本文關(guān)鍵詞：成骨細(xì)胞復(fù)合TCP異體移植成骨情況及免疫學(xué)檢測　出處：《山東大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 成骨細(xì)胞 組織工程 細(xì)胞培養(yǎng)

【摘要】：目的:研究不同來源成骨細(xì)胞體外培養(yǎng)的形態(tài)特征和生物學(xué)活性,探討成骨細(xì)胞分離培養(yǎng)的最佳方法及作為骨組織工程種子細(xì)胞的可行性和應(yīng)用價(jià)值,為骨組織工程的研究提供穩(wěn)定可靠的種子細(xì)胞來源。 方法:無菌條件下取新生新西蘭兔脛骨骨膜和骨髓,PBS沖洗3次,0.25%胰蛋白酶消化20分鐘,以徹底去除殘留在組織塊上的血污,再將骨膜剪成大小約為1×1mm的組織塊,將骨膜組織塊放入25ml培養(yǎng)瓶中,加入含10%小牛血清的高糖DMEM培養(yǎng)液2ml,細(xì)胞長滿瓶底后進(jìn)行傳代培養(yǎng)。注射器肝素化后抽取完全DMEM培養(yǎng)液沖洗骨髓腔,將混合DMEM培養(yǎng)液加入到無菌離心管內(nèi),1000轉(zhuǎn)/分鐘離心,去除最上面的脂肪層,以1×10~6/cm~2密度接種于100ml培養(yǎng)瓶中進(jìn)行原代細(xì)胞培養(yǎng)。3天后全量換液,細(xì)胞匯合成單層后用0.25%胰蛋白酶消化細(xì)胞進(jìn)行傳代培養(yǎng)。細(xì)胞傳代后改用含地塞米松(1×10(-8)mol/l)、β-甘油磷酸鈉(10mmol/l)、維生素C(50mg/ml)的條件培養(yǎng)液培養(yǎng)。取第5代生長旺盛的成骨細(xì)胞,胰蛋白酶消化后,以10~7/ml的濃度封入凍存管進(jìn)行超低溫凍存,一個(gè)月后復(fù)蘇,以10~6/ml的濃度接種在含有5mlDMEM培養(yǎng)液的培養(yǎng)瓶中培養(yǎng)。每種方法培養(yǎng)的細(xì)胞每日在相差顯微鏡下觀察細(xì)胞的形態(tài),通過Gomori鈣鈷法進(jìn)行堿性磷酸酶染色,von Kossa法進(jìn)行鈣結(jié)節(jié)染色,觀察細(xì)胞體外基質(zhì)分泌和骨化過程。 結(jié)果:骨膜培養(yǎng)第3天可見少量的梭形細(xì)胞從組織塊周圍游出,隨培養(yǎng)時(shí)間延長細(xì)胞數(shù)量逐漸增多,圍繞組織塊周圍呈放射狀生長,10天時(shí)細(xì)胞融合成單層。傳代后的細(xì)胞呈梭形、多角形,帶有粗大的突起,4天時(shí)
[Abstract]:Objective: to study the morphological characteristics and biological activity of osteoblasts from different sources in vitro, and to explore the best method for isolation and culture of osteoblasts and the feasibility and application value of osteoblasts as seed cells for bone tissue engineering. To provide a stable and reliable source of seed cells for the study of bone tissue engineering. Methods: the tibia periosteum and bone marrow of newborn New Zealand rabbits were washed with 0.25% trypsin for 20 minutes after washing under aseptic condition. Then the periosteum was cut into a tissue about 1 脳 1mm in size, and the periosteum tissue mass was put into 25ml culture bottle, and the high sugar DMEM culture medium containing 10% calf serum was added to 2ml. After heparinization of the syringe, the complete DMEM culture fluid was extracted to wash the medullary cavity, and the mixed DMEM culture medium was added into the aseptic centrifuge tube to centrifuge for 1000 rpm / min. The top adipose layer was removed and the primary cells were incubated in 100ml culture flask at a density of 1 脳 10 ~ (6) / cm ~ (-2) for 3 days. After synthesis of monolayer, the cells were subcultured with 0.25% trypsin. The cells were transferred to the cells containing dexamethasone (1 脳 10 ~ (-8) mol / L). 尾 -glycerophosphate (10 mmol 路L ~ (-1)), vitamin C ~ (50 mg / ml) was cultured in the conditioned medium. The osteoblasts of the 5th generation were harvested and digested by trypsin. The cryopreservation was carried out with a concentration of 10 / 7 / ml and was resuscitated one month later. The cells were cultured in a culture flask containing 5 ml DMEM at a concentration of 106% ml. The cells cultured in each method were observed under phase contrast microscope every day. Calcium nodules were stained by alkaline phosphatase (ALP) staining von Kossa method with Gomori calcium cobalt method to observe the process of extracorporeal matrix secretion and ossification. Results: on the 3rd day of periosteum culture, a small number of fusiform cells could be seen swimming out from around the tissue mass, and the number of cells gradually increased with the extension of culture time, and the cells grew radially around the tissue mass. The cells were fused into monolayers at 10 days. After passage, the cells were fusiform, polygonal, with a thick protuberance for 4 days.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329;R392

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