中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
19期
2968-2974
,共7页
张良明%王璇%高文杰%黄东生%戎利民
張良明%王璇%高文傑%黃東生%戎利民
장량명%왕선%고문걸%황동생%융이민
干细胞%骨髓干细胞%间充质干细胞%褪黑素%骨髓%脂肪细胞%脂肪分化%褪黑素受体%视黄酸相关孤儿受体α%国家自然科学基金
榦細胞%骨髓榦細胞%間充質榦細胞%褪黑素%骨髓%脂肪細胞%脂肪分化%褪黑素受體%視黃痠相關孤兒受體α%國傢自然科學基金
간세포%골수간세포%간충질간세포%퇴흑소%골수%지방세포%지방분화%퇴흑소수체%시황산상관고인수체α%국가자연과학기금
melatonin%receptors,melatonin%orphan nuclear receptors%celldifferentiation
背景：前期研究发现，褪黑素能够抑制骨髓间充质干细胞向脂肪细胞诱导分化，但其作用机制有待进一步研究。<br> 目的：探索褪黑素是否通过核受体视黄酸相关孤儿受体α介导抑制骨髓间充质干细胞向脂肪细胞诱导分化的作用机制。<br> 方法：无菌采集骨髓样本，采用密度梯度离心结合贴壁培养分离培养人骨髓间充质干细胞，用流式细胞仪鉴定细胞表面表型。然后将细胞分为褪黑素组、CGP52608组和对照组，分别以含褪黑素、CGP52608脂肪维持液和正常的脂肪诱导液进行诱导，用荧光定量PCR和Western blot法检测核受体视黄酸相关孤儿受体α在骨髓间充质干细胞以及其成脂肪分化过程中的表达特征。用受体特异性激活剂 CGP52608，考察核受体视黄酸相关孤儿受体α激活状态下，骨髓间充质干细胞成脂肪分化的影响。<br> 结果与结论：原代分离的骨髓间充质干细胞生长旺盛，呈纤维梭状，流式鉴定间充质干细胞表型结果示，CD34和CD45表达阴性，CD29，CD44与CD105表达阳性。荧光定量PCR发现骨髓间充质干细胞高表达褪黑素核受体视黄酸相关孤儿受体α，而且褪黑素能够呈浓度依赖性促进骨髓间充质干细胞表达视黄酸相关孤儿受体α；Western blot检测结果也证实褪黑素呈浓度依赖性促进视黄酸相关孤儿受体α在蛋白水平的表达。在成脂肪分化过程中，褪黑素在早期明显上调视黄酸相关孤儿受体α表达，但在中后期就维持在相对低水平。最后，用CGP52608激活视黄酸相关孤儿受体α后，骨髓间充质干细胞成脂肪分化被抑制。因此，褪黑素通过核受体视黄酸相关孤儿受体α抑制骨髓间充质干细胞成脂肪分化，褪黑素在调节脂肪细胞分化过程中发挥重要作用。
揹景：前期研究髮現，褪黑素能夠抑製骨髓間充質榦細胞嚮脂肪細胞誘導分化，但其作用機製有待進一步研究。<br> 目的：探索褪黑素是否通過覈受體視黃痠相關孤兒受體α介導抑製骨髓間充質榦細胞嚮脂肪細胞誘導分化的作用機製。<br> 方法：無菌採集骨髓樣本，採用密度梯度離心結閤貼壁培養分離培養人骨髓間充質榦細胞，用流式細胞儀鑒定細胞錶麵錶型。然後將細胞分為褪黑素組、CGP52608組和對照組，分彆以含褪黑素、CGP52608脂肪維持液和正常的脂肪誘導液進行誘導，用熒光定量PCR和Western blot法檢測覈受體視黃痠相關孤兒受體α在骨髓間充質榦細胞以及其成脂肪分化過程中的錶達特徵。用受體特異性激活劑 CGP52608，攷察覈受體視黃痠相關孤兒受體α激活狀態下，骨髓間充質榦細胞成脂肪分化的影響。<br> 結果與結論：原代分離的骨髓間充質榦細胞生長旺盛，呈纖維梭狀，流式鑒定間充質榦細胞錶型結果示，CD34和CD45錶達陰性，CD29，CD44與CD105錶達暘性。熒光定量PCR髮現骨髓間充質榦細胞高錶達褪黑素覈受體視黃痠相關孤兒受體α，而且褪黑素能夠呈濃度依賴性促進骨髓間充質榦細胞錶達視黃痠相關孤兒受體α；Western blot檢測結果也證實褪黑素呈濃度依賴性促進視黃痠相關孤兒受體α在蛋白水平的錶達。在成脂肪分化過程中，褪黑素在早期明顯上調視黃痠相關孤兒受體α錶達，但在中後期就維持在相對低水平。最後，用CGP52608激活視黃痠相關孤兒受體α後，骨髓間充質榦細胞成脂肪分化被抑製。因此，褪黑素通過覈受體視黃痠相關孤兒受體α抑製骨髓間充質榦細胞成脂肪分化，褪黑素在調節脂肪細胞分化過程中髮揮重要作用。
배경：전기연구발현，퇴흑소능구억제골수간충질간세포향지방세포유도분화，단기작용궤제유대진일보연구。<br> 목적：탐색퇴흑소시부통과핵수체시황산상관고인수체α개도억제골수간충질간세포향지방세포유도분화적작용궤제。<br> 방법：무균채집골수양본，채용밀도제도리심결합첩벽배양분리배양인골수간충질간세포，용류식세포의감정세포표면표형。연후장세포분위퇴흑소조、CGP52608조화대조조，분별이함퇴흑소、CGP52608지방유지액화정상적지방유도액진행유도，용형광정량PCR화Western blot법검측핵수체시황산상관고인수체α재골수간충질간세포이급기성지방분화과정중적표체특정。용수체특이성격활제 CGP52608，고찰핵수체시황산상관고인수체α격활상태하，골수간충질간세포성지방분화적영향。<br> 결과여결론：원대분리적골수간충질간세포생장왕성，정섬유사상，류식감정간충질간세포표형결과시，CD34화CD45표체음성，CD29，CD44여CD105표체양성。형광정량PCR발현골수간충질간세포고표체퇴흑소핵수체시황산상관고인수체α，이차퇴흑소능구정농도의뢰성촉진골수간충질간세포표체시황산상관고인수체α；Western blot검측결과야증실퇴흑소정농도의뢰성촉진시황산상관고인수체α재단백수평적표체。재성지방분화과정중，퇴흑소재조기명현상조시황산상관고인수체α표체，단재중후기취유지재상대저수평。최후，용CGP52608격활시황산상관고인수체α후，골수간충질간세포성지방분화피억제。인차，퇴흑소통과핵수체시황산상관고인수체α억제골수간충질간세포성지방분화，퇴흑소재조절지방세포분화과정중발휘중요작용。
BACKGROUND:Our previous studies have demonstrated that melatonin inhibits the adipogenic differentiation of bone marrow mesenchymal stem cels. However, the mechanism underlying the effect of melatonin on adipogenesis is stil unknown. <br> OBJECTIVE:To determine whether melatonin can inhibit the adipogenesis of bone marrow mesenchymal stem cels through retinoid-related orphan receptor α. <br> METHODS:Bone marrow mesenchymal stem cels were isolated and purified by density gradient centrifugation combined with cellattachment culture. The phenotype was investigated by flow cytometry. Then, cels were induced for adipogenic differentiation with melatonin, CGP52608 and normal adipose tissue, respectively. The levels of retinoid-related orphan receptor α mRNA and protein were investigated by real-time RT-PCR and western blot assay, respectively. Further, the effect of retinoid-related orphan receptor α on the dipogenic differentiation of bone marrow mesenchymal stem cels was investigated by CGP52608. <br> RESULTS AND CONCLUSION:The primary isolated bone marrow mesenchymal stem cels were spindle-shaped fibroblast-like cels. These cels did not express hematopoietic stem cels markers: CD34 and CD45; and highly expressed MSC markers: CD29, CD44, and CD10. The result of RT-PCR demonstrated that melatonin nuclear receptor, retinoid-related orphan receptor α, was highly expressed in bone marrow mesenchymal stem cels and the expression of retinoid-related orphan receptor α was further enhanced by melatonin in a dose-dependent manner, which was confirmed at protein level by western blot assay. During adiogenesis, the expression of retinoid-related orphan receptor αmRNA was up-regulated in the early stage, but maintained at a low level in the mild-later stage. While the retinoid-related orphan receptor α was activated by agonist CGP52608, the adipogenic differentiation of bone marrow mesenchymal stem cels was inhibited, which was similar to the inhibitory effect of melatonin. Therefore, melatonin inhibited the adipogenic differentiation of bone marrow mesenchymal stem cels through retinoid-related orphan receptor α, suggesting that melatonin plays an important role in the differentiation of adipocytes.
