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目的运用RNA干扰技术沉默黑色素浓集激素受体1(Melanin-Concentrating Hormone Receptor 1,MCHR1)基因的表达,观察其对3T3-L1细胞诱导分化的影响,为肥胖症的基因治疗提供新思路。方法用含绿色荧光蛋白的重组载体sh-MCHR1,通过脂质体法转染3T3-L1细胞。细胞诱导分化的同时用黑色素浓集激素(Melanin-Concentrating Hormone,MCH)干预,并在不同时点对脂滴进行油红O染色。采用RT-PCR以及Western blot分别检测过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptorγ,PPARγ)、CCAAT/增强子结合蛋白-α(CCAAT/Enhancer-Binding Proteinα,C/EBPα)和脂肪酸结合蛋白2(adipocyte protein 2,ap2)m RNA和蛋白的表达。结果重组载体sh-MCHR1成功转染;与阴性转染组相比,sh-MCHR1转染组d 10后,油红O染液相对OD510值显著下降(P<0.05);PPARγ、C/EBPα和ap2 m RNA的表达量分别在d 6、d 8和d 8后显著下降(P<0.05);3者蛋白的表达量均在d 8后显著下降(P<0.05)。结论 sh RNA沉默MCHR1基因可减缓3T3-L1细胞诱导分化,其可能通过抑制PPARγ、C/EBPα和ap2的表达而实现。
Objective To silence the expression of melanin-concentrating hormone receptor 1 (MCHR1) gene by RNA interference (RNAi) technology and observe its effect on the induction and differentiation of 3T3-L1 cells, so as to provide new ideas for the gene therapy of obesity. Methods 3T3-L1 cells were transfected with recombinant plasmid sh-MCHR1 containing green fluorescent protein by lipofectamine. Cells were induced to differentiate with Melanin-Concentrating Hormone (MCH) intervention, and lipid droplets were subjected to Oil Red O staining at different time points. Peroxisome Proliferator-Activated Receptor γ (PPARγ), CCAAT / Enhancer-Binding Protein α (C / EBPα) were detected by RT-PCR and Western blot respectively. And adipocyte protein 2 (ap2) m RNA and protein expression. Results sh-MCHR1 recombinant vector was transfected successfully. Compared with the negative transfected group, the expression of oily red O dye OD510 decreased significantly (P <0.05) after d 10 sh-MCHR1 transfected group; the expression of PPARγ, C / EBPα and The expression of ap2 mRNA decreased significantly after d 6, d 8 and d 8 (P <0.05). The expression of ap2 mRNA decreased significantly after d 8 (P <0.05). Conclusion The silencing of MCHR1 gene by sh RNA can slow the induction of differentiation of 3T3-L1 cells, which may be achieved by inhibiting the expression of PPARγ, C / EBPα and ap2.