本文選題：臍帶間充質(zhì)干細(xì)胞　切入點(diǎn)：衰老　出處：《山東大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的： 1.觀察并檢測(cè)人臍帶間充質(zhì)干細(xì)胞體外培養(yǎng)衰老過(guò)程中細(xì)胞形態(tài)學(xué)、表型、生長(zhǎng)動(dòng)力學(xué)及誘導(dǎo)分化能力等生物學(xué)特性的改變； 2.檢測(cè)人臍帶間充質(zhì)干細(xì)胞體外培養(yǎng)衰老過(guò)程中基因表達(dá)譜的改變并探討其可能的機(jī)制。 方法 1.細(xì)胞培養(yǎng)：用組織塊貼壁法培養(yǎng)人臍帶間充質(zhì)干細(xì)胞,取第3代細(xì)胞作為對(duì)照組,連續(xù)培養(yǎng)到第15代的細(xì)胞為衰老組,倒置顯微鏡下觀察細(xì)胞形態(tài),并分別制成細(xì)胞爬片掃描電鏡觀察并拍照。 2.生長(zhǎng)增殖：分別取對(duì)照組和衰老組細(xì)胞,接種于96孔培養(yǎng)板中,各設(shè)8復(fù)孔,培養(yǎng)6天。每日檢測(cè)在450nm波長(zhǎng)處測(cè)吸光值(OD值),繪制細(xì)胞生長(zhǎng)曲線。 3.表型檢測(cè)：對(duì)照組和衰老組細(xì)胞消化后充分吹打成單細(xì)胞懸液,使用流式細(xì)胞儀檢測(cè)并分析兩組細(xì)胞CD34、CD45、CD44和CD105等表型變化。 4.誘導(dǎo)分化能力鑒定：將對(duì)照組和衰老組細(xì)胞置于不同的誘導(dǎo)培養(yǎng)液中向成骨細(xì)胞及成脂細(xì)胞誘導(dǎo)分化,誘導(dǎo)分化14天后,進(jìn)行固定,并分別進(jìn)行相應(yīng)的染色觀察誘導(dǎo)分化差異。 5.基因表達(dá)譜分析：細(xì)胞洗滌后加入’Trizol試劑,提取總RNA,進(jìn)行基因表達(dá)譜芯片雜交、檢測(cè)和數(shù)據(jù)分析,并用DNA芯片掃描儀行微陣列掃描。將掃描結(jié)果進(jìn)行標(biāo)準(zhǔn)化分析,與對(duì)照組相比log2Ratio|2(4-fold change)為差異表達(dá),并進(jìn)行GO及KEGG pathway分析。 6.PCR驗(yàn)證：根據(jù)基因表達(dá)譜的結(jié)果,選取了多個(gè)典型的差異基因進(jìn)行實(shí)時(shí)定量PCR驗(yàn)證。檢測(cè)對(duì)照組和衰老組細(xì)胞BAX, FADD, JAK2, IGF3BP5, PDK1, Col-Ⅲ, Col-Ⅳ, SMAD2, SAMD4, BMPR2, CCND2等基因的差異表達(dá)。 結(jié)果 1.細(xì)胞形態(tài)：對(duì)照組細(xì)胞呈典型的成纖維狀,每平方毫米可達(dá)500個(gè)細(xì)胞；衰老組細(xì)胞個(gè)體差異較大,細(xì)胞變得寬大,每平方毫米僅有約150個(gè)細(xì)胞,衰老細(xì)胞核質(zhì)比減小。掃描電鏡下可見(jiàn)年輕組細(xì)胞形態(tài)飽滿,表面微絨毛分布均勻,而衰老組細(xì)胞偽足增多,整體扁平。 2.增殖情況：對(duì)照組和衰老組生長(zhǎng)曲線在接種后都有一個(gè)停滯期(12-24小時(shí)),然后進(jìn)入增殖期,對(duì)照組細(xì)胞能更快的進(jìn)入增殖期,并在第5天達(dá)到更高的細(xì)胞數(shù)量后進(jìn)入平臺(tái)期。而衰老組停滯期長(zhǎng),進(jìn)入增殖期后第3天進(jìn)入平臺(tái)期,最終的細(xì)胞數(shù)量也少。 3.表型檢測(cè)：根據(jù)流式細(xì)胞儀檢測(cè)結(jié)果,對(duì)照組和衰老組細(xì)胞CD34和CD45均為陰性表達(dá),CD44和CD105均為高表達(dá),組間陽(yáng)性率表達(dá)并無(wú)顯著差異。 4.分化能力：在適當(dāng)?shù)姆只瘲l件誘導(dǎo)下,對(duì)照組和衰老組細(xì)胞均能向成骨和脂肪細(xì)胞分化：向成骨細(xì)胞誘導(dǎo)14天后大部分細(xì)胞出現(xiàn)鈣化胞外基質(zhì),茜素紅S染色陽(yáng)性堿性磷酸酶染色陽(yáng)性；向脂肪細(xì)胞誘導(dǎo)分化14天后約50%的細(xì)胞內(nèi)出現(xiàn)油紅O陽(yáng)性脂肪顆粒。這些結(jié)果證明所獲得的貼壁細(xì)胞符合間充質(zhì)干細(xì)胞的特征,與對(duì)照組細(xì)胞相比,衰老組細(xì)胞誘導(dǎo)分化能力顯著降低。 5.差異基因信號(hào)通路及基因聚類分析：將衰老組細(xì)胞與對(duì)照組差異表達(dá)的基因進(jìn)行KEGG pathway分析(p0.05),可見(jiàn)衰老組顯著上調(diào)通路有20條,其中包括自身免疫病、退行性疾病相關(guān)通路,下調(diào)通路49條,涉及通路包括合成代謝、細(xì)胞外基質(zhì)、物質(zhì)代謝和細(xì)胞增殖等。對(duì)41000+條基因進(jìn)行檢測(cè),差異基因多達(dá)8000個(gè),對(duì)顯著差異的2000個(gè)基因進(jìn)行聚類分析,顯示差異基因涉及轉(zhuǎn)錄、翻譯、生物合成、物質(zhì)代謝、信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞增殖、細(xì)胞遷移、細(xì)胞間連接等多方面。 6.PCR驗(yàn)證：熒光定量PCR示衰老組細(xì)胞與對(duì)照組細(xì)胞相比,BAX, FADD, JAK2, IGF3BP5, PDK1和MGMT等基因在衰老細(xì)胞中表達(dá)上調(diào),而Col-Ⅲ, Col-Ⅳ, SMAD2, SAMD4, BMPR2, CCND2和PAK2等基因表達(dá)下調(diào)。 結(jié)論 1.第3代對(duì)照組細(xì)胞及第15代衰老組細(xì)胞具有增殖潛能、相同的細(xì)胞表型,并能向成骨、脂肪細(xì)胞誘導(dǎo)分化,但衰老組細(xì)胞在形態(tài)學(xué)、增殖速度和誘導(dǎo)分化的能力均較對(duì)照組降低。 2.體外培養(yǎng)人臍帶間充質(zhì)干細(xì)胞的過(guò)程中,細(xì)胞隨傳代次數(shù)的增加形態(tài)發(fā)生改變、增殖減慢、分化及合成代謝能力均減退,細(xì)胞趨于老化。 3.衰老組細(xì)胞與對(duì)照組細(xì)胞比較,下調(diào)表達(dá)的基因和信號(hào)通路涉及細(xì)胞增殖、細(xì)胞遷移、代謝、生物合成、細(xì)胞外基質(zhì)、細(xì)胞連接等方面,而免疫紊亂及退行性相關(guān)基因表達(dá)明顯上調(diào)。
[Abstract]:Objective:
1. to observe and detect the morphological changes, phenotypes, growth kinetics and differentiation ability of human umbilical cord mesenchymal stem cells in vitro.
2. the changes of gene expression profiles in human umbilical cord mesenchymal stem cells during the process of senescence in vitro were detected and the possible mechanisms were discussed.
Method
1. cell culture: human umbilical cord mesenchymal stem cells were cultured by tissue block adherence method. The third generation cells were taken as control group, and the fifteenth generation cells were continuously cultured into the aging group. The cell morphology was observed under inverted microscope, and then the cell slides were made and observed by SEM.
2. growth and proliferation: cells from control group and aging group were inoculated in 96 hole culture plate, and 8 holes were set up for 6 days. The daily absorbance (OD) value was measured at 450nm wavelength, and cell growth curve was drawn.
3. phenotypes: control group and aging group were digested fully into monoplast suspension, using flow cytometry and analysis of two groups of cells CD34, CD45, CD44 and CD105 phenotypes.
4. identification of differentiation ability: the control group and the aging group were induced to differentiate into osteoblasts and adipocytes in different induction medium, and then differentiated after induced differentiation for 14 days.
Expression analysis of 5. genes: after washing the cells' adding Trizol reagent, extraction of total RNA by microarray hybridization, detection and data analysis, and use the DNA chip scanner for microarray scanning. The scan results were standardized analysis, compared with the control group log2Ratio|2 (4-fold change) for differential expression, and analysis of GO the KEGG and pathway.
6.PCR validation: according to the results of gene expression profiles, a number of typical differentially expressed genes were selected for real-time quantitative PCR validation. The differential expression of BAX, FADD, JAK2, IGF3BP5, PDK1, Col- III, Col-, IV, SMAD2, SAMD4, SMAD2, etc. in control group and senescence group were detected.
Result
1. cell morphology: the control group cells were fibrous into typical, per square millimeter of up to 500 cells; cell aging group individual differences, cells become large, per square millimeter of only about 150 cells, aging cytoplasmic ratio decreases. Under a scanning electron microscope group young cells were plump, microvilli on the surface of uniform distribution, and aging group cell pseudopodia increased, the overall flat.
2. proliferation: control group and aging group growth curve have a period of stagnation after inoculation (12-24 hours), and then enter the proliferating cells in the control group, can quickly enter the proliferation period, and in fifth days to reach a higher number of cells into the platform. After the long period of stagnation and aging group, plateau in third days the proliferation after the final cell number is less.
3. phenotypic detection: according to the results of flow cytometry, CD34 and CD45 were negative in the control group and aging group. CD44 and CD105 were all highly expressed. There was no significant difference in the positive rate between the two groups.
4.: in the induction of differentiation ability of appropriate differentiated condition, control group and aging group cells were able to differentiate into osteoblasts and adipocytes: after 14 days of osteogenic induction. Most of the cells have calcified extracellular matrix, alizarin red S staining positive alkaline phosphatase staining; oil red O positive fat particle induced differentiation after 14 days about 50% to fat cell cells. These results demonstrated that the adherent cells with mesenchymal stem cell characteristics, compared with the control cells, aging group decreased significantly induced cell differentiation.
Analysis of 5. different gene signal pathway and gene clustering: a group of differentially expressed genes will be aging group with control cells by KEGG pathway analysis (P0.05), aging group was significantly up-regulated in 20 pathways were, including autoimmune diseases, degenerative disease pathway, down 49 pathways, including anabolic pathways involved in extracellular. The matrix, substance metabolism and cell proliferation. Detection of 41000+ gene, genetic differences of up to 8000, clustering analysis of 2000 genes showed significant differences, differences in genes involved in transcription, translation, biosynthesis, metabolism, signal transduction, cell proliferation, cell migration, cell junctions and other aspects.
6.PCR validation: fluorescent quantitative PCR showed that the expression of BAX, FADD, JAK2, IGF3BP5, PDK1 and MGMT increased in senescent cells compared with the control group, while Col-, III, Col-, IV, SMAD2, Col-, mRNA, and mRNA were down regulated.
conclusion
1., the third generation of the control group and the 15 generation of aging group had proliferative potential, the same cell phenotype, and could differentiate into osteoblasts and adipocytes, but the morphology, proliferation rate and differentiation ability of the aging group were all lower than those of the control group.
2. in the process of culturing human umbilical cord mesenchymal stem cells in vitro, cell morphology changes, proliferating and slowing down, the ability of differentiation and anabolism decrease, and cells tend to be aging as the number of passages increases.
3., compared with the control group, the down regulated genes and signaling pathways involved in cell proliferation, cell migration, metabolism, biosynthesis, extracellular matrix and cell connection.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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本文編號(hào)：1625242