口腔颌面外科杂志
口腔頜麵外科雜誌
구강합면외과잡지
CHINESE JOURNAL OF ORAL AND MAXILLOFACIAL SURGERY
2015年
2期
102-107
,共6页
釉基质蛋白%脂肪源性干细胞%细胞增殖%成骨分化
釉基質蛋白%脂肪源性榦細胞%細胞增殖%成骨分化
유기질단백%지방원성간세포%세포증식%성골분화
enamel matrix derivative%adipose derived stem cells%proliferation%osteogenic differentiation
目的：通过配制不同浓度的釉基质蛋白（enamel matrix proteins，EMPs）培养液，体外培养SD大鼠脂肪源性干细胞（adipose-derived stem cells, ADSCs），初步探究EMPs对ADSCs体外增殖活性及成骨分化能力的影响。方法：酶消化法获得大鼠脂肪间充质细胞，通过流式细胞仪技术分离、筛选干细胞，多向诱导分化对所得细胞进行鉴定；乙酸法制备EMPs干粉并通过蛋白免疫印记技术鉴定其成分，配制不同浓度的诱导液以备用；采用MTT法检测EMPs对ADSCs增殖活性的影响，并用实时荧光定量PCR及蛋白免疫印迹的方法检测EMPs诱导ADSCs成骨分化标志物的mRNA及蛋白表达变化情况。结果：釉基质蛋白（EMPs）对大鼠脂肪源性干细胞(ADSCs)的增殖具有明显的促进作用(P<0.05)，并呈现剂量和时间依赖性； EMPs培养ADSCs 14 d后，成骨分化标志物Runx-2、Osteocalcin(OCN)、Collagen (Col-I)在mRNA和蛋白水平表达均增加，并且与EMPs成正比（P<0.05）。结论：釉基质蛋白可作为一种成骨诱导剂，能有效增强大鼠脂肪源性干细胞体外增殖活性并诱导其成骨分化。
目的：通過配製不同濃度的釉基質蛋白（enamel matrix proteins，EMPs）培養液，體外培養SD大鼠脂肪源性榦細胞（adipose-derived stem cells, ADSCs），初步探究EMPs對ADSCs體外增殖活性及成骨分化能力的影響。方法：酶消化法穫得大鼠脂肪間充質細胞，通過流式細胞儀技術分離、篩選榦細胞，多嚮誘導分化對所得細胞進行鑒定；乙痠法製備EMPs榦粉併通過蛋白免疫印記技術鑒定其成分，配製不同濃度的誘導液以備用；採用MTT法檢測EMPs對ADSCs增殖活性的影響，併用實時熒光定量PCR及蛋白免疫印跡的方法檢測EMPs誘導ADSCs成骨分化標誌物的mRNA及蛋白錶達變化情況。結果：釉基質蛋白（EMPs）對大鼠脂肪源性榦細胞(ADSCs)的增殖具有明顯的促進作用(P<0.05)，併呈現劑量和時間依賴性； EMPs培養ADSCs 14 d後，成骨分化標誌物Runx-2、Osteocalcin(OCN)、Collagen (Col-I)在mRNA和蛋白水平錶達均增加，併且與EMPs成正比（P<0.05）。結論：釉基質蛋白可作為一種成骨誘導劑，能有效增彊大鼠脂肪源性榦細胞體外增殖活性併誘導其成骨分化。
목적：통과배제불동농도적유기질단백（enamel matrix proteins，EMPs）배양액，체외배양SD대서지방원성간세포（adipose-derived stem cells, ADSCs），초보탐구EMPs대ADSCs체외증식활성급성골분화능력적영향。방법：매소화법획득대서지방간충질세포，통과류식세포의기술분리、사선간세포，다향유도분화대소득세포진행감정；을산법제비EMPs간분병통과단백면역인기기술감정기성분，배제불동농도적유도액이비용；채용MTT법검측EMPs대ADSCs증식활성적영향，병용실시형광정량PCR급단백면역인적적방법검측EMPs유도ADSCs성골분화표지물적mRNA급단백표체변화정황。결과：유기질단백（EMPs）대대서지방원성간세포(ADSCs)적증식구유명현적촉진작용(P<0.05)，병정현제량화시간의뢰성； EMPs배양ADSCs 14 d후，성골분화표지물Runx-2、Osteocalcin(OCN)、Collagen (Col-I)재mRNA화단백수평표체균증가，병차여EMPs성정비（P<0.05）。결론：유기질단백가작위일충성골유도제，능유효증강대서지방원성간세포체외증식활성병유도기성골분화。
Objective: This study aimed to explore the impact of enamel matrix proteins (EMPs) on proliferation activity and osteogenic differentiation capacity of SD rat adipose-derived stem cells in vitro. Methods:Adipose-derived stem cells (ADSCs) were isolated from subcutaneous fat pads of healthy male SD rats aged 4 weeks by enzyme digestion method . Stem cells are separated and identified by flow cytometry technology. EMPs were extracted from tooth germs of heathy male pigs aged 4-6 months. SDS-PAGE electrophoresis technique was used to identify its characteristics, and frozen-dried powder were prepared for subsequent experiments. Effects of EMPs on cell proliferation were assessed by MTT assay. Ef-fects of EMPs on osteogenic differentiation of rat ADSCs were analyzed by Q-PCR and Western-Blot technique.Osteogenic differentiation markers of Osteocalcin (OCN), Runx-2, Collagen I (Col-I) were detected. Results: MTT assay showed that EMPs could enhance rat ADSC proliferation in a dose-dependent and time-dependent manner(P<0.05). Results of Q-PCR and Western-blot technique indicated that EMPs promoted expressions of Runx-2, Col-I and OCN at the mRNA level and protein level without osteo-induction medium (P<0.05). Conclusion: Enamel matrix proteins may be used as an osteoin-ductive agent, due its enhancing effect on adipose-derived stem cells proliferation and osteogenic differentiation.
