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目的:构建携带小鼠脂联素(Acrp30)siRNA腺病毒载体,并检测其对小鼠脂肪细胞Acrp30表达以及对3T3-L1脂肪细胞基础葡萄糖转运的影响。方法:设计并化学合成小鼠脂肪细胞Acrp30 siRNA片断,将其亚克隆入AdEaxy XL腺病毒载体系统,在293细胞内包装扩增为重组腺病毒。用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达。采用2 Deoxy-[3H]D-glucose掺入法测定脂肪细胞葡萄糖转运。结果:设计并构建了小鼠Acrp30基因特异性siRNA腺病毒载体,该载体感染脂肪细胞后,能显著抑制Acrp30 mRNA和蛋白表达,影响3T3-L1脂肪细胞基础葡萄糖的转运,与对照组相比,差异有显著性意义(P<0.05)。结论:构建的Acrp30基因特异性siRNA腺病毒载体能有效的抑制脂联素在3T3-L1脂肪细胞中的表达,从而影响3T3-L1脂肪细胞基础葡萄糖转运。
OBJECTIVE: To construct adenovirus vector carrying mouse Acrp30 siRNA and to detect its effect on Acrp30 expression in mouse adipocytes and basal glucose transport in 3T3-L1 adipocytes. Methods: Acrp30 siRNA fragment of mouse adipocytes was designed and chemically synthesized, subcloned into AdEaxy XL adenovirus vector system, and packaged into 293 cells for amplification of recombinant adenovirus. The recombinant adenovirus was used to infect 3T3-L1 adipocytes, and the expression of Acrp30 mRNA and protein was detected by RT-PCR and ELISA. Adipose cell glucose transport was measured using 2 Deoxy- [3H] D-glucose incorporation. Results: The mouse Acrp30 gene-specific siRNA adenovirus vector was designed and constructed. The vector infected with adipocytes significantly inhibited the expression of Acrp30 mRNA and protein, and affected the basal glucose transport of 3T3-L1 adipocytes. Compared with the control group, The difference was significant (P <0.05). CONCLUSION: The Acrp30 gene-specific siRNA adenovirus vector can effectively inhibit the expression of adiponectin in 3T3-L1 adipocytes and thus affect the basic glucose transport of 3T3-L1 adipocytes.