摘要
为了对瘦素及长型瘦素受体在小鼠胚胎附植前后各组织中的表达情况进行研究,本试验采用蛋白免疫印迹实验方法对下丘脑、子宫、卵巢、胃、十二指肠组织中的瘦素受体进行了分析,采用免疫组织化学SP染色法对长型瘦素受体在雌性小鼠生殖周期不同阶段的下丘脑、卵巢、子宫、胃、十二指肠细胞定位和分布进行了研究,并用ELISA方法对小鼠血清中的瘦素浓度进行了检测。皮下脂肪组织冰冻切片用苏丹(Ⅲ)染色计量脂肪细胞数量和大小的变化,结果显示:瘦素受体六个亚型的分子量大约分别为120KDa、90 KDa、77 KDa、66.2 KDa、54 KDa、47 KDa;下丘脑神经元胞质中有棕褐色阳性颗粒,且阳性细胞数量随妊娠日龄增加逐渐增加,妊娠期与间情期阳性细胞数差异显著(P<0.05);在卵巢中,卵母细胞胞质中有长型瘦素受体表达,随卵泡的发育,卵泡细胞中免疫反应阳性细胞数量逐渐增加;在胚泡附植期,瘦素受体在子宫腺和子宫内膜上皮细胞中大量表达。胃底腺中下部分胞质和细胞核有棕褐色阳性颗粒,且阳性率随妊娠日龄的增加而增加;十二指肠腺中胞质和细胞核有棕褐色阳性颗粒,且阳性率随妊娠日龄的增加而增加。与单位面积内脂肪细胞数量的变化趋势一致。妊娠7 d,单位面积内脂肪细胞比间情期少,单个细胞面积大。妊娠期血清瘦素浓度均高于间情期,且瘦素浓度从间情期到妊娠4 d,呈上升趋势,妊娠5 d稍下降,后随妊娠日龄逐渐增加。结论:瘦素及其长型受体可能促进卵泡发育,有利于小鼠胚泡的附植。血清瘦素浓度变化趋势与单位面积内脂肪细胞数量呈正相关,即在小鼠胚泡附植前后脂肪组织越多血清瘦素水平越高。推测:小鼠胚胎附植前后瘦素及其长型受体通过中枢神经和消化器官对机体的能量平衡进行调节,性腺轴针对妊娠母体生理条件的改变进行代谢调节,从而促进胚胎的附植。
The paper aimed to study the expression of leptin and long-leptin receptor positive cells in the tissues pre- and post- implantation in mice. We investigated the expression of the leptin receptor in the hypothalamus, uterus, ovary, stomach, duodenum tissues by the Western blot, and also the long-leptin receptor positive cells were located by immunohistochemistry staining method in the hypothalamus, ovarian, uterine at different stages of reproductive cycle in the female KM mouse. The concentration of serum leptin was tested by ELISA (enzymelinked immunosorbentassy) method. Additionaly, adipose tissue was made into frozen section with Sudan (Ⅲ) staining for measurement of fat cells.The results showed: The molecular weight of the six bands of leptin receptor isoforms are about 120KDa, 90 KDa, 77 KDa, 66.2 KDa, 54 KDa, 47 KDa; There are many tan-positive particles in the neuron cytoplasm of hypothalamus, and the tan-positive cells increase with pregnant days. We found that there was significant difference between dioestrus and pregnancy (P < 0.05); The expression of long-leptin receptor gradually increase in ovarian oocyte cytoplasm with the development of follicle. At the same time, the numbers of the surrounding positive follicle cells are gradually enhanced; during the period of implantation, a large number of positive cells are founded in columnar cells of the uterine gland and uterine endometrial epithelium. In the lower part of the stomach fundic gland, there are more positive brown particles in the cytoplasm and nucleus of pregnancy mice than that of the dioestrus mice. In duodenal glands, there are more positive brown particles in the cytoplasm and nucleus of pregnancy mice than that of the dioestrus. The size of unit area of fat cells in 7-day-old pregnancy is less than that of the dioestrus, but the concentrations of serum leptin in 7-day-old pregnancy is higher than that of the dioestrus. It is concluded that Leptin and its long leptin receptor regulate energy balance through the central nervous system and digestive organs, and gonad axis regulate the metabolism according to the physiological conditions of pregnant materal status, which promote embryo implantation in mice.
引文
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