Screening Of The Genes Associated With Porcine Preadipocytes Differentiation By Mrna Differential Display

Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from preadipocytes. During the preadipocyte differentiation process, is many specific genes are expressed and the cell morphology are changed accordingly. Porcine is one of the animal which has greatest fat deposit capacity, and the fat excessive accumulation in subcutaneous tissue and internal organs is an important factor which influences pork quality. In the present study, DDRT-PCR was used to screen genes associated with the porcine preadipocyte differentiation, to provide a scientific basis for the further study of regulation mechanism of fat metabolism in porcine.Experiment 1. Subcutaneous Adipose Tissue from cervical part was removed from 3 d newborn crossbred piglets and Preadipocyte was obtained by collagenaseⅣDigestion. The isolated preadipocytes were cultured in 37℃, 5%CO2 and saturation humidity and subsequently identified by morphology observation, i.e, MTT or Oil Red O staining and detection of Lipoprotein Lipase( LPL ) activity. The results showed that the amount of preadipocytes tend to be exponentially increased. 3 days later, differentiating adipocytes filled with fatty droplet appeared. Henceforth, more adipocytes containing fatty droplet were observed, and the morphology of the cells had been changed from fibroblast-like to round. At the same time, the activities of lipoprotein lipase (LPL) tend to descend from higher level. Therefore, the cells came from subcutaneous adipose tissue digested by collagenaseⅣ, possess the typical characteristics of preadipocytes.Experiment 2. In order to study the differential genes in which express on the process of proliferation and differentiation in the porcine preadipocytes cultured for 3, 5, 7 and 10 days were collected, and Trizol reagent was used to extract the total RNA of the cells. With RT-PCR and 6% non-polyacrylamide gel electrophoresis, the differential bands of the gene expression during the proliferation and differentiation period of the porcine preadipocytes were displayed on the silver stainined PAGE gel. Then the differential bands were sliced from the gel and the DNA segments were eluted and purified, the recovered products were subsequently used as template to amplify the fragments with PCR. Finanlly, the amplified segments were cloned into pMD18-T vector and sequenced, and the nucleotide sequence homology of the differential expressed gene and associated gene in Genbank were compared. The results showed that:(1) 20 ESTs of the differential expressed gene were identified, and the result of blast analysis on line indicated that 8 ESTs, A22, A23, A33, A43, A55, A73, B42 and C44 shared highly nucleotide sequence homology with associated genes reported in Genbank, 4 ESTs, A21, A32, A45 and B41 shared relatively lower nucleotide sequence homology, and their founction remained unclear, and it was found for the first time that 8 ESTs, A31, A53, A24, B31, B71, C21, C62 and C81, which were not reported in Genbank, were associated with proliferation and differentiation of the cultured porcine preadipocytes in vitro.(2) 10 ESTs, A21, A23, A31, A32, A43, A53, B31, B71, C21 and C62 mainly expressed in the pocine preadipocytes culured for 3 and 5 days, implied that genes had ESTs above were associated with preadipocyte proliferation; A22 expressed in the pocine preadipocytes culured for 3, 5 and 7 days, and the expression level was very low to be detected in cells cultured for 10 days, and presumed that A22 expression was related to the conversion and regulation process of proliferation and differentiation of the porcine preadipocytes in vitro. B41 expressed in the pocine preadipocytes culured for 5 and 7 days, implied that genes had ESTs above were associated with preadipocyte differentiation; B42, C81 expressed in the pocine preadipocytes culured for 5, 7 and 10 days,implied that genes had ESTs above were associated with preadipocyte proliferation; Furthmore, A24, A33, A45, A73 and C44 expression were found in adipocytes cultured for 7 and 10 days, which were relevant to preadipocyte differentiation. The nucleotide sequence homology of A55 and PURB originated from human came to 93%, and it as not clear that A55 was involved in regulatiuon of proliferation and differentiation of the porcine preadipocytes.