2. 青岛农业大学动物科技学院, 青岛 266109
2. Qingdao Agricultural University, Qingdao 266109, China
鹿茸是生长在鹿科动物额外脊上的一种附属性器官,每年都经历从鹿茸至鹿角的软骨内成骨过程[1]。在生茸期,鹿的机体会发生严重的生理性骨质疏松症,体骨流失近30%,鹿茸却可在该生理状态下进行着快速生茸与骨化,短短3个月最高可形成重达30 kg骨组织,待鹿角形成后,体骨骨质疏松消失,第二年夏季,鹿体骨质疏松现象再一次触发[2-3]。有学者认为,生理性骨质疏松症属于特殊的鹿茸成骨补偿,鹿机体骨质疏松的发生与鹿茸成骨具有密切的相关性,即使在生茸期饲喂高剂量的钙、磷也不能够阻断骨质疏松的发生[4]。众所周知,动物机体的骨骼始终处于成骨与破骨的平衡状态,动态平衡的打破也就面临着骨质疏松的发生,但在鹿茸成骨机制研究中,尚不明了其特殊的分子机制[5-6]。因此,探讨鹿茸的成骨机制能够为提高鹿茸产量、改善动物福利健康乃至为医学领域中骨质疏松的治疗提供新思路。
1 鹿茸逆向成骨在每年的快速生茸期,鹿茸的发育需要从机体骨骼大量重吸收矿物质,此时,鹿机体性激素水平极低,鹿科动物骨骼骨质流失并伴随生理性骨质疏松现象,将在低性激素水平且鹿体骨骼处于骨质疏松状态下鹿茸的快速生长发育现象称为鹿茸逆向成骨。
每年夏季(北半球)为鹿茸的快速生长季节,即生茸期。快速生长中心在鹿茸的顶端[7],根据Li等[8]的鹿茸分层方法,即将鹿茸由远心端至近心端依次分为真皮层(dermis, D)、间充质层(reserve mesenchyme, RM)、前软骨层(precartilage, PC)、过渡区(transition zone, TZ)、软骨层(cartilage, C)和骨组织。由图 1(该图为本团队试验制作)改良番红O-固绿与阿尔新蓝-核固红染色可见,鹿茸在快速生长的过程中伴随着从骨腔隙周围开始的快速骨化现象,Faucheux等[9]也证实了在过渡区骨腔隙间破骨细胞(osteoclast,OC)的存在。鹿茸快速生长的同时,鹿茸中大量血管[10]、神经[11]也在发育。根据相关数据统计,鹿茸从开始生长至生长完全仅需2个月[12],对比人类发育至成年后(18岁)骨骼才完成生长,其成骨速度约是人类的108倍[13]。
研究表明,生茸期的鹿体骨骼会发生不同程度的骨质流失,肋骨具有皮质变薄,重吸收窦增多,骨质多孔、骨小梁不完全骨化的特点,这些现象待鹿茸骨化后消失,雌性驯鹿同公鹿一样,存在鹿体骨质流失与逆转现象[14-16],目前关于鹿机体的骨质流失机制鲜见报道,由于鹿茸成骨过程中需要大量的钙、磷原料用于骨生成,探索骨质疏松的触发机制,有助于鹿茸逆向成骨的机制研究。在鹿茸成骨的相关研究中,雄激素的缺失会延缓鹿茸完全骨化过程[17],梅花鹿外周血甲状旁腺素(parathyroid hormone, PTH)水平在生茸期显著升高[18],在钙、磷代谢研究中[2],钙、磷的均衡摄入可提高产茸量,但生茸所需的更多矿物质仍来源于机体骨骼。除上述研究外,在其它物种干细胞成骨研究中,骨保护素(osteoprotegerin, OPG)、胰岛素样生长因子-1(insulin-like growth factor 1,IGF-1)、白细胞介素1(interleukin-1, IL-1)、转化生长因子β(transforming growth factor, TGF-β)等均在成骨过程中发挥促成骨作用[19]。综上,本文结合当前鹿茸成骨机制与细胞因子的研究现状进行综述,旨在为鹿茸成骨相关研究提供新见解。
2 成骨细胞与破骨细胞分化成骨细胞(osteoblast,OB)与OC的成熟标志着鹿茸成骨的开始。OB最初起源于骨髓间充质干细胞,通过细胞增殖分化发挥骨生成作用,在骨的生成过程中,OB与OC相互作用、相辅相成,OB通过分泌M-CSF、RANKL、Wnt5A和OPG、Wnt16来促进或抑制OC的分化和发育;OC通过分泌S1P、SEMA4D、CTHRC1和C3促进OB的分化[20]。有研究发现,鹿茸间充质干细胞(reserve mesenchyme cells, RMCs)具备成骨分化的特点,经体外诱导可生成钙结节,可被茜素红着色[21-22]。OB作为一种内分泌细胞,能够产生骨钙素、碱性磷酸酶和Ⅰ型胶原等细胞外蛋白,一直以来也被认为是成骨分化的标志。
鹿茸生长是基于鹿茸顶端RMCs的增殖、分化作用[7],RMCs来源于角柄骨膜干细胞(pedicle periosteum cells, PPCs),两者干性相似,以软骨内成骨的方式成骨[22]。孙红梅等[21]体外诱导试验表明,采用PPCs进行成骨(微粒体)诱导,先经软骨诱导可分化为软骨细胞,紧接着更换成骨诱导液可再次分化为OB,因此,RMCs具备软骨细胞向OB的转分化能力[23]。由此推测,鹿茸中的大量OB可能是通过该渠道分化形成。伴随着RMCs不断向软骨细胞、OB的分化,近心段的细胞优先分化为OB发挥骨生成作用,在外周血激素及旁分泌因子的刺激下率先骨化,这可能是鹿茸生茸期不同区段骨化程度不均的原因之一。
OC起源于骨髓中的原单核细胞和幼单核细胞,属单核/巨噬细胞系[24],可经诱导分化而来,不能传代培养,行使完骨吸收功能后在OB的作用下逐步凋亡[25]。成熟的OC直径可达100 μm,位于哈佛斯系统内骨膜的内表面[26-27],胞质嗜酸性、多核。通过分泌H+、Cl-、组织蛋白酶K、基质金属蛋白酶来降解骨骼,在骨吸收过程中分解胶原和其它基质蛋白[28-30]。OC成熟过程分为髓样分化、髓样细胞前体向OC前体分化、OC前体向前OC分化、融合成熟,随后发挥骨吸收作用。分化过程中OC前体分别表达M-CSF、RANKL、RANK和c-Fms[31]。
OC在软骨内成骨过程中主要通过降解胶原与软骨基质进而为骨质转换提供空间[31]、分泌促OB分化因子来促进成骨[20]。在鹿茸研究中,Faucheux等[9]将马鹿源性鹿茸软骨细胞离体培养为OC,RT-PCR结果显示,有标志物M-CSF、RANKL的表达,抗酒石酸酸性磷酸酶染色阳性,证实了OC的形成;在后续研究中,鹿茸中的OC形成受PTHrP正向调控,可通过激活PTH1R/PTHrP通路来促进OC形成,在培养基中联合添加甲状旁腺素相关蛋白(Parathyroid hormone-related protein,PTHrP)与RANKL可显著增加OC的生成数量[32],除上述研究外,尚未在鹿茸研究中发现有关OC的报道,有待进一步佐证。由于OC在软骨内成骨过程中发挥着不可或缺的作用,探索鹿茸中OC的分化与调控机制能进一步推动鹿茸成骨机制的研究。
综上,鹿花盘脱落后,来源于PPCs的RMCs开始向软骨细胞分化形成软骨组织,此时在软骨细胞的旁分泌作用下促进血管生成来供给营养,软骨细胞分化为OC与OB,OC通过降解胶原与软骨基质为骨转换提供空间,OB发挥骨生成作用。因鹿茸不同区段的细胞分化程度不同,故出现鹿茸骨化程度不均的现象。由于鹿茸基部最早成骨,在骨密度不断提高的过程中导致静脉回流逐步受阻,直至完全被阻隔,最后在得不到血液供应下,茸皮脱落,成为死组织。
PTH是甲状旁腺的主要分泌产物,在低钙条件下,可增强肾内钙的重吸收,增加活性维生素D的产生,以增加肠内钙吸收和动员骨钙的作用[33]。在PTH介导的成骨过程中,其活性形式PTH(1-34)发挥着主要功能,也是骨质疏松药物“特立帕肽”的主要成分[34];PTHrP由细胞旁分泌作用产生,因其N-末端与PTH高度同源,两者可共享Ⅰ型PTH/PTHrP受体来促进骨生成[35]。有研究对白尾鹿成骨相关血清指标检测发现,Ca2+水平在每年5~7月份上升,7~9月份下降,ALP自4月份开始至7月份达峰值,PTH水平存在两次上升现象,分别出现在3~5月份和8~10月份,据此推测,生茸开始时PTH的增加可能是动员骨钙为骨沉积提供原料的重要原因,与鹿茸的骨化密切相关,6月份时Ca2+增加可能通过PTH的负反馈机制来降低PTH水平防止过度骨吸收[36]。PTHrP及其受体广泛表达于鹿茸组织[37-38],对鹿茸软骨细胞增殖具有促进作用,PTHrP可通过JNK信号通路下调MMP9、p38 MAPK和PKC信号通路下调MMP13的表达来抑制鹿茸软骨细胞向成骨细胞分化,参与软骨细胞的成熟和软骨基质的降解过程[39-40]。在对鹿茸OC研究中,外源性PTHrP可促进鹿茸软骨细胞向OC分化[9],添加抑制剂OPG,可显著降低OC的生成数量[32]。由此可见,PTHrP在鹿茸成骨与破骨过程中具有双重调节作用。然而,生茸期外周血PTH水平的升高除增加机体重吸收和动员骨钙外,尚未在鹿茸研究中发现有关PTH参与鹿茸干细胞和软骨细胞的分化过程,PTHrP介导的破骨机制还有待进一步被阐明。
3.2 雄激素与雌激素睾酮(testosterone, T)和雌二醇(estradiol, E2)是性激素的两种主要成分。研究表明,雄激素与雌激素在骨代谢过程中发挥着重要作用,T可经芳香化酶代谢生成E2[41]。在对人类和啮齿类动物的研究中发现,两者均可通过减少OC的生成数量来抑制松质骨与皮质骨内表面的骨吸收,而且在维持骨骼健康方面E2作用大于T[42]。通过放射免疫分析法对梅花鹿外周血T与E2进行测定[43],两者在花盘脱落前均存在峰值,而生茸期都保持较低水平,骨化期两者均达全年最高水平,这与Weerasekera等[44]测定内源性(粪便)T水平趋势相一致。对去势白尾鹿,使用抑制剂醋酸环丙孕酮处理,可以明显延长鹿茸生长期,出现茸皮不脱落、骨小梁不完全骨化的现象;后续在狍子[45]、赤鹿[46]和黇鹿[47-48]等研究中均验证了雄激素的缺失可以延长鹿茸生长期(不完全骨化)并影响鹿茸的形态发生。雄性激素在鹿茸发生和再生过程中起着截然相反的作用,刘振[49]研究表明,低水平的雄性激素激活了鹿茸的再生过程,高水平的雄性激素激活了鹿茸的发生过程(角柄和初角茸的形成)。在体外细胞试验中,T对RMCs的增殖无显著影响,但可通过上调标志基因Col Ⅱ、AGC、COMP的表达来促进RMCs向软骨细胞分化,还可通过IHH信号通路抑制IHH、Gli1的表达,解除对Smo的抑制,进而促进Gli3的表达来增加Runx2转录促进软骨细胞分化[50]。
雌激素的缺失是导致女性绝经后骨质疏松的原因所在,雌激素在骨代谢过程中通过ERK通路调控OB的转录活性,抑制OB凋亡[51];可通过上调骨形态发生蛋白的mRNA表达来促进OB的增殖与分化[52];还可激活Fas/FasL通路诱导OC凋亡[53]。驯鹿卵巢缺失后,E2治疗组与正常生长组可实现同步骨化(驯鹿雌雄均生茸),这表明E2在雌性驯鹿鹿茸骨化过程中起着促成骨作用,与雄激素介导的公鹿鹿茸成骨作用相似。但截至目前在公鹿中尚未发现雌激素介导鹿茸骨化过程的证据。综上,可以推测这两种激素均可参与鹿茸的成骨过程,但在生茸期雄性鹿体内E2水平升高,有研究表明[54],雄激素与雌激素存在着芳香化作用,两者可相互转化,这种雄激素与雌激素在动物体内的芳香化机制有待被阐明,对鹿茸逆向成骨的机制研究与骨质疏松治疗具有重要意义。
4 鹿茸成骨相关的细胞因子 4.1 OPGOPG又名OC抑制因子,属肿瘤坏死因子受体家族成员,在OC成熟的OPG/RANKL/RANK信号通路中起着关键调控作用,能够明显抑制OC的形成,OPG的作用机制是与RANKL竞争结合OC前体细胞膜上的RANK,从而阻断RANKL/RANK通路来抑制OC分化成熟,影响机体骨质的动态平衡[55-61]。OPG来源广泛,可由OB、髓质胸腺上皮细胞、微折细胞、各类脏器等分泌[62]。靶向缺失OPG,小鼠会出现严重的早发性骨质疏松症[63]。过表达OPG,转基因小鼠则会得骨化病[64]。综上,OPG在动物骨形成中对OC的成熟起着重要的调节作用但在鹿茸研究中尚未检索到OPG相关报道,OPG作为OC分化的关键因子之一,有可能在鹿茸成骨中发挥着关键作用,具体调控机制还有待被阐明。
4.2 IGF-1IGF-1作为一种碱性多肽,是鹿茸的主要活性成分之一,高表达于鹿茸组织,在对梅花鹿外周血水平与鹿茸生长相关性研究中,IGF-1的浓度变化与鹿茸生长和体重正相关[65]。同人类IGF-1序列比对,鹿科动物的蛋白序列与人类的比对效率高于小鼠,过表达IGF-1,鹿茸软骨细胞可通过上调标志基因Comp、Cdk4、Pcna、Fgfr3的mRNA表达来促进鹿茸软骨细胞增殖,上调Col10a1, Omd, Smpd3和Pth1r表达来促进分化、还可通过下调MMP13、MMP3、MMP9、ADAMTS5的表达从而降低细胞外基质降解来促进成骨[66-69]。在鹿茸软骨细胞中,IGF-1可上调下游胰岛素样受体底物1/2,通过增加转录因子Runx1的转录来促进细胞分化[66]。
4.3 IL-1IL-1主要由巨噬细胞分泌,存在IL-1α和IL-1β两种亚型,具有调节OB、OC分化的能力。研究发现,IL-1β作用于OB,具有抑制碱性磷酸酶活性和下调OPG表达来抑制成骨的作用,还能够促进OB分泌IL-6、TNF-α、M-CSF、RANKL等OC分化因子,进一步加速OC前体的融合、分化来增强骨吸收能力[19, 70-73]。IL-1也可直接作用于OC前体来促进其增殖与分化,提高OC活性进而增强骨吸收[74-75]。Wnt经典信号通路是OB分化过程中的一条重要通路,在成纤维滑膜细胞中IL-1β可通过抑制Dickkopf相关蛋白1来促进Wnt信号的激活[76],还可上调软骨细胞中BMP2的表达[77]。Yang等[78]使用IL-1α对MC3T3-E1细胞系进行干预,IL-1α通过激活JNK和p38 MAPK通路诱导OB凋亡,抑制OB分化。综上,IL-1对成骨具有抑制作用,在多方面可促进OC的分化来提高骨吸收能力,但在鹿茸软骨内成骨过程中,尚未有关IL-1对OB与OC成骨相关的报道。
4.4 TGF-βTGF-β家族有TGF-β1、TGF-β2、TGF-β3三种亚型,通过调节细胞增殖、分化和迁移,在胚胎发生和成体组织稳态中发挥作用,TGF-β1通过激活Smad或TAK1信号通路,在PTH刺激下,通过其受体Ⅰ将信号传递给TGF-β2,最终均通过上调Runx2的表达来促进成骨[79]。TGF-β的三种亚型均有表达于鹿茸组织,其中以TGF-β3表达量最高,主要表达于软骨小梁内正在成熟和已经成熟的软骨细胞[80]。Liu等[81]发现, TGF-β1在鹿茸软骨组织相对其它层更高表达,敲除鹿茸软骨细胞TGF-β1减缓了细胞增殖,促进了细胞迁移,但上调了BMP4的mRNA表达,由此推测,当TGF-β1受阻时,可能激活了BMP信号通路介导的成骨补偿。最新研究表明,长链非编码RNA可以通过负向调控TGF-β/Smad通路抑制细胞的增殖与分化[82],重组人TGF-β蛋白的添加,可上调成骨标志物Runx2、COL-X、ALP的表达[83]。综上,TGF-β1在鹿茸软骨细胞中发挥着促进细胞增殖、分化和抑制细胞迁移的作用,可通过Smad信号通路来促进软骨细胞向成骨方向分化。
5 前景展望本文探讨了鹿茸逆向成骨相关研究的最新进展。迄今为止,鹿机体骨质流失的机理研究较少,但还存在许多待解决的问题。例如:RMCs分化为软骨细胞后,OB与OC的形成机制。在鹿茸软骨内成骨过程中,OC成熟降解软骨基质为成骨提供空间是成骨的开始,因此这两种细胞显得格外重要,由于在鹿茸中OB、OC的作用机制不清楚,激素调节研究更多的局限在动物试验中。另外,T与E2的芳香化机制均有待被阐明。在鹿茸成骨机制研究中,对细胞因子的研究更多的是在促成骨方面,其抑制破骨和促进破骨作用还有待更进一步的研究。在未来,伴随着宏基因组、单细胞测序等技术研究的深入,或将打开这其中的奥妙,为提高产茸量、药物开发甚至为骨质疏松的治疗提供理论依据。
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(编辑 范子娟)