悬滴培养促进人脂肪间充质干细胞的成骨分化

发布时间：2018-01-01 16:28

本文关键词：悬滴培养促进人脂肪间充质干细胞的成骨分化　出处：《辽宁医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的由于外伤、肿瘤及感染等原因造成的骨组织缺损是口腔颌面外科常见的临床问题。组织工程的发展为骨缺损的治疗带来了新的希望。寻找理想的种子细胞一直是骨组织工程基础研究和临床应用所面临的重大挑战之一。本研究通过探讨人脂肪组织使用酶消化法得到具有分化潜能的人脂肪来源间充质干细胞（adipose-derived stem cells, ADSCs）；研究悬滴三维培养对hADSCs成骨诱导分化的影响，以期在骨组织工程种子细胞方面取得一定突破。方法利用酶消化法分离人体脂肪组织，获得并培养hADSCs，通过相差显微镜观察其形态；流式细胞术检测其干细胞表面标志的表达，并使用不同诱导剂检测其成骨、成脂的多向分化潜能。对hADSCs进行悬滴培养，通过相差显微镜观察hADSCs悬滴培养时三维球形聚集物的形成过程；使用HE染色观察球形聚集物结构；对不同细胞量的球形聚集物（10,000、25,000、50,000），悬滴培养24h、72h、96h后进行比较、分析细胞活性（Live/dead染色）和球形聚集物直径；通过流式细胞术检测hADSCs悬滴培养后其干细胞表面标志表达的变化情况，验证hADSCs悬滴培养后成骨、成脂分化的潜能。利用MTT比色法和克隆分析hADSCs三维悬滴培养与二维平板培养对细胞增殖的影响。在成骨诱导分化的7d、14d、21d，利用荧光实时定量PCR（real time fluorescence quantitativepolymerase chain reaction）检测三维悬滴培养与二维培养hADSCs的成骨分化标志基因Collagen type I、Osteopontin及Osteocalcin的表达水平，研究悬滴培养对hADSC成骨诱导分化的影响。结果通过酶消化法分离培养的hADSCs，表达干细胞表面标志CD44、CD90、CD105，不表达CD34、CD45，诱导剂诱导后，具有成骨和成脂分化潜能；悬滴培养72h的hADSCs可形成大小均一的球状三维结构，形成的球形聚集物直径随着时间的延长直径逐渐变小，细胞聚集越紧密，形成的球形聚集物也越致密；细胞活性检测发现，球形聚集物中hADSCs细胞量为25,000，悬滴培养72h时细胞活性最好，随着时间的延长球形聚集物中的细胞活性逐渐降低；通过悬滴培养的hADSCs，，在消散后培养的细胞增殖和克隆形成能力均明显较直接二维平板培养高；流式细胞术检测结果表明hADSCs悬滴培养后其干细胞表面标志与二维培养相比无明显差异，且具有成骨、成脂分化能力，表明悬滴培养不影响脂肪间充质干细胞的基本干细胞特性；荧光实时定量PCR结果表明，在成骨诱导分化的不同时间点，hADSCs悬滴培养组的成骨基因Collagen type I,Osteopontin及Osteocalcin表达均比普通二维培养组高，表明悬滴培养可促进hADSCs的成骨诱导分化。结论通过酶消化法可以从脂肪组织中分离培养出状态优良的hADSCs；25,000hADSCs悬滴培养后形成的三维球状聚集物中的细胞活性最为优良。悬滴培养不影响hADSCs的干细胞特性，且有利于hADSCs的增殖及成骨诱导分化，有助于提高其成骨分化诱导效率。
[Abstract]:objective
Due to trauma, tumor and infection caused by reasons such as the bone tissue defect is a common clinical problem of oral and maxillofacial surgery. The development of tissue engineering has brought new hope for the treatment of bone defects. Seed cells has been searching for the ideal bone tissue engineering research foundation and clinical application are facing one of the big challenges. This study through the discussion human adipose tissue using enzymatic digestion method to obtain the differentiation potential of human adipose derived mesenchymal stem cells (adipose-derived stem cells, ADSCs); Study on the hanging drop three-dimensional culture of osteogenic differentiation of hADSCs, in order to achieve a breakthrough in seed cells for bone tissue engineering.
Method
Isolation of human adipose tissue by digestion, and cultured for hADSCs, the phase contrast microscope.; to detect the expression of cell surface markers by flow cytometry, and the use of different inducers to detect the osteogenic differentiation potential, into fat. The hADSCs hanging drop culture, the formation process of 3D spherical aggregation difference of hADSCs microscope when dropculture; staining of spherical aggregates structures using HE; spherical aggregates of different cell volume (10000,25000,50000), 24h 72h, dropculture, 96h after the comparison, analysis of cell activity (Live/dead staining) and spherical aggregates with diameter; flow cytometry hADSCs hanging drop culture after stem cell surface marker expression and osteogenic verification of hADSCs hanging drop culture after adipogenic differentiation. By MTT colorimetric assay and cloning and analysis of hADSCs 3D hanging drop Effect of culture on cell proliferation and plate culture. In the two-dimensional osteogenic differentiation of 7D, 14d, 21d, using real-time quantitative PCR (real time fluorescence quantitativepolymerase chain reaction) detection of three-dimensional hanging drop culture and two-dimensional culture of bone differentiation marker gene Collagen type I into hADSCs, the expression level of Osteopontin and Osteocalcin, research hanging drop culture and osteogenic differentiation of hADSC.
Results by enzyme digestion from cultured hADSCs, the expression of stem cell surface markers CD44, CD90, CD105, the expression of CD34 CD45 induced with osteogenic and adipogenic differentiation potential; globular three-dimensional structure dropculture 72h hADSCs can form uniform size, the formation of spherical aggregates with diameter the extended diameter becomes smaller, cell aggregation more closely, the formation of spherical aggregates is more compact; cell activity detection, spherical aggregates in hADSCs cells was 25000, cell activity best hanging drop culture of 72h, with the extension of long spherical aggregation in cell activity decreased time; by hanging drop culture hADSCs, cell proliferation and clone culture in dissipate after forming ability was significantly higher than that of direct two-dimensional plate culture; flow cytometry showed that hADSCs dropculture after the stem cell surface marker and Er Weipei Have no obvious difference compared with osteogenic, adipogenic differentiation, show that the hanging drop culture does not affect adipose derived mesenchymal stem cells characteristics of stem cells; real-time quantitative PCR showed that in different time points of bone differentiation, hADSCs hanging drop culture group based Collagen type I for osteogenesis Osteopontin, and Osteocalcin expression were higher than normal group culture showed that the two-dimensional, hanging drop culture can promote the osteogenic differentiation of hADSCs.
conclusion
To cultivate excellent state of hADSCs isolated from adipose tissue by enzyme digestion method; three-dimensional spherical form 25000hADSCs hanging drop culture after aggregation of cell activity in the most excellent. Drop culture does not affect the stem cell characteristics of hADSCs, and is conducive to the proliferation of hADSCs and induce osteogenic differentiation, help to improve the osteogenic differentiation efficiency.

【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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