本文選題：退變椎間盤 + 髓核��； 參考：《暨南大學(xué)》2017年碩士論文

【摘要】：目的:運(yùn)用組織塊貼壁法從脊柱內(nèi)鏡微創(chuàng)手術(shù)摘除的退變椎間盤髓核組織中高效分離培養(yǎng)間充質(zhì)干細(xì)胞樣細(xì)胞,并從細(xì)胞形態(tài)、細(xì)胞表型、增殖能力、三系分化潛能、多能性基因和髓核細(xì)胞標(biāo)志基因表達(dá)等方面對(duì)從不同個(gè)體退變椎間盤髓核組織樣本來源的髓核干細(xì)胞(nucleus pulposus derived stem cells,NPSCs)進(jìn)行深入研究。方法:組織塊貼壁法高效分離人退變椎間盤髓核組織來源干細(xì)胞。將獲得的細(xì)胞通過傳代進(jìn)行純化和擴(kuò)增培養(yǎng),進(jìn)行形態(tài)學(xué)觀察,取第2代生長(zhǎng)良好的干細(xì)胞,流式細(xì)胞術(shù)檢測(cè)NPSCs表面標(biāo)志物的表達(dá)情況,并進(jìn)行成骨成脂分化潛能鑒定及成軟骨分化潛能鑒定。分別取生長(zhǎng)良好的第2代的不同個(gè)體退變椎間盤髓核組織來源的干細(xì)胞,利用流式細(xì)胞術(shù)檢測(cè)間充質(zhì)干細(xì)胞表面標(biāo)志物、髓核細(xì)胞表面標(biāo)志物CD24的表達(dá),以及細(xì)胞周期和細(xì)胞凋亡情況。利用CCK8試劑盒(Cell Counting Kit,CCK-8)檢測(cè)比較細(xì)胞活力,細(xì)胞計(jì)數(shù)法檢測(cè)比較細(xì)胞的倍增時(shí)間,并繪制細(xì)胞生長(zhǎng)曲線。qRT-PCR檢測(cè)比較髓核細(xì)胞相關(guān)標(biāo)志基因COL2A1、Aggrecan和SOX9,髓核干細(xì)胞標(biāo)志基因Tie2,以及多能性基因NANOG和OCT4的表達(dá)水平。茜素紅S染色法鑒定比較成骨分化潛能,油紅O染色法鑒定比較成脂分化潛能。阿利新藍(lán)染色法鑒定比較成軟骨分化潛能。使用Image J軟件計(jì)算向成骨成脂成軟骨誘導(dǎo)分化后茜素紅S、油紅O和阿利新藍(lán)染色定量分析。結(jié)果:組織塊貼壁法分離的NPSCs,形態(tài)多為典型梭形細(xì)胞,少數(shù)為多邊形或圓形;流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面標(biāo)志物CD29、CD44、CD73、CD90和CD105呈陽性表達(dá),細(xì)胞表面標(biāo)志物CD11b、CD14、CD34、CD45和HLA-DR呈陰性表達(dá);經(jīng)過21天的成骨成脂成軟誘導(dǎo)分化后,茜素紅染色、阿利新藍(lán)染色及油紅O染色均呈陽性,說明NPSCs具有向成骨、成軟骨和成脂肪誘導(dǎo)分化的能力。進(jìn)一步對(duì)不同個(gè)體退變椎間盤髓核組織來源的干細(xì)胞比較研究發(fā)現(xiàn),根據(jù)MSC特異性表面標(biāo)志物的表達(dá)水平可分為兩組:MSC表面標(biāo)志物高表達(dá)組(H-NPSCs)和MSC表面標(biāo)志物低表達(dá)組(L-NPSCs),流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示CD29、CD44、CD73、CD90和CD105表面標(biāo)志物表達(dá)率均大于95%,與之相應(yīng)的是其傳代后的細(xì)胞形態(tài)均一,呈成纖維細(xì)胞樣的長(zhǎng)梭形。而L-NPSCs的MSC特異性表面標(biāo)志物CD29和CD105表達(dá)率顯著低于H-NPSCs,CD29的表達(dá)率為36.52±0.14%,CD105的表達(dá)率為46.58±0.15%,細(xì)胞形態(tài)表現(xiàn)多樣性,除典型梭形細(xì)胞外,可見多邊形或圓形出現(xiàn)。兩組都不表達(dá)CD11b、CD14、CD24、CD34、CD45和HLA-DR(表達(dá)率低于1%)。CCK8檢測(cè)的細(xì)胞活性和細(xì)胞周期結(jié)果表明:與L-NPSCs相比,H-NPSCs具有更強(qiáng)的增殖能力。與細(xì)胞周期結(jié)果分析一致,L-NPSCs中G0/G1和G2/M期停滯增加,S期的進(jìn)入量低于H-NPSCs,細(xì)胞凋亡、細(xì)胞倍增時(shí)間結(jié)果分析表明H-NPSCs的凋亡率和倍增時(shí)間都要低于L-NPSCs。qRT-PCR檢測(cè)這三種細(xì)胞髓核細(xì)胞標(biāo)志基因COL2A1、Aggrecan、SOX9的表達(dá)情況顯示:L-NPSCs的三種標(biāo)志基因的表達(dá)量都顯著高于H-NPSCs的表達(dá)量。髓核干細(xì)胞標(biāo)志基因Tie2的表達(dá)情況顯示:H-NPSCs的表達(dá)量的顯著高于L-NPSCs的表達(dá)量。而多能性基因NANOG和OCT4的表達(dá)情況顯示:L-NPSCs的表達(dá)量顯著高于H-NPSCs的表達(dá)量。誘導(dǎo)分化后特異性染色檢測(cè)顯示,L-NPSCs的多向分化能力明顯弱于H-NPSCs。結(jié)論:1.組織塊貼壁法可從人退變椎間盤髓核組織中分離出間充質(zhì)干細(xì)胞樣細(xì)胞,表達(dá)間充質(zhì)干細(xì)胞表面標(biāo)志物CD29、CD44、CD73、CD90和CD105,不表達(dá)造血干細(xì)胞表面標(biāo)志物CD11b、CD14、CD34、CD45和HLA-DR,并具有向成骨成脂及成軟骨多向分化潛能。2.退變椎間盤髓核組織來源的NPSCs可根據(jù)其間充質(zhì)干細(xì)胞表面標(biāo)志物的表達(dá)水平分為高表達(dá)組(H-NPSCs)與低表達(dá)組(L-NPSCs)。與H-NPSCs相比,L-NPSCs的增殖能力、細(xì)胞活力和多向分化潛能明顯減弱,而L-NPSCs的髓核細(xì)胞標(biāo)志基因表達(dá)水平顯著高于H-NPSCs。髓核干細(xì)胞的MSC表面標(biāo)志物的表達(dá)水平與細(xì)胞增殖能力、多向分化潛能和髓核細(xì)胞特異性標(biāo)志基因表達(dá)水平相關(guān)。
[Abstract]:Objective: to isolate and culture mesenchymal stem cell like cells from degenerative intervertebral discs removed from spinal endoscope minimally invasive surgery by tissue block adherence, and to degenerate intervertebral discs from different individuals in terms of cell morphology, cell phenotype, proliferation, three lineage differentiation potential, pluripotent gene and gene expression of nucleus pulposus cells. Nucleus pulposus derived stem cells (NPSCs) of nucleus pulposus samples. Methods: the tissue block adherence method was used to efficiently separate the stem cells from the human degeneration intervertebral disc nucleus. The obtained cells were purified and expanded through the passage, and the morphological observation was carried out for the second generations of fine stem cells. Cell, flow cytometry was used to detect the expression of NPSCs surface markers, identification of osteogenic differentiation potential and identification of chondrogenic differentiation potential. Stem cells from different individuals with good growth of second generations of degenerative intervertebral disc nucleus were taken respectively. Flow cytometry was used to detect the surface markers of mesenchymal stem cells and the cell surface of nucleus pulposus cells. The expression of CD24, cell cycle and cell apoptosis. The cell viability was compared with the CCK8 Kit (Cell Counting Kit, CCK-8). Cell counting method was used to detect the doubling time of the cells, and the cell growth curve.QRT-PCR was plotted and compared with the nucleus pulposus related marker gene COL2A1, Aggrecan and SOX9, and the nucleus pulposus stem cells. The marker gene Tie2, as well as the expression level of the pluripotent gene NANOG and OCT4. The alizarin red S staining was used to identify the osteogenic differentiation potential. The oil red O staining method was used to identify the lipid differentiation potential. Alizarin red S and oil red after differentiation of osteogenic adipogenic into cartilage by Image J software were used to calculate the differentiation of alizarin red S and oil red. Quantitative analysis of O and alicaln blue staining. Results: the morphology of NPSCs separated by tissue block adherence was mostly spindle cells with a few polygons or circles; flow cytometry was used to detect the cell surface markers CD29, CD44, CD73, CD90 and CD105, and the cell surface markers CD11b, CD14, CD34, CD45 and HLA-DR were negative expression; after 21 days After a soft induction of osteogenesis, alizarin red staining, alizarin blue staining and oil red O staining were positive, indicating that NPSCs has the ability to induce differentiation into osteogenesis, cartilage and adipose tissue. Further research on stem cells from different individual degenerative intervertebral discs has been found to be based on the expression of specific surface markers of MSC. The level can be divided into two groups: MSC surface markers high expression group (H-NPSCs) and low expression group (L-NPSCs) of MSC surface markers. Flow cytometry results showed that the expression rates of CD29, CD44, CD73, CD90 and CD105 surface markers were more than 95%, corresponding to the cell morphology of the cells after its passage, and the long spindle shape of fibroblast like, and L-NPSCs. The expression rate of MSC specific surface markers, CD29 and CD105, was significantly lower than that of H-NPSCs, the expression rate of CD29 was 36.52 + 0.14%, the expression rate of CD105 was 46.58 + 0.15%, and the cell morphology was varied. The polygon or round appearance was visible except for the typical spindle cells. The two groups did not express CD11b, CD14, CD24, CD34, CD45 and HLA-DR (the expression rate under 1%). The results of cell activity and cell cycle test showed that compared with L-NPSCs, H-NPSCs had stronger proliferation ability. In accordance with the analysis of cell cycle results, the stagnation of G0/G1 and G2/M in L-NPSCs increased, and the entry of S stage was lower than H-NPSCs, cell apoptosis, and the result of cell doubling time results showed that the apoptosis rate and doubling time of H-NPSCs were lower than L-NP. The expression of the three cell marker genes COL2A1, Aggrecan, and SOX9 showed that the expression of the three markers of L-NPSCs was significantly higher than the expression of H-NPSCs. The expression of Tie2 in the stem cell marker gene of the nucleus pulposus showed that the expression of H-NPSCs was significantly higher than the expression of L-NPSCs, while the pluripotent gene N was expressed as N. The expression of ANOG and OCT4 showed that the expression of L-NPSCs was significantly higher than that of H-NPSCs. After induction of differentiation, specific staining showed that the multidirectional differentiation ability of L-NPSCs was significantly weaker than that of H-NPSCs. conclusion: 1. tissue block adherent method can separate mesenchymal stem like cells from the nucleus of human degeneration intervertebral disc and Express mesenchymal stem cells. Cell surface markers, CD29, CD44, CD73, CD90 and CD105, do not express the surface markers of hematopoietic stem cells, CD11b, CD14, CD34, CD45 and HLA-DR, and NPSCs can be divided into high expression groups according to the expression level of the surface markers of mesenchymal stem cells. Compared with the low expression group (L-NPSCs), the proliferation ability, cell viability and pluripotent differentiation potential of L-NPSCs were significantly lower than that of H-NPSCs, while the expression level of L-NPSCs was significantly higher than the expression level and proliferation ability of MSC surface markers in H-NPSCs. nucleus pulposus stem cells, the pluripotent differentiation potential and the specific markers of nucleus pulposus cells. The expression level of the chronicles is correlated.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.5

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