【作者】 岳恒学；

【导师】 刘瑞芬；

【作者基本信息】 山东中医药大学 ， 中医妇科学， 2013， 博士

【摘要】 目的：评价IUD避孕大鼠无创建模法及探讨宫宁颗粒调控IUD模型大鼠子宫血管重塑机制方法：方法一：以SD雌性大鼠为实验对象，将50只大鼠随机分为5组：无创模型组、开腹模型组、无创假手术组、开腹假手术组、正常对照组。制作IUD无创模型、IUD开腹模型、IUD无创假手术模型、IUD开腹假手术模型。开腹观察IUD放置成功率、IUD脱落率、避孕率、子宫炎症率。方法二:以SD雌性大鼠为实验对象，将50只大鼠随机分为5组，分组方法及制作IUD模型方法同上述方法一。用酶联免疫吸附法（ELISA）检测各组大鼠腹腔冲洗液肿瘤坏死因子-α（TNF-α）、转化生长因子-β（TGF-β）、细胞间粘附分子-1（ICAM-1）的浓度，检测宫腔冲洗液血栓素（TXA2）代谢产物血栓烷（TXB2）、前列腺素（PGI2）代谢产物6-酮-前列腺素(6-keto-PGF_1α)、纤溶酶原激活物（t-PA）的浓度及检测子宫组织匀浆碱性磷酸酶（AKP）、碳酸酐酶（CA）的浓度。方法三：以SD雌性大鼠为实验对象，将50只大鼠随机分为5组，模型组、消炎痛组、宫宁颗粒组、假手术组、正常对照组，制作IUD无创模型及IUD无创假手术模型。用免疫组化S-P法检测各组大鼠子宫血管平滑肌细胞（VSMC）表型标志物α-SM-肌动蛋白（α-SM-action）、VSMC增殖细胞核抗原（PCNA）的表达，计算子宫VSMC增殖率（PCNA/α-SM-action阳性细胞数×100%）。结果：结果一：无创模型组、开腹模型组IUD放置成功率分别为90%、80%；IUD脱落率分别为0、10％；避孕率均为100％；子宫炎症率分别为10％（无创模型组）、40％（开腹模型组）、30％（开腹假手术组）。结果二：①无创模型组大鼠腹腔冲洗液TNF-α的浓度极显著低于开腹模型组浓度，差异有极显著性（P<0.01）。②无创模型组大鼠腹腔冲洗液TGF-β浓度极显著低于开腹模型组浓度，差异有极显著性（P<0.01）。③无创模型组大鼠腹腔冲洗液ICAM-1浓度接近开腹模型组浓度，差异无显著性（P>0.05）。④无创模型组大鼠宫腔冲洗液TXB2的浓度极显著高于开腹模型组浓度,差异有极显著性（P<0.01）。⑤无创模型组大鼠宫腔冲洗液6-keto-PGF_1α的浓度极显著低于开腹模型组浓度，差异有极显著性（P<0.01）。⑥无创模型组大鼠宫腔冲洗液t-PA的浓度极显著低于开腹模型组浓度，差异有极显著性（P<0.01）。⑦无创模型组大鼠宫腔冲洗液6-keto-PGF1a/TXB2比率极显著低于开腹模型组比率，差异有极显著性（P<0.01）。⑧无创模型组大鼠子宫组织匀浆AKP浓度接近开腹模型组浓度，差异无显著性（P>0.05）。⑨无创模型组大鼠子宫组织匀浆CA浓度接近开腹模型组浓度，差异无显著性（P>0.05）。结果三：①模型组大鼠子宫内膜VSMC表型标志物α-SM-action的表达较正常对照组极显著降低，治疗后各组α-SM-action表达已显著升高及极显著升高。其中，宫宁颗粒组α-SM-action的表达与模型组比较，极显著升高，已接近正常对照组表达，差异无显著性（P>0.05）；而消炎痛组虽显著升高α-SM-action的表达，但与宫宁颗粒组比较，差异有极显著性（P<0.01）。②模型组大鼠子宫内膜VSMC增殖细胞核抗原PCNA的表达较正常对照组极显著升高，治疗后各组PCNA的表达均已极显著降低。其中，宫宁颗粒组PCNA表达与消炎痛组比较，显著性降低，差异有显著性（P<0.05），表明宫宁颗粒组较消炎痛组表达更接近正常对照组表达。③模型组大鼠子宫内膜VSMC增殖率PCNA/a-SM-action的比率较正常对照组极显著升高，治疗后各组VSMC增殖率PCNA/a-SM-action的比率已极显著降低。其中，宫宁颗粒组增值率与消炎痛组增值率比较，差异有极显著性（P<0.01），表明宫宁颗粒组增值率较消炎痛组更接近正常对照组。结论：结论一：从行为学、子宫形态学两方面对无创模型组大鼠、开腹模型组大鼠进行了仔细的肉眼观察和对比，可以看出开腹模型组大鼠患炎症的程度明显高于无创模型组大鼠。通过对比分析看出，IUD放置成功率开腹模型组略低于无创模型组，差异无显著性（P>0.05）；IUD脱落率开腹模型组略高于无创模型组，差异无显著性（P>0.05）；避孕率相同；子宫炎症率开腹模型组明显高于无创模型组，差异有显著性（P<0.05）。所以无创IUD造模方法优于开腹IUD造模方法。结论二：从腹腔冲洗液（TNF-α、TGF-β、ICAM-1）、宫腔冲洗液(TXB2、6-keto-PGF_1α、t-PA)、子宫组织匀浆（AKP、CA）各指标的显著变化说明开腹模型组比无创模型组炎症高，对子宫损伤重，更易引起IUD出血副反应。实验一、实验二分别从宏观、微观两方面对无创模型组、开腹模型组各指标进行了对比分析，从实验一、实验二的结论中看出，IUD避孕大鼠无创建模法优于开腹建模法，IUD无创建模法是理想的IUD避孕大鼠建模法。结论三：具有祛瘀清热止血调经作用的宫宁颗粒，通过调控IUD模型大鼠子宫内膜VSMC的α-SM-action、PCNA表达，促使VSMC由合成表型VSMC（病理性）逆转为收缩表型VSMC（生理性），从而使受损的模型大鼠子宫内膜血管由病理性重塑逆转为适应性重塑，减轻了模型大鼠子宫内膜VSMC的显微、超微结构的形态学损伤，消除了模型大鼠子宫内膜血管构筑的异常，达到调控IUD模型大鼠子宫血管重塑的目的，从而恢复了子宫内膜血管正常生理功能。更多还原

【Abstract】 Purpose: To evaluate the non-incision modeling method of rat IUD and explore theregulation of Gongning granules on the remodeling mechanism of uterine vessels.Methods:Method I: Use SD female rat as the experimental subject. Randomly divide50ratsinto5groups: non-incision model group, abdomen-incision model group, non-incision fakeoperation model group, abdomen-incision fake operation group and normal control group.Make the IUD non-incision model, IUD abdomen-incision model, IUD non-incision fakeoperation model group and IUD abdomen-incision fake operation model and incise theabdomen of the rats to observe the successful placing rate, falling rate and effectivecontraception rate and rate of IUD inflammation.Method II: Use SD female rat as the experimental subject. Randomly divide50ratsinto5groups as the method I and duplicate IUD modeling method also as described in themethod I. Test the concentration of the tumor necrosis factor-α（TNF-α）, transforminggrowth factorβ（TGF-β）and intercellular adhesion molecule-1（ICAM-1） of theperitoneal flushing fluid of the five groups by enzyme linked immunosorbent assay. Thenby the same method, test the concentration of thromboxane （TXB2）, metabolite ofthromboxane （TXA2）, concentration of6-keto-PGF_1α, metabolite of prostaglandin（PGI2）and concentration of plasminogen activator （t-PA） as well as the concentration ofuterine tissue homogenate, alkaline phosphatase （AKP） and carbonic anhydrase（CA）.Method III: Use SD female rats as the experimental subject. Divide50rats into5groups at random: model group, fake operation group, Gongning granula group,indomethacin group and normal control group. Duplicate the IUD non-incision model and IUD non-incision fake operation model. Test the expression of the surface indicator,α-SM-action of vascular smooth muscle cell （VSMC） and proliferating cell nuclearantigen （PCNA）by Immunohistochemical S-P method to calculate the proliferating rateof uterine vascular smooth muscle cell（VSMC）（PCNA/α-SM-action positive cells*100%）.Result:Result I: The successful IUD placing rate of non-incision model group andabdomen-incision model group is90%and80%respectively and the falling rate ofnon-incision model group and abdomen-incision model group is0%and10%respectively.The contraception rate of both groups is100%and the inflammation rate of non-incisionmodel group is10%, that of abdomen-incision model group is40%and that ofabdomen-incision fake operation group is30%.Result II:1. The concentration of the rat Peritoneal flushing liquid TNF-αofnon-incision model group is apparently lower than that of abdomen-incision model groupwith significant difference（P<0.01）.2. The concentration of the rat peritoneal flushingfluid of non-incision model group is apparently lower than that of abdomen-incision modelgroup with significant difference（P<0.01）.3. The concentration of non-incision modelgroup is close to that of abdomen-incision model group without significant difference（P>0.05）.4. The concentration of rat peritoneal flushing liquid TXB2of non-incisionmodel group is apparently higher than that of abdomen-incision model group withsignificant difference（P<0.05）.5.The concentration of the rat uterine cavity lavage fluid,6-keto-PGF_1α of non-incision model group is apparently lower than that ofabdomen-incision model group with significant difference（P<0.05）.6. The concentrationof the rat uterine cavity lavage fluid, t-PA of non-incision model group is apparently lowerthan that of abdomen-incision model group with significant difference（P<0.01）.7. Thenrate of6-keto-PGF1a/TXB2of rat uterine cavity lavage fluid of non-incision model isapparently lower than that of abdomen-incision model group with significant difference（P<0.01）.8. The concentration of the rat uterine tissue homogenate,AKP of non-incisionmodel group is close to that of abdomen-incision model group without significantdifference（P>0.05）.9. The CA concentration of rat uterine cavity tissue homogenate ofnon-incision model group is close to that of abdomen-incision model group without significant difference（P>0.05）.Result III:1. The indicator of VSMC of model group, α-SM-action decreasessignificantly compared with that of normal control group; After treatment, the expressionof α-SM-action of all the groups has increased obviously and extremely obviously. Theexpression of α‐SM‐action of Gongning granules group increases apparentlycompared with that of model group. It is close to that of normal control group withoutsignificant difference（P>0.05）. Although the α‐SM‐action expression of indomethacingroup increases apparently but it still significantly differs from that of Gongning Granules.2. The expression of PCNA of VSMC of model group increases apparently compared withthat of normal control group. After treatment, the PCNA expression of all the groups hasdecreased apparently. The expression of Gongning granules decreases apparentlycompared with that of indomethacin group with significant difference（P<0.05）, whichindicates that the expression of Gongning granules group is closer to that of normal controlgroup than that of indomethacin group.3. The rate of PCNA/a-SM-action of rat VSMC ofmodel group increases apparently compared with normal control group. After treatment,the rate of PCNA/a-SM-action has decreased apparently. The appreciation rate ofGongning granules differs significantly from that of indomethacin group（P<0.01）, whichindicates that the appreciation rate of Gongning granules is closer to that of normal controlgroup than that of indomethacin group.Conclusion:Conclusion I: By observing visually and comparing the rats of non-incision modelgroup and that of abdomen-incision model group, it is found that the inflammation of therats of abdomen-incision model group is apparently more serious than that of non-incisionmodel group. It can be concluded by analysis that the abdomen-incision model group has aslightly lower successfully IUD placing rate than non-incision model group withoutsignificant difference （P>0.05）However, the IUD falling rate of abdomen-incision modelgroup is slightly higher than that of non-incision model group without significantdifference（P>0.05） but the inflammation rate of abdomen-incision model group isapparently higher than that of non-incision model group with significant difference（P<0.05）. Therefore, the non-incision IUD model is better than the abdomen-incisionIUD model.Conclusion II: The significant changes of the various indicants of peritoneal flushing liquid （TNF-α、TGF-β、ICAM-1），Uterine cavity lavage liquid(TXB2、6-keto-PGF_1α、t-PA)and uterine tissue homogenate（AKP、CA）demonstrate that the abdomen-incisionmodel group has a higher inflammation rate than non-incision model group with seriousinjury to uterine. Thus the abdomen-incision model group is more likely to cause IUDnegative reaction.The various index of non-incision model group and abdomen-incision model groupare analyzed by comparing with each other in Experiment I and Experiment II atmacro-level and micro-level respectively. It can be seen from Experiment I andExperiment II that the IUD non-incision modeling is better than abdomen-incisionmodeling and non-incision modeling is the ideal IUD method for rats.Conclusion III: The Gongning granula with the effect of removing stasis and clearingheat and menstruation-regulating helps transfer VSMC from synthetic phenotype（pathologic）into contractile phenotype（physiologic） by relieving the morphologicaldamage of micro-structure and ultromicor-structure of endometrium VSMC andregulating the expression of VSMC、α-SM-action、 PCNA so that the impairedendometrium vascular changes from pathologic remodeling to adaptive remodeling toeliminate the abnormal angioarchitecture of uterine vascular so as to achieve the purposeof remodeling of uterine vascular and preventing and curing blooding after IUD placement.Furthermore, the effect of Gongning granule in curing IUD blooding is more obvious thanthat of indomethacine.更多还原