外源性NO对脂肪干细胞成软骨分化的影响  

Influence of Exogenous Nitric Oxide on Chondrogenic Differentiation of Adipose Derived Stem Cells

作　　者：刘爽[1,2,3] 黄海森[2,3] 于湄[2,3] 罗小昕 郭维华[2,3,5] 田卫东[2,3,6] 

机构地区：[1]四川大学生命科学学院,成都610064 [2]四川大学口腔再生医学国家地方联合工程实验室,成都610041 [3]四川大学口腔疾病研究国家重点实验室,成都610041 [4]四川大学材料科学与工程学院,成都610064 [5]四川大学华西口腔医院儿童口腔科,成都610041 [6]四川大学华西口腔医院口腔颌面外科,成都610041

出　　处：《四川大学学报（医学版）》2015年第1期6-10,21,共6页Journal of Sichuan University(Medical Sciences)

基　　金：国家重点基础研究发展计划(973计划)(No.2010CB944802);国家自然科学基金(No.30973348)资助

摘　　要：目的通过观察不同浓度外源性一氧化氮(nitric oxide,NO)供体硝普钠(SNP)对脂肪干细胞(adipose-derived stem cells,ADSCs)增殖状况及相关基因表达的影响,探讨外源性NO在ADSCs成软骨分化中的作用。方法获取并培养ADSCs,分别进行成骨和成脂诱导,采用茜素红染色和油红O染色进行多向分化鉴定。NO检测试剂盒检测ADSCs成软骨分化过程中NO的变化;采用细胞计数试剂盒-8(CCK8)法检测不同浓度SNP(0.25mmol/L、1.00mmol/L、4.00mmol/L)下ADSCs的增殖情况。以Real time-PCR(RT-PCR)方法检测ADSCs在不同浓度SNP下,表达转化生长因子-β1(TGF-β1)以及成软骨分化特异基因——信号传导蛋白Smad3、Ⅱ胶原蛋白(Col-Ⅱα1)基因的变化。结果培养的细胞经成骨和成脂诱导后茜素红染色和油红O染色呈阳性;ADSCs成软骨分化过程中,培养上清液中NO浓度始终比对照组高(P<0.05);与对照组相比,低浓度SNP(0.25mmol/L、1.00mmol/L)对ADSCs的增殖无明显影响(P>0.05),高浓度SNP(4.00mmol/L)抑制ADSCs增殖,差异有统计学意义(P<0.05)。RT-PCR检测发现SNP能抑制ADSCs自身TGF-β1mRNA的表达,同时抑制Smad3mRNA和Col-Ⅱα1mRNA的表达。结论在ADSCs成软骨分化过程中,外源性NO可以通过抑制ADSCs自身产生TGF-β1,并且抑制TGF-β下游信号通路,从而抑制成软骨分化。Objective To determine the effects of exogenous nitric oxide （NO） donor sodium nitroprusside （SNP） on the proliferation and expression of related-gene of adipose-derived stem cells（ADSCs） and its role in chondrogenic differentiation of ADSCs. Methods Rat ADSCs were harvested and cultured, and then induced to osteogenic and adipogenic differentiations, detected with Alizarin red stained and Oil red O stain, respectively. The change of NO during chondrogenic differentiation of ADSCs was tested by NO detection kit. Cell counting kit-8 （CCK8） was used to detect the proliferation of ADSCs under different concentrations of SNP （0. 25 mmol/L, 1.00 mmol/L, 4.00 mmol/L） . Gene expression level of transformation growth factor （TGF-β1） and specific gene of chondrogenic differentiation-signaling protein Smad3 and Collage Ⅱ α1 （Col-Ⅱα1）, were detected by Real time- PCR （RT-PCR） method. Results Positive alizarin red staining and Oil red O staining were found after osteogenic and adipogenic induction of cultured ADSCs. Higher concentrations of NO were found in the supernatant of the experimental group with ADSCs chondrogenic differentiations compared with the controls （P〈0. 05）. Low concentrations （0. 25 mmoi/L, 1. 00 mmol/L） of SNP showed no significant effects on ceil proliferations （P〉 0.05）, whereas high concentration （4. 00 mmol/L） of SNP inhibited cell proliferation （P〈0. 05）. RT-PCR revealed that SNP inhibited the gene expression of TGF-fll mRNA and chondrogenic differentiation of specific geneSmad3 mRNA, Col- Ⅱa1 mRNA. Conclusion SNP can inhibit chondrogenic differentiations by suppressing the production of TGF-β1 and inhibiting downstream of TGF-β1 signaling pathways, thereby inhibiting ADSCs differentiation into chondrocytes.

关 键 词：外源性一氧化氮 脂肪干细胞 成软骨分化 TGF-Β1 

分 类 号：R681.3[医药卫生—骨科学]