發(fā)布時(shí)間：2018-03-06 19:29

  本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：年齡相關(guān)　出處：《中南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：由于骨髓間充質(zhì)干細(xì)胞(Mesenchymal Stromal Cells, MSCs)具有多向分化潛能,而且易于獲得,所以近年來隨著基因工程興起,不少學(xué)者把骨髓間充質(zhì)干細(xì)胞作為基因治療靶細(xì)胞,治療某些難治疾病,比如骨缺損、椎間盤退行性疾病、心力衰竭等等,而這些疾病的患病人群多為老年患者,可能跟衰老機(jī)體中的骨髓間充質(zhì)干細(xì)胞的生長和分化發(fā)生一定改變有關(guān),因此,探討間充質(zhì)干細(xì)胞的生物學(xué)特性,以及其功能隨著年齡增長是否發(fā)生改變尤為重要。本次實(shí)驗(yàn)通過對不同年齡層SD大鼠的骨髓間充質(zhì)干細(xì)胞的生物學(xué)特性進(jìn)行比較分析,探討年齡對間充質(zhì)干細(xì)胞的影響。目的： 研究不同年齡SD大鼠的骨髓間充質(zhì)干細(xì)胞的生物學(xué)特性,并探討不同年齡SD大鼠骨髓間充質(zhì)干細(xì)胞生物學(xué)特性的影響。方法： 分別取幼年(21天)、中年(3個(gè)月)、老年(14個(gè)月)的SD大鼠,取四肢骨髓,采用沖骨髓細(xì)胞貼壁培養(yǎng)法,用含10%體積分?jǐn)?shù)胎牛血清(FCS)的低糖DMEM (L-DMEM)培養(yǎng)基,貼壁、傳代、培養(yǎng)、純化出MSC,使其在體外擴(kuò)增生長,在倒置顯微鏡下觀察生長特性,瑞氏染色觀察細(xì)胞形態(tài)。流式細(xì)胞術(shù)檢測細(xì)胞表面抗原。分別取每個(gè)年齡階段細(xì)胞的第3代、第6代、第9代、第12代和第15代做MTT,繪制生長曲線,比較細(xì)胞的增殖能力。取每個(gè)年齡層的第3代的MSCs做成骨誘導(dǎo)分化和成脂肪誘導(dǎo)分化,觀察年齡差異對其成成骨細(xì)胞的能力和成脂肪細(xì)胞的能力的影響。通過茜素紅半定量法比較鈣鹽沉積情況。通過油紅O定量法比較3個(gè)年齡層SD鼠MSCs的成脂能力。通過檢測ALP的值,來比較年齡對MSCs成骨能力的影響。結(jié)果： (1)經(jīng)流式細(xì)胞術(shù)鑒定出細(xì)胞表面抗原95.27±0.52CD29, CD44為陽性,4.06±0.29CD34, CD45為陰性,證明通過沖骨髓細(xì)胞貼壁培養(yǎng)法培養(yǎng)出的細(xì)胞為骨髓間充質(zhì)干細(xì)胞。 (2)倒置顯微鏡下觀察細(xì)胞形態(tài),幼年組,中年組,老年組的SD大鼠的MSC的原代都是呈集落樣生長,并且其中有許多雜細(xì)胞,經(jīng)過一次胰酶消化傳代后,細(xì)胞均呈梭形,不再有集落產(chǎn)生,第3,4代,細(xì)胞形態(tài)趨于穩(wěn)定,雜細(xì)胞逐漸減少,各組間沒有差異。 (3)MTT結(jié)果顯示,第3、6代的3個(gè)年齡組比較,增殖能力無顯著差異,但在第9代、第12代和第15代,幼年組的增殖能力顯著高于中年和老年組。 (4)成脂肪細(xì)胞誘導(dǎo)和成骨細(xì)胞誘導(dǎo)的結(jié)果顯示,幼年SD大鼠的間充質(zhì)干細(xì)胞誘導(dǎo)出的鈣結(jié)節(jié)明顯,且數(shù)目顯著多于中年組和老年組,而中年組多于老年組。誘導(dǎo)出的成脂肪細(xì)胞,脂泡在數(shù)量上幼年組顯著多于中年組和老年組,并且更加飽滿圓潤。 (5)ALP檢測結(jié)果得出幼年組MSCs的成骨能力優(yōu)于中年組,中年組要優(yōu)于老年組。結(jié)論： 幼年鼠,中年鼠和老年鼠的MSC的生物學(xué)特性都具有較強(qiáng)的增殖能力,及成骨、成脂誘導(dǎo)分化能力,但是幼年組的MSC的增殖能力、成脂和成骨誘導(dǎo)分化能力要明顯優(yōu)于中年組和老年組。圖9幅,參考文獻(xiàn)106篇
[Abstract]:Because bone marrow mesenchymal Stromal cells (MSCs) have multidirectional differentiation potential and are easy to obtain, in recent years, with the rise of genetic engineering, many scholars have used bone marrow mesenchymal stem cells as target cells for gene therapy to treat some refractory diseases. For example, bone defects, intervertebral disc degenerative diseases, heart failure and so on, and most of the patients with these diseases are elderly patients, which may be related to the growth and differentiation of bone marrow mesenchymal stem cells in the aging organism, so, It is particularly important to investigate the biological characteristics of mesenchymal stem cells and whether their functions change with age. The biological characteristics of bone marrow mesenchymal stem cells in SD rats of different age groups were compared and analyzed. Objective: to investigate the effect of age on mesenchymal stem cells (MSCs). To study the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in SD rats of different ages and to explore the effects of different ages on the biological characteristics of BMSCs. Sprague-Dawley rats with 21 days of infancy, middle age (3 months, 14 months) were taken from bone marrow of limbs. Bone marrow cells were flushed and cultured on low sugar DMEM L-DMEM medium containing 10% volume fraction fetal bovine serum. MSCI was purified to grow in vitro, growth characteristics were observed under inverted microscope, cell morphology was observed by Rayleigh staining, and cell surface antigen was detected by flow cytometry. MTTs of the 12th and 15th generations were used to draw the growth curve to compare the proliferation ability of the cells. The third generation of MSCs from each age group was used to induce bone differentiation and adipogenic differentiation. To observe the effect of age difference on the ability of osteoblasts and adipoblasts. To compare the calcium deposition by alizarin red semi-quantitative method, and to compare the fat-forming ability of MSCs of three age groups SD rats by oil red O method. By detecting the value of ALP, To compare the effect of age on MSCs osteogenesis. Results:. Cell surface antigen 95.27 鹵0.52CD29, CD44 positive 4.06 鹵0.29CD34, CD45 negative, and bone marrow mesenchymal stem cells (BMSCs) identified by flow cytometry were proved to be bone marrow mesenchymal stem cells. (2) the morphology of the cells was observed under inverted microscope. The primary MSC of SD rats in juvenile group, middle age group and aged group all had colony like growth, and there were many mixed cells in them. After one passage of trypsin digestion, the cells were all fusiform. There was no colony formation, but in the 3rd and 4th generation, the cell morphology tended to be stable, and the hybrids decreased gradually, but there was no difference among the three groups. The results of MTT showed that there was no significant difference in proliferative ability among the three age groups in the 3rd and 6th generation, but in the 9th, 12th and 15th generation, the proliferative ability of the young group was significantly higher than that of the middle age group and the old age group. 4) the results of adipoblast induction and osteoblast induction showed that the number of calcium nodules induced by mesenchymal stem cells in young SD rats was significantly higher than that in middle-aged and elderly groups. But the number of adipoblasts induced in middle age group was more than that in old age group, and the number of lipid bubble in juvenile group was significantly more than that in middle age group and old age group, and it was more full and round. The results showed that the osteogenic ability of MSCs in young group was superior to that in middle age group, and that in middle age group was superior to that in old group. The biological characteristics of MSC in juvenile, middle-aged and aged mice have strong proliferative ability, osteogenesis and adipogenic differentiation ability, but the proliferation ability of MSC in juvenile group. The ability of adipogenic and osteogenic induction was better than that of middle age group and old age group.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R329

【共引文獻(xiàn)】

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