本文選題：高齡生育 + SD大鼠��； 參考：《鄭州大學》2017年碩士論文

【摘要】：隨著經(jīng)濟社會的發(fā)展,女性受教育程度的提高,婚育年齡逐漸推遲,高齡生育逐漸增加成為一個社會問題。特別是我國全面二胎政策的開放,高齡生育已經(jīng)成為一種社會趨勢,越來越受到社會各界的重視。高齡生育除了對于產(chǎn)婦本身和生育結(jié)局具有不利影響外,同時也對嬰幼兒的生長發(fā)育具有負面作用。但是,眾多研究報道中,母親高齡生育對子代神經(jīng)發(fā)育和智力的影響尚存在爭議。本實驗室已采用Morris水迷宮實驗證明雌鼠高齡生育子代的學習記憶能力比適齡生育子代較低,并通過基因芯片、q RT-PCR和Western blot證明一系列基因在雌鼠高齡生育子代新生期海馬中差異性表達。為了進一步探討高齡生育對子代生長發(fā)育,特別是對神經(jīng)智力方面的影響,我們擬采用q RT-PCR法和免疫印跡法檢測雌性高齡生育子代海馬發(fā)育過程中S期激酶相關(guān)蛋白2(S-phase kinase-associated protein 2,Skp2)基因動態(tài)表達特性,及Skp2下游底物蛋白p27和p21表達特性。同時我們也記錄比較了高齡生育子代與青年生育子代發(fā)育過程中的相關(guān)指標和成年海馬組織內(nèi)神經(jīng)發(fā)育相關(guān)基因的表達特性,在分子水平上進一步探索高齡生育對子代海馬發(fā)育的影響。目的研究雌鼠高齡生育對子代發(fā)育過程中相關(guān)發(fā)育指標和海馬組織內(nèi)Skp2及p21、p27的影響,觀測成年后海馬組織內(nèi)神經(jīng)發(fā)育相關(guān)基因的表達特性,探討其在海馬神經(jīng)發(fā)育過程中的作用,以及對子代的成年后的學習、記憶、認知的可能影響。方法方法1.實驗動物和分組:選用清潔級SD(Sprague Dawley)大鼠(購自鄭州大學實驗動物中心)。青年雌鼠(3月齡)和高齡雌鼠(12月齡)。分別同隨機選擇的青年雄鼠(3月齡)合籠,所生仔鼠分別為青年組和高齡組。2.生育能力檢測:記錄合籠-分娩天數(shù)、每窩產(chǎn)仔數(shù)和雌雄比例。3.仔鼠發(fā)育過程中體重的檢測:記錄仔鼠出生第1、3、7、14、21、28、60天的體重。4.Skp2及相關(guān)基因的檢測:仔鼠出生第1、3、7、14、21、28、60天處死后取海馬組織,通過實時熒光定量PCR法檢測Skp2 m RNA表達,通過免疫印跡法檢測Skp2及其底物p27、p21蛋白的表達。5.成年子代海馬內(nèi)學習記憶相關(guān)基因的檢測:通過實時熒光定量PCR法檢測海馬內(nèi)神經(jīng)發(fā)育相關(guān)基因Edn1、Hspa4、Fos、Bcl2、Grin1 m RNA表達。6.統(tǒng)計學分析:用SPSS17.0統(tǒng)計學軟件進行數(shù)據(jù)分析。對于青年組和高齡組體重變化采用單因素方差分析。對于兩組仔鼠雌雄比例、發(fā)育過程中各時間點的發(fā)育指標及Skp2的表達量采用獨立樣本的t檢驗。檢驗水準取α=0.05。結(jié)果1.雌鼠生育能力:青年雌鼠和高齡雌鼠分別同隨機選擇的青年雄鼠合籠,高齡組的合籠至分娩天數(shù)比青年組顯著增加,產(chǎn)仔數(shù)則明顯降低(P0.01),兩組子代雌雄比例無顯著差異(P0.05)。2.仔鼠發(fā)育過程中體重檢測:分別在仔鼠出生后第1、3、7、14、21、28、60天記錄體重。發(fā)現(xiàn)仔鼠出生后體重隨時間的推移顯著增加,高齡組與青年組在各個時間點體重差異無統(tǒng)計學意義(P0.05)。3.發(fā)育過程中Skp2、p27和p21表達檢測:高齡組和青年組子代海馬內(nèi)Skp2的m RNA表達量隨發(fā)育時間的延長而下降。高齡組中第7天Skp2的m RNA表達顯著低于青年組(P0.05),其余各時間點兩組間無顯著性差異(P0.05)。高齡組和青年組中Skp2的蛋白表達量均隨發(fā)育時間的延長逐漸下降,出生第1~14天兩組間無顯著差異(P0.05),第21~60天高齡組子代海馬中Skp2表達顯著高于青年組(P0.05);高齡組和青年組中p27的蛋白表達量隨發(fā)育時間的延長逐漸下降,兩組間無顯著差異(P0.05);高齡組和青年組中p21的蛋白表達量隨發(fā)育時間的延長增加,高齡組第28、60天p21表達顯著高于青年組(P0.05)。4.高齡組成年子代海馬內(nèi)神經(jīng)功能的相關(guān)基因m RNA檢測:高齡組成年子代(60天)海馬內(nèi)Edn1和Hspa4的表達量均顯著低于青年組(Edn1 P0.05,Hspa4 P0.01),Fos、Bcl2和Grin1的表達量則無顯著差異(P0.05)。結(jié)論1.Skp2和p21可能是雌鼠高齡生育對子代神經(jīng)功能及學習和記憶的影響因素,但Skp2-p27/p21調(diào)控不是高齡生育對子代海馬神經(jīng)發(fā)育的調(diào)控方式。2.高齡組成年子代海馬內(nèi)Edn1和Hspa4的m RNA呈低表達狀態(tài),可能與高齡生育子代神經(jīng)功能改變相關(guān)。
[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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R714

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