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山西医科大学
硕士学位论文
双酚A对子宫肌瘤细胞体外增殖的研究
申请学位级别:硕士
专业:妇产科学
指导教师:李美蓉
1双酚对子宫肌瘤细胞体外增殖的研究
通过研究环境雌激素双酚
,对体外培养的人子宫肌瘤细胞增殖的
影响,及其对,,.表达的影响,来探讨双酚能否促进子
宫肌瘤的生长,及其可能的作用机制,为子宫肌瘤的预防及治疗提供实验依据。
应用胶原酶消化法分离、获得子宫肌瘤细胞,免疫细胞化学法进行细胞学鉴定。
用不同浓度双酚×。/,×~/,×。/干预后,分别于、、、
应用法检测细胞的增殖活性,同时为后续研究筛选出有效作用时间点;应用.
检测经不同浓度干预后的各组肌瘤细胞的矾,壬,删埘的表达
.成功培养了人子宫肌瘤细胞,并经免疫细胞化学鉴定证实。
.各组子宫肌瘤细胞的体外生长曲线形态良好,溶剂对照组与子宫肌瘤组相比较,差异无
显著性尸.。以剂量.时间依赖性的方式促进子宫肌瘤细胞的增殖。作用时效
果不明显胗.,时开始稳步性升高.,时增殖效应最显著氏.。故
在后续实验中选取为研究的有效作用时间点。×Ⅵ组对肌瘤细胞
,,的表达无明显影响,在体外促进细胞增殖的作用微弱
胗.;浓度为一儿时,肌瘤细胞,酬,状的
表达显著上升,细胞的增殖活力也相应的上升尸.;当浓度达×~/时,
,斟,的表达进一步上升,肌瘤细胞的体外增殖能力显著上
.能够增加肌瘤细胞体外增殖活性,且存在着剂量.时间依赖性。
.,矾及在子宫肌瘤中高表达,诱导细胞异常增殖。
.用干预后,增加了肌瘤细胞,和的表达水平,增加
了肌瘤细胞的增殖活性,且对呈时间和浓度依赖性。
关键词:环境雌激素:双酚:子宫肌瘤
2山西医科大学硕十学位论文
: 之,,锄虢仆
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SULT1E1和雌,孕激素受体蛋白在子宫肌瘤中的表达及其临床意义
·中文论著摘要·
SULT1E1和雌,孕激素受体蛋白在子宫肌瘤中的表达及其临床意义
目的子宫肌瘤作为妇科最常见的良性肿瘤，对其病因的研究至今尚无定论。但目前普遍认为:子宫肌瘤是一种卵巢激素依赖性肿瘤，其生长与雌激素、孕激素、催乳素等内分泌激素有关，并且受M到局部多肽类生长因子的介导。但应用激素疗法及选择性使用激素受体调节剂及生长因子调节剂，均不能彻底治愈子宫肌瘤，因此提示还有其他因素参与子宫肌瘤的发生。长期以来，雌激素，孕激素一直被认为是子宫肌瘤发生和发展的促进剂，但与子宫肌瘤发生的关系迄今为止，尚不清楚。近年来硫酸基转移酶(SULTs)成为肿瘤研究的热点。SULTS超家族主要包括酚(SULT1)和羟类固醇(SULT2)两大亚族。SULT1E1属于硫酸基转移酶超家族一员，对雌激素催化作用最强，可使雌激素硫酸化，失去生物活性[1]。因此考虑SULT1E1可能通过调节性激素及其受体含量在子宫肌瘤的发生、发展中起着非常重要的作用。本研究采用免疫组织化学链霉菌抗生物素蛋白-过氧化酶染色法(S-P免疫组化法)，检测SULT1E1、雌激素受体亚型(ERα、ERβ)、孕激素受体亚型(PRα、PRβ)在子宫肌瘤中的表达，探讨SULT1E1、ERα、ERβ、PRα和PRβ与子宫肌瘤的发生、发展之间的关系。
方法 收集子宫肌瘤组织标本30例,相应肌瘤包膜外正常子宫肌层组织标本30例,因宫颈CINⅢ级或子宫脱垂行子宫切除术的正常子宫肌层组织标本10例,应用免疫组织化学链霉素抗生物素蛋白-过氧化酶连接法(S-P法),检测SULT1E1、ERα、ERβ和PRα ,PRβ在子宫肌瘤组织、肌瘤包膜外正常子宫肌层组织及正常子宫肌层组织中的表达。统计学处理采用spss11.0软件包。阳性率比较及相关性分析采用x~2检验和spearman等级相关分析。
结果1、SULT1E1的表达:在子宫肌瘤、肌瘤包膜外正常子宫肌层组织及正常子宫肌层组织SULT1E1表达的比较:30例子宫肌瘤组织中20例SULT1E1阳性表达(20/30)，30例肌瘤包膜外正常子宫肌层组织中27例阳性表达(27/30)，10例正常子宫肌层组织SULT1E1均阳性表达(10/10)，三组不同组织SULT1E1阳性率比较有非常显著性差异(χ2=8.06，P0.004);子宫肌瘤组与肌瘤包膜外正常子宫肌层组阳性率比较有非常显著性差异χ2=4.81，p=0.037);子宫肌瘤组与正常子宫肌层组织组SULT1E1阳性率比较亦有显著性差异(χ2=4.44，p=0.045)。2、ERα蛋白的表达:在子宫肌瘤、肌瘤包膜外正常子宫肌层组织及正常子宫肌层组织中ERα蛋白表达的比较:30例子宫肌瘤组织中29例ER;蛋白阳性表达(29/30)，30例肌瘤包膜外正常子宫肌层组织中21例阳性表达(21/30)，10例正常子宫肌层组织中7例阳性表达(7/10)，三组不同组织ERα蛋白阳性率比较有非常显著性差异χ2=8.06，p=0.004);子宫肌瘤组与肌瘤包膜外正常子宫肌层组织组ERα蛋白阳性率比较有非常显著性差异(χ2=7.68，p=0.008);子宫肌瘤组与正常子宫肌层组织组阳性率比较亦有显著性差异(χ2=5.92，p=0.018)。3、ERβ蛋白的表达:在子宫肌瘤、肌瘤包膜外正常子宫肌层组织及正常子宫肌层组织中ERβ蛋白表达的比较:30例子宫肌瘤组织中20例ERβ蛋白阳性表达(20/30)，30例肌瘤包膜外正常子宫肌层组织中10例阳性表达(10/30)，10例正常子宫肌层组织中3例阳性表达(3/10)，三种不同组织ERβ蛋白阳性率比较有非常显著性差异(χ2=7.95，p=0.003);子宫肌瘤组与肌瘤包膜外正常子宫肌层组织组ERβ蛋白阳性率比较有非常显著性差异(χ2=6.67，P=0.010);子宫肌瘤组与正常子宫肌层组 ERβ蛋白阳性率比较亦有显著性差异(χ2=4.126，P=0.042)。4、PR-A蛋白的表达:在子宫肌瘤、肌瘤包膜外正常子宫肌层组织及正常子宫肌层组织中PR-A蛋白表达的比较:30例子宫肌瘤组织中28例PR-A蛋白阳性表达(28/30)，30例肌瘤包膜外正常子宫肌层组织中21例阳性表达(21/30)，10例正常子宫肌层组织中6例阳性表达(6/10)，三种不同组织PR-A蛋白阳性率比较有非常显著性差异(χ2=7.24，P=0.009);子宫肌瘤组与肌瘤包膜外正常子宫肌层组PR-A蛋白阳性率比较有非常显著性差异(χ2=5.54，P=0.019);子宫肌瘤组与正常子宫肌层组阳性率比较亦有显著性差异(χ2=6.53，P=0.012)。5、PR-B蛋白的表达:在子宫肌瘤、肌瘤包膜外正常子宫肌层组织及正常子宫肌层组织中PR-B蛋白表达的比较:30例子宫肌瘤组织中28例PR-B蛋白阳性表达(28/30)，30例肌瘤包膜外正常子宫肌层组织中21例阳性表达(21/30)，10例正常子宫肌层组织中6例阳性表达(6/10)，三种不同组织PR-A蛋白阳性率比较有非常显著性差异(χ2=7.24，P=0.009);子宫肌瘤组与肌瘤包膜外正常子宫肌层组PR-B蛋白阳性率比较有非常显著性差异(χ2=5.54，P=0.019);子宫肌瘤组与正常子宫肌层组阳性率比较亦有显著性差异(χ2=6.53，P=0.012)。6、SULT1E1与ERα蛋白在子宫肌瘤组织中表达的相关性:相关系数R=-0.827，(P=0.001)。说明在子宫肌瘤组织中，SULT1E1与ERα蛋白之间存在负相关关系。7、SULT1E1与ERβ蛋白在子宫肌瘤组织中表达的相关性:相关系数r=0.192， (P=0.309)。说明在子宫肌瘤组织中，SULT1E1与ERβ蛋白之间无相关关系。8、SULT1E1与PR-A蛋白在子宫肌瘤组织中表达的相关性:相关系数r=-0.493， (P=0.006)。说明在子宫肌瘤组织中，SULT1E1与PR-A蛋白之间存在负相关关系。9、SULT1E1与PRβ蛋白在子宫肌瘤组织中表达的相关性:相关系数r=-0.538， (P=0.004)。说明在子宫肌瘤组织中，SULT1E1与PR-B蛋白之间存在负相关关系。
结论1、SULT1E1、雌激素受体ERα、ERβ和孕激素受体PRα、PRβ在子宫肌瘤、肌瘤包膜外肌层组织和正常子宫肌层组织中均有表达。2、SULT1E1在肌瘤包膜外肌层组织和正常子宫肌层组织中高表达，而雌激素受体ERα、ERβ、孕激素受体PRα、PRβ在肌瘤组织中高表达。说明SULT1E1可能是子宫肌瘤发生过程中的抑制因素。3、在子宫肌瘤组织中SULT1E1的表达与ERα、PRβ表达有相关关系且均呈负相关。SULT1E1与ERβ和PR-B蛋白之间无相关关系。4、在子宫肌瘤组织中ERα与PRα表达有相关关系且呈正相关。ERβ与PRβ蛋白之间无相关关系 5、SULT1E1和雌、孕激素受体蛋白的表达与子宫肌瘤的发生、发展有关。其机制可能为:SULT1E1可催化内源性雌激素发生硫酸化反应，形成雌激素无活性形式——硫酸化雌激素，这种复合物不能与雌激素受体结合而丧失活性，可降低循环及靶组织雌激素暴露水平。SULT1E1在子宫肌瘤组织中的低表达可以导致内源性雌激素水平升高，雌激素有上调PR含量的作用，PR与ER蛋白的表达强度呈正相关，间接升高ERα含量，促进子宫肌瘤生长。
关键词子宫肌瘤；雌激素受体α；雌激素受体β；孕激素受体α；孕激素受体β；SULT1E1；免疫组化。
·参考文献·
1. Hayashi S，Miharu N，Okamoto E et al. Detection of chromosomal abnormalities in Uterine leiomyoma using conventional cytogenetic method and interphase fluorescence in situ hybridization. Cancer Genet Cytogenet，1996，89(2):98
2. KovacsKA，Oszter A，Szabo I. Comparative analysis of cyelin Dl and oestrogen receptor (α and β)levels in human leiomyoma and adjacent myometrium. Mol Hum Report，2001，7(11):277-294
3. Bmdon DD，Bethea CL，Strawn EY. Progesterone receptor mesenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas.Am J Obs Gyn，2003，169:78-85
4. Bord S，Horner A，Beavan S， et al． Estrogen receptors alpha and beta are differentially expressed in developing human bone.J Clin Endocrinol Metab，2001，86( 5) : 2 309 －2&341
5. Zhang Guixiang, Zhao Xuedong, Estrogen receptor subtypes in the status quo. Foreign Medical Sciences (Obstetrics and Gynecology Volume), ): 352-355.
6. Pasqualini JR ,Chetrite GS.Estrone sulfatase versus estrone sulfotransferase in human breast cancer: potential clinical applications .J Steroid Biochem Mol Biol. -6):287-292.
7. Keiko Ka ,Hironobu Sasano ,Nobuhiro Harada ,Masahiko Ozaki , Shinji Sato and Akira Yajima. Aromatase in human common epithelial ovarian neoplasms . Am j Pathol. -50
8. Gurates B, Sebstian S, Yang S, et al. W T1 and DAX21 inhibit aromatase P450 expression in human endometrial and endometriotic stromal cell. J Clin Endocrinol Meta. ) :.
9. Papadim itriou CA ,M arkak i S, Siapkaras J , et al. Hormonal therapy withletrozole for relapsed epithelial ovarian cancer. Onco logy. ) : 112-117.
10.Seeger H,W allw iener D, Kraemer E, et al. Estradio l metabolites are potentmitogenic substances for human ovarian cancer cells. Eur J Gynaecol Onco l. ) : 383-385.
11. Vihko P, Harknen P, Oduwole O,et al.17beta-hydroxysteriod dehydrog-enases and cancers. J Steriod Biochem Mol Biol.-5):119-122.
12. Yu Wen, Jiang Ying, Wang Xiaoling. Uterine leiomyoma tissue ,oestrogene receptor& , progesterone& receptor
13. himomnra MabsuoH, SemotoT.UP a Regulation by Progesterone of Proliferating cell nuclear&antigen and epidermal growth factor expression in human uterine leiomyoma .Jclinendoemal Metab, 2 - 2198.
14. Min Wang SULT1A1 gene polymorphisms Arg213 His research in gynecological tumors progress Chinese Journal of Gynecology and Obstetrics, 2007, 02
15. Zhang Chuanying, Min. SULT1E1 gene in endometrial adenocarcinoma and its clinical significance [A]. Northeast Conference Proceedings Fourth Obstetrics and Gynecology [C], 200816 Min Wang, Christopher etc.Sulfation of tibolone metablites by human postmenopausal liver and small intestinal sulfotransferases.STEROIDS 71 (2006) :343-351.
17.郁雯，姜英，王晓玲。子宫肌瘤组织雌、孕激素受体和表皮生长因子受体的研究。肿瘤研究与临床，2002，14(4):234-236
·English abstrat·
The Study on the Expression of Sulfotransferases1E1、Estrogen Receptor α、Estrogen Receptorβ、Progesterone Receptorα，Progesterone Receptorβ in Uterine Leiomyoma and Its Clinical Significance
Objective Uterine leiomyoma is the most common gynecologic benign tumor. The case for the disease is unknown. A it is widely believed that uterine leiomyoma is Ovarian hormone-dependent tumor. Its growth is related to endocrine hormones such as estrogen、progesterone prolactin and it is mediated by local multi-peptide growth factor. But hormone therapy or the use of hormone receptor regulator agent can not cure uterine leiomyoma completely. So it points out that other factors are related to the formation of uterine leiomyoma. With the method of immune histochemistry (streptobacteria anti organism protein-peroxidase staining), we examine the expression of sulfotransferases1E1, estrogen receptorα、estrogen receptorβ and progesterone receptorα, progesterone receptorβ in uterine leiomyoma occurs, the relationship between their development.
Method Collect uterine leiomyoma specimen of 30 cases, normal myometrium tissues which are over 2cm far away the wrapping membrane of myoma 30 cases, and normal myometrium tissue of 10 cases. Examine the expression of sulfotransferases1E1、estrogen receptorα、estrogen receptorβ, progesterone receptorα and progesterone receptorβ in uterine leiomyoma tissue、normal myometrium out of uterine leiomyoma tissue and normal myometrium tissue with the method of immune histochemistry (streptobacteria anti organism protein-peroxidase staining).Using SPSS 17.0 software to do statistical analysis, using Test x~2 to compare the positive ratios and spearman analyze the correlation coefficieent
Results1.The expression of sulfotransferases1E1 (SULT1E1) protein:The positive ratios between uterine leiomyoma tissue and normal myometrium tissue show the significant difference(x~2=4.44 P =0.045). 2. The expression of estrogen receptorα (ERα) protein:The positive ratios of ERα protein show the very significant difference (χ 2 = 8.06, P=0.004).The positive ratios between uterine leiomyoma tissue and normal myometrium tissues far away the wrapping membrane tissue show the significant difference (χ 2 = 7.68, p = 0.008).The positive ratios between uterine leiomyoma tissue and normal myometrium tissue show the significant difference(x~2=5.92 P =0.018). 3. The expression of estrogen receptorβ (ERβ)protein:The positive ratios of ERβ protein is relatively very significant difference (χ 2 = 7.95, p = 0.003).The positive ratios between uterine leiomyoma tissue and normal myometrium tissues far away the wrapping membrane tissue show the significant difference(x~2=6.67 P=0.010).The positive ratios between uterine leiomyoma tissue and normal myometrium tissue show the significant difference(x~2=4.12 P =0.042). 4. The expression of progesterone receptorα (PRα) protein: There was a significant difference between the three groups on the expression rate .The positive ratios of PRα protein show the significant difference (χ 2 = 7.24, P = 0.009). The positive ratios between uterine leiomyoma tissue and normal myometrium tissues far away the wrapping membrane tissue show the significant difference(x~2=5054 P=0.019).The positive ratios between uterine leiomyoma tissue and normal myometrium tissue show the significant difference(x~2=6.53 P =0.012). 5. The expression of progesterone receptorβ (PRβ):There was a significant difference between the three groups on the expression rate .The positive ratios of PRβ protein show the very significant difference (χ 2 = 7.24, P = 0.009). The positive ratios between uterine leiomyoma tissue and normal myometrium tissues far away the wrapping membrane tissue show the significant difference(x~2=5054 P=0.019).The positive ratios between uterine leiomyoma tissue and normal myometrium tissue show the significant difference(x~2=6.53 P =0.012). 6. The correlative expression of SULT1E1 and ERα in uterine leiomyoma tissue:The rank correlation coefficient r=-0.827, (P=0.001).show SULT1E1 and ERα significantly negative correlated in uterine leiomyoma tissue. 7. The correlative expression of SULT1E1 and ERβ in uterine leiomyoma tissue:The rank correlation coefficient r=0.192, (P=0.309).show SULT1E1 and ERβ no correlated in uterine leiomyoma tissue. 8. The correlative expression of SULT1E1 and PRα in uterine leiomyoma tissue:The rank correlation coefficient r=-0.493,(P=0.006).show SULT1E1 and PR significantly negative correlated in uterine leiomyoma tissue.9. The correlative expression of SULT1E1 and PRβ in uterine leiomyoma tissue:The rank correlation coefficient r=-0.538,(P=0.510)。.show ERα and ERβ negative correlated in uterine leiomyoma tissue.
Conclusion 1. Sulfotransferases1E1, Estrogen receptor α, β, progesterone receptor α, β express in uterine leiomyoma tissue、normal myometrium tissue far away the wrapping membrane tissue and normal myometrium tissue. 2. Sulfotransferases1E1 expresses more in normal myometrium tissue. Estrogen receptorα, β, and progesterone receptorα, β express more in tissue. Sulfotransferases1E1 maybe play a suppression role in uterine myoma. (Inhibiting factor) 3. The Significant correlation in the expression of sulfotransferases1E1 and estrogen receptorα、PRα.in uterine myoma, Sulfotransferases1E1, estrogen receptorβ and progesterone receptor β no correlated in uterine leiomyoma tissue.4. The expression of correlation of ERα and PR ，PR has the positive correlation relationship in uterine leiomyoma tissue, ERβ and PR no correlated in uterine leiomyoma tissue. 5. The expression of sulfotransferases1E1、estrogen receptor and progesterone receptor is related to the invasion of uterine leiomyoma.
Keyword& U estrogen receptor α ; estrogen receptor β; progesterone receptor α ; progesterone receptor α ,progesterone receptor β ; SULT1E1; immunohistochemistry.
References
1. Hayashi S，Miharu N，Okamoto E et al. Detection of chromosomal abnormalities in Uterine leiomyoma using conventional cytogenetic method and interphase fluorescence in situ hybridization. Cancer Genet Cytogenet，1996，89(2):98
2.KovacsKA，Oszter A，Szabo I. Comparative analysis of cyelin Dl and oestrogen receptor (α and β)levels in human leiomyoma and adjacent myometrium. Mol Hum Report，2001，7(11):277-294
3.Bmdon DD，Bethea CL，Strawn EY. Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas.Am J Obs Gyn，2003，169:78-85
4.Bord S，Horner A，Beavan S， et al． Estrogen receptors alpha and beta are differentially expressed in developing human bone.J Clin Endocrinol Metab，2001，86( 5) : 2 309 －2&341
5.Zhang Guixiang, Zhao Xuedong, Estrogen receptor subtypes in the status quo. Foreign Medical Sciences (Obstetrics and Gynecology Volume), ): 352-355.
6. Pasqualini JR ,Chetrite GS.Estrone sulfatase versus estrone sulfotransferase in human breast cancer: potential clinical applications .J Steroid Biochem Mol Biol. -6):287-292.
7. Keiko Ka ,Hironobu Sasano ,Nobuhiro Harada ,Masahiko Ozaki , Shinji Sato and Akira Yajima. Aromatase in human common epithelial ovarian neoplasms . Am j Pathol. -50
8. Gurates B, Sebstian S, Yang S, et al. W T1 and DAX21 inhibit aromatase P450 expression in human endometrial and endometriotic stromal cell. J Clin Endocrinol Meta. ) :.
9. Papadim itriou CA ,M arkak i S, Siapkaras J , et al. Hormonal therapy withletrozole for relapsed epithelial ovarian cancer. Onco logy. ) : 112-117.
10.Seeger H,W allw iener D, Kraemer E, et al. Estradio l metabolites are potentmitogenic substances for human ovarian cancer cells. Eur J Gynaecol Onco l. ) : 383-385.
11. Vihko P, Harknen P, Oduwole O,et al.17beta-hydroxysteriod dehydrog-enases and cancers. J Steriod Biochem Mol Biol.-5):119-122.
12. Yu Wen, Jiang Ying, Wang Xiaoling. Uterine leiomyoma tissue ,oestrogene receptor& , progesterone& receptor
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