摘要
目的研究高浓度全反式视黄酸(atRA)抑制大鼠骨髓间充质干细胞(r BMSCs)成脂分化过程中,视黄酸核受体γ(RARγ)对过氧化物酶体增殖激活受体γ2(PPARγ2)与CCAAT增强子结合蛋白α(C/EBPα)调控的机制。方法体外分离、培养、诱导r BMSCs。成脂分化诱导液中,加入0.5、1.0μmol/L atRA持续作用15 d后,real-time PCR和Western blotting分别检测RARγ、C/EBPα、PPARγ2的mRNA和蛋白表达水平。重组腺病毒过表达RARγ(over-RARγ)、沉默RARγ(si-RARγ)分别感染BMSCs,采用real-time PCR及Western blotting检测RARγ、C/EBPα、PPARγ2的mRNA及蛋白表达水平。Co-IP检测1.0μmol/L atRA持续成脂诱导15 d后,明确RARγ与PPARγ2两者之间是否有相互作用。结果诱导15 d结束时,与对照组相比,加入0.5、1.0μmol/L atRA后,RARγ表达明显增加(P<0.01,P<0.001),而PPARγ2、C/EBPα表达明显降低(P<0.001)。over-RARγ组:RARγmRNA和蛋白表达均较对照组明显增加(P<0.01),而PPARγ2、C/EBPαmRNA和蛋白表达均较对照组明显降低(P<0.05,P<0.01)。si-RARγ组:RARγ蛋白表达较对照组明显减低,但PPARγ2、C/EBPα却无明显变化。Co-IP结果提示:RARγ与PPARγ2蛋白之间有直接作用,形成复合物,未发现其与视黄酸反应元件(RARE)相结合证据。结论高浓度atRA抑制r BMSCs成脂分化,是通过激活视黄酸信号通路RARγ而实现的。RARγ下调成脂分化通路PPARγ2、C/EBPα表达是通过直接作用于下游的PPARγ2蛋白而实现的。
Objective To investigate the mechanism of regulating peroxisome proliferator-activated receptor y2 （PPARy2） and CCAAT enhancement binding protein ct （C/EBPct） by retinoic acid receptory（RARey） during the course of inhibiting adipogenic differentiation of rat bone marrow mesenchymal stem cells （rBMSCs） by high concentration of aU-trans retinoic acid （atRA）. Methods The rBMSCs were isolated, cultured, and induced in vitro. The atRA solution of 0.5 and 1.0 μmol/L was added to the medium of adipogenic induction. After being cultured for 15 d, mRNA and protein expressions of RARyPPARy2, and C/EBPet were detected by the real-time quantitative polymerase chain reaction （RTPCR） and Western blotting. After being infected with the over-express RARey （over-RARy） adenovirus and silence RARy （si-RARy） adenovirus, mRNA and protein expressions of RARy PPARy2, and C/EBPet were detected by the RTPCR and Western blotting. After being inducted by atRA of 1.0 μmnol/L for 15 d, the coimmunoprecipitation （Co-IP） technique was employed to investigate the interaction between RAR-y and PPARy2. Results Compared with the control group, the expression of RARe/ after being induced by atRA of 0.5 and 1.0 μmol/L for 15 d significantly increased （ P〈 0.01, P〈0. 001）, while expressions of PPARy2 and C/EBP（x significantly decreased （P〈0. 001）. The mRNA and protein expressions of RARy after being infected by over-RAR-y adenovirus significandy increased （P 〈 0. 01）, while mRNA and protein expressions of PPARy2 and C/EBPa significantly decreased （P〈0.05, P〈 0.01）. The protein expression of RAR3, after being infected by si-RARy adenovirus significantly decreased, while expressions of PPARy2 and C/EBPa did not change significandy. The results of Co-IP showed that RARey directly interacted with PPARy2 and complex was formed. The combination of RAReyand retinoic acid response element （RARE） was not found. Conclusion High concentration of atRA can inhibit the adipogenic differentiation of rBMSCs by activating the signaling pathway of retinoic acid. RARey downregnlates the expressions of PPARy2 and C/EBPa of signaling pathway of adipogenic differentiation by directly interacting with downstream PPARy2.
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2015年第5期682-687,共6页
Journal of Shanghai Jiao tong University:Medical Science
