CN103710387B - A method of bovine skeletal muscle satellite cell proliferation promoting - Google Patents

A method of bovine skeletal muscle satellite cell proliferation promoting Download PDF

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CN103710387B
CN103710387B CN 201310745773 CN201310745773A CN103710387B CN 103710387 B CN103710387 B CN 103710387B CN 201310745773 CN201310745773 CN 201310745773 CN 201310745773 A CN201310745773 A CN 201310745773A CN 103710387 B CN103710387 B CN 103710387B
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bovine
muscle satellite
skeletal muscle
mir
133b
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CN103710387A (en )
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丁向彬
王轶敏
刘新峰
郭宏
代阳
聂永伟
陈微微
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天津农学院
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Abstract

本发明涉及一种促进牛骨骼肌卫星细胞体外增殖的方法,包括步骤有:包含牛miR-133b基因序列的目的序列的获取:表达载体pEGFP-C1-miR133b的构建:pEGFP-C1-miR133b转染牛骨骼肌卫星细胞:一种促进牛骨骼肌卫星细胞体外增殖的方法的应用,通过牛骨骼肌卫星细胞过表达微小RNA miR-133b的获取,利用miR-133b的作用,提高牛骨骼肌卫星细胞的体外增殖能力,并有效抑制其分化。 The present invention relates to a method for promoting the proliferation of bovine skeletal muscle satellite cells in vitro, comprising the steps of: bovine gene sequence comprising the sequence of acquisition of miR133b: Construction of vector pEGFP-C1-miR133b expression: pEGFP-C1-miR133b transfection bovine skeletal muscle satellite cells: An application method of a cell proliferation promoting skeletal muscle satellite bovine, bovine skeletal muscle satellite cells by micro RNA miR-133b acquiring, by the action of miR-133b, of bovine skeletal muscle satellite cells overexpressing proliferation in vitro, and to inhibit their differentiation. 本发明利用pEGFP-C1构建miR-133b的过表达载体,利用微小RNA对牛肌卫星细胞的增殖和分化进行调控,得到高纯度的牛肌卫星细胞,为研究各调控因子在肌卫星细胞增殖与分化过程中的功能和影响提供良好的细胞模型。 The present invention utilizes pEGFP-C1 constructs miR-133b overexpression vector on proliferation and differentiation of bovine muscle satellite cells is regulated with a minute an RNA, high purity bovine muscle satellite cells, for studying various regulatory factor in muscle satellite cell proliferation and during differentiation and function to provide a good influence cell model.

Description

一种促进牛骨骼肌卫星细胞体外増殖的方法 A method of promoting in vitro bovine skeletal muscle satellite cells colonize zo

技术领域 FIELD

[0001] 本发明属于生物技术领域的细胞工程技术,具体涉及一种促进牛骨骼肌卫星细胞体外增殖的方法。 [0001] The present invention belongs to the technical field of cell biological engineering technology, particularly to a method of promoting the proliferation of bovine skeletal muscle satellite cells in vitro.

背景技术 Background technique

[0002] 骨骼肌卫星细胞(skeletal muscle satellite cell)是骨骼肌中具有分化增殖潜能的肌源性干细胞,通常以静息状态存在于肌纤维肌膜与基底膜之间,在一定的条件下可以被激活,发生增殖和分化,形成骨骼肌细胞,肌卫星细胞在动物出生后肌肉的生长发育和再生过程中发挥着十分重要的作用。 [0002] skeletal muscle satellite cells (skeletal muscle satellite cell) is a skeletal muscle-derived stem cells having differentiation and proliferation potential, typically present in the resting state between the base film and the sarcolemma of muscle fibers, under certain conditions can be activated, proliferate and differentiate to form skeletal muscle cells, muscle satellite cells play an important role in the growth and regeneration of muscle in an animal born. 肌卫星细胞的激活、增殖与分化过程受多种因素的调控,了解骨骼肌卫星细胞的发生调控模式,可以增加人为控制肌细胞形成的可能性。 Muscle satellite cell activation, proliferation and differentiation are regulated by many factors, understanding the regulation of the occurrence of mode skeletal muscle satellite cells can increase the likelihood of human control muscle cell formation. 目前骨骼肌卫星细胞的研宄工作主要集中于肌卫星细胞的分离培养、鉴定及影响其增殖和分化的机制等方面。 Work is currently in a Subsidiary skeletal muscle satellite cells isolated and cultured mainly in muscle satellite cells, the identification and impact mechanisms of proliferation and differentiation, and the like. 在分离肌卫星细胞时往往掺杂着其它细胞,而且骨骼肌卫星细胞在低血清浓度下会自发地分化为骨骼肌细胞,使得要获得大量高纯度的肌卫星细胞具有很大的挑战性,如何提高肌卫星细胞的体外增殖能力并抑制其分化成为高效并经济地获取足量高纯度肌卫星细胞的关键。 When isolated muscle satellite cells are often mixed with other cells, and skeletal muscle satellite cells under low serum concentrations spontaneously into skeletal muscle cells, so that to obtain a large amount of high purity muscle satellite cells having great challenge, how improve the ability of muscle satellite cell proliferation and inhibit differentiation become critical efficiently and economically obtain a sufficient amount of high purity muscle satellite cells.

[0003] 骨骼肌增殖和分化过程涉及多基因的表达、信号途径及网络式调控,过程极其复杂。 [0003] skeletal muscle proliferation and differentiation process involves expression of multiple gene-regulatory signaling pathways and networks, extremely complex process. 已有研宄表明微小RNA (microRNAs,miRNAs)在肌肉发育和肌细胞增殖与分化中发挥了关键性的调控作用。 Research has shown that traitor small RNA (microRNAs, miRNAs) play a key role in regulating muscle development and muscle cell proliferation and differentiation. miRNAs是一类广泛存在于真核细胞当中由基因组编码的长度约22nt的高度保守的内源性非编码单链RNA,它们通过与对应的靶mRNA的3'非翻译区(3'untranslated reg1n, 3'UTR)的非完全或完全配对结合,阻止其翻译或破坏其稳定性,实现对基因表达的转录后调控。 miRNAs are a class of widely distributed among eukaryotic cells by the length of the group of highly conserved genes encoding endogenous noncoding about 22nt single-stranded RNA, which by the 3 'untranslated region of the corresponding target mRNA (3'untranslated reg1n, 3'UTR) regulation after incomplete binding or exact match, prevent or destroy translation stability, effect transcription of the gene expression. 可以利用表达载体将miRNAs转染导入细胞或动物体内,用于研宄miRNAs的功能,一般导入细胞或动物体内的miRNAs基因,应使用包含该miRNAs基因前体的一段序列,研宄发现所选择的片段至少应该在miRNAs基因前体两侧各自延伸40nt,这样可以使转入基因更有效的被剪切加工成为成熟的miRNAs。 MiRNAs may utilize expression vector transfected into a cell or an animal for study based on the function of miRNAs, miRNAs generally introduced gene or animal cells, it should be used comprising a sequence of the genome before the miRNAs, a Subsidiary selected found fragments should at least before the respective sides thereof extending miRNAs genes 40nt, so that the transgene can be more effectively sheared into mature miRNAs. miRNAs作用的靶基因几乎覆盖细胞每一条信号通路,参与细胞生长、增殖、发育、分化和细胞凋亡等多个生命活动环节。 MiRNAs target gene effect, covering almost every cellular signaling pathways involved in cell growth, proliferation, a plurality of links life activities development, differentiation and apoptosis. miR-133是肌肉组织特异性miRNAs,它在心肌和骨骼肌均表达,它参与了肌细胞的增殖与分化、肌肉的生理病理学等过程,具有重要功能。 miR-133 is a muscle-specific of miRNAs that were expressed in cardiac and skeletal muscle, it is involved in the proliferation and differentiation of muscle cells, muscle and other physiopathological processes, have important functions. 研宄发现miR-133在小鼠C2C12成肌细胞分化过程中迅速上调,进一步的功能研宄证明miR-133促进成肌细胞的增殖并抑制其分化。 A Subsidiary found miR-133 in mouse C2C12 myoblast differentiation during shoot up, further demonstrate the function of miR-133 in a Subsidiary promote muscle cell proliferation and inhibits differentiation. 研宄也证明miR-133能促进大鼠和人胚胎干细胞增殖并抑制其分化。 A Subsidiary miR-133 also proved to promote rat and human embryonic stem cell proliferation and inhibit their differentiation. 过表达miR-133也能抑制血管平滑肌细胞向成骨样细胞的分化。 Overexpression of miR-133 also inhibits vascular smooth muscle cells to osteoblast-like cell differentiation. miRNAs不仅在结构上保守而且在物种间具有高度的进化保守性,预示着某些miRNAs在牛肌肉发育分化中可能具有非常重要的调控作用,因此,验证并确定利用牛miR-133来促进牛肌卫星细胞的增殖并抑制其分化效果,建立牛肌卫星细胞高效的体外培养和纯化方法就成了本领域技术人员亟待解决的课题。 miRNAs conserved not only in structure but also a high degree of evolutionary conservation across species, suggesting some miRNAs may have a very important role in cattle muscle development and differentiation, therefore, to verify and determine the use of bovine cattle miR-133 promotes muscle inhibition of proliferation of satellite cells and their differentiation effect, establish efficient bovine muscle satellite cells in vitro and purification methods become those skilled urgent subject.

发明内容 SUMMARY

[0004] 鉴于miR-133对肌细胞增殖和分化的影响,本发明的目的在于提供一种通过过表达miR-133b来促进牛骨骼肌卫星细胞的体外增殖能力,并有效抑制其分化的方法,借助此方法并对肌卫星细胞进行纯化可以得到高纯度的牛肌卫星细胞,为研宄各调控因子在肌卫星细胞增殖与分化过程中的功能和影响提供良好的细胞模型。 [0004] Given the impact of miR-133 on muscle cell proliferation and differentiation, an object of the present invention is to provide a by overexpression of miR-133b ability to promote proliferation of bovine skeletal muscle satellite cells, and methods effective to inhibit their differentiation, with this method and muscle satellite cells can be purified with high purity bovine muscle satellite cells, for the study based on the respective regulators to provide a good model of cell function and muscle satellite cells influence proliferation and differentiation processes.

[0005] 本发明的技术方案如下: [0005] aspect of the present invention is as follows:

[0006] 一种促进牛骨骼肌卫星细胞体外增殖的方法,包括以下步骤: [0006] A method of in vitro cell proliferation promoting bovine skeletal muscle satellite, comprising the steps of:

[0007] 第一步、包含牛miR_133b基因序列的目的序列的获取: [0007] The first step, object miR_133b bovine gene sequence comprising obtaining sequence:

[0008] 根据牛miR-133b基因序列设计一对PCR引物,在两个引物的5'端分别引入XhoI酶切位点和BamHI酶切位点,利用该对引物从牛基因组中扩增出包含miR-133b基因的全长为31 Ibp的一段序列; [0008] to design a pair of PCR primers according bovine miR-133b gene sequence, were introduced into the XhoI restriction site and BamHI restriction sites at the 5 'ends of two primers, using this pair of primers amplified from bovine genome comprising miR-133b full-length gene is a sequence of 31 Ibp;

[0009] 第二步、表达载体pEGFP-Cl-miR133b的构建: [0009] The second step, the expression vector pEGFP-Cl-miR133b of:

[0010] 用XhoI和BamHI双酶切第一步中所得序列及pEGFP_Cl载体,通过连接酶连接,使所述序列插入质粒载体pEGFP-Cl的XhoI和BamHI酶切位点,构建出牛miR_133b基因的表达载体pEGFP-Cl-miR133b ; [0010] double digestion with XhoI and BamHI resulting in a first step and pEGFP_Cl vector sequence by a ligase, the sequence inserted into the plasmid pEGFP-Cl vector into the XhoI and BamHI restriction sites, the gene construct bovine miR_133b The expression vector pEGFP-Cl-miR133b;

[0011] 第三步、pEGFP-Cl-miR133b转染牛骨骼肌卫星细胞: [0011] The third step, pEGFP-Cl-miR133b bovine skeletal muscle satellite cells transfected with:

[0012] 用得到的表达载体pEGFP-Cl-miR133b,采用脂质体转染试剂转染牛骨骼肌卫星细胞,使牛骨骼肌卫星细胞过表达微小RNA miR-133b。 [0012] with an expression vector pEGFP-Cl-miR133b obtained using Lipofectamine transfection reagent bovine skeletal muscle satellite cells, bovine skeletal muscle satellite cells overexpressing micro RNA miR133b.

[0013] 而且,所述第一步中包含miR-133b基因的全长为311bp的一段序列进一步包含牛mir-133b基因84bp,基因前序列95bp,基因后序列132bp,总共311bp。 [0013] Further, the entire length of the first step comprising miR-133b gene is a sequence of 311 bp further comprising the 84 bp bovine mir-133b gene, gene sequences 95bp before, the 132-bp gene sequence, total 311bp.

[0014] 而且,所述第三步采用脂质体转染试剂转染牛骨骼肌卫星细胞是在转染前一天把牛骨骼肌卫星细胞培养到六孔板内,使第二天细胞能达到约70%〜80%满,在进行转染前,每孔更换成新鲜的无血清细胞培养液2ml。 [0014] Further, the third step using Lipofectamine transfection reagent in bovine skeletal muscle satellite cells the day before transfection the culture of bovine skeletal muscle satellite cells into six-well plates, the cells can be achieved so that the next day about 70% ~ 80% full, before performing transfection, each well was replaced with fresh serum-free cell culture medium 2ml.

[0015] —种促进牛骨骼肌卫星细胞体外增殖的方法的应用,通过牛骨骼肌卫星细胞过表达微小RNA miR-133b的获取,利用miR_133b的作用,提高牛骨骼肌卫星细胞的体外增殖能力,并有效抑制其分化。 [0015] - A method of promoting proliferation of human species bovine skeletal muscle satellite applications, bovine skeletal muscle satellite cells acquired by micro RNA miR-133b overexpression, by the action of miR_133b improve the in vitro proliferation of bovine skeletal muscle satellite cells, and inhibit their differentiation.

[0016] 本发明的优点及效果: [0016] The advantages and effects of the present invention:

[0017] 1、本发明利用pEGFP-Cl构建miR_133b的过表达载体,可以利用EGFP标签对转染效果进行监测。 [0017] 1, the present invention is constructed using miR_133b overexpression vector pEGFP-Cl, transfection efficiency can be monitored using the EGFP tag.

[0018] 2、本发明利用微小RNA对牛肌卫星细胞的增殖和分化进行调控,miR-133b对细胞增殖和分化的微调作用对细胞的其它生物学特性影响较小。 [0018] 2, the present invention utilizes a micro RNA of bovine muscle satellite cell proliferation and differentiation regulation, miR-133b trim cell proliferation and differentiation smaller effect on the biological properties of other cells.

[0019] 3、利用本发明在牛骨骼肌卫星细胞过表达微小RNA miR-133b的方法得到的牛骨骼肌卫星细胞的体外增殖能力强,可以得到高纯度的牛肌卫星细胞,为研宄各调控因子在肌卫星细胞增殖与分化过程中的功能和影响提供良好的细胞模型。 [0019] 3, high proliferative ability in vitro bovine skeletal muscle satellite using the present invention, overexpression of miR-133b in a micro Bovine an RNA satellite cells of skeletal muscle cells obtained by the method can be obtained with high purity bovine muscle satellite cells, each for the study based on Effect on the function and regulation factors muscle satellite cell proliferation and differentiation in cells provide a good model.

附图说明 BRIEF DESCRIPTION

[0020] 图1是本发明所用质粒载体pEGFP-Cl的结构示意图; [0020] FIG. 1 is a schematic diagram of plasmid pEGFP-Cl vector used in the present invention;

[0021] 图2是本发明构建出的表达载体pEGFP-Cl-miR133b的结构示意图; [0021] FIG. 2 is a schematic diagram of the expression vector pEGFP-Cl-miR133b structure of the present invention is constructed;

[0022] 图3是转染miR-133b48h后牛骨骼肌卫星细胞中miR_133b表达水平与未转染组的对比图片; [0022] Figure 3 is transfected with miR-133b48h bovine skeletal muscle satellite cells with the expression level compared picture miR_133b untransfected group;

[0023] 图4是转染miR_133b后牛骨骼肌卫星细胞在添加诱导分化培养基后第3天图片; [0023] FIG. 4 is a rear transfection miR_133b bovine skeletal muscle satellite cells 3 days after addition of differentiation medium induced image;

[0024] 图5是未转染牛骨骼肌卫星细胞在添加诱导分化培养基后第3天图片。 [0024] FIG. 5 untransfected bovine skeletal muscle satellite cells after addition of differentiation medium on day 3 induction picture.

具体实施方式 detailed description

[0025] 下面结合具体实施方案对本发明进一步说明,其具体实施方案应该理解为仅为举例说明,不是限定性的,不能以下述举例说明来限定本发明的保护范围。 [0025] The following embodiment with reference to specific embodiments of the present invention is further described, it should be understood that the specific embodiments are illustrative only, not limiting, the following illustrate not to limit the scope of the present invention.

[0026] 本发明的设计思路是: [0026] The design concept of the present invention are:

[0027] 根据牛miR_133b基因序列设计一对PCR引物,在两个引物的5'端分别引入XhoI酶切位点和BamHI酶切位点,利用该对引物从牛基因组中扩增出包含miR_133b基因的全长为31 Ibp的一段序列,利用引入的酶切位点将该序列插入载体pEGFP-Cl,构建出牛miR-133b基因的表达载体pEGFP-Cl_miR133b ;然后用得到的表达载体pEGFP-Cl_miR133b转染牛骨骼肌卫星细胞,使牛骨骼肌卫星细胞过表达微小RNA miR-133b,利用miR_133b的作用,提高牛骨骼肌卫星细胞的体外增殖能力,并有效抑制其分化。 [0027] to design a pair of PCR primers according bovine miR_133b gene sequence, were introduced into the XhoI restriction site and BamHI restriction sites at the 5 'ends of two primers, using this pair of primers amplified from bovine genome comprising miR_133b gene the total length of a sequence of 31 Ibp, using the restriction sites introduced the sequence into the vector pEGFP-Cl, miR-133b bovine construct expression vector pEGFP-Cl_miR133b; then with an expression vector pEGFP-Cl_miR133b turn obtained transfection bovine skeletal muscle satellite cells, bovine skeletal muscle satellite cells overexpressing micro RNA miR-133b, by the action miR_133b improve the in vitro proliferation of bovine skeletal muscle satellite cells, and to inhibit their differentiation.

[0028] 下面结合附图和具体实施例对本发明作进一步的详细说明: Drawings and specific embodiments of the present invention will be further described in detail [0028] below with:

[0029] 一种促进牛骨骼肌卫星细胞体外增殖的方法,包括以下步骤: [0029] A method of in vitro cell proliferation promoting bovine skeletal muscle satellite, comprising the steps of:

[0030] 第一步,包含miR_133b基因序列的目的序列的获取: [0030] The first step, object miR_133b gene sequence comprising obtaining sequence:

[0031]根据牛 miR-133b 基因序列(GeneID: 100312996 ;miRBase:M10009734)设计一对PCR引物,在两个引物的5'端分别引入XhoI酶切位点和BamHI酶切位点,利用该对引物从牛基因组中扩增出包含miR-133b基因的全长为311bp的一段序列(Chromosome:23;AC_000180.1, 24312235-24312545),其中包含牛mir-133b前体基因序列84bp,基因前序列95bp,基因后序列132bp,总共311bp。 [0031] The gene sequence of bovine miR-133b (GeneID: 100312996; miRBase: M10009734) to design a pair of PCR primers were introduced into the XhoI restriction site and BamHI restriction sites at the 5 'ends of two primers, using this pair primers from the full length bovine genome comprising miR-133b gene is a sequence of 311bp (Chromosome: 23; AC_000180.1, 24312235-24312545), which contains gene sequences bovine mir-133b 84bp, before the sequence of the gene 95bp, after the gene sequence 132bp, a total of 311bp. 将该目的序列连入pUCm-T载体测序验证; The sequence of interest ligated into pUCm-T vector and sequenced to verify;

[0032] 具体步骤是: [0032] The specific steps are:

[0033] (I)牛全血基因组DNA的提取。 [0033] Genomic DNA from whole blood (I) bovine extract. 冷冻血样室温解冻,取0.5mL血液,用基因组DNA提取试剂盒提取DNA,琼脂糖凝胶电泳检测。 Blood samples were frozen thawed at room temperature, 0.5mL of blood taken, DNA Extraction Kit genomic DNA, agarose gel electrophoresis.

[0034] (2)PCR扩增。 [0034] (2) PCR amplification. 设计一对含有XhoI和BamHI酶切位点的特异性引物SEQ ID N0.1、SEQ ID N0.2, SEQ ID N0.1:5' -CCGCTCGAGAGCTCCTTCTGTGTTGCAGG-3' (下划线处碱基表示XhoI 酶切位点),SEQ ID N0.2:5' -CGGGATCCGGGCTCCCTTTTTCTCCCTT-3?(下划线处碱基表示BamHI酶切位点),以牛基因组为模板,按下列条件进行PCR扩增。 To design a pair of primers comprising SEQ ID N0.1 XhoI and BamHI restriction site, SEQ ID N0.2, SEQ ID N0.1: '-CCGCTCGAGAGCTCCTTCTGTGTTGCAGG-3' (underlined bases represent an XhoI 5 point), SEQ ID N0.2:? 5 '-CGGGATCCGGGCTCCCTTTTTCTCCCTT-3 (underlined bases indicate restriction sites BamHI), to bovine genome as a template, PCR amplification was performed under the following conditions.

[0035]反应体系:25yL 反应体系,2x Es Taq MasterMixl2.5 μ L,模板DNA2 μ L,引物SEQID N0.1 (10 μΜ) I μ L,引物SEQ ID N0.2 (10 μΜ) I μ L,灭菌超纯水8.5 μ L。 [0035] The reaction system: 25yL reaction system, 2x Es Taq MasterMixl2.5 μ L, template DNA2 μ L, primer SEQID N0.1 (10 μΜ) I μ L, primer SEQ ID N0.2 (10 μΜ) I μ L , sterilized ultrapure water to 8.5 μ L.

[0036] 反应条件:94°C预变性2min ;94°C变性30s,62°C退火30s,72°C延伸45s,循环35次;72°C 延伸2min。 [0036] Reaction conditions: 94 ° C denaturation for 2min; 94 ° C denaturation 30s, 62 ° C annealing 30s, 72 ° C extension 45s, 35 cycles; 72 ° C extending 2min.

[0037] 琼脂糖凝胶电泳检测扩增产物,得到包含miR_133b基因的全长为311bp的一段序列SEQ ID N0.3,并在其5'端引入了一个XhoI酶切位点,3'端引入了一个BamHI酶切位点,SEQ ID N0., 3:5'-agctccttctgtgttgcaggcttgaaccagggatgctcgggacacaccaagaatcctcgccctagaggctgcagtc acctcccccaggagctgccccctgctctggctggtcaaacggaaccaagtccgtcttcctgagaggtttggtccccttcaa ccagctacagcagggctggcaaagcccagtccttggagaaacagaagagattcgccttctgtggctgaaagtacctacta ccgtttctcttaagaaaatgactgattttgttatcacactctcatgctctcaggaagggagaaaaagggagccc-3'。 [0037] The amplification product is detected by agarose gel electrophoresis, full-length genes are miR_133b comprising a sequence SEQ ID N0.3 311bp and at its 5 'end an XhoI restriction site, 3' end a BamHI restriction site, SEQ ID N0, 3:. 5'-agctccttctgtgttgcaggcttgaaccagggatgctcgggacacaccaagaatcctcgccctagaggctgcagtc acctcccccaggagctgccccctgctctggctggtcaaacggaaccaagtccgtcttcctgagaggtttggtccccttcaa ccagctacagcagggctggcaaagcccagtccttggagaaacagaagagattcgccttctgtggctgaaagtacctacta ccgtttctcttaagaaaatgactgattttgttatcacactctcatgctctcaggaagggagaaaaagggagccc-3 '.

[0038] (3)胶回收。 [0038] (3) Gel Extraction. 琼脂糖凝胶电泳扩增产物,在紫外光灯下切胶,按照胶回收试剂盒操作说明进行回收。 Agarose gel electrophoresis, the amplified products, under ultraviolet light cut plastic, recycled in a gel extraction kit instructions.

[0039] (4)感受态细胞的制备:取ImL DH5a菌液用CaC12处理,制成100 μ L感受态细胞。 [0039] (4) Preparation of competent cells: take ImL DH5a bacteria treated with CaC12, made of 100 μ L of competent cells.

[0040] (5)连接与转化:采用10 μ L连接反应体系,无菌水2 μ L,纯化后的PCR产物4 μ L,1XLigat1n Bufferl μ L,50%PEG1 μ L,pUCm-T 载体I μ L,T4DNA Ligasel μ L。 [0040] (5) connected to the conversion: The ligation reaction 10 μ L sterile water 2 μ L, PCR product was purified 4 μ L, 1XLigat1n Bufferl μ L, 50% PEG1 μ L, pUCm-T vector I μ L, T4DNA Ligasel μ L. 上述混合液轻轻震荡后再短暂离心,然后置于16°C PCR温仪中保温过夜(16h),然后将10 μ L连接产物转入100 μ L感受态细胞。 Centrifuged briefly and then the liquid mixture shaken gently, then placed in a 16 ° C PCR Thermometers incubated overnight (for 16 h), and then 10 μ L ligation products into 100 μ L of competent cells. 将细菌涂布在氨苄青霉素平板上,培养过夜。 The bacteria were plated on ampicillin plates and incubated overnight. 挑取克隆至含氨苄青霉素的液体培养基中过夜,提取质粒测序,测序正确的质粒命名为pT-miR133b。 Clones were picked into broth containing ampicillin, plasmids were extracted sequencing, sequencing correct plasmid was designated pT-miR133b.

[0041] 第二步,表达载体pEGFP-Cl-miR133b的构建: [0041] The second step, the expression vector pEGFP-Cl-miR133b of:

[0042] 用XhoI和BamHI双酶切第一步中所得pT_miR133b及pEGFP_Cl载体,通过连接酶连接,使311bp的目标序列插入质粒载体pEGFP-Cl的XhoI和BamHI酶切位点,构建出牛miR-133b基因的表达载体pEGFP-Cl-miR133b,如图1和图2所示; [0042] double digestion with XhoI and BamHI and pEGFP_Cl pT_miR133b obtained in the first step vector by a ligase, the target sequence 311bp insertion in the plasmid vector pEGFP-Cl XhoI and BamHI restriction sites, constructed bovine miR- 133b gene expression vector pEGFP-Cl-miR133b, 1 and 2;

[0043] 具体步骤是: [0043] The specific steps are:

[0044] (I)XhoI 和BamHI 双酶切步骤I 中所得pT_miR133b。 [0044] (I) XhoI and BamHI double digestion step I obtained pT_miR133b.

[0045]反应体系:20 yL 反应体系,超纯水11 yL,10XBuffer2yL,pT_miR133b5 μ L,XhoI 酶I yL,BamHI 酶I yL。 [0045] The reaction system: 20 yL reaction system, ultrapure water 11 yL, 10XBuffer2yL, pT_miR133b5 μ L, XhoI enzyme I yL, BamHI enzyme I yL.

[0046]反应条件:37°C lh,80°C 5min ; [0046] Reaction conditions: 37 ° C lh, 80 ° C 5min;

[0047] 胶回收酶切产物; [0047] Gel Extraction cleavage product;

[0048] (2) XhoI 和BamHI 双酶切pEGFP-Cl 载体。 [0048] (2) XhoI and BamHI double digested pEGFP-Cl vector.

[0049]反应体系:20 μ L 反应体系,超纯水llμL,10XBuffer2μL,质粒pEGFP-C15μL,XhoI 酶I yL,BamHI 酶I yL。 [0049] The reaction system: 20 μ L of the reaction system, ultrapure water llμL, 10XBuffer2μL, plasmid pEGFP-C15μL, XhoI enzyme I yL, BamHI enzyme I yL.

[0050]反应条件:37°C Ih,80°C 5min。 [0050] Reaction conditions: 37 ° C Ih, 80 ° C 5min.

[0051] 胶回收酶切产物。 [0051] The cleavage product recovered gum.

[0052] ( 3 )连接两种胶回收酶切产物。 [0052] (3) connecting the two cleavage product recovered gel.

[0053] 反应体系:20yL反应体系,目的序列回收产物5yL,质粒pEGFP-Cl回收产物5 μ L,T4DNA Ligasel μ L,T4 连接酶缓冲液2 μ L,超纯水7 μ L0 [0053] Reaction system: 20yL reaction system, the sequence of recovered product 5yL, the product is recovered plasmid pEGFP-Cl 5 μ L, T4DNA Ligasel μ L, T4 ligase buffer, 2 μ L, 7 μ L0 ultrapure

[0054]反应条件:22°C 4h。 [0054] Reaction conditions: 22 ° C 4h.

[0055] 转化大肠杆菌DH5 α,酶切鉴定连接效果。 [0055] The transformation of E. coli DH5 α, enzyme digestion is connected effect.

[0056] 进一步经测序证实为正确连接,得到表达载体pEGFP-Cl-miR133b (图2)。 [0056] further confirmed by sequencing is properly connected, resulting in the expression vector pEGFP-Cl-miR133b (FIG. 2).

[0057] 第三步,pEGFP-Cl-miR133b转染牛骨骼肌卫星细胞: [0057] The third step, pEGFP-Cl-miR133b bovine skeletal muscle satellite cells transfected with:

[0058] 在转染前一天把牛骨骼肌卫星细胞(实验室自存)培养到六孔板内,使第二天细胞能达到约70%〜80%满,在进行转染前,每孔更换成新鲜的无血清细胞培养液2ml ; [0058] The day before transfection in bovine skeletal muscle satellite cells (from the laboratory stored) into the culture six-well plates so that the next day the cells reach about 70% ~ 80% full, before performing transfection per well replaced with fresh serum-free cell culture fluid 2ml;

[0059] 用得到的表达载体pEGFP-Cl-miR133b,采用脂质体转染试剂转染牛骨骼肌卫星细胞,按照脂质体转染试剂说明书进行转染操作,使牛骨骼肌卫星细胞过表达微小RNAmiR_133b0 [0059] with an expression vector pEGFP-Cl-miR133b obtained using overexpressing liposome transfection reagent transfection bovine skeletal muscle satellite cells, in accordance with Lipofectamine transfection reagent manual operation, bovine skeletal muscle satellite cells small RNAmiR_133b0

[0060] 具体步骤为: [0060] Specific steps of:

[0061] 把Lipofectamine2000脂质体转染试剂轻轻混勾。 [0061] The liposomes Lipofectamine2000 transfection reagent hook gently mixed. 在一洁净无菌离心管内加入240 μ I无血清培养基,然后加入10 μ I Lipofectamine2000 ;在另一洁净无菌离心管内加Λ 245 μ I无血清培养基,然后加入5 μ I质粒(4 μ g);两管液体温育5min后混合,室温下静置20min ;然后将混合液逐滴加入六孔板的一个孔内,摇动培养板轻轻混匀,二氧化碳培养箱中培养6h ;6h后更换为含血清培养基培养。 Add 240 μ in a clean sterile centrifuge tube I serum-free medium, followed by addition of 10 μ I Lipofectamine2000; within another clean sterile centrifuge tube Λ serum-free medium plus 245 μ I, followed by addition of 5 μ I plasmid (4 μ G); after two liquid mixing incubated 5min, 20min standing at room temperature; the mixture was then added dropwise a hole six well plate, mix gently shaking the culture plate, the carbon dioxide incubator 6h; for 6h culture medium containing serum replaced.

[0062] 第四步,转染效果检测: [0062] The fourth step, detecting transfection efficiency:

[0063] (I)转染后miR_133b表达水平的qRT_PCR检测。 [0063] (I) after transfection, the expression levels qRT_PCR miR_133b detected.

[0064] 转染48h后检测转染水平,采用TRIzol法提取未转染肌卫星细胞和转染后肌卫星细胞总RNA,按照GeneCopoeia miRNA qRT-PCR检测试剂盒说明进行操作: [0064] 48h after transfection, transfection levels using TRIzol extraction method of total RNA and muscle satellite cells transfected muscle satellite cells untransfected, follow GeneCopoeia miRNA qRT-PCR detection kit instructions:

[0065]① miRNA First-Strand cDNA 合成。 [0065] ① miRNA First-Strand cDNA synthesis.

[0066]反应体系:25yL 反应体系,提取的总RNA5yL,2.5u/yL Poly A Polymerase [0066] Reaction system: 25yL reaction system, the extracted total RNA5yL, 2.5u / yL Poly A Polymerase

1 μ L,RTase Mixl μ L,5XPAP/RT Buffer5 μ L,超纯水13 μ L,总体积25 μ L。 1 μ L, RTase Mixl μ L, 5XPAP / RT Buffer5 μ L, ultrapure water 13 μ L, total volume of 25 μ L.

[0067]反应条件:37°C lh,85°C 5min。 [0067] Reaction conditions: 37 ° C lh, 85 ° C 5min.

[0068]② miR_133b qPCR 检测。 [0068] ② miR_133b qPCR detection.

[0069]反应体系:20 4 1^反应体系,2父六11-111-01^^]\1 qPCR MixlO μ L,miR_133b上游引物(引物序列与成熟miR_133b 序列相同,2 μ Μ)2 μ L,Universal Adaptor PCR Primer (2 μ M) [0069] Reaction system: 2041 ^ reaction system, 2 six parent 11-111-01 ^^] \ 1 qPCR MixlO μ L, miR_133b upstream primer (primer sequence identical to the sequence of the mature miR_133b, 2 μ Μ) 2 μ L , Universal Adaptor PCR Primer (2 μ M)

2 μ L,First-strand cDNA (按1:5 用超纯水稀释)2 μ L,超纯水4 μ L0 2 μ L, First-strand cDNA (1: was diluted with ultrapure water 5) 2 μ L, ultra pure water 4 μ L0

[0070] 反应条件:95°C预变性1min ;95°C变性10s,60°C退火20s,72°C延伸15s,40个循环;溶解曲线制作温度范围65。 [0070] Reaction conditions: 95 ° C denaturation for 1min; 95 ° C denaturation 10s, 60 ° C annealing 20s, 72 ° C extension 15s, 40 cycles; 65 dissolution curve preparation temperature range. . 〜95。 ~ 95. . , 0.5°C /time,6s/time。 , 0.5 ° C / time, 6s / time.

[0071] 结果:转染后48h,转染后肌卫星细胞中miR_133b表达量比未转染肌卫星细胞高出6.39倍,说明pEGFP-Cl-miR133b转染成功并提高了目标基因的表达量(图3)。 [0071] Results: After transfection 48h, transfected muscle satellite cells than in non-transfected expression miR_133b muscle satellite cells transfected 6.39 times higher, indicating pEGFP-Cl-miR133b successful transfection and increase the amount of target gene expression ( image 3).

[0072] (2)转染后miR_133b促进肌卫星细胞增殖及抑制分化的效果。 [0072] (2) after transfection miR_133b promote muscle satellite cell proliferation and differentiation inhibiting effect.

[0073] 转染48h后将未转染肌卫星细胞和转染后肌卫星细胞更换为含2%马血清的分化培养基进行培养,观察肌卫星细胞的增殖和诱导分化效果。 After 48h after [0073] transfection muscle satellite cells non-transfected and transfected muscle satellite cells are cultured to replace differentiation medium containing 2% horse serum, proliferation and induce differentiation observed effects of muscle satellite cells.

[0074] 分化培养基添加后第3天,未转染组肌卫星细胞分化明显,形成了粗大的肌管(图4),而转染miR-133b组未见明显粗大肌管出现,仅见零星小肌管出现(图5),说明了miR-133b过表达可以促进牛骨骼肌卫星细胞增殖,并有效抑制其分化。 [0074] 3 days after the differentiation medium was added, non-transfected group was significantly differentiate muscle satellite cells, the formation of coarse myotubes (FIG. 4), miR-133b transfected group showed no coarse myotubes appeared, see only sporadic myotubes appears small (FIG. 5), described the overexpression of miR-133b can facilitate bovine skeletal muscle satellite cell proliferation and inhibit their differentiation.

Claims (2)

  1. 1.一种促进牛骨骼肌卫星细胞体外增殖的方法,其特征在于包括以下步骤: 第一步、包含牛miR-133b基因序列的目的序列的获取: 设计一对PCR引物,在两个引物的5'端分别引入XhoI酶切位点和BamHI酶切位点,利用该对引物从牛基因组中扩增出包含miR-133b基因的全长为311bp的目的序列,该对引物序列为:5' -CCGCTCGAGAGCTCCTTCTGTGTTGCAGG-3',5' -CGGGATCCGGGCTCCCTTTTTCTCCCTT-3',包含miR_133b 基因的全长为311bp 的目的序列为agctccttctgtgttgcaggcttgaaccagggatgctcgggacacaccaagaatcctcgccctagaggctgcagtcacctcccccaggagctgccccctgctctggctggtcaaacggaaccaagtccgtcttcctgagaggtttggtccccttcaaccagctacagcagggctggcaaagcccagtccttggagaaacagaagagattcgccttctgtggctgaaagtacctactaccgtttctcttaagaaaatgactgattttgttatcacactctcatgctctcaggaagggagaaaaagggagccc ; 第二步、表达载体pEGFP-Cl-miR133b的构建: 用XhoI和BamHI双酶切第一步中所得序列及pEGFP-Cl载体,通过连接酶连接,使所得序列插入 1. A method of in vitro cell proliferation promoting bovine skeletal muscle satellite, comprising the steps of: a first step comprising object bovine miR-133b gene sequence acquisition sequence: to design a pair of PCR primers, two primers 5 'ends are introduced into the XhoI restriction site and BamHI restriction sites, using the pair of primers from bovine genome comprising a full-length gene of miR-133b 311bp of the sequence, the sequence of the primer: 5' -CCGCTCGAGAGCTCCTTCTGTGTTGCAGG-3 ', 5' -CGGGATCCGGGCTCCCTTTTTCTCCCTT-3 ', containing the full-length sequence for the purpose miR_133b 311bp gene was agctccttctgtgttgcaggcttgaaccagggatgctcgggacacaccaagaatcctcgccctagaggctgcagtcacctcccccaggagctgccccctgctctggctggtcaaacggaaccaagtccgtcttcctgagaggtttggtccccttcaaccagctacagcagggctggcaaagcccagtccttggagaaacagaagagattcgccttctgtggctgaaagtacctactaccgtttctcttaagaaaatgactgattttgttatcacactctcatgctctcaggaagggagaaaaagggagccc; a second step, the expression vector pEGFP-Cl-miR133b construct: a first double digested with XhoI and BamHI the resulting sequence of step and pEGFP-Cl vector by ligase, the resulting sequence is inserted 粒载体pEGFP-Cl的XhoI和BamHI酶切位点,构建出牛miR_133b基因的表达载体pEGFP-Cl-miR133b ; 第三步、pEGFP-Cl-miR133b转染牛骨骼肌卫星细胞: 用得到的表达载体pEGFP-Cl-miR133b,采用脂质体转染试剂转染牛骨骼肌卫星细胞,使牛骨骼肌卫星细胞过表达miR-133b ; 而且,所述第一步中包含miR-133b基因的全长为311bp的目的序列由牛mir_133b基因84bp、基因前序列95bp、基因后序列132bp组成,总共311bp ; 而且,所述第三步采用脂质体转染试剂转染牛骨骼肌卫星细胞是在转染前一天把牛骨骼肌卫星细胞培养到六孔板内,使第二天细胞能达到70%〜80%满,在进行转染前,每孔更换成新鲜的无血清细胞培养液2ml。 PEGFP-Cl plasmid vector into the XhoI and BamHI restriction sites, bovine miR_133b constructed expression vector pEGFP-Cl-miR133b; a third step, pEGFP-Cl-miR133b bovine skeletal muscle satellite cells transfected with: an expression vector obtained by pEGFP-Cl-miR133b, liposome transfection reagent transfection bovine skeletal muscle satellite cells, bovine skeletal muscle satellite cells overexpressing miR133b; and full-length, comprising the first step of gene miR133b sequence of 311 bp of the 84 bp bovine mir_133b gene, gene sequences before 95bp, 132bp sequence consisting of the gene, a total of 311 bp; and, said third step using lipofectamine transfection reagent bovine skeletal muscle satellite cells prior to transfection one day culture the bovine skeletal muscle satellite cells into six-well plates so that the next day the cells reach 70% ~ 80% full, before performing transfection, each well was replaced with fresh serum-free cell culture medium 2ml.
  2. 2.—种权利要求1所述的促进牛骨骼肌卫星细胞体外增殖的方法的应用,其特征在于:利用miR-133b的作用,提高牛骨骼肌卫星细胞的体外增殖能力,并有效抑制牛骨骼肌卫星细胞的分化。 Application of method of promoting cell proliferation of bovine skeletal muscle satellite 2.- species according to claim 1, wherein: by the action of miR-133b and improve the in vitro proliferation of bovine skeletal muscle satellite cells, effective to inhibit bovine bones and muscle satellite cell differentiation.
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