本文關(guān)鍵詞：兔同種異體脫鈣骨復(fù)合骨髓間充質(zhì)干細(xì)胞關(guān)節(jié)腔內(nèi)培養(yǎng)軟骨與同腔軟骨的性狀對(duì)比研究　出處：《安徽醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 兔骨髓間充質(zhì)干細(xì)胞 生長(zhǎng)因子 成軟骨 腔內(nèi)培養(yǎng)

【摘要】：目的采用兔骨髓間充質(zhì)干細(xì)胞(BMSC)復(fù)合同種異體脫鈣骨(DBM)體外培養(yǎng)和關(guān)節(jié)腔內(nèi)培養(yǎng)組織工程軟骨與同腔軟骨的性狀對(duì)比研究。 方法1.采用同源雄性新西蘭幼兔(4～6周齡)全骨髓貼壁篩選法培養(yǎng)原代BMSC,第三代細(xì)胞融合至約80%時(shí)培養(yǎng)基中加入成軟骨誘導(dǎo)液:含轉(zhuǎn)化生長(zhǎng)因子(Transforming Growth Factor,TGF)-β1 10ng/ml、胰島素樣生長(zhǎng)因子(Insulin-like Growth Factor,IGF)-1 20ng/ml、維生素C 50ng/ml。2.根據(jù)Urist提供的方法制作DBM并做掃描電鏡(SEM)觀察。3.將誘導(dǎo)后的BMSC種植于30個(gè)DBM支架培養(yǎng)并分別在體外(A組)及關(guān)節(jié)腔內(nèi)(B組)培養(yǎng)。于4、8、12周分別取材,同時(shí)取同腔軟骨(C組)作為對(duì)照,行SEM等大體形態(tài)觀察并制石蠟切片行蘇木素-伊紅染色(Hematoxylin-Eosin Staining,HE)、甲苯胺藍(lán)(Toluidine Blue,TB)染色、Masson三色染色、Ⅱ型膠原免疫組化等組織學(xué)觀察并進(jìn)行比較。 結(jié)果1.本實(shí)驗(yàn)方法分離和培養(yǎng)的BMSC形態(tài)特征明顯,呈長(zhǎng)梭形,原代培養(yǎng)10～12天細(xì)胞融合約90%,增殖傳代的細(xì)胞6～7天可達(dá)約90%融合。2.BMSC經(jīng)定向誘導(dǎo)后表現(xiàn)出軟骨細(xì)胞的特性。3.制備得到的DBM為三維立體疏松多孔的海綿樣結(jié)構(gòu)。4.A、B、C三組培養(yǎng)12w后,取出標(biāo)本行SEM等大體觀察、組織學(xué)切片觀察及Ⅱ型膠原免疫組化檢測(cè):A組:SEM:標(biāo)本表面凹凸不平,內(nèi)部尚有部分空隙;HE染色見(jiàn)軟骨細(xì)胞少量增生,胞核藍(lán)染;甲苯胺藍(lán)染色見(jiàn)軟骨細(xì)胞排列無(wú)序,少量周圍基質(zhì)包繞;Masson染色陽(yáng)性區(qū)域小,細(xì)胞排列無(wú)序;Ⅱ型膠原免疫組化見(jiàn)軟骨細(xì)胞胞漿及胞外基質(zhì)少量黃色顆粒。B組:SEM:標(biāo)本表面較光滑;HE染色見(jiàn)軟骨細(xì)胞增生,胞核藍(lán)染;甲苯胺藍(lán)染色見(jiàn)軟骨細(xì)胞成串排列,軟骨陷窩形成,周圍基質(zhì)包繞;Masson染色陽(yáng)性,軟骨細(xì)胞多,基質(zhì)藍(lán)染,按一定應(yīng)力方向排列;Ⅱ型膠原免疫組化見(jiàn)細(xì)胞外基質(zhì)中出現(xiàn)較多棕黃色顆粒,Ⅱ型膠原染色陽(yáng)性。 結(jié)論兔BMSC與同種異體DBM制備的細(xì)胞-支架復(fù)合物可在體外及膝關(guān)節(jié)腔內(nèi)培養(yǎng)出組織工程軟骨,關(guān)節(jié)腔內(nèi)培養(yǎng)的軟骨比體外培養(yǎng)的軟骨更接近正常軟骨。
[Abstract]:Objective to compare the characteristics of rabbit bone marrow mesenchymal stem cells (BMSC) combined with allogenic decalcified bone (DBM) cultured in vitro and articular cavity culture tissue engineered cartilage and the same cavity cartilage.
Methods 1. male New Zealand rabbits by homology (4 ~ 6 weeks) the whole bone marrow adherence screening method of primary cultured BMSC cells of the third generation fusion to about 80% when added to the medium of chondrogenic media containing transforming growth factor (Transforming Growth, Factor, TGF) 10ng/ml beta 1 and insulin-like growth factor (Insulin-like Growth Factor -1 20ng/ml, IGF), vitamin C 50ng/ml.2. according to the methods provided by Urist DBM and scanning electron microscopy (SEM) observation of.3. after induction BMSC grown in 30 DBM scaffold and respectively in vitro (A group) and intra-articular (B group). Cultured in 4,8,12 weeks were drawn at the same time take the same cavity cartilage (group C) as control, SEM for gross observation and paraffin sections were stained with hematoxylin eosin staining (Hematoxylin-Eosin Staining, HE (Toluidine), Blue, TB) toluidine blue staining, Masson staining, type II collagen immunohistochemical and histological view Compare and compare.
The results of the 1. experiment methods to isolate and culture BMSC morphology obviously, fusiform, cultured for 10~12 days about 90% cell fusion, cell proliferation and passage of 6~7 days can reach about 90%.2.BMSC fusion after oriented induction showed cavernous structure of.4.A, prepared by the cartilage cell characteristics of.3. DBM for the three dimensional system the three-dimensional porous B, C three groups after 12W, remove the specimens SEM, gross observation, histological observation and detection of type II collagen group: A group: SEM: specimen surface is uneven, there are some internal voids; HE staining of cartilage cell proliferation of small amount, blue stained nuclei; toluidine blue staining showed chondrocytes a small amount of disorder arrangement, surrounded by the matrix; Masson staining area is small, cells arranged in disorder; type II collagen immunohistochemical cartilage cell cytoplasm and extracellular matrix of small yellow particles in.B group: SEM: specimen surface is smooth; HE staining The proliferation of cartilage cells, blue stained nuclei; toluidine blue staining showed that the cartilage cells arranged in clusters, cartilage lacuna formation, surrounded by the matrix; Masson staining, cartilage cells, matrix stained blue, arranged in a certain stress direction; type II collagen immunohistochemical see more brown granules in the extracellular matrix, collagen staining positive.
Conclusion rabbit BMSC and allogenic DBM preparation of cell scaffold composite can produce tissue engineered cartilage in vitro and knee joint cavity, articular cartilage culture than in vitro cartilage closer to normal cartilage.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 趙廷寶,范清宇,周勇,張殿忠,裘秀春,文艷華;復(fù)合骨形成蛋白骨修復(fù)材料的生物相容性研究[J];中國(guó)矯形外科雜志;2002年02期

2 孔清泉,項(xiàng)舟,鮮思平,汪金平,楊志明;自體骨髓間充質(zhì)干細(xì)胞與外源性透明質(zhì)酸鈉修復(fù)兔膝關(guān)節(jié)軟骨缺損的研究[J];中國(guó)修復(fù)重建外科雜志;2004年04期

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本文編號(hào)：1402657