【摘要】：目的:構(gòu)建人Sox9基因慢病毒載體,體外Sox9慢病毒載體轉(zhuǎn)染人臍帶間充質(zhì)干細胞(hUC-MSCs),觀察Sox9基因轉(zhuǎn)染對hUC-MSCs生物學(xué)特性的影響,以及經(jīng)Sox9基因修飾的hUC-MSCs在單層培養(yǎng)條件下向軟骨細胞分化的能力,為軟骨組織損傷及退變性疾病的細胞學(xué)及基因治療提供實驗依據(jù)。 方法:通過PCR的方法獲得人Sox9基因編碼區(qū)片段,將該片段克隆入慢病毒載體Pwpxl-MOD中產(chǎn)生Pwpxl-MOD/Sox9 ,通過酶切測序鑒定慢病毒載體,將Pwpxl-MOD/Sox9、pRsv-REV、pMDlg-pRRE、pMD2G共轉(zhuǎn)染293T細胞,包裝成含有Sox9基因的重組慢病毒。采用酶消化法從人臍帶基質(zhì)中獲取間充質(zhì)干細胞,流式細胞儀進行細胞表型鑒定,誘導(dǎo)向成骨、脂肪分化以觀察其多向分化潛能。包裝好的慢病毒體外轉(zhuǎn)染hUC-MSCs,利用熒光倒置相差顯微鏡觀察轉(zhuǎn)基因細胞的形態(tài)變化、綠色熒光表達(GFP)情況,轉(zhuǎn)染后48h確定基因轉(zhuǎn)染效率。逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)法及免疫印跡(Western-blot)檢測Sox9在hUC-MSCs中的表達;四甲基偶氮唑鹽比色法(MTT)法測定慢病毒對細胞增殖能力的影響。在高密度單層培養(yǎng)條件下,不增加生長因子、地塞米松等軟骨誘導(dǎo)液,將Sox9基因修飾的hUC-MSCs培養(yǎng)分化21d, RT-PCR、Western-blot、免疫組化及免疫熒光染色、阿利新蘭染色檢測特異性軟骨細胞分子標志Ⅱ型膠原、Agrrecan的表達以及蛋白多糖的分泌情況。 結(jié)果:酶切、測序鑒定證實慢病毒載體Pwpxl-MOD中插人片段為基因Sox9。經(jīng)檢測包裝好的慢病毒滴度為6.0×10~7TU/ml;從人臍帶基質(zhì)分離出的hUC-MSCs形態(tài)類似于成纖維樣細胞,CD29(95.9%)、CD44(96.5%)表達陽性,CD34(3.0%)、CD45(2.6%)造血干細胞表型標志表達為陰性。向脂肪及成骨方向誘導(dǎo)培養(yǎng)21d,油紅O染色、堿性磷酸酶染色、Von kossa染色均為強陽性,表明hUC-MSCs具有較強的多向分化潛能�；蜣D(zhuǎn)染48h后觀察到較強的綠色熒光表達,Lenti-GFP-Sox9及Lenti-GFP的轉(zhuǎn)染率均達到90%以上。RT-PCR、Western-blot檢測到目的基因Sox9在hUC-MSCs中出現(xiàn)過表達(P0.001),細胞生長曲線顯示轉(zhuǎn)染對hUC-MSCs的增殖活力無明顯影響(P0.05)。Sox9基因修飾的hUC-MSCs在單層培養(yǎng)下7d開始出現(xiàn)自發(fā)性的細胞聚集現(xiàn)象,到14d細胞集聚明顯增大,21d左右,有較大的軟骨結(jié)節(jié)形成。而Lenti-GFP及未轉(zhuǎn)染的細胞無明顯的細胞集聚現(xiàn)象產(chǎn)生。RT-PCR、Western-blot、免疫組化及免疫熒光染色檢測到Ⅱ型膠原、Agrrecan有較高水平的表達,阿利新蘭染色示大量的蛋白多糖的在軟骨結(jié)節(jié)中結(jié)聚。 結(jié)論:1、成功構(gòu)建了含有Sox9基因慢病毒載體。2、人臍帶間充質(zhì)干細胞體外培養(yǎng)擴增容易,具有多向分化潛能,易于被外源性基因修飾。2、在單層培養(yǎng)條件下,無生長因子及其他軟骨誘導(dǎo)因子的刺激的情況下,經(jīng)Sox9基因修飾的HU-MSCs具有較強的向軟骨細胞分化的能力,表明Sox9基因在促進間充質(zhì)干細胞向軟骨細胞分化過程中發(fā)揮了重要的作用。
[Abstract]:Aim: to construct lentivirus vector of human Sox9 gene and transfect Sox9 lentivirus vector into human umbilical cord mesenchymal stem cells (hUC-MSCs) in vitro to observe the effect of Sox9 gene transfection on the biological characteristics of hUC-MSCs. The ability of hUC-MSCs modified by Sox9 gene to differentiate into chondrocytes in monolayer culture provides experimental basis for cytology and gene therapy of cartilage tissue damage and degeneration. Methods: the encoding region of human Sox9 gene was obtained by PCR. The fragment was cloned into lentivirus vector Pwpxl-MOD to produce Pwpxl-MOD/Sox9. The lentivirus vector was identified by restriction endonuclease sequencing, and Pwpxl-MOD/Sox9,pRsv-REV,pMDlg-pRRE, was identified. PMD2G co-transfected 293T cells and packaged into recombinant lentivirus containing Sox9 gene. Mesenchymal stem cells were obtained from human umbilical cord stroma by enzyme digestion. The phenotype of mesenchymal stem cells was identified by flow cytometry. After transfection of lentivirus into hUC-MSCs, in vitro, the morphological changes of transgenic cells were observed by fluorescence phase contrast microscope, the expression of (GFP) was detected by green fluorescence, and the efficiency of gene transfection was determined 48 hours after transfection. The expression of Sox9 in hUC-MSCs was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (Western-blot), and the effect of lentivirus on cell proliferation was determined by (MTT) assay. Under the condition of high density monolayer culture, the Sox9 gene modified hUC-MSCs was cultured for 21 days, RT-PCR,Western-blot, immunohistochemistry and immunofluorescence staining, without increasing the growth factor, dexamethasone and other cartilage inducers. The expression of type 鈪，

本文編號：2362072