【作者】 朱坤；

【导师】 陈晓蓉；

【作者基本信息】 安徽医科大学 ， 人体解剖与组织胚胎学， 2008， 硕士

【摘要】 目的①研究去甲二氢愈创木酸(nordihydroguaiaretic acid, NDGA)对小鼠胚胎早期发育的影响;②用NDGA处理植入前期小鼠后观察子宫内膜光镜和超微结构变化;③研究NDGA处理植入前期小鼠后子宫内膜基质金属蛋白酶-9(matrix metalloproteinase-9, MMP-9)与基质金属蛋白酶组织抑制物-1(tissue inhibitor of metalloproteinase-1, TIMP-1)表达的变化。方法以成年未经产雌性昆明种小鼠为研究对象,雌雄鼠3:1合笼,第二天清晨查到阴栓记作孕1d,将120只孕2d小鼠随机分4组(低剂量、中剂量、高剂量和对照组),每组30只,低剂量、中剂量、高剂量组分别按10 mg/kg, 30 mg/kg, 90 mg/kg剂量皮下注射NDGA,对照组注射等量PBS溶液;孕4d,每组各取15只孕鼠,颈椎脱臼处死,取出子宫,刮取约1 mm3子宫内膜组织,戊二醛固定,透射电镜分析子宫内膜结构的变化,余下子宫组织4%多聚甲醛固定,HE染色、免疫组化SP法检测MMP-9、TIMP-1表达,显微成像系统拍摄图像,进行计算机图像分析;孕8d,处死另半数孕鼠检测子宫脏器系数和胚胎着床数。结果①NDGA对小鼠子宫脏器系数、植入胚胎数的影响:对照组小鼠子宫脏器系数(2.387±0.376)%,植入胚胎数15.87±1.92,低剂量组小鼠子宫脏器系数(2.341±0.106)%,植入胚胎数14.93±1.79,与对照组相比无显著差异(P>0.05);中剂量组小鼠子宫脏器系数(1.350±0.010)%,植入胚胎数8.27±0.96,与对照组相比,有不同程度的减少(P<0.05),高剂量组小鼠子宫脏器系数(0.850±0.287)%,与对照组相比,显著下降(P<0.01),未见胚胎植入。②NDGA对小鼠子宫内膜形态学的影响:光镜观察结果显示,对照组与低剂量组小鼠子宫内膜发育明显,基质疏松、基质细胞肥大,腺上皮细胞与腔上皮细胞柱状。中剂量组与高剂量组小鼠子宫内膜基质结构较为紧密、基质细胞变小,子宫内膜腺上皮细胞与腔上皮细胞变矮。透射电镜结果表明,对照组与低剂量组小鼠子宫内膜基质细胞胞膜完整,胞质内富含粗面内质网、线粒体,腺上皮与腔上皮富含线粒体,表面可见短小微绒毛;中剂量组与高剂量组小鼠子宫内膜基质细胞边界不清,腺上皮细胞与腔上皮细胞线粒体空泡变性。③NDGA对小鼠子宫内膜MMP-9与TIMP-1表达的影响:免疫组织化学SP法染色结果显示,MMP-9与TIMP-1在对照组小鼠子宫内膜基质细胞、腺上皮细胞与腔上皮细胞中均有阳性表达;与对照组相比,低剂量组小鼠子宫内膜基质细胞、腺上皮与腔上皮细胞MMP-9与TIMP-1免疫阳性细胞的平均光密度值(mean optical density, MOD)差异无显著性(P>0.05),中剂量组与高剂量组小鼠子宫内膜基质细胞、腺上皮与腔上皮细胞MMP-9与TIMP-1免疫阳性细胞的MOD值与对照组相比,差异具有显著性(P<0.05)。结论①NDGA可能具有抑制小鼠胚胎植入的作用且此作用具有一定的剂量依赖性;②NDGA可以使植入前期小鼠子宫内膜基质细胞、腺上皮与腔上皮细胞发生不同程度的变性;③NDGA可能对植入前期小鼠子宫内膜基质细胞、腺上皮细胞与腔上皮细胞MMP-9, TIMP-1的表达具有抑制作用;④植入前期小鼠子宫内膜MMP-9与TIMP-1表达失衡可能是NDGA诱导胚泡着床障碍的机制之一。更多还原

【Abstract】 Objective①To investigate the effects of nordihydroguaiaretic acid (NDGA) on the mice embryos growth;②To observe the change of light microscope structure and ultramicrostructure in pre-implantation mice endometrium after treating with NDGA;③To investigate the change of the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in pre-implantation mice endometrium after treating with NDGA. Methods We took mature and nulliparous Kunming female mice as subject, three Kunming adult female mice were caged with a male overnight. The following morning, females were checked for the presence of a vaginal sperm plug. The day of vaginal plug was designated as day 1 of pregnancy. 120 Kunming pregnant mice were randomly divided into four groups (low-dose group, middle-dose group, high-dose group and control); low- dose group, middle-dose group, high-dose group were given NDGA (10, 30, 90 mg/kg) by s.c. injection on the day 2 of pregnancy and the control group were given the equal volume of PBS. Half of mice were put to death through cervical vertebrae luxation on the day 4 of pregnancy, then the uteri were removed, scratched one cubic centimeters of endometrium and fixed with glutaraldehyde to observe the change of ultramicrostructure in it. The remaining mice uteri fixed with 4% paraformaldehyde; HE staining was used to observe the chang of structure in mice endometrium on the day 4 of pregnancy and immunohistochemical SP staining was used to measure the expression of MMP-9 and TIMP-1 in mice endometrium on day 4 of pregnancy. Microscopic imaging system took the pictures and then analysed by computer. On the day 8 of pregnancy, the other half was killed to calculate the uterine organ coefficient and the number of the embryos. Results①In the control group, the uterine organ coefficient was (2.387±0.376)% and the number of the embryos were 15.87±1.92; In the low-dose group, the uterine organ coefficient was (2.341±0.106)% and the number of the embryos were 14.93±1.79; There were no significant differences in the uterine organ coefficient and the number of the embryos between the low-dose group and the control; In the middle-dose group, the uterine organ coefficient was (1.350±0.010)% and the number of the embryos were 8.27±0.96; Compared with the control, the uterine organ coefficient and the number of the embryo were decreased in the middle- dose group; In high-dose group, the uterine organ coefficient was(0.850±0.287)%;Compared with the control, the uterine organ coefficient were distinctly decreased in high-dose group; No embryo was observed in high-dose group.②Effect of NDGA on morphological change of mice endometrium: HE staining showed distinctly endometrial development with loosened stroma, big stromal cell, high columnar glandular epithelial cell and luminal epithelial cell in the control and low-dose groups mice endometrium; Compared with the control, the mice endometrium showed clear charges with compactible stroma, small stromal cell, low glandular epithelial cell and luminal epithelial cell in the middle-dose and high-dose groups; Under TEM, In the control and low-dose groups, the membrane of mice endometrial stromal cell was normal; Rough endoplasmic reticula(RER) and mitochondria enriched in mice endometrial stromal cell; Mitochondria enriched in glandular epithelial cell and luminal epithelial cell with short microvilli. Compared with the control, the membrane of mice endometrial stromal cell was broken and mitochondria in most glandular epithelial cell and luminal epithelial cell exhibited vacuole degeneration in the middle-dose and high-dose groups.③Effect of NDGA on the expression of MMP-9 and TIMP-1 in mice endometrium: The result of immunohistochemisty showed MMP-9 and TIMP-1 expressed in the control group mice endometrial stromal cell, glandular epithelial cell and luminal epithelial cell. Compared with the control, the mean optical density (MOD) of MMP-9 and TIMP-1 of the low-dose groups mice endometrial stromal cell, glandular epithelial cell and luminal epithelial cell had no significant difference. Compared with the control, the mean optical density (MOD) of MMP-9 and TIMP-1 of the middle-dose and high-dose groups mice endometrial stromal cell, glandular epithelial cell and luminal epithelial cell had significant difference. Conclusion①NDGA may inhibite mice blastocysts implantation with a dose-dependent manner.②After treating with NDGA, endometrial stromal cell, glandular epithelial cell and luminal epithelial cell in mice endometrium were degeneration in varying degrees.③NDGA may inhibit the expression of MMP-9 and TIMP-1 in the endometrial stromal cell, glandular epithelial cell and luminal epithelial cell.④The expression of MMP-9 and TIMP-1 were imbalance in mice endometrium after treating with NDGA and it may be one of the mechanism induced the failure of implantation.更多还原