AbstractUsing semi-quantitative reverse transcriptase polymerase chain reaction (semi-qRT-PCR), cell transfection, Hoechst 33342, acridine orange (AO) and flow cytometry methods, apoptotic changes in porcine pre-adipocytes treated with 9-cis retinoic acid (9-cisRA)–retinoid X receptor α (RXRα) ligand, transfected with pEnhanced green fluorescent proteins C2–RXRα (pEGFPC2–RXRα) or RXRα–small interfering RNA (RXRα–siRNA), respectively, were investigated. Observations of morphologic changes showed changes in cell structure, cellular shrinkage and condensation of chromatin, pyknosis of the nucleus into a compact body, complete fragmentation within the nuclear membrane, continuous budding and shedding, and, finally conversion of the cell into several apoptotic bodies wrapped in membranes of different sizes. Compared with the control group, the apoptotic rate was significantly lowered in the 10 nmol/l 9-cisRA-treated group, and significantly higher in the 10 μmol/l 9-cisRA-treated group (both P<0.05). Gene expression of RXRα was significantly up-regulated and down-regulated when cultured pre-adipocytes were transfected with pEGFPC2–RXRα or RXRα–siRNA (P<0.05), respectively; the apoptotic rate decreased in the pEGFPC2–RXRα group and significantly increased in the RXRα–siRNA group when compared with a control group (P<0.05). This implies that RXRα inhibits the apoptosis of porcine pre-adipocytes.

The effects of retinoic acid X receptor α (RXRα) overexpression were studied during the differentiation of porcine adipocytes by various assays, such as plasmid transfection, oil red O extraction, GPDH activity measurement and semi-qRT-PCR. The results showed that with the increased level of pRXRα-EGFP following transfection to primary cultured porcine preadipocytes, preadipocytes differentiation with morphological changes of adipocytes was observed. Meanwhile, GPDH activity and the expression level of differentiation transcription factors PPARγ (peroxisome proliferator-activated receptor-γ) and C/EBPα (CCAAT/enhancer binding proteins) were significantly increased (P<0.05). The data implies that RXRα promotes differentiation of porcine adipocytes by enhancing the PPARγ and C/EBPα mediated pathway.

The elucidation of the molecular mechanism of porcine adipocyte differentiation is important not only to domestic animal industry, but also to medical science because of the similarities in the trait of body lipid deposition between human and pigs. In this study, we investigated the roles of RXRalpha in porcine (Sus scrofa) adipocyte differentiation by using RNAi method and primary cultured porcine adipocytes. With the transfection of RXRalpha-siRNA, the gene and protein expressions of RXRalpha were successfully inhibited in differentiating porcine adipocytes. By the RXRalpha gene silencing, the adipocyte differentiation, as characterized by lipid accumulation and GPDH activity, was suppressed. Furthermore, in association with the decrease in RXRalpha gene expression level, PPARgamma and C/EBPalpha genes expression level also decreased, suggesting that RXRalpha is involved in their expression. Thus, our study clearly demonstrated the pivotal roles of RXRalpha in porcine adipocyte differentiation.

To identify the optimal concentration of vitamin C on the proliferation and differentiation of porcine preadipocytes,the expression of five adipogenesis-related genes:peroxisome proliferator-activated receptor gamma(PPARγ),retinoid X receptor alpha(RXRα),lipoprotein lipase(LPL),phosphoenolpyruvate carboxykinase(PEPCK),stearoyl-CoA desaturase(SCD)mRNA were studied during differentiation of porcine preadipocytes induced by vitamin C(Vit C).There were two groups in the study divided by control and Vit C:(1)control group:incubating the cells without any inducer;(2)Vit C-treated group:incubating the cells with Vit C.Porcine preadipocytes were induced to differentiate by different concentration of 25,100,250,500 μmol/L Vit C.The optimal concentration of Vit C was used to treat the porcine preadipocytes.Then these cells were harvested on the 2nd,4th,6th and 8th days after differentiation,and the total RNA were abstracted.The genes PPARγ,RXRα,LPL,PEPCK,SCD mRNA expressions were assayed by SQ RT-PCR.Two results obtained from the studies:(1)100 μmol/L Vit C can promote the proliferation of porcine preadipocytes.(2)During the differentiation induced by 250 μmol/L Vit C group,the expression of PPARγ in the porcine cells was low on the 2nd day after inducing differentiation.The expression was increased lightly on the 4th day after inducing differentiation,and it was kept on the high level on the 6th and 8th day after induction.But RXRα mRNA expression increased on the 6th day,and then kept on the 8th day;The LPL,PEPCK,SCD mRNA were expressed at low level on the 2nd day after inducing differentiation,they increased significantly on the 4th day,also increased on the 6th and 8th day significantly after inducting.The conclusion showed that the sequential expression of five adipogenesis-related genes induced by VitC may be the important mechanism for the role of VitC to induce the porcine preadipocytes to differentiation.

The processing parameters of concentrated tomato juice by orthogonal experiments including soluble solid content, sterilized time, and preservative quantity were studied. The result showed that: additing 0.7% NaCl was helpful for improving the ratio of expressing juice.The optimum processing conditions were: vacuum pressure 87～93kPa at 50℃; soluble solid content 23%, sterilized time 10min and no-preservative needed. The lycopene content was 97.20g/100g and VC 5.85mg/100g.