【作者】 李雁；

【导师】 张绍芬； 刘瑞梓； 夏贤；

【作者基本信息】 复旦大学 ， 妇产科学， 2010， 博士

【摘要】 子宫腺肌病是一种子宫内膜腺体及间质侵入子宫肌层,伴随子宫局灶或弥漫性增大的良性疾病。此病多发生于40岁以上的经产妇,患病率为5%-70%,子宫切除后腺肌病灶的检出率约为30%-60%。临床表现主要为经量增多和经期延长,以及逐渐加剧的进行性痛经。子宫腺肌病患者的痛经率高达64.8%-77.8%,此外还可以出现慢性盆腔痛、排便痛及性交痛。疼痛严重影响了女性的生殖健康和生活质量,但其机制尚未完全明确。神经生长因子(nerve growth factor,NGF)是一类在疼痛过程中起到关键调节作用的神经因子,主要通过低亲和力的P75神经营养因子受体(p75 neurotrophin receptor, p75NTR)与高亲和力的酪氨酸激酶受体A (tyrosine kinase A, trkA)发挥生物学作用,参与疼痛发生、发展的各个环节。研究表明NGF在内异症患者异位内膜高表达,且与疼痛相关,可能成为内异症及腺肌病疼痛研究中的关键环节。本课题检测NGF在腺肌病患者或动物模型子宫及脊髓背根神经节的表达变化,研究NGF表达与腺肌病本身及疼痛程度的关系,分析可能诱发NGF变化的原因,并通过检测NGF相关的神经植入、疼痛受体、局部炎症、中枢敏感性的变化,及NGF对腺肌病灶内膜间质细胞增殖、凋亡、雌激素合成关键酶表达的作用,探讨NGF参与子宫腺肌病疼痛的机制。第一部分子宫腺肌病患者腺肌灶NGF表达与神经植入及疼痛程度的关系目的研究NGF在子宫腺肌病患者病灶内膜的表达及其与患者疼痛评分及病灶局部神经植入密度的关系。方法选择2009年3月-10月在复旦大学附属妇产科医院院行子宫切除的腺肌病患者45例,子宫切除且否认疼痛的宫颈上皮内瘤变(CIN)Ⅲ期及子宫肌瘤患者共26例,所有病例均经病理组织学确诊。于术中取腺肌病患者病灶内膜或子宫肌瘤及CIN患者在位内膜,免疫组化法显示神经生长因子(NGF)表达；免疫荧光法显示蛋白基因产物9.5(PGP9.5)阳性神经纤维植入；视觉评分系统(VAS)评测腺肌病患者痛经、慢性盆腔痛及性交痛的程度。结果腺肌病疼痛组患者病灶NGF表达强度及PGP9.5阳性神经纤维植入密度高于腺肌病无痛组及对照组(p<0.05)；腺肌病痛经重痛组NGF表达强度及PGP9.5阳性神经纤维植入密度高于腺肌病痛经轻中痛组及对照组(p<0.05)；NGF表达强度及PGP9.5阳性神经纤维植入密度与腺肌病患者是否出现慢性盆腔痛及性交痛无关；腺肌病患者病灶NGF表达强度与PGP9.5阳性神经纤维密度变化具有相关性(r=0.31,p<0.05)。结论腺肌病患者病灶处NGF高表达可能参了患者痛经的发生机制,而促进病灶内膜神经纤维植入是其机制之一。第二部分NGF及其受体在子宫腺肌病小鼠子宫及脊髓背根神经节的表达目的使用口服他莫昔芬法建立ICR小鼠子宫腺肌病模型并鉴定；检测NGF及其受体在腺肌病小鼠模型子宫及脊髓背根神经节(DRGs)的表达随动情周期及病情进展的变化。方法新生ICR小鼠连续4d滴喂他莫昔芬为造模组,并与同龄对照小鼠分别于42 d、85-95 d、135-145 d、185-195 d、235-245 d龄处死(每组4-5只),取双子宫及T13至L2脊髓背根神经节。使用苏木素-伊红染色检测子宫病理改变；阴道脱落细胞法检测动情周期变化；逆转录-聚合酶链反应(RT-PCR)检测子宫缓激肽受体1(BKR-1)、神经激肽1受体(NK1-R)的基因表达；免疫组化补体31(CD31)染色计算子宫微血管的密度、直径及所占面积比；免疫组化PGP9.5及SP染色计算神经纤维密度；免疫组化检测NGF及其受体在子宫的表达；蛋白印迹(Western-bolt)检测子宫NGF及其受体蛋白水平随动情周期及鼠龄增加的变化；实时定量逆转录聚合酶链反应(Real-time RT PCR)检测DRGs中NGF受体mRNA水平随鼠龄增加的变化。结果使用口服他莫昔芬法建立ICR小鼠子宫腺肌病模型的造模率为100%,且疾病严重程度随鼠龄增加进展；85-95d及135-145d龄造模小鼠子宫肌层微血管密度和面积比均高于对照小鼠(p<0.05)；135-145d造模小鼠子宫内膜及肌层PGP9.5阳性神经纤维、内膜SP阳性神经纤维植入密度高于对照组(p<0.05)；135-145d造模小鼠子宫BKR-1、NK1-R的基因表达较对照组明显升高(p<0.05)；免疫组化结果显示NGF在小鼠子宫内膜上皮细胞和间质细胞表达,p75NTR在子宫内膜间质细胞表达,trkA在子宫内膜上皮细胞及神经纤维表达；Western-bolt结果表明135-145d腺肌病鼠及对照鼠子宫NGF及其受体的表达随动情周期变化波动,NGF及p75表达水平在动情前期达高峰,trkA在动情期达高峰(p<0.05)；85-95d腺肌病小鼠NGF及其受体表达强度与对照组无差异,135-145d腺肌病小鼠子宫出现NGF前体及p75NTR表达增高(p<0.05),且随鼠龄增长腺肌病鼠子宫NGF、NGF前体及NGF受体的蛋白水平(p<0.05), DRGs中trkA的mRNA水平均增高(p<0.05),对照鼠未见此变化。结论口服他莫昔芬法可有效建立ICR小鼠子宫腺肌病模型,模型小鼠出现腺肌病特征性病理改变及血管、神经、炎症、疼痛相关受体的表达异常,病情随鼠龄进展；腺肌病及对照小鼠子宫NGF及受体的表达受动情周期调节,随疾病进展增强；腺肌病小鼠DRGs中trkA表达增强可能是中枢神经敏感性增强的原因之一；腺肌病子宫NGF表达与BKR-1及NK1-R所反映的局部组织、神经炎症变化一致。第三部分腺肌病患者异位内膜间质细胞NGF表达的影响因素及其对间质细胞的调控作用研究目的研究影响腺肌病患者子宫内膜间质细胞NGF表达的因素及NGF对子宫内膜间质细胞增殖、凋亡、雌激素合成的作用。方法选择2009年5月-10月在复旦大学附属妇产科医院院行子宫切除的腺肌病患者为实验组,子宫切除宫颈上皮内瘤变(CIN)Ⅲ期及子宫肌瘤患者为对照组。于术中取实验组病灶内膜或对照组在位内膜分离子宫内膜间质细胞(ESC)并行原代培养。免疫组化法显示ESC的NGF及其受体表达；于培养体系中分别加入不同浓度17β-雌二醇、肿瘤坏死因子(TNF)及氯化钴(CoCl2),western-bolt检测实验组及对照组ESC合成NGF蛋白水平的变化；于培养体系中加入不同浓度NGF, MTT法检测两组ESC增殖变化,real time RT-PCR与western-bolt法分别检测两组ESC合成芳香化酶的：mRNA及蛋白水平变化；培养体系中加入H2O2或CoCl2诱导ESC凋亡并加入不同浓度NGF, MTT法检测ESC凋亡变化。结果腺肌病及对照组ESC均表达NGF及其受体p75NTR,均不表达trkA。外源性17β-雌二醇可促进腺肌病ESC合成NGF,但对对照组ESC无此作用；外源性TNF可促进腺肌病组与对照组NGF的合成；CoCl2缺氧可降低对照组ESC合成NGF,但对腺肌病ESC无此作用；外源性NGF可明显促进腺肌病ESC增殖及芳香化酶的合成,但对对照组ESC无此作用；外源性NGF对两组ESC H2O2及CoCl2诱导的凋亡均无保护作用。结论17β-雌二醇与TNF均可促进腺肌病患者ESC表达NGF; NGF可促进腺肌病患者ESC增殖及雌激素合成关键酶的表达。更多还原

【Abstract】 Adenomyosis is defined as the presence of ectopic endometrial glands and stroma within the myometrium. This disorder mainly affects women more than 40 years old. The prevalence of adenomyosis reported in the literature ranges from 5 to 70%, and largely varies in relation to the number of tissue sections analysed. It was found that adenomysis in 30-60% of hysterectomy specimens. Pain, which could be divided into dysmenorrhoea, dyspareunia, and chronic pelvic pain, is one of the common and severe complaints in symptomatic women with adenomyosis. The prevalence of adenmyosis patients complained dysmenorrhoea was about 64.8%-77.8%. The patho-genic mechanism responsible for adenomyosis and its pain has not been well known as yet. nerve growth factor (NGF) is a key factor in pain producing and transfer. Previous studies indicated that the level of NGF was higher in ectopic endometnrium. It may take part in the important part of the mechanism of adenomyosis, especially for the pain.PartⅠThe expression of NGF in the ectopic endometrium of adenomyosis and its relationship with pain scale and innervationObjective:To investigate the expression of NGF in the ectopic endometrium from adenomyosis patients, and explore the relationship between NGF expression and innervation or pain scale.Methods:45 patients who had adenomyosis and underwent hysterectomy were chosen as experimental group,26 patients who had leiomyoma or Cervical intraepithelial neoplasiaⅢand underwent hysterectomy were chosen as control group. Ectopic endometrium from experimental group and eutopic endometrium from control group were collected in the surgery. The expression of NGF was examined by immunohistochemistry. The density of PGP9.5 positive nerve fibers was detected by immuno-fluorescence. The degree of dysmenoreal, chronic pelvic pain and dyspareunia was detected by Visual rating scale (VAS).Results:The expression of NGF and the density of PGP9.5 positive nerve fibers were higer in adenomyosis pain group than adenomyosis painless and control group. The NGF level and density of PGP9.5 positive nerve fibers in severe dysmenorrheal group were higher than light dysmenorrheal group and painless group in adenmyosis patients. There was no difference of NGF expression in chronic pelvic pain group and no chronic pelvic pain group, so was dyspareunia group and no dyspareunia group. The expression of NGF was correlated with the density of PGP9.5 positive nerve fibers in adenomyosis nodules (r=0.31, p<0.05)Conclusions:the enhanced expression of NGF in ectopic endometrium of adenomyosis might take part in the mechanism of pian, and improving innervation may be one of its ways.PartⅡThe expression of NGF and its receptors in uteri and dorsal root ganglias in mice model with adenomyosisObjective:first, to establish ICR mouse model of adenomyosis with tamoxifen and characterize the adenomyosis nodules, angiogenesis and uterine inflammation in this animal model. Then, examine the expression of NGF and its receptors in uteri or dorsal root ganglias (DRGs) at different stages of estrous cycle and course of adenomyosis.Methods:ICR mice with adenomyosis were induced by orally administration of tamoxifen on days 2-5 after birth. Adenomyosis modle mice and control mice were killed on day 42,85-95,135-145,185-195 and 235-245, respectively. Hematoxylin and eosin (HE) staining was performed to observe the adenomyosis nodules. Microvessel density, diameter and the ratio of area in the uterus were assessed by immunohistochemistrial staining with CD31. The gene expression of bradykinin receptor-1 (BKR-1) and neurokinin1 receptor (NK1-R) was detected by RT-PCR. The expression of NGF and its receptors, tyrosine kinase receptor (trkA) and p75 neurotrophin receptor (p75NTR), in the uteri and dorsal root ganglias (DRGs) of adenomyosis and control mice in 4 age groups of 85-95,135-145,185-195 and 235-245 by western bolt, immunochemistry and real time reverse transcription PCR.Results:All mice dosed with tamoxifen developed adenomyosis with a incidence rate of 100%. The microvessel density and the ratio of area in myometrium of experimental mice were significantly higher than those of the controls (P<0.05). In the uterus of 135-135 d dosage mice, the mRNA expression of BKR-1 and NK1-R was significantly higher than that of controls (P<0.05). In uteri of adenomyosis and control mice, NGF immunoreactivity was detected predominantly in the luminal, glandular epithelial cells and stromal cells of endometrium. Meanwhile, immunolocalization of p75NTR was detected mainly in stromal cells of endometrium and trkA in nerve fibers as well as luminal and glandular epithelial cells. The protein level of NGF, trkA and p75NTR in uteri and mRNA expression of trkA in DRGs were higher in aggravated adenomyosis mice than controls. Based on the data of western blot, the expression of NGF and its receptors in uteri were fluctuated as the estrous cycle changed, and increased gradually while age growing for adenomyosis mice but changed little in control mice. Results of real time reverse transcription PCR also revealed that the mRNA level of trkA in DRGs was increasing as age growing in adenomyosis mice.Conclusions:Adenomyosis induced in mice by tamoxifen has abnormal angiogenesis, inflammation and pain related receptor expression in the uterus. The reinforced expression of NGF and its receptors in uteri and DRGs of adenomyosis mice and the gradual increasing in aggravated adenomyosis might hint their participation in the mechanism of adenomyosis, especially for the pain.PartⅢthe impact factors of NGF expression by adenomyosise endometrium stromal cell and the effect of NGF on adenomyosis endometrium stromal cellObjective:To investigate the factors which may impact the NGF expression of adenomyosis endometrial stromal cell (ESC), and the effect of NGF on ESC from the way of cell proliferation, apoptosis and estrogen synthesisMethods:Patients who had adenomyosis and underwent hysterectomy were chosen as experimental group. Patients who had leiomyoma or cervical intraepithelial neoplasiaⅢand underwent hysterectomy were chosen as control group. Ectopic endometrium from experimental group and eutopic endometrium from control group were collected in the surgery. ESC was separated and cultured. The expression of NGF and its two receptors(p75NTR and trkA) were displayed by Immuno-fluorescence.17β-estradiol, TNF and CoCl2 were added in the culture system, respectively. The expression of NGF by adenomyosis and control ESC was examined by western-bolt. NGF was added in the culture system and the proliferation of ESC was detected by MTT. Then, the mRNA and protein level of aromatase were detected by real time RT-PCR and western-bolt, respectively. H2O2 or CoCl2, as well as NGF were added in the culture system, and than the apoptosis rate of ESC was examined by MTTResults:Both adenomyosis ESC and control ESC expressed NGF and its receptor p75NTR. Neither of them expressed trkA.17β-estradiol could promote the NGF expression by adenomyosis ESC but not control ESC. TNF could promote the NGF synthesis by both adenomyosis and control ESC. CoCl2 could inhibit the control ESC to synthesize NGF, but have no effect on NGF synthesis by adenomyosis ESC. NGF could promote the proliferation and the synthesis of aromatase of ESC from adenomyosis patients. However, NGF had no effect on the protection of adenomyosis and control ESC from H2O2 and CoCl2 induced apoptosis.Conclusions 17β-estradiol and TNF added in the culture system of ESC from patients with adenomyosis could stimulate the expression of NGF; and NGF could promote the proliferation and the expression of key enzyme in estrogen synthesis of ESC from in patients with adenomyosis.更多还原