川芎嗪对早期胚胎发育和植入过程中IL-6表达的影响
摘要
目的：检测盐酸川芎嗪处理小鼠妊娠着床期的孕激素和雌激素、子宫和胚胎白细胞介素-6(interleukine-6，IL-6)的表达情况，观测盐酸川芎嗪对体外胚胎早期发育的影响，评估盐酸川芎嗪对早期妊娠的安全性。
方法：从妊娠第1天开始，将妊娠小鼠分为3个用药组，按0.067mg／g换算，以不同浓度的盐酸川芎嗪(10mg／d.ml，2mg／d.ml，0.4mg／d.ml)持续灌胃，连续4天，同时设立对照组，灌胃酸性生理盐水，分别于第4天和5天取血，测血清中孕酮(Progesterone，P)和雌二醇(estradiol，E2)浓度；同时第5天取子宫，通过免疫组化和逆转录聚合酶链式反应分析IL-6蛋白和IL6mRNA的表达情况；取第6天的子宫观测胚胎数和胚胎与子宫的发育情况。另取妊娠2-细胞、4-细胞和8-细胞的胚胎，用高中低浓度的盐酸川芎嗪(20ug／ml，2ug／ml，0.2ug／ml)进行体外培养，设立对照，观察胚胎发育情况。
结果：第4天用药组小鼠血清雌二醇浓度与对照组相比无明显差异(P＞0.05)，用药组血清孕酮浓度与对照组相比差异显著升高(P＜0.05～P＜0.01)；第5天的高浓度用药组血清雌二醇浓度与对照组相比显著升高(P＜0.05)，各用药组孕酮浓度与对照组相比均显著降低(P＜0.05～P＜0.01)。第6天中浓度胚胎数与对照组相比差异显著减少(P＜0.05)，低浓度组和高浓度组与对照组相比无明显差异(P＞0.05)；低浓度组与对照组相比胚胎发育无明显差异，中浓度和高浓度与对照组相比胚胎发育迟缓。在体外培养的胚胎中，高浓度组、中浓度组和低浓度组与对照组相比能明显抑制2-细胞、4-细胞的发育；对于8-细胞的发育，4个组之间无明显差异(P＞0.05)。免疫组化显示，低浓度组与对照组相比，IL-6蛋白表达表达无明显差异(P＞0.05)，然而中浓度组与高浓度组IL-6表达显著降低(P＜0.05与P＜0.01)。但各浓度组间IL-6 mRNA的表达量无明显差异。
结论：盐酸川芎嗪在体内，可通过降低血清孕酮、升高雌二醇浓度和减弱子宫上皮IL-6蛋白表达，使胚胎发育迟缓、子宫血液循环受阻；在体外能阻抑2-细胞、4-细胞和8-细胞的发育。
Object: In order to evaluate the safty of Ligustrazine Hydrochloride in the early pregnancy, estrogen and progesterone in mice serum as well as interleukine-6(IL-6) in uterus and embryos,which treat with Ligustrazine Hydrochloride were investigated. The effect of Ligustrazine Hydrochloride on the development of early embryos was also observed in vitro and in vivo.
Methods: The pregnant mice were treated intragastrically with Ligustrazine Hydrochloride (10mg/d.ml,2mg/d.ml,0.4mg/d.ml) for 4 days from the first day of pregnancy (0.067mg/g), and the control group was treated with acidic saline. The content of progesterone and estradiol in serum were measured by RIA(radioimmunoassay) at 4th and 5th day of pregnancy, the expression of IL-6 at the protein and mRNA level were detected by immunohistochemistry and RT-PCR(reverse transcription-polymerase chain reaction).The number of embryos in each uterus were counted at 6th day of pregnancy. For investigation of the direct effect of Ligustrazine Hydrochloride, the two-cell four-cell and eight-cell of mouse embryos were cultured in the M16medium and treated with different concentrations (20ug/ml,2ug/ml,0.2ug/ml) of Ligustrazine Hydrochloride in for three days, the morphology of embryos was observed and took pictures under microscope.
Result: The level of the serum estradiol in the 3 experimental groups is similar to the control group at 4th day of pregnancy (P>0.05) ,while the level of progesterone in 0.4mg/d.ml 2mg/d.ml and 10mg/d.ml groups is distinctly increased dependent with the dose of Ligustrazine Hydrochloride (P<0.05-P<0.01) .Compared with the control group,the level of serum estradiol distinctly increased in 10mg/d.ml group (P<0.05) . The level of serum progesterone in 0.4mg/d.ml 2mg/d.ml and 10mg/d.ml groups distinctly increased compared with the dose of Ligustrazine Hydrochloride (P<0.05-P<0.01) at 5th day of pregnancy. The number of embryos in 2mg/d.ml group is much less than that of the control group at 6th day of pregnancy (P<0.05), The number of embryos in 0.4mg/d.ml and 10mg/d.ml groups are similar to the control group (P>0.05); the development of embryos in 2mg/d.ml and 10mg/d.ml groups are slower than the control group.Compared with the control group,the development of 2-cell embryos in 20ug/ml is significantly inhibited in vitro. During the period of later development of 4-cell, 20ug/ml and 2ug/ml groups could significantly inhibitor development of 8-cell compared with the control group. There are not significantly difference among four groups. The results also show that the expression of IL-6 protein but not the IL-6 mRNA in uterus and embryos could be inhibited by 10mg/d and 2mg/d of Ligustrazine Hydrochloride.
Conclusion: The Ligustrazine Hydrochloride could inhibitors the development of embryos through decrease the level of the serum estradiol and increase the level of serum progesterone,and inhibitors the circulation of uterus blood through decrease the expression of IL-6 protein in uterus epitheliumin in vivo; inhibitors the development of 2-cell 4-cell and 8-cell in vitro.
方法：从妊娠第1天开始，将妊娠小鼠分为3个用药组，按0.067mg／g换算，以不同浓度的盐酸川芎嗪(10mg／d.ml，2mg／d.ml，0.4mg／d.ml)持续灌胃，连续4天，同时设立对照组，灌胃酸性生理盐水，分别于第4天和5天取血，测血清中孕酮(Progesterone，P)和雌二醇(estradiol，E2)浓度；同时第5天取子宫，通过免疫组化和逆转录聚合酶链式反应分析IL-6蛋白和IL6mRNA的表达情况；取第6天的子宫观测胚胎数和胚胎与子宫的发育情况。另取妊娠2-细胞、4-细胞和8-细胞的胚胎，用高中低浓度的盐酸川芎嗪(20ug／ml，2ug／ml，0.2ug／ml)进行体外培养，设立对照，观察胚胎发育情况。
结果：第4天用药组小鼠血清雌二醇浓度与对照组相比无明显差异(P＞0.05)，用药组血清孕酮浓度与对照组相比差异显著升高(P＜0.05～P＜0.01)；第5天的高浓度用药组血清雌二醇浓度与对照组相比显著升高(P＜0.05)，各用药组孕酮浓度与对照组相比均显著降低(P＜0.05～P＜0.01)。第6天中浓度胚胎数与对照组相比差异显著减少(P＜0.05)，低浓度组和高浓度组与对照组相比无明显差异(P＞0.05)；低浓度组与对照组相比胚胎发育无明显差异，中浓度和高浓度与对照组相比胚胎发育迟缓。在体外培养的胚胎中，高浓度组、中浓度组和低浓度组与对照组相比能明显抑制2-细胞、4-细胞的发育；对于8-细胞的发育，4个组之间无明显差异(P＞0.05)。免疫组化显示，低浓度组与对照组相比，IL-6蛋白表达表达无明显差异(P＞0.05)，然而中浓度组与高浓度组IL-6表达显著降低(P＜0.05与P＜0.01)。但各浓度组间IL-6 mRNA的表达量无明显差异。
结论：盐酸川芎嗪在体内，可通过降低血清孕酮、升高雌二醇浓度和减弱子宫上皮IL-6蛋白表达，使胚胎发育迟缓、子宫血液循环受阻；在体外能阻抑2-细胞、4-细胞和8-细胞的发育。
Object: In order to evaluate the safty of Ligustrazine Hydrochloride in the early pregnancy, estrogen and progesterone in mice serum as well as interleukine-6(IL-6) in uterus and embryos,which treat with Ligustrazine Hydrochloride were investigated. The effect of Ligustrazine Hydrochloride on the development of early embryos was also observed in vitro and in vivo.
Methods: The pregnant mice were treated intragastrically with Ligustrazine Hydrochloride (10mg/d.ml,2mg/d.ml,0.4mg/d.ml) for 4 days from the first day of pregnancy (0.067mg/g), and the control group was treated with acidic saline. The content of progesterone and estradiol in serum were measured by RIA(radioimmunoassay) at 4th and 5th day of pregnancy, the expression of IL-6 at the protein and mRNA level were detected by immunohistochemistry and RT-PCR(reverse transcription-polymerase chain reaction).The number of embryos in each uterus were counted at 6th day of pregnancy. For investigation of the direct effect of Ligustrazine Hydrochloride, the two-cell four-cell and eight-cell of mouse embryos were cultured in the M16medium and treated with different concentrations (20ug/ml,2ug/ml,0.2ug/ml) of Ligustrazine Hydrochloride in for three days, the morphology of embryos was observed and took pictures under microscope.
Result: The level of the serum estradiol in the 3 experimental groups is similar to the control group at 4th day of pregnancy (P>0.05) ,while the level of progesterone in 0.4mg/d.ml 2mg/d.ml and 10mg/d.ml groups is distinctly increased dependent with the dose of Ligustrazine Hydrochloride (P<0.05-P<0.01) .Compared with the control group,the level of serum estradiol distinctly increased in 10mg/d.ml group (P<0.05) . The level of serum progesterone in 0.4mg/d.ml 2mg/d.ml and 10mg/d.ml groups distinctly increased compared with the dose of Ligustrazine Hydrochloride (P<0.05-P<0.01) at 5th day of pregnancy. The number of embryos in 2mg/d.ml group is much less than that of the control group at 6th day of pregnancy (P<0.05), The number of embryos in 0.4mg/d.ml and 10mg/d.ml groups are similar to the control group (P>0.05); the development of embryos in 2mg/d.ml and 10mg/d.ml groups are slower than the control group.Compared with the control group,the development of 2-cell embryos in 20ug/ml is significantly inhibited in vitro. During the period of later development of 4-cell, 20ug/ml and 2ug/ml groups could significantly inhibitor development of 8-cell compared with the control group. There are not significantly difference among four groups. The results also show that the expression of IL-6 protein but not the IL-6 mRNA in uterus and embryos could be inhibited by 10mg/d and 2mg/d of Ligustrazine Hydrochloride.
Conclusion: The Ligustrazine Hydrochloride could inhibitors the development of embryos through decrease the level of the serum estradiol and increase the level of serum progesterone,and inhibitors the circulation of uterus blood through decrease the expression of IL-6 protein in uterus epitheliumin in vivo; inhibitors the development of 2-cell 4-cell and 8-cell in vitro.
引文
[1] 王敏康，刘冀珑，陈永福等．哺乳动物早期胚胎体外发育阻滞的研究进展．遗传，2001，23(4)：391-395．
[2] 吴嫦丽，张守全，高淑静等．不同培养液对昆明小鼠体外受精率及早期胚胎体外发育的影响．华南农业大学学报，2006，27(1)：107-109．
[3] 陈系古，李智，俞生．小鼠胚胎体外培养条件的研究．中国实验动物学报，1998，6(1)：42-47．
[4] Devgan V, Seshagiri PB.Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media. Molecular Reproduction and Development,2003,65: 269-277.
[5] 肖文伍，邹亚芬，刘登华．小鼠早期胚胎的体外培养研究．华中科技大学学报，2003，32 (6)：593-596．
[6] 刘莹，武迪迪，宗志宏．昆明系小鼠体外受精方法及一种新培养基的应用．中国医科大学学报，1999，28(1)：8-10．
[7] Mognetti B,Leppens G,SakkasD. The development of preimplantation mouse parthenogenones in vitro in absence of glucose:influence of the maternally inherited components. Molecular Reproduction and Development, 1996,43:421-427.
[8] Ain R, Seshagiri PB. Succinate and malate improve development of hamster eight-cell embryos in vitro:confirmation of viability by embryo transfer. Molecular Reproduction and Development, 1997,47:440-447.
[9] 叶新红，严敬明，徐国江．羊输卵管上皮细胞对小鼠受精卵体外发育的影响．上海实验动物科学，2001，21(1)：17-21．
[10] Chatot C. L, Lawry J. R, Germain B, etal. Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos. Molecular Reproduction and Development, 1997,47:248-254.
[11] Gardner DK, Lane MW, Lane M. EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation. Molecular Reproduction and Development,2000,57: 256-261.
[12] 安志兴，李雪峰，李向臣．昆明小鼠原核胚在不同培养液中的体外发育．中国实验动物学报，2003，11(4)：233-236．
[13] 王敏康，李光鹏，陈大元等．M16添加牛磺酸和EDTA支持昆明白小鼠体外受精并发育至 囊胚．遗传，2000，22(5)：301-302．
[14] 王敏康，张田，王晓燕等．几种克服昆明小鼠2细胞胚胎发育阻滞的培养液研究．动物学报，2000，46(1)：81-87．
[15] Carlos Simon, Julio Cesar and Antonio Pellicer. Paracrine regulators of implantation. Best Practice & Research Clinical Obstetrics &Gynaecology, 2000, 14(5):815-826.
[16] Demayo FJ and Lee KY. Animal models of implantation.Reproduction, 2004, 128(6):679-695.
[17] Joseph S,Souvik C,Wang YP, etal.DNA methylation controls the responsiveness of hepatoma cells to leukemia inhibitory factor.Hepatology.2003,38(6): 1516-1528.
[18] 张玲华，郭勇，田兴山等．白细胞介素-6的分子生物学研究进展及其在畜禽业中的应用前景．中国畜牧兽医，2005，32(2)：31-33．
[19] Bornstein SR, Rutkowski H,Vrezas I.Cytokines and steroidogenesis.Molecular and Cellular Endocrinology,2004,215:135-141.
[20] 傅瑞全．几种细胞因子的临床研究热点．右江民族医学院学报，2002，24(1)：122-123．
[21] 宋伦，任蕴芳．白细胞介素6及其受体结构与功能关系研究进展．生物技术通讯，1997，8(1)：29-32．
[22] Stephen AD,Zhong Z,Wen ZL, etal. Stat signaling is active during early mammalian development. Developmental Dynamics, 1997,208:190-198.
[23] David RH,Elaine MH,William LF. The role of IL-6 and STAT3 in inflammation and cancer. European Journal of Cancer,2005,41:2502-2512.
[24] 张炜，周剑萍，张俊慧．细胞间粘附分子-1在着床前小鼠胚胎培养液中的表达．生殖医学杂志，1999，3(8)：33-36．
[25] Evsikov SV, Vagyna IN,Solomko AP. Mechanisms of cell number regulation in the peri-implantation mouse blastocyst.The Journal of experimental Zoology, 1996, 276:201-208.
[26] 刘承权，袁瑶，王忠兴等．雌、孕激素对IL-6基因在子宫内膜表达的影响．生殖医学杂志，1997，6(4)：207-210．
[27] Jacobs AL,Sehgal PB, Julian J, etal.Secretion and hormonal regulation of interleukine-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro. Endocrinology, 1992,131:1037-1046.
[28] Jasper MJ, Trremellen KP, Robertson SA.Reduced expression of IL-6 and IL-1 alpha mRNAs in secretory phase endometrium of women with recurrent miscarriage. Journal of Reproductive Immunology,2007,73 (1):74-84.
[29] Eiichi H,Kokubu KJ,Kiso Y, etal. Localization of interleukin-6 receptor mRNA in pregnant and non-pregnant mouse uterus.Journal of Reproduction and Development,2005,51 (6):777-781.
[30] Nicolas MO,Ekbote UV,Gopichandran N, etal. Uterine and serum cytokine arrays in the mouse during estrus. Animal Reproduction Science,2006,3163:10-11.
[31] Chabot V,Lambert RD,Lessard M,etal.Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts.Animal Reproduction Science, 2004,81:137-149.
[32] Stefan F, Abrahams VM,Alvero A,etal.Trophoblast-macrophage interaction:a regulatory for the protection of preganacy. American Journal of Reproductive Immunology,2006,57:55-66.
[33] Sharkey AM,Cameo P, Mavrogiania PM, etal. Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation.Endocrinology,2007,148(2):618-626.
[34] 孙树梅，王永午．白细胞介素6(IL-6)的免疫调节与临床疾病．医学综述，1996，2(3)：134-136．
[35] 阳春霞，冯大力，郎巍．白细胞介素-6与妊娠的相互关系．中国妇幼保健，1998，13 (6)：362-363．
[36] Hava G, Vered L, Mordechai,etal. Alterations in behavior in adult offspring mice following maternal inflammation during pregnancy. Prenatal Maternal Inflammation Reduced Anxiety. 2006,48:162-168.
[37] 林红，沈倍奋，肖培根．白细胞介素6及其受体与自身免疫性疾病．细胞与分子免疫学杂志，1997，13(2)：51-54．
[38] 王艳萍，李文兰，范玉奇．川芎嗪药理作用的研究进展．药品评价，2006，3(2)：144-146．
[39] 张鹤玲，张英．川芎嗪的作用机理与临床．医学理论与实践，2004，14(8)：809-810．
[40] 陈玉清．盐酸川芎嗪治疗急性脑梗死的疗效观察．医学理论与实践，2006，19(12)：1411-1412．
[41] 单萍，王建平．川芎嗪治疗急性心梗溶栓时再灌注损伤的疗效观察及护理．心脑血管病防治，2003，6(3)：146．
[42] 周亚伟．盐酸川芎嗪注射液治疗慢性间质性肾炎的效果观察．右江民族医学院学报，2006，4：551-552．
[43] 朱红倩，潘红平，汪漫江等．川芎嗪联合雄激素和皮质激素治疗苯中毒性再生障碍性贫血临床观察．临床荟萃，2004，10(20)：1184-1185．
[44] 周银莉，刘文励，孙汉英等．川芎嗪对骨髓移植小鼠早期造血重建作用的研究．中华血液学杂志，2002，4(23)：207-208．
[45] 祝领旗，袁朋，王军等．川芎嗪注射液治疗膜性肾病疗效观察．现代中西医结合杂 志，2004，13(21)：2841-2842．
[46] 刘晓丽，霍展样，张金莲．盐酸川芎嗪体外抗氧化作用的研究．中国航天医药杂志，2003，4(5)：37-39．
[47] 石韫珍，郝文科，董光富等．川芎嗪对狼疮肾炎血栓素B2与TNF-α及IL-6的影响．中国中西医结合肾病杂志，2006，7(8)：406-408．
[48] 包旭．川芎嗪的药理学研究．四川生理科学杂志，1999，21(2)：20-25．
[49] 李有田，李赫，许丹等．盐酸川芎嗪的药理作用及临床应用概述．吉林中医药，2004，24 (6)：56-58．
[50] 王敏康，张田，刘冀珑等．一种改进的胚胎吸管．动物学杂志，1999，33(6)：34-35．
[51] Cangi MG, Cukor B, Loda M, etal.Role of the cdc25A phosphatase in human breast cancer.The journal of clinical investigation,2000,106:753-761.
[52] 黄志力，桂常青，孙瑞元．盐酸川芎嗪在大鼠体内的分布．中国药理学通报，1994，10 (4)：297-300．
[53] 李振良，郭春红，韦丽红．四甲基吡嗪的药代动力学特点．张家口医学院学报，2000，17 (4)：113-115．
[54] 闫双银．川芎嗪注射液的临床应用进展．中国药事，2003，17(12)：772-774．
[55] 黄义昆，张志荣，杜娟等．盐酸川芎嗪脂质体的制备及其在小鼠体内分布的研究．中国药学杂志，2004，41(2)：129-131．
[56] 黄义昆，张志荣，杜娟等．盐酸川芎嗪脂质体的研制及其质量评价．中国药房，2004，15 (6)：343-345．
[57] Stewart C, Cullinan E. Preimplantation development of the mammalian embryo and its regulation by growth factors. Developmental Genetics, 1997,21:91-101.
[58] 李明文，刘辉，陈大元等．小鼠卵母细胞成熟和受精过程中Ca~(2+)分布变化的研究．动物学报，1997，43(1)：80-84．
[59] Lane M, Boatman DB, Bavister BD,etal. Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo. Molecular Reproduction and Development, 1998,50:443-450.
[60] Matsumoto H, Jiang JY, Sato E, etal. Distribution and gene expression of cytoskeletal proteins in two-cell rat embryos and developmental arrest. Journal of Experimental Zoology,2002,293,641-648.
[61] Barnett DK, Clayton MK, Kimura J,etal. Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Molecular Reproduction and Development,1997, 48: 227-237.
[62] Cardellini P,Davanzo G,Citi S.Tight junctions in early amphibian development :detection of junctional eingulin from the 2-cell stage and its localization at the boundary of distinct membrane domains in dividing blastomeres in low calcium. Developmental dynamics, 1996,207:104-113.
[63] Vejlsted M, Avery B, Poul MH,etal.Effect of leukemia inhibitory factor(LIF) on in vitro produced bovine embryos and their outgrowth colonies. Molecular Reproduction and Development,2005,70:445-454.
[64] 蔡惠兰，张端莲，熊彦娥等．维生素A酸对小鼠囊胚Fas蛋白表达的影响．武汉大学学报，2005，26(6)：734-737．
[65] 张艳萍，武玉玲，邴鲁军等．降钙素受体在小鼠早期胚胎及围植入期子宫内膜的表达．山东大学学报，2005，43(12)：1104-1107．
[66] Lane M,Ludwig TE, Bavister BD. Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis. Molecular Reproduction and Development, 1999,54:410-417.
[67] Scott L,Whittingham DG. Influence of genetic background and media components on the development of mouse embryos in vitro. Molecular Reproduction and Development, 1996,43:336-346.
[68] Nishikimi A,Uekawa N, Yamada M. Involvement of glycolytie metabolism in developmental inhibition of rat two-cell embryos by phosphate. Journal of Experimental Zoology,2000,287: 503-509
[69] Gao SR, Priddle H, Wilmut I, etal.Effects of donor oocytes and culture conditions on development of cloned mice embryos. Molecular Reproduction and Development,2003,66:126-133.
[70] Kamjoo M,Brison DR, Kimber SJ. Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture. Molecular Reproduction and Development,2002,61:67-77.
[71] 孙新明，魏泓，赵永聚等．昆明小鼠成熟卵母细胞体外受精及受精卵体外培养的研究．中国比较医学杂志，2005，15(3)：129-132．
[72] 安铁洙，谭建华，岳占碰．小鼠体外受精及胚胎发育的影响因素．中国兽医学报，2002，22(2)：191-193．
[73] 钟瑜，朱伟杰，张春雪．克服昆明小鼠早胚2-细胞阻滞的研究．暨南大学学报，2000，21 (5)：105-110．
[74] Lee CS, Fang NZ,Lee, K, etal. Potent and stage-specific action of glutathione on the development od goat early in vitro. Molecular Reproduction and Development,2000,57:48-54.
[75] Edwards LJ, Williams DA, Gardner DK. Intracellular pH of the preimplantation mouse embryo:effects of extracellular pH and weak acids. Molecular Reproduction and Development,1998,50:434-442.
[76] Kawagishi R, Tahara M,Sawada K, etal.Na~+/H~+ exchanger-3 is involved in mouse blastocyst formation. Journal of Experimental Zoology,2004,301:767-775.
[77] Lane M, Gardner DK.Inhibiting 3-phosphoglycerate kinase by EDTA stimulates the development of the cleavage stage mouse embryo. Molecular Reproduction and Development,2001,60:233-240.
[78] Mitchell LA. Parvovirus B19 nonstructural(NS1) protein as a transactivator of interleukin-6 synthesis:common pathway in inflammatory sequelae of human parvovirus infections?.Journal of Medical Virology,2002,67:267-274.
[79] Doria A,Iaccarino A,Maurizio C,etal. Pregnancy, cytokines,and disease activity in systemic lupus erythematosus.Arthfitis and Rheumatism,2004,51 (6):989-995.
[80] 郑辉，李洪义，王自能等．白细胞介素-6mRNA在内异症子宫内膜的表达变化．中国病理生理杂志，2002，18(5)：553-555．
[81] Ding NZ,Michael JK, Yang ZM,etal.Expression of leukemia inhibitory factor receptor and gp130 in mouse uterus during early pregnancy. Molecular Reproduction and Development,2002, 63:143-150.
[82] Teng CB,Diao HL,Ma XH,etal.Differential expression and activation of Stat3 during mouse embryo implantation and decidualization.Molecular Reproduction and Development,2004,69:1-10.
[83] 卢静，江静，吴瑞芳等．米非司酮和孕酮对子宫内膜异位症患者子宫内膜白细胞介素6分泌的影响．中华妇产科杂志，2001，36(10)：603-605．
[84] Tetsuya Yamamoto, Noriko Sato,Tadashi Matsuda, etal.Molecular interaction between STAT3 and protein inhibitor of activated STAT3,and androgen receptor. Biochemical and Biophysical Research communications,2003,306:610-615.
[85] Tetsuya Yamamoto,Tadashi Matsuda,Hiroyuki Kishi,etal.Cross-talk between signal transducer and activator of transcription 3 and estrogen receptor signaling.Federation of Europaen Biochemical Societies,2000,486:143-148.
[86] Kapiteijn K, Plaisier M,Koolwijk P, etal.Human embryo-conditioned medium stimulates in vitro endometrial angiogenesis.Fertility and Sterility,2006,85(supply. 1): 1232-1239.
[87] Gershon H,Mordechai H,Mahrnoud H,etal.Different effects of magnesium sulfate and angiotensin Ⅱ on the capacity of the fetal and maternal compartments of normal human placenta to secrete TNF-α and IL-6. Journal of ReproductiveImmunology,2006,69(2): 115-125.
[2] 吴嫦丽，张守全，高淑静等．不同培养液对昆明小鼠体外受精率及早期胚胎体外发育的影响．华南农业大学学报，2006，27(1)：107-109．
[3] 陈系古，李智，俞生．小鼠胚胎体外培养条件的研究．中国实验动物学报，1998，6(1)：42-47．
[4] Devgan V, Seshagiri PB.Successful development of viable blastocysts from enhanced green fluorescent protein transgene-microinjected mouse embryos: comparison of culture media. Molecular Reproduction and Development,2003,65: 269-277.
[5] 肖文伍，邹亚芬，刘登华．小鼠早期胚胎的体外培养研究．华中科技大学学报，2003，32 (6)：593-596．
[6] 刘莹，武迪迪，宗志宏．昆明系小鼠体外受精方法及一种新培养基的应用．中国医科大学学报，1999，28(1)：8-10．
[7] Mognetti B,Leppens G,SakkasD. The development of preimplantation mouse parthenogenones in vitro in absence of glucose:influence of the maternally inherited components. Molecular Reproduction and Development, 1996,43:421-427.
[8] Ain R, Seshagiri PB. Succinate and malate improve development of hamster eight-cell embryos in vitro:confirmation of viability by embryo transfer. Molecular Reproduction and Development, 1997,47:440-447.
[9] 叶新红，严敬明，徐国江．羊输卵管上皮细胞对小鼠受精卵体外发育的影响．上海实验动物科学，2001，21(1)：17-21．
[10] Chatot C. L, Lawry J. R, Germain B, etal. Analysis of glutaminase activity and RNA expression in preimplantation mouse embryos. Molecular Reproduction and Development, 1997,47:248-254.
[11] Gardner DK, Lane MW, Lane M. EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation. Molecular Reproduction and Development,2000,57: 256-261.
[12] 安志兴，李雪峰，李向臣．昆明小鼠原核胚在不同培养液中的体外发育．中国实验动物学报，2003，11(4)：233-236．
[13] 王敏康，李光鹏，陈大元等．M16添加牛磺酸和EDTA支持昆明白小鼠体外受精并发育至 囊胚．遗传，2000，22(5)：301-302．
[14] 王敏康，张田，王晓燕等．几种克服昆明小鼠2细胞胚胎发育阻滞的培养液研究．动物学报，2000，46(1)：81-87．
[15] Carlos Simon, Julio Cesar and Antonio Pellicer. Paracrine regulators of implantation. Best Practice & Research Clinical Obstetrics &Gynaecology, 2000, 14(5):815-826.
[16] Demayo FJ and Lee KY. Animal models of implantation.Reproduction, 2004, 128(6):679-695.
[17] Joseph S,Souvik C,Wang YP, etal.DNA methylation controls the responsiveness of hepatoma cells to leukemia inhibitory factor.Hepatology.2003,38(6): 1516-1528.
[18] 张玲华，郭勇，田兴山等．白细胞介素-6的分子生物学研究进展及其在畜禽业中的应用前景．中国畜牧兽医，2005，32(2)：31-33．
[19] Bornstein SR, Rutkowski H,Vrezas I.Cytokines and steroidogenesis.Molecular and Cellular Endocrinology,2004,215:135-141.
[20] 傅瑞全．几种细胞因子的临床研究热点．右江民族医学院学报，2002，24(1)：122-123．
[21] 宋伦，任蕴芳．白细胞介素6及其受体结构与功能关系研究进展．生物技术通讯，1997，8(1)：29-32．
[22] Stephen AD,Zhong Z,Wen ZL, etal. Stat signaling is active during early mammalian development. Developmental Dynamics, 1997,208:190-198.
[23] David RH,Elaine MH,William LF. The role of IL-6 and STAT3 in inflammation and cancer. European Journal of Cancer,2005,41:2502-2512.
[24] 张炜，周剑萍，张俊慧．细胞间粘附分子-1在着床前小鼠胚胎培养液中的表达．生殖医学杂志，1999，3(8)：33-36．
[25] Evsikov SV, Vagyna IN,Solomko AP. Mechanisms of cell number regulation in the peri-implantation mouse blastocyst.The Journal of experimental Zoology, 1996, 276:201-208.
[26] 刘承权，袁瑶，王忠兴等．雌、孕激素对IL-6基因在子宫内膜表达的影响．生殖医学杂志，1997，6(4)：207-210．
[27] Jacobs AL,Sehgal PB, Julian J, etal.Secretion and hormonal regulation of interleukine-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro. Endocrinology, 1992,131:1037-1046.
[28] Jasper MJ, Trremellen KP, Robertson SA.Reduced expression of IL-6 and IL-1 alpha mRNAs in secretory phase endometrium of women with recurrent miscarriage. Journal of Reproductive Immunology,2007,73 (1):74-84.
[29] Eiichi H,Kokubu KJ,Kiso Y, etal. Localization of interleukin-6 receptor mRNA in pregnant and non-pregnant mouse uterus.Journal of Reproduction and Development,2005,51 (6):777-781.
[30] Nicolas MO,Ekbote UV,Gopichandran N, etal. Uterine and serum cytokine arrays in the mouse during estrus. Animal Reproduction Science,2006,3163:10-11.
[31] Chabot V,Lambert RD,Lessard M,etal.Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts.Animal Reproduction Science, 2004,81:137-149.
[32] Stefan F, Abrahams VM,Alvero A,etal.Trophoblast-macrophage interaction:a regulatory for the protection of preganacy. American Journal of Reproductive Immunology,2006,57:55-66.
[33] Sharkey AM,Cameo P, Mavrogiania PM, etal. Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation.Endocrinology,2007,148(2):618-626.
[34] 孙树梅，王永午．白细胞介素6(IL-6)的免疫调节与临床疾病．医学综述，1996，2(3)：134-136．
[35] 阳春霞，冯大力，郎巍．白细胞介素-6与妊娠的相互关系．中国妇幼保健，1998，13 (6)：362-363．
[36] Hava G, Vered L, Mordechai,etal. Alterations in behavior in adult offspring mice following maternal inflammation during pregnancy. Prenatal Maternal Inflammation Reduced Anxiety. 2006,48:162-168.
[37] 林红，沈倍奋，肖培根．白细胞介素6及其受体与自身免疫性疾病．细胞与分子免疫学杂志，1997，13(2)：51-54．
[38] 王艳萍，李文兰，范玉奇．川芎嗪药理作用的研究进展．药品评价，2006，3(2)：144-146．
[39] 张鹤玲，张英．川芎嗪的作用机理与临床．医学理论与实践，2004，14(8)：809-810．
[40] 陈玉清．盐酸川芎嗪治疗急性脑梗死的疗效观察．医学理论与实践，2006，19(12)：1411-1412．
[41] 单萍，王建平．川芎嗪治疗急性心梗溶栓时再灌注损伤的疗效观察及护理．心脑血管病防治，2003，6(3)：146．
[42] 周亚伟．盐酸川芎嗪注射液治疗慢性间质性肾炎的效果观察．右江民族医学院学报，2006，4：551-552．
[43] 朱红倩，潘红平，汪漫江等．川芎嗪联合雄激素和皮质激素治疗苯中毒性再生障碍性贫血临床观察．临床荟萃，2004，10(20)：1184-1185．
[44] 周银莉，刘文励，孙汉英等．川芎嗪对骨髓移植小鼠早期造血重建作用的研究．中华血液学杂志，2002，4(23)：207-208．
[45] 祝领旗，袁朋，王军等．川芎嗪注射液治疗膜性肾病疗效观察．现代中西医结合杂 志，2004，13(21)：2841-2842．
[46] 刘晓丽，霍展样，张金莲．盐酸川芎嗪体外抗氧化作用的研究．中国航天医药杂志，2003，4(5)：37-39．
[47] 石韫珍，郝文科，董光富等．川芎嗪对狼疮肾炎血栓素B2与TNF-α及IL-6的影响．中国中西医结合肾病杂志，2006，7(8)：406-408．
[48] 包旭．川芎嗪的药理学研究．四川生理科学杂志，1999，21(2)：20-25．
[49] 李有田，李赫，许丹等．盐酸川芎嗪的药理作用及临床应用概述．吉林中医药，2004，24 (6)：56-58．
[50] 王敏康，张田，刘冀珑等．一种改进的胚胎吸管．动物学杂志，1999，33(6)：34-35．
[51] Cangi MG, Cukor B, Loda M, etal.Role of the cdc25A phosphatase in human breast cancer.The journal of clinical investigation,2000,106:753-761.
[52] 黄志力，桂常青，孙瑞元．盐酸川芎嗪在大鼠体内的分布．中国药理学通报，1994，10 (4)：297-300．
[53] 李振良，郭春红，韦丽红．四甲基吡嗪的药代动力学特点．张家口医学院学报，2000，17 (4)：113-115．
[54] 闫双银．川芎嗪注射液的临床应用进展．中国药事，2003，17(12)：772-774．
[55] 黄义昆，张志荣，杜娟等．盐酸川芎嗪脂质体的制备及其在小鼠体内分布的研究．中国药学杂志，2004，41(2)：129-131．
[56] 黄义昆，张志荣，杜娟等．盐酸川芎嗪脂质体的研制及其质量评价．中国药房，2004，15 (6)：343-345．
[57] Stewart C, Cullinan E. Preimplantation development of the mammalian embryo and its regulation by growth factors. Developmental Genetics, 1997,21:91-101.
[58] 李明文，刘辉，陈大元等．小鼠卵母细胞成熟和受精过程中Ca~(2+)分布变化的研究．动物学报，1997，43(1)：80-84．
[59] Lane M, Boatman DB, Bavister BD,etal. Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo. Molecular Reproduction and Development, 1998,50:443-450.
[60] Matsumoto H, Jiang JY, Sato E, etal. Distribution and gene expression of cytoskeletal proteins in two-cell rat embryos and developmental arrest. Journal of Experimental Zoology,2002,293,641-648.
[61] Barnett DK, Clayton MK, Kimura J,etal. Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Molecular Reproduction and Development,1997, 48: 227-237.
[62] Cardellini P,Davanzo G,Citi S.Tight junctions in early amphibian development :detection of junctional eingulin from the 2-cell stage and its localization at the boundary of distinct membrane domains in dividing blastomeres in low calcium. Developmental dynamics, 1996,207:104-113.
[63] Vejlsted M, Avery B, Poul MH,etal.Effect of leukemia inhibitory factor(LIF) on in vitro produced bovine embryos and their outgrowth colonies. Molecular Reproduction and Development,2005,70:445-454.
[64] 蔡惠兰，张端莲，熊彦娥等．维生素A酸对小鼠囊胚Fas蛋白表达的影响．武汉大学学报，2005，26(6)：734-737．
[65] 张艳萍，武玉玲，邴鲁军等．降钙素受体在小鼠早期胚胎及围植入期子宫内膜的表达．山东大学学报，2005，43(12)：1104-1107．
[66] Lane M,Ludwig TE, Bavister BD. Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis. Molecular Reproduction and Development, 1999,54:410-417.
[67] Scott L,Whittingham DG. Influence of genetic background and media components on the development of mouse embryos in vitro. Molecular Reproduction and Development, 1996,43:336-346.
[68] Nishikimi A,Uekawa N, Yamada M. Involvement of glycolytie metabolism in developmental inhibition of rat two-cell embryos by phosphate. Journal of Experimental Zoology,2000,287: 503-509
[69] Gao SR, Priddle H, Wilmut I, etal.Effects of donor oocytes and culture conditions on development of cloned mice embryos. Molecular Reproduction and Development,2003,66:126-133.
[70] Kamjoo M,Brison DR, Kimber SJ. Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture. Molecular Reproduction and Development,2002,61:67-77.
[71] 孙新明，魏泓，赵永聚等．昆明小鼠成熟卵母细胞体外受精及受精卵体外培养的研究．中国比较医学杂志，2005，15(3)：129-132．
[72] 安铁洙，谭建华，岳占碰．小鼠体外受精及胚胎发育的影响因素．中国兽医学报，2002，22(2)：191-193．
[73] 钟瑜，朱伟杰，张春雪．克服昆明小鼠早胚2-细胞阻滞的研究．暨南大学学报，2000，21 (5)：105-110．
[74] Lee CS, Fang NZ,Lee, K, etal. Potent and stage-specific action of glutathione on the development od goat early in vitro. Molecular Reproduction and Development,2000,57:48-54.
[75] Edwards LJ, Williams DA, Gardner DK. Intracellular pH of the preimplantation mouse embryo:effects of extracellular pH and weak acids. Molecular Reproduction and Development,1998,50:434-442.
[76] Kawagishi R, Tahara M,Sawada K, etal.Na~+/H~+ exchanger-3 is involved in mouse blastocyst formation. Journal of Experimental Zoology,2004,301:767-775.
[77] Lane M, Gardner DK.Inhibiting 3-phosphoglycerate kinase by EDTA stimulates the development of the cleavage stage mouse embryo. Molecular Reproduction and Development,2001,60:233-240.
[78] Mitchell LA. Parvovirus B19 nonstructural(NS1) protein as a transactivator of interleukin-6 synthesis:common pathway in inflammatory sequelae of human parvovirus infections?.Journal of Medical Virology,2002,67:267-274.
[79] Doria A,Iaccarino A,Maurizio C,etal. Pregnancy, cytokines,and disease activity in systemic lupus erythematosus.Arthfitis and Rheumatism,2004,51 (6):989-995.
[80] 郑辉，李洪义，王自能等．白细胞介素-6mRNA在内异症子宫内膜的表达变化．中国病理生理杂志，2002，18(5)：553-555．
[81] Ding NZ,Michael JK, Yang ZM,etal.Expression of leukemia inhibitory factor receptor and gp130 in mouse uterus during early pregnancy. Molecular Reproduction and Development,2002, 63:143-150.
[82] Teng CB,Diao HL,Ma XH,etal.Differential expression and activation of Stat3 during mouse embryo implantation and decidualization.Molecular Reproduction and Development,2004,69:1-10.
[83] 卢静，江静，吴瑞芳等．米非司酮和孕酮对子宫内膜异位症患者子宫内膜白细胞介素6分泌的影响．中华妇产科杂志，2001，36(10)：603-605．
[84] Tetsuya Yamamoto, Noriko Sato,Tadashi Matsuda, etal.Molecular interaction between STAT3 and protein inhibitor of activated STAT3,and androgen receptor. Biochemical and Biophysical Research communications,2003,306:610-615.
[85] Tetsuya Yamamoto,Tadashi Matsuda,Hiroyuki Kishi,etal.Cross-talk between signal transducer and activator of transcription 3 and estrogen receptor signaling.Federation of Europaen Biochemical Societies,2000,486:143-148.
[86] Kapiteijn K, Plaisier M,Koolwijk P, etal.Human embryo-conditioned medium stimulates in vitro endometrial angiogenesis.Fertility and Sterility,2006,85(supply. 1): 1232-1239.
[87] Gershon H,Mordechai H,Mahrnoud H,etal.Different effects of magnesium sulfate and angiotensin Ⅱ on the capacity of the fetal and maternal compartments of normal human placenta to secrete TNF-α and IL-6. Journal of ReproductiveImmunology,2006,69(2): 115-125.