CAJ | 학술논문

目的探讨Notch信号和血管内皮生长因子(VEGF165)基因对大鼠骨髓间充质干细胞(MSCs)诱导分化后内皮细胞功能的影响.方法分离、培养大鼠MSCs,用含VEGF165和碱性成纤维细胞生长因子(bFGF)的细胞培养液培养大鼠MSCs 2周诱导其向内皮细胞分化；用脂质体将携带有VEGF165基因的质粒转染诱导内皮细胞并对转染效果进行鉴定,逆转录-聚合酶链反应(RT-PCR)检测转染前后细胞上Notch信号受体Notch 1和配体Jagged 1的表达变化；用γ-内分泌酶抑制剂L-685458阻断细胞Notch信号通路的转导,划痕实验检测细胞迁移能力；将细胞接种在半固体培养基上,观察其形成毛细血管样结构的能力.结果转染VEGF165基因的内皮细胞上表达有VEGF165 mRNA,说明实验成功地将VEGF165基因导入诱导后内皮细胞中.转染VEGF165基因后,细胞上Notch信号配体Jagged1 mRNA表达增强(1.08±0.01比1.01±0.02,P＜0.01),Notch1 mRNA表达无明显变化(0.60±0.02比0.59±0.01,P＞0.05)；细胞的迁移能力增强(划痕空白处细胞个数:46.45±4.46比41.61±1.42,P＜0.05),形成毛细血管样结构能力无明显变化(细胞分级:3.00±0.89比2.00±0.89,P＞0.05).内皮细胞转染VEGF165基因后,以γ-内分泌酶抑制剂L-685458阻断细胞Notch信号通路的转导,则细胞迁移能力(划痕空白处细胞个数:51.72±3.47比46.45±4.46)和形成毛细血管样结构能力(细胞分级:4.17±0.75比3.00±0.89)均进一步增强(均P＜0.05).结论转染VEGF165基因可增强大鼠MSCs诱导分化内皮细胞的功能,在此基础上阻断Notch信号通路转导可进一步增强细胞功能.
목적 탐토Notch신호화혈관내피생장인자(VEGF165)기인대대서골수간충질간세포(MSCs)유도분화후내피세포공능적영향.방법 분리、배양대서MSCs,용함VEGF165화감성성섬유세포생장인자(bFGF)적세포배양액배양대서MSCs 2주유도기향내피세포분화；용지질체장휴대유VEGF165기인적질립전염유도내피세포병대전염효과진행감정,역전록-취합매련반응(RT-PCR)검측전염전후세포상Notch신호수체Notch 1화배체Jagged 1적표체변화；용γ-내분비매억제제L-685458조단세포Notch신호통로적전도,화흔실험검측세포천이능력；장세포접충재반고체배양기상,관찰기형성모세혈관양결구적능력.결과 전염VEGF165기인적내피세포상표체유VEGF165 mRNA,설명실험성공지장VEGF165기인도입유도후내피세포중.전염VEGF165기인후,세포상Notch신호배체Jagged1 mRNA표체증강(1.08±0.01비1.01±0.02,P＜0.01),Notch1 mRNA표체무명현변화(0.60±0.02비0.59±0.01,P＞0.05)；세포적천이능력증강(화흔공백처세포개수:46.45±4.46비41.61±1.42,P＜0.05),형성모세혈관양결구능력무명현변화(세포분급:3.00±0.89비2.00±0.89,P＞0.05).내피세포전염VEGF165기인후,이γ-내분비매억제제L-685458조단세포Notch신호통로적전도,칙세포천이능력(화흔공백처세포개수:51.72±3.47비46.45±4.46)화형성모세혈관양결구능력(세포분급:4.17±0.75비3.00±0.89)균진일보증강(균P＜0.05).결론 전염VEGF165기인가증강대서MSCs유도분화내피세포적공능,재차기출상조단Notch신호통로전도가진일보증강세포공능.
Objective To explore the effects of Notch signaling pathway and the vascular endothelial growth factor(VEGF165)gene on the functions of endothelial cells derived from rat bone marrow mesenchymal stem cells (MSCs).Methods Isolated and cultivated rat bone marrow MSCs in vitro,then the cells were treated by VEGF165 and basic fibroblast growth factor(bFGF)for 2 weeks to induce them to differentiate into endothelial cells.The gene of VEGF165 was transfected into differentiated endothelial cells to promote the functions of the cells.The receptor Notch 1 and the ligand Jagged1 of the Notch signaling were detected by reverse transcription-polymerase chain reaction (RT-PCR)before and after the transfection.γ-seeretase inhibitor L-685458 was used to block Notch pathway.Migration ability of cells was detected by scarification test.Cells were inoculated on semisolid gel to study their ability of forming capillary-like structure.Results After transfection,VEGF165 mRNA could be detected on the differentiated endothelial cells.The expression of Jagged1 mRNA was up regulated(1.08 ± 0.01 vs.1.01 ± 0.02,P＜0.01)and there was no change in Notch 1 mRNA(0.60 ± 0.02 vs.0.59 ± 0.01,P＞0.05).The ability of migration was enhanced(number of cells on the scratched area:46.45 ±4.46 vs.41.61 ± 1.42,P＜0.05),and the ability of forming capillary-like structure on semisolid gel showed no change(cells classification:3.00 ± 0.89 vs.2.00 ± 0.89,P＞0.05).After the tranfection,using the γ-secretase inhibitor L-685458 to block the Notch signaling transduction,the ablility of migration of the differentiated endothelial cells(number of cells on the scratched area:51.72 ± 3.47 vs.46.45 ± 4.46,P＜0.05),and that of forming capillary-like structure(cells classification:4.17 ± 0.75 vs.3.00 ± 0.89,P＜0.05),was also further enhanced.Conclusion Transfection of the gene of VEGF165 into the differentiated endothelial cells can reinforce the function of these cells,and when Notch signaling was blocked,this effect can be further amplified.