| Adipose tissue is an important organ in charge of energy deposition and endocrine in organisms,which is involved in regulating the physiological processes such as fatty acid(FA)metabolism,carbohydrate metabolism and cholesterol metabolism of body.Adipose-derived mesenchymal stem cells(ADSCs)are the pluripotent stem cells existing in adult adipose tissue,which is ideal model for not only differentiation of adipocyte but also energy metabolism or signaling pathways.The composition of FAs in adipose tissue has significant effect on flavor and taste of meat products of livestock,in which monounsaturated FAs(MUFA)is considered to be one of key factors.The rate-limiting enzyme stearoyl-CoA desaturase 1(SCD1),which catalyzed synthesis of MUFA in the animal tissues,participates in the regulation of FA metabolism and energy balance and therefore has a direct effect on quality of livestock meat products.This study was conducted to isolate and identify bovine adipose-derived mesenchymal stem cells(bADSCs),and to assess the effect of overexpression of SCD1 gene on the expression of FA metabolism related genes and the composition of FAs,aiming to explore the method of elevating MUFA content in bovine adipose by gene modification.1.Isolation,cultivation and identification of bADSCsADSCs were isolated from cattle subcutaneous fat,and the biological characteristics were detected including growth curve,chromosome number and clone formation capability and the marker molecular and features of multipotential were identified by immunofluorescence,flowcytometry and differentiation induction in vitro.Results showed that the obtained bADSCs proliferated rapidly in vitro and had the capability for clone formation,and the biological characteristics of bADSCs were kept after continuously cultured to passage 55 such as stable chromosome number;the cells of P5 expressed CD13,CD44,CD49d,CD90,CD105,and Vimentin while none expressed CD34.The positive cells of CD44 and CD90 were 92.24±2.12%and 15.33±1.49%,respectively,accompanied by gradually decreased ratio along with passaging.The cells of P5 underwent adipogenic,osteogenic or chondrogenic induction,respectively,followed by positive detection of specific marker by histochemical staining or RT-PCR.Results above showed that isolation and identification of bADSCs were successfully achieved.Then genes involved in FA metabolism were detected by Realtime PCR during the adipogenetic induction of bADSCs.The transcriptional level of SCD1 and other genes related to lipogenesis increased significantly at day 7 of induction and then declined(P<0.05);that of genes related to FA deposition were increased to the peak at 14d and then declined;while,the transcriptional peak of genes related to FA oxygenolysis varied(P<0.05).The FA composition of bADSCs was examined by gas chromatograph-mass spectrometer during adipogenetic differentiation,and the content of palmitoleic acid(C16:1),oleic acid(C18:1),MUFA and polyunsaturated FA(PUFA)increased significantly at day 7,then declined,as well as desaturation index(C16:1/C16:0 and C18:1/C18:0)(P<0.05).Moreover the content of palmitic acid(C16:0),stearic Acid(C18:0),and saturated FA(SFA)decreased at day 7(P<0.05)and increased slightly from day 14 to day 21(P>0.05).These indicated that day 7-14 after induction is an active stage for lipogenesis and day 7 is the peak stage of unsaturated FA synthesis and SCD1 is critical to the lipogenesis.2.Effect of SCD1 overexpression on genes related to FA metabolism and FA composition in bovine fetal fibroblasts(bEFs)and bovine adipose-derived mesenchymal stem cells(bADSCs)A 5.9kb adipose specific promoter of mouse FABP4 gene(mFABP4)was shortened to 2.3kb by EcoT22I digestion.The 5.9kb and 2.3kb fragment were ligated to pDs-Red2-1 respectively,and then expression of red fluorescence protein was examined in transfected bEFs and bADSCs by Realtime PCR.Results showed that the efficiency of 2.3kb fragment increased about 1 fold than that of 5.9kb fragment.The constructed vector pFS was consisted of SCD1 driven by 2.3kb fragment of mFABP4 promoter.The vector was transfected into bEFs and bADSCs,respectively,and the transfection condition was determined by transfection efficiency analysis through flowcytometry.The stable transfected cell clones selected by G418 were further surveyed by PCR,and the copy number of DNA integration was detected by Realtime PCR.One bEFs transgenic clone was identified with the copy number as 0.99 ± 0.05;while transgenic clones from bADSCs were identified as 2.00±0.23,6.07±0.37 and 3.94±0.29,respectively.Genes related to FA metabolism were detected by Realtime PCR and the FA composition were detected by gas chromatograph-mass spectrometer in the transient transfected cells after 24h,48h and72h and in the stable transfected cell clones.Results were as follows:1)In transient transfected bEFs cells,after 24h of transfection,the transcriptional level of SCD1 and sterol regulatory element binding protein-1(SREBP1)which is important transcription factor for lipogenesis increased significantly and then gradually reduced(P<0.05).And the transcriptional level of genes related to FA oxygenolysis were steadily higher than that of control group(P<0.05).And majority of other genes detected showed the trend as it raised firstly and then declined.In the transient transfected bADSCs cells,the transcriptional level of SCD1,peroxisome proliferator-activated receptor gamma(PPARg)which is a key transcription factor to adipogenesis,and adipocyte specific marker gene,fatty acid-binding protein(FABP4)increased continuously(P<0.05).Genes related to FA oxygenolysis were steadily higher than that of control group(P<0.05),and other genes detected had no significant change.2)In stable transfected bEFs cells,the expression of SCD1 mRNA in clone 1#were significantly lower than that of control group(P<0.05)and the expression of other genes changed diversely.Compared to the control group,C16:0,C18:0 and SFA were significantly raised(P<0.05),and C16:1,C18:1,desaturation index and MUFA had no difference(P>0.05),and PUFA decreased significantly(P<0.05).In stable transfected bADSCs cells,the expression of SCD1 and the bulk of other detected genes related to FA metabolism in clone 1#were lower than that of control,whereas PPARg increased significantly(P<0.05).C16:0,C16:1,C18:0,C16:1/C16:0 were similar to the control group,C18:1 and C18:1/C18:0 were significantly lower(P<0.05).Transgenic clone 2#showed that transcription level of SCD1 gene as well as other genes related to FA metabolism increased significantly.C16:1 and desaturation index were the highest.C16.0 and C18:0 were significantly lower than other groups of cells.Transgenic clone 3#showed that mRNA level of SCD1 gene and the bulk of genes related to FAs metabolism decreased except for PPARg.Nonetheless C16:0,C18:0 and desaturation index had no difference with control.C16:1 is significantly higher than control and C18:1 was significantly lower.Among the transgenic clones of bADSCs,clone 1#had the highest content of SFA,lowest content of MUFA and moderate PUFA;clone 2#had the lowest content of SFA and the highest content of MUFA and PUFA;the content of SFA,MUFA and PUFA of clone 3#were all close to the top value.These results indicated that transcription of SCD1 gene were different between non-adipocyte and ADSCs,as well as their FA composition.3)The expression of genes related to FA metabolism and the FA composition were further detected in bADSCs transgenic clones during the process of adipogenic induction.At Day 7 of induction,all the bADSCs transgenic clones expressed lower or equivalent mRNA level of SCD1 and the majority of genes detected.Transgenic clone 1#had the highest value of C16:1,C18:1,desaturation index and MUFA,while clone 2#and 3#had no significant difference with control group.At Day 14 of induction,mRNA level of SCD1 was as follow:clone 2#,clone 3#,clone 1#and control(P<0.05).All the bADSCs transgenic clones had higher C16:1 and MUFA but lower SFA compared to control group.There were no difference of the transcription of SCD1 gene among the 3 transgenic clones,but after adipogenic induction,it was coincidentally increased in the 3 clones,accompanied by elevation of MUFA content.These results indicated that specifically overexpression of SCD1 in adipocyte could improve the intracellular MUFA content.In conclusion,bADSCs were isolated from subcutaneous fat of cattle,and were identified with the characteristics of adipose derived mesenchymal stem cells in this study.Transfection of exogenous SCD1 can instantaneously increase the expression of genes related to intracellular lipogenesis,but in stable transfected cell lines the expression of SCD1 gene were different between bEFs and bADSCs.However,after adipogenic induction,the expression of SCD1 increased in coincidence with its product(C16:1,C18:1),desaturation index and MUFA.Therefore,transfection of SCD1 gene driven by adipose cell-specific promoter(mFABP4)could elevate the content of MUFA in adipocyte specifically,although the expression features and the regulation mechanism still need to be clarified.These results laid a foundation for further study of regulation mechanism of SCD1 over lipid metabolism,and provided possible method for improving the beef quality through genetic modification. |
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