本文關(guān)鍵詞：懸滴培養(yǎng)促進人脂肪間充質(zhì)干細(xì)胞的成骨分化　出處：《遼寧醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： ADSCs 種子細(xì)胞 懸滴培養(yǎng) 球形聚集物 成骨分化

【摘要】：目的 由于外傷、腫瘤及感染等原因造成的骨組織缺損是口腔頜面外科常見的臨床問題。組織工程的發(fā)展為骨缺損的治療帶來了新的希望。尋找理想的種子細(xì)胞一直是骨組織工程基礎(chǔ)研究和臨床應(yīng)用所面臨的重大挑戰(zhàn)之一。本研究通過探討人脂肪組織使用酶消化法得到具有分化潛能的人脂肪來源間充質(zhì)干細(xì)胞（adipose-derived stem cells, ADSCs）；研究懸滴三維培養(yǎng)對hADSCs成骨誘導(dǎo)分化的影響，以期在骨組織工程種子細(xì)胞方面取得一定突破。 方法 利用酶消化法分離人體脂肪組織，獲得并培養(yǎng)hADSCs，通過相差顯微鏡觀察其形態(tài)；流式細(xì)胞術(shù)檢測其干細(xì)胞表面標(biāo)志的表達，并使用不同誘導(dǎo)劑檢測其成骨、成脂的多向分化潛能。對hADSCs進行懸滴培養(yǎng)，通過相差顯微鏡觀察hADSCs懸滴培養(yǎng)時三維球形聚集物的形成過程；使用HE染色觀察球形聚集物結(jié)構(gòu)；對不同細(xì)胞量的球形聚集物（10,000、25,000、50,000），懸滴培養(yǎng)24h、72h、96h后進行比較、分析細(xì)胞活性（Live/dead染色）和球形聚集物直徑；通過流式細(xì)胞術(shù)檢測hADSCs懸滴培養(yǎng)后其干細(xì)胞表面標(biāo)志表達的變化情況，驗證hADSCs懸滴培養(yǎng)后成骨、成脂分化的潛能。利用MTT比色法和克隆分析hADSCs三維懸滴培養(yǎng)與二維平板培養(yǎng)對細(xì)胞增殖的影響。在成骨誘導(dǎo)分化的7d、14d、21d，利用熒光實時定量PCR（real time fluorescence quantitativepolymerase chain reaction）檢測三維懸滴培養(yǎng)與二維培養(yǎng)hADSCs的成骨分化標(biāo)志基因Collagen type I、Osteopontin及Osteocalcin的表達水平，研究懸滴培養(yǎng)對hADSC成骨誘導(dǎo)分化的影響。 結(jié)果通過酶消化法分離培養(yǎng)的hADSCs，表達干細(xì)胞表面標(biāo)志CD44、CD90、CD105，不表達CD34、CD45，誘導(dǎo)劑誘導(dǎo)后，具有成骨和成脂分化潛能；懸滴培養(yǎng)72h的hADSCs可形成大小均一的球狀三維結(jié)構(gòu)，形成的球形聚集物直徑隨著時間的延長直徑逐漸變小，細(xì)胞聚集越緊密，形成的球形聚集物也越致密；細(xì)胞活性檢測發(fā)現(xiàn)，球形聚集物中hADSCs細(xì)胞量為25,000，懸滴培養(yǎng)72h時細(xì)胞活性最好，隨著時間的延長球形聚集物中的細(xì)胞活性逐漸降低；通過懸滴培養(yǎng)的hADSCs，，在消散后培養(yǎng)的細(xì)胞增殖和克隆形成能力均明顯較直接二維平板培養(yǎng)高；流式細(xì)胞術(shù)檢測結(jié)果表明hADSCs懸滴培養(yǎng)后其干細(xì)胞表面標(biāo)志與二維培養(yǎng)相比無明顯差異，且具有成骨、成脂分化能力，表明懸滴培養(yǎng)不影響脂肪間充質(zhì)干細(xì)胞的基本干細(xì)胞特性；熒光實時定量PCR結(jié)果表明，在成骨誘導(dǎo)分化的不同時間點，hADSCs懸滴培養(yǎng)組的成骨基因Collagen type I,Osteopontin及Osteocalcin表達均比普通二維培養(yǎng)組高，表明懸滴培養(yǎng)可促進hADSCs的成骨誘導(dǎo)分化。 結(jié)論 通過酶消化法可以從脂肪組織中分離培養(yǎng)出狀態(tài)優(yōu)良的hADSCs；25,000hADSCs懸滴培養(yǎng)后形成的三維球狀聚集物中的細(xì)胞活性最為優(yōu)良。懸滴培養(yǎng)不影響hADSCs的干細(xì)胞特性，且有利于hADSCs的增殖及成骨誘導(dǎo)分化，有助于提高其成骨分化誘導(dǎo)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.

【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

【參考文獻】

相關(guān)期刊論文 前2條

1 Brian E.Grottkau;Claudia C.Friedrich;;DAPT Enhances the Apoptosis of Human Tongue Carcinoma Cells[J];International Journal of Oral Science;2009年02期

2 鞠曉東,婁思權(quán),田華,王衛(wèi)國,劉延青;脂肪間充質(zhì)干細(xì)胞的基本生物學(xué)特性及向成骨細(xì)胞誘導(dǎo)分化的實驗研究[J];中華實驗外科雜志;2004年06期

本文編號：1365294