摘要
干细胞按来源不同可分为胚胎干细胞(embryonic stem cells,ESCs)和成体干细胞(adult stem cells,ASCs)。ESCs由于存在伦理学争议、有形成畸胎瘤的可能、移植的ESCs可诱发宿主的免疫反应等问题,使其临床应用受到一定限制,而ASCs中的间充质干细胞(Mesenchymal stem cells, MSCs)可从成熟个体皮肤真皮、脂肪、肌肉、骨髓、外周血等多种组织分离且体外扩增较为方便,具有跨胚层的多向分化能力,较强的归巢和迁移到受伤组织能力和免疫调节功能,避免了一直以来存在的伦理学争议。MSCs还能释放一些生物活性物质抑制局部缺血引起的凋亡,抑制疤痕组织的形成,刺激血管生成,创造良好的再生微环境。MSCs分泌物还是宿主体内干细胞的有丝分裂刺激物并有利于宿主体内干细胞分化。MSCs已被认为是理想的治疗遗传性疾病和退行性疾病的细胞来源,还可控制和治疗移植物抗宿主病及炎症性肠道疾病(如Crohn’s disease)、治疗自身免疫性疾病(如糖尿病)、皮肤大面积烧伤或损伤后无疤痕再生、中风和脊髓损伤、急慢性心血管事件及急性肾衰或肝衰。MSCs对辐射损伤后造血恢复、骨和软骨组织置换、骨骼肌修复及血管修复等治疗也是前景良好,是目前组织工程和再生医学研究的热点。但是利用MSCs进行临床治疗必须解决两个问题:一是高效、安全的得到大量扩增的干细胞,二是精确控制干细胞朝着特定的组织细胞分化,解决这两个问题均依赖于对MSCs增殖与分化的分子机制的认识。一般认为,细胞的增殖与分化主要是受细胞内基因不同时间和空间精细的表达调控,新的细胞表型的建立及细胞增殖均需要相关调控基因的准确的打开或关闭,一旦这种基因调控出现异常就会出现细胞异常增生甚至恶性转化等问题。阐明MSCs增殖与分化基因表达调控机制,既需要实验结果的积累,更需要技术方法的突破。近年来出现的一种前瞻性基因扫描研究技术即基因捕获,基本原理是用一含报告基因(既可作为内源基因的标签,也可使随后的基因克隆变得可行)的DNA载体随机插入细胞基因组,产生内源基因失活突变,通过报告基因的表达提示插入突变的存在及突变内源基因的表达特点。它用于MSCs多向分化研究有其独特的优点:基因捕获载体转染MSCs后,在一个实验中就可产生大量的随机插入突变体,载体中的报告基因可随时监测被捕获基因的表达时间、蛋白分布和水平,而用cDNA末端快速扩增法(rapid amplification of cDNA ends,RACE)等技术可分离被捕获基因。前瞻性基因扫描技术既可以验证以往的MSCs分化研究成果,发现一些已知基因的新功能,也可能发现新基因,从而丰富MSCs分化机制理论,提高临床干预措施的有效性。
严重创伤往往因修复细胞的功能障碍和/或缺乏而难以得到有效修复,细胞替代治疗成为近年研究的热点。MSCs因其组织分布的广泛性和具有多向分化能力的特点,是细胞替代治疗的最佳候选种子细胞。揭示MSCs分化为组织细胞的基因调控机制将有助于在体内外定向诱导细胞分化,还有利于体内外细胞重编程变为干细胞(如怎样高效、安全的诱导出iPS细胞)以用于有效的细胞治疗及提高其促进创伤愈合的有效性。C3H/10T1/2细胞(简称10T1/2细胞)是Reznikoff等从C3H小鼠胚胎分离建立的间充质干细胞株,可分化为平滑肌、内皮、骨、软骨、脂肪、骨骼肌和神经元细胞,在研究胚层发育和细胞分化中可发挥重要作用。TGF-β1是胚胎发育和创伤愈合中极其重要的生长因子之一,参与和促进多种细胞表型的变化和转换。因此,本课题拟以10T1/2细胞为间充质干细胞模型,重点研究基因捕获技术捕获的在MSCs有转录活性的基因,并以TGF-β1为细胞分化诱导因子,研究MSCs的增殖、多向分化及相关机制,丰富MSCs促进创伤愈合的理论基础。本研究获得的主要结果和结论如下:
1. C3H/10T1/2细胞表型鉴定及多向分化的实验研究
通过流式细胞仪鉴定10T1/2细胞不表达内皮细胞表面抗原CD31,造血干细胞表面抗原CD34,造血细胞表面抗原CD45,但表达CD44、CD73、CD90、CD105等干细胞表面抗原。用TGF-β1诱导10T1/2细胞向平滑肌分化,24 h后诱导细胞出现平滑肌细胞(smooth muscle cells,SMCs)特征性的“峰谷”样生长外观,RT-PCR证实αSMA、SM22a、SM-MHC、SRF等平滑肌分化相关基因表达增强,免疫荧光实验证实αSMA蛋白表达增强,Western blot证实αSMA、SM22a蛋白表达增强,说明成功建立TGF-β1诱导10T1/2细胞平滑肌分化模型。用VEGF和bFGF联合诱导10T1/2细胞成内皮分化,9天后诱导细胞出现内皮细胞特征性“鹅卵石”样外观,RT-PCR发现内皮分化相关基因Flt1、Flk1、Ang2、Tie2、VE-cadherin、CD31、Ang1、Vezf1、Notch1、Jagged1和EphB4/EphrinB2诱导后表达逐渐增强,但不表达淋巴内皮细胞标志Flt4,说明诱导后细胞不是淋巴内皮细胞。免疫细胞荧光染色显示诱导后细胞表达内皮细胞特异性标志CD31、FactorⅧ和VE-cadherin。透射电镜下观察诱导后细胞胞浆存在Weibel-Palade小体(简称W-P小体)和丰富的胞饮小泡。Dil-ac-LDL吞噬实验表明分化后细胞具有吞噬ac-LDL的功能,Ⅰ型胶原三维成血管实验显示分化后细胞在体外三维培养中能形成血管样结构,说明诱导后细胞不但表达内皮细胞标志且具有内皮细胞功能。三维成血管后αSMA/CD31免疫荧光双标实验显示10T1/2细胞自身表达中等强度αSMA,但在分化诱导后表达逐渐降低,而CD31表达逐渐增强,说明VEGF和bFGF联合诱导10T1/2细胞成内皮分化时并不存在同时向平滑肌分化的现象。用5-氮杂胞苷诱导10T1/2细胞17天后可见粗大肌管形成,但此诱导并非特异性诱导,诱导15天后少量细胞出现了微小脂滴,并逐步融合变大,25天后形成典型的脂肪细胞,油红染色可见脂滴。用维生素C、β-磷酸甘油、地塞米松诱导10T1/2细胞成骨分化,第21天茜素红染色可见红色骨结节。用3-异丁基-1-甲基黄嘌呤( 3-isobutyl-1-methylxanthine,IBMX)、地塞米松、胰岛素诱导10T1/2细胞成脂分化,第15天油红染色可见脂滴。用bFGF、β-巯基乙醇、二甲基亚砜(DMSO)诱导10T1/2细胞成神经元分化,第3天出现类似神经元样细胞,15天时免疫细胞化学检测神经元标志物神经元特异性烯醇化酶(neuron-specific enolase,NSE)仍为阳性,说明诱导后细胞为神经元样细胞。体外培养贴壁生长、细胞表面抗原鉴定及有多向分化的潜能说明10T1/2细胞是良好的间充质干细胞模型。
2.多用途基因捕获C3H/10T1/2细胞阳性克隆库的建立及鉴定
用含LacZ为报告基因的PolyA基因捕获载体ROSAFARY转染包装细胞Phoenix,收集病毒上清,感染10T1/2细胞,并以潮霉素筛选整合了基因捕获载体的阳性细胞克隆。测试不同浓度(50、100、150、200、300、400、600、800 mg/l)潮霉素对10T1/2细胞的毒性,以7天杀死全部细胞确立潮霉素对10T1/2的最佳筛选浓度为150 mg/l。以150 mg/l潮霉素筛选共获得103个克隆,经以插入载体ROSAFARY的LacZ为目的基因的细胞基因组DNA PCR及LacZ染色鉴定最终建立了含64个不同克隆的10T1/2细胞基因捕获阳性克隆库,其中6个LacZ染色阳性,说明捕获到部分干细胞中高表达的基因,其余细胞克隆LacZ染色呈阴性,说明捕获的是干细胞低或不表达的基因。
3. TGF-β1诱导后表达下调捕获基因的分离及生物信息学分析
TGF-β1诱导前期建立的10T1/2细胞基因捕获阳性克隆库平滑肌分化,对分化前后的细胞进行LacZ染色筛选获得3个平滑肌分化后表达下调克隆,通过RACE等分子生物学技术及电子克隆分离获得差异表达序列,使用GenBank网站nblast进行生物信息学分析,结果表明为一个已知基因Mrps6和两个新基因(命名为mgt-6和mgt-16)。mgt-6位于14号染色体,包括4个转录本,翻译2个蛋白质,用GenBank网站在线投递软件BankIt提交后获ID号(FJ744746、FJ860514、FJ860513和FJ748867),转录本1、2和3含3个外显子,编码短蛋白(命名为MGT-6S),用pI/Mw批量计算软件计算蛋白分子量为1414.68,等电点为5.75,转录本4含2个外显子,编码长蛋白(命名为MGT-6L),计算其分子量为4073.57,等电点为6.52。mgt-16基因位于19号染色体,含2个外显子,编码一个93个氨基酸的蛋白(命名为MGT-16),计算其分子量为9772.02,等电点为6.04,提交GenBank网站获ID号(GU266552)。最后提取6、16号基因捕获克隆RNA进行RT-PCR后测序验证了捕获载体ROSAFARY插入在mgt-6、mgt-16第一个内含子中,并获取ROSAFARY载体精确的剪接受体(splicing acceptor,SA)位点在其2754 nt处。
4.新基因mgt-16的克隆及功能研究
以逆转录病毒载体pL-EGFP-N1为基础经测序证实成功构建mgt-16融合EGFP的过表达载体pL-EGFP-N1-16,转染包装细胞Phoenix后收集病毒感染10T1/2细胞,以400μg/ml G418筛选建立了稳定表达mgt-16融合EGFP的细胞克隆,荧光显微镜下观察绿色荧光只出现在胞浆,证实MGT-16蛋白在细胞胞浆表达,与16号克隆正常培养下LacZ染色结果一致。
以载体pL-EGFP-N1和pIRES2-EGFP-N1为基础经测序证实成功构建含内核糖体插入序列(internal ribosome entry site,IRES)逆转录病毒载体pL-IRES-EGFP-N1,再以pL-IRES-EGFP-N1为基础成功构建mgt-16融合6×His标签的逆转录病毒载体pL-IRES-EGFP-16-His,转染包装细胞Phoenix后收集病毒感染10T1/2细胞,400μg/ml G418筛选获得mgt-16融合6×His且带IRES-EGFP的稳定表达克隆。以pL-IRES-EGFP-N1感染的10T1/2细胞为对照,GAPDH为内参,Western blot检测平滑肌分化相关基因αSMA和SM22a,结果表明过表达mgt-16可抑制TGF-β1诱导的10T1/2细胞平滑肌分化相关基因的表达。
以16号基因捕获克隆为mgt-16基因低表达模型,稳定表达mgt-16融合EGFP蛋白的10T1/2细胞克隆和稳定表达mgt-16融合6×His并带IRES-EGFP元件的10T1/2细胞克隆为mgt-16基因过表达模型。CCK-8实验检测培养细胞OD值反映细胞增殖情况,结果显示:16号克隆较正常10T1/2细胞增殖减慢(P <0.05),说明mgt-16基因被打断后可使细胞增殖减慢;而无论是mgt-16融合EGFP还是mgt-16融合His的过表达10T1/2细胞均比其对照组增殖加快(P <0.05),说明mgt-16基因过表达后可使细胞增殖加快。细胞划痕实验结果显示:16号克隆24 h细胞伤口愈合面积百分率较正常10T1/2细胞低(P <0.05),说明mgt-16基因被打断后可使细胞迁移减慢;而无论是mgt-16融合EGFP还是mgt-16融合His的过表达10T1/2细胞24 h细胞伤口愈合面积百分率均比其对照组高(P <0.05),说明mgt-16基因过表达后可使细胞迁移加快。通过比较mgt-16基因低表达和过表达两种细胞模型的效应差异,均表明mgt-16基因为细胞增殖、迁移正相关基因。
5. TGF-β1下调mgt-16表达参与平滑肌分化的分子机制
16号克隆用TGF-β1、p38/RK抑制剂、PI3K/AKT抑制剂、ERK抑制剂、mTOR抑制剂、NF-κB抑制剂,以及单一抑制剂联合TGF-β1处理24 h,LacZ染色及RT-PCR结果均表明TGF-β1是通过激活P38信号通路引起mgt-16表达降低。接着我们发现:①p38抑制剂(SB203580)可抑制TGF-β1对细胞形态学的影响;②Western blot检测TGF-β1可使P38磷酸化蛋白增加及SB203580可抑制此效应;③RT-PCR证实SB203580可抑制TGF-β1诱导的平滑肌分化相关基因SM22a的表达。此三点表明TGF-β1通过激活P38信号通路诱导10T1/2细胞平滑肌分化。至此,提出假说:TGF-β1可能通过激活P38信号通路下调mgt-16基因而调控10T1/2细胞平滑肌分化。
6.新基因mgt-6的克隆及功能研究
6号克隆正常培养及5-氮杂胞苷处理后LacZ染色均显示正常情况下mgt-6在细胞胞浆表达,而5-氮杂胞苷处理后可发生核转位,并由构建mgt-6融合EGFP的真核表达载体pEGFP-N1-6转染10T1/2细胞观察绿色荧光亚细胞定位所证实,提示新基因mgt-6可能参与了细胞DNA低甲基化效应。此外,RT-PCR结果表明mgt-6含四个转录本且在小鼠不同组织及不同细胞系有不同表达。皮肤、脾、小肠、肾脏、心脏和骨骼肌主要表达转录本4,而肝脏主要表达转录本1,脑和肺脏4个转录本均有较强表达。C2C12主要表达转录本4,而10T1/2细胞和Lewis细胞4个转录本均有较强表达且转录本1、2、4在Lewis细胞表达较10T1/2细胞表达要高。小鼠骨骼肌成肌细胞系C2C12和成年小鼠骨骼肌组织均主要表达转录本4(编码MGT-6L)而弱表达转录本1、2、3(编码MGT-6S),提示MGT-6L或MGT-6S可能与骨骼肌形成或分化有关。6号克隆正常培养、TGF-β1、p38/RK抑制剂、PI3K/AKT抑制剂、ERK抑制剂处理24 h后LacZ染色以及10T1/2细胞正常培养、TGF-β1及不同抑制剂处理24 h后RT-PCR,结果均表明TGF-β1及PD98059(ERK抑制剂)处理可使mgt-6表达下调,而SB203580及LY294002(PI3K/AKT抑制剂)处理可使mgt-6表达上调,提示mgt-6可能与平滑肌分化有关,机制可能是TGF-β1通过激活P38、PI3K和抑制ERK信号通路。
总之,本研究着眼于MSCs应用于创伤愈合研究中的瓶颈问题,即MSCs增殖、分化调控的分子机制尚不十分清楚,创新性地把主要用于研究发育相关基因的前瞻性基因扫描方法之一,基因捕获技术,应用到MSCs的研究中,建立了有广泛用途的基因捕获阳性克隆库;拓展建立的10T1/2细胞多向分化的实验条件对诱导MSCs定向分化有参考价值;发现了两个新基因(mgt-6和mgt-16)并初步研究了其功能;对TGF-β1信号通路及其调控MSCs平滑肌分化的分子机制有了新的认识,为探索诱导MSCs定向分化的新策略,提高其在创伤愈合中应用的有效性提供了理论指导和实验依据。
Based on their origins, stem cells fall into two categories, embryonic stem cells (ESCs, ES) and adult stem cells (ASCs). Currently, the clinical application of ESCs is limited by the ethical dispute, the formation of teratomas and the possibility of provoking immune reaction after transplantation of ESCs into a new host. In contrast, mesenchymal stem cells (MSCs), one kind of ASCs, which reside in but not limited to dermis, adipose tissue, muscle, bone marrow and peripheral blood, can be easily isolated and amplified in vitro. In addition, MSCs has the capacity to differentiate or transdifferentiate into endodermal, mesodermal, and ectodermal lineages, preferentially home to the sites of injured tissue and modulate host immunoresponses, and devoid of the ethical concern. Moreover, MSCs secrete bioactive factors that inhibit ischemia-caused apoptosis, prevent the formation of scar tissue, stimulate angiogenesis, and establish a favourable regenerative microenvironment. These factors are mitogenic to tissue-specific stem cells and can enhance the host-mediated differentiation of host-intrinsic stem cells. Therefore, MSCs are ideal cell soureces for treating hereditary or degenerative disease, graft-versus-host disease and inflammatory disease (such as Crohn’s disease), autoimmune diseases (like diabetes), scarless regeneration of skin following massive burns or injury, stroke and spinal cord contusion or excision injuries, acute and chronic cardiac events, and acute renal or liver failure. MSCs also has great potential and promising future in the applications in the improving the hematopoietic recovery after radiation injury, tissue replacement with tissue-engineered bone/cartilage, repair of skeletal muscle and vascular injury, leading these cells the hot research focus in tissue engineering and regenerative medicine. There are two critical issues that need to be resolved before the safe and effective clinical application. One is how to obtain enough stem cell more effectively and safely, the other is how to control the differentiation of MSCs into committed effector cells, of which the answers depend on our understanding of the molecular mechanisms that regulate MSCs proliferation and differentiation. The processes of cell proliferation and differentiation are under the spatial and temporal control of cellular restricted expression pattern of genes. Establishing a new cellular phenotype or stimulating the normal cell proliferation requires switching precisely on and off the genes related to cell proliferation and differentiation. Abnormal cell proliferation or even cell transformation may occur once the gene regulation is disorder or deregulated. To understand the molecular mechanisms of MSCs proliferation and differentiation more in detail, it needs not only the accumulation of more experimental results but also the breakthrough of the technology. In recent years an approach based on forward genetic scaning called gene trapping is widely used to identify the location, sequence, expression and function of the trapped genes. Gene trapping is initially designed for random insertional mutagenesis in mouse embryonic stem cells by introducing a DNA vector with a promoterless reporter gene into the host genome. The reporter gene in the gene trap vector can not only trace the expression pattern of the trapped gene but also provide the starting site for the cloning sequences of the trapped genes. Furthermore, the success of insertion mutation and the responses of the trapped genes to various exogenous factors can be readily examined by the reporter gene. There are some distinct advantages when gene trapping is used to study the molecular mechanisms of MSCs differentiation: there are large amount of random insertional mutants after the gene trap constructs are introduced into MSCs chromosomes in one experiment; the expression pattern of endogenous gene can be monitored by the reporter gene at any time, and its sequences can readily be identified by RACE (rapid amplification of cDNA ends); gene trap mutagenesis-based forward genetic approach can be used to explore novel genes, identify the new function of known genes and validate the previous research of MSCs differentiation. So, the results from such experiment will provide more understanding of MSCs differentiation and help to make the clinical intervention more effective.
Severe injury leads to dramatically reduced repopulation and dysfunction of tissue-repairing cells, which can impair healing, and cell replacement therapy is one of the feasible strategies in the management of wound healing. Due to their multipotency and widely distribution in various tissues, MSCs represent the most promising candicate cell source for cell replacement therapy. Through the study on the molecular mechanisms of MSC’s committed differentiation, we will learn how to direct the cells to generate stem cells (iPS cells) and appropriate specialized cells to improve cell therapies as well as wound healing. The mesenchymal stem cell line C3H10T1/2 (also termed 10T1/2 cells) was isolated from C3H mouse embryo by Reznikoff, and can differentiate into smooth muscle cells, endothelial cells, osteoblasts, chondrocytes, adipocytes, myogenic cells and neurocytes. The multipotential 10T1/2 cells provide a good model for the molecular genetic analysis of mesodermal determination and cell differentiation, especially under the induction of growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-β(TGF-β). TGF-β1 is one of cytokines which play very important roles during embryonic development and wound healing by modulating many kinds of cytodifferentiation and cell phenotype change. Therefore, in the present study, 10T1/2 cells are adopted as a model of MSC and TGF-β1 as an inducer of cell differentiation in order to explore the molecular mechanisms of MSC proliferation and differentiation, and provide theoretical basis for MSC application in promoting wound healing, The trapped genes with active transcription will be the key interest.
The main results and conclusions are summarized as follows:
1. The immunophenotype and multilineage differentiation potential of murine embryonic mesenchymal stem cell line C3H/10T1/2 cells.
After analyzed by flow cytometry, the immunophenotype of 10T1/2 cells could be summarized as CD44+, CD73+, CD90+ and CD105+, but CD31-, CD34- and CD45-, which was similar to the reported cell surface markers of mesenchymal stem cells. The model of smooth muscle cell (SMC) differentiation was successfully established based on following findings: 10T1/2 cells tended to be an peak valley-like appearance, an smooth muscle cell-featured growth pattern, and SMC-specific genes such asαSMA, SM22a, SM-MHC, SRF, were upregulated after treated with transforming growth factor-beta1 (TGF-β1) for 24 hours by RT-PCR analysis,αSMA upregulation by immunofluorescence staining analysis, andαSMA, SM22a upregulation by Western blot analysis. After induced by VEGF and bFGF for 9 days, 10T1/2 cells tended to be an obvious cobblestone-like appearance, an endothelial cell-featured growth pattern. Endothelial cell specific markers such as Flt1, Flk1, Ang2, Tie2, VE-cadherin, CD31, Ang1, Vezf1, Notch1, Jagged1 and EphB4/EphrinB2 were upregulated but not Flt4 (a marker of lymphatic endothelial cells) detected by RT-PCR, which suggested the induced endothelial differentiation may not lymphatic endothelial committed. Moreover, the endothial differentiation was successfully established and rendered the cells with endothelial cell function of mature endothelial phenotype as shown by positive immunofluorescence staining for CD31, FactorⅧand VE-cadherin, abundant pinocytotic vesicles and Weibel-Palade bodies under transmission electron microscope, the uptake of Ac-LDL and formation of tubular structures in three-dimensional culture in collagen I gel. Additionally, the constitutively expressedα-SMA in 10T1/2 cells decreased, while significantly CD31 expression occurred after the induction, which did not support the existence of smooth muscle differentiation. When induced by 5-azacytidine, myotube-like cells appeared at day 17. Meanwhile, adipose globelets were also observed in a small proportion of cells at day 17, and Oil Red O staining confirmed adipose accumulation in cytoplasm day 25. After induced to osteoblast differentiation by combining ascorbic acid,β-glycerophosphate, and dexamethasone, bone-like nodules were identified by alizarin red staining at day 21. After induced to adipogenic differentiation by 3-isobutyl-1- methylxanthine (IBMX), dexamethasone, and insulin, Oil Red O staining confirmed adipose accumulation in cytoplasm at day 15. After treated with bFGF,β-mercaptoethanol (β-ME) in combination with DMSO, cytoplasm retracted towards the nucleus and the cell processes prolonged at day 3, and immunofluorescence staining showed that differentiated cells expressed neuron-specific enolase (NSE) at day 15, which indicated the phenotype conversion into neurocytes. Plastic-adherent under culture conditions in vitro, cell surface markers analyzed by flow cytometry, and the potential of multilineage differentiation defined that 10T1/2 cells can be used as an alternative source of mesenchymal stem cells.
2. The establishment and identification of gene trap clones from C3H/10T1/2 cells transfected with ROSAFARY vector.
The retroviral polyA gene-trap vector with resporter gene LacZ, ROSAFARY, was transfected into packaging cell line Phoenix by lipofectamine. Then the retrovirus particles containing gene-trap construct were collected to infect 10T1/2 cells, and the cell clones with integrated gene trapping vector was selected by hygromycin. The sensitivity of 10T1/2 cells to different concentrations of hygromycin (50, 100, 150, 200, 300, 400, 600 and 800 mg/l) was tested, and the optimized concentration for hygromycin selection on 10T1/2 cells was 150 mg/l because all 10T1/2 cells were killed after cultured with this concentration of hygromycin for 7 days. After the drug selection of 150 mg/l hygromycin, individual drug-resistant colonies were picked and expanded. As a result, totally 103 clones with hygromycin resistance were obtained. Among them, 64 clones were confirmed by genomic DNA PCR that targets LacZ sequence in ROSAFARY vector, including 6 clones with positive LacZ staining, which indicated the active promoter activity of trapped genes, while the other clones with negtive LacZ staining indicated the weak or inactive promoter activity of trapped genes.
3. cDNA Cloning and bioinformatic analysis of trapped genes downregulated after transforming growth factor beta1 treatment.
Three gene trapped clones with differential expression patterns were obtained by LacZ staining before and after TGF-β1 induced phenotypic modulation of 10T1/2 gene trapped clones to smooth muscle cells. It was found that the trapped genes were Mrps6, a known gene, and two novel genes (mgt-6 and mgt-16) when the RACE-obtained sequences were searched in GenBank using BLAST algorithm. Mgt-6 (murine gene trap clone 6) is mapped to mouse chromosome 14, encodes two protein and has 4 transcripts, which have been confirmed by RT-PCR and submitted to GenBank by its online software BankIt (GenBank accession no. FJ744746, FJ860514, FJ860513 and FJ748867). Transcript 1, 2, 3 have 3 exons but encoded the shorter protein (MGT-6S), whose molecular weight is 1414.68 and isoelectric point is 5.75 calculated by pI/Mw software. Transcript 4 has 2 exons that encoded the longer protein (MGT-6L), whose molecular weight is 4073.57 and isoelectric point is 6.52. Mgt-16 (murine gene trap clone 16) is mapped to mouse chromosome 19, has 2 exons and encodes a protein, whose length is 93 amino acids, molecular weight is 9772.02 and isoelectric point is 6.04. Mgt-16 has been submitted to GenBank (GenBank accession no. GU266552). In order to confirm gene trap vector insertion, proper adaption to the upstream exon of the insertion point, and obtain the precise splicing acceptor(SA) site in ROSAFARY, RT-PCR was performed to detect the fusion transcripts. It was found that ROSAFARY construct were inserted into the intron 1 of both mgt-6 and mgt-16, and the precise splicing acceptor (SA) site is at 2754 nt in ROSAFARY.
4. Cloning and molecular characterization of a novel gene, mgt-16, from multipotent 10T1/2 cells.
The EGFP fused mgt-16 retroviral vector, pL-EGFP-N1-16, was constructed based on retroviral vector, pL-EGFP-N1, and verified by sequencing. Then they were transfected into packaging cell line Phoenix by lipofectamine. The retrovirus particles were collected to infect 10T1/2 cells and 400μg/ml G418 continuous selection was conducted. The overexpressed EGFP fused mgt-16 in 10T1/2 cells showed the green fluorescence in pan-cytoplasmic distribution which indicated the subcellular localization of EGFP-fused MGT-16 protein expressed in the cytoplasm. This result was consistent with LacZ staining pattern of pan-cytoplasmic distribution in gene trap clone 16.
To exclude any possible interference of EGFP on the function of MGT-16, 6×His was used as a small tag to trace MGT-16. The retroviral vector containing the internal ribosome entry site (IRES), pL-IRES-EGFP-N1, was constructed based on two vectors, pL-EGFP-N1 and pIRES2-EGFP-N1, and verified by sequencing. Then 6×His fused mgt-16 retroviral vector, pL-IRES-EGFP-16-His, was constructed based on plasmid pL-IRES-EGFP-N1. Then the two constructed vectors were transfected into packaging cell line Phoenix by lipofectamine. The retrovirus particles were collected to infect 10T1/2 cells and stably overexpressed 6×His fused mgt-16 containing IRES-EGFP construct clones were obtained by 400μg/ml G418 continuous selection. Western blot analysis showed that the overexpression of mgt-16 in 10T1/2 cells attenuated the expression of the SMC-specific markers,αSMA and SM22a which was induced by TGF-β1.
Using gene trap clone 16 as a model of mgt-16 gene disrupted, overexpressed 6×His-fused mgt-16 or EGFP-fused mgt-16 10T1/2 cell clones as a model of mgt-16 gene overexpressed, the influence of mgt-16 on the cell proliferation was determined using WST-8 dye (Cell Counting Kit-8) and cell migration by scratch wound on monolayer cells. According to the optical density value measured using WST-8 dye which is proportional to the number of viable cells in the medium, gene trap clone 16 grew more slowly than normal 10T1/2 cells did (P <0.05), which meant the disruption of mgt-16 gene would inhibit cell proliferation. however, the 10T1/2 cells with overexpressed 6×His-fused mgt-16 or EGFP-fused mgt-16 grew more rapidly compared with the corresponding control group (P <0.05), indicating the overexpressed mgt-16 gene would accelerate cell proliferation. The scratch wound assay showed that gene trap clone 16 migrated more slowly than normal 10T1/2 cells did (P <0.05), which implied the disruption of mgt-16 gene could slow down cell migration, while the cell clones with overexpressed 6×His-fused mgt-16 or EGFP-fused mgt-16 migrated more rapidly compared with the corresponding control group (P <0.05), suggesting overexpressed mgt-16 gene would accelerate cell migration. According to the results in disrupted and overexpressed model in cell proliferation and migration tests, it is assumed that mgt-16 gene can positively regulate cell proliferation and migration in 10T1/2 cells.
5. The molecular mechanisms of mgt-16 downregulated by TGF-β1
In order to elucidate the molecular mechenisms of how mgt-16 is downregulated by TGF-β1, gene trap clone 16 was treated with TGF-β1, or a single inhibitor of p38/RK, PI3K/AKT, ERK, mTOR and NF-κB, or TGF-β1 combined with individual inhibitors for 24 hours. Revealed by LacZ staining and RT-PCR, p38 pathway was demonstrated to be involved in the downregulation of mgt-16 by TGF-β1. Further study showed that p38 pathway was involved in TGF-β1-induced phenotypic conversion of 10T1/2 cells to smooth muscle cells as supported by following evidences: the p38 inhibitor (i.e., SB203580) can suppress the morphological changes caused by TGF-β1; the phosphorylation of p38 MAPK increased after TGF-β1 treatment and the p38 inhibitor (i.e., SB203580) attenuated the phosphorylation of p38 MAPK as determined by Western blot analysis; SB203580 suppressed TGF-β1-induced SMC-specific gene SM22a expression in 10T1/2 cells as confirmed by RT-PCR. Linking above data together, we proposed a hypothesis: TGF-β1 downregulates mgt-16 by activating p38 pathway in 10T1/2 cells to promote smooth muscle gene expression.
6. Cloning and molecular characterization of a novel gene, mgt-6, from mutipotent 10T1/2 cells.
Gene trap clone 6 exhibited a LacZ staining pattern of pan-cytoplasmic distribution. When treated with 5-azacytidine, the localization ofβ-galactosidase activity in cells of clone 6 translocated from the cytoplasm to the cell nucleus. This result was also demonstrated by overexpressed EGFP fused MGT-6 protein in 10T1/2 cells, which gives the clues that the novel gene mgt-6 may participate in the process of DNA hypomethylation. Additionally, the four splice variants of mgt-6 had different expression patterns in different murine tissues and 10T1/2, C2C12, Lewis cells as shown by RT-PCR. The transcript 4 was the only one expressed in skin, spleen and intestine, and predominantly presented in kidney, heart and skeletal muscle, while the transcript 1 predominantly expressed in liver. All four transcripts expressed in brain and lung. C2C12 predominantly expressed the transcript 4, while all four transcripts expressed in both 10T1/2 and Lewis cells. The transcript 1, 2 and 4 were expressed more strongly in Lewis than in 10T1/2 cells. C2C12 mouse myoblasts and adult murine skeletal muscle predominantly expressed the transcript 4 which endcoded MGT-6L protein, but weakly expressed the transcript 1, 2, 3 which endcoded MGT-6S protein. As for whether MGT-6L or MGT-6S protein is related with the development of skeletal muscle, further study is needed. After gene trap clone 6 or 10T1/2 cells was treated with TGF-β1, p38/RK inhibitor, PI3K/AKT inhibitor or ERK inhibitor, as shown by LacZ staining and RT-PCR, the mgt-6 gene expression was downregulated when treated with TGF-β1 or PD98059 (the ERK inhibitor) but upregulated when treated with LY294002 (the PI3K/AKT inhibitor), which indicated mgt-6 may be involved in the process of SMC differentiation and this differentiation may be positively correlated with TGF-β1-evoked P38 and PI3K but repressed ERK signaling pathway.
In conclusion, focusing on the key unsolved problems on the molecular mechanisms of mesenchymal stem cell proliferation and differentiation, the forward genetic approach gene trapping was applied to scan the active genes in MSCs for the first time. Established were a large number of gene trap clones and some differentiation models of 10T1/2 cells, which were valuable of comprehensive application. In addition, we cloned and characterizated two novel genes. These results expand our understanding of TGF-β1 signaling pathway and the molecular mechenisms of smooth muscle cell differention, and provide theoretical support and expremental basis for further research on the committed differentiation of MSCs in order to more effectively improve wound healing by MSCs.
引文
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