本文選題：組織工程　切入點(diǎn)：組織再生　出處：《第三軍醫(yī)大學(xué)》2005年碩士論文

【摘要】： 目的:探討兔髓核細(xì)胞體外單層培養(yǎng)的適宜條件,并觀察一種自行構(gòu)建的II型膠原支架上立體培養(yǎng)兔髓核細(xì)胞的變化和10ng/ml rhTGF-β1對其生物學(xué)行為的影響。方法: 單層培養(yǎng):改變單層培養(yǎng)條件(培養(yǎng)瓶是否鋪被PLL、培養(yǎng)液pH、胎牛血清濃度、rhTGF-β1濃度),利用MTT法和細(xì)胞計數(shù)法,決定兔髓核細(xì)胞體外單層培養(yǎng)的適宜條件。 支架培養(yǎng):將兔髓核細(xì)胞以5×10~6/ml接種到II型膠原支架上進(jìn)行立體培養(yǎng)1w,利用倒置顯微鏡和掃描電鏡觀察細(xì)胞形態(tài)學(xué)變化;分別利用RT-PCR、3H-proline摻入法觀察單層培養(yǎng)、立體培養(yǎng)和10 ng/ml rhTGF-β1刺激24小時后兔髓核細(xì)胞基因表達(dá)水平和總膠原合成的變化。 結(jié)果: 單層培養(yǎng): (1)兔髓核細(xì)胞對培養(yǎng)條件要求較高,其中培養(yǎng)介質(zhì)是否鋪被PLL、培養(yǎng)液的pH、胎牛血清濃度、rhTGF-β1對細(xì)胞影響較為明顯。鋪被PLL明顯促進(jìn)兔髓核細(xì)胞貼壁和生長(1.12倍,p0.01);pH在6.8～7.2之間,兔髓核細(xì)胞生長較好,pH7.0為較適宜培養(yǎng)液pH;rhTGF-β1在5～10 ng/ml范圍內(nèi)呈劑量依賴性促進(jìn)細(xì)胞生長。在確立的適宜條件中培養(yǎng)時,細(xì)胞生長良好,電鏡結(jié)果示細(xì)胞器增多。 (2)原代兔髓核細(xì)胞指數(shù)生長期是接種后的8～10d,第一代兔髓核細(xì)胞指數(shù)生長期是接種后的6～8d。兔髓核細(xì)胞體外培養(yǎng)時增殖能力較弱,第一代活性最高。 (3)兔髓核細(xì)胞單層培養(yǎng)時II型膠原和集聚蛋白聚糖免疫組化染色陽性。 支架培養(yǎng) (1)兔髓核細(xì)胞可以在II型膠原支架上黏附生長,細(xì)胞由單層培養(yǎng)時的多角形變?yōu)轭悎A形。 (2)兔髓核細(xì)胞在II型膠原支架上培養(yǎng)1周,其II型膠原、集聚蛋白聚糖基因表達(dá)水平和總膠原合成較單層培養(yǎng)增加(1.21、1.12、1.11倍,p0.05、p0.05、p0.01)。
[Abstract]:Aim: to investigate the suitable conditions for monolayer culture of rabbit nucleus pulposus cells in vitro, and to observe the changes of rabbit nucleus pulposus cells cultured on a self-constructed type II collagen scaffold and the effect of 10ng/ml rhTGF- 尾 1 on its biological behavior.Methods:Monolayer culture: changing the conditions of monolayer culture (whether the culture bottle was coated with PLL, the pH of culture medium, the concentration of rhTGF- 尾 1 in fetal bovine serum), the suitable conditions for monolayer culture of rabbit nucleus pulposus cells in vitro were determined by MTT method and cell counting method.Scaffold culture: rabbit nucleus pulposus cells were seeded on type II collagen scaffold with 5 脳 10~6/ml for 1 week. Morphological changes were observed by inverted microscope and scanning electron microscope, and monolayer culture was observed by RT-PCRN 3H-proline incorporation method.The changes of gene expression and total collagen synthesis in rabbit nucleus pulposus cells stimulated by 10 ng/ml rhTGF- 尾 1 and stereoscopic culture for 24 hours.Results:Monolayer culture:1) Rabbit nucleus pulposus cells require higher culture conditions, among them, whether the culture medium is covered with PLL, the pH of the culture medium, the concentration of fetal bovine serum and rhTGF- 尾 1 have obvious effects on the cells.When cultured in suitable conditions, the cells grew well and the organelles increased under electron microscope.(2) the growth period of primary rabbit nucleus pulposus cell index was 810 days after inoculation, and that of the first generation rabbit nucleus pulposus cell index was 6 days after inoculation.The proliferative ability of rabbit nucleus pulposus cells was weak in vitro and the first generation activity was the highest.Type II collagen and agglutinin were positive in monolayer culture of rabbit nucleus pulposus cells.Scaffold culture1) Rabbit nucleus pulposus cells could grow on type II collagen scaffold, and the cells changed from polygonal shape to round shape in monolayer culture.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329

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本文編號：1721524