摘要
目的:临床实验研究主要从基因及蛋白水平探讨肾虚型早期自然流产与孕激素受体(PR)、转化生长因子-beta1(TGF-β1)的相关性以及外周血单个核细胞TGF-β 1mRNA与蜕膜TGF-β 1mRNA的相关性;动物实验研究主要从免疫调控角度进一步探讨补肾健脾中药助孕3号方防治自然流产的作用机理。
方法:
1 肾虚型早期自然流产患者蜕膜黄体效应、TGF-β1表达的相关研究:
收集在广州中医药大学第一附属医院就诊的流产患者,选择肾虚型早期自然流产清宫孕妇23例为实验组,设立非肾虚型早期自然流产清宫孕妇40例(血瘀型20例,血热型20例)和正常早期妊娠的孕妇要求人工流产者20例为对照组。于吸宫前刮取5克蜕膜组织,运用RT-PCR方法检测蜕膜PRmRNA、TGF-β 1mRNA及外周血单个核细胞TGF-β 1mRNA的表达,同时采用免疫组化法(LSAB)检测蜕膜PR、TGF-β1的表达,并在光镜下观察蜕膜组织形态结构。
2 助孕3号方对肾虚黄体抑制妊娠流产大鼠血清TGF-β1影响的研究:
雄性10-12周龄SPF级SD大鼠,雌性8-10周龄SPF级SD大鼠,体重约200～250克。将SD大鼠雌雄合笼,每日晨阴道涂片,以发现大量精子者作为妊娠第1天。以羟基脲与米非司酮建立肾虚黄体抑制大鼠流产模型,妊娠第1至7天羟基脲灌胃450mg/Kg/天,用生理盐水配制,1ml/日;第7天时给予米非司酮(3.75mg/kg),用生理盐水配制,1ml/日。共分5组:A多力玛组(羟基脲+米非司酮+多力玛),造模的同时,用多力玛1.5mg/kg/日灌胃,用生理盐水配制,2ml/日,连续7天;B造模组(羟基脲+米非司酮);C助孕3号方高剂量组(羟基脲+米非司酮+助孕3号方高剂量组),造模同时,用助孕3号浓缩干粉7.08g/kg/日灌胃(成人等效剂量2倍),用生理盐水配制,3ml/日,连续7天;D助孕3号方低剂量组(羟基脲+米非司酮+助孕3号方低剂量组),造模同时,用助孕3号浓缩干粉3.54g/kg/日灌胃(成人等效剂量)用生理盐水配制,2ml/日,连续7天;E空白对照组(生理盐水),用生理盐水灌胃,4ml/日,连续7天。观察孕鼠的一般情况、流产率等。灌服米非司酮24h后眼眶后静脉丛取血,后处死动物。运用ELISA法进行血清TGF-β1含量的检测。
结果:
Objective:1. To study the Progestogen Receptor and Transforming Growth factor-beta1 expression in the deciduas so as to explore the mechanism of the kidney deficiency early pregnant women with spontaneous abortion.2. To examine the further mechanism of Pill ZHUN YUN III on kidney deficiency and the luteal inhibition aborting rat model.Methods:l.Research on the deciduas effect to the Luteal and transforming growth factor-betal expression of the kidney deficiency early pregnant women with spontaneous abortion: 23 patients were diagnosed as spontaneous abortion and the kidney deficiency in the first trimester in the 1st Affiliated Hospital, Guangzhou University of TCM. These patients' deciduas and the other deciduas from 40 women diagnosed as spontaneous abortion and the blood stasis ( or blood fever) in the same period as well as 40 normal early pregnancy women who desired conducting induced abortion were collected. AT the same time, their serum were collected.RT-PCR and LSAB were used to detect the expression of PRmRNA and TGF-β1 mRNA in the deciduas. RT-PCR also was used to detect the expression of TGF-β1mRNA in the PBMC.2. Study on the Herbal Medicine to Interfere the Serum TGF-β1 of Aborting Rat Model:
SPF grade male SD rats, aged 10-12 weeks and female SD rats , aged 8-10 weeks.weight 200-250gm, were mated. Pregnant rats were daily fed with hydroxyurea450mg/Kg from the first day to the seventh day. daily for. On the eighth day, theywere fed with RU486 3.75 mg/kg to create a kidney deficiency and the lutealinhibition aborting rat model. The rats were divided into 5 groups:A."model withDuoLiMa,B: model without herbs, C: model with high dose herbal medicine,D: model with low dose herbal medicine ,E:controlled group(fed with salineinstead of medicine). There were 8 days for creating model and treatment.Different groups were observed at the same period for the weight, the rateof abortion, the lever of serum TGF-3 1.Results:1. On the gene or protein lever, the expression of PRmRNA and TGF-5 lmRNA inthe deciduas every group were different, the different wassignificant(P<0. 01), the expression of the kidney deficiency early pregnantwomen with spontaneous abortion was lowest(P<0. 01).2.Group A, B, C and D showed some symptoms of kidney deficiency, such aswithered fur, slow action and less ability. The body weight of rats in eachgroup were all increased from Dl to D8, the different wassignificant (P<0. 01), group E was the highest and group B was the lowest. Therate of abortion was decreased, the different was significant (P<0. 01), groupC and group A was lowest. Pill ZHUN YUN III could increase the serum leverof TGF-0 1.Conclusion:1.In the clinical research, the normal expression of PRmRNA and TGF-P lmRNAin the deciduas are important in maintaining early pregnancy. The lowerexpression is related closely to the kidney deficiency early pregnant womenwith spontaneous abortion.2.The expression of TGF-3 lmRNA in the PBMC and deciduas has the positivecorrelation.
3. Pill ZHUN YUN III could increase the serum lever of TGF- p 1 of the kidney deficiency and the luteal inhibition aborting rat model. Pill ZHUN YUN III can regulate immune function.
引文
[1] 牛建昭等,现代中西医妇科学.1996年版506.
[2] 罗元恺.罗元恺医著选.广州中医学院妇科教研室.广州:广东科技出版社,1980版.63.5
[3] 哈荔田著.哈荔田妇科医案医话选天津:天津科学技术出版社1982:17.
[4] 罗元恺,夏桂成等.习惯性流产的防治.中医杂志,1988,(4):5.
[5] 赵延楼.逐瘀法治疗滑胎212例[J].辽宁中医杂志,1986,(9):26.
[6] 宋文武.宋鸿元老中医治疗习惯性流产经验.河南中医,1995,15(4):228-229.
[7] 曾诚,岳明明.试论先兆流产的中医证型分布规律.中国中医药信息杂志,2002,9(8):5-7.
[8] 许钧,归绥琪.自然流产病因分类与中医证型相关性研究.上海中医药杂志,37(3):10-11.
[9] 苏延华,吴效科.近代PCOS研究的若干进展.第三届全国妇产科内分泌学术会议论文汇编,1996,10.
[10] Okon MA, haird SM, Tuckerman, et al. Serum androgen levels in women who have recurrent miscarriages and their correlation with markers of endometrial function. Fertil Steril, 1998, 69(4): 682.
[11] 王梦玖主编.生殖免疫学.北京:中国展望出版社,1986,88.
[12] 闫春芳,于学文,崔绪琴,等.反复自然流产的免疫学研究进展.国外医学·妇幼保健分册.2000,11(4):164—167.
[13] 王秀丽,王树松,宗文萍..早期自然流产患者的5种抗体分析[J].中国妇幼保健,2003,18(2):92—93.
[14] 林羿,曾耀英,曾山,等.在反复自然流产模型CBA/J×DBA/2小鼠血清中未检测出抗精子抗体[J].暨南大学学报(自然科学版),2002,23(3):121-124.
[15] 罗颂平,张玉珍,梁国珍,等.自然流产的免疫性因素与中医药治疗.中国医药学报,1996,11(4):27-30.
[16] 王越,屈野,陈莉,等.早孕妇女外周血Th1/Th2细胞亚群的测定[J].免疫学杂志,2001,17(2):159-160.
[17] 邱丽华,林其德,洪燕,等.原因不明习惯性流产患者主动免疫治疗前后T辅助细胞1、2型细胞因子的变化[J].中华妇产科杂志,2001,36:408-410.
[18] Christiansen OB[J]. Hum Reprod Update, 2000, 2(10): 171-179.
[19] 李洁,庄广伦.子宫内膜功能异常与反复自然流产.中国实用妇科与产科杂志,1999,15(3):141-143.
[20] Edward JK: The physiological and pathophysiological significance of steroid hormone receptors. Gynecol Obstet 5 (4): 9 1983.
[21] Caslbtcaum AJ, mastroianni LJ, Wheeler JM, et al. Timing of the endometrial may be critical for accurate diagnosis of luteal phase deficiency. Fertil Steril, 1994, 61(3): 443.
[22] 徐苗厚,张振国,江森,等.黄体功能缺陷与子宫内膜效应不良患者的内分泌特征.中华妇产科杂志,1997,32(3):141.
[23] Cook ID and Lambadarios C. The endometrium. Clin Obstet Oynecol. 1974, 1: 369.
[24] Keller DW, et al. Pseudocorpus luteum insufficiency: a local defect of progesterone action on endometrial stroma. J Clin Endocrinol Metab. 1979, 48: 127.
[25] 徐苗厚,江森,许燕雪,等.重复性早期自然流产病人非孕期血清激素水平和子宫内组织ER、PR含量变化的研究.现代妇产科进展,1995,4(3):217-219.
[26] 岳明明,罗颂平,谭宇蕙.补肾中药对肾虚黄体抑制流产大鼠的实验研究.中医药学报,2005,33(1):47-49.
[27] 颜建华.母胎免疫机制的研究进展.中国免疫学杂志,1994,10(Suppl):208-211.
[28] Massage J, Cheifetzs S, haiho M, etal. Transforming growth factor-beta[J]. Cancer surveyes, 1992, 12: 81-103.
[29] Buner KL, Rodgers WH, Gold LI, etal. Transforming growth factor beta mediates the progesterone suppression of epithelial metalloproteinase by adiacents tromain human endpmetrium. Proc Nat 1 Sci USA, 1995, 92: 7362-7366.
[30] Clark DA, Coker R. Transforming growth factor-beta (TGF-beta). Int. J Biochem Cell Biol, 1998.30(3): 293-298。
[31] Prudhomme GJ, Piccirillo CA. The inhibitory effects of transforming growth factor-beta-1(TGF—betal) in autoimmune disease[J]. J Autoimmnue, 2000, 14: 23-42.
[32] Chung IB, Yelian FD, Zaher FM, et aI. Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line[J]. Placenta, 2000, 21: 320-324.
[33] 邹丽,黄瑛,徐可树,等.转化生长因子在孕鼠胚胎及胎盘发育中的作用.中华妇产科杂,1999,34(4):239-240.
[34] 邱丽华,林其德,李东至,等.转化生长因子β1与原因不明习惯性流产关系的 初步研究.现代妇产科进展,2002,11(3):203-205
[35] 姜学强,吕娜,包洪初,等.早期自然流产与转化生长因子-β1的相关性研究.中国生育健康杂志,2004,15(2):96-98.
[36] Piccinni MP, Romagnani S. Regulation of fetal allograft survival by a hormonecontrolled Th1-and Th2-type cytokines. Immunol Res, 1996, 15(2): 141-50. Review.
[37] 李昭荣,韩香,吕海侠,等.Th1/Th2型细胞因子与反复自然流产的关系.西安交通大学学报,2003,24(3):254.
[38] 张玉珍,刘菊芳,罗颂平.罗元恺教授经验方滋肾育胎丸临床总结.新中医,1983,(3):11.
[39] 归绥琪,许钧,余而慨,等.中药治疗自然流产对封闭抗体-hCG,孕酮、雌二醇的影响[J].中国中西医结合杂志,1997,17(11):645—648.
[40] 罗颂平,等.免疫性自然流产与免疫性不孕的中医治疗[J].医学通讯.1999,28(4):10—11.
[41] 刘芳.助孕3号丸治疗早孕先兆流的临床疗效与免疫—内分泌调节机理研究,广州中医药大学2004届硕士毕业论文.
[42] 梁雪芳,黄晋琐.补肾法治疗先兆流产62例.现代中西医结合杂志,1999,8(10):1627.
[43] 李改样.寿胎丸和四君子汤加减治疗滑胎98例[J].山西中医,1990,6(6):23.
[44] 邯郸市中医院妇科课题组.芪丝安胎散治疗胎漏、胎动不安和滑胎108例临床观察[J]。河北中医,1992,14(6):26—27.
[45] 许丽娜.固肾保胎饮治疗滑胎20例[J].新中医,2001,33(1):63.
[46] 刘迪加.泰山磐石散治疗习惯性流产132例[J].内蒙古中医药,1999,18(2):13.
[47] 杨晓海.先兆流产从肾虚气血失调论治.福建中医药,1998,29(1):5.
[48] ParandM, ChedidL. Protective effects of chlorpromazine against endoinduced abortion. Proc Soc Exp Bio Med, 1964, 116: 906.
[49] 李小球,罗颂平.“助孕3号丸”对妊娠大鼠孕激素及受体的影响.暨南大学学报(医学版),1999,20(6):111-114.
[50] 宁艳,罗颂平,梁国珍.补肾健脾中药复方对肾虚黄体抑制妊娠大鼠流产模型的实验研究.中国医药学报,2000,15(6):28-29.
[51] 罗颂平,梁国珍,张玉珍,等.助孕3号方防治大鼠自然流产的机理研究.中国中西医结合杂志,2003,23(7):522-525.
[52] Ning Y, Luo SP, Liang GZ&Xu LM. The Experiment about the Treatment of Kindey-Deficiency and Luteal Phase Defect Aborting Rat Model with Prescription Zhuyun. (in Chinese). Guangzhou: Oraduation Thesis of The Graduate Student in Guangzhou Traditional Chinese Medicine University, 1999, 44.
[53] 刘昱磊,罗颂平,梁国珍,等.助孕3号方及拆方防治肾虚黄体抑制动物流产模型的实验研究.生殖与避孕.2003,23(1):17-22.
[54] 李昆明,归绥琪,蒋立辉,等.补肾益气清热方对溴隐亭致低PRL大鼠流产模型蜕膜PRLR、PR、ERmRNA的影响.生殖与避孕.2003,23(4).209-213.
[55] 蒋立辉,归绥琪.中药对溴隐亭致流产大鼠Th2/Th1细胞因子的影响.浙江中西医结合杂志,2003,13(3):155-157.
[56] 曹立幸,韩冰,李同玺,等.益气养血、固肾安胎法对肾虚流产免疫因素影响的实验研究.天津中医,2001,18(5):35。
[57] 归绥琪,许钧.自然流产的中西医结合诊治规律初探.上海中医药大学学报,2001,15(2):25-27.
[58] 许钧,归绥琪.自然流产病因分类与中医证型相关性研究——附310例临床资料分析.上海中医药杂志,2003,37(3):10-11.
[59] 冯蓓,张丽珠.早孕妇女血清孕酮、雌二醇水平和蜕膜组织孕酮受体的变化及其在早孕维持中的作用.生殖与避孕,1984,4(2):40-44.
[60] 孙慧清,盖凌,张立宏,等.重复性早期流产患者雌、孕激素及其受体研究.生殖与避孕,1995,15(3):184-185.
[61] Baulieu EE. On the mechanism of action of RU486, In: seppala M, Hamberger L, editors. Frontiers in humanreproduction. Ann N Y Acad Sci 1991;626: 545—6.
[62] Bouchard P, Mairaoui J, Massai MR, Medalie DA, De Ziegler D, Perrot-Applanat M, Frydman R, Bergeron C. Immunocytochemical localization of oestradiol and progesterone receptors in human endometrium: a tool to assess endometrial maturation. Baillieres Clin Obstet Gynaecol 1991 Mar;5(1): 107-115.
[63] Tan J, Paria BC, Dey SK, Das SK: Differential uterine expression of estrogen and progesterone receptors correlates with uterine preparation for implantation and decidualization in the mouse. Endocrinology 1999 Nov;140(11):5310-21.
[64]Stavreus-Evers A, Nikas G, Sahlin L, Eriksson H, Landgren BM: Formation of pinopodes in human endometrium is associated with the concentrations of progesterone and progesterone receptors. Fertil Steril 2001 Oct;76 (4): 782-91.
[65] Jensen Evand Desomber ER. Mechanism of action of the female sex hormones[J]. Annual Review of Biochemistry, 1972,41:203.
[66]Schrader WT. New model for steroid hormone receptors [J]. Nature, 1984, 308(5954): 17.
[67] Chen Y, Kuchroo VK, Inobe J, et al. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science, 1994, 265:1237.
[68]Delarco J, E, Todaro G J. Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci USA, 1978, 75:4001-4005.
[69]Letterio JJ, Roberts AB. Regulation of immune responses by TGF-beta.Annu Rev Immunol, 1998, 16:137.
[70]Stoeck M, Ruegg C, Miescher S. Comparison of the immunosuppressive properties of milk growth factor and transforming growth factors beta 1 and beta 2. [J] Immunol, 1989,143:3258.
[71]Grotendorst GR, Smale G, Pencev D. Production of transforming growth factor beta by human peripheral blood monocytes and neutrophils. [J] Cell Physiol, 1989,140:396.
[72] Inge TH, Hoover SK, Susskind BM, et al. Inhibition of tumor-specific cytotoxic T-lymphocyte responses by transforming growth factor beta 1 .Cancer Res, 1992,52:1386.
[73]Czarniecki CW, Chiu HH, Wong GH, et al. Transforming growth factor-beta 1 modulates the expression of class Ⅱ histocompatibility antigens on human cells. J Cell Physiol, 1988,140:4217.
[74]Graham CH. Effect of transforming growth factor beta on the plasminogen activator system incultured first trimester human cytotrophoblasts [J]. Placenta, 1997,8(2 3): 137 143.
[75]Kubota T, Taguchi M, Kobayashi K, etal. Relationship between the release of prolactin and endothelin 1 in human decidualized endometrial cells[J].Eur J Endocrinol. 1997,137(2):200-204.
[76] Good win VJ, Sato TA, Mitche 11 MD, etal. Anti-inflammatory effects of interleukin-4, interleukin-10 and transforming growth factor on human placental cells in vitro. Am [J] Reprod Immunol, 1998, 40: 319-325.
[77] 徐瑞荣,张会平,崔兴.补肾益髓方对慢性再生障碍性贫血骨髓分泌造血细胞因子的影响.山东中医杂志,2005,24(1):13-15.
[78] Choi BC, Hill JA. Th1(IFN-gamma), Th2(IL-10) and TGF betal Secretion in Response to Trophbblast and Progesterone in Women with Unexplained Recurrent Pregnancy Loss and Fertile Controls[J]. Fertil Steril, 1997, 68(1S): 17-18.
[79] Clark DA, McDermott M, Sczewzuk MR. Impairment of host vs graft reaction in pregnant mice. Ⅱ. selective suppression of cytotoxic cell generation correlates with soluble suppression activity and with successful allogeneic pregnancy. Cell Immunol, 1984;52: 146-114.
[80] Parand M, Chedid L. Protective effects of chlorpromazine against endo induced abortion. Proc Soc Exp Bio Med, 1964, 116: 906.
[81] 王钦茂,姚道云,杨耀芳,等.孕康口服液保胎作用的实验研究.中国中西医结合杂志,1994,14(7):418-419.
[82] 蒋立辉,归绥琪,李昆明,等.中药对溴隐亭致流产大鼠保胎机理的实验研究.中国免疫学杂志,2002,18(10):705-707.
[83] 林羿,曾耀英,何贤辉.淋巴细胞免疫治疗对小鼠自然流产模型母胎界面CD80~+表达及妊娠结局的影响.生殖与避孕,2002,22(6):332-337.
[84] 周跃华,朱晓勇,李大金,等.孕早期干预CD80/CD86协同刺激信号对自然流产模型免疫活性细胞协同刺激分子表达的影响.中国免疫学杂志,2004,20(8):537-543.
[85] 魏彦明.中药成分对LPS诱导大鼠流产作用机理的研究.甘肃农业大学,2003届博士学位论文.
[86] Irving M, Spilz MD, Bardin CW. Clinical phartnacology of Ru486-anantiprogestin and antiglnocorticoid. Contraception, 1993;48(5): 408.
[87] 陈可冀.新编抗衰老中药学.北京:人民卫生出版社,1998.
[88] 肖锦松,崔风军,宁廷选,等.玉竹、菟丝子醇提物对烧伤小鼠免疫功能的影响.中国中药杂志,1990:15(9):45-47.
[89] 张秀敏,刘冉,许津.中药桑寄生的抗Ⅰ型变态反应作用.中国药师,2005,8(1):5-7
[90] 储大同,孙燕,林娟如.黄芪提取成分对癌症患者淋巴细胞免疫功能恢复及对大鼠 免疫抑制逆转的作用.中西医结合杂志,1989,9(6):351-354.
[91] 金虹,梁曼若,王建华.从IL-2水平探讨黄芪及黄芪多糖的免疫调节作用.中国免疫学杂志,1989,5(5):308-310.
[92] 高向东,吴梧桐.五种抗衰老中药对小鼠T淋巴细胞增殖与IL-2产生的影响.中国药科大学学报,1990,21(1):43-45.
[93] 张启凤.五味子、党参、黄芪复方液对小白鼠红细胞超氧化物歧化酶活力的影响.陕西中医学院学报,1990,13(3):45.
[94] 龚晓健,季晖,王青,等.川续断总生物碱对妊娠大鼠子宫的抗致痉及抗流产作用.中国药科大学学报,1998,29(6):459-461.
[95] 龚晓健,吴知行,陈真,等.川续断DA303对大鼠子宫的作用.中国药科大学学报,1996,27(1):48-51.
[96] 石扣兰,李丽芬,李月英,等.川续断对小鼠免疫功能的影响.中药药理与临床,1998,14(1):36-37.
[97] 骆和生,罗鼎辉主编.中药免疫学——中药免疫药理与临床.北京:北京医科大学,中国协和医科大学联合出版社,1999.
[98] 任艳玲,郑洪新,杜松.补肾健脾药物血清对大鼠成骨细胞TGF-β1表达的影响.辽宁中医杂志,2005,32(2):100-102.
[99] 任艳玲,郑洪新,杜松.密骨颗粒对去卵巢大鼠TGF-β1表达的影响.中国骨质疏松杂志,2004,10(2):221-225.