鹿茸干细胞内[Na^+]测定及其影响因素鉴定

Measurement of the intracellular [Na^+] of antler stem cells and identification of regulation factors

为探索鹿茸的形态发生与鹿茸干细胞(包括发生干细胞和再生干细胞)内[Na^+]的关系有关,以鹿茸干细胞为模型,研究不同干性的哺乳动物干细胞内[Na^+]的差异,并鉴定引起这种差异的电压门控性钠离子通道(NaV)因素。采集并培养具有不同干性的梅花鹿鹿茸干细胞,包括生茸区骨膜细胞(AP细胞,鹿茸发生干细胞)和角柄骨膜细胞(PP细胞,鹿茸再生干细胞,其干性低于AP细胞),以鹿普通脸部骨膜细胞(FP细胞)作为对照,并采用CoroNa染色方法,通过荧光强度来分析不同类型细胞内[Na^+]的差异,结合PCR方法鉴定转录水平上NaV基因的差异,并分别用睾酮和MS-222对细胞进行处理,观测其对细胞内[Na^+]的影响。结果表明:鹿茸干细胞内[Na^+]高于FP细胞,其中AP细胞内[Na^+]高于PP细胞;NaV1.1基因在AP细胞中特异性转录;睾酮对这3种细胞内[Na^+]水平没有显著影响;但是,MS-222处理能够在一定程度上降低细胞内[Na^+]。本研究发现:鹿茸干细胞内[Na^+]与其干性一致,干性高的AP细胞内[Na^+]也相对较高;NaV1.1基因在转录水平上的差异,可能是造成AP细胞内[Na^+]高的主要原因;干扰NaV的MS-222能够在一定程度上降低细胞内[Na^+]。说明哺乳动物器官的发生和再生可能与低等动物器官上的发现类似,都与细胞内[Na^+]有关。

To investigate the relationship between deer antler morphogenesis and intracellular [Na^+] of antler stem cells including antlerogenic periosteum (AP) stem cells and pedicle periosteum (PP) stem cells,AP tissue and the facial periosteum (FP) tissue (used as a control) were collected from 10-month-old male sika deer calves at the time of pedicle initiation through surgery.The PP tissue was collected from 2-year-old male sika deer.All of the AP stem cells,PP stem cells,FP cells were cultured in vitro.The intracellular sodium ion of AP cells,PP cells and FP cells were stained with CoroNa Green indicator dye.Voltage-gated sodium channel (NaV) genes were identified through PCR.The difference of intracellular [Na^+] in the three cell types was analyzed with fluorescent density.Influence of testosterone and MS-222 on intracellular [Na^+] in the three cell types was tested.The results showed that the descending order of the intracellular [Na^+] in the three cell types was:AP cells>PP cells>FP cells.It was consistent with that of the plasticity of these cells.NaV1.1 gene was transcript specifically in AP cells.The intracellular [Na^+] of the three cell types was not influenced by testosterone treatment.The MS-222 treatment decreased the intracellular [Na^+] concentration of the three cell types but did not alter the order of the intracellular [Na^+] in the three cell types.The president study implies that morphogenesis of deer antlers may be similar to that of lower animal organs.It is regulated by intracellular sodium ion of antler stem cells.Transcription of NaV1.1 gene in AP cells might contribute to the high level of intracellular [Na^+].No evidence showed that testosterone had direct effects on intracellular [Na^+] of antler stem cells,though both of them were closely related with antler morphogenesis.In conclusion,the generation and regeneration of mammalian organ were similar to those of lower formed animals,which were related to intracellular [Na^+].