【摘要】： 背景與目的 骨髓間充質(zhì)干細胞(bone marrow mesenchymal stem cells, BMSCs)來源于中胚層,主要存在于全身的結(jié)締組織與器官間質(zhì)中,以骨髓中的含量最多。骨髓間充質(zhì)干細胞具有自我更新的能力與多向分化、增殖的潛能。骨髓間充質(zhì)干細胞因為具有可于自體獲得、易培養(yǎng)、移植后能夠長期存活并且不會出現(xiàn)排斥反應(yīng),可被外源基因轉(zhuǎn)染并長期表達的優(yōu)點,近年來成為組織工程、細胞治療、基因治療及器官移植的研究熱點,在多學(xué)科多領(lǐng)域做為一種理想的種子細胞得到了廣泛應(yīng)用。但是骨髓間充質(zhì)干細胞雖然在體內(nèi)的來源比較廣泛,但是數(shù)量極少,并且隨著身體機能的下降與個體的衰老,數(shù)量會逐漸減少。而組織工程與細胞工程開展的首要條件是種子細胞的獲取和培養(yǎng)傳代,如何能夠在體外獲得足夠數(shù)量和較高純度的種子細胞極其重要。而且目前尚沒有對骨髓間充質(zhì)干細胞進行直接鑒定的方法。通過本研究擬建立一種體外分離培養(yǎng)、擴增骨髓間充質(zhì)干細胞的理想方法,并且觀察其生長特性,探討其鑒定方法,為其在組織工程學(xué)與細胞工程學(xué)等學(xué)科的應(yīng)用奠定基礎(chǔ)。材料與方法 選用健康中國青山羊,骨髓穿刺法抽取髂骨骨髓,應(yīng)用密度梯度離心法從中分離出骨髓間充質(zhì)干細胞,結(jié)合貼壁篩選法進行培養(yǎng),顯微鏡下觀察細胞形態(tài)與生長情況,測定細胞成活率及傳代細胞貼壁率,對第2、4、6代細胞采用連續(xù)計數(shù)法繪制出生長曲線,計算細胞倍增時間,免疫細胞化學(xué)染色法鑒定細胞表面抗原標志。結(jié)果 1.骨髓間充質(zhì)干細胞原代細胞培養(yǎng)的形態(tài)觀察：培養(yǎng)后24h可見部分細胞貼壁,貼壁細胞多呈橢圓形。48 h后可見更多的細胞貼壁,貼壁細胞有伸展變形現(xiàn)象,細胞伸出偽足,形態(tài)漸變?yōu)樗笮巍⒍嘟切巍?～6d后可見數(shù)個細胞集落形成,顯微鏡下見貼壁細胞的細胞膜及細胞核清晰。隨培養(yǎng)進行細胞集落進一步增多,細胞生長逐漸融成片狀,12～14d時可長滿培養(yǎng)瓶底80%。 2.骨髓間充質(zhì)干細胞傳代細胞培養(yǎng)的形態(tài)：細胞傳代后6～8d即可長滿瓶底,細胞融合率可達100%。傳代細胞鏡下呈均一長梭形,細胞膜及細胞核清晰,細胞密度較高時呈放射狀、漩渦狀排列。 3.骨髓間充質(zhì)干細胞的鑒定結(jié)果：第3代骨髓間充質(zhì)干細胞免疫組化染色,細胞表面抗原CD29,CD44呈陽性表達(胞漿呈黃褐色染色),CD34,CD45呈陰性表達。 4.骨髓間充質(zhì)干細胞的生長曲線與倍增時間：生長曲線呈S型,1-3d為潛伏期,生長速度較為緩慢；2-3d后進入對數(shù)增長期,生長速度明顯加快；6-7d后進入平臺期,生長速度趨于穩(wěn)定。第2、4、6代細胞平均倍增時間分別為(25.37±1.04)h,(27.45±0.91)h,(29.49±2.02)h,總體平均倍增時間為(27.44±2.18)h。結(jié)論 1.本研究采用密度梯度離心法從山羊骨髓中分離骨髓間充質(zhì)干細胞,結(jié)合貼壁培養(yǎng)法進行體外培養(yǎng)擴增,細胞生長狀態(tài)良好,體外增殖能力較強。 2.通過鑒定細胞表面抗原標志,結(jié)合形態(tài)學(xué)觀察,證實研究中所分離培養(yǎng)細胞是骨髓間充質(zhì)干細胞。
[Abstract]:Background and purpose
Bone marrow mesenchymal stem cells (BMSCs) are derived from the mesoderm and mainly exist in the connective tissue and organ stroma of the whole body, with the highest content in bone marrow. In recent years, it has become a research hotspot in tissue engineering, cell therapy, gene therapy and organ transplantation. It has been widely used as an ideal seed cell in many subjects and fields. Although mesenchymal stem cells have a wide range of sources in vivo, their number is very small, and the number will gradually decrease with the decline of body function and the aging of individuals. Daughter cells are extremely important, and there is no direct identification method for bone marrow mesenchymal stem cells. through this study, we intend to establish an ideal method of isolation and culture in vitro, amplification of bone marrow mesenchymal stem cells, and observe their growth characteristics, explore its identification methods, for its tissue engineering and cell engineering disciplines. Laying the foundation for application. Materials and methods
Bone marrow was extracted from iliac bone marrow of healthy Chinese goats by bone marrow puncture. Bone marrow mesenchymal stem cells were isolated by density gradient centrifugation and cultured by adherence screening. Cell morphology and growth were observed under microscope. Cell survival rate and adherence rate of passaged cells were measured. Cells of the 2nd, 4th and 6th passages were drawn by continuous counting method. The growth curve was made, the doubling time was calculated, and the surface antigen markers were identified by immunocytochemical staining.
1. Morphological observation of primary cell culture of bone marrow mesenchymal stem cells: Some cells adhered to the wall 24 hours after culture, and most of the adherent cells were elliptical. After 48 hours, more cells adhered to the wall, the adherent cells had stretching deformation, the cells extended pseudopodia, the morphology gradually changed into spindle shape, and several cell colonies formed after 4-6 days, microscopically. The cell membrane and nucleus of adherent cells were clear. With the further increase of cell colony, cell growth gradually melted into sheets. At 12-14 days, 80% of the bottom of the culture bottle could be grown.
2. The morphology of bone marrow mesenchymal stem cells: the cells could grow to the bottom of the bottle 6-8 days after passage, and the fusion rate could reach 100%. Under the passage microscope, the cells were uniform spindle-shaped, the cell membrane and nucleus were clear, the cells were radial and arranged in a vortex when the density was high.
3. Identification of bone marrow mesenchymal stem cells: Immunohistochemical staining of the third generation of bone marrow mesenchymal stem cells showed positive expression of cell surface antigen CD29 and CD44 (cytoplasm was yellow-brown staining), negative expression of CD34 and CD45.
4. The growth curve and doubling time of bone marrow mesenchymal stem cells: the growth curve was S-shaped, 1-3 days was incubation period, and the growth rate was slower; 2-3 days later entered the logarithmic growth period, the growth rate was significantly accelerated; 6-7 days later entered the plateau phase, the growth rate tended to be stable. The average doubling time of the 2nd, 4th and 6th generation cells were (25.37 + 1.04) h, (27.45 + 0.91) h, respectively. H, (29.49 + 2.02) h, the overall average doubling time was (27.44 + 2.18) H. conclusion.
1. Bone marrow mesenchymal stem cells (BMSCs) were isolated from goat bone marrow by density gradient centrifugation and cultured in vitro with adherent culture. The cells grew well and had strong proliferation ability in vitro.
2. By identifying the markers of cell surface antigen and morphological observation, it was confirmed that the cells isolated and cultured in this study were bone marrow mesenchymal stem cells.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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相關(guān)期刊論文 前10條

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