雌鼠高龄生育子代海马Skp2和神经发育相关基因的表达

发布时间：2018-06-15 15:25

本文选题：高龄生育 + SD大鼠　；参考：《郑州大学》2017年硕士论文

【摘要】：随着经济社会的发展,女性受教育程度的提高,婚育年龄逐渐推迟,高龄生育逐渐增加成为一个社会问题。特别是我国全面二胎政策的开放,高龄生育已经成为一种社会趋势,越来越受到社会各界的重视。高龄生育除了对于产妇本身和生育结局具有不利影响外,同时也对婴幼儿的生长发育具有负面作用。但是,众多研究报道中,母亲高龄生育对子代神经发育和智力的影响尚存在争议。本实验室已采用Morris水迷宫实验证明雌鼠高龄生育子代的学习记忆能力比适龄生育子代较低,并通过基因芯片、q RT-PCR和Western blot证明一系列基因在雌鼠高龄生育子代新生期海马中差异性表达。为了进一步探讨高龄生育对子代生长发育,特别是对神经智力方面的影响,我们拟采用q RT-PCR法和免疫印迹法检测雌性高龄生育子代海马发育过程中S期激酶相关蛋白2(S-phase kinase-associated protein 2,Skp2)基因动态表达特性,及Skp2下游底物蛋白p27和p21表达特性。同时我们也记录比较了高龄生育子代与青年生育子代发育过程中的相关指标和成年海马组织内神经发育相关基因的表达特性,在分子水平上进一步探索高龄生育对子代海马发育的影响。目的研究雌鼠高龄生育对子代发育过程中相关发育指标和海马组织内Skp2及p21、p27的影响,观测成年后海马组织内神经发育相关基因的表达特性,探讨其在海马神经发育过程中的作用,以及对子代的成年后的学习、记忆、认知的可能影响。方法方法1.实验动物和分组:选用清洁级SD(Sprague Dawley)大鼠(购自郑州大学实验动物中心)。青年雌鼠(3月龄)和高龄雌鼠(12月龄)。分别同随机选择的青年雄鼠(3月龄)合笼,所生仔鼠分别为青年组和高龄组。2.生育能力检测:记录合笼-分娩天数、每窝产仔数和雌雄比例。3.仔鼠发育过程中体重的检测:记录仔鼠出生第1、3、7、14、21、28、60天的体重。4.Skp2及相关基因的检测:仔鼠出生第1、3、7、14、21、28、60天处死后取海马组织,通过实时荧光定量PCR法检测Skp2 m RNA表达,通过免疫印迹法检测Skp2及其底物p27、p21蛋白的表达。5.成年子代海马内学习记忆相关基因的检测:通过实时荧光定量PCR法检测海马内神经发育相关基因Edn1、Hspa4、Fos、Bcl2、Grin1 m RNA表达。6.统计学分析:用SPSS17.0统计学软件进行数据分析。对于青年组和高龄组体重变化采用单因素方差分析。对于两组仔鼠雌雄比例、发育过程中各时间点的发育指标及Skp2的表达量采用独立样本的t检验。检验水准取α=0.05。结果1.雌鼠生育能力:青年雌鼠和高龄雌鼠分别同随机选择的青年雄鼠合笼,高龄组的合笼至分娩天数比青年组显著增加,产仔数则明显降低(P0.01),两组子代雌雄比例无显著差异(P0.05)。2.仔鼠发育过程中体重检测:分别在仔鼠出生后第1、3、7、14、21、28、60天记录体重。发现仔鼠出生后体重随时间的推移显著增加,高龄组与青年组在各个时间点体重差异无统计学意义(P0.05)。3.发育过程中Skp2、p27和p21表达检测:高龄组和青年组子代海马内Skp2的m RNA表达量随发育时间的延长而下降。高龄组中第7天Skp2的m RNA表达显著低于青年组(P0.05),其余各时间点两组间无显著性差异(P0.05)。高龄组和青年组中Skp2的蛋白表达量均随发育时间的延长逐渐下降,出生第1~14天两组间无显著差异(P0.05),第21~60天高龄组子代海马中Skp2表达显著高于青年组(P0.05);高龄组和青年组中p27的蛋白表达量随发育时间的延长逐渐下降,两组间无显著差异(P0.05);高龄组和青年组中p21的蛋白表达量随发育时间的延长增加,高龄组第28、60天p21表达显著高于青年组(P0.05)。4.高龄组成年子代海马内神经功能的相关基因m RNA检测:高龄组成年子代(60天)海马内Edn1和Hspa4的表达量均显著低于青年组(Edn1 P0.05,Hspa4 P0.01),Fos、Bcl2和Grin1的表达量则无显著差异(P0.05)。结论1.Skp2和p21可能是雌鼠高龄生育对子代神经功能及学习和记忆的影响因素,但Skp2-p27/p21调控不是高龄生育对子代海马神经发育的调控方式。2.高龄组成年子代海马内Edn1和Hspa4的m RNA呈低表达状态,可能与高龄生育子代神经功能改变相关。
[Abstract]:With the development of the economy and society, the age of marriage and childbearing is gradually postponed and the age of older children is becoming a social problem. In particular, the opening of the overall two child policies in our country has become a social trend, and more and more attention has been paid to the social circles. The outcome has negative effects and negative effects on the growth and development of infants. However, there are many controversies on the influence of the mother's age on the neurodevelopment and intelligence of the offspring. In this laboratory, the Morris water maze test has been used to prove that the learning and memory ability of the female offspring is better than that of the older offspring. The differential expression of a series of genes in the newborn hippocampus of the female rats was demonstrated by gene chip, Q RT-PCR and Western blot. In order to further explore the growth and development of the offspring, especially the effects on the neural intelligence, we should use the Q RT-PCR method and the immunoblotting method to detect the female older generation. The dynamic expression of S phase kinase related protein 2 (S-phase kinase-associated protein 2, Skp2) and the expression characteristics of p27 and p21 of the downstream substrate protein of the offspring during the development of the offspring hippocampus, as well as the expression characteristics of the p27 and p21 of the downstream substrate protein of the Skp2. Meanwhile, we also recorded and compared the related indicators in the development of the offspring and the young offspring of the young offspring and the nerve hair in the adult hippocampus. The effect of the expression of the related genes on the development of the hippocampal formation of the offspring at the molecular level was further explored. Objective to study the related developmental indexes and the effects of Skp2 and p21, p27 in the hippocampus of the female rats during the development of the offspring, and to observe the expression characteristics of the neural development related genes in the adult hippocampus. Its role in the development of hippocampal neural development and the possible influence on the learning, memory and cognition of the adulthood of the progeny. Methods and methods 1. experimental animals and groups: selected SD (Sprague Dawley) rats (from the experimental animal center of Zhengzhou University). Young female rats (3 month old) and older female rats (12 month old). The male rats (3 month old) were caged, and the offspring rats were tested for.2. fertility in young and old age groups, respectively. The number of birth days, the number of litter and the male and female offspring in the development of.3. offspring were measured. The weight of.4.Skp2 and related genes were recorded on day 1,3,7,14,21,28,60 of the offspring of the offspring: the birth of the offspring was 1,3,7,14,21,28,60. The expression of Skp2 m RNA was detected by real-time fluorescence quantitative PCR, Skp2 and its substrate p27 were detected by immunoblotting, and the expression of p21 protein was detected by immunoblotting. The detection of learning and memory related genes in the hippocampus of.5. adult progeny was detected by the immunoblotting method: Edn1, Hspa4, Fos, and Edn1 were detected by real-time fluorescent quantitative PCR method. M RNA expression.6. statistical analysis: using SPSS17.0 statistical software for data analysis. Single factor variance analysis was used for the body weight change of young and elderly groups. For two groups of male and female male and female male and female sex ratio, the development index and the expression of Skp2 in the development process were independent sample based t test. The test level took the alpha =0.05. result 1. female. The reproductive capacity of the rats: young female rats and older female rats were coaged with young male rats, respectively. The number of cages in the aged group increased significantly than that in the young group, and the number of baby litter decreased significantly (P0.01). There was no significant difference between the two groups of offspring and male and female sex (P0.05) in the development of.2. offspring: 1,3,7,14,21,2 after the birth of the offspring, respectively. Weight was recorded at 8,60 days. It was found that the weight of the offspring increased significantly with time, and there was no significant difference between the age group and the young group at all time points (P0.05), and the expression of Skp2, p27 and p21 in the development of.3.: the m RNA expression of Skp2 in the hippocampus of the elderly and young groups decreased with the prolongation of the development time. The expression of M RNA of Skp2 in the last seventh days was significantly lower than that in the young group (P0.05), and there was no significant difference between the two groups at the rest of the time (P0.05). The protein expression of Skp2 in the elderly and young groups decreased gradually with the prolongation of the development time. There was no significant difference between the two groups at day 1~14 (P0.05), and the expression of Skp2 in the offspring of the offspring of the first 21~60 day group was significantly higher. In the young group (P0.05), the protein expression of p27 in the age group and the young group gradually decreased with the development time, and there was no significant difference between the two groups (P0.05). The protein expression of p21 in the elderly and young groups increased with the prolongation of the development time. The expression of p21 in the 28,60 days of the elderly group was significantly higher than that of the young group (P0.05).4. senior. M RNA detection of the related genes of internal nerve function: the expression of Edn1 and Hspa4 in the hippocampus of the older generation (60 days) was significantly lower than that of the young group (Edn1 P0.05, Hspa4 P0.01), and there was no significant difference in the expression of Fos, Bcl2 and Grin1 (P0.05). Factors, however, Skp2-p27/p21 regulation is not a way of regulating the development of the hippocampal nerve in the offspring of the elderly..2. is a low expression of M RNA in the hippocampal Edn1 and Hspa4, which may be related to the changes in the neurologic function of the offspring of the older generation.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714

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