中华创伤骨科杂志
中華創傷骨科雜誌
중화창상골과잡지
CHINESE JOURNAL OF ORTHOPAEDIC TRAUMA
2014年
5期
427-432
,共6页
李富航%靳宇飞%毕龙%樊俊俊%户刚%裴国献
李富航%靳宇飛%畢龍%樊俊俊%戶剛%裴國獻
리부항%근우비%필룡%번준준%호강%배국헌
骨髓细胞%P物质%成骨细胞%内皮细胞%共同培养技术
骨髓細胞%P物質%成骨細胞%內皮細胞%共同培養技術
골수세포%P물질%성골세포%내피세포%공동배양기술
Bone marrow cells%Substance P%Osteoblasts%Endothelial cells%Coculture techniques
目的 探讨P物质对骨髓基质干细胞(BMSCs)来源的成骨细胞(OB)与内皮细胞(EC)体外联合培养的影响. 方法 分别建立BMSCs来源的OB与EC单独培养体系,以及按2∶1的比例体外直接或间接共培养体系.实验组:各培养体系细胞内同时加入1×10-8 mol/L P物质(n=6),对照组:各培养体系不添加P物质(n=6).培养48 h后采用流式细胞仪分析细胞的增殖与活性,并采用酶联免疫吸附法测定碱性磷酸酶(ALP)的表达,实时定量聚合酶链式反应检测骨钙素(OC)mRNA基因的表达.比较两组不同培养方式细胞功能的变化. 结果 与对照组比较,实验组OB单独培养、间接共培养时细胞G1期减少,G2/M期明显增高,差异均有统计学意义(P＜0.05).实验组OB单独培养、间接共培养、直接共培养的细胞ALP增殖活性[(2.877 ±0.188)、(3.053±0.179)、(4.345±0.305)]及OCmRNA的表达量[(1.682±0.155)、(3.188±0.213)、(3.720 ±0.194)]均明显高于对照组[(2.600±0.233)、(2.842±0.120)、(3.220±0.191);(1.360±0.141)、(2.272±0.143)、(2.507 ±0.176)],差异均有统计学意义(P＜0.05);而EC单独培养的ALP增殖活性和OC mRNA的表达量两组间比较差异均无统计学意义(P＞0.05).实验组间接共培养、直接共培养的ALP增殖活性及OC mRNA的表达量较OB单独培养明显增高,且直接共培养较间接共培养也有所提高,差异均有统计学意义(P＜0.05). 结论 BMSCs来源的OB与EC共培养细胞相容性良好,P物质可显著促进共培养体系中OB的增殖与活性.
目的 探討P物質對骨髓基質榦細胞(BMSCs)來源的成骨細胞(OB)與內皮細胞(EC)體外聯閤培養的影響. 方法 分彆建立BMSCs來源的OB與EC單獨培養體繫,以及按2∶1的比例體外直接或間接共培養體繫.實驗組:各培養體繫細胞內同時加入1×10-8 mol/L P物質(n=6),對照組:各培養體繫不添加P物質(n=6).培養48 h後採用流式細胞儀分析細胞的增殖與活性,併採用酶聯免疫吸附法測定堿性燐痠酶(ALP)的錶達,實時定量聚閤酶鏈式反應檢測骨鈣素(OC)mRNA基因的錶達.比較兩組不同培養方式細胞功能的變化. 結果 與對照組比較,實驗組OB單獨培養、間接共培養時細胞G1期減少,G2/M期明顯增高,差異均有統計學意義(P＜0.05).實驗組OB單獨培養、間接共培養、直接共培養的細胞ALP增殖活性[(2.877 ±0.188)、(3.053±0.179)、(4.345±0.305)]及OCmRNA的錶達量[(1.682±0.155)、(3.188±0.213)、(3.720 ±0.194)]均明顯高于對照組[(2.600±0.233)、(2.842±0.120)、(3.220±0.191);(1.360±0.141)、(2.272±0.143)、(2.507 ±0.176)],差異均有統計學意義(P＜0.05);而EC單獨培養的ALP增殖活性和OC mRNA的錶達量兩組間比較差異均無統計學意義(P＞0.05).實驗組間接共培養、直接共培養的ALP增殖活性及OC mRNA的錶達量較OB單獨培養明顯增高,且直接共培養較間接共培養也有所提高,差異均有統計學意義(P＜0.05). 結論 BMSCs來源的OB與EC共培養細胞相容性良好,P物質可顯著促進共培養體繫中OB的增殖與活性.
목적 탐토P물질대골수기질간세포(BMSCs)래원적성골세포(OB)여내피세포(EC)체외연합배양적영향. 방법 분별건립BMSCs래원적OB여EC단독배양체계,이급안2∶1적비례체외직접혹간접공배양체계.실험조:각배양체계세포내동시가입1×10-8 mol/L P물질(n=6),대조조:각배양체계불첨가P물질(n=6).배양48 h후채용류식세포의분석세포적증식여활성,병채용매련면역흡부법측정감성린산매(ALP)적표체,실시정량취합매련식반응검측골개소(OC)mRNA기인적표체.비교량조불동배양방식세포공능적변화. 결과 여대조조비교,실험조OB단독배양、간접공배양시세포G1기감소,G2/M기명현증고,차이균유통계학의의(P＜0.05).실험조OB단독배양、간접공배양、직접공배양적세포ALP증식활성[(2.877 ±0.188)、(3.053±0.179)、(4.345±0.305)]급OCmRNA적표체량[(1.682±0.155)、(3.188±0.213)、(3.720 ±0.194)]균명현고우대조조[(2.600±0.233)、(2.842±0.120)、(3.220±0.191);(1.360±0.141)、(2.272±0.143)、(2.507 ±0.176)],차이균유통계학의의(P＜0.05);이EC단독배양적ALP증식활성화OC mRNA적표체량량조간비교차이균무통계학의의(P＞0.05).실험조간접공배양、직접공배양적ALP증식활성급OC mRNA적표체량교OB단독배양명현증고,차직접공배양교간접공배양야유소제고,차이균유통계학의의(P＜0.05). 결론 BMSCs래원적OB여EC공배양세포상용성량호,P물질가현저촉진공배양체계중OB적증식여활성.
Objective To study the effect of substance P (SP) on the co-cultured osteoblasts (OB) and endothelial cells (EC) induced from bone marrow stromal cells (BMSCs) in vitro in rabbits.Methods Four culture systems (OB,EC,indirect and direct co-culture of OB and EC at the ratio of 2∶ 1) were respectively established.Of each system,the experimental group was simultaneously added with 1 × 10-8mol/L SP (n =6) while the control was not.After culture for 48 hours,the cell cycles were analyzed by flow cytometry to detect the cell proliferation and activity; the expression of ALP (Alkaline phosphatase) was measured by ELISA; the mRNA expression of OC (osteocalcin) gene in osteoblasts was detected by real-time quantitative PCR.The changing of cell function was compared between the 2 groups in each cell culture system.Results In systems of mere OB culture and indirect co-culture,the cells were significantly decreased at phase G1 and significantly increased at phase G2/M in the experimental groups compared with the controls (P ＜ 0.05).In systems of mere OB culture and indirect and direct co-cuhures,the experimental groups had significantly higher ALP expression (2.877 ±0.188,3.053 ±0.179 and 4.345 ±0.305) and mRNA expression of OC (1.682 ±0.155,3.188 ± 0.213 and 3.720 ± 0.194) than the controls respectively (2.600±0.233,2.842 ±0.120 and 3.220 ±0.191; 1.360 ±0.141,2.272 ±0.143 and 2.507 ±0.176) (P ＜ 0.05).In the system of mere EC culture,there were no significant differences in ALP expression or mRNA expression of OC between the 2 groups (P ＞ 0.05).In the experimental groups,indirect and direct co-cultures produced significantly higher ALP expression and mRNA expression of OC than mere OB culture,and so did the direct co-culture than the indirect co-culture (P ＜ 0.05).Conclusions There is good cellular compatibility between the endothelial cells and osteoblasts induced from BMSCs when the 2 kinds of cells are co-cultured.SP can significantly promote the activity and proliferation of OB in a co-culture system.
