發(fā)布時(shí)間：2018-08-03 18:00

【摘要】：目的: 通過堿性成纖維生長因子(bFGF)慢病毒載體感染大鼠骨髓間充干細(xì)胞(rBM-MSCs),觀察bFGF基因修飾的rBM-MSCs在體外的分化現(xiàn)象,研究bFGF基因修飾的rBM-MSCs是否具有向血管內(nèi)皮細(xì)胞樣細(xì)胞分化的傾向性及優(yōu)越性。 方法: 體外分離、培養(yǎng)并鑒定rBM-MSCs,將bFGF慢病毒載體感染rBM-MSCs,通過ELSIA、RT-PCR等方法檢測bFGF的表達(dá)及分泌;MTT法檢測細(xì)胞活力。將細(xì)胞分成正常組(普通的rBM-MSCs組)、空對(duì)組(不含bFGF基因含有GFP的慢病毒載體感染組)、目的組(bFGF基因慢病毒載體感染組),用含有血管內(nèi)皮生長因子(VEGF)的培養(yǎng)液培養(yǎng)28d,觀察細(xì)胞形態(tài)的改變,采用免疫細(xì)胞化學(xué)技術(shù)(IHC)檢測細(xì)胞表面標(biāo)記分子CD31、CD34及Ⅷ因子的表達(dá)及Dil-AC- LDL吞噬實(shí)驗(yàn)等。 結(jié)果: 1.第3代rBM-MSCs表面分子陽性率CD11b/c(:14.2±0.7)%,CD34(:1.3±0.5)%,CD44:(97.8±0.9)%,CD90:(96.9±1.5)%。 2.攜帶有GFP的慢病毒載體感染rBM-MSCs后,綠熒光表達(dá)率高,細(xì)胞傳代后沒有減弱,且大部分細(xì)胞皆有表達(dá)。 3.目的組能夠大量分泌bFGF,而正常組及空對(duì)組分泌較少,其中目的組:(5.500±0.124)μg/ml,空對(duì)組:(2.650±0.402)μg/ml,正常組:(2.763±0.253)μg/ml,三組bFGF表達(dá)有顯著差異(P =0.0000.01)。 4. MTT實(shí)驗(yàn),正常組:0.392±0.189;空對(duì)組:0.394±0.243;目的組:0.398±0.186,三組OD值無明顯差異(P=0.8670.05)。 5.原代rBM-MSCs呈條索狀或多角形,分布不均勻;三組細(xì)胞誘導(dǎo)后皆表現(xiàn)為多角形或鵝卵石樣,細(xì)胞連接緊密,分布均勻,但之間差異不明顯。 6.誘導(dǎo)后目的組能有效地吞噬Dil- AC-LDL,表達(dá)熒光,而正常組及空對(duì)組只有微量表達(dá);平均光密度值,目的組: 0.518±0.227,正常組:0.047±0.027,空對(duì)組:0.071±0.019,目的組明顯高于其他兩組(P=0.0000.01)。 7.誘導(dǎo)后目的組能部分表達(dá)CD31、CD34及Ⅷ因子,而空對(duì)組及正常組只能微弱表達(dá)。CD31,目的組:(22.73±5.42)%,正常組:(2.57±1.21)% ,空對(duì)組:(7.85±1.83)%;CD34,目的組:(26.32±3.95)%,正常組:(2.41±0.97)% ,空對(duì)組:(8.93±1.37)%; FⅧ,目的組:(30.34±6.62)%,正常組:(1.97±0.52)%,空對(duì)組:(8.93±17.37)%,目的組的表達(dá)明顯高于其他兩組,有顯著差異(P0.01)。 結(jié)論: 采用密度梯度離心法能獲取較純的rBM-MSCs,rBM-MSCs獲得bFGF的穩(wěn)定遺傳后,細(xì)胞相對(duì)活力沒有影響,并能分泌bFGF,在VEGF的誘導(dǎo)下bFGF基因修飾的rBM-MSCs能夠更有效地向血管內(nèi)皮細(xì)胞樣細(xì)胞分化。
[Abstract]:Aim: to investigate the differentiation of rat bone marrow mesenchymal stem cells (rBM-MSCs) infected with basic fibroblast growth factor (bFGF) lentivirus vector in vitro. To study whether rBM-MSCs modified by bFGF gene has the tendency and superiority to differentiate into vascular endothelial cell-like cells (VECs). Methods: rBM-MSCs were isolated, cultured and identified in vitro. RBM-MSCs were infected with bFGF lentivirus vector. The expression of bFGF and the activity of rBM-MSCs were detected by Elisa RT-PCR. The cells were divided into normal group (normal rBM-MSCs group), empty pair group (lentivirus vector group without bFGF gene containing GFP), objective group (bFGF gene lentivirus vector infection group) and cultured in culture medium containing vascular endothelial growth factor (VEGF). At 28 days, the changes of cell morphology were observed. The expression of CD31, CD34 and 鈪，

本文編號(hào)：2162527