Inhibitory effect of Cyrtomium falcatum on melanogenesis in α-MSH-stimulated B16F10 murine melanoma cells

Objective:To explore the anti-melanogenic potential of Cyrtomium falcatum.Methods:The effects of Cyrtomium falcatum crude extract and its solvent fractions on tyrosinase activity,melanin content,and the expressions of melanogenesis-related genes and proteins were analyzed inα-melanocyte-stimulating hormone(α-MSH)-stimulated B16F10 cells.Results:α-MSH treatment significantly increased tyrosinase activity,and extracellular and intracellular melanin content,as well as the expression levels of tyrosinase,microphthalmia-associated transcription factor(MITF),tyrosinase-related protein(TRP)-1,and TRP-2 in B16F10 cells.Treatment with Cyrtomium falcatum crude extract and its solvent fractions reduced tyrosinase activity and extracellular and intracellular melanin content and downregulated the expression levels of tyrosinase,MITF,TRP-1,and TRP-2 in a dose-dependent manner.Conclusions:Cyrtomium falcatum has potential anti-melanogenesis effects and can be used as a potential source material in cosmeceutical industry for the research and development of novel lead molecules with whitening properties.

23-羟基桦木酸对B_(16)细胞系的诱导分化作用

目的评价 2 3 羟基桦木酸对黑色素瘤B16细胞的抑瘤作用。方法以MTT法测定细胞增殖的抑瘤率 ,并以B16细胞形态、黑色素含量、细胞周期变化及体内致瘤能力的测定作为观察指标。结果用 10～ 2 0 μg/ml的2 3 羟基桦木酸作用肿瘤细胞 ,见有不同程度的抑瘤作用 (P <0 .0 0 1)。表现为黑色素生成能力增加 ,细胞生长缓慢。可使B16细胞阻断在G1期 ,肿瘤体积明显缩小。结论 2 3 羟基桦木酸低剂量 (10～ 2 0 μg/ml)对B16细胞有明显的分化诱导作用 。

苯乙酸钠对小鼠B_(16)黑色素瘤细胞的分化诱导作用

目的 评价苯乙酸钠对黑色素瘤 B1 6 的抑瘤作用。方法 以 MTT法测定细胞增殖的抑制率。B1 6 胞形态 ,黑色素含量 ,细胞周期变化及体内致瘤能力的测定作为观察指标。结果 用 7.5 m M～ 17.5 m M的苯乙酸钠作用于肿瘤细胞 ,见有不同程度的抑瘤作用 P<0 .0 1。 MTT法测得IC5 0 为 2 .6 7～ 3.6 3m M。在 7.5～ 17.5 m M浓度下 ,对 B1 6 细胞都有较强的分化诱导作用 ,表现为黑色素颗粒生成能力明显增多 ,生长缓慢。细胞动力学研究表明 ,苯乙酸钠可使 B1 6 细胞阻断在 G1 期因而 S期明显减少 ,体内致瘤表明成瘤能力明显降低。结论 苯乙酸钠对 B1 6 细胞有强力分化诱导作用 ,而对体内外黑色素瘤增殖有明显抑制。

转导白细胞介素2基因的B_(16)细胞对机体免疫反应的影响

应用XM6逆转录病毒载体将人IL-2cDNA转入小鼠B16黑色素瘤细胞。丝裂霉素C处理的转染前后B16细胞作为瘤苗对小鼠进行免疫接种。结果显示，接种B16-IL-2细胞可明显排斥再移植黑色素瘤的生长。对脾淋巴细胞检测的结果表明，MLTR引起的淋巴细胞增殖与特异性CTL杀伤活性，以及NK、LAK活性与诱生的IL-2水平几项指标，B16-IL-2细胞免疫组均明显高于B16免疫组及对照组。提示转基因肿瘤细胞在体内分泌IL-2增强了机体的特异性与非特异性免疫功能。

基于光动力疗法的四苯基卟啉脂质体制备及体外抗肿瘤活性评价

目的制备四苯基卟啉脂质体(TPP-Lip)以提高游离四苯基卟啉的水溶性、稳定性和靶向性,同时对该脂质体进行体外抗肿瘤活性评价。方法测定光敏剂四苯基卟啉的单线态氧,采用薄膜分散超声法制备TPP-Lip,并通过细胞摄取和细胞毒性实验考察TPP-Lip光动力疗法对黑色素瘤细胞B_(16)F_(10)和乳腺癌细胞4T1的抗肿瘤活性。结果制备得到形态均匀的球形或类球形脂质体TPP-Lip,其包封率为(88.84±2.64)%,粒径为(115.84±3.89)nm。体外实验考察得到B_(16)F_(10)细胞和4T1细胞最佳光照时间分别为3、6 min,两者的最佳孵育时间均为4 h。设定TPP-Lip浓度梯度为1.25~30μg/mL,在此范围内脂质体对B_(16)F_(10)和4T1细胞有较好的光毒性,IC_(50)值分别为13.73、16.30μg/mL,并对2种细胞的暗毒性均较小。同时,CFPE标记的脂质体在2种细胞中的摄取程度差异不大。结论成功制备出四苯基卟啉脂质体并具有良好的稳定性,TPP-Lip通过光动力疗法发挥体外抗肿瘤作用,为疏水性光敏剂的应用提供参考。

Isolation and Identification of Cancer Stem-Like Cells from Murine Melanoma Cell Lines

In current study, cancer stem-like cells in the murine melanoma B16F10 cells were investigated. CD phenotypes of the B16F10 cells were analyzed by flow cytometry, and the specific CD phenotype cells from the B16F10 cells were isolated by MACS. Then we used colony formation assay in soft agar media, the cell growth assay in serum-free culture media as well as the tumorigenicity investigation of the specific CD phenotype cells in C57BL/6 mice, respectively, to identify cancer stem-like cells in the B16F10 cells. The results showed that the B16F10 cells could form spherical clones in serum-free culture media, and the rate of clonegenesis of CD133^＋, CD44^＋ and CD44^＋CD133^＋ cells was higher than that of CD133^-, CD44^- and CD44^＋CD133^＋ cells in soft agar media, respectively. The tumorigenic potential of CD133^＋, CD44^＋, CD44^＋CD133^＋ cells and CD44^＋CD133^＋CD24^＋ cells was stronger than that of CD133^-, CD44^-, CD44^＋CD133^- cells and CD44^＋CD133^＋CD24^- cells in mice, respectively. In conclusion, the CD44^＋CD133^＋CD24^＋ cells have some biological properties of cancer stem-like cells or are highly similar to the characteristics of cancer stem cells （CSC）. These results provide an important method for identifying cancer stem-like cells in B16F10 cells and for further cancer target therapy. Cellular ＆ Molecular Immunology.

肿瘤坏死因子对淋巴激活素活化的杀伤细胞抗肿瘤活性的影响

用B_(16)W_(10)黑色素瘤作靶瘤,C_(57)BL/6小鼠为荷瘤动物,小鼠脾细胞经白细胞间介质-2培养3d为效应细胞即LAK细胞,观察了肿瘤坏死因子对LAK细胞抗肿瘤活性的影响。体外实验结果表明:单独肿瘤坏死因子(500U/ml)或低剂量白细胞间介质-2(10U/ml)均不影响LAK细胞的杀瘤活性;肿瘤坏死因子能增强亚适剂量白细胞间介质-2(100U/ml)诱导的LAK细胞活性。而不增强最适剂量白细胞间介质-2(1000U/ml)诱导的LAK细胞活性。体内实验结果表明:对局部肿瘤,瘤内同时注射肿瘤坏死因子和白细胞间介质-2和LAK细胞时,6只荷瘤鼠4只瘤块消失。其中2只存活期超过60d。对全身肺转移癌、肿瘤坏死因子、白细胞间介质-2和LAK细胞联合静脉注射亦显抗瘤作用增强,该组小鼠肺表面瘤结节数和肺结节发生率减少。

A strategy for uncovering germline variants altering anti-tumor CD8 T cell response

Among many factors known to alter the outcomes of T cell receptor(TCR)-induced proximal signaling,the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address.Here,we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations(pTyr-SNVs)that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways.We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin(OVA)antigens and retrovirally engineered syngeneic major histocompatibility complex(MHC)Class I restricted OVA TCR-transgenic CD8 T cells(OT-I).Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4(ITGA4)p.S1027I variant as a prototype,we show that under identical TCR stimulation conditions,genetically determined membrane-proximal immunoreceptor tyrosin activation motif(ITAM)results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein(ZAP70)and the levels of cytotoxic effector molecule granzyme B(GZMB),which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line.This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.