碱性成纤维生长因子基因修饰大鼠骨髓间充质干细胞的体外分化实验研究
发布时间：2018-08-03 18:00
【摘要】：目的: 通过碱性成纤维生长因子(bFGF)慢病毒载体感染大鼠骨髓间充干细胞(rBM-MSCs),观察bFGF基因修饰的rBM-MSCs在体外的分化现象,研究bFGF基因修饰的rBM-MSCs是否具有向血管内皮细胞样细胞分化的倾向性及优越性。 方法: 体外分离、培养并鉴定rBM-MSCs,将bFGF慢病毒载体感染rBM-MSCs,通过ELSIA、RT-PCR等方法检测bFGF的表达及分泌;MTT法检测细胞活力。将细胞分成正常组(普通的rBM-MSCs组)、空对组(不含bFGF基因含有GFP的慢病毒载体感染组)、目的组(bFGF基因慢病毒载体感染组),用含有血管内皮生长因子(VEGF)的培养液培养28d,观察细胞形态的改变,采用免疫细胞化学技术(IHC)检测细胞表面标记分子CD31、CD34及Ⅷ因子的表达及Dil-AC- LDL吞噬实验等。 结果: 1.第3代rBM-MSCs表面分子阳性率CD11b/c(:14.2±0.7)%,CD34(:1.3±0.5)%,CD44:(97.8±0.9)%,CD90:(96.9±1.5)%。 2.携带有GFP的慢病毒载体感染rBM-MSCs后,绿荧光表达率高,细胞传代后没有减弱,且大部分细胞皆有表达。 3.目的组能够大量分泌bFGF,而正常组及空对组分泌较少,其中目的组:(5.500±0.124)μg/ml,空对组:(2.650±0.402)μg/ml,正常组:(2.763±0.253)μg/ml,三组bFGF表达有显著差异(P =0.0000.01)。 4. MTT实验,正常组:0.392±0.189;空对组:0.394±0.243;目的组:0.398±0.186,三组OD值无明显差异(P=0.8670.05)。 5.原代rBM-MSCs呈条索状或多角形,分布不均匀;三组细胞诱导后皆表现为多角形或鹅卵石样,细胞连接紧密,分布均匀,但之间差异不明显。 6.诱导后目的组能有效地吞噬Dil- AC-LDL,表达荧光,而正常组及空对组只有微量表达;平均光密度值,目的组: 0.518±0.227,正常组:0.047±0.027,空对组:0.071±0.019,目的组明显高于其他两组(P=0.0000.01)。 7.诱导后目的组能部分表达CD31、CD34及Ⅷ因子,而空对组及正常组只能微弱表达。CD31,目的组:(22.73±5.42)%,正常组:(2.57±1.21)% ,空对组:(7.85±1.83)%;CD34,目的组:(26.32±3.95)%,正常组:(2.41±0.97)% ,空对组:(8.93±1.37)%; FⅧ,目的组:(30.34±6.62)%,正常组:(1.97±0.52)%,空对组:(8.93±17.37)%,目的组的表达明显高于其他两组,有显著差异(P0.01)。 结论: 采用密度梯度离心法能获取较纯的rBM-MSCs,rBM-MSCs获得bFGF的稳定遗传后,细胞相对活力没有影响,并能分泌bFGF,在VEGF的诱导下bFGF基因修饰的rBM-MSCs能够更有效地向血管内皮细胞样细胞分化。
[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，
本文编号：2162527
[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，
本文编号：2162527
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