中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
CHINESE JOURNAL OF ANESTHESIOLOGY
2012年
9期
1126-1129
,共4页
林群%雷立华%林财珠%林献忠%林健清%郑辉哲%蔡宏达%杨庆%高友光
林群%雷立華%林財珠%林獻忠%林健清%鄭輝哲%蔡宏達%楊慶%高友光
림군%뢰립화%림재주%림헌충%림건청%정휘철%채굉체%양경%고우광
肝细胞生长因子%生物,基因修饰%干细胞%骨髓
肝細胞生長因子%生物,基因脩飾%榦細胞%骨髓
간세포생장인자%생물,기인수식%간세포%골수
Hepatocyte growth factor%Organisms,genetically modified%Stem cells%Bone marrow
目的 构建人肝细胞生长因子(hHGF)基因修饰大鼠骨髓间充质干细胞系(MSCs).方法 采用脂质体法将hHGF基因慢病毒载体质粒(lenti-hHGF)转染至293FT细胞,收集病毒上清液感染大鼠MSCs细胞,经过G418筛选得到稳定分泌hHGF的MSCs细胞株(hHGF-MSCs细胞).以转染空载体细胞(eGFP-MSCs)、未转染慢病毒载体的MSCs细胞作为对照.采用免疫印迹法检测hHGF的表达.hHGF-MSCs细胞分别加入成骨诱导剂或成脂诱导剂培养,分别采用茜素红染色和油红O染色鉴定成骨细胞和脂肪细胞表型.结果 与未转染慢病毒载体的MSCs细胞和eGFP-MSCs细胞比较,hHGF-MSCs细胞hHGF表达上调(P＜0.01).hHGF-MSCs细胞体外诱导培养后具有明显的成骨和成脂表型,可向成骨细胞和脂肪细胞分化.结论 成功构建hHGF基因修饰大鼠MSCs.
目的 構建人肝細胞生長因子(hHGF)基因脩飾大鼠骨髓間充質榦細胞繫(MSCs).方法 採用脂質體法將hHGF基因慢病毒載體質粒(lenti-hHGF)轉染至293FT細胞,收集病毒上清液感染大鼠MSCs細胞,經過G418篩選得到穩定分泌hHGF的MSCs細胞株(hHGF-MSCs細胞).以轉染空載體細胞(eGFP-MSCs)、未轉染慢病毒載體的MSCs細胞作為對照.採用免疫印跡法檢測hHGF的錶達.hHGF-MSCs細胞分彆加入成骨誘導劑或成脂誘導劑培養,分彆採用茜素紅染色和油紅O染色鑒定成骨細胞和脂肪細胞錶型.結果 與未轉染慢病毒載體的MSCs細胞和eGFP-MSCs細胞比較,hHGF-MSCs細胞hHGF錶達上調(P＜0.01).hHGF-MSCs細胞體外誘導培養後具有明顯的成骨和成脂錶型,可嚮成骨細胞和脂肪細胞分化.結論 成功構建hHGF基因脩飾大鼠MSCs.
목적 구건인간세포생장인자(hHGF)기인수식대서골수간충질간세포계(MSCs).방법 채용지질체법장hHGF기인만병독재체질립(lenti-hHGF)전염지293FT세포,수집병독상청액감염대서MSCs세포,경과G418사선득도은정분비hHGF적MSCs세포주(hHGF-MSCs세포).이전염공재체세포(eGFP-MSCs)、미전염만병독재체적MSCs세포작위대조.채용면역인적법검측hHGF적표체.hHGF-MSCs세포분별가입성골유도제혹성지유도제배양,분별채용천소홍염색화유홍O염색감정성골세포화지방세포표형.결과 여미전염만병독재체적MSCs세포화eGFP-MSCs세포비교,hHGF-MSCs세포hHGF표체상조(P＜0.01).hHGF-MSCs세포체외유도배양후구유명현적성골화성지표형,가향성골세포화지방세포분화.결론 성공구건hHGF기인수식대서MSCs.
Objective To construct F344 rat bone marrow mesenchymal stem cell line (MSC) modified with human hepatocyte growth factor (hHGF) gene.Methods Recombinant virus containing hHGF was obtained by transfecting the packaging cell line 293 FT with lentiviral vector pLV/EF1α-hHGF-IRES-eGFP.MSCs derived from F344 rat bone marrow were then tranfected with packed lentiviral vector.Purified MSCs expressing hHGF was obtained by screening culture with G418.MSCs and MSCs transfected with empty vector were used as control.The expression of hHGF protein was detected by Western blot (eGFP-MSCs).The hHGF-transfected MSCs were cultured in osteoblast-inducing culture medium and osteoblast phenotype was assayed by alizarin Red staining.The cells were also cultured in adipogenesis medium and stained with Oil Red O for identification.Results The expression of hHGF protein was significantly up-regulated in the hHGF-MSCs as compared with MSCs and eGFP-MSCs.hHGF-MSCs readily differentiated into mineralizing cells or adipocytes when incubated in differentiation medium.Conclusion A F344 rat MSC line that stably expresses HGF is successfully established.
