【作者】 杜娟；

【导师】 丛慧芳；

【作者基本信息】 黑龙江中医药大学 ， 中医妇科学， 2014， 博士

【摘要】 目的：1采用自体移植法建立子宫内膜异位症(EMs)大鼠模型并动态观察其异位内膜病理学改变,确定经痛愈舒预防及治疗EMs的给药时间。2观察经痛愈舒治疗性用药对EMs大鼠异位内膜组织病理学改变的影响,及其对NF-KB蛋白、RNA表达的干预效果,探讨经痛愈舒治疗EMs的可能机制,并为其临床应用提供实验依据。3通过比较研究经痛愈舒预防性与治疗性用药干预EMs大鼠病理学表现及NF-KB蛋白、RNA表达的效果,探讨经痛愈舒预防性用药对EMs的影响及潜在作用机制。方法：1实验第一部分选用20只雌性未孕SD大鼠,随机分为5组；模型3天组、模型5天组、模型4周组、模型6周组及模型8周组,每组4只。均采用自体移植法建立EMs模型,各组分别于造模后第3天、第5天、第4周、第6周及第8周处死,苏木素伊红(HE)染色法观察异位内膜组织病理形态改变,并与正常SD大鼠子宫内膜对照2实验第二部分选用造模成功的EMs大鼠72只,随机分为模型4周组、模型8周组,经痛愈舒低、中、高剂量组及西药组,每组12只,另取12只雌性未孕SD大鼠作为正常对照。各组大鼠分别予生理盐水或对应药物灌胃。其中模型4周组于造模4周后处死,其他各组均于造模后8周处死。HE染色观察各组大鼠异位内膜病理形态改变,免疫组.化法观察异位内膜NF-KB表达,Western blot法、RT-PCR法观察异位内膜NF-κB蛋白及RNA表达。3实验第三部分选用36只EMs模型大鼠,随机分为模型组、经痛愈舒预防性用药组及经痛愈舒治疗性用药组,每组12只,另取12只雌性未孕SD大鼠作为正常对照。经痛愈舒预防性用药组于造模后5天给药,治疗性用药组于造模后5天予生理盐水,至造模后4周后再予经痛愈舒中药。各组大鼠均于造模后8周处死。HE染色观察各组大鼠异位内膜病理形态改变变化,免疫组化法、Westernblot法、RT-PCR法观察NF-κB表达。结果：1实验第一部分自体移植EMs大鼠模型造模成功率约70%。HE染色结果显示：与正常对照组比较,造模后3天异位内膜仅有少量炎性细胞侵润。造模后5天异位内膜间质结构松散、血供较差,尚未形成典型的内膜组织结构。造模后4周可观察到典型异位内膜组织形成。造模后6周及8周异位内膜病理表现与4周相似。2实验第二部分异位内膜HE染色可见：经痛愈舒高剂量组及西药组内膜上皮细胞柱状增生,内膜间质松散,较多腺体萎缩,血供明显变差。3免疫组化结果显示：模型4周组、8周组、经痛愈舒各组及西药组NF-κB表达明显高于正常对照组；经痛愈舒各组及西药组NF-κB表达明显低于模型8周组,且经痛愈舒高剂量组效果优于西药组。4Western blot结果显示：模型4周组、8周组、经痛愈舒各组及西药组NF-κB蛋白表达明显高于正常对照组；经痛愈舒各组及西药组NF-κB蛋白表达明显低于模型8周组,且经痛愈舒中、高剂量组效果优于西药组。5RT-PCR结果显示：模型4周组、模型8周组、经痛愈舒各组及西药组NF-κB RNA含量明显高于正常对照组；经痛愈舒各组及西药组NF-κB蛋白表达明显低于模型8周组,其中经痛愈舒中、高剂量组效果优于西药组。6实验第三部分HE染色结果显示：经痛愈舒预防性及治疗性用药组内膜上皮萎缩变薄,间质细胞数量减少,异位内膜组织出现萎缩退变,其中预防性用药组改变更明显。7免疫组化结果显示：模型组、经痛愈舒治疗组及预防组NF-κB表达明显高于正常对照组；经痛愈舒治疗组及预防组NF-κB表达明显低于模型组,但经痛愈舒治疗组及预防组NF-κB表达未见明显差异。8Western blot结果显示：模型组、经痛愈舒治疗组及预防组NF-κB表达明显高于正常对照组；经痛愈舒治疗组及预防组NF-κB表达明显明显低于模型组,但经痛愈舒治疗组及预防组NF-κB表达未见明显差异。9RT-PCR结果显示：模型组、经痛愈舒治疗组及预防组NF-κB表达明显高于正常对照组；经痛愈舒治疗组及预防组NF-κB表达明显明显低于模型组,但经痛愈舒治疗组及预防组NF-κB表达未见明显差异。结论：1通过自体移植法可成功建立EMs大鼠模型,造模后5天可作为预防性用药干预时间点,造模后4周可作为治疗性用药干预时间点。2经痛愈舒治疗性用药可在一定程度上促使EMs大鼠异位内膜组织萎缩退变,并抑制其NF-κB蛋白及RNA表达。3经痛愈舒预防性用药亦可在一定程度上促进EMs大鼠异位内膜组织萎缩,并抑制其NF-κB表达,但与治疗性用药比较无明显差异。更多还原

【Abstract】 Object i ve:1To study the pathologic changes of the transplanted endometriotic tissues of the endometriosis rat model established by the method of autoplastic transplantation, to decide the best dosing time of prevention and therapy.2To study the effect of "Jingtongyushu" on the pathologic changes as well as the protein and RNA expression of NF-κB in the transplanted endometriotic tissues of the endometriosis rat model, providing experimental foundation for using "Jingtongyushu" to remedy endometriosis.3To study the effect of "Jingtongyushu" on the pathologic changes and the expression of NF-κB in the transplanted endometriotic tissues of the endometriosis rat model with preventive and therapy dosing separately, discussing the therapeutic mechanism of preventive dosing of "Jingtongyushu".Methods:1In the first part of animal research, female rat without offspring were divided into model3days group, model5days group, model4weeks group, model6weeks group and model8weeks group. There were4rats in each group. They were executed at3days,5days,4weeks,6weeks and8weeks after became endometriosis model established by the method of autoplastic transplantation. Then the pathologic changes of transplanted endometriotic tissues were compared with normal rats by the method of HE stain.2In the second part of animal research,72endometriosis rats were divided into model4weeks group, model8weeks group,"Jingtongyushu" group (low-,medium-,high-dosage) and danazol group, using female rats without offspring as control group. There were12rats in each group. The4weeks group were executed after4weeks, other groups were executed after8weeks. The changes of transplanted endometriotic tissues, as well as the protein and RNA expressions of NF-κB were detected by HE stain, Immunohistochemical stain, Western blot and RT-PCR.3In the third part of animal research,36endometriosis rats were divided into model group, preventive group and therapeutic group, using female rats without offspring as control group. There were12rats in each group. They were executed after8weeks, The changes of transplanted endometriotic tissues, as well as the protein and RNA expressions of NF-κB were detected by HE stain, Immunohistochemical stain, Western blot and RT-PCR.Results:1Endometriosis model were established satisfactorily by the method of autoplastic transplantation, the success rate was70percent. Model3days and model5days group didn’t show mature transplanted endometriotic tissues, but model4weeks,6weeks and8weeks groups had already builds mature transplanted endometriotic tissues.2"Jingtongyushu" high dosage group and danazol group effectively reduced the number of the endometrial stromal cells, and shrunk transplanted endometriotic tissues.3The Immunohistochemical stain in first part showed that, compared with control group, NF-κB expression significantly increased in model groups,"Jingtongyushu" groups and danazol group. Compared with model8weeks group,"Jingtongyushu" high-dosage group and danazol group significantly decreased NF-κB expression, and "tongjingtong" high-dosage group had better effectiveness.4The Western blot in first part showed that, compared with control group, the protein expression of NF-κB significantly increased in model groups,"Jingtongyushu" groups and danazol group. Compared with model8weeks group,"Jingtongyushu" high dosage group and danazol group significantly decreased protein expression, and "Jingtongyushu" medium,high-dosage groups had better effectiveness.5The RT-PCR in first part showed that, compared with control group, the RNA expression of NF-κB significantly increased in model groups,"Jingtongyushu" groups and danazol group. The effect of "Jingtongyushu" and danazol groups was similar with the results of Western blot.6In the third part of animal research,"Jingtongyushu" preventive and therapeutic groups had successfully shrink transplanted endometriotic tissues, the preventive group had better effectiveness.7The Immunohistochemical stain in third part showed that, compared with control group, NF-κB expression significantly increased in model group, preventive and therapeutic groups. Compared with model group, preventive group and therapeutic group significantly decreased NF-κB expression, but there were no difference in these two groups.8The Western blot showed that, compared with control group, the protein expression of NF-κB significantly increased in model group, preventive and therapeutic groups. Compared with model group, preventive group and therapeutic group significantly decreased the protein expression of NF-κB, but there were no difference in these two groups.9The RT-PCR showed that, compared with control group, the RNA expression of NF-κB significantly increased in model group, preventive and therapeutic groups. The effect of preventive and therapeutic groups was similar with the results of Western blot.Conclusion:1Endometriosis model can be established successfully with the method of autoplastic transplantation. Choosing5days and4weeks after model built as the preventive, therapeutic dosing time.2"Jingtongyushu" can shrink the transplanted endometriotic tissues of endometriosis model by decreasing its protein and RNA expressions of NF-κB.3Preventive using of " Jingtongyushu" can also shrink the transplanted endometriotic tissues of endometriosis model, as well as decrease the protein and RNA expressions of NF-κB, but there was no difference between preventive and therapeutic groups.更多还原