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Effect of Small Interfering RNA Targeting AGT on Differentiation on Mouse Preadipocytes

Author: WuLiHua
Tutor: YangRuDe；ZhangLiJun；HuangZhiLi
School: South China University of Technology
Course: Microbiology
Keywords: RNAi AGT 3T3-L1 preadipocytes differentiation RAS PsiRNAT-U6.1/neo
CLC: Q343
Type: Master's thesis
Year: 2010
Downloads: 24
Quote: 0
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Abstract


The primary reason of obesity is hypertrophy and hyperplasia in adipocyte. Studies on proliferation and differentiation of preadipocytes will help to reveal the physiological and pathophysiological mechanisms underlying adipose tissue development. RAS is a peptidergic system with endocrine characteristic which is considered having important effect on obesity and other correlative diseases. But some of the researches have shown contradictory results. There for further researches on the effect of RAS during preadipocytes differentiation still need to be elucidated.1. The biological characteristic of growth and differentiation of 3T3-L1Preadipocytes were propagated in DMEM containing 10% calf serum, then were induced with adipogenesis initiation media including IBMX, dexamethasone and insulin. then were refeeded with DMEM containing 10% fetal calf serum. The morphological changes of 3T3-L1 was observed on stage different. Intracytoplasmic lipids were quantitated by Oil Red O staining and GPDH activity methods. The result indicated that intracytoplasmic lipids increased along with the differentiation time, and the maximum amount of increase was observed four days later. GPDH activity appeared the same regularity which increased quickly at the 4th day. The maximum value of GPDH activity was achieved at 8th day and maintained high level.2. The construction of AGT RNAi PlasmidThe plasmid containing short hairpin RNA(shRNA)of Psi-AGT was constructed to suppress the expression of angiotensinogen in 3T3-L1 preadipocytes. Two reverse repeated motifs targeting different regions of AGT gene were designed and synthesized respectively. Besides, one reverse repeated motifs without targeting any region were designed and synthesized as negative control. All these small fragments were inserted into plasmid PsiRNAT-U6.1/neo to generate the plasmids Psi-AGT0, Psi- AGT1and Psi-AGT2.3. The effect of Psi-AGT on Differentiation on 3T3-L1 PreadipocytesAll the recombinant plasmids of Psi-AGT0,Psi-AGT1,Psi-AGT2 were transfected into 3T3-L1 preadipocytes. The stable transfection was obtained by G418 with the selective concentration of 600μg/ml. Each group of transfected cell was induced to differentiate into an adipocyte phenotype by low concentration adipogenesis initiation media with 5%FCS. We investigated changes of intracytoplasmic lipids content and GPDH activity after transfection with the Psi-AGT1,2 and negative control Psi-AGT0 according to its physiological concentration. Compared to negative control Psi-AGT0, transfection Psi-AGT1 lipid content and GPDH activity decreased 12.5% and 6.6%. Transfection Psi-AGT2 lipid content and GPDH activity decreased 20% and 15%. According to Significance analysis, GPDH activity showed a significant difference between Psi-AGT2 and Psi-AGT0 after 8d and 12d. Lipid content activity showed a difference between Psi-AGT2 and Psi-AGT0.from the beginning to 8d and a significant difference between Psi-AGT2 and Psi-AGT0 from 10d to 12d.

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