川芎嗪对早期胚胎发育和植入过程中IL-6表达的影响

【作者】 王伟；

【导师】 杨秀萍；

【作者基本信息】 首都师范大学 ， 细胞生物学， 2007， 硕士

【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。更多还原

【Abstract】 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.更多还原