【作者】 刘志能；

【导师】 姚珍薇；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2008， 博士

【摘要】 子宫内膜异位症(EMs)是妇科的常见病、多发病,约有10%~15%的育龄妇女罹患此病。EMs的发病机制尚未阐明,现有的手术治疗及药物治疗方案均难以达到满意的效果。因此进一步探讨EMs的发病机制及寻求新的治疗靶点尤为必要。EMs具有类似肿瘤的粘附、侵袭、转移等生物学行为,二者在发病机制上可能具有一定的相似性,而间隙连接细胞间通讯(GJIC)功能缺陷或低下是肿瘤的普遍特征,因此我们推测在EMs的发病过程中可能也存在GJIC功能的异常。GJIC是相邻细胞间传递信息的主要方式,它在维持机体组织内环境的稳定、调节正常细胞的生长等方面极为重要。细胞GJIC功能受其结构单位连接子的调控,圆柱状的连接子含有一个亲水通道,它允许离子(Na+、K+、Ca2+等)及分子量小于1000的小分子(cAMP、cGMP、IP3等)自由通过。每个连接子由6个蛋白亚单位(Cx)组成,啮齿动物和人的子宫内膜中表达的连接蛋白主要有Cx43、Cx32、Cx26三种。在EMs的发病过程中,间质细胞起着重要的主导作用,正常间质细胞具有完善的GJIC功能,并且能通过间隙连接影响上皮细胞的GJIC功能状态,故EMs内膜间质细胞的GJIC功能理应受到更多的关注。研究发现全反式维甲酸(ATRA)具有上调肿瘤细胞Cx表达和GJIC功能的作用,从而诱导肿瘤细胞恢复良性表型。ATRA对正常子宫内膜间质细胞也有类似的上调作用,但尚不清楚ATRA是否能调节EMs内膜间质细胞Cx表达及GJIC功能。探讨这些问题将为ATRA尝试用于EMs的治疗提供理论依据。本研究分为三个部分:(1)建立EMs在位、异位和正常内膜的间质细胞和上皮细胞体外培养模型;(2)通过体外培养的内膜细胞分析EMs内膜间质细胞Cx表达及GJIC功能的差异;采用反义寡聚核苷酸技术抑制正常内膜间质细胞Cx的表达,了解GJIC功能抑制后,正常内膜间质细胞的生长、粘附及侵袭能力是否增强,以此全面分析间质细胞的GJIC功能在EMs发病中的作用;(3)探讨ATRA对EMs在位及异位内膜间质细胞GJIC功能的调节作用及相关机制,以初步解答ATRA用于EMs治疗的可能性。第一部分EMs在位、异位及正常内膜间质细胞和上皮细胞的体外培养目的:探索一种获得高产率及高纯度子宫内膜间质细胞和上皮细胞的分离培养方法,同时建立EMs在位、异位及正常内膜细胞的体外培养模型,并尝试细胞的冻存与复苏,为后续实验奠定基础。方法:选取重庆医科大学附属第一医院妇产科2006年2月~2007年1月住院行手术治疗的内异症患者41例,其中合并单侧或双侧卵巢巧克力囊肿24例;取同期因子宫肌瘤行子宫全切术的患者30例作对照。刮取靠近子宫底部的内膜组织或剥取卵巢巧克力囊肿的内壁。借鉴传统的细胞分离方法并加以改良,采用机械碎化、多种酶联合消化、细胞滤网分选、不同细胞贴壁时间差而将间质与上皮细胞分离纯化。结果:间质细胞与上皮细胞同时成功培养率分别为:异位内膜组33.3%(8/24)、EMs在位内膜组92.7%(38/41)、对照组93.3%(28/30),两组在位内膜间质细胞纯度达98%,异位内膜间质细胞纯度为95%;两组在位内膜上皮细胞纯度达95%,异位内膜上皮细胞纯度为92%。上皮细胞和间质细胞均能成功地冻存与复苏。结论:改良的分离方法可提高间质细胞与上皮细胞的产率与活力,而且两种细胞的纯度较高,能完全满足后续实验的要求。第二部分子宫内膜间质细胞的Cx表达及GJIC功能在EMs发病中的意义目的:找出EMs内膜间质细胞Cx表达及GJIC功能的差异,探讨间质细胞GJIC功能异常在EMs发病过程中的意义。方法:(1)以免疫荧光法检测EMs异位、在位内膜以及正常子宫内膜间质细胞和上皮细胞Cx43、Cx32、Cx26的表达。(2)采用荧光漂白后恢复技术分析三组内膜间质细胞和上皮细胞的GJIC功能,并探讨共培养体系中EMs内膜间质细胞对不同上皮细胞GJIC功能的调节作用。(3)通过Lipo介导下的反义寡聚核苷酸技术(ASODNs),特异性下调正常间质内膜细胞中Cx43蛋白表达,分析间质细胞的生长、粘附及侵袭能力的改变。结果:Cx43在对照组间质细胞、EMs在位内膜间质细胞、异位内膜间质细胞中表达依次降低;Cx32、Cx26仅表达于两组在位内膜的上皮细胞中,且两组间的表达量相同;异位内膜上皮细胞出现Cx43的异常表达。对照组、EMs在位内膜及异位内膜三组的上皮细胞GJIC功能无明显差异,其间质细胞GJIC功能则按对照组→EMs在位组→EMs异位组依次减弱。EMs在位、异位内膜间质细胞对不同上皮细胞GJIC的调节作用均较对照组减弱。间质细胞的GJIC功能以及对上皮细胞的调节能力与其Cx43的表达量密切相关。ASODNs+Lipo的转染效率较高,对目的蛋白Cx43的抑制作用明显,撤除ASODNs+Lipo后,抑制效果至少可持续48h以上。GJIC功能被抑制后,正常间质细胞表现出生长加速,粘附、侵袭能力增强。结论:间质细胞Cx43表达降低、GJIC功能减弱与EMs发病关系密切,间质细胞的Cx43或GJIC功能可能是EMs潜在的治疗靶点。第三部分ATRA对EMs内膜间质细胞Cx43表达、GJIC功能的调节作用及相关机制目的:研究全反式维甲酸(ATRA)对EMs在位及异位内膜间质细胞Cx43表达和GJIC功能的调节作用及其相关机制,初步探讨ATRA用于EMs治疗的可能性。方法:(1)采用不同浓度的ATRA处理EMs在位及异位内膜间质细胞,并检测其GJIC功能及Cx43 mRNA和蛋白的表达。(2)引入雌激素受体(ER)拮抗剂ICI 182 780,分析ATRA的作用与ER的关系。(3)引入TPA(佛波酯,系Cx磷酸化诱导剂),分析ATRA的作用与Cx43蛋白磷酸化状态的关系。结果:ATRA浓度为1μmol/L和10μmol/L时,均能有效上调EMs内膜间质细胞的GJIC功能,但上调作用至72h以后不再继续增加;ATRA浓度为0.1μmol/L时则没有上调作用。1μmol/L ATRA能诱导间质细胞Cx43 mRNA及蛋白的表达。不同浓度的ICI 182 780均不影响ATRA对间质细胞GJIC功能的上调作用。TPA能抑制ATRA对间质细胞GJIC功能的上调作用。结论:ATRA可上调EMs在位及异位内膜间质细胞的GJIC功能,且上调效果与ATRA的作用时间及浓度有关; ATRA能从基因和翻译水平促进Cx43的表达,诱导或维持Cx43蛋白的去磷酸化形式,从而上调EMs内膜间质细胞的GJIC功能。更多还原

【Abstract】 Endometriosis (EMs) is a common gynecological disorder, with an incidence of 10-15% in reproductive aged women. The pathogenesis of endometriosis remains unclear, and current therapeutic effects are not satisfied. New strategies against EMs are urgently needed.Similar to tumors’biological behaviors, EMs has the nature of adhesion, invasion and metastasis, which suggests that EMs and some tumors share the same mechanisms of pathogenesis. The dysfunction of gap junctional intercellular communication (GJIC) occurred in cancer cells, suggesting that aberrant GJIC might involve in the development of EMs.GJIC is the main way for adjacent cells to communicate and transmit signal, which is necessary for maintenance of tissue homeostasis. Functional GJIC is mediated by connexons consisting of proteinaceous cylinders with a hydrophilic channel. The channels allow ions (Na+, K+, Ca2+, etc) and small molecules (bellow 1000Da, such as cAMP, cGMP, IP3) to travel freely. Each connexon, structural units of GJIC, was composed of a hexamer of connexin (Cx). There are three Cxs, Cx43, Cx32, Cx26, expressing in rodents and human endometrium.Endometrial stromal cells may play an important role in the development of EMs. Normal stromal cells are with consummate function of GJIC, and they can modulate the status of GJIC in endometrial epithelial cells by gap junction.All-trans-retinoic acid (ATRA) can enhance Cx expression and up-regulate GJIC in many tumor cells, even induce the differentiation of cancers. ATRA can also up-regulate functional GJIC in normal stromal cells, but it is still ambiguity whether ATRA can modulate Cx expression and GJIC in endometrial stromal cells from patients with EMs.This study composed of three sections: (1) Stromal cells and epithelial cells were isolated from the ectopic endometrial lesions located in ovaries, eutopic endometria from women with EMs and normal endometria. (2) Endometrial stromal cells and epithelial cells ware cultured in vitro to explore the expression of Cx and functional GJIC. Antisense oligodeoxyribonucleotides (ASODNs) was used to suppress Cx expression in normal stomal cells. Cell growth, adhesion and invasion were investigated in normal stromal cells after treatment with ASODNs. The correlation between deficient GJIC in endometrial stromal cells and pathogenesis of EMs was analyzed. (3) ATRA was used to modulate the function of GJIC in stromal cells from EMs. The objective was to determine whether ATRA be an agent to cure EMs.PARTⅠIN VITRO CULTURE OF ENDOMETRIAL STROMAL AND EPITHELIAL CELLS FROM EMS AND NORMAL ENDOMETRIUMObjective: To establish an approach to purify and culture human endometrial stromal and epithelial cells from endometriotic lesions loctated in ovaries, eutopic endometria with EMs and normal endometria, respectively.Methods: The cases were randomly chosen from patients hospitalized receiving surgical interventions in Feb 2006-Jan 2007. Samples from eutopic endometria or endometriotic lesions were collected from 41 cases suffered from endometriosis, in all, 24 cases were accompanied with unilateral or bilateral chocolate cysts in ovaries. 30 cases received hysterectomy for uterine myoma were recruited as control. Endometrial tissue near the bottom of the uterus was scraped, and the lining of chocolate cyst located in ovary was striped. Digestion, purification and culture of endometrial stromal and epithelial cells were performed.Results: The success rate of isolation and culture was 33.3% (8/24) of endometriotic stromal and epithelial cells, 92.7% (38/41) of eutopic endometrial stromal and epithelial cells with EMs, and 93.3% (28/30) of normal endometrial stromal and epithelial cells. The purities of eutopic stromal and epithelial cells from endometrium with or without EMs were 98% and 95% respectively. The purities of ectopic stromal and epithelial cells were 95% and 92% respectively. Both stromal cells and epithelial cells could be cryopreservated and thawed successfully.Conclusion: The higher yields and viabilities were realized through this multistep procedure.PARTⅡTHE EXPRESSION OF CONNEXIN AND GJIC IN ENDOMETRIAL STROMAL CELLS IN THE DEVELOPMENT OF ENDOMETRIOSISObjective: To investigate the different expression of Cx and functional GJIC in stromal cells in endometriotic lesion and eutopic endometrium with EMs from those of in normal endometrium, and to reveal the correlation between deficient GJIC in endometrial stromal cells and the pathogenesis of EMs.Methods: (1) The immumofluorescence assay manifested the expression of Cx43, Cx32 and Cx26 protein in stromal and epithelial cells from endometriotic lesions, eutopic endometria with EMs and normal endometria. (2) Fluorescence recovery after photobleaching (FRAP) was applied to analyze the function of GJIC in stromal and epithelial cells with and without EMs. Furthermore, GJIC status in epithelial cells regulated by different stromal cells was evaluated in co-culture system in vitro. (3) The abilities of growth, adhesion and invasion were investigated in normal stromal cells after downregulating Cx43 with antisense oligodeoxyribonucleotides (ASODNs).Results: The level of Cx43 in endometrial stromal cells was gradually decreased from normal endometrium, eutopic endometrium with EMs to endometriotic tissue. Cx32 and Cx26 protein were observed in normal endometrial epithelial cells and eutopic epithelial cells from endometrium with EMs. There was no difference of Cx32 protein expression in epithelial cells between normal endometrium and eutopic endometrium with EMs, nor did Cx26 protein. Cx43 was aberrant expression in endometriotic epithelial cells. There was similar functional GJIC status in epithelial cells from control, eutopic endometrium with EMs and endometriotic lesion. However, the function of GJIC in stromal cells from ectopic endometrial tissues was lower than those from the other two groups, the highest functional GJIC was observed in normal endometrial stromal cells, and the distinctions among these groups were significant. The ability of regulation of GJIC in epithelial cells by stromal cells from endometriotic lesion or eutopic endometrium with EMs was weaker compared with those stromal cells from control group. Stromal cells’GJIC and ability to modulate epithelial cells were closely related to their Cx43 protein. Cx43 protein and functional GJIC were suppressed when normal stromal cells were transfected with ASODNs+Lipo, resulting in a faster growth, enhanced adhesion and promoted invasion.Conclusion: The down-regulation of Cx43 and attenuated GJIC were related to the pathogenesis of EMs. Cx43 or GJIC in endometrial stromal cells might be used as potent target for treatment of EMs. PARTⅢMECHANISMS OF REGULATION OF CONNEXIN43 EXPRESSION AND GJIC IN STROMAL CELLS FROM ENDOMETRIOTIC LESIONS AND ENDOMETRIA WITH ENDOMETRIOSIS BY ATRA IN VITROObjective: To explore mechanisms involving in the regulation of GJIC by ATRA in stromal cells from endometriotic lesions and eutopic endometria with EMs.Methods: (1) Stromal cells from endometriotic lesions and eutopic endometria with EMs were treated with ATRA with concentrations of 0.1μmol/L, 1μmol/L and 10μmol/L for 24, 48, 72, 96 and 120 h, respectively, Laser scanning confocal microscope was applied to determine the function of GJIC in cells. The mRNA and protein levels of Cx43 were investigated. (2) ICI 182 780, an estrogen receptor downregulator, was introduced to analyze the relationship between the effects of ATRA and estrogen receptor. (3) An inductor, 12-o-tetradecanoylphorbol-13-acetate (TPA), was used to judge that the dephosphorylated form or phosphorylated species of Cx43 would contribute to the up-regulation of functional GJIC in stromal cells.Results: GJIC was enhanced significantly either in ectopic stromal cell or eutopic stromal cell from endometrium with EMs after being exposed to 1μmol/L and 10μmol/L ATRA. The up-regulation persisted up to 72h. No evidence for enhanced GJIC was confirmed in these stromal cells treatment with 0.1μmol/L ATRA. Treatment of stromal cells with 1μmol/L ATRA up regulated the expressions of Cx43 mRNA and protein. TPA inhibited the effect of ATRA on modulation of functional GJIC in ectopic stromal cells and eutopic stromal cells from endometrium with EMs. However, ICI 182 780 didn’t have the inhibitory effect similar to TPA.Conclusions: GJIC in ectopic stromal cells and eutopic stromal cells from endometrium with EMs was up-regulated by ATRA in a manner of time- and dosedependent. ATRA enhanced Cx43 expression on both gene and protein levels, and induced or maintained the dephosphorylated form of Cx43 protein in these stromal cells.更多还原