發(fā)布時(shí)間：2018-08-31 13:15

【摘要】： 研究背景和目的 衰老(Senescence)是隨著年齡增長(zhǎng)，機(jī)體各器官發(fā)生生物學(xué)功能退化的過程，表現(xiàn)為老年相關(guān)疾病的發(fā)病率增高，細(xì)胞更新的能力下降，死亡的可能性增加。熱量限制(Calorie Restriction，CR)是一種目前公認(rèn)的有效延緩衰老的方法。CR不僅能延緩衰老，還能延緩和預(yù)防一些與年齡相關(guān)疾病的發(fā)生發(fā)展。衰老的大腦在功能上主要表現(xiàn)為學(xué)習(xí)記憶能力減退，而CR能夠減少與認(rèn)知能力相關(guān)的疾病(如Alzheimer's病)的發(fā)病率，但是CR對(duì)自然衰老引起的學(xué)習(xí)記憶能力衰減的延緩作用仍缺乏行為學(xué)和組織學(xué)依據(jù)，并且多數(shù)的研究結(jié)果是基于對(duì)動(dòng)物進(jìn)行終生飲食限制獲得的，研究周期較長(zhǎng)。對(duì)于CR是否能夠延緩老年早期的大鼠學(xué)習(xí)記憶能力的衰退，還存有爭(zhēng)議，甚至有人對(duì)成年或老年早期的大鼠進(jìn)行飲食限制是否能夠提高大鼠的生存率提出質(zhì)疑。在明確CR能否對(duì)認(rèn)知能力的下降有延緩作用時(shí)，確定認(rèn)知相關(guān)的腦區(qū)(如海馬和額葉皮質(zhì))神經(jīng)元的衰老狀態(tài)是揭示CR延緩學(xué)習(xí)記憶能力下降的細(xì)胞學(xué)基礎(chǔ)，衰老細(xì)胞的鑒定是確定細(xì)胞是否發(fā)生衰老的標(biāo)志。盡管有報(bào)道，在培養(yǎng)的細(xì)胞，衰老細(xì)胞表現(xiàn)為細(xì)胞變大，變扁平，但在組織學(xué)，衰老細(xì)胞僅從形態(tài)學(xué)上難以與其它細(xì)胞區(qū)分。生物學(xué)標(biāo)記為衰老細(xì)胞的鑒定提供了一個(gè)重要方法，衰老相關(guān)的β半乳糖苷酶(Senescence associatedβ-galactosidase，SA-β-GAL)是目前被廣泛采用的一種生物學(xué)標(biāo)記物，它在衰老細(xì)胞中表達(dá)升高，因此成為鑒定細(xì)胞衰老的一個(gè)標(biāo)志。值得提出的是，有些衰老的生物學(xué)標(biāo)記并不具有普遍性，SA-β-GAL作為一個(gè)衰老的生物學(xué)標(biāo)記在體外檢測(cè)復(fù)制衰老的細(xì)胞已經(jīng)得到了廣泛的認(rèn)可，但其是否能夠應(yīng)用于腦組織，作為檢測(cè)衰老神經(jīng)元的生物學(xué)標(biāo)記，目前還未得到證實(shí)，因此在應(yīng)用上應(yīng)加以驗(yàn)證。本研究選定老年早期的大鼠，對(duì)其進(jìn)行飲食控制，即選在認(rèn)知能力衰退的年齡段進(jìn)行觀察，旨在明確限制飲食對(duì)老年早期的大鼠的生存狀態(tài)的影響和通過行為學(xué)檢測(cè)觀察學(xué)習(xí)記憶能力的改變，利用SA-β-GAL在學(xué)習(xí)記憶產(chǎn)生的主要部位海馬檢測(cè)衰老神經(jīng)元。探討認(rèn)知能力的提高是否與限制飲食引起的延緩神經(jīng)元衰老有關(guān)，同時(shí)驗(yàn)證SA-β-GAL作為衰老的生物標(biāo)記在腦組織檢測(cè)衰老神經(jīng)元的可靠性，為進(jìn)一步探討CR延緩海馬衰老的生物學(xué)機(jī)制提供行為學(xué)和組織學(xué)依據(jù)。 材料與方法 實(shí)驗(yàn)動(dòng)物選用18月齡的健康雄性Sprague Dawley大鼠，根據(jù)進(jìn)食情況分為兩組，一組為CR組(n=29)給予對(duì)照組進(jìn)食量的60％的食量喂養(yǎng)，對(duì)照組為ad libitum(AL)組(n=31)自由進(jìn)食，觀察6個(gè)月，在此期間，記錄大鼠的生存狀況，每周測(cè)定大鼠體重；6個(gè)月后，進(jìn)行開放場(chǎng)實(shí)驗(yàn)(open-field test)觀察自發(fā)活動(dòng)能力；利用Morris水迷宮(Morris water maze，MWM)檢測(cè)大鼠的空間學(xué)習(xí)能力和參考記憶能力。選用6月齡(n=10)，18月齡(n=6)和24月齡(n=7)的SD雄性大鼠，進(jìn)行SA-β-GAL的組織化學(xué)檢測(cè)；同時(shí)海馬神經(jīng)元原代培養(yǎng)，取培養(yǎng)6天，12天和20天的神經(jīng)元進(jìn)行SA-β-GAL的細(xì)胞化學(xué)檢測(cè)。 結(jié)果 CR組給予60％的飲食控制后1-10周內(nèi)體重增長(zhǎng)率下降明顯，低于對(duì)照組的近30％，之后CR組的體重增長(zhǎng)率有緩慢的升高，并維持在低于對(duì)照組約20％左右(p＜0.001)。CR組的生存率明顯高于AL組(p=0.039)；在開放場(chǎng)實(shí)驗(yàn)中測(cè)定的大鼠白發(fā)活動(dòng)路程明顯長(zhǎng)于AL組(p=0.021)，而自發(fā)活動(dòng)速度也明顯高于AL組(p=0.021)，并且CR組大鼠具有在中央?yún)^(qū)域的活動(dòng)距離長(zhǎng)(p=0.048)和在周邊區(qū)域活動(dòng)速度快(p=0.012)的特點(diǎn)。Morris水迷宮定向航行實(shí)驗(yàn)中，CR組的逃避潛伏期(Escape latency，EL)明顯比AL組短(p=0.038)，而在空間探索實(shí)驗(yàn)中，比較兩組的穿環(huán)次數(shù)無統(tǒng)計(jì)學(xué)差異(p=0.232)，兩組大鼠在目的象限Ⅲ中的時(shí)間和路程差別無統(tǒng)計(jì)學(xué)意義(p=0.324和p=0.367)，但在象限Ⅱ中的時(shí)間和路程都有明顯統(tǒng)計(jì)學(xué)差異(p=0.027和p=0.029)。與AL大鼠相比，CR組大鼠海馬CA3區(qū)錐體細(xì)胞層細(xì)胞排列相對(duì)整齊、緊密，細(xì)胞計(jì)數(shù)明顯增多(p=0.039)。SA-β-GAL在CR大鼠的神經(jīng)元染色微弱(p＜0.001)。18月齡和24月齡的大鼠海馬組織的SA-β-GAL染色較6月齡增加(P＜0.001)，培養(yǎng)12天和20天的海馬神經(jīng)元SA-β-GAL染色較培養(yǎng)6天的海馬神經(jīng)元明顯增多(P＜0.001)。 結(jié)論： 1．對(duì)老年早期的大鼠給予6個(gè)月的限制飲食(CR)可以提高大鼠的生存率。 2．CR能夠提高大鼠的活動(dòng)能力，反映了大鼠的健康狀況改善。 3．CR延緩衰老引起的空間學(xué)習(xí)能力減弱，提高參考記憶力的準(zhǔn)確性。 4．大鼠空間學(xué)習(xí)記憶能力的提高可能與CR延緩了大鼠海馬CA3區(qū)神經(jīng)元的衰老有關(guān)。 5．SA-β-GAL是可靠的檢測(cè)海馬CA3區(qū)海馬衰老神經(jīng)元的生物學(xué)標(biāo)記物。 限制飲食(calorie restriction，CR)能夠不同程度的延長(zhǎng)多種生物的壽命，有效延緩的衰老和減少老年相關(guān)疾病的發(fā)生。了解CR引起這些效應(yīng)的作用機(jī)制有助于指導(dǎo)人類的飲食和生活方式，起到延緩衰老，預(yù)防疾病發(fā)生的作用。然而，CR是如何發(fā)揮抗衰老的作用，參與其調(diào)節(jié)的分子機(jī)制還遠(yuǎn)未了解清楚。 沉默信息調(diào)節(jié)因子2(silence information regulator 2，Sir2)是一種煙酰胺腺嘌呤核苷酸(nicotinamide adenine dinucleotide，NAD)依賴性去乙�；�，研究發(fā)現(xiàn)，在CR能夠引起Sir2的上調(diào)，并且這一現(xiàn)象從低等生物酵母到高等生物如哺乳類動(dòng)物，靈長(zhǎng)類動(dòng)物等普遍存在，因此，Sir2被認(rèn)為是介導(dǎo)CR發(fā)揮效應(yīng)的一個(gè)關(guān)鍵調(diào)節(jié)因子。哺乳動(dòng)物的同源基因(sirtuin 1，SirT1)能夠與多種底物作用，如p53，NF-kappaB和叉頭框轉(zhuǎn)錄因子(Forkhead transcription factors，F(xiàn)oxO3a)參與細(xì)胞對(duì)氧化應(yīng)激的抵抗力和基因穩(wěn)定性的調(diào)節(jié)。 在哺乳動(dòng)物生長(zhǎng)激素和IGF-1也參與了壽命調(diào)節(jié)，對(duì)于信號(hào)通路中FOXO家族的一員，F(xiàn)oxO3a參與調(diào)節(jié)多種生物學(xué)功能，包括細(xì)胞周期停滯，修復(fù)受損的DNA和衰老。CR能夠在多種生物中引起胰島素／胰島素樣生長(zhǎng)因子-1(insulin／insulin-like growth factor 1，insulin／IGF-1)信號(hào)的抑制，其中FOXO家族是這個(gè)信號(hào)通路下游的一個(gè)主要靶點(diǎn)。因此，F(xiàn)oxO3a在CR調(diào)節(jié)中可能參與了重要的介導(dǎo)作用，同時(shí)FoxO3a在大腦中大量表達(dá)，尤其在發(fā)育和成年的海馬，這提示FoxO3a可能參與了學(xué)習(xí)記憶的過程和(或)調(diào)節(jié)神經(jīng)元的衰老。但對(duì)于FoxO3a在CR的大鼠海馬的表達(dá)變化及與SirT1變化的關(guān)系，仍是一個(gè)值得探討的問題。 為探討限制飲食引起的延緩神經(jīng)元衰老的調(diào)節(jié)機(jī)制，本研究選用結(jié)合觀察SirT1及FoxO3a及相關(guān)信號(hào)因子，同時(shí)，利用SirT1的激動(dòng)劑白藜蘆醇作用于PC12細(xì)胞，觀察對(duì)培養(yǎng)細(xì)胞的生物學(xué)和分子生物學(xué)改變，進(jìn)一步探討SirT1與FoxO3參與CR的調(diào)控機(jī)制。 材料與方法： 實(shí)驗(yàn)動(dòng)物選用18月齡的健康雄性Sprague Dawley(SD)大鼠，根據(jù)進(jìn)食情況分為兩組，一組為CR組(n=29)給予60％的進(jìn)食量，另一組為AL組(n=31)自由進(jìn)食，采用免疫組織化學(xué)和Westem blot方法觀察海馬CA3區(qū)SirT1和FoxO3a蛋白水平的改變。在培養(yǎng)的PC12細(xì)胞中，給予不同濃度的白藜蘆醇，觀察細(xì)胞形態(tài)學(xué)改變，利用MTT試驗(yàn)觀察細(xì)胞增殖情況，利用RT—PCR觀察SirT1，F(xiàn)oxO3a及細(xì)胞周期素依賴性激酶(Cyclin-dependent Kinase Inhibitors，Cdk)抑制蛋白p27~(KIP1)的mRNA水平的改變，利用流式細(xì)胞術(shù)觀察白藜蘆醇對(duì)PC12細(xì)胞周期的影響。 結(jié)果： 免疫組織化學(xué)和Westem blot檢測(cè)SirT1的表達(dá)結(jié)果顯示，CR組明顯升高(p=0.018)，免疫組織化學(xué)觀察到FoxO3a在兩組中呈現(xiàn)細(xì)胞定位的不同，，CR組FoxO3a在細(xì)胞核內(nèi)的表達(dá)減少(p=0.002)而轉(zhuǎn)移至胞漿(p=0.005)。 在培養(yǎng)的PC12細(xì)胞中，隨著白藜蘆醇濃度增加(5-50μmol／L)出現(xiàn)明顯的細(xì)胞生長(zhǎng)抑制(p＜0.001)，在細(xì)胞周期的檢測(cè)中，發(fā)現(xiàn)10μmol／L的白藜蘆醇使G0／G1期的細(xì)胞比例增加(P=0.023)。RT-PCR結(jié)果顯示，10μmol／L的白藜蘆醇能夠使SirT1表達(dá)升高(p=0.02)，而p27~(KIP1)在20μmol／L和50μmol／L白藜蘆醇刺激下表達(dá)升高(p＜0.001)，而FoxO3a的改變不明顯(p=0.09)。 結(jié)論： 1．在SirT1的上調(diào)和FoxO3a的失活參與了限制飲食延緩海馬神經(jīng)元衰老的調(diào)節(jié) 2．SirT1的激活能夠使PC12細(xì)胞G0／G1期停滯
[Abstract]:Research background and purpose
Senescence is a process in which the biological functions of various organs degenerate with age. It is characterized by an increased incidence of age-related diseases, a decreased ability to regenerate cells, and an increased likelihood of death. Old age also delays and prevents the development of age-related diseases. The aging brain is functionally characterized by impaired learning and memory, while CR can reduce the incidence of cognitive-related diseases such as Alzheimer's disease. However, CR's delaying effect on the impairment of learning and memory caused by natural aging is still lacking. It is controversial whether CR can delay the decline of learning and memory in rats in the early years of old age, and whether dietary restriction in adult or early old rats can improve the growth of rats. The survival rate of rats was questioned. In determining whether CR could delay cognitive decline, determining the aging state of neurons in cognitive-related brain areas (such as the hippocampus and frontal cortex) was the cytological basis for revealing that CR delayed the decline in learning and memory, and the identification of aging cells was a marker for determining whether cells were aging. It is reported that in cultured cells, senescent cells become larger and flatter, but histologically, senescent cells are difficult to distinguish from other cells only by morphology. Biological markers provide an important method for identifying senescent cells. Senescent associated beta-galactosidase (SA-beta-GAL) is an order. It is worth mentioning that some biological markers of aging are not universal, and SA-beta-GAL as a biological marker of aging has been widely used in vitro to detect replicative aging cells. This study selected early-aged rats for dietary control, i.e. at the age of cognitive decline, in order to clearly restrict diet to the elderly. The effect of early survival state on rats and the change of learning and memory ability were observed by behavioral tests. Aging neurons were detected by SA-beta-GAL in the hippocampus, the main part of learning and memory production. The reliability of biomarkers in detecting senescent neurons in brain tissue provides behavioral and histological evidence for further study of the biological mechanism of CR delaying senescence in hippocampus.
Materials and methods
Eighteen-month-old healthy male Sprague Dawley rats were divided into two groups according to their dietary intake. One group was CR group (n=29) fed 60% of the control group, and the other group was ad libitum (AL) group (n=31) fed freely. The rats were observed for six months. During this period, the survival status of the rats was recorded and the weight of the rats was measured weekly for six months. Then, open-field test was used to observe the spontaneous activity, Morris water maze (MWM) was used to detect the spatial learning ability and reference memory ability of rats, and 6-month-old (n = 10), 18-month-old (n = 6) and 24-month-old (n = 7) SD male rats were selected to carry out SA-beta-GAL histochemical detection; meanwhile, the hippocampal neurons were detected by histochemistry. Cultured neurons were cultured for 6 days, 12 days and 20 days to carry out cytochemical detection of SA- beta -GAL.
Result
The body weight growth rate of CR group decreased significantly within 1-10 weeks after 60% dietary control, which was lower than 30% of the control group. After that, the body weight growth rate of CR group increased slowly and remained about 20% lower than that of the control group (p < 0.001). The survival rate of CR group was significantly higher than that of AL group (p = 0.039). The escape latency (EL) of the CR group was significantly shorter than that of the AL group (p = 0.021), and the spontaneous activity speed was significantly higher than that of the AL group (p = 0.021), and the CR group had longer distance in the central region (p = 0.048) and faster speed in the peripheral region (p = 0.012). In the space exploration experiment, there was no significant difference in the number of perforations between the two groups (p = 0.232). There was no significant difference in time and distance between the two groups (p = 0.324 and P = 0.367), but there was significant difference in time and distance between the two groups (p = 0.027 and P = 0.029). SA-beta-GAL staining was weaker in CR rats (p < 0.001). The SA-beta-GAL staining in hippocampus of 18-month-old and 24-month-old rats was higher than that of 6-month-old rats (P < 0.001). The SA-beta-GAL staining in hippocampus of 12-day-old and 20-day-old rats was higher than that of 6-day-old rats (P < 0.001). Hippocampal neurons increased significantly (P < 0.001).
Conclusion:
1. 6 months restriction diet (CR) for early aged rats can improve the survival rate of rats.
2.CR can improve the activity of rats and reflect the improvement of the health status of rats.
3.CR slowed down the spatial learning ability caused by aging and improved the accuracy of reference memory.
4. The improvement of spatial learning and memory in rats may be related to CR delaying the senescence of neurons in hippocampal CA3 area.
5.SA- beta -GAL is a reliable biomarker for detecting hippocampal aging neurons in hippocampal CA3 region.
Calorie restriction (CR) can prolong the life span of various organisms, effectively delay aging and reduce the incidence of diseases related to aging. Understanding the mechanism of these effects can help guide human diet and lifestyle, delay aging and prevent disease. The molecular mechanism underlying the role of anti aging and its involvement in regulation is far from clear.
Silence information regulator 2 (Sir2) is a nicotinamide adenine dinucleotide (NAD) dependent deacetylase. It has been found that CR can induce the up-regulation of Sir2, and this phenomenon can be seen from lower yeast to higher organisms such as mammals, primates and so on. Sir2 is considered to be a key regulator of CR response. Mammalian homologous genes (sirtuin 1, SirT1) can interact with a variety of substrates, such as p53, NF-kappaB and Forkhead transcription factors (FoxO3a) involved in cell resistance to oxidative stress and regulation of gene stability. Festival.
In mammals, growth hormone and IGF-1 are also involved in life-span regulation. For FOXO family members in the signaling pathway, FoxO3a is involved in regulating a variety of biological functions, including cell cycle arrest, repair of damaged DNA and aging. CR can cause insulin/insulin-like growth factor-1 (insulin/insulin-like growth facts) in a variety of organisms. Therefore, FoxO3a may play an important role in CR regulation. At the same time, FoxO3a is highly expressed in the brain, especially in the developing and adult hippocampus, suggesting that FoxO3a may be involved in the learning and memory process and/or modulation. Aging of ganglion neurons. However, the expression of FoxO3a in the hippocampus of CR rats and the relationship between FoxO3a and SirT1 is still a problem worth discussing.
To investigate the mechanism of dietary restriction in delaying neuronal senescence, SirT1 and FoxO3a and related signaling factors were observed. At the same time, resveratrol, an agonist of SirT1, was used to treat PC12 cells to observe the biological and molecular biological changes of cultured cells, and to further explore the role of SirT1 and FoxO3 in the regulation of CR. System.
Materials and methods:
Eighteen-month-old male Sprague Dawley (SD) rats were divided into two groups according to their dietary status. One group was given 60% of the food intake in CR group (n=29) and the other group was given free food in AL group (n=31). Immunohistochemistry and Western blot were used to observe the changes of SirT1 and FoxO3a protein levels in hippocampal CA3 region. Resveratrol was given at different concentrations to observe the morphological changes of cells. MTT assay was used to observe the proliferation of cells. RT-PCR was used to observe the mRNA levels of SirT1, FoxO3a and Cyclin-dependent Kinase Inhibitors (Cdk) inhibitor protein p27~ (KIP1) and flow cytometry was used to observe the effect of resveratrol on P C12 cell cycle.
Result:
Immunohistochemistry and Western blot showed that the expression of SirT1 in CR group was significantly increased (p=0.018). Immunohistochemistry showed that FoxO3a was differentially localized in the two groups. The expression of FoxO3a in the nucleus of CR group was decreased (p=0.002) and transferred to the cytoplasm (p=0.005).
In cultured PC12 cells, significant cell growth inhibition (p < 0.001) was observed with the increase of resveratrol concentration (5-50 micromol/L). 10 micromol/L of resveratrol increased the proportion of G0/G1 phase cells (P = 0.023). RT-PCR results showed that 10 micromol/L of resveratrol could increase the expression of SirT1 (p = 0.02). However, the expression of p27~ (KIP1) was increased (p < 0.001) when stimulated by 20 and 50 micromol/L resveratrol, while the change of FoxO3a was not significant (p = 0.09).
Conclusion:
1. up regulation of SirT1 and inactivation of FoxO3a are involved in regulating the aging of hippocampal neurons by restricting diet.
Activation of 2.SirT1 can arrest the G0 / G1 phase of PC12 cells.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王寶恒;傅玉才;史桂芝;許銘炎;耿義群;徐小虎;許錦階;;LASS1基因克隆及其在大鼠腦皮層的表達(dá)與神經(jīng)元衰老的相關(guān)性初步研究(英文)[J];生物化學(xué)與生物物理進(jìn)展;2006年08期

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