摘要
背景:睫状神经因子通过细胞表面的受体可以使成肌细胞去分化而转变为多能性干细胞,而p44/p42激酶可以进一步激活睫状神经因子下游基因的表达,与细胞去分化密切相关。目的:观察丹参注射液诱导成肌细胞分化成神经元的细胞模型中p42/p44激酶磷酸化的情况。设计、时间及地点:细胞分子水平实验,于2008-03/06在辽宁医学院附属第二医院口腔科学实验室完成。材料:清洁级新生1周SD大鼠1只,由辽宁医学院实验动物中心提供。丹参注射液为四川三精升和制药有限公司产品,批号080717,主要成分为丹参。睫状神经营养因子为Sigma产品。方法:体外分离培养大鼠成肌细胞,传至第5代接种于6孔板内,诱导组使用50μg/L睫状神经因子预诱导72h,再更换含复方丹参注射液的无血清DMEM培养基诱导10h;对照组使用不含复方丹参注射液的无血清DMEM培养基处理。取诱导后的成肌细胞,加入p44/p42激酶抑制剂PD98059进行干预处理。主要观察指标:Western blotting法检测诱导后成肌细胞蛋白标记物MyoD,Myf5的表达,神经元蛋白标记物酪氨酸羟化酶、神经丝的表达,以及p44/p42激酶在成肌细胞蛋白标记物下调过程中的作用。结果:与未诱导的对照组比较,培养的成肌细胞经睫状神经因子预诱导、再加入丹参注射液诱导的过程中,成肌细胞蛋白标记物MyoD,Myf5的表达均明显下调;而诱导后期神经元特异蛋白标记物酪氨酸羟化酶、神经丝的表达增高。丹参诱导后成肌细胞p44/p42激酶发生磷酸化,同时MyoD和Myf5的表达量下调;在p44/p42激酶抑制剂PD98059存在情况下,p44/p42激酶磷酸化及MyoD,Myf5表达量下调均被抑制。结论:经睫状神经因子预诱导后逐渐去分化的成肌细胞,在丹参注射液的诱导下能够成功分化为表达神经元特异蛋白标记物的神经元样细胞,p44/p42激酶通过磷酸化参与了成肌细胞的再分化过程。
BACKGROUND： Ciliary neurotrophic factor by cell surface receptor can induce myoblast dedifferentiation and change into pluripotent stem cells. The p44/p42 kinase can further activate expression of downstream gene of ciliary neurotrophic factor, which is closely associated with cell dedifferentiation. OBJECTIVE： To study p42/p44 kinase phosphorylation in cell models of myoblast differentiation into neurons induced by Salvia miltiorrhiza injection in vitro. DESIGN, TIME AND SETTING： A cell molecular level study was performed at the Oral Science Laboratory of Second Affiliated Hospital of Liaoning Medical University from March to June 2008. MATERIALS： One clean Sprague Dawley rats aged 1 week were provided by Experimental Animal Center, Liaoning Medical University. Salvia miltiorrhiza injection was purchased from Sanjingshenghe, Sichuan, China （lot number 080717）, and the main component was Salvia miltiorrhiza. Ciliary neurotrophic factor （Sigma, USA） was used in this study. METHODS： In vitro isolated rat myoblasts at passage 5 were incubated in a 6-well plate. In the induction group, cells were induced using 50 ~ g/L ciliary neurotrophic factor for 72 hours, and then with serum-free DMEM containing compound Salvia miltiorrhiza injection for 10 hours. In the control group, cells were treated with serum-free DMEM without compound Salvia miltiorrhiza injection. Cells after induction were intervened with p44/p42 kinase inhibitor PD98059. MAIN OUTCOME MEASURES： MyoD and Myf5 as two biomarkers of muscle-derived stem cells, expression of neurofilament and tyrosine hydroxylase as two biomarkers of neuron-like cells, and p44/p42 kinase effect during downregulation of myoblast protein markers were measured by Western blot assay. RESULTS： Compared with the control group, MyoD, Myf5 expression level was downregulated, but the expression of neurofilament and tyrosine hydroxylase, two biomarkers of neuron, was upregulated, after the myoblast was induced with ciliary neurotrophic factor and Salvia miltiorrhiza injection. Following Salvia miltiorrhiza induction, p44/p42MAPK was phosphorylated, and MyoD, Myf5 expression was downregulated. The p44/p42 kinase inhibitor PD98059 could inhibit p44/p42MAPK phosphorylation and MyoD and Myf5 expression downregulation. CONCLIUSION： Human myoblast could be induced to neuron-like cells treated with ciliary neurotrophic factor and Salvia miltiorrhiza injection. The p44/p42 kinase participates in redifferentiation of myoblasts by phosphorylation.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第19期3656-3660,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30772190/c1607)~~
