地塞米松在家兔脂肪基质干细胞定向诱导为成骨细胞的作用

Role of dexamethasone in the differentiation of adipose-derived stromal stem cells into osteoblasts in rabbits

目的:观察不同浓度地塞米松对家兔脂肪基质干细胞生长、增殖及向成骨细胞分化的影响。方法:实验于2006-11/2007-01在南方医科大学组织工程研究中心完成。实验材料:成年新西兰大白兔购于南方医科大学实验动物中心(机构许可证号SYXK(粤)2005-0056)。实验方法:①脂肪基质干细胞获取:取成年新西兰大白兔腹股沟皮下脂肪组织一二毫升,分离培养脂肪基质干细胞,并按1∶3进行传代培养。选取生长良好的P3代细胞进行免疫组织化学染色测定CD44抗原表达。并于第3,7,14天观察细胞增殖情况。②脂肪基质干细胞成骨诱导:成骨诱导液由不同浓度地塞米松、50mg/L的维生素C、10mmol/L的β-磷酸甘油和DMEM完全培养基组成。实验分6组:0mol/L地塞米松组、10-8mol/L地塞米松组、10-7mol/L地塞米松组、10-6mol/L地塞米松组、10-5mol/L地塞米松组、对照组。选择生长良好P3代兔脂肪基质干细胞,用0.25%胰酶消化3～5min,制成单细胞悬液,调整细胞密度为1×104,接种于25cm2的培养瓶中,在体积分数为0.05的CO2饱和湿度、37℃恒温培养箱培养。24h后所有诱导组更换为成骨诱导培养液,进行成骨细胞诱导分化处理,记为诱导第0天,对照组换DMEM完全培养液。诱导期间,每隔二三天换1次成骨诱导液,对照组换1次常规DMEM完全培养液。③第7,14,21天茜素红染色鉴定成骨细胞特性,观察不同浓度地塞米松对兔脂肪基质干细胞的生长、增殖及向成骨细胞分化的影响。结果:①地塞米松对脂肪基质干细胞增殖的影响:与对照组相比较,10-6,10-5mol/L组细胞数目明显减少(t=2.8623,3.4245,3.5203,3.9594,2.2641,2.7012,P<0.05)。②地塞米松对脂肪基质干细胞成骨分化的影响:光镜下观察整个诱导分化期,对照组未观察到钙化结节形成,0mol/L地塞米松组只观察到个别钙化结节形成。在诱导第14天,10-5,10-6,10-7,10-8mol/L地塞米松组均可观察到少数钙化结节形成,10-5,10-6,10-7mol/L组间钙化结节数差异无显著性意义(t=0.5571,0.5929,1.1142,P>0.05),但钙化结节数目明显多于10-8mol/L组(t=5.4960,6.2811,6.3640,P<0.05)。在诱导第21天,10-5,10-6,10-7mol/L组均可观察到大量钙化结节形成,均明显多于10-8mol/L组(t=6.2703,6.4065,5.6349,P<0.05)。结论:>10-7mol/L高浓度地塞米松对脂肪基质干细胞的生长、增殖有明显抑制作用;在其他诱导条件一致的情况下,10-7mol/L地塞米松最有利于脂肪基质干细胞向成骨细胞分化。

AIM： To observe the influence of dexamethasone of different concentrations on growth towards the osteoblast in adipose-derived stromal stem cells of rabbits. METHODS： The experiment was performed at the Tissue Engineering Research Center, Southem Medical University from November 2006 to January 2007. Experimental materials： the adult New Zealand rabbits were purchased from Experiment Animal Center of Southem Medical University [license of institution SYXK （Yue） 2005-0056]. Experimental methods： （1）Collection of adipose-derived stromal stem cells： 1-2 mL subcutaneous adipose tissues were harvested from inguinal groove of rabbits. Adipose-derived stromal stem cells were isolated, cultured and passaged at 1：3. The expression of CD44 from well P3 cells was assayed by immunohistochemicel staining. Proliferation was observed at days 3, 7 and 14. （2）Osteoblast induction in adipose-derived stromal stem cells： Osteoblast induction liquor was composed of dexamethasone of different concentrations, 50 mg/L vitamin C, 10 mmol/L 13-glycerophosphate and DMEM complete medium. There were 6 groups, namely 0 mol/L dexamethasone group, 10^-8 mol/L dexamethasone group, 10^-7 mol/L dexamethasone group, 10^-8 mol/L dexamethasone group, 10^-5 mol/L dexamethasone group and control group. Well P3 stromal stern cells were digested with 0.25% trypsin for 3-5 minutes and made into monoplast suspension. Cells＇were adjusted into 1×10^4 and then inoculated in 25 cm^2 culture flask and cultured in incubator with CO2 saturated humidity of 0.05 volume fraction at 37 %. 24 hours later, culture medium was changed into osteoblast induction medium in all the induction groups, marking the 0 day, and it was DMEM complete culture medium in the control group. Osteoblast induction medium was Changed once every 2 or 3 days, whereas routine DMEM complete culture medium was changed once in the control group. （3）Osteoblast and its characteristics were identified by alizarin straining at days 7, 14 and 21. Influences of dexamethasone of different concentrations on growth, proliferation and differentiation into osteoblast were observed. RESULTS： （1）Influence of dexamethasone on proliferation of adipose-derived stromal stem cells： Compared with the control group, number of adipose-derived stromal stem-cells was significantly decreased in the 10^-6 and 10^-5 mol/L groups （t =2.862 3, 3.424 5, 3.520 3, 3.959 4, 2.264 1, 2.701 2, P 〈 0.05）. （2）Influence of dexamethasone on differentiation towards the osteoblast from adipose-derived stromal stem cells： The whole induction and differentiation were observed under light microscope. No calcification node was formed in the control group, whereas few calcification node was found in the 0 mol/L dexamethasone group. On the 14^th day, several calcification node was seen in the 10^-5, 10^-6,10^-7,10^-8 mol/L dexamethasone groups. There was no significant difference of calcification node in the 10^-5,10^-6,10^-7 mol/L dexamethasone groups（t =0.557 1, 0.592 9, 1.114 2, P 〉 0.05）, but number of calcification node was obviously more than that in the 10^-6 mol/L dexamethasone group （t =5.496 0, 6.281 1, 6.364 0, P〈 0.05）. On the 21^st day, a mass of calcification node was seen in the 10^-5,10^-6 ,10^-7 mol/L dexamethasone groups, which was markedly more than that in the 10^-6 mol/L dexamethasone group （t =6.270 3, 6.406 5, 5.634 9, P〈 0.05）. CONCLUSION： Dexamethasone of over 10^-7 mol/L concentration has obviously inhibitory effect on growth and proliferation of adipose-derived stromal stem cells. Under the same induction condition, 10^-7 mol/L dexamethasone is effective to differentiate into osteoblast from adipose-derived stromal stem cells.