摘要
目的：采用大鼠子宫内膜异位症(Endometriosis,EMS)动物模型，研究中药制剂小柴胡汤(Xiao Chai Hu Tang,XCHT)对EMS大鼠异位内膜(Endometriotic Tissue,EOS)的影响及其可能的机制。
方法：(1)采用自身子宫内膜移植法建立大鼠EMS模型，分组给予不同剂量XCHT、丹那唑(Danazol)治疗4周。(2)通过观察、测量EOS治疗前后体积，了解药物对EOS的作用。(3)医学图像定量分析EOS子宫内膜上皮高度(H)、腺细胞核体积(V)、核体积积分光密度(Viod)。(4)透射电子显微镜下观察各组在位子宫内膜(Endometrium，EM)、EOS超微病理学特征。(5)免疫组化观察、分析各用药组EM及EOS的Caspase-3、Fas蛋白表达特征。
结果：(1)成功建立了大鼠EMS模型。(2)与对照组比较，中、高剂量XCHT组及丹那唑组对EOS的抑制作用明显，EOS体积较治疗前明显减小(P＜0.01)。(3)医学图像定量分析结果显示，中、高剂量XCHT组及丹那唑组子宫内膜上皮高度、核体积、核体积积分光密度较对照组明显减小(P＜0.01)。(4)透射电镜下可见中、高剂量XCHT组治疗后较多腺上皮细胞出现凋亡特征，表现为细胞体积变小、核固缩、胞浆和核染色质凝集，密度增高，细胞间凋亡小体，同时基质细胞中可见一些坏死细胞。(5)免疫组化结果显示，对照组EM中凋亡相关蛋白Caspase-3、Fas的表达水平明显高于EOS；而XCHT组恰恰相反，丹那唑组差异不明显。
结论：(1)EMS发生、发展与EOS本身细胞凋亡水平异常有关。(2)XCHT对EMS大鼠EOS的生长、增殖具有抑制作用。(3)XCHT治疗EMS的机理可能通过免疫调节方式诱导EOS腺上皮细胞凋亡、坏死而起作用。(4)XCHT对EMS大鼠EOS生长增殖的抑制作用与丹那唑的作用机理不尽相同。
Objective: To investigate the effects of Xiao Chai Hu Tang (XCHT) on endometriotic tissue (EOS) and explore the probable mechanisms by means of the Endometriosis (EMS) rat model.
Methods: (1) The rat EMS model established by surgical operation is divided into groups to give different concentration XCHT and Danazol to cure 4 weeks. (2) By observing and measuring EOS' volume before and past treatment, to explore the inhibition of XCHT and Danazol. (3)height of endometrial epithelium(H) , volume of glandular nucleus(V) and volume integrated optical density(Viod) of glandular nucleus in EOS were investigated by the morphometry method.(4) Ultrastructural change in EM and EOS of each group were observed by electron microscopy. (5)Caspase-3 and Fas protein expression in EM and EOS of each group were observed and analyzed by immunohistochemistry method.
Results: (1) The rat models of surgically induced endometriosis was established successfully.(2)Compared with control group , the EOS'volume of middle or high-concentration XCHT and Danazol group was obviously reduced(P<0.01).(3)the diminution of the H and V and Viod in the middle or high-concentration XCHT and Danazol groups were more significant than the control group(P<0.01).(4) the glandular cell in the endometrial implant after therapy with middle or high-concentration XCHT showed characteristic features of apoptosis which displayed by the cell decreased in size, karyopyknosis , cytoplasmic and nuclear chromatin condensation , density increased and a lot of apoptotic bodies among cell. Whereas some stromal cell displayed degeneration and necrosis. (5)The protein expression of Caspase-3 and Fas in control group's EM were higher than it EOS; while XCHT group was on the other way round and the difference was not obvious in Danazol group.
Conclusions: (1)The abnormal apoptosis level play a important role in Endometriosis (2)XCHT could inhibit the growth and proliferation of EOS in rat EMS model. (3) The probable mechanisms that XCHT inhibit EMS was that cell apoptosis and necrosis had induced by XCHT immuno-modulation.
引文
[1] 连利娟．林巧稚妇科肿瘤学．北京：人民卫生出版，1996年第3版：757
[2] Brincat M, Galea R, Buhagiar A. Polycystic ovaries andendomet rlosis: a possible connection. Br J Obstet Gynecol, 1994: 101(4): 346
[3] Vander Linden PJ, Dunselman GA, De Goeij AF, et al. Eipithelial cells in peritoneal fluid of endometrial origin. Am J Obstet Gynecol, 1995, 173:566
[4] 皇甫俊惠．子宮内膜异位症病因研究进展．国际医药卫生导报，2003，9(7)：113
[5] Rajkumar K, Schott PW, Simpson CW, et al. The rat as an animal model for endometriosis to examine recurrence of ectopic endometrial tissue after regression. Fertil Steril, 1990, 53(5):921
[6] 纪巍，宋晖，陈必良．子宮内膜异位症的发病机制研究．国外医学妇幼保健分册，2000，11(4)：145
[7] Oosterlynck DJ, Meuleman C, Lacquet FA, et al. Flow cytometry analysis of lymphocyte subpopulations in peritoneal fluid of women with endometriosis. Am J Reprod Immunol, 1994, 31(1):25
[8] 令狐华，徐小蓉，卞度宏．子宫内膜异位症患者外周血及腹腔液中自然杀伤细胞活性的测定．中华妇产科杂志，1996，31(10)：586
[9] 殷红梅、王苏荣、戴淑真，等．子宮内膜异位症患者T辅助细胞亚群功能失衡的研究．中华妇产科杂志，2000，35(6)：327
[10] Kennedy S. Is there a genetic basis to endometrlosis. Semin Repord Bndocrin, 1997, 15(3):309
[11] Koninckx PR, Barlow D, Kennedy S. Implantation versus infiltration: the sampson versus the endometriotic disease theory. Gynecol Obstet Invest, 1999, 47(Suppll):3
[12] Fukunaga M, Nomura K, Ishikawa E, et al. Ovarian atypical endometriosis: its close association with malignant epithelial tumours. Histopathology, 1997, 30(3):249
[13] Gebel HM, Braun DP, Tambur A, et al. Spontaneous apoptosis of endometrial
tissue is impaired in women with endometriosis. Fertil Steril, 1998,69(6):1042
[14] Harada M, Suganuma N, Furuhahi M, et al. Detection of apoptosis in human endometriosis tissues. Mol Hum Reorod, 1996, 2(5):307
[15] Braun DP, Ding J, Shen J, et al. Relationship between apoptosis and the number of macrophages in eutopic endometrium from women with and without endometriosis. Fertil Steril, 2002, 78(4):830
[16] Shawki O, Bahnasi A. Apoptosis and angiogenesis in endometriosis: relationship to development and progression. Fertil Steril, 2002, 77(2suppll):s44
[17] Meresman GF, Vighi S, Buquet RA, et al. Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis. Fertil Steril, 2000, 74(4):760
[18] Goumenou A, Panayiotides I, Matalliotakis I, et al. Bcl-2 and Bax expression in human endometriotic and adenomyotic tissues. Eur J Obstet Gynecol Reprod Biol, 2001, 99(2):256
[19] 王云霞，李亚里，黄靖香．凋亡调节基因bcl-2，bax和fas在子宫内膜异位症中的表达．解放军医学杂志，2001，26(1)：69
[20] 高颖，罗丽兰，何福仙．异位子宫内膜细胞的凋亡与增殖．中华妇产科杂志，1999，34(9)：536
[21] 陈杰，潘晓玲．EM药物进展．中国处方药，2002，11(9)：48
[22] 陈德甫，吴宇芬，狄文．子宫内膜异位症用雷公腾多甙治疗．上海中医药杂志，1994，28(9)：6
[23] 李继俊．棉酚引起雌鼠实验性子宫内膜异位症的快速消退．现代妇产科进展，1994，3(1)：15
[24] 杨洪艳，欧阳惠卿，罗颂平，等．丹那唑、罗氏内异方对实验性子宫内膜异位症大鼠血液流变性影响．安徽中医学院学报，2001，20(2)：45
[25] 周真．小柴胡汤与肿瘤坏死因子、干扰素、细胞白介素关系的研究．中医研究，1999，12(6)：15
[26] 李玉梅．小柴胡汤在肝癌防治中的研究进展．中西医结合肝病杂志，1999，9(6)：60
[27] 计惠民，徐归燕．肝硬变的汉方治疗．国外医学·中医中药分册，1998，20(4)：28
[28] Okita K, Li Q, Murakamio T, et al. Anti-growth effects with components of Sho-saiko-to (TJ-9) on cultured human hepatoma cells. Eur J Cancer Prey, 1993, 2(2):169
[29] Yano H, Mizoguchi A, Fukuda K, et al. The herbal medicine sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G_0/G_1 phase. Cancer Res, 1994, 54(2):448
[30] 王雅新．小柴胡汤抑制黑素瘤发病．国外医学·中医中药分册，2001，23(4)：245
[31] Ueda S, Nakamura H, Masutani H, et al. Baicalin induces apoptosis via mitochondrial pathway as prooxidant. Molecular Immunology, 2002, 38(10):781
[32] Leonard S, Robert S, Nelson, et aI. PC-SPES: herbal formulation for prostate cancer. Urology, 2002, 60(3):369
[33] Chenn S. In vitro mechanism of PC spes. Urology, 2001, 58(2s1):28
[34] Liu W, Kato M, Akhand AA, et al. The herbal medicine sho-saiko-to inhibits the growth of malignant melanoma cells by upregulating Fas-mediated apoptosis and arresting cell cycle through downregulation of cyclin dependent kinases. Int J Oncol, 1998, 12(6):1321
[35] 杨奎，张德波，史焱，等．含黄芩血清及黄芩甙影响内生致热原产生的研究．中药药理与临床，1994，10(6)：13
[36] 王雪峰，刘芳，魏克伦．小柴胡汤及其分解剂对柯萨奇B_(3Ⅲ)病毒感染乳鼠心肌保护及细胞免疫调节作用的研究．中国中西医结合杂志，2000，20(8)：599
[37] 同心．口服小柴胡汤对肺局部免疫反应有调节作用．国外医学·中医中药分册，2001，23(4)：封3
[38] 阿部博子．小柴胡汤对大鼠和小鼠肾上腺及免疫作用的研究．国外医学·中医中药分册，1995，3(5)：1861
[39] 山辅昌由，等．小柴胡汤对癌症患者末梢单核细胞IL-1、IL-6及GM-CSF产生功能的影响．[日]和汉医学会志，199l，8(3)：254
[40] Masaono I．小柴胡汤和大柴胡汤对大耳白兔实验性动脉粥样硬变的影响．国外医学中医药分册，1990，12(1)：21。
[41] 徐瑶，卞国武，吴敏毓．小柴胡汤对小鼠腹腔巨噬细胞释放NO的影响．中药新药药理与临床，2000，11(9)：289
[42] 周红．继发于造血干细胞移植后的免疫缺陷与小柴胡汤的免疫调控．中医药研究，1999，15(2)：4
[43] 长谷川格．小柴胡汤对人末梢血单核细胞产生IL-4以及小鼠脾细胞产生IL-6的影响．[日]和汉医学会志，1990，7(3)：280
[44] 张纪立．小柴胡汤的药理研究与临床应用．浙江中西医结合杂志，1994，4(2)：9
[45] 竹林治夫．小柴胡汤对脾有丝分裂刺激活性的影响．[日]国外医学中医学分册，1994，10(2)：299
[46] 唐已婷，陈家旭．小柴胡汤实验研究进展．中国医药学报，2000，15(6)：59
[47] 景浩．小柴胡汤治疗变应鼻炎的实验研究．辽宁中医杂志，2001，28(2)：124
[48] 郑辉，李洪义，王自能，等．丹那唑治疗大鼠子宫内膜异位症的时间相关作用．中国病理生理杂志，2001，17(5)：422
[49] Sakata M,Terakawa N, Mizutani T, et al. Effects of danazol, gonadotropin-releasing hormone agonist, and a combination of danazol and gonadotropin-releasing hormone agonist on experimental endometriosis. Am J Osstet Gynecol, 1990, 163(5pt1):1679
[50] 夏潮涌，夏志坚．睾丸组织原位三维测量和分析生精细胞核的DNA含量与倍体．中国体视学与图像分析，1998，3(1)：22
[51] Gundersen HJ. The nucleator. J Microsc, 1988, 151(pt1):3
[52] Gundersen HJ, Bagger P, Bendtsen TF, et al. The new stereological tools:disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS, 1988, 96(10):857
[53] 毛熙光，彭芝兰，郑莹，等．FAS基因蛋白在人子宫内膜的表达及意义泸州医学院学报，1998，21(5)：361
[54] 刘学军，何援利．子宫内膜异位症动物模型的建立及应用．中国实用妇科与产科杂志，2002，18(7)：444
[55] Vinatier D, Orazi G, Cosson M, et al. Theories of endometriosis. Eur J Obstet Gynecol Reprod Biol, 2001, 96(1):21
[56] D'Hooghe TM. Clinical relevance of the baboon as a model for the study of
endometriosis. Fertil Steril, 1997, 68(4):613
[57] D'Hooghe TM, Bambra CS, Raeymaekers BM, et al. Intrapelvic injection of menstrual endometrium causes endometriosis in baboons (Papio cynocephalus and Papio anubis).Am J Obstet Gynecol, 1995, 173(1):125
[58] Jones RC. The effect of a luteinizing hormone-releasing hormone antagonist on experimental endometriosis in the rat. Acta Endocrinol(Copenh), 1987, 114(3):379
[59] Rajkumar K; Schott PW; Simpson CW, et al. The rat as an animal model for endometriosis to examine recurrence of ectopic endometrial tissue after regression. Fertil Steril, 1990, 53(5):921
[60] Vinatier D, Cosson M, Dufour P, et al. Is endometriosis an endometrial disease? Eur J Obstet Gynecol Reprod Biol, 2000, 91(2):113
[61] Johannisson E, Brosens I, Cornillie F, et al. Morphometric study of the human endometrium following continuous exposure to levonorgestrel released from vaginal rings during 90 days. Contraception, 1991, 43(4):361
[62] Johannisson E, Landgren BM, Diczfalusy E, et al. Endometrial morphology and peripheral steroid levels in women with and without intermenstrual bleeding during contraception with the 300 microgram norethisterone (NET) minipill. Contraception, 1982, 25(1):13
[63] Donnez J. Today's treatments:medical, surgical and in partnership. Int J Gynaecol Obstet, 1999, 64(Suppl 1):s5
[64] Matsuzaki Y, Kurokawa N, Terai S, et al. Cell death induced by baicalein in human hepatocellular carcinoma cell lines. Jpn J Cancer Res, 1996, 87(2):170
[65] 王淳，陈誉华，陈澄．Paraptosis—一种新的细胞死亡方式．解剖科学进展，2002，8(3)：257
[66] 梅菠．GnRHa治疗子宫内膜异位症机制的实验研究．中山大学医学院2002届硕士学位论文，2002年，第26页
[67] 赵建武，令狐华，徐小荣．子宫内膜异位症患者在位内膜Fas、FasL的表达及其意义．贵州医药 2001，25(4)：296
[68] Dmowski WP, Ding J, Shen J, et al. Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis. Hum Reprod, 2001, 16(9):1802
[69] Kusuki I, Kitawaki J, Ishihara H, et al. Immunohistochemical localization of aromatase and apoptosis-associated proteins in ovarian serous cystadenocarcinoma arising from ovarian endometriosis. Eur J Obstet Gynecol Reprod Biol, 2001, 98(1):114
