去血清饥饿条件下肌卫星细胞增殖及自噬蛋白LC3、Beclin1的表达
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摘要
背景:自噬可促进低氧环境下肌卫星细胞的存活,然而基于自噬有明显的时效性,不同时间的自噬水平与肌卫星细胞增殖能力间的关系如何未见探讨。目的:探讨不同时间去血清饥饿对大鼠肌卫星细胞增殖能力及自噬微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)及Beclin1表达的变化。方法:原代分离培养雄性SD大鼠(广州中医药大学动物实验中心提供,体质量约120 g)多裂肌卫星细胞,取第3代细胞,培养至对数生长期后,分别设置正常血清组(体积分数10%胎牛血清)、空白血清(仅含培养基,无胎牛血清)6 h组、空白血清12 h组、空白血清24 h组,CCK-8检测各组细胞的增殖情况,并采用Western-blot法和RT-PCR法检测LC3及Beclin1蛋白及基因的表达。结果与结论:(1)细胞增殖能力:空白血清诱导12 h后细胞增殖能力较正常血清组及诱导6 h时下降(P <0.01),诱导24h后细胞增殖能力较诱导12h下降(P<0.05);(2)LC3及Beclin1蛋白及基因的表达:空白血清诱导6 h后,LC3Ⅱ/LC3Ⅰ及Beclin1蛋白及基因的表达较正常血清组明显升高(P <0.05),诱导12 h及诱导24 h后LC3Ⅱ/LC3Ⅰ蛋白表达较诱导6 h时明显升高(P <0.01),Beclin1的表达较诱导6 h时升高(P <0.05),LC3及Beclin1基因的表达较6 h均升高(P <0.05);(3)结果证实,去血清饥饿可以诱导肌卫星细胞发生自噬,自噬在去血清早期(12 h内)可保护细胞的增殖能力,12 h后细胞可出现过度自噬,不利于细胞的增殖。
BACKGROUND: Autophagy can promote the survival of muscle satellite cells in hypoxic environment. However, based on the obvious time-dependent nature of autophagy, the relationship between the level of autophagy and the proliferation ability of muscle satellite cells at different time has not been explored. OBJECTIVE: To observe the effect of different time of serum-free starvation on the proliferative capacity and expression of autophagy-related proteins, microtubule-associated protein 1 light chain 3(LC3) and Beclin1, in rat muscle satellite cells. METHODS: Primary multifidus muscle satellite cells were isolated and cultured from male Sprague-Dawley rats(provided by the Animal Experimental Center, Guangdong University of Chinese Medicine in China, about 120 g of weight). Passage 3 cells cultured in logarithmic phase were assigned into normal serum group(10% fetal bovine serum), 6-hour blank serum group, 12-hour blank serum, and 24-hour blank serum group. Proliferative capacity of cells was detected by cell counting kit-8, while the expression of LC3 and Beclin1 at protein and mRNA levels was determined by western blot and RT-PCR. RESULTS AND CONCLUSION:(1) No difference was found between the 6-hour blank serum group and the normal serum group in term of the proliferative capacity(P > 0.05), while the proliferative capacity of cells in the 12-hour blank serum group was lower than that in the normal serum group and 6-hour blank serum group(P < 0.01). The proliferative capacity of cells in the 24-hour blank serum group was lower than that in the 12-hour blank serum group(P < 0.05).(2) Compared with the normal serum group, the protein and mRNA expressions of LC3 II/LC3 I and Beclin1 in the 6-hour blank serum group increased significantly(P < 0.05), while compared with the 6-hour blank serum group, the protein and mRNA expressions of LC3 II/LC3 I and Beclin were increased significantly in the 12-hour and 24-hour blank serum groups(P < 0.01 or P < 0.05). To conclude, serum-free starvation can induce autophagy in muscle satellite cells, and the autophagy can protect the cell proliferation within 12 hours. However, excessive autophagy maybe occurs after 12 hours, which is detrimental to the cell proliferation.
BACKGROUND: Autophagy can promote the survival of muscle satellite cells in hypoxic environment. However, based on the obvious time-dependent nature of autophagy, the relationship between the level of autophagy and the proliferation ability of muscle satellite cells at different time has not been explored. OBJECTIVE: To observe the effect of different time of serum-free starvation on the proliferative capacity and expression of autophagy-related proteins, microtubule-associated protein 1 light chain 3(LC3) and Beclin1, in rat muscle satellite cells. METHODS: Primary multifidus muscle satellite cells were isolated and cultured from male Sprague-Dawley rats(provided by the Animal Experimental Center, Guangdong University of Chinese Medicine in China, about 120 g of weight). Passage 3 cells cultured in logarithmic phase were assigned into normal serum group(10% fetal bovine serum), 6-hour blank serum group, 12-hour blank serum, and 24-hour blank serum group. Proliferative capacity of cells was detected by cell counting kit-8, while the expression of LC3 and Beclin1 at protein and mRNA levels was determined by western blot and RT-PCR. RESULTS AND CONCLUSION:(1) No difference was found between the 6-hour blank serum group and the normal serum group in term of the proliferative capacity(P > 0.05), while the proliferative capacity of cells in the 12-hour blank serum group was lower than that in the normal serum group and 6-hour blank serum group(P < 0.01). The proliferative capacity of cells in the 24-hour blank serum group was lower than that in the 12-hour blank serum group(P < 0.05).(2) Compared with the normal serum group, the protein and mRNA expressions of LC3 II/LC3 I and Beclin1 in the 6-hour blank serum group increased significantly(P < 0.05), while compared with the 6-hour blank serum group, the protein and mRNA expressions of LC3 II/LC3 I and Beclin were increased significantly in the 12-hour and 24-hour blank serum groups(P < 0.01 or P < 0.05). To conclude, serum-free starvation can induce autophagy in muscle satellite cells, and the autophagy can protect the cell proliferation within 12 hours. However, excessive autophagy maybe occurs after 12 hours, which is detrimental to the cell proliferation.
引文
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[29]Chazaud B,Sonnet C,Lafuste P,et al.Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth.J Cell Biol.2003;163(5):1133-1143.
[30]江媛媛.超声造影评价兔骨骼肌损伤修复动态变化的实验性研究[D].泰安:泰山医学院,2013.
[2]Pagotto A,Pilotto G,Mazzoldi EL,et al.Autophagy inhibition reduces chemoresistance and tumorigenic potential of human ovarian cancer stem cells.Cell Death Dis.2017;8(7):e2943.
[3]Nagy P,Sándor GO,Juhász G.Autophagy maintains stem cells and intestinal homeostasis in drosophila.Sci Rep.2018;8(1):4644.
[4]Ho TT,Warr MR,Adelman ER,et al.Autophagy maintains the metabolism and function of young and old stem cells.Nature.2017;543(7644):205-210.
[5]邵蕾,朱晓萍.自噬-溶酶体途径与骨骼肌和膈肌萎缩的研究进展[J].复旦学报(医学版),2017,44(1):99-104.
[6]贡歌,杨翔,殷建等.自噬和凋亡在肌少症中的研究进展[J].中国医学科学院学报,2018,40(1):112-115.
[7]Garc??A-Prat L,Mart??Nez-Vicente M,Perdiguero E,et al.Autophagy maintains stemness by preventing senescence.Nature.2016;529(7584):37-42.
[8]刘通,于佳妮,邹德辉,等.电针血清对多裂肌卫星细胞增殖及Pax-7、成肌分化抗原、磷酸化蛋白激酶B表达的影响[J].针刺研究,2016,41(5):402-409.
[9]刘通,于佳妮,陈玉佩,等.单根肌纤维法分离培养多裂肌卫星细胞及鉴定[J].中国组织工程研究,2018,22(25):4065-4070.
[10]Buccarelli M,Marconi M,Pacioni S,et al.Inhibition of autophagy increases susceptibility of glioblastoma stem cells to temozolomide by igniting ferroptosis.Cell Death Dis.2018;9(8):841.
[11]Nguyenmccarty M,Klein PS.Autophagy is a signature of a signaling network that maintains hematopoietic stem cells.PLoSOne.2017;12(5):e0177054.
[12]Liu S,Jin Y,Shi S.Autophagy guarantees stemness of muscle stem cells by maintaining quiescence.Oral Dis.2016;23(2):139-140.
[13]Pagano TB,Wojcik S,Costagliola A,et al.Age related skeletal muscle atrophy and upregulation of autophagy in dogs.Vet J.2015;206(1):54-60.
[14]Nakashima A,Aoki A,Kusabiraki T,et al.Role of autophagy in oocytogenesis,embryogenesis,implantation,and pathophysiology of pre-eclampsia.J Obstet Gynaecol Res.2017;43(4):633-643.
[15]Mizushima N,Levine B,Cuervo AM,et al.Autophagy fights disease through cellular self-digestion.Nature.2008;451(7182):1069-1075.
[16]Noack M,Richter-Landsberg C.Activation of autophagy by rapamycin does not protect oligodendrocytes against protein aggregate formation and cell death induced by proteasomal inhibition.J MolNeurosci.2015;55(1):99-108.
[17]Levine B,Yuan J.Autophagy in cell death:an innocent convict?JClin Invest.2005;115(10):2679-2688.
[18]Loos B,Genade S,Ellis B,et al.At the core of survival:autophagy delays the onset of both apoptotic and necrotic cell death in a model of ischemic cell injury.Exp Cell Res.2011;317(10):1437-1453.
[19]Urbani L,Piccoli M,Franzin C,et al.Hypoxia increases mouse satellite cell clone proliferation maintaining both in vitro and in vivo heterogeneity and myogenic potential.PLoS One.2012;7(11):e49860.
[20]江丽斌,秦文,刘文佳,等.低氧环境下自噬对颏舌肌卫星细胞增殖与凋亡的影响[J].口腔医学研究,2015,31(3):209-212.
[21]Zhang D,Jia S,Wang H,et al.Effect of sustained hypoxia on autophagy of genioglossus muscle-derived stem cells.Med Sci Monit.2018;24:2218-2224.
[22]Li G,Wang G,Ma L,et al.miR-22 regulates starvation-induced autophagy and apoptosis in cardiomyocytes by targeting p38α.Biochem Biophys Res Commun.2016;478(3):1165-1172.
[23]安洋,张慧宇,郭俊峰,等.降钙素基因相关肽对血清饥饿作用下MC3T3-E1成骨细胞凋亡和自噬的影响[J].华西口腔医学杂志,2017,35(2):133-138.
[24]Kim SN,Kwon HJ,Akindehin S,et al.Effects of epigallocatechin-3-gallate on autophagic lipolysis in adipocyte.Nutrients.2017;9(7):680.
[25]Xin W,Klionsky DJ.Autophagy is a key factor in maintaining the regenerative capacity of muscle stem cells by promoting quiescence and preventing senescence.Autophagy.2016;12(4):617-618.
[26]Yi C,Tong J,Lu P,et al.Formation of a Snf1-Mec1-Atg1 module on mitochondria governs energy deprivation-induced autophagy by regulating mitochondrial respiration.Deve Cell.2017;41(1):59-71.
[27]Xu DQ,Wang Z,Wang CY,et al.PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14Lassociated PI3Kactivity.EMBO J.2016;35(5):496-514.
[28]Paolini A,Omairi S,Mitchell R,et al.Attenuation of autophagy impacts on muscle fibre development,starvation induced stress and fibre regeneration following acute injury.Sci Rep.2018;8(1):1-12.
[29]Chazaud B,Sonnet C,Lafuste P,et al.Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth.J Cell Biol.2003;163(5):1133-1143.
[30]江媛媛.超声造影评价兔骨骼肌损伤修复动态变化的实验性研究[D].泰安:泰山医学院,2013.