一株罗布泊盐湖链霉菌的鉴定及其代谢产物对酪氨酸酶活性、黑色素生成的抑制效应CSCD

本研究采用多相分类法对一株分离自新疆罗布泊盐湖链霉菌89-2-2进行鉴定,通过CCK-8法、L-多巴氧化法和NaOH溶解法检测该菌株乙酸乙酯提取物对小鼠黑色素瘤B16细胞增殖、细胞内酪氨酸酶活性及黑色素含量的影响。以抑制酪氨酸酶活性为指标,应用LC-MS代谢组学方法检测链霉菌89-2-2发酵条件优化前后所产生的差异代谢物,分析可能具有抑制活性的代谢产物类型。结果初步鉴定该菌株为西唐氏链霉菌(Streptomyces setonii)89-2-2。S.setonii 89-2-2乙酸乙酯提取物在100~1000μg/mL浓度范围内几乎无细胞毒性,但能有效抑制B16细胞内酪氨酸酶活性和黑色素生成,与空白组相比均具有显著性差异(P<0.05)。代谢组学实验的结果显示S.setonii 89-2-2产生的差异代谢物主要为维生素类化合物、芳香类化合物和羧酸类化合物。本研究为从新疆罗布泊链霉菌中开发酪氨酸酶抑制剂和黑色素合成抑制剂提供重要的科学依据。     

荧光标记糖电泳在新琼寡糖定性和定量分析中的应用

目的:建立荧光辅助糖电泳(FACE)对系列新琼寡糖进行定性、定量分析的方法。方法:将系列新琼寡糖用7-氨基-1,3-萘胺二磺酸钾(AGA)氨化还原衍生后,在浓度梯度为18%-25%的聚丙烯酰胺凝胶中电泳分离,于波长264nm直接检测寡糖衍生物,得到聚合度为4-14的新琼寡糖衍生物电泳分析图谱。结果:寡糖衍生物的相对电迁移率与其分子量的负三分之二次方成线性关系;运用图像分析软件对电泳图谱进行数字化转换,发现寡糖衍生物的灰度积分面积与样品浓度呈线形关系。结论:建立了快速、精确的新琼寡糖微量定性、定量分析的方法,为新琼寡糖的质量分析提供了技术支撑。     

一株转化琼胶为新琼寡糖菌株的筛选及鉴定

摘要:【目的】筛选一株可以将琼胶转化为新琼寡糖的菌株,并对该菌株进行鉴定。【方法】从紫菜生长区域采集紫菜和该区域海水,用含1‰琼胶的培养基富集培养,逐级稀释涂布、平板划线进行初筛,液体培养进行复筛,DNS法测定琼胶降解产物中还原糖的含量。通过16S rDNA序列分析,结合菌体形态、菌落特征及生理生化特性,确立该菌的系统发育学地位。【结果】从紫菜振荡液中筛选出一株可以产琼胶酶的菌株HJPHYXJ-1,该菌属于革兰氏阴性菌,16S rDNA序列同源性与需钠弧菌(Vibrio natriegens)的达到了99%,结合形态特征和生理生化实验结果鉴定该菌为需钠弧菌。HPLC法测定酶解产物为新琼寡糖。【结论】HJPHYXJ-1被筛选用于转化琼胶,酶解产物的聚合度在2－12 之间,是以二糖为单位的新琼寡糖,该菌产生的酶为β-琼胶酶。     

黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响

为研究黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响,以及初步研究其作用机制,采用磺酰罗丹明B(SRB)法测黄泥螺提取物对B16细胞的生长抑制作用,得到48 h后半数抑制浓度(IC50)为68.56 ug/ml.当黄泥螺提取物浓度为70ug/ml时,台盼蓝排斥试验显示有部分细胞死亡.经Hoechest 33258染色并用荧光显微镜观察,发现培养的细胞中出现凋亡小体,细胞核发生固缩;DNA琼脂糖凝胶电泳呈现出DNA大片段;用流式细胞仪进一步检测表明,细胞生长周期发生变化并检测到凋亡峰.结果表明,体外培养的B16细胞经过黄泥螺提取物处理后,B16细胞增殖受到抑制,细胞周期被阻滞,B16细胞受到诱导后存在凋亡.因此,黄泥螺提取物对小鼠黑色素瘤细胞B16生长有明显的影响.     

氧化鱼油对黄颡鱼黑色素合成酶及内分泌激素的影响

实验模拟自然氧化条件制备氧化鱼油, 替代普通鱼油加入饲料中, 研究其对黄颡鱼(Pelteobagrus fulvidraco)表皮黑色素、酪氨酸酶活力、血清与脑内分泌激素含量的影响。处理组分别为未替代组(C组)、50%氧化鱼油替代组(Y50组)和100%氧化鱼油替代组(Y100组), 结果表明: 随着氧化鱼油替代普通鱼油的比例提高, 黄颡鱼5级(黑色素占细胞面积80%以上)黑色素细胞比例上升; 酪氨酸酶活力上升, 但各组差异不显著(P>0.05); 皮质酮(CORT)在血清和脑组织中各组含量差异均不显著(P>0.05); 肾上腺皮质激素(ACTH)在脑组织中含量增加 (P<0.05), 而血清中Y50组含量高于C组及Y100组(P<0.05); 血清中促肾上腺皮质激素释放激素(CRH)含量降低, Y50与Y100组之间差异不显著(P>0.05), 而脑组织中各组差异不显著(P>0.05); 血清中α-黑素细胞刺激素(α-MSH)含量, Y100组高于其他组(P<0.05), 而脑组织中各组差异不显著(P>0.05)。上述结果表明, 随着氧化鱼油替代普通鱼油的比例提高, 5级黑色素细胞比例上升, 黄颡鱼血清和脑组织中能互相转换的ACTH与α-MSH总量提高, 变化趋势与表皮黑色素含量和酪氨酸酶活性相一致。     

一种新型补骨脂素衍生物BSP-1诱导B16细胞中黑色素合成的作用机制

8-甲氧基补骨脂素（8-MOP）和5-甲氧基补骨脂素（5-MOP）等补骨脂素类药物在临床上常用于治疗白癜风,但同时具有诸多副作用。因此,发掘作用更强、毒性更小的补骨脂素类化合物用以治疗白癜风成为研究热点。在我们的前期研究中,本课题组设计合成了一系列结构新颖的补骨脂素席夫碱衍生物,并评价了它们的抗白癜风活性。本论文选取了其中一个补骨脂素席夫碱衍生物（BSP-1）,研究了它对小鼠B16细胞中黑色素合成的作用及其信号通路。利用CCK 法、L-Dopa 氧化法、NaOH溶解法及Western印迹法分别分析BSP-1对细胞增殖、黑色素含量、酪氨酸酶(TYR)活性及相关蛋白表达的影响。结果显示,BSP-1能够促进B16细胞内黑色素生成和TYR活性,上调 TYR、TRP-1、TRP-2和MITF的蛋白表达,并呈浓度依赖性。机制研究发现,BSP-1通过提高Akt和GSK-3β的磷酸化水平,上调细胞核中β 联蛋白的含量,最终使得小眼相关转录因子（MITF）的蛋白表达增加。综上所述,本研究提示BSP-1可通过调节Wnt/β-联蛋白信号通路来促进B16细胞内的黑色素合成。     

LncRNA00067110通过靶向Cabyr调控B16-F10细胞增殖、凋亡和黑色素生成

长非编码RNA(lncRNA)是一类转录长度大于200个核苷酸的非编码RNA。现已证明,多个lncRNA是潜在的癌症治疗靶点。LncRNA00067110是从小鼠黑色素瘤B16-F10细胞和正常黑色素细胞转录物组图谱中发现的差异表达基因。为研究lncRNA00067110是否调控B16-F10细胞的增殖、凋亡和黑色素生成,本文通过LncTar预测和双荧光酶活性验证了钙结合酪氨酸磷酸化调节蛋白(Cabyr) 和lncRNA00067110存在靶向关系。通过构建lncRNA00067110的过表达载体,转染B16-F10细胞,经过对B16-F10细胞的转录图谱分析,并对细胞增殖、凋亡和黑色素生成的表型以及相关基因表达变化进行了检测。结果显示,lncRNA00067110靶向Cabyr,在过表达lncRNA00067110的B16细胞中,17个基因呈差异表达。其中,Cabyr的表达被上调,细胞增殖相关基因MEK/ERK/MNK/CREB和黑色素生成相关基因TYR家族成员及CREB的mRNA和蛋白质水平显著被下调,凋亡相关基因AKT和Bcl-2的mRNA水平和蛋白质丰度被上调。进一步通过细胞增殖和凋亡的表型的变化验证了lncRNA00067110的功能。结果提示,lncRNA00067110通过靶向Cabyr,调控相关基因表达,从而抑制B16-F10细胞的增殖和黑色素生成,并诱导黑色素瘤细胞的凋亡,可能成为治疗和抑制黑色素瘤的新的靶点。     

WIP1对小鼠B细胞及T细胞发育的影响

目的研究WIP1基因对小鼠骨髓B细胞发育及胸腺T细胞发育的影响。方法流式细胞术测定小鼠骨髓B细胞及胸腺T细胞发育中各阶段的细胞比例。结果虽然WIP1缺失小鼠骨髓B细胞发育各阶段比例正常,但骨髓总体B细胞比例下降;WIP1基因敲除小鼠胸腺发育障碍,CD8/CD4双阴性细胞比例增高,CD8/CD4双阳性细胞比例降低。结论 WIP1基因在小鼠骨髓B细胞及胸腺T细胞的发育过程中起重要作用。     

小刺猴头菌发酵浸膏寡糖对荷瘤小鼠免疫功能的影响

通过建立S180荷瘤小鼠试验动物模型,研究小刺猴头菌发酵浸膏寡糖对S180荷瘤小鼠免疫功能的影响。选择灌胃的方法将不同组别小刺猴头菌发酵浸膏寡糖喂服到荷瘤小鼠体内,观察肿瘤生长情况,对其进行免疫功能的测定。结果表明:小刺猴头菌发酵浸膏寡糖可增加荷瘤小鼠免疫器官脾脏和胸腺质量,提高荷瘤小鼠血清的IL-2和TNF-α含量,抑制肿瘤生长,提高机体免疫功能。     

MiR-186-5p对绵羊黑色素细胞黑色素生成的调控

黑色素合成是极其复杂的过程,其中涉及多种基因和miRNAs的参与.miR-186-5p在绵羊不同毛色皮肤中差异表达,说明其可能与毛色形成有关,生物信息学预测和绵羊不同毛色皮肤中miR-186-5p和Mitf的差异表达间接说明二者可能存在靶向关系,双荧光报告直接证实了二者的靶向关系.为了证实miR-186-5p在黑色素形...     

A novel form of melanoma apoptosis resistance: melanogenesis up-regulation in apoptotic B16-F0 cells delays ursolic acid-triggered cell death

Melanoma is one of the most aggressive forms of cancer with a continuously growing incidence worldwide and is usually resistant to chemotherapy agents, which is due in part to a strong resistance to apoptosis. The resistance mechanisms are complex and melanoma cells may have diverse possibilities for regulating apoptosis to generate apoptotic deficiencies. In this study, we investigated the relationship between melanogenesis and resistance to apoptosis induced by ursolic acid, a natural chemopreventive agent, in B16-F0 melanoma cells. We demonstrated that cells undergoing apoptosis are able to delay their own death. It appeared that tyrosinase and TRP-1 up-regulation in apoptotic cells and the subsequent production of melanin were clearly implicated in an apoptosis resistance mechanism; while TRP-2, a well known mediator of melanoma resistance to cell death, was repressed. Our results confirm the difficulty of treating melanomas, since, even undergoing apoptosis, cells are nevertheless able to trigger a resistance mechanism to delay death.     

Involvement of calpain in melanogenesis of mouse B16 melanoma cells

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to α-melanocyte-stimulating hormone (α-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following α-MSH-treatment, and restored to the initial level in 6–12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on α-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked α-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16melanoma cells.     

Alteronol inhibits the invasion and metastasis of B16F10 and B16F1 melanoma cells in vitro and in vivo

Aims

The purpose of this study is to evaluate the anti-metastatic effects of alteronol on melanoma B16F10 and B16F1 cells in vitro and in vivo.

Main methods

Melanoma B16F1 and B16F10 cells were cultured in vitro. Cell proliferation was analyzed via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The cell migration and invasion were evaluated via wound healing and transwell chamber assays. The activity of matrix metalloproteinase 2 (MMP-2) in culture supernatants was assessed via gelatin zymography. The expression of MMP-2 and TIMP-2 were detected via enzyme-linked immunosorbent assay (ELISA) assay. The anti-metastatic ability in vivo was detected through experimental lung metastasis.

Key findings

The data indicate that alteronol can inhibit the proliferation, invasion, and migration of B16F1 and B16F10 cells in vitro and in vivo, decrease the activity and expression of MMP-2, enhance the expression level of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), and inhibit the experimental lung metastasis of B16F1 and B16F10 cells.

Significance

Although alteronol and taxol are obtained from the same source, these substances do not destroy the rare resource; the mechanisms of them on tumor growth inhibition are different. Conversely, alteronol treatment had lesser effects on normal cells revealing for a selective property and a strong competitive advantage.     

Diarylheptanoids with inhibitory effects on melanogenesis from the rhizomes of Curcuma comosa in B16 melanoma cells

The methanolic extract from the dried rhizomes of Curcuma comosa cultivated in Thailand was found to inhibit melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extract, three new diarylheptanoids, diarylcomosols I–III, were isolated together with 12 known diarylheptanoids. Their chemical structures were elucidated on the basis of chemical and physicochemical evidence. The diarylheptanoids inhibited melanogenesis, and several structural requirements of the active constituents for the inhibition were clarified. In particular, (3R)-1,7-bis(4-hydroxyphenyl)-(6E)-6-hepten-3-ol exhibited stronger inhibitory effect [IC₅₀ = 0.36 μM] without inducing cytotoxicity. The biological effect was much stronger than that of a reference compound, arbutin [IC₅₀ = 174 μM]. We conclude that diarylheptanoid analogs are promising therapeutic agents for the treatment of skin disorders.     

Ketonethiosemicarbazones: structure-activity relationships for their melanogenesis inhibition

A series of 2-(1-phenylalkylidene)hydrazinecarbothioamides 2, 2-(1-phenylalkyl)hydrazinecarbothioamides 3, 2-(3,4-dihydronaphthalen-1(2H)-ylidene)hydrazinecarbothioamide (4), and 2-(1-(thiophen-2-yl)ethylidene)hydrazinecarbothioamide (5) were synthesized for their melanogenesis inhibition in melanoma B16 cells. The SAR of these ketonethiosemicarbazones revealed that the benzylidene hydrogen in aldehydethiosemicarbazones 1 can be replaced by hydrophobic moiety and substitutions with alkyl group for the terminal amino hydrogen of ketonethiosemicarbazones improved the activity appreciably. In addition, the double bond in thiosemicarbazones is an important factor for the increment of hydrophobicity. Thus hydrophobic ketonethiosemicarbazones are excellent inhibitors of melanogenesis like aldehydethiosemicarbazones.     

Human placental lipid induces mitogenesis and melanogenesis in B16F10 melanoma cells

A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in anin vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0.01 to 200 μg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 μg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C₂ ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.     

Increase of melanogenesis by retinoic acid: an ultrastructural and morphometric study

Melanin is a dark pigment protecting the skin against UV radiation in some organisms. Studies on invasion and metastasis using retinoic acid as inhibitor agent are well known, but its role in melanin production (melanogenesis), especially at ultrastructural level and using morphometry were not well studied. In the present study, we analyzed the effects of retinoic acid on the melanosomes in B16F10 melanoma cells. These organelles were identified and quantified using routine electron microscopy and the specific HMB45 antibody. Other approaches such as immunofluorescence, and flow cytometry were also used. Our results indicated that retinoic acid increased the melanogenesis process in B16F10 melanoma cells. Furthermore, this work also provided evidence that this substance interferes at the subcellular level altering the numerical density of melanosomes, as well as the relative volume of the nucleus and nucleolus. In addition, the cells displayed altered morphology and an increase in the percentage of the relative volume of melanosomes, mainly the stages II-III and IV, leading to melanin formation. Furthermore, a decrease in the cells number after retinoic acid treatment was also observed.     

Effects of trace metals on mouse B16 melanoma cells in culture

The effects of fourteen metal ions (As³⁺, As⁵⁺, Cd²⁺, Co²⁺, Cr³⁺, Cr⁶⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, Se⁴⁺, V⁵⁺, VO²⁺) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10⁻⁴ M, metal ions such as As³⁺, As⁵⁺, Cd²⁺, Cr⁶⁺, Se⁴⁺, V⁵⁺, VO²⁺, and, to a minor extent, Li⁺, Hg²⁺, and Co²⁺ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg²⁺>Cr⁶⁺=Cd²⁺>As³⁺, As⁵⁺>V⁵⁺, VO²⁺>Se⁴⁺=Ni²⁺=Co²⁺=Li⁺. An increased synthesis of melanin in B16 cells was noted after incubation with Co²⁺, Ni²⁺, Cd²⁺, and Li⁺, whereas Se⁴⁺ had, on the contrary, an inhibiting effect on melanogenesis.