本文選題：大鼠 + 胼胝體　； 參考：《重慶醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 衰老是不可避免的生理過(guò)程,與衰老有關(guān)的疾病給家庭和社會(huì)帶來(lái)了許多問(wèn)題。這其中,大腦的衰老又尤其引人關(guān)注。長(zhǎng)久以來(lái)的觀點(diǎn)是老年大腦存在著神經(jīng)元的大量死亡,然而,隨著新的體視學(xué)方法的建立和應(yīng)用,Pakkenberg和Gundersen發(fā)現(xiàn)正常老年腦并不存在顯著性大腦皮質(zhì)神經(jīng)元的死亡,而老年大腦白質(zhì)體積下降了28%,隨后大量的研究也證實(shí)了此發(fā)現(xiàn),對(duì)大腦白質(zhì)的研究開始越來(lái)越深入和廣泛。由于大腦白質(zhì)不同區(qū)域的解剖結(jié)構(gòu)和功能不同,老年大腦白質(zhì)特定區(qū)域改變與老年大腦功能損害的確切關(guān)系仍需深入探究。胼胝體作為兩大腦半球?qū)?yīng)腦皮質(zhì)之間聯(lián)絡(luò)清晰的、解剖和功能都較為清楚的連合纖維束,越來(lái)越多的受到研究者的關(guān)注。許多采用影像學(xué)技術(shù)的研究發(fā)現(xiàn),正常老年和異常老年中都存在著胼胝體的萎縮。他們推測(cè)這種萎縮可能是纖維束丟失所導(dǎo)致的。上述研究說(shuō)明了胼胝體神經(jīng)纖維尤其是有髓神經(jīng)纖維對(duì)相關(guān)腦葉神經(jīng)元之間的神經(jīng)沖動(dòng)相互傳導(dǎo)的完整性起著至關(guān)重要的作用,因此,胼胝體神經(jīng)纖維的改變很可能是影響大腦認(rèn)知功能的結(jié)構(gòu)基礎(chǔ)之一。但是即往研究都沒(méi)有對(duì)胼胝體內(nèi)神經(jīng)纖維進(jìn)行準(zhǔn)確的定量研究,以反映纖維丟失的情況,原因之一在于缺乏準(zhǔn)確定量在三維空間內(nèi)無(wú)序排列的神經(jīng)纖維的手段。Tang等在國(guó)際上首次應(yīng)用了新的體視學(xué)方法對(duì)大腦白質(zhì)神經(jīng)纖維進(jìn)行定量研究,從而為神經(jīng)纖維的三維定量提供了有效的研究手段。因此,本研究運(yùn)用TEM和體視學(xué)技術(shù)研究Long-Evans大鼠胼胝體及其內(nèi)有髓神經(jīng)纖維的老年性改變。眾多研究發(fā)現(xiàn),有髓神經(jīng)纖維受損、丟失的同時(shí),髓鞘蛋白也存在一定變化,并與認(rèn)知功能降低相關(guān)。MBP是CNS特有的一種髓鞘蛋白,對(duì)髓鞘的發(fā)生和形成具有重要的生理作用。因此,本研究運(yùn)用Western-blot技術(shù)研究大鼠胼胝體MBP的老年性改變,以及MBP的改變與有髓神經(jīng)纖維改變之間的關(guān)系。 第一部分 1材料與方法 1.1雌性Long-Evans大鼠,年輕組(7月齡)5只、中老年組(18月齡)5只和老年組(27月齡)6只,體重280±20 g。 1.2每只大鼠隨機(jī)抽取一側(cè)大腦半球,從中隨機(jī)抽取6塊胼胝體組織塊作成超薄切片,從每張切片上隨機(jī)抽取4個(gè)視野在透射電鏡下放大6000倍拍照,每只動(dòng)物共拍攝24張照片。 1.3運(yùn)用體視學(xué)方法分別計(jì)算出每只大鼠胼胝體體積、胼胝體有髓神經(jīng)纖維長(zhǎng)度密度和總長(zhǎng)度、胼胝體有髓神經(jīng)纖維體積密度和總體積、髓鞘體積密度和總體積、有髓神經(jīng)纖維平均直徑。 2結(jié)果 2.1電鏡下,老年組大鼠胼胝體有髓神經(jīng)纖維存在明顯的脫髓鞘的改變,可見雙層髓鞘形成、髓鞘板層分離和髓鞘內(nèi)空泡化。 2.2年輕組大鼠胼胝體體積為70.7±5.6 mm3,中老年組為63.2±4.2 mm3,老年組為51.6±2.6 mm3。與年輕組相比,中老年組顯著降低了10.6% (p0.01),老年組顯著降低了26.9% (p0.01)。老年組與中老年組相比,胼胝體體積顯著降低了18.3% (p0.01)。 2.3年輕組大鼠有髓神經(jīng)纖維總長(zhǎng)度為78.3±7.7 km,中老年組為75.7±11.0 km,老年組為62.4±5.8 km。與年輕組相比,中老年組的下降沒(méi)有顯著性(p0.05),老年組顯著下降了20.3% (p0.05)。老年組與中老年組相比,有髓神經(jīng)纖維總長(zhǎng)度顯著下降了17.5% (p0.05)。 2.4年輕組大鼠有髓神經(jīng)纖維總體積為46.6±3.7 mm3,中老年組為43.4±3.0 mm3,老年組為39.4±2.5 mm3。與年輕組相比,中老年組的下降沒(méi)有顯著性(p0.05),老年組顯著下降了15.5% (p0.01)。老年組與中老年組相比,下降沒(méi)有顯著性(p0.05)。 2.5年輕組有髓神經(jīng)纖維髓鞘總體積為21.3±2.5 mm3,中老年組為19.7±2.6 mm3,老年組為18.4±1.4 mm3。三者之間沒(méi)有顯著性改變(p0.05)。 2.6年輕組有髓神經(jīng)纖維平均直徑為0.62±0.03μm,中老年組為0.66±0.53μm,老年組為0.70±0.04μm。與年輕組相比,中老年組有髓神經(jīng)纖維直徑的增加沒(méi)有顯著性(p0.05),老年組顯著增加了13.0% (p0.01)。老年組與中老年組相比,沒(méi)有顯著性差異(p0.05)。 2.7與年輕組相比,老年組大鼠直徑為0.2 - 0.4μm的有髓神經(jīng)纖維總長(zhǎng)度顯著下降了47.3% (p0.01),直徑為0.4 - 0.6μm的有髓神經(jīng)纖維總長(zhǎng)度顯著下降了35.6% (p0.01)。中老年組與年輕組相比,0.2 - 0.4μm和0.4 - 0.6μm直徑段的有髓神經(jīng)纖維總長(zhǎng)度的變化沒(méi)有顯著性(p0.05)。 第二部分 1材料與方法 1.1雌性Long-Evans大鼠,7月齡10只,隨機(jī)分為兩組;27月齡10只,隨機(jī)分為兩組。 1.2取7月齡和27月齡大鼠各一組,進(jìn)行電子顯微鏡切片制作和體視學(xué)分析,方法同第一部分。 1.3取7月齡和27月齡大鼠各一組,從每只動(dòng)物大腦胼胝體內(nèi)提取MBP,Western-blot檢測(cè)MBP表達(dá),實(shí)驗(yàn)至少重復(fù)5次。 1.4 Quantity One圖像分析軟件分析蛋白條帶OD值,以各蛋白條帶與β-actin的灰度值之比表示該蛋白的表達(dá)量。 2結(jié)果 2.1老年組與年輕組相比,大鼠胼胝體有髓神經(jīng)纖維總長(zhǎng)度下降了20.3% (p 0.01)。有髓神經(jīng)纖維總體積下降了15.5 (p 0.01)。髓鞘總體積雖有下降,但沒(méi)有顯著性(p0.05)。 2.2與年輕組相比,老年組胼胝體內(nèi)各分子量的MBP表達(dá)量都有降低,但只有Mr 21.5×104的MBP降低具有顯著性(p0.05)。對(duì)Mr 21.5×104的MBP含量改變與胼胝體有髓神經(jīng)纖維總長(zhǎng)度、總體積和髓鞘總體積作Spearman相關(guān)分析,Mr 21.5×104MBP的降低與有髓神經(jīng)纖維總體積的降低存在正相關(guān)(r = 0.770;p0.01),與有髓神經(jīng)纖維髓鞘的總體積的降低存在正相關(guān)(r = 0.636;p0.05),與有髓神經(jīng)纖維總長(zhǎng)度沒(méi)有明顯相關(guān)性(r = 503;p0.05)。做多重線性回歸分析后發(fā)現(xiàn),隨著年齡的增加,MBP的降低與有髓神經(jīng)纖維髓鞘總體積的降低具有顯著的相關(guān)性(β= -1.105;p0.05),與有髓神經(jīng)纖維總長(zhǎng)度及有髓神經(jīng)纖維總體積的降低不具有顯著相關(guān)性(β= -0.438, p0.05;β= -1.306, p0.05)。 全文結(jié)論 1.本研究首次把新的體視學(xué)方法和電子顯微鏡技術(shù)結(jié)合起來(lái)定量研究大鼠胼胝體及其有髓神經(jīng)纖維的老年改變。 2.首次獲得了年輕、中老年及老年雌性大鼠胼胝體體積、胼胝體有髓神經(jīng)纖維的總長(zhǎng)度和總體積、髓鞘總體積及纖維平均直徑的基本數(shù)值,這些數(shù)值為今后運(yùn)用該動(dòng)物模型探討CNS退化性疾病對(duì)胼胝體有髓神經(jīng)纖維的影響提供了重要的參考數(shù)據(jù)。 3.證實(shí)了以前在人胼胝體的研究發(fā)現(xiàn),即存在老年性胼胝體萎縮。 4.首次發(fā)現(xiàn)老年雌性Long-Evans大鼠大腦胼胝體有髓神經(jīng)纖維總長(zhǎng)度和總體積顯著性降低,且總長(zhǎng)度的降低主要是由于細(xì)小直徑的有髓神經(jīng)纖維降低所致。 5.證實(shí)了老年胼胝體MBP的表達(dá)降低,并得出胼胝體MBP的表達(dá)降低與胼胝體有髓神經(jīng)纖維的老年改變之間存在相關(guān)性。
[Abstract]:Aging is an inevitable physiological process, and aging related diseases have brought many problems to family and society. Among them, brain aging is particularly attractive. The long-standing view is that there is a large number of neuronal deaths in the brain of the elderly. However, with the establishment and application of new stereological methods, Pakkenberg and Gundersen have found that There is no significant cerebral cortical neuron death in the normal aged brain, and the volume of white matter in the aged brain drops by 28%. Subsequently, a large number of studies have also confirmed the discovery that the study of white matter in the brain is becoming more and more deep and extensive. The specific areas of white matter in the brain are changed because of different anatomical structures and functions in different regions of the brain white matter. The exact relationship between the changes and the impairment of brain function in the elderly still needs to be explored. The corpus callosum is more and more concerned with the researchers' attention as a clear, clear, anatomical and functional connection between the two cerebral hemispheres that correspond to the cerebral cortex. Many studies using imaging techniques have found that both normal and abnormal old age are stored. They speculate that the atrophy of the corpus callosum may be caused by loss of fiber bundles. These studies show that the corpus callosum fibers, especially the myelinated nerve fibers, play a vital role in the integrity of the mutual conduction of the nerve impulses between the related lobar neurons. Therefore, the changes in the corpus callosum fibers are likely to be altered. It is one of the structural foundations that affect the cognitive function of the brain. However, there is no accurate quantitative study of the nerve fibers in the corpus callosum to reflect the loss of fibers. One of the reasons is that the lack of accurate quantitative and unordered neural fibers in the three-dimensional space of the hand segment.Tang, for the first time, has applied new stereopsis to the world for the first time. A quantitative study of white matter nerve fibers in the brain provides an effective method for the three-dimensional quantification of nerve fibers. Therefore, this study uses TEM and stereological techniques to study the age-related changes in the corpus callosum and its intramedullary nerve fibers in Long-Evans rats. At the time, the myelin protein is also changed, and associated with the decrease of cognitive function,.MBP is a kind of myelin protein that is unique to CNS. It has an important physiological role in the occurrence and formation of myelin sheath. Therefore, this study uses Western-blot technique to study the age-related changes of the corpus callosum MBP in rats, and the change of MBP with the changes of myelinated nerve fibers. Relationship.
Part one
1 materials and methods
1.1 female Long-Evans rats, young group (7 month old) 5, middle and old age group (18 month old) 5 and old age group (27 month old) 6, weight 280 280 20 G.
1.2 each rat was randomly selected from one side of the cerebral hemisphere. 6 pieces of corpus callosum were randomly selected for ultrathin sections. From each slice, 4 fields were randomly selected to enlarge 6000 times under transmission electron microscope. Each animal was photographed with 24 photographs.
1.3 the volume of corpus callosum, the length density and total length of the myelinated nerve fibers in the corpus callosum, the volume density and total volume of the myelinated nerve fibers in the corpus callosum, the medullary volume density and the total volume, and the average diameter of the myelinated nerve fibers were calculated by the stereological method.
2 Results
2.1 under electron microscope, there were obvious demyelinating changes in the callosal nerve fibers of the aged rats, and the formation of the double myelin sheath, the separation of the myelin sheath and the vacuolation in the myelin sheath.
The volume of corpus callosum in 2.2 young rats was 70.7 + 5.6 mm3, middle and old age group was 63.2 + 4.2 mm3, aged group was 51.6 + 2.6 mm3., compared with young group, the middle and old age group decreased significantly (P0.01), and the elderly group decreased by 26.9% (P0.01). The volume of corpus callosum was significantly reduced by 18.3% (P0.01) in the elderly group compared with the middle and old age group.
The total length of the myelinated nerve fibers in the 2.3 young rats was 78.3 + 7.7 km, the middle and old age group was 75.7 + 11 km, the elderly group was 62.4 + 5.8 km., compared with the young group, the decrease of the elderly group was not significant (P0.05), and the elderly group decreased significantly (P0.05). The total length of the myelinated nerve fibers in the elderly group was significantly decreased by 17.5% (p0.) compared with the middle and old age group. 05).
The total volume of myelinated nerve fibers in 2.4 young rats was 46.6 + 3.7 mm3, middle and old age group was 43.4 + 3 mm3, and aged group was 39.4 + 2.5 mm3., compared with young group, the decrease of middle aged and elderly group was not significant (P0.05), and the elderly group decreased significantly (P0.01). The decline was not significant (P0.05) in the elderly group compared with the middle and old age group.
The total medullary sheath volume of myelinated nerve fibers in the 2.5 young groups was 21.3 + 2.5 mm3, and the middle and old age group was 19.7 + 2.6 mm3, and the elderly group was 18.4 + 1.4 mm3. three. There was no significant change (P0.05).
The average diameter of the myelinated nerve fibers in the 2.6 young group was 0.62 + 0.03 m, the middle and old age group was 0.66 + 0.53 mu m, the elderly group was 0.70 + 0.04 micron M., compared with the young group, there was no significant increase in the diameter of the myelinated nerve fibers in the middle and old age group (P0.05), and the elderly group increased by 13% (P0.01). There was no significant difference in the elderly group compared with the middle and old age group (P0.05).
2.7 compared with the young group, the total length of the myelinated nerve fibers in the aged group of 0.2 - 0.4 mu m was significantly decreased by 47.3% (P0.01). The total length of the myelinated nerve fibers with the diameter of 0.4 - 0.6 m decreased significantly by 35.6% (P0.01). The total length of the myelinated nerve fibers in the middle and old age group was compared with the young group, 0.2 to 0.4, m and 0.4 - 0.6 m diameter segments. The change was not significant (P0.05).
The second part
1 materials and methods
1.1 female Long-Evans rats, 7 month old 10, were randomly divided into two groups. 27 month old 10 rats were randomly divided into two groups.
1.2 each group of 7 month old and 27 month old rats was subjected to electron microscope sectioning and stereology analysis, the same as the first part.
1.3 take 7 month old and 27 month old rats in each group, extract MBP from the corpus callosum of each animal, detect the expression of MBP by Western-blot, and test at least 5 times.
1.4 the Quantity One image analysis software was used to analyze the OD value of protein bands, and the ratio of the protein bands to the gray value of the beta -actin was used to express the protein expression.
2 Results
2.1 the total length of the myelinated nerve fibers in the corpus callosum of the aged group decreased by 20.3% (P 0.01) compared with the young group. The total volume of myelinated nerve fibers decreased by 15.5 (P 0.01). Although the total volume of myelin sheath decreased, the total medullary volume decreased, but was not significant (P0.05).
2.2 compared with the young group, the MBP expression of each molecular weight in the corpus callosum in the elderly group decreased, but only the Mr 21.5 x 104 MBP decreased significantly (P0.05). The MBP content of Mr 21.5 x 104 was associated with the total length of the myelinated nerve fibers in the corpus callosum, the total volume of the myelin sheath and the total medullary volume, and the decrease of Mr 21.5 x 104MBP and myeloid God. There was a positive correlation (r = 0.770; P0.01) with the decrease of total volume of fiber (r = 0.636; P0.05), and there was no significant correlation with the total length of myelinated nerve fibers (r = 503; P0.05). After multiple linear regression analysis, the decrease of MBP and myelinated nerve fibers as age increased. The reduction of total medullary volume has a significant correlation (beta = -1.105; P0.05). There is no significant correlation with the total length of myelinated fibers and the reduction of total medullary nerve fibers (beta = -0.438, P0.05; beta = -1.306, P0.05).
Full text conclusion
1. in this study, the new stereology method and electron microscopy technique were first applied to quantitatively study the aging changes of corpus callosum and myelinated nerve fibers in rats.
2. the corpus callosum volume, the total volume and total volume of the callosal nerve fibers, the total volume of myelin sheath and the average diameter of the fibers were obtained for the first time in young, middle-aged and old female rats. These values provide important implications for the future use of the animal model to explore the effects of CNS degenerative diseases on the callosum myelinated nerve fibers. Reference data.
3. it is confirmed that previous studies in human corpus callosum showed age-related atrophy of corpus callosum.
4. the total length and total volume of the medullary nerve fibers in the corpus callosum of the aged female Long-Evans rats decreased significantly for the first time, and the decrease in the total length was mainly due to the decrease of the myelinated nerve fibers in the small diameter.
5. it was confirmed that the expression of MBP in the corpus callosum of aged patients was reduced, and the correlation between the decrease of MBP expression in corpus callosum and the age-related changes of corpus callosum myelinated nerve fibers was found.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329.54

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