牛原代肌肉卫星细胞和前体脂肪细胞不同混合比例共培养细胞活性的比较
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摘要
为探索不同细胞混合比例对牛原代肌肉卫星细胞和前体脂肪细胞共培养体系细胞活性的影响,本研究采集新生秦川犊牛背最长肌和肾周脂肪,分离提取肌卫星细胞和前体脂肪细胞,采用DMEM/F12培养基,建立不同血清浓度(5%、10%、15%、20%的胎牛血清,FBS)的牛原代肌肉卫星细胞和前体脂肪细胞的共培养体系,然后通过分别调整各共培养体系中肌卫星细胞和前体脂肪细胞的混合比例(肌肉细胞∶脂肪细胞=10∶1、5∶1、2∶1),来研究细胞混合比例对各共培养体系的影响。共培养14d,每两天更换培养基,并采用MTT染色法测定细胞活性。统计分析后发现∶随着共培养体系中前体脂肪细胞比例增加,共培养细胞活性增加;共培养10d以后不同混合比例细胞活性有着显著差异(P<0.05);混合比例为2∶1时共培养细胞活性最高。综上,为了获得最好的牛肌卫星细胞和前体脂肪细胞共培养效果,达到较高的细胞培养活性,建议牛肌卫星细胞和前体脂肪细胞按5∶1或2∶1的混合比例进行共培养。
To explore the effect of cellular viability in different cells mixed proportion of co-culture bovine muscle satellite cells and preadipocytes, there were longissimus dorsi and perirenal fat collected from new-born Qinchuan calf to isolate the muscle satellite cells and preadipocytes. Using the common culture medium of DMEM/F12 with different proportions of serum(fetal bovine serum∶ 5%, 10%, 15%, 20%(v/v)), it was established a co-culture system of 14-days cultivation. In each co-culture system, we changed the cellular mixed ratio(the ratio of SMSC to preadipocytes were 2∶1, 5∶1, and 10∶1), and changed the medium and determined cellular viability by MTT assay in every 2 days, to compare the cellular viability under different mixed proportion. After the statistical analysis, the results showed that, 1) with the increase of the proportion of preadipocytes in this co-culture system, the co-culture cellular viability increased. 2) after 10-day, there was significant difference between different mixed ratio. 3)there was the highest cellular viability at the 2∶1 mixing ratio of muscle satellite cells and preadipocytes. So that, in order to obtain the highest cellular viability of co-culture bovine muscle satellite cells and preadipocytes, it was suggested that the mixture ratio were 5∶1 or 2∶1.
To explore the effect of cellular viability in different cells mixed proportion of co-culture bovine muscle satellite cells and preadipocytes, there were longissimus dorsi and perirenal fat collected from new-born Qinchuan calf to isolate the muscle satellite cells and preadipocytes. Using the common culture medium of DMEM/F12 with different proportions of serum(fetal bovine serum∶ 5%, 10%, 15%, 20%(v/v)), it was established a co-culture system of 14-days cultivation. In each co-culture system, we changed the cellular mixed ratio(the ratio of SMSC to preadipocytes were 2∶1, 5∶1, and 10∶1), and changed the medium and determined cellular viability by MTT assay in every 2 days, to compare the cellular viability under different mixed proportion. After the statistical analysis, the results showed that, 1) with the increase of the proportion of preadipocytes in this co-culture system, the co-culture cellular viability increased. 2) after 10-day, there was significant difference between different mixed ratio. 3)there was the highest cellular viability at the 2∶1 mixing ratio of muscle satellite cells and preadipocytes. So that, in order to obtain the highest cellular viability of co-culture bovine muscle satellite cells and preadipocytes, it was suggested that the mixture ratio were 5∶1 or 2∶1.
引文
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[2]PANDURANGAN M,HWANG I.Application of cell co-culture system to study fat and muscle cells [J].Appl Microbiol Biot,2014,98(17)∶ 7359-64.
[3]PENG X D,XU P Z,CHEN M L,et al.Dwarfism,impaired skin development,skeletal muscle atrophy,delayed bone developement,and impeded adipogenesis in mice lacking Akt1 and Akt2 [J].Genes & development,2003,17(11)∶ 1352-65.
[4]VIDALPUIG A J,CONSIDINE R V,JIMENEZLINAN M,et al.Peroxisome proliferator-activated receptor gene expression in human tissues - Effects of obesity,weight loss,and regulation by insulin and glucocorticoids [J].Journal of Clinical Investigation,1997,99(10)∶ 2416-22.
[5]SEALE P,BJORK B,YANG W L,et al.PRDM16 controls a brown fat/skeletal muscle switch [J].Nature,2008,454(7207)∶ 961-U27.
[6]GU W,SCHNEIDER J W,CONDORELLI G,et al.INTERACTION OF MYOGENIC FACTORS AND THE RETINOBLASTOMA PROTEIN MEDIATES MUSCLE-CELL COMMITMENT AND DIFFERENTIATION [J].Cell,1993,72(3)∶ 309-24.
[7]FLORINI J R,EWTON D Z,COOLICAN S A.Growth hormone and the insulin-like growth factor system in myogenesis [J].Endocrine reviews,1996,17(5)∶ 481-517.
[8]ARNOLD L,HENRY A,PORON F,et al.Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis [J].Journal of Experimental Medicine,2007,204(5)∶ 1057-69.
[9]WEISBERG S P,MCCANN D,DESAI M,et al.Obesity is associated with macrophage accumulation in adipose tissue [J].Journal of Clinical Investigation,2003,112(12)∶ 1796-808.
[10]YAMAUCHI T,KAMON J,WAKI H,et al.The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity [J].Nature medicine,2001,7(8)∶ 941-6.
[11]HAWKE T J,GARRY D J.Myogenic satellite cells∶ physiology to molecular biology [J].Journal of applied physiology,2001,91(2)∶ 534-51.
[12]CHARGE S B P,RUDNICKI M A.Cellular and molecular regulation of muscle regeneration [J].Physiological Reviews,2004,84(1)∶ 209-38.
[13]BLAIS A,TSIKITIS M,ACOSTA-ALVEAR D,et al.An initial blueprint for myogenic differentiation [J].Genes & development,2005,19(5)∶ 553-69.
[14]GOODELL M A,JACKSON K A,MAJKA S M,et al.Stem cell plasticity in muscle and bone marrow [M].ORLIC D,BRUMMENDORF T H,SHARKIS S J,et al.Hematopoietic Stem Cells 2000 Basic and Clinical Sciences.New York;New York Acad Sciences.2001∶ 208-20.
[15]RYAN K J P,DANIEL Z C T R,CRAGGS L J L,et al.Dose-dependent effects of vitamin D on transdifferentiation of skeletal muscle cells to adipose cells [J].Journal of Endocrinology,2013,217(1)∶ 45-58.
[16]ZHANG J,CUI X W,SHEN Y H,et al.Distinct expression profiles of LncRNAs between brown adipose tissue and skeletal muscle [J].Biochemical and biophysical research communications,2014,443(3)∶ 1028-34.
[17]CHU W W,WEI W,YU S G,et al.C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene [J].Biochemical and biophysical research communications,2016,472(1)∶ 68-74.
[18]PANDURANGAN M,RAVIKUMAR S.Impact of stress hormone on adipogenesis in the 3T3-L1 adipocytes [J].Cytotechnology,2014,66(4)∶ 619-24.
[19]ZDYCHOVA J,CEJKOVA S,LESNA I K,et al.Co-Cultivation of Human Aortic Smooth Muscle Cells With Epicardial Adipocytes Affects Their Proliferation Rate [J].Physiol Res,2014,63(S419-S27.
[20]YAN J,GAN L,YANG H,et al.The proliferation and differentiation characteristics of co-cultured porcine preadipocytes and muscle satellite cells in vitro [J].Molecular biology reports,2013,40(4)∶ 3197-202.
[21]HAUSMAN G J,POULOS S P.A method to establish co-cultures of myotubes and preadipocytes,from collagenase digested neonatal pig semitendinosus muscles [J].Journal of Animal Science,2005,83(5)∶ 1010-6.
[22]CHOI S H,CHUNG K Y,JOHNSON B J,et al.Co-culture of bovine muscle satellite cells with preadipocytes increases PPAR gamma and C/EBP beta gene expression in differentiated myoblasts and increases GPR43 gene expression in adipocytes [J].Journal of Nutritional Biochemistry,2013,24(3)∶ 539-43.
[23]OHYAMA M,MATSUDA K,TORII S,et al.The interaction between vitamin A and thiazolidinedione on bovine adipocyte differentiation in primary culture [J].J Anim Sci,1998,76(1)∶ 61-5.
[24]SURYAWAN A,HU C Y.Effect of retinoic acid on differentiation of cultured pig preadipocytes [J].J Anim Sci,1997,75(1)∶ 112-7.
[25]GAO Q,ZHAO L,SONG Z,et al.Expression pattern of embryonic stem cell markers in DFAT cells and ADSCs [J].Molecular biology reports,2012,39(5)∶ 5791-804.
[26]CHUNG K Y,JOHNSON B J.Melengestrol acetate enhances adipogenic gene expression in cultured muscle-derived cells [J].J Anim Sci,2009,87(12)∶ 3897-904.
[27]WU X,WANG S,CHEN B,et al.Muscle-derived stem cells∶ isolation,characterization,differentiation,and application in cell and gene therapy [J].Cell and tissue research,2010,340(3)∶ 549-67.
[28]TREMP M,SALEMI S,LARGO R,et al.Adipose-derived stem cells (ADSCs) and muscle precursor cells (MPCs) for the treatment of bladder voiding dysfunction [J].World journal of urology,2014,32(5)∶ 1241-8.
[29]NI X C,SUN W,SUN S P,et al.Therapeutic Potential of Adipose Stem Cells in Tissue Repair of Irradiated Skeletal Muscle in a Rabbit Model [J].Cellular reprogramming,2014,16(2)∶ 140-50.