等级资料的比较采用非参数分析,多组计量资料的比较采用单因素分析。结果：临床研究：盆腔炎性疾病患者解脲支原体阳性率为42/124例(33.87%),合并其它微生物感染的有7例,分别是光滑假丝酵母菌、粪肠球菌、热带假丝酵母菌、无乳链球菌(B群)、白假丝酵母菌、沙眼衣原体、人型支原体各1例。人型支原体阳性1/124例(0.81%)。沙眼衣原体阳性1/136例(0.81%)。普通细菌培养阳性21/124例(16.94%),其中念珠菌6例(白假丝酵母菌3例,光滑假丝酵母菌、热带假丝酵母菌、克柔假丝酵母菌各1例),大肠埃希菌5例,粪肠球菌3例,金黄色葡萄球菌2例,链球菌2例,细球菌属、棒杆菌属、变形菌属各例。盆腔炎性疾病患者感染uu对四环素类最敏感,大环内酯类次之,而喹诺酮类耐药最为严重。不孕症患者uu阳性率为36/92例(39.13%),合并大肠埃希菌感染2例,光滑假丝酵母菌1例,人型支原体感染2例；人型支原体2/92例(2.17%)；衣原体未见感染者(0%)；普通细菌感染者6/92例(6.52%),包括大肠埃希菌2例、凝固酶阴性葡球菌、光滑假丝酵母菌、金黄色葡球菌、溶血链球菌各1例。TFI患者感染uu对四环素类最敏感,大环内酯类次之,而喹诺酮类耐药最为严重。PID患者与TFI患者感染uu的构成比以及与年龄差异均无统计学意义。实验研究：第一部分：空白组阴道、宫颈、子宫、输卵管四个部位组织结构清晰,可有少量炎细胞。模型组：阴道组织鳞状上皮部分缺损,炎细胞浸润,以中性粒细胞为主,阴道内可见炎性渗出物及脱落上皮,固有层可见充血及炎细胞浸润。宫颈组织粘膜上皮细胞变性、坏死脱落,上皮及固有层、肌层炎细胞浸润,以中性粒细胞为主,宫颈腔内可见大量炎性渗出物及坏死组织。子宫组织内膜上皮破碎,出现复层核和空泡状,大量炎细胞浸润,以中性粒细胞为主,少量浆细胞,粘膜下可见充血、水肿,肌层可见炎细胞浸润,腺体不同程度的增生、扩张及鳞状上皮化生。输卵管组织炎细胞浸润不明显,以粘膜层病变为显著,部分纤毛显著变短或消失,粘膜皱襞变平、减少、疏松水肿。四个部位均可见少量肥大细胞,并有脱颗粒现象。中西药治疗组四个部位的病理均有改善,病理评分后阴道、子宫、输卵管组织炎症病变改善具有统计学意义(p<0.05)。妇科Ⅰ号方对于小鼠血清细胞因子、胸腺指数和脾指数的升高或降低均无统计学意义。第二部分：空白对照组和雌激素对照组阴道、宫颈、子宫、输卵管结构正常,未见充血、肥大、水肿、渗出。模型组可见外阴脱毛、红肿、阴道分泌物较多,宫颈肥大,子宫和输卵管不同程度水肿。光镜下观察空白对照组小鼠生殖器官结构清晰,可有少量炎症细胞,上皮角化不全或过度,腺体内可见均匀粉染的分泌物。雌激素对照组宫颈部分呈中度炎症改变,其余组织同空白对照组。模型组可见粘膜层以及间质中性粒细胞的不同程度的浸润,管腔内有不同程度的脓性渗出,腺体有扩张或增生,间质有疏松水肿。输卵管部分纤毛结构模糊或消失,呈矮柱状上皮。中西药治疗组四个部位的病理均有改善,病理评分各组差异有统计学意义。妇科Ⅰ号方对于支原体致小鼠盆腔炎症性疾病胸腺指数和脾指数的影响：模型组的胸腺指数与空白组比较降低,差异具有统计学意义。阿齐霉素组和妇科Ⅰ方组的胸腺指数均较模型组升高,但差异无统计学意义,阿齐霉素组的胸腺指数仍较空白组低,差异具有统计学意义。脾指数各组无统计学意义。妇科Ⅰ号方能够升高模型小鼠血清IL-2,降低血清IL-6、IL-10和TNF-α浓度,第一项指标差异具有统计学意义。中药复方对于T细胞亚群的影响：模型组和雌激素组CD4+/CD8+与空白组相比均有显著降低,中药及西药干预后能够提高这一比值,但与模型组相比,差异无显著性。单纯雌激素组的CD4+和CD8+与模型组相比均明显增高,差异有统计学意义,但其比值与模型组相比有降低,差异无统计学意义,与空白组相比降低,差异有统计学意义。中西药对于小鼠脾组织T-bet/GATA-3mRNA的影响无统计学意义。结论：1我科盆腔炎性疾病及其后遗症住院患者性传播疾病的病原体以解脲支原体为主,普通细菌以大肠杆菌为主,喹诺酮类抗生素耐药情况严重。2妇科Ⅰ号方能够改善大肠杆菌致小鼠盆腔炎性疾病模型的炎性病变,但对其血清细胞因子等免疫方面未见影响。3妇科Ⅰ号方能够改善解脲支原体致小鼠盆腔炎性疾病模型的炎性病变,对其免疫机制的影响仍需进一步探索。4不同病原体致小鼠盆腔炎性疾病模型有不同病理特点,其致炎机理、发生发展过程、免疫系统的参与机制以及中药复方的作用机制仍需进一步的实验证实。更多还原

【Abstract】 Objective:To study the infection of Ureaplasma urealyticum(uu) and other pathogenic microorganism of cervical discharge in patients with pelvic inflammatory disease (PID) and tubal factor infertility (TFI) in order to instruct the diagnose and treatment. To study the effects of Modified Sini San decoction on histopathology changes of the genital organism, cytokines of spleen organism and cytokines and T lymph cells of blood in the Balb/C mice with pelvic inflammatory disease infected by ureaplasma urealyticum or colibacillus. To study the characteristics of two PID mice model infected by ureaplasma urealyticum or colibacillus.Method:Clinical research:from the inpatients of our department in China-Japan Friendship Hospital, PID and TFI cases were investigated by different pathogens: uu, Mycoplasma huminis(Mh), Chlamydia trachomatis (CT), and common bateriacs. A retrospective analysis of resistance to antibiotic drugs of these pathogens also was done. Experimtantal studies:The first mice model of PID was infected one of uterine by colibacillus. Chinese herbals were intragastuic administrated for 3 weeks as treated group and antibiotics Levofloxacin was administrated for 2 weeks as one of controlled group.Mice blood cytokines, the index of thymus gland and spleen were calculated and analyzed. The second mice model of PID was Oestradiaol-treated female BALB/c mice, which were inoculated intravaginally by serotype 8 of ureaplasma urealyticum. Chinese herbals were intragastric administrated for 3 weeks as treated group and Azithromycin as one of controlled groups. T lymphocyte subgroup of peripheral blood were analyzed by flow cytometry, cytokines of IFN-γ、TNF-α、IL-4 of mice spleen by ELISA, T-bet/GATA3 mRNA by real-time PCR.Result:Clinical research:In PID cases, the positive rates for uu tests, Mh tests, CT tests and common bactials tests were 33.87%(42/124),0.81%(1/124), 0.81%(1/136),16.94%(21/124). In bacterial culture, mainly candida, followed by colibacillus. In TFI cases, the positive rates for uu tests, Mh tests, CT tests and common bactials tests were 39.13%(36/92),2.17%(2/92); 0%(0/92); 6.52%(6/92). Exper imtantal studies:The PID mice modal infected by colibacillus showed infiltration of neutrophil in vagina, cervix, uterine, fallopian tube. After treatment of Chinese herbal compound FukeyihaoFang, the inflammatory pathological changes of PID mice improved well. But either the blood cytokines IL-2, IL-6, IL-10 and TNF-αof different groups or index of spleen and thymus gland showed no statistical significance. In the PID mice modal inoculated by uu, the vulvae turned red, the vaginal secretions became profuse, and uterines and oviducts even showed edema. Ureaplasmas can be sought in all model mice group. There were obvious infiltrations of neutrophil in 4 positions. And Chinese herbal compound FukeyihaoFang showed the same good improvement about it. The ratio of CD4+ and CD8+ of mice model were found to decline. The ratio of CD4+ and CD8+ and inflammatory cytokines in Chinese herbal group and Azithromicin group increased compared with model group, but the difference is not statistically significant. In addition, the T-bet/GATA3 mRNA of spleen in different groups showed no statistical significance.Conclusion:The pathogen of PID are mainly uu. Colibacillus is the most common pathogen for bacterial infection. Resistance for quinolone drugs is most serious. Intravaginal inoculation with ureaplasma urealyticum serotype 8 can induce pelvic inflammatory disease of mice; result in decreasing of immune function and genital tract acute inflammation. Chinese herbal compound FukeyihaoFang can improve the inflammatory pathological changes of PID mice model infected by either Escherichia coli or uu, although these two pathological models have different characteristics and formation mechanisms respectively. The mechanisms of Chinese herbals relieving inflammatory pathological changes on immunological network regulation need more experimental proof.更多还原