DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Phosphorus in interstitial water induced by redox potential in sediment of Dianchi Lake, China

The sediment redox potential was raised in the laboratory to estimate reduction of internal available phosphorus loads, such as soluble reactive phosphorus (SRP) and total phosphorus (TP), as well as the main elements of sediment extracts in Dianchi Lake. Several strongly reducing substances in sediments, which mainly originated from anaerobic decomposition of primary producer residues, were responsible for the lower redox potential. In a range of -400 to 200 mV raising the redox potential of sediments decreased TP and SRP in interstitial water. Redox potentials exceeding 320 mV caused increases in TP, whereas SRP maintained a relatively constant minimum level. The concentrations of Al, Fe, Ca2+, Mg2+, K+, Na+ and S in interstitial water were also related to the redox potential of sediments, suggesting that the mechanism for redox potential to regulate the concentration of phosphorus in interstitial water was complex.

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Delta psi m collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-kappa B activation and intracellular Ca2+ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.

Effects of salt stress on carbohydrate metabolism in desert soil alga Microcoleus vaginatus Gom.

The effects of salt stress on carbohydrate metabolism in Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crusts, were investigated in the present study. Extracellular total carbohydrates and exopolysaccharides (EPS) in the culture medium produced by M. vaginatus increased significantly during the growth phase and reached a maximum during the stationary phase. The production of extracellular carbohydrates also significantly increased under higher salt concentrations, which was attributed to an increase in low molecular weight carbohydrates. In the presence of NaCl, the production of cellular total carbohydrates decreased and photosynthetic activity was impaired, whereas cellular reducing sugars, water-soluble sugars and sucrose content and sucrose phosphate synthase activity increased, reaching a maximum in the presence of 200 mmol/L NaCl. These parameters were restored to original levels when the algae were transferred to a non-saline medium. Sodium and K+ concentrations of stressed cells decreased significantly and H+-ATPase activity increased after the addition of exogenous sucrose or EPS. The results suggest that EPS and sucrose are synthesized to maintain the cellular osmotic equilibrium between the intra- and extracellular environment, thus protecting algal cells from osmotic damage, which was attributed to the selective exclusion of cellular Na+ and K+ by H+-ATPase.

A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish

Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca2+-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development. (C) 2003 Elsevier Inc. All rights reserved.

Structure of macrozoobenthos of the East Dongting nature reserve, with emphasis on relationships with environmental variables

Ecological survey of macrozoobenthos assemblages was carried out at 32 sites in the East Dongting Nature Reserve, located in the northern region of the East Dongting Lake in the middle basin of the Yangtze River, China. All total 51 taxa including 18 oligochaetes, 15 mollusks, 14 insects and four other animals were recorded. Mollusks composed the dominant group and accounted for more than 70% of the total abundance. Assemblages were composed mainly of scrapers (66.7%) and collector-gatherers (nearly 20%), and to a lesser extent collector-filterers (roughly 12%), predators (ca. 7%), and shredders (ca. 6%). Two-way indicator species analysis, detrended correspondence, and canonical correspondence analysis (CCA) were employed to identify the relationships between macrozoobenthos assemblages and environmental variables. Thirty-two sites were separated into four site groups based on composition and relative abundance of benthic macroinvertebrates. CCA detected that water depth, pH, conductivity, SiO2, total nitrogen, total phosphorus, alkalinity, hardness, and Ca2+, were significant environmental factors influencing the pattern of macozoobenthos. In this minimal subset, water depth, pH, alkalinity and hardness were the most influential variables.

Distribution of epilithic algae in the Xiangxi River system and their relationships with environmental factors

The spatial pattern of epilithic algae in the Xiangxi River system was studied in relation to several environmental factors by two-way indictor species analysis (TWINSPAN), detrended correspondence analysis (DCA), and canonical correspondence analysis (CCA). Eighty-nine taxa including diatoms, green algae, and blue-green algae were observed. Diatoms were dominant, and Cocconeis placentula, Cymbella minuta, Diatoma vulgare, and Gomphonema angustatum appeared in most of sampling sites. By TWINSPAN and DCA, thirty-one sites were divided into three groups based on composition and relative richness of benthic algae. CCA indicated that SiO2, pH, total phosphorus, Ca2+, velocity, elevation, and Cl- were significant environmental factors affecting the distribution of algae communities. In this minimal subset, SiO2 and pH were the most influential variables.

Cell responses of Dunaliella salina FACHB 435 (green alga) to microgravitational stimulation by clinorotation

Clinorotation experiments were established to simulate microgravity on ground. It was found that there were obvious changes of Dunaliella salina FACHB435 cells and their metabolic characteristics during clinorotation. The changes included the increases of glycerol content, the rate of H+ secretion and PM H+-ATPase activity, and the decrease of ratio of the plasma membrane (PM) phospholipid to PM protein. These results indicated that microgravity was a stress environment to Dunaliella salina. It is deduced that it would be possible to attribute the effect of microgravity on algal cells to the secondary activation of water stress.

板栗壳对水中Cu~(2+)吸附性能的研究

研究食品加工剩余物板栗壳对水中Cu2+的吸附性能,为其用于含铜废水的处理提供理论依据。【方法】研究吸附质溶液pH、Cu2+质量浓度、吸附剂用量、粒径、吸附温度和时间对板栗壳吸附Cu2+效果的影响,探讨吸剂和吸附剂循环利用次数对解吸和再生的影响;并采用穿透曲线和洗脱曲线对动态吸附进行了分析。【结果】吸附质溶液pH值为6、Cu2+起始质量浓度为20 mg/L、吸附剂粒径为0.25 mm时的吸附效果较好,该吸附为放热过程,升高温度虽然可以加快吸附进程,但却降低了吸附量和去除率。Na+和Ca2+对Cu2+的解吸置换能力较弱,0.1mol/L HCl可使96.1%的Cu2+得以解吸回收。通过Thomas模型预测,在固定床柱吸附条件下饱和吸附量为10.94mg/g。【结论】板栗壳对水中Cu2+的吸附性能较好,因而具有很好的应用前景。

p21Waf1/Cip1的表达纯化以及与其靶分子的相互作用

p21Waf1/Cip1，是目前具有最广泛激酶抑制活性的细胞周期抑制蛋白。随着研究的发展，发现它在细胞增殖、分化、凋亡等方面具有越来越多的功能，并且这些功能很大程度上依赖于它能够与各种各样的靶蛋白相互作用。p21与靶分子的作用与它的结构特点密切相关， 因而从分子水平上研究p21蛋白的构象特点、以及其与靶分子的相互作用具有非常重要的意义。 在本文中，我们首先运用分子克隆技术在大肠杆菌中成功地构建了由温度控制的pBV220-p21表达体系。经30℃培养、42℃诱导表达以后，p21蛋白以包涵体形式得到高效表达，通过离子交换和分子筛两步纯化得到电泳纯的、具有生物活性的p21蛋白。利用生物化学、生物物理学等方法和手段我们首次较为系统地表征了p21蛋白的构象特点， 发现其具有典型的无规卷曲的二级结构；色氨酸很大程度上暴露于溶液中，处在正电荷的微环境中；首次发现p21蛋白能够与一些阴离子配合物（如suramin、Bis-ANS等）以近10-7数量级的解离常数直接相互作用，化学计量比为：1：1，进一步研究表明suramin在p21上的结合位点可能是p21的C-末端。在此基础上，我们通过丹磺酰氯标记过的钙调蛋白（CaM）的荧光光谱、p21141-164突变的色氨酸的内源荧光光谱及荧光猝灭方法、圆二色谱、非变性蛋白电泳等方法和手段表征了p21及p21141–164与CaM的作用。首次发现p21与CaM结合的解离常数在10-7数量级，p21与CaM之间除了疏水作用，还有较强的静电作用，以及一定的H键作用。深入研究表明p21141-164在CaM上的结合位点在CaM的Ca2+诱导的疏水区域，p21141–164的150和151位突变的色氨酸在结合后离CaM的疏水区域较近，而159位的色氨酸离的较远。同时发现p21141–164与CaM结合后只诱导出约14%的α螺旋结构，即p21141–164基本上是以无结构的状态与CaM结合的。

人类带3蛋白胞质片段的表达纯化及其性质表征

带3蛋白胞质片段(cdb3)具有多种生理功能：它可以将膜和膜骨架蛋白相联，起着维持细胞形状以及沟通内外环境的作用。而且，它还通过与多个糖酵解酶的相互作用来调节红细胞内糖酵解速率。它的这些功能很大程度上归因于它的分子柔性大，可以通过各种构象与膜蛋白、糖酵解酶等竞争性结合。 在本文中，我们首先运用分子克隆技术在大肠杆菌BL21(DE3)中成功地构建pET28b-cdb3表达体系。经37℃培养至OD600达0.6时，加入1mM IPTG ，于30℃诱导表达cdb3蛋白。经离子交换和亲和层析两步纯化得到电泳纯的具有生物活性的cdb3蛋白。之后，我们首次较为系统地表征了cdb3蛋白的构象特点：在常规的生理环境下, cdb3蛋白呈现典型的α-螺旋、β折叠二级结构， cdb3的四个Trp残基很大程度地包埋于疏水环境中；随溶液GuHCl浓度增加，cdb3 的四个Trp残基逐渐暴露于极性环境中；当pH值从6.0升高到10.0时，cdb3的Tm值逐渐降低约15℃，其内源荧光强度增加两倍，并在pH 7.2和pH9.2处呈现拐点，而蛋白的二级结构却没有发生变化。金属离子Cd2+、Ca2+、Cu2+、Co2+、Mg2 、Zn2+的结合位点在cdb3蛋白的Trp残基附近，而50μM的金属离子对重组cdb3蛋白的二级结构影响微小。最后，我们合成了cdb3蛋白N端肽段1－23，发现其在水溶液中呈无规卷曲结构，TFE可诱导其形成α螺旋，当TFE浓度增至80%时α螺旋含量达最高。无规卷曲的肽段不与醛缩酶相结合，其以α螺旋形式与醛缩酶相结合。 ATP可与肽段、重组cdb3蛋白结合，并对其光谱学性质有一定影响。当ATP浓度达到3mM时，重组cdb3蛋白发生聚集。

DNA结构的扫描探针显微学研究

1．利用原子力显微镜，我们研究了DNA与邻菲咯琳钻之间的反应。乙醇溶液中DNA复合物的形貌改变很好地说明了静电力在引起DNA凝集中的重要地位。随着醇浓度增加，对应更低的介电常数，静电反应增强。当介电常数降低，平衡离子就会在DNA的磷酸骨架上聚集，并中和负电荷到能引起凝集的程度。2，为了深入认识DNA-表面活性剂复合物的形貌特征，我们对阳离子表面活性剂CTAB引起的DNA凝集体进行了研究。由环状凝集体的体积分析，我们认为DNA发生单分子凝集。除环状体之外，许多不规则的环状体有助于说明DNA环状体的形成过程。部分绕转体和核小体状环状体对应于绕转模型而棒状中间体显示DNA分子也可能采取棒状体到环状体的转化模型。3．DNA分子在形成稳定的凝集体之前会经过一系列的中间体。因为温度是DNA结构的一个重要的决定因素，在其他凝集条件一定时，我们观察了DNA凝集中间体在温度升高时的结构转变。AFM分析得出质粒DNA pBR322的凝集结构受温度的影响很大。低温时，单个分子花束体是DNA分子采取的主要凝集结构。较高温度时，分子变得舒展，但链上有许多可能利于多分子凝集的小环。继续升高温度，DNA分子大范围地发生分子间的单中心、交联。4．在Mg2+，Ca2＋，Sr2和Ba2+分别作为DNA在云母表面的平衡离子时，我们观察了DNA分子在云母表面扩散平衡或被动力俘获的形貌特征。末端距和伸展长度可由原子力显微镜的数据分析结果得到。实验结果表明当Mg2＋，Ca2+和Sr2+为平衡离子时，DNA分子能够在云母表面扩散平衡。然而Ba2+作用时，严重交接的DNA结构说明DNA分子不易在云母表面反应平衡，俘获的程度也随实验条件而改变。在醇溶液中，我们还观察了B-A构象转变对平衡离子种类的依赖关系。四种碱土金属引起B-A构象转变的能力不同，其中Sr2-导致B-A构象转变的程度最大。

猕猴胚胎干细胞的神经谱系分化及胶质祖细胞迁移机理研究

由于伦理和材料来源的限制，目前对灵长类早期神经发育缺乏深入地了解。与啮齿类动物相比，猕猴在遗传和生理上与人类更接近，因此猕猴胚胎干细胞(rESCs)研究具有重要的研究价值，不仅能为研究发育生物学基础理论提供良好的模型，而且可为细胞替代性治疗提供大量的供体细胞。本文以rESCs为主要研究对象，在rESCs定向分化为神经细胞的基础上着重研究神经谱系分化及调控胶质祖细胞迁移的机理。主要结论如下：1) rESCs来源的神经上皮干/前体细胞(NEPs)主要变为辐射状胶质细胞(RG)后再通过中间类型的祖细胞——神经元祖细胞(NPs)和胶质祖细胞(GPs)——分别分化为神经元和胶质细胞。同时，NEPs/RG细胞群具有早期神经管背-腹和前-后轴空间特性。NEPs/RG的维持受Notch和FGFR信号作用。此外，实验中还纯化和鉴定了猕猴胶质限定性前体细胞(GRPs)。结果表明，rESCs的神经谱系分化能够模拟体内发育过程，并与啮齿类动物早期神经谱系变化过程相似。2) 气体信号分子NO(由10μM—250μM SNP供体释放)促进rESCs来源的A2B5+/Nestin/PSA-NCAM胶质祖细胞迁移。进一步研究发现Netrin-DCC信号通路介导了NO启动的细胞迁移过程。同时，Ca2也参与调控胶质祖细胞的迁移。此外，细胞外基质和整合素α6亦可能与Netrin-DCC相互作用调控细胞迁移。结果显示，NO通过激活一个复杂的信号网络系统调控胶质祖细胞迁移。本实验的研究结果有助于揭示灵长类中枢神经系统发育的机理，同时也能为治疗神经系统退行性疾病提供阶段特异性的供体细胞。

几类“无线粒体”原生生物的DNA拓扑异构酶II基因及进化地位研究

以贾第虫、毛滴虫、内变形虫和微抱子虫等为代表的几类原生生物，不仅因为他们的寄生致病性而在医学上长期备受关注，它们的进化地位也是一个十分令人注目的问题。因为曾认为它们不具线粒体等细胞器，再加上一些分子系统学研究表明它们处在真核生物的最基部，因此不少人认为它们是在线粒体产生之前即已分化的极原始真核生物，其进化地位是处在原核生物向真核生物的过渡阶段，并有人称之为achezoa。这一发现一度被认为对探讨真核细胞（生物）的起源进化极为重要，是进化生物学上的重要突破。然而，近年来不断有新的证据对此提出质疑，其进化地位也就存在较大争议。本文首先利用PCR扩增、测序和基因组数据库搜索等技术方法鉴定了蓝氏贾第虫（Giardialamblia）、阴道毛滴虫（Trichomonasvaginalis）和痢疾内变形虫（entamoebahistolytica）的II型DNA拓扑异构酶基因序列。RT-PCR和序列分析表明它们均不具内含子。蛋白质序列搜索的结果表明它们与其它真核生物的DNA拓扑异构酶H是高度同源的。用生物信息学的方法，我们还对这些酶的性质进行了初步分析。分析还表明蓝氏贾第虫的DNA拓扑异构酶H具有一些不同于其．宿主的特征，如在ATPase区和中间区有六个插入，中间区要长大约100个氨基酸，而C端区又短大约200个氨基酸且富含带电荷的氨基酸残基。这些结果对研制以该酶为靶分子的专一性抗贾第虫药物具有指导意义。其次，将上述获得的序列数据结合GenBank数据库中已有的脑炎微抱子虫（Encephalitozooncuniculi）和其它一系列处在不同进化地位的真核生物的相应序列数据，用多种方法构建出分子系统树，对这些"无线粒体"原生生物的进化地位进行了探讨，并对"长枝吸引"对系统树的影响进行了分析。结果表明，由于DNA拓扑异构酶H的特点和可以克服"长枝吸引"等以往分子系统分析中的不足，所构建的系统树不仅能有效地反映出已普遍接受的真核生物各主要类群的系统关系，而且显示出这些"无线粒体"原生动物不同于以前系统树所反映的进化地位：它们并非是最早分支出来的真核生物，而是在具有线粒体的生物如动基体类或菌虫类等之后才分化的、分别属于不同进化地位的类群。结合近来它们中发现了类似线粒体细胞器等证据，我们认为这些所谓"无线粒体"的原生生物虽然其中有些种类（如以贾第虫为代表的双滴虫类）进化地位很低等，对探讨真核细胞的早期进化具有一定意义，但总体上它们并非过去所认为的那么极端原始，它们应该是线粒体产生之后才分别分化出来的不同生物类群

雨蛙皮肤分泌液中两种活性蛋白结构和功能的研究

在本研究中我们首次从雨蛙皮肤分泌液中分离得到了一种神经毒素（命名为Anntoxin）和一种干细胞自我更新支持因子（命名为AnSF）。随后，我们通过构建雨蛙皮肤cDNA 文库，利用特异引物筛选到Anntoxin 和AnSF 的cDNA 编码序列，前者的Gene Bank 登录号为FJ598043，后者还在等待分配登录号。Anntoxin 具有60 个氨基酸，是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，构建Anntoxin 的3D-NMR 溶液结构，证实Anntoxin 不同于有三对二硫键（键组合模式：1-6，2-4，3-5）的Kunitz 类型丝氨酸蛋白酶抑制剂，它只有两对二硫键（组合模式：1-4，2-3）。AnSF 具有123 个氨基酸，在C 端具有和Calmadolin 同源的两个EF 手指结构，能够支持人类胚胎干细胞（hESC）和猴神经干细胞（rNSC）的自我更新。为了进行Anntoxin 的生物活性和结构分析，我们在体外成功表达了 Anntoxin，获得了大量的重组Anntoxin（rAnntoxin）。经过生物活性分析， rAnntoxin 和天然分离到的Anntoxin 生物活性相当，都具有很强的胰蛋白酶抑制剂活性。Anntoxin 是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，和来源于芋螺（Cone Snail）的神经毒素Conkunitzin-S1，黑色眼镜蛇毒液（black cobra, Dendroaspis polylepis polylepis）的树突毒素δ-DaTX 或蛋白酶抑制剂K 分别具有32.8%和36.7%的相同序列，和鱼类（fish）来源的Stonustoxin 也有一定的同源性。利用膜片钳技术分别检测Anntoxin 对大鼠背根神经节（rat DRG）上Na+通道，K+通道，Ca2+通道的作用，结果证明Anntoxin 对河豚毒素敏感（TTX-S）的钠离子通道（Nav）有较强的抑制活性，对 K+通道，Ca2+通道作用不明显。随后我们在非洲爪蟾卵母细胞上表达几种典型和常用于测试对亚型K+通道作用的Kv1.1，Kv1.2，Kv1.3，Kv2.1 和 Kv4.2，Kv4.3，Anntoxin 对这些亚型K+通道上的K+电流都没有明显影响。我们成功构建了Anntoxin 的3D-NMR 溶液结构（NMR 号：PDB ID 2KCR， BMRB ID 16094），证实Anntoxin 具有典型的Kunitz 结构，由反向平行的 β–折叠片和α–螺旋及转角组成梨形结构。利用RT-PCR，WesternBlot 以及 ELISA 技术，发现在皮肤、脑、肝、胃和肠中都能检测Anntoxin mRNA 转录，但只在皮肤、脑、肝和胃中有蛋白表达，表达量分别为29.5、5.39、 4.80 和2.02 微克/克鲜重，可以看出Anntoxin 在皮肤中大量表达，是皮肤分泌液中非常重要的组成部分。因为皮肤是雨蛙接触外界的第一屏障，雨蛙的生存环境中存在很多潜在威胁，比如微生物、吸血昆虫、鸟类、爬行动物、哺乳动物等，所以Anntoxin 有可能是雨蛙适应环境的重要化学武器，于是我们测试了Anntoxin 对甜菜夜蛾幼虫（Laphygma exigua Hubner）、水蛇（Enhydris plumbea）、鹌鹑（Coturnix coturnix）、昆明小鼠（Kunming mice）的急性毒性，其LD50 分别为50，450，2500 和3000 微克/千克体重，说明在华西雨蛙皮肤中大量表达的Anntoxin 对几类潜在天敌确实有较强的杀灭作用。为了检测AnSF 的生物学活性，我们在体外成功表达了AnSF，获得了大量rAnSF。设计三个浓度梯度10、100 和500ng/ml，把AnSF 和hESC 共培养，发现在10~100 ng/ml 浓度时对hESC 的自我更新有支持作用；设计三个浓度梯度10、100 和500ng/ml，把AnSF 和rNSC 共培养，发现在 10ng/ml 时对rNSC 的自我更新有较强的支持作用。在超过500ng/ml 高浓度时，AnSF 对hESC 和rNSC 都有明显的细胞毒性作用，对rNSC 的毒性作用更明显。利用RT-PCR 技术，我们检测了雨蛙的皮肤、肌肉、肝脏、胰脏、胃、肠、心脏和脑，AnSF 只在皮肤中有少量表达。这表明AnSF 可能只参与雨蛙皮肤干细胞库的维持，保持皮肤内环境稳定，因为蛙类的皮肤细胞要负责产生大量活性物质参与先天免疫和抗氧化等重要的生理活动，需要经常更新，而AnSF 的存在可能保证雨蛙皮肤干细胞库容量稳定，不断分化出各种成熟的皮肤细胞来使皮肤能够得到足够和及时的更新，保证其功能的正常行使。所以AnSF 是维持华西雨蛙皮肤内环境稳定的重要物质。