【摘要】： 骨髓基質(zhì)干細(xì)胞(Bone marrow stromal cells,BMSCs)是一類具有多向分化潛能的組織干細(xì)胞,在體內(nèi)外適當(dāng)?shù)恼T導(dǎo)環(huán)境下可以分化成為骨、軟骨、脂肪、肌肉、神經(jīng)、肌腱及韌帶等多種細(xì)胞和組織。該細(xì)胞來源充足,取材方便,增殖能力強,可以在體外大規(guī)模擴增而不丟失多向分化潛能,并且異體移植免疫排斥反應(yīng)小。目前,已成為應(yīng)用較廣泛的重要種子細(xì)胞之一。為了獲得充足的骨髓基質(zhì)干細(xì)胞來源,我們建立了豬骨髓基質(zhì)干細(xì)胞的分離方法,通過體外培養(yǎng)和生物學(xué)鑒定,證實其為骨髓基質(zhì)干細(xì)胞,并具有成骨分化性能。通過體外轉(zhuǎn)導(dǎo)pEGFP-C1-TGF-β1入骨髓基質(zhì)干細(xì)胞,在條件培養(yǎng)基作用下誘導(dǎo)其向軟骨細(xì)胞分化。以期為骨組織工程和軟骨損傷修復(fù)的研究提供新的策略,獲得了以下結(jié)果: 1.無菌采取豬股骨頭,采用骨鉗鉗破骨髓腔,收集骨髓液于無菌離心管,沉淀制成懸液接種于培養(yǎng)皿,于37℃、5 % CO2條件下培養(yǎng)。培養(yǎng)的細(xì)胞為單層貼壁生長,呈成纖維樣形態(tài)。CD44因子免疫組化鑒定為陽性;條件培養(yǎng)基誘導(dǎo)成骨分化,ALP染色陽性,并有礦化結(jié)節(jié)生成;結(jié)合細(xì)胞形態(tài)學(xué)觀察,結(jié)果表明,分離培養(yǎng)的細(xì)胞為骨髓基質(zhì)干細(xì)胞。開始時大多為細(xì)長梭形,增殖速度快,細(xì)胞傳代周期延短,待傳至第7、8代時細(xì)胞鋪展得寬大而扁薄,增殖速度減慢,細(xì)胞傳代周期延長,細(xì)胞內(nèi)顆粒物質(zhì)增多。 2.從豬外周血淋巴細(xì)胞中抽提總RNA,并用TGF-β1特異性引物擴增獲得TGF-β1全基因,并將TGF-β1全基因亞克隆到帶有綠色熒光蛋白報告基因的真核表達(dá)質(zhì)粒pEGFP-C1。 3.并用脂質(zhì)體法轉(zhuǎn)染BMSCs,通過直接熒光觀察pEGFP-C1-TGF-β1融合蛋白在細(xì)胞中的分布定位,結(jié)果在轉(zhuǎn)染后觀察到綠色熒光,間接免疫熒光法檢測TGF-β1表達(dá)均為陽性,轉(zhuǎn)染后細(xì)胞失去BMSCs典型的成纖維形態(tài),而呈現(xiàn)出三角、多角形態(tài)。Ⅱ型膠原免疫組化染色檢測顯示,轉(zhuǎn)染TGF-β1基因后的BMSCs開始表達(dá)軟骨細(xì)胞表面特異性標(biāo)志物Ⅱ型膠原。 綜上所述,本研究在成功分離培養(yǎng)豬BMSCs的基礎(chǔ)上,將TGF-β1基因轉(zhuǎn)入BMSCs后,表達(dá)了軟骨細(xì)胞表面標(biāo)志物,說明BMSCs已向軟骨細(xì)胞分化。
[Abstract]:Bone marrow stromal cell (Bone marrow stromal cells,BMSCs) is a kind of tissue stem cells with multiple differentiation potential, which can differentiate into bone, cartilage, fat, muscle, nerve, tendons and ligaments in vivo and in vitro. The cells were abundant in source, convenient in selecting materials, strong in proliferative ability, and could be expanded in vitro without losing the potential of multidirectional differentiation, and the immune rejection of allograft was small. At present, it has become one of the most important seed cells. In order to obtain sufficient bone marrow mesenchymal stem cells (BMSCs), we established a method for isolation of porcine BMSCs, which was proved to be bone marrow mesenchymal stem cells (BMSCs) by in vitro culture and biological identification. Bone marrow mesenchymal stem cells (BMSCs) were transfected with pEGFP-C1-TGF- 尾 1 in vitro and induced to differentiate into chondrocytes. In order to provide a new strategy for bone tissue engineering and cartilage repair, the following results are obtained: 1. The porcine femoral head was sterilized. Bone forceps were used to break the medullary cavity, and bone marrow fluid was collected in aseptic centrifuge tube. The suspensions were precipitated and inoculated in culture dish at 37 鈩，

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