CN1880452A - 人sema 4c基因能够抑制成肌细胞增殖并促进其向肌管分化 - Google Patents
人sema 4c基因能够抑制成肌细胞增殖并促进其向肌管分化 Download PDFInfo
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Abstract
本发明公开了一个人Semaphorin家族成员Sema4C的新功能,该基因能够抑制小鼠成肌细胞C2C12的增殖并促进其向肌管分化。本研究利用小鼠成肌细胞系C2C12和原代大鼠成肌细胞体外分化培养模型,发现Sema 4C的表达量随着成肌分化的进行而逐渐增强。此外,我们又对Sema 4C的亚细胞定位进行了研究,发现全长Sema 4C基因在胞浆和胞膜上广泛分布,而对缺失突变体的研究发现,缺失胞外段的Sema 4C基因失去精确的亚细胞定位能力,在全细胞中呈弥散性表达;组织表达发现,Sema 4C基因主要在神经系统中高表达,此外,在E13.5小鼠胚胎上肢的肢芽中也有一过性地表达,在成年小鼠的视网膜和肾小管上皮组织中也检测到了明显的阳性信号。进一步深入研究又证实Sema 4C基因能够抑制成肌细胞的增殖并促进其向肌管的分化与成熟。由此推测,该基因不仅在神经系统发育过程中发挥作用,而且还很可能参与了肌肉发育和损伤修复的过程,并在该过程中发挥某种关键的调控作用。
Description
技术领域
本发明涉及一个在成肌细胞增殖分化过程中具有重要调控作用的新基因Sema 4C。该基因及其编码的蛋白涉及生物医学高技术中新基因、新蛋白的开发与应用领域。
发明背景
骨骼肌在胚胎阶段的发育是非常复杂的过程,包括对骨骼肌前体细胞迁移、增殖和分化在时间和空间上进行有序的调节。骨骼肌的再生是成年骨骼肌受到损伤后出现的一种生理性反应,主要依赖骨骼肌组织中的肌卫星细胞受到刺激信号发生活化,进入细胞周期进行增殖分化形成新生肌纤维。可见,骨骼肌卫星细胞在骨骼肌的发育和再生过程中都占据着极其重要的地位。同时,骨骼肌卫星细胞因其自身良好的体外增殖和分化潜能,为我们对参与细胞增殖与组织分化复杂分子机制的研究提供了一个完美细胞模型(Definingthe regulatory networks for muscle development.Curr.Opin.Gen.Dev.,1996,6:445-453.)。但是,目前有关肌卫星细胞活化、增殖和分化的详尽调控机制仍然不甚了解。因此,发现并证实更多的参与成肌细胞增殖分化调节的基因,对于探讨骨骼肌的发育和再生的分子机制,显得极为迫切和必要。
我们通过对小鼠成肌细胞系C2C12和原代大鼠成肌细胞体外分化培养的研究发现,Sema 4C基因随着成肌分化的进行而表达逐渐增加。由于很多肌肉分化相关的基因在肌细胞分化前后都存在着表达的明显差异,比如成肌转录因子MRF4和MEF2C,参与细胞周期调控的CDK抑制蛋白p21WAF1/Cip1,p27kip1,p57,p18等(Coordinate regulation of cell cycle and apoptosis during myogenesis.Prog.Cell Cycle Res.,1997,3:53-58.)(Resistance to apoptosis conferred by Cdkinhibitors during myocyte differentiation.Science,1996,273:359-361.),由此提示,Sema 4C可能是又一个新的在骨骼肌细胞增殖分化过程中起到调控作用的分子。
Semaphorin家族成员是一大类轴突导向分子,在神经系统发育过程中对轴突导向起着重要的调节作用。但越来越多的研究发现,Semaphorin家族成员除了在胚胎神经发育过程中发挥作用之外,还在细胞迁移、免疫系统和血管生成等方面发挥功能。小鼠Sema 4C基因最初被称作M-Sema F,于1995年由Iwahashi领导的实验小组率先克隆成功(Identification of a member ofmouse semaphorin family.FEBS Lett.,1995,370:269-272)。到目前为止,Madline上尚检索不到有关Sema 4C基因确切生物学功能的研究报道,研究仅局限于其相互作用蛋白的寻找及发现方面,对其生物学功能还停留在分析预测的水平上。因此从这个角度看,明确Sema 4C的表达特点,探讨Sema 4C的生理功能并阐述其作用机制,不仅对于明确该基因的独特生物学功能具有重要意义,同时也为深入了解和揭示Semaphorin其他家族成员在机体生长发育过程中的生物学功能提供了线索和依据。
接下来,我们对Sema 4C的细胞和组织表达进行了初步的探讨;重点对体外转染得到稳定高表达Sema 4C的阳性克隆进行了增殖分化能力的研究。结果显示,高表达Sema 4C的细胞克隆增殖速度较对照组的确明显下降;分化为肌管的能力增强。我们通过实验观察得到的Sema 4C能够抑制成肌细胞增殖并促进其向骨骼肌分化与成熟的现象迄今未见报道。
发明目的
通过对过表达Sema 4C的阳性克隆细胞增殖分化能力的研究,明确该基因在成肌细胞增殖分化过程中发挥的作用,并初步探讨其可能的作用机制。通过对Sema 4C在成肌细胞增殖分化过程中作用的研究,不仅能够为阐明骨骼肌发育和再生的复杂网络调节机制开辟新的思路,同时也为临床上各种萎缩性及营养不良性肌病的治疗提供了新的契机。
内容与要求
本发明利用了RT-PCR、Western Blot、Wholemount、原位杂交、激光共聚焦、免疫组化、流式细胞仪等实验技术对Sema 4C的组织表达、亚细胞定位和初步功能进行了探讨,从而发现并证实了Sema 4C是一个全新的在成肌细胞增殖分化过程中发挥重要作用的调控基因。
该基因/蛋白达到的技术指标:
1、通过RT-PCR和Western Blot的方法均证实,不管是成肌细胞系C2C12还是原代大鼠成肌细胞,在成肌分化过程中Sema 4C在转录和翻译水平的表达都发生了上调。
2、激光共聚焦进行亚细胞定位观察证实,Sema 4C-FL基因定位于胞浆和胞膜上,而缺失胞外段的突变体Sema 4C-ID则丧失亚细胞定位能力,在全细胞内呈弥散性分布。
3、Wholemount和原位杂交显示Sema 4C主要在发育的神经系统中高表达。此外在E13.5小鼠胚胎的上肢肢芽中也有部分表达,在成年小鼠的肾小管上皮细胞中也检测到高表达。
4、经转染高表达Sema 4C的C2C12阳性克隆细胞增殖能力减慢,G0/G1期细胞比例较对照组明显升高,而S期比例明显下降。
5、对高表达Sema 4C的C2C12细胞克隆进行分化培养发现其分化为肌管的时间提前,成肌细胞的融合率增加;成肌转录因子MRFs及成熟骨骼肌相关的标志物表达时间提前,表达量增加。
6、我们的结果证实,Sema 4C在成肌细胞增殖分化过程中具有重要调控作用。此现象重复性强,结果可靠,为阐明骨骼肌发育和再生的复杂网络调节机制提供了新的视角。
附图说明
图1.为C2C12细胞分化前后的免疫组化鉴定。其中A:分化前增殖状态的细胞MyoD阳性。B:分化前增殖状态的细胞Myf-5阳性。C:分化前Myogenin鉴定结果为阴性。D:分化前MHC(Myosin Heavy Chain)的染色结果为阴性。
E:分化后的细胞Myogenin的染色结果。F:分化后的细胞MHC的鉴定结果。
图2.A:为采用混合酶消化,反复差速贴壁的方法,获得纯化的原代大鼠成肌细胞相差照片。B&C:免疫荧光鉴定成肌细胞的纯度。其中B为DAPI复染所有的细胞核结果,C红色为Desmin阳性细胞染色结果。
图3.A:为半定量RT-PCR的方法检测C2C12细胞不同分化阶段分化相关基因及Sema 4C的表达情况。B:为Sema 4C在原代大鼠成肌细胞不同分化时期的表达结果的半定量RT-PCR分析。
图4.为采用Western Blot的方法进一步验证RT-PCR的结果。
图5.为激光共聚焦显微镜观察Sema 4C及其缺失突变体在COS7细胞中的亚细胞定位结果。其中A为融合了GFP后的Sema 4C全长蛋白在细胞中定位结果,B为融合了GFP后的Sema 4C胞内段在细胞中的定位结果。
图6.为原位杂交的方法检测Sema 4C在小鼠胚胎和成年小鼠组织中的表达结果。A为E11.5的小鼠胚胎整体原位杂交(Wholemount)结果。B为E13.5的小鼠胚胎Wholemount结果。C为Sema 4C在成年大鼠大脑皮层中的表达结果。D为Sema 4C在成年大鼠小脑组织中的表达结果。E为Sema 4C在成年大鼠视网膜节细胞层中的表达结果。F为Sema 4C在成年大鼠肾组织中的表达结果。
图7.A为RT-PCR检测挑取的稳定单克隆细胞中转染外源性基因和抗性基因结果。B为Western Blot检测C2C12[Sema 4C]克隆10中融合Myc蛋白的外源Sema 4C表达结果。
图8.A为转染Sema 4C组和空载体组C2C12细胞克隆增殖曲线结果。B为流式细胞仪检测细胞周期结果。
图9.A为空载体组C2C12细胞在不同增殖分化阶段的形态学变化结果(100×),B为转染Sema 4C的C2C12细胞组在不同增殖分化阶段的形态学变化结果(100×)。
图10.RT-PCR检测不同分化阶段C2C12[Sema 4C]和C2C12[Vector]细胞分化相关基因的表达变化结果。其中A为外源人Sema 4C基因及内源小鼠Sema 4C基因的转录表达结果。B为成肌分化相关的细胞周期调控因子的转录表达变化结果。C为成肌转录因子MRFs的表达变化结果。D为骨骼肌发育相关的分子标志物表达变化结果。
具体实施方式
实施例一稳定可靠的成肌细胞分化培养模型的建立及成肌分化相关
功能基因的筛选
1、C2C12细胞分化培养模型的建立及鉴定
将野生型C2C12细胞按2×104/cm2密度接种到60mm细胞培养皿中,用生长液(DMEM/15%FBS/1%青链霉素/1%谷氨酰胺)进行增殖培养,待细胞生长至亚融合状态时,更换为分化液(DMEM/2%HS)进行分化培养,每日各取2皿,分别用TRIzol(Invitrogen)和蛋白裂解液提取总RNA和细胞全蛋白,用于RT-PCR和Western Blot分析。同时,取分化前增殖状态和分化后7天的C2C12细胞用4%的多聚甲醛固定,0.3%过氧化氢消除内源性过氧化物酶活性后,进行MyoD,myogenin和MHC(均为Santa Cruz公司产品)的免疫组化染色鉴定。MyoD和myf-5为兔多抗(1∶500),myogenin和MHC为小鼠单抗(1∶1000),4℃过夜,1×PBS洗涤,3×10min/次,用生物素标记的二抗(Vector,1∶200),室温反应2小时后,1×PBS洗涤,3×10min/次,再加入ABC复合物(1∶100),37℃反应1小时,用DAB法显色,阳性部位为棕黄色的沉淀物。结果显示(见图1),增殖状态的C2C12细胞myf-5,MyoD呈阳性,而myogenin和MHC阴性,当其分化至第7天时,可见有融合的多核的肌管形成,这些分化的细胞myogenin和MHC阳性。由此证实,我们对C2C12进行分化培养的过程是可靠的,能够用于成肌分化相关基因的筛选。
2、原代大鼠成肌细胞分化培养模型的建立及鉴定
原代大鼠成肌细胞取自Wistar大鼠胫前肌,称重并剪成约1mm3碎片,按每克组织加入2ml混合消化酶液(分散酶2.4U/ml,胶原酶0.2%,2.5mMCaCl2)进行消化,200目滤网过滤,滤液350g离心5min,收集底层细胞用生长液重悬,通过反复差速贴壁的方法去除成纤维细胞的污染。通过成肌细胞特异的表面标志物Desmin(Zymed)(1∶500)进行免疫荧光鉴定(二抗采用FITC标记的羊抗鼠IgG),获得纯度在99%以上的原代大鼠成肌细胞(如图2)。随后对分离纯化的原代大鼠成肌细胞进行连续分化培养3天,在不同分化阶段提取RNA及蛋白,并采用免疫组化的方法鉴定其分化的效率和比例。方法及过程同小鼠成肌细胞系C2C12(结果未显示)。分化培养后多数成肌细胞拉伸变长,相互融合形成多核的肌管。亦可用于成肌分化相关基因的筛选。
3、RT-PCR和Western-blot分析sema4c在C2C12细胞及原代大鼠成肌细
胞分化过程中的表达变化
首先,采用半定量RT-PCR的方法,对提取的不同分化阶段的C2C12细胞的RNA进行逆转录,具体为:不同分化阶段的细胞RNA经Beckman DU800定量和测定A260/280比值后,取2μg RNA,加入1μl Oligo dT,70℃加热5分钟后冰浴,采用TaKaRa公司提供的反转录试剂盒,进行反转录。以反转录后获得的cDNA为模板,PCR检测候选基因的表达变化。本试验中小鼠Sema4C基因采用的上下游引物分别为:5’AGA CAA TGG CTA CAC CAG TT3’和5’CAC GAC AGC AAC AAG GTA G3’;大鼠Sema 4C基因采用的上下游引物分别为:5’ATC AAG AAG CAC CCG CTG A3’和5’AAA GGA AGG AGCCAG GTTGT3’。PCR的反应条件为:94℃1’--58℃30”--72℃30”,30 cycles。PCR产物采用1.2%琼脂糖凝胶电泳,MultiimageTM(Alpha Innotech Co.)凝胶成像系统扫描电泳结果,并采用Bio-Rad灰度分析软件对电泳结果进行半定量分析。结果发现,在转录水平Sema 4C基因随着C2C12细胞分化时间的延长,其表达逐渐增加,其表达量的变化趋势同成肌转录因子和成熟肌管标志性分子相一致。原代大鼠成肌细胞分化培养也发现有类似的结果(见图3)。
随后,为了在蛋白水平进一步验证该结果,我们将由细胞裂解液(50mMTris-HCl pH 7.2,150mM NaCl,0.5%NP-40,1mM EDTA,1%Trition X-100,0.1%脱氧胆酸钠,0.1%SDS,1mM PMSF,0.1%Leupeptin,0.1%Aprotin)裂解得到的不同分化阶段的细胞全蛋白加入到5×Loading Buffer中100℃变性5分钟,进行10%SDS-PAGE电泳,Semi-dry转膜仪20V转膜1小时后,5%脱脂奶粉室温封闭PVDF膜2小时,分别加入一抗鼠源Sema 4C(BDCo.)(1∶1000),鼠源MHC(Santa Cruz)(1∶500),兔源MyoD(Santa Cruz)(1∶500)和内标羊源Actin(Santa Cruz)(1∶1000),1×TBST洗涤,3×10min/次,再分别加入HRP标记的羊抗鼠,羊抗兔和兔抗羊二抗(1∶1000),室温作用2小时,1×TBST洗涤,3×10min/次,随后,采用DAB Kit(中杉公司)显色法显色。结果可见(如图4),在蛋白水平Sema 4C的表达同MyoD、MHC的表达呈正相关,随着成肌分化的进行其表达量逐渐增加。
实施例二对Sema 4C基因进行亚细胞定位和组织表达的研究
1.对Sema 4C基因进行亚细胞定位的研究
将Sema 4C全长和胞内段(2218-2658)分别克隆至pEGFP-C1绿色荧光蛋白表达载体中,测序正确后,提取质粒,将C2C12,COS7,HeLa,A204等细胞提前一天按70%密度接种到共聚焦专用的35mm培养皿中(Nunc),第二天,采用Lipofectamine 2000试剂盒进行瞬时高效转染,48小时后利用激光共聚焦显微镜观察荧光表达区域,均得到类似结果(如图5)。即Sema 4C-FL基因定位于胞浆和胞膜上,而缺失胞外段的突变体Sema 4C-ID则丧失亚细胞定位能力,在全细胞内呈弥散性分布。结果表明,Sema 4C基因的胞外段的SEMA结构域及PSI结构域对于该基因精确的亚细胞定位是必需的。
2.对Sema 4C基因进行组织表达的研究
1)RNA探针的制备 采用如下引物5’AGA CAA TGG CTA CAC CAGTT3’和5’CAC GAC AGC AAC AAG GTA G3’从C2C12细胞cDNA中扩出814bp的产物,回收产物并将其连接到pGEM-T载体中,分别经Sal I和Sac II酶切后进行体外转录,制备出DIG标记的单链正反义RNA探针。将获得正反义探针分别同整体小鼠胚胎和成年小鼠的多组织芯片进行杂交。
2)整体小鼠胚胎原位杂交(Wholemount)将蛋白酶K消化处理过的E11.5和E13.5的小鼠胚胎63℃预杂交2h。再用含有0.5μg/ml小鼠Sema4C基因RNA探针的杂交液取代预杂液,63℃杂交16-18h。用含50%甲酰胺的2×SSC63℃漂洗2次,每次30min,20μg/ml RNaseA(溶于Buffer II中)37℃处理2次,每次30min;再用2×SSC(含0.1%Tween-20)63℃漂洗4次,每次20min。浸入Buffer I中室温放置20min,含5%羊血清和0.1%TritonX-100的BufferII室温封闭4h。按1∶2000加入抗DIG-AP抗体(Roche公司,用含5%羊血清和0.1%Triton X-100的Buffer II配制),4℃过夜。免疫组化步骤按常规操作进行。并采用NBT/BCIP(Roche公司,1∶50)显色试剂,室温避光显色2-10h,待显色至满意程度后终止。结果显示(见图6)Sema 4C主要在发育的神经系统中高表达。小鼠Sema 4C基因在E11.5以后的小鼠胚胎的前脑,眼原基,背根神经节和动脉弓中均有表达,E13.5小鼠胚胎除以上部位以外在上肢肢芽中也有部分表达。
3)成年大鼠多组织芯片原位杂交将制备好的多组织载玻片进行脱蜡和乙醇梯度复水后,蛋白酶K(10ng/μl),37℃作用30min,4%多聚甲醛(预冷)室温下固定20min;0.1mol/L PBS洗3×5min,2×SSC洗5min。70℃预杂8min,37℃预杂1hr,2×SSC洗5min,两次,乙醇梯度脱水。预杂后,再将探针加入到预杂液中95℃变性5min。43℃杂交过夜。杂交后采用梯度SSC洗脱非特异结合的探针,并采用Roche公司DIG-AP抗体和NBT/BCIP显色系统显色。待显色至满意程度后用TE Buffer终止反应。风干后室温保存,乙醇常规梯度脱水,二甲苯透明,中性树脂封片。结果显示(见图6),Sema 4C在成年小鼠的大脑皮层,小脑蒲肯野纤维层,视网膜视神经节细胞层和肾小管上皮细胞层中均有较高表达,而在肌肉,心脏,肺,脾脏,肝脏等组织中表达量比较低或未检测到表达(结果未显示)。
实施例三 过表达Sema 4C基因对C2C12细胞增殖分化能力的影响
1.细胞稳定转染和单细胞克隆的筛选
C2C12在转染前一天接种于25cm2培养瓶中,密度控制在2.0×105左右,18-24小时后,采用Lipofectamine 2000转染细胞。质粒量为8μg,两天后,细胞传代至3个25cm2培养瓶中,同时加入300μg/ml的Zeocin进行筛选。筛选过程持续2-3周,中间每2天更换一次新鲜培养基。细胞克隆形成后,采用克隆盘(Sigma)挑取单个细胞克隆进行培养。通过RT-PCR检测外源性人Sema 4C基因以及抗性基因Zeo的表达,并通过抗Myc标签抗体,采用Western Blot的方法挑选出转染后表达最高的阳性克隆10用于后续试验研究。(结果见图7)
2.过表达Sema 4C基因对C2C12细胞增殖能力的影响
将高表达Sema 4C和空载体的阳性克隆分别按相同的细胞密度(8000cells/well)接种到六孔板中,连续培养六天,每天以0.25%胰酶将细胞消化,用血细胞计数板进行计数,将计数结果进行统计后绘制细胞的增殖曲线。结果显示(见图8),高表达Sema 4C的细胞克隆增殖速度较对照组的确明显下降。为进一步证实此结果,我们将实验组和对照组按相同的密度接种到60mm培养皿中,48小时后收集样品,PBS洗涤后进行胰酶消化,75%乙醇固定过夜,洗涤后重悬于0.5ml的PBS中,RNaseA消化30min后进行PI染色,采用流式细胞仪的方法分析细胞周期并计算其增殖指数。结果表明(见图8)高表达Sema 4C的阳性克隆G0/G1期细胞比例较对照组明显升高,而S期比例明显下降;Sema 4C阳性克隆细胞的增殖指数为33.9%明显低于对照组的45.9%(P<0.01)。
3.过表达Sema 4C基因对C2C12细胞分化能力的影响
将高表达Sema 4C和空载体的阳性克隆分别按相同的细胞密度(2×104/cm2)密度接种到60mm细胞培养皿中进行培养(DMEM/15FBS,1%双抗,1%谷氨酰胺,FBS购自于Hyclone公司),待细胞生长至亚融合状态时更换为分化液(DMEM/2%HS,HS购自于Hyclone公司)进行分化培养,观察细胞融合成多核肌管的比例和融合产生肌管的大小粗细。同对照组相比可见(如图9),在分化培养条件下,过表达Sema 4C基因的成肌细胞融合率增加,形成的肌管粗且大,胞核也明显比对照组多。同时,在分化培养过程中,每日各取一皿细胞提取总RNA用于RT-PCR分析,比较成肌转录因子、细胞周期相关因子及成熟肌管表面标志物的表达情况。RT-PCR采用的反应体系同前,循环次数为28次。所采用的引物序列如下:M-Cadherin:5’TTG GAC TTGGGT GGC TCT AC 3’和5’CAT ACT GCT CAC GGC TCT CA3’;myogenin:5′GCT CAG CTC CCT CAA CCA G3′和5′ATG TGA ATG GGG AGT GGGGA3′;myoD:5′GAT GGC ATG ATG GAT TAC AGC 3′和5′GAC TAT GTCCTT TCT TTG GGG 3′;myf-5:5’GAG CCA AGA GTA GCA GCC TTC G3’和5’GTT CTT TCG GGA CCA GAC AGG G 3’;Myosin light chain2(MLC2):5’ATG GCA CCC AAG AAG GCC AA 3’和5’CTA TTC CTG GTC CTT AGCATC 3’;Muscle Creatine Kinase(MCK):5’ACC TGG ACC CCA ACT ATG TG 3’和5’ATC ATG TCG TCG ATG GAC TG 3’;p21WAF1/Cip1:5′AAT CCT GGT GATGTC CGA CCT G 3′和5′TCC GTG ACG AAG TCA AAG TTC C 3′;p27kip1:5′GCT TGC CCG AGT TCT ACT ACA 3’和5’TTC CTC ATC CCT GGA CACTG 3’;p53:5’TAT GGC TTC CAC CTG GGC TT3’和5’GCA CAA ACA CGAACC TCA AAG 3’;Cdk4:5’TCT GTG CTA CTT CCC GAA CTG3’和5’AGACTC CTC CAT CTC TGG CA 3’;Cyclin D3:5’ATG CTG GAG GTG TGT GAGGA 3’和5’GGA GGA TAC ATC GCA AAG GTG 3’;小鼠Sema4C:5’AGACAA TGG CTA CAC CAG TT 3’和5’CAC GAC AGC AAC AAG GTA G 3’;人Sema 4C:5’GTG GAG TCC GAC TGC TAT GC 3’和5’AGG TTG CAGATG CCT GAA GT3’。对每个靶基因的扩增都重复了3次,均得到相似的结果,取其中的一次结果为代表。结果(见图10)可见,高表达Sema 4C的C2C12细胞中促分化的成肌转录因子MyoD,myogenin的表达量明显增加;作为细胞周期调控因子的CDK抑制蛋白p21WAF1/Cip1和p27kip1的表达明显上调;而成熟骨骼肌相关的分子MLC2和MCK的表达量也明显增加。以上结果更进一步证实了,Sema 4C能够抑制成肌细胞的增殖且能促进其向骨骼肌的分化与成熟。
Claims (5)
1、本发明涉及一个能够抑制小鼠成肌细胞C2C12增殖并促进其向肌管分化成熟的基因Sema 4C,其特征在于:互补脱氧核苷酸(cDNA)全长3776bp,含一个2658bp的不含信号肽的开放阅读框,编码886个氨基酸,分子量为97.46kDa的蛋白质。
2、根据权利要求1所述,Sema 4C为一跨膜蛋白,全长的Sema 4C表达在胞浆和胞膜上。该基因及其编码的蛋白在神经系统高表达,从E11.5开始,小鼠Sema 4C基因在胚胎的前脑,眼原基,背根神经节,动脉弓中均有表达。Sema 4C胚胎发育阶段的骨骼肌组织中一过性表达,在E13.5小鼠胚胎的上肢肢芽中能检测到部分Sema 4C的表达。在成年小鼠的大脑皮层,小脑蒲肯野纤维层,视网膜视神经节细胞层和肾小管上皮细胞层中均有较高表达。而在肌肉,心脏,肺,脾脏,肝脏等组织中表达量比较低或未检测到表达。
3、根据权利要求1或2所述之Sema 4C,其特征在于:所编码的蛋白具有典型的SEMA Domain(316-1602),Ig-like C2 Domain(1825-2070),Poly-Proline Domain(2281-2478),PDZ Binding Domain(2647-2658)。
4、根据权利要求1、2、3所述之能够抑制成肌细胞增殖并促进其向肌管分化成熟的基因Sema 4C,其特征在于:该基因能降低体外培养的小鼠成肌细胞C2C12的增殖能力并促进其向肌管分化成熟。
5.根据权利要求1、2、3、4所述之能够抑制成肌细胞增殖并促进其向肌管分化成熟的基因Sema 4C,不仅在神经系统发育过程中发挥作用,还可能参与了骨骼肌的再生过程并在成肌细胞的增殖分化过程中发挥某种调控作用,这些发现对于某些肌萎缩性疾病的病理机制探讨、相关药物开发和临床治疗都具有重要的理论意义和应用价值。
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