中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2015年
11期
675-680
,共6页
刘蓉%刘江峰%郭吕华%田爱峰
劉蓉%劉江峰%郭呂華%田愛峰
류용%류강봉%곽려화%전애봉
骨髓间充质干细胞%内皮细胞%血管内皮生长因子%碱性成纤维细胞因子%共培养
骨髓間充質榦細胞%內皮細胞%血管內皮生長因子%堿性成纖維細胞因子%共培養
골수간충질간세포%내피세포%혈관내피생장인자%감성성섬유세포인자%공배양
Bone marrow mesenchymal stem cells%Endothelial cells%Vascular endothelial growth factor%Basic fibroblast growth factor%Co-culture system
目的 观察血管内皮细胞与骨髓间充质干细胞共培养时,不同比例共培养系统的成骨性能,为体外构建血管化骨组织工程奠定基础.方法 全骨髓离心贴壁法分离大鼠骨髓间充质干细胞.骨髓间充质干细胞的内皮向诱导(M199+体积分数为0.1的胎牛血清+体积分数为0.01的青霉素/链霉素+2 μg/L的碱性成纤维细胞生长因子bFGF+10 μg/L的血管内皮生长因子VEGF).建立不同比例骨髓间充质干细胞和血管内皮细胞的共培养系统(10∶0、10∶1、8∶2、7∶3、5∶5、3∶7、2∶8、1∶10、0∶10).通过干细胞和内皮细胞的形态、免疫荧光、碱性磷酸酶活性、成骨基因表达等从酶学、组织学、基因等不同方面观察各个比例血管内皮细胞对骨髓间充质干细胞成骨活性的影响.结果 第3代骨髓间充质干细胞的生长曲线显示:骨髓间充质干细胞的细胞倍增时间为39.9 h.免疫荧光染色结果显示,骨髓间充质干细胞表面特异性标志、内皮细胞表面特异性标志免疫荧光染色阳性.倒置相差显微镜观察结果显示,联合培养组不同比例的骨髓间充质干细胞和血管内皮细胞混合生长良好.共培养组茜素红染色结果显示,共培养比例为7∶3时钙结节计数为19.0±3.0,共培养比例为5∶5时钙结节计数为20.8±2.9,两者之间差异无统计学意义(P＞0.05);但均显著高于其他共培养组(P＜0.01).成骨诱导7d,共培养比例为10∶0、10∶1、8∶2、7∶3和5∶5共培养组酶活性分别为:16.84±0.82、15.86±3.10、16.37±1.33、17.99±1.98和17.49±0.87,差异无统计学意义(P＞0.05),但均显著高于其他共培养组(P＜0.05);随着诱导时间延长,碱性磷酸酶活性整体升高,7:3共培养组(33.74±0.99)略高于5∶5共培养组(31.09±0.87),二者均显著高于其他共培养组(P＜0.01).定量PCR检测结果显示,7∶3共培养组成骨相关基因OCN和RUNX2的表达均显著高于其他共培养组(P＜0.05).结论 由骨髓间充质干细胞诱导的内皮细胞能增强骨髓间充质干细胞的成骨活性;当骨髓间充质干细胞与血管内皮细胞以7∶3比例混合时,骨髓间充质干细胞碱性磷酸酶活性最强,成骨相关基因表达量最高,成骨能力最强.
目的 觀察血管內皮細胞與骨髓間充質榦細胞共培養時,不同比例共培養繫統的成骨性能,為體外構建血管化骨組織工程奠定基礎.方法 全骨髓離心貼壁法分離大鼠骨髓間充質榦細胞.骨髓間充質榦細胞的內皮嚮誘導(M199+體積分數為0.1的胎牛血清+體積分數為0.01的青黴素/鏈黴素+2 μg/L的堿性成纖維細胞生長因子bFGF+10 μg/L的血管內皮生長因子VEGF).建立不同比例骨髓間充質榦細胞和血管內皮細胞的共培養繫統(10∶0、10∶1、8∶2、7∶3、5∶5、3∶7、2∶8、1∶10、0∶10).通過榦細胞和內皮細胞的形態、免疫熒光、堿性燐痠酶活性、成骨基因錶達等從酶學、組織學、基因等不同方麵觀察各箇比例血管內皮細胞對骨髓間充質榦細胞成骨活性的影響.結果 第3代骨髓間充質榦細胞的生長麯線顯示:骨髓間充質榦細胞的細胞倍增時間為39.9 h.免疫熒光染色結果顯示,骨髓間充質榦細胞錶麵特異性標誌、內皮細胞錶麵特異性標誌免疫熒光染色暘性.倒置相差顯微鏡觀察結果顯示,聯閤培養組不同比例的骨髓間充質榦細胞和血管內皮細胞混閤生長良好.共培養組茜素紅染色結果顯示,共培養比例為7∶3時鈣結節計數為19.0±3.0,共培養比例為5∶5時鈣結節計數為20.8±2.9,兩者之間差異無統計學意義(P＞0.05);但均顯著高于其他共培養組(P＜0.01).成骨誘導7d,共培養比例為10∶0、10∶1、8∶2、7∶3和5∶5共培養組酶活性分彆為:16.84±0.82、15.86±3.10、16.37±1.33、17.99±1.98和17.49±0.87,差異無統計學意義(P＞0.05),但均顯著高于其他共培養組(P＜0.05);隨著誘導時間延長,堿性燐痠酶活性整體升高,7:3共培養組(33.74±0.99)略高于5∶5共培養組(31.09±0.87),二者均顯著高于其他共培養組(P＜0.01).定量PCR檢測結果顯示,7∶3共培養組成骨相關基因OCN和RUNX2的錶達均顯著高于其他共培養組(P＜0.05).結論 由骨髓間充質榦細胞誘導的內皮細胞能增彊骨髓間充質榦細胞的成骨活性;噹骨髓間充質榦細胞與血管內皮細胞以7∶3比例混閤時,骨髓間充質榦細胞堿性燐痠酶活性最彊,成骨相關基因錶達量最高,成骨能力最彊.
목적 관찰혈관내피세포여골수간충질간세포공배양시,불동비례공배양계통적성골성능,위체외구건혈관화골조직공정전정기출.방법 전골수리심첩벽법분리대서골수간충질간세포.골수간충질간세포적내피향유도(M199+체적분수위0.1적태우혈청+체적분수위0.01적청매소/련매소+2 μg/L적감성성섬유세포생장인자bFGF+10 μg/L적혈관내피생장인자VEGF).건립불동비례골수간충질간세포화혈관내피세포적공배양계통(10∶0、10∶1、8∶2、7∶3、5∶5、3∶7、2∶8、1∶10、0∶10).통과간세포화내피세포적형태、면역형광、감성린산매활성、성골기인표체등종매학、조직학、기인등불동방면관찰각개비례혈관내피세포대골수간충질간세포성골활성적영향.결과 제3대골수간충질간세포적생장곡선현시:골수간충질간세포적세포배증시간위39.9 h.면역형광염색결과현시,골수간충질간세포표면특이성표지、내피세포표면특이성표지면역형광염색양성.도치상차현미경관찰결과현시,연합배양조불동비례적골수간충질간세포화혈관내피세포혼합생장량호.공배양조천소홍염색결과현시,공배양비례위7∶3시개결절계수위19.0±3.0,공배양비례위5∶5시개결절계수위20.8±2.9,량자지간차이무통계학의의(P＞0.05);단균현저고우기타공배양조(P＜0.01).성골유도7d,공배양비례위10∶0、10∶1、8∶2、7∶3화5∶5공배양조매활성분별위:16.84±0.82、15.86±3.10、16.37±1.33、17.99±1.98화17.49±0.87,차이무통계학의의(P＞0.05),단균현저고우기타공배양조(P＜0.05);수착유도시간연장,감성린산매활성정체승고,7:3공배양조(33.74±0.99)략고우5∶5공배양조(31.09±0.87),이자균현저고우기타공배양조(P＜0.01).정량PCR검측결과현시,7∶3공배양조성골상관기인OCN화RUNX2적표체균현저고우기타공배양조(P＜0.05).결론 유골수간충질간세포유도적내피세포능증강골수간충질간세포적성골활성;당골수간충질간세포여혈관내피세포이7∶3비례혼합시,골수간충질간세포감성린산매활성최강,성골상관기인표체량최고,성골능력최강.
Objective To evaluate the effect of co-culture system of bone marrow mesenchymal stem cells(BMSC) and vascular endothelial cells(EC) on osteogenesis.Methods BMSC were isolated by whole bone marrow centrifugal adherent method.Then BMSC were induced into EC with induced medium.Co-culture system in different proportions of BMSC and EC(10∶0, 10∶1, 8∶2, 7∶3, 5∶5, 3∶7, 2∶8, 1∶10, 0∶10) were further evaluated.The cell growth level of BMSC was examined.The CD44 expression of BMSC and von willebrand factor(vWF) expression of vascular EC were examined by immunofluorescence.Furthermore,calcium nodules exhibited by alizarin red staining, alkaline phosphatase activity, and the expression of osteogenic genes by reverse transcription-quantitative PCR(RT-qPCR) were observed to validate the osteogenesis of co-culture system.Results The growth curve of P3 passage of BMSC demonstrated the doubling time of BMSC was 39.9 h.The positive specific markers of BMSC and EC showed efficient induction.Although the calcium nodules ratio of the co-culture[group 7∶3(19.0±3.0) and group 5∶5(20.8±2.9)] was not significantly different(P＞0.05), but higher than that of other co-culture groups with a significant difference(P＜0.01).Alkaline phosphatase activity was increased with prolonged induction of osteogenic medium.While alkaline phosphatase activity of group 10: 0(16.84±0.82), group 10∶1(15.86±3.10), group 8∶2(16.37± 1.33), group 7∶3(17.99± 1.98), and group 5∶5(17.49±0.87) did not show significant difference after osteogenic induction for 7 days(P＞0.05), but significantly higher than that of other co-culture groups(P＜0.05).The co-culture ratio of 7∶3(33.74±0.99) was slightly higher than that of 5∶5 (31.09±0.87), but significantly higher than that of other groups(P＜0.01).Moreover, the osteocalcin(OCN) and runt-related transcription factor 2(RUNX2) expression of group 7∶3 was significantly higher than that of other groups.Conclusions The EC that derived from BMSC can promote the BMSC differentiate into osteoblasts.The co-culture system of BMSC and EC with the ratio of 7∶3 increases the alkaline phosphatase activity and facilitates the expression of osteogenic genes.
