秦本源, 杨阳, 张燕伟, 刘敏, 张万锋, 王海珍, 吴怡琦, 张雪莲, 蔡春波, 高鹏飞, 郭晓红, 李步高, 曹果清^,山西农业大学动物科技学院,山西太谷 030801

Isolation, Culture, Identification and Biological Characteristics of Pig Skeletal Muscle Satellite Cells

QIN BenYuan, YANG Yang, ZHANG YanWei, LIU Min, ZHANG WanFeng, WANG HaiZhen, WU YiQi, ZHANG XueLian, CAI ChunBo, GAO PengFei, GUO XiaoHong, LI BuGao, CAO GuoQing^,College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi

通讯作者: 曹果清,Tel：13403665105;E-mail: anniecao710502@aliyun.com

秦本源与杨阳为同等贡献作者。
责任编辑: 林鉴非
收稿日期:2019-09-29接受日期:2020-01-13网络出版日期:2020-04-16

基金资助:

国家自然科学基金.31872336 三晋****支持计划专项经费资助.2016 三晋****支持计划专项经费资助.2017 山西省“1331”工程资助 山西省农业重点研发项目.201803D221022-1 山西农业大学畜牧学学科建设专项课题资助

Received:2019-09-29Accepted:2020-01-13Online:2020-04-16
作者简介 About authors
秦本源,Tel：18503442342;E-mail: 923726358@qq.com。

杨阳,Tel：13007050956;E-mail: yangyangh@163.com。














摘要
【目的】建立猪骨骼肌卫星细胞体外分离、纯化及鉴定的方法,并对其生物学特性进行探讨,为进一步研究猪肌肉生长发育提供良好的细胞模型。【方法】选取1日龄仔猪背最长肌为材料,无菌状态下将背最长肌剪碎为肉糜状。此后采用浓度为0.2%的Ⅰ型胶原酶消化90 min,再加入浓度为0.25%的胰蛋白酶37 ℃联合消化30 min。经终止消化、过滤、重悬后将分离得到的细胞置于37 ℃、5% CO₂细胞培养箱中培养。选用反复差速贴壁法对骨骼肌卫星细胞进行纯化,第一次纯化选择在细胞接种2 h后,将未贴壁细胞转移至新培养皿。上清液继续培养18 h后,对卫星细胞进行第二次差速贴壁纯化。当细胞密度达70%—80%时可对细胞进行传代或冻存处理。利用细胞免疫荧光技术检测P2代卫星细胞标志基因Pax7、MyoD的蛋白表达情况,并绘制卫星细胞生长曲线。分别添加不同诱导分化液使卫星细胞定向分化为肌细胞、脂肪细胞、成骨细胞,检测成肌分化标志基因MHC的免疫荧光,鉴定卫星细胞肌管形成情况;油红O染色及油红O定量鉴定卫星细胞诱导成脂分化效果;茜素红染色鉴定卫星细胞成骨分化能力,qRT-PCR检测成肌、成脂、成骨进程中关键基因的表达。【结果】通过两步酶消化和反复差速贴壁法分离纯化得到了纯度较高的卫星细胞,刚分离的细胞折光性强,贴壁后呈梭形或纺锤形,此后细胞延展并开始快速增殖。卫星细胞特异标志蛋白Pax7、MyoD细胞免疫荧光鉴定结果呈阳性,表明分离细胞为骨骼肌卫星细胞。骨骼肌卫星细胞增殖过程经潜伏期、生长期最终达到平台期,细胞生长曲线呈“S”型。当细胞生长至90%密度时,卫星细胞会出现自融合现象。对分离的骨骼肌卫星细胞成肌诱导分化后,可见邻近卫星细胞融合形成大量粗长肌管,多核肌管呈方向性排列,成肌标志蛋白MHC染色呈阳性。qRT-PCR结果显示标志基因MyoD、MyoG在成肌诱导分化过程中二者均呈先上升后下降趋势。经成脂诱导后细胞形态变为三角形,连续诱导发现脂滴出现并聚集成大脂滴,油红O染色可见大量红色葡萄样脂滴。油红O定量检测结果表明,成脂诱导过程中甘油三酯含量呈稳步上升趋势,各时间点均存在极显著差异（P<0.01）。qRT-PCR结果显示,PPARγ基因表达量在诱导中后期高表达;FABP4在诱导分化第6天达到最高,极显著高于其余时间点（P<0.01）;CEBP/β和HSL表达趋势一致,均呈先升高后降低趋势。诱导成骨分化后,发现细胞形态变为不规则状,诱导后期细胞复层生长形成骨钙结节,茜素红染色可见圆形不透明钙化结节,数量和密度较未诱导时期都明显增加,结果表明细胞出现成骨向分化。成骨标志基因BGLAP、RUNX2的表达量也随诱导进程呈稳步上升趋势,相比未诱导细胞差异极显著（P<0.01）。【结论】建立了基于联合酶消化和反复差速贴壁实现猪骨骼肌卫星细胞分离和纯化的方法,所得细胞增殖能力强且具有多向分化潜能,为猪骨骼肌卫星细胞作为种子细胞用于未来组织工程研究提供了技术平台。
关键词： 猪;卫星细胞;分离培养;鉴定;分化

Abstract
【Objective】 The aim of this study was to establish a method for isolation, purification and identification of porcine skeletal muscle satellite cells in vitro, and to explore its biological characteristics, in order to provide a reliable cell model for further research of muscle growth and development in pigs. 【Method】 In this study, the longissimus dorsi muscle of 1 day old pig was selected and cut into meat emulsion in aseptic state. After that, it was digested with 0.2% type I collagenase for 90 min, and then digested with 0.25% trypsin for 30 min at 37 ℃. After termination of digestion, filtration and resuspension, the isolated cells were cultured in a 37 ℃ and 5% CO₂ cell incubator. The skeletal muscle satellite cells were purified by repeated differential adherence technique. The first purification selection was performed after cell culturing for 2 h, and non-adherent cells were transferred to a new culture dish. After the supernatant was further cultured for 18 h, the satellite cells were purified again. The cells were subcultured or frozen when the cell density reached 70% - 80%. Cell immunofluorescence technique was used to detect the protein expression of marker genes of Pax7 and MyoD of P2 satellite cell, and the growth curve was determined. The satellite cells were differentiated into myocytes, adipocytes and osteoblasts by adding different inducing differentiation fluids. The protein expression of myoblast differentiation marker gene MHC was detected by using cell immunofluorescence to identify myotube formation in satellite cells. Oil red O staining and triglyceride content were quantified to identify the adipogenic differentiation effect in satellite cells. Alizarin red staining was used to identify the osteogenic differentiation ability in satellite cells, which were detected the expression of key genes during myogenesis, adipogenesis and osteogenesis by qRT-PCR. 【Result】 The results showed that the satellite cells with higher purity were isolated and purified by two-step enzymatic digestion and repeated differential adherence technique. The cells that were originally isolated with highly refractive, and were fusiform or spindle-shaped after adherence, after which the cells extended and began to proliferate. The results of cell immunofluorescence identification of satellite cells specific marker proteins Pax7 and MyoD were positive, indicating that the isolated cells were skeletal muscle satellite cells. Skeletal muscle satellite cell proliferation underwent the incubation period, and the growth period and finally reached the plateau phase. The satellite cells were self-fusion when the cells grew to 90% density. After myogenic induction and differentiation of the satellite cells, a large number of myotubes were formed by the adjacent satellite cells. Multinucleated myotubes were regularly arranged and the myoblast marker protein MHC staining was positive. The qRT-PCR results showed that the marker genes of MyoD and MyoG both increased first and then decreased during the process of myoblast differentiation. After adipogenic induction, the cell morphology changed into triangle, and lipid droplets appeared and aggregated into large lipid droplets with continuous induction. Oil red O staining observed a large number of red grape-like lipid droplets. Oil red O staining quantitative results showed that the triglyceride content steadily increasing during the adipogensis. There were extremely significant differences among each time point (P<0.01). The qRT-PCR results showed that the expression level of PPARγ gene was high in the middle and the late stage of induction. The FABP4 gene reached the highest at the 6th day of induction and was significantly higher than that at other time points (P<0.01). A similar dynamic was observed with the relative expression level of CEBP/β and HSL in the differentiating cells. Their expression tended to increase first and then decrease. After inducing osteogenic differentiation, it was found that the cell morphology became irregular. Cells formed bone nodules after inducing, compared with the period without induction, the alizarin red staining showed that the number and density of round opaque calcified nodules were significantly increased. The results showed that the cells appeared osteogenic differentiation. The expression levels of osteogenic marker genes BGLAP and RUNX2 also showed a steady upward trend during the inducing procession, which was significantly different from that without inducing cells (P<0.01). 【Conclusion】 This study established a method for isolation and purification of pig skeletal muscle satellite cells based on combined enzyme digestion and differential adherent technique. The obtained cells had strong proliferation ability and multi-directional differentiation potential. The results provided a technical platform for pig skeletal muscle satellite cells as seed cells for future tissue engineering research.
Keywords：pig;satellite cell;isolation culture;identification;differentiation


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秦本源, 杨阳, 张燕伟, 刘敏, 张万锋, 王海珍, 吴怡琦, 张雪莲, 蔡春波, 高鹏飞, 郭晓红, 李步高, 曹果清. 猪骨骼肌卫星细胞分离培养、鉴定及其生物学特性[J]. 中国农业科学, 2020, 53(8): 1664-1676 doi:10.3864/j.issn.0578-1752.2020.08.015
QIN BenYuan, YANG Yang, ZHANG YanWei, LIU Min, ZHANG WanFeng, WANG HaiZhen, WU YiQi, ZHANG XueLian, CAI ChunBo, GAO PengFei, GUO XiaoHong, LI BuGao, CAO GuoQing. Isolation, Culture, Identification and Biological Characteristics of Pig Skeletal Muscle Satellite Cells[J]. Scientia Acricultura Sinica, 2020, 53(8): 1664-1676 doi:10.3864/j.issn.0578-1752.2020.08.015

0 引言

【研究意义】骨骼肌卫星细胞（satellite cells, SCs）位于肌纤维膜与基底膜之间,属于肌源性干细胞^[1],具有较强的增殖、分化潜能。正常哺乳动物体内,骨骼肌卫星细胞通常处于静息状态,但当肌肉组织受到高强度训练、损伤或其他外界刺激时,卫星细胞便被激活^[2]。此后卫星细胞进入细胞周期,通过增殖、迁移、分化、融合等一系列复杂的生物学过程,最后形成新的肌纤维,以恢复正常的组织结构,维持骨骼肌的生长发育^[3,4,5]。因此,卫星细胞可作为组织工程的种子细胞对机体相关疾病的治疗具有重大意义。猪骨骼肌卫星细胞是探究猪肌肉形成过程的良好模型,建立体外肌卫星细胞的分离培养体系对探索猪肌肉生长发育过程具有指导意义,也可为卫星细胞移植在临床中的应用提供参考。【前人研究进展】骨骼肌卫星细胞最先由MAURO于1961年在青蛙的胫前肌中发现,电子显微镜下观察到其位置分布紧贴于肌细胞的表面^[6]。卫星细胞的数量随年龄的增加而逐渐减少,细胞核总数在出生后一段时期内明显下降,当达到一定程度后可终身维持^[7,8]。相较于动物幼龄期而言,成年后卫星细胞含量较低,约占1%—5%^[9]。先前研究已表明,各种分子标记是不同阶段细胞鉴定的基础,骨骼肌卫星细胞的特异性基因可作为骨骼肌卫星细胞鉴定的常用方法。判定卫星细胞最常用的标志基因是Pax7、MyoD和MyoG^[10]。配对盒基因7（pired box 7, Pax7）作为骨骼肌卫星细胞的标志基因,在静息期和增殖期均有表达^[11],已有的研究结果证明Pax7对卫星细胞的成肌分化是必须的,只有当Pax7正常表达时才具备对肌肉损伤的修复功能^[12]。生肌决定因子（myogenic determination gene, MyoD）可引导卫星细胞向成肌细胞分化,MyoD缺陷型小鼠的卫星细胞无法参与分化进程,阻碍肌纤维的修复^[13]。肌细胞生成素（myogenin, MyoG）主要在分化的中后期表达,可作为成肌分化的标志,当卫星细胞融合形成肌管时,MyoG表达量的升高会促进肌卫星细胞终末分化。肌球蛋白重链（myosin heavy chain, MHC）基因最后表达,标志着分化已进入终末时期,肌管形成^[14]。目前,关于卫星细胞的分离主要有单根肌纤维法^[15]和酶消化法^[16]。其中酶消化法又可分为链霉蛋白酶消化法^[17]和两步酶消化法^[18],即采用胶原酶胰酶联合消化,此法可将肌纤维中的卫星细胞释放,分离得到较多卫星细胞。尽管酶消化法提高了卫星细胞得率,但不可避免地引入了其它的非肌源性细胞,如成纤维细胞、红细胞等^[19]。因此,科学家们又对卫星细胞的纯化进行了改良,当前常用的纯化方法主要有差速贴壁法^[20]、Percoll梯度密度离心法^[21]、流式细胞分选术^[22]和免疫磁珠细胞分选术^[23]等。骨骼肌卫星细胞自被证实具有干细胞特性以来便引起了广大研究者的关注,已有的研究结果发现,骨骼肌卫星细胞除了可以向成肌方向分化外,在不同诱导环境下也可以成脂分化形成脂滴或成骨分化形成钙沉淀^[24,25]。【本研究切入点】快速简洁高效地分离高纯度骨骼肌卫星细胞成为了当前研究的热点,体外骨骼肌卫星细胞的分离已在小鼠^[26]、鸡^[27]、牛^[28]、羊^[29]等动物上成功获得,但在猪上研究较少,对猪骨骼肌卫星细胞生物学特性及多向分化潜能研究鲜有报道。【拟解决的关键问题】故本研究选取1日龄仔猪背最长肌为材料,采用两步酶消化法成功分离猪骨骼肌卫星细胞并对其鉴定,此后分别诱导成肌、诱导成脂、诱导成骨检测其多向分化潜能。分离培养高纯度的骨骼肌卫星细胞,有利于探明其细胞生物学特性,也为家畜遗传资源改良和相关疾病治疗奠定了良好的基础。

1 材料与方法

1.1 主要试剂

1.1.1 试验动物 选取1日龄大白猪,体重1 kg,购自山西省清徐县天禄丰种猪育种有限公司。试验于2019年3—7月在山西农业大学动物科技学院动物遗传育种与繁殖实验室完成。

1.1.2 主要试剂 DMEM高糖培养基、胎牛血清FBS、马血清HS均购自Gibco公司;胰蛋白酶、青链霉素混合液、PBS、油红O染液、茜素红染液（pH= 4.2）、4%多聚甲醛、Triton X-100、DAPI均购自Solaibio公司;2%即用型山羊血清封闭液购自博士德生物工程有限公司;Ⅰ型胶原酶、地塞米松（DEX）、胰岛素、3-异丁基-1-甲基黄嘌呤（IBMX）、罗格列酮、β-甘油磷酸钠、抗坏血酸均购自美国Sigma公司;Anti-Pax7、Anti-MHC、Anti-MyoD购自Abcam公司;FITC标记羊抗鼠IgG二抗购自武汉三鹰生物技术有限公司;反转录和实时荧光定量试剂盒购自日本TaKaRa公司;引物合成于上海生工生物工程有限公司。

1.2 骨骼肌卫星细胞的分离及纯化

无菌采集仔猪背最长肌组织,先后置于75%乙醇、含2%双抗的PBS中清洗数次,在预冷的PBS中除去肉眼可见的血管、筋膜等结缔组织,剪碎为1 mm³的组织块。转移组织块至无菌离心管中,用含0.2%的Ⅰ型胶原酶置于摇床消化90 min,期间每隔15 min吹打消化液一次。再用0.25%胰蛋白酶37 ℃消化30 min,期间每隔10 min晃动混匀一次。消化结束后,用等体积完全培养基（10% FBS+1%双抗+DMEM高糖）终止消化,依次过70目、200目细胞筛。滤液1 000 r/min离心5 min,重悬细胞并接种至60 mm细胞培养皿中。置于体积分数为5% CO₂、37 ℃培养箱中培养2 h后将上清吸至新皿中,记为P01。P01细胞继续培养18—24 h后再次转移上清,记为P02。其中,P01、P02即为纯化后的骨骼肌卫星细胞,培养48 h后首次换液,之后每2 d更换一次完全培养基。

1.3 骨骼肌卫星细胞的传代与冻存

待培养皿中细胞生长汇合至70%—80%时,按照1﹕2或1﹕3的比例传代培养,隔天换液,倒置显微镜下观察。卫星细胞的冻存取消化后的细胞用细胞冻存液（20% FBS+10% DMSO+DMEM）重悬,吹打混匀后加入冻存管中,-80℃冰箱过夜,取出后投入液氮中保存。复苏时从液氮中取出细胞,立刻放入37℃水浴锅中快速融化,离心去上清后用新鲜的完全培养基重悬接入新培养皿。

1.4 骨骼肌卫星细胞的免疫荧光染色鉴定

选P2代骨骼肌卫星细胞接种于24孔细胞培养板,待细胞密度达到50%左右时开始细胞免疫荧光染色。PBS漂洗细胞3次后,用4%多聚甲醛固定30 min,蒸馏水冲洗3次,每次5 min。0.1% Triton X-100通透细胞30 min,蒸馏水洗涤3次,每次5 min。2%即用型山羊血清封闭1 h,直接滴加一抗（Anti-Pax7, 1﹕300;Anti-MyoD, 1﹕200）覆盖孔底,4 ℃孵育过夜。移除一抗后用蒸馏水清洗,加入荧光二抗（羊抗鼠,1﹕100）,室温避光孵育1 h。吸去二抗后每孔滴加250 μL DAPI染液,荧光显微镜下观察。

1.5 骨骼肌卫星细胞生长曲线

取生长状态良好的P3代卫星细胞,0.25%胰酶消化制成单细胞悬液,以1×10⁴个/孔接种于24孔细胞培养板。每天取3孔细胞消化,血球计数板计数,连续8 d,以培养时间为X轴,细胞密度为Y轴,绘制细胞生长曲线。

1.6 骨骼肌卫星细胞成肌诱导及鉴定

选P3代骨骼肌卫星细胞接种于6孔板,待细胞生长至80%—90%汇合时,改用成肌分化培养基（2% HS+1%双抗+DMEM高糖培养基）继续培养。隔2 d换一次液,观察细胞生长状况和肌管融合情况。

连续诱导8 d,视野中看到大量细胞融合时进行细胞免疫荧光染色。经固定、通透、封闭后,加入一抗（Anti-MHC, 1﹕200）4 ℃过夜,第二天加入荧光二抗避光孵育1 h。滴加DAPI染液后,荧光显微镜观察。

1.7 骨骼肌卫星细胞成脂诱导及鉴定

取P3代骨骼肌卫星细胞接种于6孔板,当细胞培养至80%融合时,更换完全培养基为成脂诱导分化液（10% FBS+5 μg·mL^-1 胰岛素+1 μmol·L^-1 DEX +0.5 mmol·L^-1 IBMX +1μmol·L^-1罗格列酮+1%双抗+DMEM高糖培养基）诱导3 d,之后改用成脂诱导维持液（10% FBS+5 μg·mL^-1 胰岛素+1%双抗+DMEM高糖培养基）维持9 d。每隔3 d更换新鲜诱导维持培养液。

油红O染色鉴定细胞成脂分化能力。PBS缓冲液漂洗细胞3次,用4%多聚甲醛室温固定10 min,弃去并加入新多聚甲醛再次固定1 h。蒸馏水冲洗3次,加入60%异丙醇静置5 min,开盖完全干燥。加入1 mL油红O染液（油红O储液﹕蒸馏水=3﹕2）,37 ℃染色30—40 min,弃去油红O染液,蒸馏水洗涤4次,显微镜下观察染色结果。

油红O定量检测,每孔加2 mL异丙醇萃取脂滴,37 ℃恒温摇床孵育15 min,酶标仪检测510 nm处OD值。

1.8 骨骼肌卫星细胞成骨诱导及鉴定

取P3代骨骼肌卫星细胞接种于6孔板,细胞密度达到90%后诱导成骨。成骨诱导培养液（10% FBS+10 mmol·L^-1 β-甘油磷酸钠+0.1 μmol·L^-1 DEX+50 mg·L^-1 抗坏血酸+1%双抗+DMEM高糖培养基）培养3周,期间每3 d换一次液,观察细胞形态变化。

对诱导细胞茜素红染色。多聚甲醛固定15 min,弃去固定液后蒸馏水洗3次,待液体完全吸干净后加入茜素红染色液,室温染色30 min。弃去染液加蒸馏水保持湿润,显微镜下观察。

1.9 RNA提取及相关基因qRT-PCR检测

成肌诱导组（收集0 d、诱导2、4、6、8 d细胞）、成脂诱导组（收集0 d、诱导3、6、9、12d细胞）、成骨诱导组（收集0 d、诱导7、14、21 d细胞）分别提取细胞RNA,并按照说明书要求反转录。采用qRT-PCR的方法分别检测成肌诱导、成脂诱导、成骨诱导后相关基因的相对表达量,以18S为内参基因,引物序列见表1。

Table 1
表1
表1qRT-PCR引物
Table 1Primers for qRT-PCR

基因名称Gene name	引物序列（5′→3′）Primer sequence	产物长度Product length (bp)	序列号Accession No.
MyoD	F: GAGAGCACTACAGCGGTGAC R: CCTCGCTGTAATAGGTGCCG	132	NM_001002824.1
MyoG	F: CTCAGCTCCCTCAACCAGGA R: GGAGTGCAGATTGTGGGCAT	195	NM_001012406.1
FABP4	F:TGGTACAGGTGCAGAAGTGG R: TTCTGGTAGCCGTGACACCT	108	NM_001002817.1
CEBP/β	F:CTGGAGACGCAGCATAAGGT R:TGCTTGAACAAGTTCCGCAG	110	NM_001199889.1
PPARγ	F:CTATTCCATGCTGTCATGGGTG R: ACCATGGTCACCTCTTGTGA	107	NM_214379.1
HSL	F:CACTGACTGCTGACCCCAAG R:TCCTCACTGTCCTGTCCTTCAC	121	NM_214315.3
BGLAP	F: GCCACACTCTGCCTTGCTG R: CTCCTGGAAGCCGATGTGA	237	NM_001164004.1
RUNX2	F: GAGTGGACGAGGCAAGAGTT R:ACTTGGTGCAGAGTTCAGGG	256	XM_003482203.4
18S	F:CCCACGGAATCGAGAAAGAG R: TTGACGGAAGGGCACCA	122	NW_018085108.1

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1.10 数据分析

所有试验均设置3个生物学重复,数据结果使用SPSS Statistics 21.0软件单因素方差分析进行显著性检验,不同时间点标志基因表达量的差异采用Duncan’s法进行多重比较,P<0.05表示差异显著,P<0.01表示差异极显著。

2 结果

2.1 骨骼肌卫星细胞形态学观察

分离的原代骨骼肌卫星细胞接种至培养皿2 h后,其中的成纤维细胞、血细胞等杂细胞已贴壁。倒置显微镜下观察,刚分离出来的卫星细胞呈球形,折光性强（图1-A）。此时,转移细胞悬液到新培养皿中,对骨骼肌卫星细胞进行第一次纯化。18 h后再次转移上清液,卫星细胞的纯度再次提升。多次纯化后,观察发现部分卫星细胞贴壁并向四周延展,部分细胞周围有小的突起（图1-B）。继续培养24 h,大多数卫星细胞已贴壁,细胞呈梭形或纺锤形,细胞饱满且折光性较强（图1-C）。首次更换新鲜培养基后,可见卫星细胞数量明显增多,且细胞形态也变得多样,相邻卫星细胞开始相互接触（图1-D）。

图1

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图1骨骼肌卫星细胞形态

A：刚分离的骨骼肌卫星细胞呈球形,折光性强;B：部分卫星细胞开始细胞贴壁,向四周延展;C：分离培养48 h卫星细胞呈梭形或纺锤形;D：分离培养72 h卫星细胞数量增加出现汇合;E：细胞生长至70%时的形态;F：培养5 d细胞拉长变细呈有规律排列;G：培养7 d卫星细胞自发融合,形成多核肌管;H：复苏第3次传代后冻存的骨骼肌卫星细胞
Fig. 1The morphology of skeletal muscle satellite cells (100×)

A: The separated skeletal muscle satellite cells are spherical and have strong refractive index; B: Some satellite cells begin to adhere and extend to the periphery; C: 48 h of satellite cells are fusiform or spindle-shaped; D: Separated and cultured for 72 h. The number of satellite cells increased and converged; E: Morphology of cells growth to 70%; F:Cells elongated and thinned regularly after cultured 5 days; G: 7 days of satellite cells spontaneously fused to form multinucleated myotubes; H: Recovery skeletal muscle satellite cells frozen after 3 passages


当卫星细胞生长达70%时,可按照1﹕1的比例传代培养或冻存（图1-E）。由于细胞增殖能力不断增强,细胞逐渐拉长变细形成有规律的平行排列（图1-F）。继续培养可见细胞方向性排列更明显,并能自发融合形成多核肌管（图1-G）。复苏后的骨骼肌卫星细胞与冻存前形态相似,细胞生长状态良好且增殖能力较强,表明试验所用的冻存体系可用来保存骨骼肌卫星细胞（图1-H）。

2.2 骨骼肌卫星细胞免疫荧光鉴定

应用细胞免疫荧光技术检测P2代骨骼肌卫星细胞蛋白Pax7、MyoD的表达情况,并用DAPI染核。结果发现,Pax7和MyoD均呈阳性表达（图2-A、2-D）,经DAPI染色后细胞核呈蓝色（图2-B、2-E）。其中,Pax7荧光强度较强,表明其在细胞内的表达量可能较高（图2-C）,MyoD荧光强度较弱,说明其在细胞中表达量可能较低（图2-F）,综上确证本次试验成功分离了骨骼肌卫星细胞,且细胞纯度较高。

图2

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图2骨骼肌卫星细胞免疫荧光鉴定

A：Pax7呈阳性表达;B：DAPI染色细胞核;C：A与B叠加图片;D：MyoD呈阳性表达;E：DAPI染色细胞核;F：D与E叠加图片
Fig. 2Immunofluorescence identification of skeletal muscle satellite cells (100×)

A: Pax7 expression was positive; B: DAPI stained nuclei; C: Image merged between A and B; D: MyoD expression was positive; E: DAPI stained nuclei; F: Image merged between D and E

2.3 骨骼肌卫星细胞生长曲线

骨骼肌卫星细胞生长曲线呈“S”型。体外培养骨骼肌卫星细胞前2 d生长较缓慢,为潜伏期。3—5 d细胞开始迅速增殖,进入指数生长期。此后由于细胞密度增大,细胞相互接触造成生长减慢,达到平台期。

图3

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图3骨骼肌卫星细胞生长曲线

Fig. 3Skeletal muscle satellite cell growth curve

2.4 骨骼肌卫星细胞成肌诱导

利用2% HS对骨骼肌卫星细胞诱导成肌分化, 加入诱导成肌分化液48 h后可见细胞融合并出现少量短粗肌管（图4-A）。随后,细胞融合更加广泛,形成大量多核肌管（图4-B）。继续诱导至4 d,显微镜下观察可见肌管相互融合,且肌管之间平行排列（图4-C）。MHC细胞免疫荧光结果为阳性（图4-D）,DAPI染色可见肌管内存在多个细胞核（图4-E）,表明本试验卫星细胞成肌诱导成功（图4-F）。

图4

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图4骨骼肌卫星细胞成肌诱导分化及免疫荧光染色

A：诱导48 h,卫星细胞融合出现少量肌管;B：诱导72 h,肌管数量增多;C：诱导4 d后,肌管之间相互融合;D：分化后MHC免疫荧光;E：DAPI染核;F：D和E叠加
Fig. 4Myogenic differentiation of skeletal muscle satellite cells and immunofluorescence (100×)

A: After 48 h induction, satellite cells fusion and a amount of myotubes were appeared; B: Induced 72 h, the number of myotubes increased; C: After 4 days induction, myotubes were fused together; D: MHC immunofluorescence; E: DAPI stained nuclei; F: Image merged between D and E


对诱导0、2、4、6、8 d细胞进行qRT-PCR检测,结果显示标志基因MyoD和MyoG在诱导的各阶段均有表达（图5）。在成肌诱导分化过程中二者均呈先升高后下降趋势,都在第4天达到最大值,且极显著高于诱导0 d（P<0.01）。

图5

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图5成肌诱导分化后MyoD、MyoG表达情况

不同大写字母表示差异极显著（P<0.01）。下同
Fig. 5Expression of MyoD and MyoG after myogenic differentiation

Different uppercase letters indicate that extremely significant differences (P<0.01). The same as below

2.5 骨骼肌卫星细胞成脂诱导

选取P2代细胞诱导成脂分化72 h后发现,细胞形态由梭形变为三角形或多边形（图6-A）。诱导6 d,显微镜下可见细胞质逐渐透亮,有少量脂滴出现（图6-B）。继续诱导至9 d,可见细胞逐渐变圆,体积增大,脂滴数量明显增多（图6-C）。随着时间的延长,诱导12 d后,脂滴聚集融合形成大脂滴（图6-D）。用油红O分别对诱导3、6、9和12 d的卫星细胞染色,结果可见大小不等的红色葡萄样脂滴（图6-E—H）,肉眼观察培养板底部也出现红色沉淀（图6-I—L）。

图6

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图6骨骼肌卫星细胞成脂诱导分化及油红O染色

A-D：分别为成脂诱导3、6、9、12 d卫星细胞形态学观察（100×）;E-H：诱导细胞油红O染色,脂滴呈红色（100×）;I-L：肉眼观察诱导后培养皿底可见红色沉淀
Fig. 6Adipogenic differentiation of skeletal muscle satellite cells and Oil red O staining

A-D: Morphological observation of satellite cells at 3, 6, 9, 12 d induced by adipogenesis (100×); E-H: The lipid droplets was positive expression under Red Oil O dyed (100×); I-L: After the induction, the red precipitate was observed at the bottom of the culture dish


油红O定量检测结果表明,随着成脂诱导的推进,甘油三酯含量呈稳步上升趋势,诱导3 d表达量最低,12 d后含量最高,且各时间点均存在极显著差异（P<0.01）。结合油红O染色及定量结果,表明卫星细胞成脂诱导分化成功（图7）。

图7

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图7油红O定量结果

Fig. 7Oil red O staining quantitative results



检测PPARγ、CEBP/β、FABP4和HSL的表达情况,结果如图8所示,分化各时间点与0 d相比差异极显著（P<0.01）。其中PPARγ表达量随着诱导分化时间的推进,该基因表达量前期逐步上升并保持稳定。FABP4主要在分化中期表达,表达量呈先升高后下降趋势。CEBP/β和HSL表达情况相似,第3天达到最高,随后逐渐下降。

图8

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图8成脂诱导分化后PPARγ、CEBP/β、FABP4和HSL表达情况

Fig. 8Expression of PPARγ, CEBP/β, FABP4 and HSL after adipogenic differentiation

2.6 骨骼肌卫星细胞成骨诱导

在成骨诱导培养基中培养7 d后,卫星细胞逐渐收缩变为不规则形态（图9-A）。诱导14 d后,细胞部分复层生长,细胞间可见钙化沉淀（图9-B）。继续诱导至21 d,细胞立体感增强,出现大量矿化结节（图9-C）。茜素红染色可见随着分化时间的延长,呈圆形不透明的钙化结节数量和密度都明显增加（图9-D—E）。染色后培养皿底部也出现了颗粒状沉淀,进一步表明成骨诱导成功（图9-F—G）。

图9

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图9骨骼肌卫星细胞成骨诱导分化及茜素红染色

A-C：成骨诱导7、14、21 d卫星细胞显微镜下观察（100×）;D-F：茜素红可将诱导后细胞形成的钙化结节染成红色（100×）;G-I：肉眼观察诱导后皿底出现红色沉淀结节
Fig. 9Osteogenic differentiation of skeletal muscle satellite cells and Alizarin Red staining

A-C: After osteogenic induction 7, 14, 21 d, satellite cells were observed under microscope (100×); D-F: Alizarin red staining can stain the calcified nodules formed after induction into red (100×); G-I: Macroscopic observation of red precipitated nodules at the bottom after the osteogenesis


诱导骨骼肌卫星细胞成骨分化,检测成骨基因骨钙素（BGLAP）和Runt相关转录因子2（RUNX2）的表达情况,如图10所示。诱导成骨分化过程中,成骨标志基因的表达水平均上调,都在第3周达到最高,与0 d相比差异极显著（P<0.01）。

图10

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图10成骨诱导后BGLAP和RUNX2表达情况

Fig. 10Expression of BGLAP and RUNX2 after osteogenic induction

3 讨论

骨骼肌卫星细胞作为静息状态的肌源性干细胞,参与骨骼肌的生长与修复过程,对维持机体稳态具有重要作用^[30]。骨骼肌卫星细胞分离培养的关键在于使数量较多且活力较好的卫星细胞释放,因此有****对卫星细胞的纯化方式进行了改进。单根肌纤维法是将完整的肌纤维从肌束中剥离,从而使卫星细胞游离出来的一种方法^[31]。但该方法分离卫星细胞所需时间较长且获得细胞数量有限,因而应用受限。鉴于骨骼肌卫星细胞存在于基膜与肌纤维之间,只有基膜分解才有助于卫星细胞的获取^[32],因此如今体外分离卫星细胞常用酶消化法。传统的单一酶消化法不能完全解离卫星细胞,选用胶原酶胰酶的两步酶消化法可弥补先前缺点,保证卫星细胞的产出率^[33]。运用酶消化法很难避免成纤维细胞的出现,可通过纯化的方式提高卫星细胞的纯度。其中流式分选术用于人和小鼠等模式动物细胞分选较多,但基于设备的限制及细胞表面特异分子标志价格昂贵,并未在家养动物中推广。Percoll梯度密度离心法步骤繁琐、易污染,多次离心后会导致细胞流失。差速贴壁法是根据卫星细胞与杂细胞粘附时间上的差异,成纤维细胞贴壁速度快,通过多次差速得到纯度较高的卫星细胞,此法简单易行且对细胞无损伤^[34]。

本试验选用Ⅰ型胶原酶和胰蛋白酶联合消化法分离骨骼肌卫星细胞,并对消化时间进行严格控制。刚分离出的骨骼肌卫星细胞呈圆形,通过反复差速贴壁两次以逐步去除成纤维细胞、血细胞等杂细胞,实现对卫星细胞的纯化。重新接种6 h后,部分卫星细胞开始贴壁延展,继续培养24 h,细胞呈现长梭形或纺锤形,接种3 d可见贴壁细胞数量明显增多,部分细胞出现汇合。接种5 d后,细胞密度达到90%,此时细胞呈有规律的方向性生长,继续培养则卫星细胞出现自融合现象,临近细胞会融合成肌管。分离纯化结果与BAQUERO等研究结果基本一致^[35,36]。

目前对骨骼肌卫星细胞的鉴定有多种方法,其中公认度最高的是免疫细胞化学染色法,该法原理就是抗原抗体反应,因灵敏度高、特异性强等优点被广泛使用^[37]。已有报道证实95%以上的卫星细胞都表达Pax7,此外Pax7还能调控卫星细胞处于静息或激活状态,因此可作为鉴定骨骼肌卫星细胞的标志物^[38]。本试验参考FENG等^[39]的方法,通过细胞免疫荧光法对卫星细胞特异性基因Pax7、MyoD的蛋白表达情况进行检测,结果可见标志物染色呈阳性,且阳性细胞多,表明分离培养的卫星细胞纯度高、活性好。

骨骼肌卫星细胞对培养要求较高,大量的研究结果表明,卫星细胞的体外培养需要添加合适浓度的血清,高浓度的血清有助于细胞增殖,血清浓度的降低会促进卫星细胞的分化。STARKEY的研究发现,低血清浓度可使卫星细胞自发向成肌细胞方向分化,但并不能自行分化为脂肪细胞、骨细胞等非成肌细胞^[40]。已有的研究结果证实,在添加碱性成纤维细胞生长因子的培养基中,有利于卫星细胞的增殖^[41]。本试验最终选择在高糖培养基中添加10%浓度胎牛血清来培养骨骼肌卫星细胞,当细胞增殖汇合到一定密度后,即使高浓度血清培养细胞也会出现不可逆分化,最终融合成肌管的现象,因此卫星细胞的传代应选择细胞密度70%左右时进行。本试验结合之前方法用2%马血清诱导卫星细胞分化,结果显示诱导4 d可见大量较长较粗肌管形成,MHC染色呈阳性,进一步确证骨骼肌卫星细胞的成肌分化。

在模式动物中的研究中,已证实体外培养的小鼠卫星细胞可直接分化成脂肪细胞、骨细胞和软骨细胞等^[42]。目前,对猪骨骼肌卫星细胞多向分化潜能的研究较少。本试验参考“激素鸡尾酒”法对卫星细胞诱导成脂,在添加胰岛素、地塞米松、IBMX和罗格列酮等诱导因子条件下培养卫星细胞^[43]。对细胞成脂能力的鉴定主要选用油红O染色,油红O染色呈阳性可作为脂滴沉积的标志^[44]。结果发现诱导3 d卫星细胞形态发生变化,此后更换为维持培养基继续诱导可见大量脂滴出现,油红O染色及定量结果都表明成脂诱导成功。对于骨骼肌卫星细胞成骨诱导,多在培养基中补充β-甘油磷酸钠、抗坏血酸和地塞米松来促进钙质形成,诱导结节钙化^[45]。利用茜素红可与钙发生显色反应的特性,可结合染色结果判定细胞成骨矿化结节积累情况,进而鉴定细胞成骨分化效果^[46]。本试验结果表明诱导卫星细胞成骨分化后,茜素红染色可见大量骨钙结节形成,同时荧光定量结果显示诱导成骨成功,这与REN等^[47]在山羊上的研究结果相似。

众所周知,骨骼肌卫星细胞作为机体干细胞的一种,具有可塑性强、多潜能等优点^[48]。卫星细胞体外分离培养体系的建立,可为猪骨骼肌生长发育、肉质改良提供研究基础。同时为畜禽遗传资源保存提供了新方法,也为今后基因组学研究提供相应的实验材料。此外本试验还证明了猪骨骼肌卫星细胞的多向分化潜能,拓宽了卫星细胞在组织工程和再生医学上的研究价值和广泛的应用前景。

4 结论

本试验成功分离猪骨骼肌卫星细胞,通过细胞免疫荧光检测骨骼肌卫星细胞标志蛋白对其进行了鉴定。建立了猪骨骼肌卫星细胞体外分离、纯化以及鉴定的方法,同时利用不同诱导剂分别对其诱导成肌、成脂、成骨,探索了骨骼肌卫星细胞的生物学特性。对研究猪肌肉生长发育过程具有一定科学意义,也为后续探究猪肌肉卫星细胞功能和分子机制提供了试验材料。

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