TRPV1及其致敏因子与子宫内膜异位症痛经的关系及补肾温阳化瘀方对其影响

发布时间：2018-04-15 03:29

本文选题：子宫内膜异位症 + 痛经　；参考：《河北医科大学》2014年博士论文

【摘要】：目的：痛经是子宫内膜异位症患者的主要症状，严重影响患者的生活质量，但其发生机制尚不明确，成为目前研究的热点问题。本研究通过分析TRPV1及其致敏因子NGF、BK/BKB1R、PGF2α在内异症患者血清、在位内膜、异位内膜的变化，以期阐明内异症痛经的部分发病机制；动物实验是研究药物治疗本病作用机制的主要手段，但内异症痛经的动物模型却未见报道，本研究尝试利用近交系BALB/c小鼠建立内异症痛经模型；采用补肾温阳化瘀方对内异症痛经模型动物进行干预，分析治疗前后TRPV1及其致敏因子的变化，进一步探讨补肾温阳化瘀方治疗内异症痛经的作用机制。方法：第一部分：TRPV1及其致敏因子与子宫内膜异位症痛经关系的研究内异症组：选自2012年9月～2013年12月于石家庄市第四医院住院并符合内异症纳入标准的患者34例；对照组来自同期住院的子宫肌瘤无痛经患者16例，所有患者术前抽取空腹肘静脉血，术中取在位及异位内膜组织，采用ELISA法检测血清NGF、BK、PGF2α的含量，采用免疫组化及Western blot方法检测NGF、BKB1R、TRPV1蛋白的表达，采用Real-timePCR方法检测NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达。第二部分：子宫内膜异位症痛经动物模型的建立将68只清洁级近交系BALB/c雌性小鼠随机分为6组，分别为假手术组、雌激素+缩宫素组、手术组、手术+雌激素组、手术+缩宫素组、手术+雌激素+缩宫素组，其中假手术组、雌激素+缩宫素组每组10只，其余每组12只。除假手术组、雌激素+缩宫素组外其余各组均采用自体移植法将小鼠自体子宫组织块移植到腹膜，复制子宫内膜异位症模型，术后1~12d采用不同方案诱发小鼠扭体反应，记录扭体潜伏期及扭体次数，并取异位灶行HE染色和病理组织学观察，筛选建立内异症痛经模型的最佳方案。第三部分：补肾温阳化瘀方对子宫内膜异位症痛经模型小鼠TRPV1及其致敏因子的影响 75只近交系BALB/c雌性小鼠随机分为5组：假手术组、模型组、中药高剂量组、中药低剂量组、西药组，每组15只。除假手术组外，其余各组均采用手术＋雌激素+缩宫素建立小鼠内异症痛经模型，并于术后1-12d同时给药，于第12天诱发扭体反应，断头取血并取子宫及异位灶组织。采用ELISA法、免疫组化法、Western blot方法及Real-time PCR方法检测各组TRPV1及其致敏因子的变化。结果：第一部分：TRPV1及其致敏因子与子宫内膜异位症痛经关系的研究 1血清NGF、BK、PGF2α的含量及相关性分析结果 1.1血清NGF、BK、PGF2α的含量内异症患者痛经组血清NGF、BK、PGF2α水平较对照组及无痛组明显升高，差异有统计学意义(P0.01或P0.05)。 1.2血清NGF、BK、PGF2α含量的相关性分析 NGF与PGF2α呈正相关关系(P0.01)，BK与PGF2α水平亦呈正相关关系(P0.01)，NGF与BK之间未发现相关关系(P0.05)。 1.3血清NGF、BK、PGF2α含量与VAS评分的相关性分析 NGF与VAS评分呈正相关(P0.01)， PGF2α与VAS评分呈正相关(P0.01)，BK含量与VAS评分呈正相关(P0.01)。 2免疫组化法检测在位、异位内膜组织中NGF、BKB1R、TRPV1蛋白的表达结果 2.1NGF的表达在位内膜痛经组NGF表达增强，与对照组及无痛组相比显著升高(P0.01，P0.05)；异位内膜痛经组NGF的表达显著高于无痛组(P0.05)。 2.2BKB1R的表达在位内膜痛经组BKB1R表达增强，与对照组及无痛组相比显著升高(均为P0.01);异位内膜痛经组BKB1R的表达显著高于无痛组(P0.01)。 2.3TRPV1的表达在位内膜痛经组TRPV1的表达增强，与对照组及无痛组相比显著升高(均为P0.01)。异位内膜痛经组TRPV1的表达显著高于无痛组(P0.01)。 3Western blot法检测在位、异位内膜组织中NGF、BKB1R、TRPV1蛋白的表达结果 3.1在位、异位内膜组织中NGF的表达在位内膜痛经组NGF的表达高于对照组及无痛组(均为P0.01)；异位内膜痛经组NGF的表达显著高于无痛组(P0.01)。 3.2在位、异位内膜组织中BKB1R的表达在位内膜痛经组BKB1R的表达高于对照组及无痛组(均为P0.01)。异位内膜痛经组显著高于无痛组(P0.01)。 3.3在位、异位内膜组织中TRPV1的表达在位内膜痛经组TRPV1的表达高于对照组及无痛组(均为P0.01)。异位内膜痛经组TRPV1的表达显著高于无痛组(P0.01)。 3.4NGF、BKB1R、TRPV1蛋白表达之间的相关性分析在位内膜、异位内膜NGF与BKB1R的蛋白表达呈正相关关系(均为P0.01)，NGF与TRPV1的表达呈正相关关系(P0.05，P0.01)，BKB1R与TRPV1的表达亦呈正相关关系(均为P0.01)。 4Real-time PCR检测内异症在位、异位内膜组织中NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达结果 4.1在位、异位内膜组织中NGFmRNA的表达在位内膜痛经组NGFmRNA的表达显著高于对照组及无痛组(均为P0.01)。异位内膜NGF mRNA的表达：痛经组显著高于无痛组(P0.01)。 4.2内异症在位、异位内膜组织中BKB1RmRNA的表达在位内膜痛经组BKB1RmRNA的表达显著高于对照组及无痛组(均为P0.01)；异位内膜BKB1RmRNA的表达痛经组高于无痛组(P0.01)。 4.3内异症在位、异位内膜组织中TRPV1mRNA的表达在位内膜痛经组TRPV1mRNA的表达显著高于对照组及无痛组(均为P0.01)；异位内膜TRPV1mRNA的表达：痛经组显著高于无痛组，差异有统计学意义(P0.01)。 4.4NGFmRNA、BKB1RmRNA、TRPV1mRNA表达之间的相关性分析在位内膜、异位内膜NGFmRNA与BKB1RmRNA的表达呈正相关关系(均为P0.01)，NGFmRNA与TRPV1mRNA的表达呈正相关关系(均为P0.01)， BKB1RmRNA与TRPV1mRNA的表达亦呈正相关关系(均为P0.01)。 4.5NGFmRNA、BKB1RmRNA、TRPV1mRNA表达与VAS评分的相关性分析在位内膜、异位内膜NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达与VAS评分呈正相关关系(均为P0.01)。第二部分：子宫内膜异位症痛经动物模型的建立与手术+缩宫素组、手术组相比，手术+雌激素+缩宫素组、手术+雌激素组移植物体积明显增大，差异有统计学意义(P0.01)；手术+雌激素+缩宫素组扭体发生率100%，雌激素+缩宫素组扭体发生率80%，手术+缩宫素组扭体发生率50%，其余组未出现扭体反应，组间差异有统计学意义(P0.01)；与手术+缩宫素组、雌激素+缩宫素组相比，手术+雌激素+缩宫素组扭体潜伏期明显缩短(P0.01，P0.05)，扭体次数明显增多(P0.01，P0.05)，差异有统计学意义。第三部分补肾温阳化瘀方对子宫内膜异位症痛经模型小鼠TRPV1及其致敏因子的影响 1补肾温阳化瘀方对内异症痛经模型小鼠扭体反应的影响与模型组相比，，各给药组扭体潜伏期明显延长(均为P0.01)，扭体次数显著减少(P0.05或P0.01)。 2补肾温阳化瘀方对内异症痛经模型小鼠异位灶体积的影响与模型组相比，各给药后异位灶体积明显缩小(均为P0.01)。 3补肾温阳化瘀方对内异症痛经模型小鼠血清NGF、BK、PGF2α的影响模型组血清NGF、BK、PGF2α含量显著高于假手术组(P0.01，P0.01，P0.05)；与模型组相比，各给药组NGF、BK、PGF2α含量显著下降，差异有统计学意义(P0.05或P0.01) 4模型小鼠血清NGF、BK、PGF2α含量与扭体反应的相关性分析血清BK、PGF2α、NGF浓度与扭体次数呈正相关关系(均为P0.01)；PGF2α浓度与扭体潜伏期呈负相关关系(P0.05)；NGF、BK与扭体潜伏期之间未发现相关关系(P0.05)；NGF与BK呈正相关关系(P0.01)，PGF2α与BK呈正相关关系(P0.05)，NGF与PGF2α亦呈正相关关系(P0.01)。 5免疫组化法检测NGF、BKB1R、TRPV1的表达结果模型组子宫NGF、BKB1R、TRPV1表达显著高于假手术组(均为P0.01)；与模型组相比，各给药组子宫及异位灶NGF、BKB1R、TRPV1表达显著降低，差异有统计学意义(P0.05或P0.01)。 6Western blot检测NGF、BKB1R、TRPV1蛋白的表达结果与假手术组相比，模型组子宫NGF、BKB1R、TRPV1蛋白表达增高(均为P0.01)；与模型组相比，各给药组子宫及异位灶NGF、BKB1R、TRPV1表达降低(均为P0.01)，且NGF、BKB1R、TRPV1蛋白的表达存在正相关关系(均为P0.01)。 7Real-time PCR检测子宫及异位灶NGFmRNA、 BKB1RmRNA、TRPV1mRNA的表达结果与假手术组相比，模型组子宫NGFmRNA、 BKB1RmRNA、TRPV1mRNA表达增高(均为P0.01)；与模型组相比，各给药组子宫及异位灶NGFmRNA、BKB1RmRNA、TRPV1mRNA表达不同程度下降(P0.01或P0.05)，且NGFmRNA、BKB1RmRNA、TRPV1mRNA的表达存在正相关关系(均为P0.01)。结论： 1NGF、BK、PGF2α在内异症患者血清中水平升高，且与痛经程度呈正相关关系，TRPV1、NGF、BKB1R蛋白在内异症患者在位内膜、异位内膜中表达增强，TRPV1mRNA、NGFmRNA、BKB1RmRNA在内异症痛经患者在位内膜及异位内膜中表达增强，且与痛经程度呈正相关。提示TRPV1及其致敏因子NGF、BK/BKB1R、PGF2α可能在内异症痛经的发生过程中起重要作用； 2手术+雌激素+缩宫素组引起的扭体次数最多，扭体潜伏期最短，方法简单易行，可用于内异症痛经发病机制及药物治疗研究，是建立内异症痛经模型的最佳方案； 3补肾温阳化瘀方可以缩小模型小鼠异位灶体积，减少扭体次数，延长扭体潜伏期，降调内异症痛经模型TRPV1及其致敏因子的表达，提示补肾温阳化瘀方可通过影响TRPV1及其致敏因子发挥治疗内异症痛经的作用。
[Abstract]:Objective : To study the changes of serum , in - situ and ectopic endometrium of patients with endometriosis , including NGF , BK / BKB1R and PGF2伪 , in order to clarify the pathogenesis of endometriosis .
Animal experiments were the main methods to study the mechanism of drug therapy . However , the animal model of endometriosis was not reported . The study attempted to establish a model of endometriosis by using inbred BALB / c mice .
The effect mechanism of Bushen Wenyang Huayu Fang on the treatment of endometriosis dysmenorrhea was further investigated by using the model animal of tonifying kidney , warming yang and removing blood stasis to intervene and analyze the changes of TRL and its sensitizing factor before and after treatment .

Method :

Study on the relationship between TRpv1 and its sensitizing factor and endometriosis

Patients with endometriosis : 34 patients who were hospitalized in the Fourth Hospital of Shijiazhuang City from September 2012 to December 2013 and included in the standard .
The expression of NGF , BK and PGF2伪 was detected by ELISA . The expression of NGF , BKB1R , TRpv1 was detected by immunohistochemistry and Western blot . The expression of NGF , BKB1R and TRp1mRNA was detected by immunohistochemistry and Western blot . The expression of NGF , BKB1R mRNA and TRPV1mRNA was detected by Real - time PCR .

Part Two : Establishment of Animal Model of endometriosis

68 clean - grade inbred BALB / c female mice were randomly divided into six groups : sham operation group , estrogen + oxytocin group , operation group , operation + estrogen group , operation + oxytocin group , operation + estrogen plus oxytocin group .

The third part : Effect of Bushen Wenyang Huayu Recipe on TRpv1 and its sensitizer in endometriosis model mice

75 inbred BALB / c female mice were randomly divided into five groups : sham operation group , model group , traditional Chinese medicine high - dose group , low - dose Chinese medicine group and western medicine group .

Results :

Study on the relationship between TRpv1 and its sensitizing factor and endometriosis

Assay results of serum NGF , BK , PGF2伪

1.1 Content of serum NGF , BK , PGF2伪

The levels of NGF , BK and PGF2伪 in the patients with endometriosis were significantly higher than those in the control group and the painless group ( P0.01 or P0.05 ) .

1.2 Correlation analysis of serum NGF , BK and PGF2伪 levels

There was positive correlation between NGF and PGF2伪 ( P0.01 ) . There was positive correlation between BK and PGF2伪 ( P0.01 ) . There was no correlation between NGF and BK ( P0.05 ) .

1.3 Correlation between the content of serum NGF , BK and PGF2伪 and VAS score

There was positive correlation between NGF and VAS score ( P0.01 ) , and PGF2伪 positively correlated with VAS score ( P0.01 ) , and BK content was positively correlated with VAS score ( P0.01 ) .

The expression of NGF , BKB1R and TRpv1 protein in ectopic and ectopic endometrium were detected by immunohistochemistry .

2.1 Expression of NGF

The expression of NGF in the ectopic endometrium increased significantly compared with the control group and the painless group ( P0.01 , P0.05 ) .
The expression of NGF in ectopic endometrium was significantly higher than that in painless group ( P0.05 ) .

2 . Expression of 2BKB1R

Compared with control group and painless group , the expression of BKB1R was significantly higher than that in control group and painless group ( P0.01 ) . The expression of BKB1R in ectopic endometrium was significantly higher than that in painless group ( P0.01 ) .

2.3 Expression of TRpv1

Compared with the control group and the painless group ( P0.01 ) , the expression of TRpv1 in the ectopic endometrium was significantly higher than that in the control group and the painless group ( P0.01 ) .

The expression of NGF , BKB1R and TRpv1 protein in ectopic and ectopic endometrium were detected by Western blot .

3.1 Expression of NGF in situ and ectopic endometrium

The expression of NGF in the endometriosis group was higher than that in the control group and the painless group ( P0.01 ) .
The expression of NGF in ectopic endometrium was significantly higher than that in painless group ( P0.01 ) .

3.2 Expression of BKB1R in situ and ectopic endometrium

The expression of BKB1R in the ectopic endometrium was higher than that in the control group and the painless group ( P0.01 ) . The ectopic endometrium was significantly higher than that in the painless group ( P0.01 ) .

3.3 Expression in place and ectopic endometrium tissue

TRv1 expression was higher in the ectopic endometrium than in the control group and in the painless group ( P0.01 ) . The expression of TRpv1 in the ectopic endometrium was significantly higher than that in the painless group ( P0.01 ) .

Analysis of the correlation between expression of 3 . 4 NGF , BKB1R and TRpv1 protein

There was positive correlation between the expression of NGF and BKB1R in the ectopic endometrium , ectopic endometrium and BKB1R ( P0.01 ) .

The expression of NGFmRNA , BKB1RmRNA and TRPV1mRNA in ectopic and ectopic endometrium tissues by 4Real - time PCR

4.1 Expression of NGFmRNA in situ and ectopic endometrium

The expression of NGFmRNA in the ectopic endometrium was significantly higher than that in the control group and the painless group ( P0.01 ) . The expression of the ectopic endometrium NGF mRNA was significantly higher than that in the painless group ( P0.01 ) .

4.2 Expression of BKB1RmRNA in the place of endometriosis and ectopic endometrium

The expression of BKB1RmRNA was significantly higher than that in control group and painless group ( P0.01 ) .
The expression of BKB1RmRNA in ectopic endometrium was higher than that of painless group ( P0.01 ) .

4.3 Expression of TRPV1mRNA in the place of endometriosis and ectopic endometrium

TRPV1mRNA expression was significantly higher than that in control group and painless group ( P0.01 ) .
The expression of TRPV1mRNA in ectopic endometrium was significantly higher than that in painless group ( P0.01 ) .

4 . Correlation analysis between expression of 4 NGFmRNA , BKB1RmRNA and TRPV1mRNA

There was positive correlation between the expression of mRNA and mRNA of BKB1RmRNA ( P0.01 ) . The expression of NGFmRNA and TRPV1mRNA was positively correlated ( P0.01 ) . The expression of BKB1RmRNA and TRPV1mRNA was positively correlated ( P0.01 ) .

Correlation analysis of 4 . 5NGFmRNA , BKB1RmRNA , TRPV1mRNA expression and VAS score

The expression of NGFmRNA , BKB1RmRNA and TRPV1mRNA was positively correlated with VAS score ( P0.01 ) .

Part Two : Establishment of Animal Model of endometriosis

Compared with the operation group and the operation group , the volume of the operation + estrogen plus oxytocin group and the operation + estrogen group was obviously increased , the difference was significant ( P0.01 ) .
The incidence of writhing was 100 % , the rate of estrogen plus oxytocin group was 80 % , the rate of torsion body was 50 % , and no torsion reaction occurred in the rest group ( P0.01 ) .
Compared with the surgery + oxytocin group and estrogen plus oxytocin group , the latency of the torsion body in the operation + estrogen plus oxytocin group was significantly shortened ( P0.01 , P0.05 ) , and the number of writhing increased significantly ( P0.01 , P0.05 ) . The difference was statistically significant .

The effect of the third part of Bushen Wenyang Huayu Recipe on TRpv1 and its sensitization factor in endometriosis model mice

Compared with the model group , the latent period of writhing was significantly prolonged ( P0.01 ) , and the number of writhing was significantly decreased ( P0.05 or P0.01 ) .

Compared with the model group , the volume of ectopic foci was significantly reduced ( P0.01 ) .

The levels of NGF , BK and PGF2伪 in serum NGF , BK and PGF2伪 in the model group were significantly higher than those in sham operation group ( P0.01 , P0.01 , P0.05 ) .
Compared with model group , the content of NGF , BK and PGF2伪 in each group was significantly decreased ( P0.05 or P0.01 ) .

The serum levels of NGF , BK and PGF2伪 in the model mice were positively correlated with the frequency of writhing reaction ( P0.01 ) .
The concentration of PGF2伪 negatively correlated with the latency of writhing ( P0.05 ) .
There was no correlation between NGF , BK and latency of writhing ( P0.05 ) .
There was positive correlation between NGF and BK ( P0.01 ) . There was positive correlation between PGF2伪 and BK ( P0.05 ) . The relationship between NGF and PGF2伪 was positively correlated ( P0.01 ) .

The expression of NGF , BKB1R , TRpv1 in the model group was significantly higher than that in sham operation group ( P0.01 ) .
Compared with the model group , the expressions of NGF , BKB1R and TRpv1 in the uterus and ectopic foci were significantly lower in each group than in the model group ( P0.05 or P0.01 ) .

Expression of NGF , BKB1R and TRpv1 Protein by Western blot

Compared with the sham operation group , the expression of NGF , BKB1R and TRpv1 in the model group increased ( all P0.01 ) .
Compared with the model group , the expressions of NGF , BKB1R , TRpv1 in the uterus and ectopic foci were decreased ( P0.01 ) , and there was positive correlation between NGF , BKB1R and TRpv1 protein ( all P0.01 ) .

Expression of NGFmRNA , BKB1RmRNA and TRPV1mRNA in uterus and ectopic foci by 7Real - time PCR

Compared with sham operation group , the expression of NGFmRNA , BKB1RmRNA and TRPV1mRNA in model group increased ( P0.01 ) .
Compared with model group , the expression of NGFmRNA , BKB1RmRNA and TRPV1mRNA decreased ( P0.01 or P0.05 ) , and the expression of NGFmRNA , BKB1RmRNA and TRPV1mRNA was positively correlated ( P0.01 ) .

Conclusion :

The expression of TRV1mRNA , NGFmRNA , BKB1RmRNA in the ectopic endometrium and ectopic endometrium of patients with endometriosis was positively correlated with the degree of dysmenorrhea . The expression of TRPV1mRNA , NGFmRNA , BKB1RmRNA in the ectopic endometrium and ectopic endometrium was positively correlated with the degree of dysmenorrhea .

2 Surgery + estrogen plus oxytocin group had the most torsion body frequency , the shortest latency was the shortest , the method is simple and easy , it can be used in the pathogenesis of endometriosis and the study of drug therapy , which is the best scheme for the establishment of the model of endometriosis ;

3 Bushen warming yang Huayu can reduce the volume of ectopic foci in the model mice , reduce the number of writhing , prolong the latent period of the writhing , and reduce the expression of the allodynia model TRL 1 and its sensitizing factor in the model mice . It is suggested that the kidney warming yang Huayu can play the role of treating endometriosis by influencing TRpv1 and its sensitizing factor .

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.71

【引证文献】