不同浓度地塞米松诱导脂肪干细胞成骨分化的实验研究  

EXPERIMENTAL STUDY ON OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS TREATED WITH DIFFERENT CONCENTRATIONS OF DEXAMETHASONE

作　　者：王伟[1] 陈磊杰[2] 张晨[1] 余强[1] 王坤正[1] 

机构地区：[1]西安交通大学医学院第二附属医院骨科,西安710004 [2]昆明医学院第二附属医院骨科

出　　处：《中国修复重建外科杂志》2011年第12期1486-1492,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：西安交通大学光华基金资助项目(0203204);西安交通大学医学与理工科交叉合作项目(08143027)~~

摘　　要：目的地塞米松是MSCs成骨诱导分化的基础试剂,探讨诱导脂肪干细胞(adipose-derived stem cells,ADSCs)成骨分化过程中地塞米松的优选浓度,为进一步骨组织工程研究提供理论依据。方法 3月龄清洁级健康新西兰大白兔5只,雌雄不限,体重2～3 kg。取腹股沟区皮下脂肪4～6 mL,采用胶原酶消化离心贴壁法分离培养ADSCs,取第3代细胞进行实验。倒置相差显微镜观察细胞形态变化;联合CD44、CD106免疫荧光染色和成脂诱导分化鉴定ADSCs。调整细胞密度为1×105个/mL,分别用普通培养液(A组)及含0(B组)、1×10-9(C组)、1×10-8(D组)、1×10-7(E组)、1×10-6(F组)、1×10-5 mol/L(G组)地塞米松的成骨诱导培养液对ADSCs进行培养。MTT法检测细胞增殖情况;RT-PCR检测诱导细胞骨钙素(osteocalcin,OC)和核心结合因子α1(core binding factorα1,Cbfα1)的表达;测定ALP活性及矿化面积百分率;对矿化结节行茜素红染色。结果 ADSCs形态多为梭形、多角形,呈"漩涡状"排列;表面抗原分子CD44呈阳性,CD106呈阴性,成脂诱导后可观察到细胞内有脂滴形成,油红O染色呈阳性。MTT检测显示随地塞米松浓度升高,吸光度(A)值呈下降趋势;其中成骨诱导5、7 d时,D、E组A值比较差异有统计学意义(P<0.05)。RT-PCR检测示,成骨诱导7 d OC和Cbfα1 mRNA的表达分别在E组和D组达高峰;成骨诱导14 d ALP活性和矿化面积百分率均在D组达高峰,随后逐渐下降。D、E组间OC和Cbfα1 mRNA的表达量、ALP活性及矿化面积百分率比较差异均无统计学意义(P>0.05),与其余各组比较差异均有统计学意义(P<0.05)。成骨诱导14 d,G组细胞均死亡;茜素红染色除A、G组外均呈阳性。结论成骨培养液中地塞米松浓度为1×10-8 mol/L时,能在减少对细胞增殖抑制的同时,更有效地诱导ADSCs成骨分化。Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells.To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederived stem cells（ADSCs） so as to provide the theoretical basis for further bone tissue engineering researches.Methods Five New Zealand rabbits（2-3 kg） of clean grade,aged 3 months and male or female,were obtained.ADSCs were isolated from the subcutaneous adipose tissue of inguinal region,and cultured with collagenase digestion,then were detected and identified by CD44,CD106 immunofluorescence staining and adipogenic differentiation.ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL,then the cells were treated with common cultural medium（group A） and osteogenic induced medium containing 0（group B）,1 × 10-9（group C）,1 × 10-8（group D）,1 × 10-7（group E）,1 × 10-6（group F）,and 1 × 10-5 mol/L（group G） dexamethasone,respectively.The cell proliferation and the mRNA expressions of osteocalcin（OC） and core binding factor α1（Cbfα1） were detected by MTT and RT-PCR,respectively.The activity of alkaline phosphatase（ALP） was measured,and the percentage of mineral area was calculated.The mineral nodules were also detected by alizarin red staining.Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106.The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium.The result of MTT revealed that the absorbance（A） value declined with the ascending of the concentration of dexamethasone,and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction（P 0.05）.The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction,respectively.The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 d

关 键 词：脂肪干细胞 细胞培养 成骨分化 地塞米松 兔 

分 类 号：R965[医药卫生—药理学]