【作者】 张慧；

【导师】 钱新华；

【作者基本信息】 南方医科大学 ， 儿科学， 2013， 博士

【摘要】 研究背景随着围产医学技术的进步,尤其是危重新生儿救治水平的提高,早产儿,特别是出生体重低于1500g的极低/超低出生体重儿(Very Low Birth Weight/Extremely Low Birth Weight, VLBW/ELBW)的存活率明显提高。虽然早产儿存活率大大提高,但神经发育障碍并未成比例的下降。早产儿中脑性瘫痪(cerebral palsy, CP)的发生率呈逐年上升趋势。存活早产儿神经发育的远期问题主要为脑性瘫痪、认知能力障碍、智力障碍以及视听觉缺陷。即使有部分早产儿并无以上的损伤,但却表现为隐性残疾(如：情绪不受控、注意力无法集中、阅读书写能力差等。早产儿神经发育障碍与窒息、病理性黄疸、颅内出血、缺氧缺血性脑病等早产儿并发症有关外,早产儿营养对神经发育结局的影响是备受各国学者关注的焦点之一,如何做到最佳的营养支持方案这一直是医学界丞待解决的问题。Lucas教授提出“营养程序化”(nutritional programming)的概念,即在发育的关键期或敏感期的营养状况将对机体或各器官功能产生长期乃至终生的影响,其临床研究也表明,在关键期给予早产配方奶(高蛋白)更有利于神经发育。Singhal等学者则发现,虽然强化营养配方喂养组比起标准配方喂养组神经发育更优,但青春期时胰岛素抵抗和高血压等代谢性疾病的发生率却更高。大脑发育是个漫长而又极其复杂的过程,涉及神经细胞的分化、增殖,神经滋养细胞的分化、增殖,脑及脑区的形成等,不同的发育阶段对营养有不同的需求,除“关键期”营养干预可影响早产儿神经发育外,是否在“关键期”后实施营养干预也能影响早产儿神经发育,并且还能降低早产儿追赶生长带来的负面效应,这是一个非常值得探讨的课题。哺乳类动物雷帕霉素靶蛋白(mammalian target of rapamyoin, mTOR)是进化上高度保守的丝氨酸/苏氨酸蛋白激酶,属于磷脂酰肌醇3-激酶相关激酶(plosphatidylin0sitol3-kinase-related kinase, PIKK)蛋白质家族的成员,在哺乳动物细胞内感受细胞外营养、能量水平以及生长因子等信号变化。mTOR不同结构域和不同蛋白结合在细胞内主要形成两种复合物,即mTOR-Raptor复合物(mTORCl)和mTOR-Richtor复合物(mTORC2)。正常情况下,生长因子(如胰岛素、胰岛素样生长因子、表皮生长因子等)诱导mTOR的活化是通过激活PI3K实现的。PI3K可磷酸化3-磷酸肌醇,磷酸化的3-磷酸肌醇(PI3Ps)与Akt的PH区域连接导致Akt转位至浆膜.从而与磷酸肌醇依赖性激酶1(PDKI)接触。PDKI负责Akt的磷酸化,从而激活Akt。激活的Akt可磷酸化结节性硬化复合体2(tuberoussclerosiscomplex2, TSC2)并减弱后者对mTORC1的抑制作用。与生长因子激活通路不同,营养信号激活通路主要经过AMPK途径。AMPK受细胞内ATP/AMP的调节当ATP/AMP比例下调时.AMPK被激活,活化的AMPK可磷酸化并激活TSC1/TSC2二聚体,抑制mTORC1,反之亦然。活化的mTORC1进一步磷酸化S6K和4EBP,从而参与基因转录、蛋白质翻译起始、核糖体生物合成、细胞周期调控等。近年来,一系列的研究发现mTOR相关信号通路与突触可塑性、学习、记忆功能,神经系统退行性病变及摄食等密切相关。mTOR信号通路在海马的学习、记忆提取和记忆巩固的蛋白翻译过程中有着重要作用；mTOR信号通路是长时程电位晚期成分形成的必要条件；脂肪贮存充足时,mTOR促进来普汀的产生,来普汀依次传递能量过剩的信息给中枢神经系,抑制摄食和促进能量消耗。然而目前的研究多集中在成年相关疾病,mTOR与新生儿特别是早产儿的生长发育相关研究较少,不同营养条件下早产儿mTOR通路的活性及其生长发育的影响还有待我们探讨。现行研究多集中在成年相关疾病,mTOR与新生儿特别是早产儿的生长发育相关研究较少,深入研究不同营养条件下mTOR通路的活性改变对于探讨早产儿生长发育尤其是神经系统发育,优化早产儿营养支持方案具有重要意义。目的与意义：本研究通过建立早产大鼠模型,断奶后给予不同的蛋白质饲料喂养至6周和8周,并通过Morris水迷宫实验,检测大鼠学习记忆能力的改变,并通过免疫组织化学染色、蛋白印迹法(western blot)技术分析mTOR/S6K通路活性的变化,探讨关键期后不同的蛋白摄入水平对早产大鼠学习记忆能力的影响及其可能机制,为优化早产儿营养支持方案提供依据。方法：通过剖宫产法获得新生SD早产大鼠,仔鼠由代乳母鼠哺乳至21日龄。将断奶后的早产SD仔鼠和足月SD仔鼠采用机数字法分为6组：早产标准蛋白组(A)、足月标准蛋白组(B)、早产低蛋白组(C)、足月低蛋白组(D)、早产高蛋白组(E)、足月高蛋白组(F),每组大鼠34只。从第22日龄起,标准蛋白组、低蛋白组和高蛋白组给予标准蛋白饲料(含18%蛋白质)、低蛋白饲料(含8%蛋白质)和高蛋白饲料(含30%蛋白质)喂养至实验结束。每组大鼠分别在第6周和第8周进行Morris水迷宫实验,检测大鼠学习记忆能力的改变。同期免疫组织化学法观察海马mTOR分布。Western blot观察海马mTOR、p-mTOR(Ser2448)、 p70S6K、p-p70S6K(Thr389、4EBP1表达和活性。研究结果(一)不同蛋白摄入水平对早产大鼠学习记忆能力的影响1定位航行实验(1)6周龄大鼠逃避潜伏期比较：各组大鼠6周龄时定位航行实验中逃避潜伏期的比较结果显示,足月大鼠(足月高蛋白组、足月低蛋白组和足月标准蛋白组)的逃避潜伏期随训练天数增加逐渐缩短(P<0.05),并且每天缩短的速度较为稳定,而早产大鼠(早产高蛋白组、早产低蛋白组和早产标准蛋白组)的逃避潜伏期随训练天数的变化趋势与对足月大鼠不同,呈现出先快后慢的变化趋势。早产大鼠在前3天的逃避潜伏期随训练天数增加逐渐缩短(P<0.05),但是在接下来的第4天其逃避潜伏期与第3天基本接近,变化不显著(P>0.05)。组间两两比较显示,与足月大鼠比较,早产大鼠前3天的逃避潜伏期无显著性差异,第4天逃避潜伏期,足月高蛋白组大鼠明显低于早产高蛋白组大鼠(P<0.05)。说明6周龄的足月大鼠学习认知能力强于早产大鼠。(2)8周龄大鼠逃避潜伏期比较：各组大鼠8周龄时定位航行实验中逃避潜伏期的比较结果显示,足月大鼠和早产大鼠的逃避潜伏期均随训练天数增加逐渐缩短。(3)早产大鼠6周龄和8周龄的逃避潜伏期比较：早产大鼠8周龄时定位航行实验中逃避潜伏期的比较结果显示,其逃避潜伏期随训练天数增加逐渐缩短(P<0.05),并且每天缩短的速度较为稳定,而在6周龄时,早产大鼠的逃避潜伏期随训练天数的变化趋势与8周龄时不同,呈现出先快后慢的变化趋势。6周龄早产大鼠在前3天的逃避潜伏期随训练天数增加逐渐缩短,但是在接下来的第4天其逃避潜伏期与第3天基本接近,变化不显著(P>0.05)。不同周龄间两两比较显示,与6周龄早产大鼠比较,8周龄早产大鼠前3天的逃避潜伏期无显著性差异；第4天逃避潜伏期,8周龄早产高蛋白组大鼠明显低于6周龄早产高蛋白组大鼠(P<0.05)。2空间探索实验(1)各组大鼠6周龄时空间探索实验的比较：在予蛋白含量相同饲料饲养的情况下,6周龄早产大鼠的原平台所在象限时间和原平台所在象限路程占总路程的百分比均明显少于6周龄足月大鼠,差异具有显著性(P<0.05)。(2)不同蛋白饲料对6周龄足月大鼠空间探索实验的影响：不同蛋白饲料组足月大鼠6周龄时,各组大鼠的原平台所在象限时间和原平台所在象限路程占总路程的百分比差异无统计学意义(P>0.05)。(3)不同蛋白饲料对6周龄早产大鼠空间探索实验的影响：不同蛋白饲料组早产大鼠6周龄时,各组大鼠的原平台所在象限时间和原平台所在象限路程占总路程的百分比差异无统计学意义(P>0.05)。(4)同种蛋白饲料对各组大鼠8周龄时空间探索实验的影响：在予蛋白含量相同饲料饲养的情况下,与8周龄足月大鼠比较,早产大鼠的原平台所在象限时间和原平台所在象限路程占总路程的百分比均无统计学差异(P>0.05)。(5)不同蛋白饲料对8周龄足月大鼠空间探索实验的影响：不同蛋白饲料组足月大鼠8周龄时,各组大鼠的原平台所在象限时间和原平台所在象限路程占总路程的百分比差异均无统计学意义(P>0.05)。(6)不同蛋白饲料对8周龄早产大鼠空间探索实验的影响：与早产高蛋白组比较,早产标准蛋白组大鼠和早产低蛋白组大鼠的原平台所在象限时间和原平台所在象限路程占总路程的百分比均明显减少,差异具有统计学意义(P<0.05)。(7)早产大鼠6周龄和8周龄时空间探索实验的比较：在予蛋白含量相同饲料饲养的情况下,8周龄早产高蛋白组大鼠的穿越原平台时间和原平台所在象限时间占总时间的百分比均明显大于6周龄早产高蛋白组大鼠,差异具有显著性(P<0.05)。(二)不同蛋白摄入水平对早产大鼠海马mTOR/S6K信号通路的影响1免疫组化检测早产大鼠海马mTOR蛋白的表达(1)同种蛋白饲料对6周龄大鼠海马CA1、CA3区1mTOR表达的影响：免疫组化结果显示,mTOR免疫阳性细胞主要分布于大脑海马CA1、CA3和齿状回(DG)。其中海马区CA1起始部及CA3起始部,阳性细胞相对海马其它部位多,着色更深。高倍镜下,mTOR阳性细胞边界清晰,主要在胞浆着色。同种蛋白饲料6周龄各组大鼠海马CA1、CA3区mTOR免疫阳性细胞计数,6周龄足月各组大鼠海马CA1、CA3区mTOR阳性细胞均比早产组多,差异具有统计学意义(P<0.05)。(2)不同蛋白饲料对6周龄足月大鼠海马CA1、CA3区mTOR表达的影响：6周龄各组足月大鼠海马CA1、CA3区mTOR免疫阳性细胞计数差异并不明显(P>0.05)。(3)不同蛋白饲料对6周龄早产大鼠海马CA1、CA3区mTOR表达的影响：6周龄各组早产大鼠海马CA1、CA3区mTOR免疫阳性细胞计数差异并不明显(P>0.05)。(4)同蛋白饲料对8周龄大鼠海马CA1、CA3区mTOR表达的影响：免疫组化结果显示,mTOR免疫阳性细胞主要分布于大脑海马CA1、CA3和齿状回(DG)。其中海马区CA1起始部及CA3起始部阳性细胞相对海马其它部位多,着色更深。高倍镜下,mTOR阳性细胞边界清晰,主要在胞浆着色。同种蛋白饲料8周龄各组大鼠海马CA1、CA3区1mTOR免疫阳性细胞计数中,8周龄足月低蛋白组大鼠海马CA1、CA3区mTOR阳性细胞均比早产低蛋白组多,差异具有统计学意义(P<0.05)。8周龄标准蛋白饲料、高蛋白饲料足月组大鼠海马CA1、 CA3区mTOR阳性细胞与同龄同饲料喂养早产组大鼠海马CA1、CA3区mTOR阳性细胞的差异不明显(P>0.05)。(5)不同蛋白饲料对8周龄足月大鼠海马CA1、CA3区mTOR表达的影响：8周龄各组足月大鼠海马CA1、CA3区mTOR免疫阳性细胞计数差异并不明显(P>0.05)。(6)不同蛋白饲料对8周龄早产大鼠海马CA1、CA3区1mTOR表达的影响：8周龄早产大鼠海马CA1、CA3区mTOR免疫阳性细胞主要分布情况同6周龄早产大鼠。经阳性细胞计数半定量分析结果显示,8周龄早产高蛋白组大鼠海马CA1、CA3区mTOR阳性细胞均比其余两组多,差异具有统计学意义(P<0.05)。(7)早产大鼠6周龄和8周龄时mTOR表达的比较：在予蛋白含量相同饲料饲养的情况下,与6周龄早产大鼠比较,8周龄早产大鼠海马CA1和CA3区mTOR阳性细胞增多,差异具有显著性(P<0.05),其中8周龄早产高蛋白组mTOR阳性细胞增加较为显著。2免疫印迹检测蛋白摄入水平对早产大鼠mTOR/S6K信号通路的影响(1)6周龄大鼠海马mTOR信号通路的变化：①早产标准蛋白(PS)、早产低蛋白(PL)、早产高蛋白(PH)组6周龄大鼠在海马中的p-p70S6K(Thr389)的表达受严重抑制甚至不表达。②早产标准蛋白(PS)、早产低蛋白(PL)、早产高蛋白(PH)组6周龄大鼠在海马中的4EBP1的表达比同龄足月标准蛋白(PS)、足月低蛋白(PL)、足月高蛋白(PH)组的弱。③早产标准蛋白、早产低蛋白、早产高蛋白组、足月标准蛋白(PS)、足月低蛋白、足月高蛋白组6周龄大鼠海马中的mTOR、p-mTOR(Ser2448)、p70S6K的表达差异不大。(2)8周龄大鼠海马mTOR信号通路的变化：①早产标准蛋白(PS)、早产低蛋白(PL)、早产高蛋白(PH)组8周龄大鼠在海马中的p-p70S6K(Thr389)表达较前明显。②早产低蛋白组8周龄大鼠在海马中4EBP1的表达较同龄的其他组(早产标准蛋白组、早产高蛋白组同龄足月标准蛋白组、足月低蛋白组、足月高蛋白组)为弱。③早产标准蛋白、早产低蛋白、早产高蛋白组、足月标准蛋白(PS)、足月低蛋白、足月高蛋白组8周龄大鼠海马中的1mTOR、 p-mTOR(Ser2448)、p70S6K的表达差异不大。(3)6周,8周龄各组大鼠海马p-p70S6K/Actin免疫活性比值比较：①6周周龄各组大鼠海马p-p70S6K/Actin免疫活性比值比较,结果显示,早产各组大鼠海马p70S6K磷酸化水平较同龄足月各组大鼠海马p70S6K磷酸化水平明显降低,以胎龄和蛋白水平为主要影响因素的双向ANOVA分析提示胎龄有显著性作用(F胎龄=8.233,P=0.014<0.05)。②8周周龄各组大鼠海马p-p70S6K/Actin免疫活性比值比较,结果显示,高蛋白组(早产高蛋白组合足月高蛋白组)大鼠海马p70S6K磷酸化水平显著升高,以胎龄和蛋白水平为主要影响因素的双向ANOVA分析提示蛋白水平有显著性作用(F蛋白水平=4.13,P=<0.05)。高蛋白组与低蛋白组的LSD事后检验P=0.021<0.05,高蛋白组与标准蛋白组的LSD事后检验P=0.042<0.05。结论1在大鼠早期生长发育过程中,早产可能严重影响大鼠大脑的功能,在大脑发育关键期后的发育过程中,短时间加强营养的补给仍不能弥补早产对大鼠学习认知能力造成的影响,其学习记忆能力明显差于足月大鼠。2在随后的生长发育过程中,高蛋白组早产大鼠的学习记忆能力得到恢复并能追赶上足月大鼠。经过长时间高蛋白营养的补充,早产高蛋白组大鼠的学习认知能力可以得到改善。3早产大鼠的大脑发育过程中可能存在另一个发育关键期。4我们的研究证实,早产可能抑制mTOR/S6K活性,可能与早产大鼠学习记忆能力差有关。5长时间摄入高蛋白营养可显著改善早产大鼠学习记忆能力,可能与上调mTOR/S6K表达有关。更多还原

【Abstract】 As the development of perinatal medicine and technology, especially in rescuing critically ill neonates, the survival rate of premature infants, were significantly improved. Especially in very low birth weight/extremely low birth weight infants (VLBW/ELBW),whose birth weight is under1500g. But at the same time, extrauterine growth retardation (EUGR), neurodevelopmental disorders and other new issues have become globally problems. Althought the survival rate of the premature infants is greatly improved, but the decline is not proportional to the neurodevelopmental disorders. Survival preterm infants face neurological development problems and long-term problems as cerebral palsy, mental retardation, cognitive impairment and audio-visual defects. Even if there are no injury in preterm infants, they may showed recessive disability (such as:the mood is not controlled, inability to concentrate, reading and writing ability, etc.)Nutrition is an important problem that has caused widespread concern of neurodevelopmental outcomes. Lucas proposed the "nutritional programming" concept, namely in the nutritional status of key developmental or sensitive period of the organ function influence the whole life. Clinical study of Lucas also shows that, given the preterm formula milk in the critical period (high protein) can affect the long-term neurodevelopmental. Singhal found that, although the fortified formula feeding group compared with the standard formula feeding group of neural development is better, but the adolescent hypertension and insulin resistance and metabolic disease incidence rate is high. Therefore, in premature infants, nutritional status and related to the critical period of neurodevelopment delay and risk of metabolic disease. How to balance and reduce these risk are the challenges we face. Brain development is a long process, understand the different stages of nutritional effects on brain development and its mechanism, has important significance to improve the neurological development of preterm infants prognosis and reduce the risk of metabolic diseases.Mammalian target of rapamycin (mTOR) is evolutionarily conserved serine/threonine protein kinase, belongs to the phosphatidylinositol3-kinase kinase (PIKK) protein family members, receptor cell nutrition, energy level and growth factor signals change in mammalian cells. mTOR play an important role in the formation of the formation and development of the brain, neural synapses occurrence, learning and memory.In this study, we established premature rat model. Then the rats were feeded to different protein feed after weaning to6weeks and8weeks, and tested the ability of learning and memory in rats through the Morris water maze test. Analyzed the changes of mTOR/S6K pathway activity in hippocampus by using immunohistochemistry and Western blot technique. Then to explore the effects of different protein intake levels on ability of learning and memory of premature birth rats and its possible mechanism.Objective:To study the influence of protein intake on learning,memory capability and mTOR expression in premature rats.Methods:The neonatal Sprague-Dawley premature rats were obtained through the method of cesarean section. Premature new born rats were lactated to21days by nursing mother. Premature rats and full-term rats were randomly divided into six groups after weaning:①preterm standard protein group(PS).②term standard protein group(TS).③preterm low protein group(PL).④term low protein group(TL).⑤preterm high protein group(PH).⑥term high protein group(TH). After weaning, the low protein groups were fed low protein diets (including8%protein) until the end of experiment, the high protein groups were provided with high protein diets (including30%protein) until the end of experiment, the standard protein groups were fed standard protein diets (content of18%protein) until the end of experiment. Within each group, the rats were respectively tested at6weeks and8weeks. Morris water maze task was performed to assess the learning and cognitive abilities of the premature rats. Immunohistochemistry and western blot were used to observe the distribution of mTOR、S6K、4EBP1in the hippocampus.Results1. Morris water maze task:1.1Directional navigation experiments:1.1.1At the age of6weeks, the escape latencies to find the platform were shortened with increased training times for full-term rats (P<0.05). The escape latencies of the premature rats did not chang significantly in the third and fourth day of the experiment.1.1.2At the age of6weeks, the number of crossing the platform f the premature rats were no significant difference (P>0.05)1.1.3At the age of8weeks,the escape latencies to find the platform were shortened with increased training times for all of the rats (P<0.05)1.1.4At the age of8weeks,the high-protein premature rats spent time at platform and the percent of time spent in target quadrant were significantly greater than the6-week-old’s (P<0.05)1.2. Probe trial test:1.2.1At the age of6weeks, prematuer rats spent significantly less time in target quadrant than full-time rats. And the percent of traveled distance in target quadrant of premature rats were significantly less than full term rats (P<0.05)1.2.2At the age of8weeks, high-protein premature rats spent significantly greater time in target quadrant than other two groups of premature rats.And the percent of traveled distance in target quadrant of high-protein premature rats were significantly higher than other two groups of premature rats (P<0.05)1.2.3At the age of8weeks,the high-protein premature rats spent time in target quadrant and the percent of traveled distance in target quadrant were higher than the high-protein full term rats’, but there was no significant difference (P>0.05)1.2.4At the age of8weeks,the high-protein premature rats spent time at platform and the percent of time spent in target quadrant were significantly greater than the6-week-old’s (P<0.05)1.3Immunohisto chemistry results:1.3.1At the age of6weeks, the number of crossing the platform and the expression of mTOR in hippocampal CA1,CA3region were no significant difference.1.3.2At the age of8weeks, the expression of mTOR in high-protein premature rats’ hippocampal CA1,CA3region were also significantly increased(P<0.05).1.3.3Compared with high-protein premature rats at the age of6weeks, the expression of mTOR in hippocampal CA1,CA3region were also significantly increased in high-protein premature rats at the age of8weeks.1.4Western blot results:1.4.1At the age of6weeks,the expressions of p-p70S6K (Thr389) were seriously inhibited in premature rats.Compare to the term rats, the expressions of4EBP1in premature rats were decreased.1.4.2At the age of8weeks,the expressions of p-p70S6K (Thr389) were increased in premature rats.Compare to the other groups, the expressions of4EBP1in premature low protein feed rats were decreased.1.4.3At the age of6weeks, Phosphorylated levels of p70S6K were significantly decreased in the premature rats(A two-way ANOVA with gestation age and protein level as main effects indicated significant effects of gestation age, Fgestation age=8.233, P=0.014<0.05).1.4.4At the age of8weeks, Phosphorylatedlevels of p70S6K were significantly increased in high protein rats.(A two-way ANOVA with gestation age and protein level as main effects indicated significant effects of protein level, Fprotein level=4.13,P=<0.05;high protein groups vs low protein groups,LSD post-hoc test,P=0.021<0.05;high protein groups vs standard protein groups,LSD post-hoc test,P=0.042<0.05).*P<0.05Conclusion:In the early growth and development process of the brain,premature birth may inhibit mTOR activity, which can lead to the learning and memory ability of the premature rats was impaired.After a long time high protein nutritional intake, high-protein premature rats impaired learning and cognitive ability can get recovery. That may be associated with upregulation of mTOR expression.更多还原