B16-F10-luc-G5黑色素瘤细胞生物学特性的研究

目的：探讨B16-F10-luc-G5黑色素瘤细胞的生物学特性.方法：倒置荧光显微镜下连续观察B16-F10-luc-G5细胞生长动态.流式细胞术检测冻存复苏后、培养基漂浮细胞、胰酶消化损伤后的细胞死亡率以评价其对实验损伤的耐受性.1×104/孔B16-F10-luc-G5细胞按1∶2梯度稀释至0.78×102/孔,加入底物荧光素后检测其生物发光特性.Babl/C和C57 bl/6雄性小鼠各10只接种该细胞,目测肿瘤生长速度和活体成像监测Babl/C小鼠移植瘤生长动态以评价其成瘤特性.结果：B16-Fl0-luc-G5细胞生长动态符合典型B16-F10细胞特性,无自发及激发荧光.B16-F10-luc-G5细胞冻存、漂浮细胞及对数生长期细胞消化后死亡率分别为23.8％、35.8％和4.8％.B16-F10-luc-G5细胞数与实测平均光子数之间存在线性回归关系,检测光子数可反映细胞数目.两组小鼠移植成瘤率均为100％,平均肿瘤出现时间差异显著（t =9.05,P＜0.05）,移植瘤病理符合B16黑色素瘤典型生长特点;活体成像监测可灵敏监测移植瘤生长动态.结论：B16-F10-luc-G5细胞具有生长快、对各种实验操作耐受性好、标记生物发光基因易追踪等优点,且其一般特性与B16-F10细胞基本相同,是肿瘤学研究良好的实验材料.     

GnRH/M2融合蛋白致敏DC疫苗对黑色素瘤B16F10细胞移植瘤的抑制

目的:制备促性腺激素释放激素(gonadotropin releasing hormone,GnRH)与M2的融合蛋白(GnRH/M2),研究由该融合蛋白致敏而成的DC疫苗对黑色素瘤B16F10细胞小鼠移植瘤的抑制作用.方法:构建表达载体pET28a-ansB-C-GnRH3-hinge-MVP-M2质粒,该质粒转化的工程菌在乳糖的诱导下,融合蛋白ansB-C-GnRH3-hinge-MVP-M2以包涵体形式表达,经超声破碎、洗涤和乙醇分级沉淀纯化后,通过酸水解将蛋白多肽GnRH3-hinge-MVP-M2释放出来,并通过DEAE-52阴离子交换层析进行分离.将此融合多肽致敏DC获得DC疫苗.构建黑色素瘤B16F10细胞小鼠移植瘤模型,按接种疫苗不同,分为:环磷酰胺组(CTX)、GnRH/M2融合蛋白致敏DC组(GDC)、肿瘤细胞裂解物致敏DC组(BDC)、GnRH/M2融合蛋白致敏DC+环磷酰胺组(GDCTX)、肿瘤细胞裂解物致敏DC+环磷酰胺组(BDCTX)和生理盐水组(NS),观察GnRH/M2疫苗对模型小鼠的移植瘤生长、CTL杀伤能力和T细胞增殖的作用.结果:成功构建pET28a-ansB-C-GnRH3-hinge-MVP-M2质粒并高效表达融合蛋白.GDC组移植瘤生长明显慢于NS组(P＜0.05),且与BDC组相似(P ＞0.05);GDCTX组抑瘤效果虽进一步提高,但与CTX组相比差异无统计学意义(P＞0.05).各实验组对B16F10细胞的杀伤作用和对T细胞增殖作用均优于阴性对照组(P ＜0.05或P＜0.01),且GDC组与BDC组间差异不显著(P＞0.05).结论:初步证明融合多肽GnRH/M2致敏的DC疫苗能有效抑制黑色素瘤B16F10细胞小鼠移植瘤的生长.     

B16黑色素瘤皮内移植瘤模型的建立

背景与目的： 建立B16黑色素瘤细胞(简称B16细胞)皮内移植瘤模型。 材料与方法： 制备商品源B16细胞悬液,按每只C57BL鼠接种0.1 ml (3×105个)B16细胞于后肢外侧皮内;分离、培养移植瘤源B16细胞,倒置显微镜下观察细胞形态。 结果： 移植瘤出现时间为(9.6±1.2)d,致瘤率为100％;皮内注射B16细胞18 d内,肿瘤形态呈圆形或类圆形,边缘清楚;移植瘤源原代B16细胞多为圆形、类圆形或短梭形,商品源B16细胞为梭形或不规则形;HE染色表明移植瘤内有大量B16细胞。 结论： B16细胞皮内移植瘤模型易于对肿瘤生长的观察和测量,是早、中期肿瘤研究的合适模型之一。     

输血对局部B16黑色素瘤生长的影响

用小鼠的输血模型，探讨输血对局部Ｂ１６黑色素瘤生长的影响，结果发现，同种异品系间的输血，可降低血鼠的ＮＫ细胞活性和淋巴细胞转化率，并能促进肿瘤的生长，肿瘤的体积及重量均明显高于输生理盐水组，而同种同品系间的输血则无此不良影响，本研究提示，Ｈ－２不相容性输血可促进肿瘤的生长，免疫功能下降可能是输血促进肿瘤生长的重要因素。     

肝脂素对小鼠S37肉瘤与B16黑色素瘤的抗肿瘤作用

目的：探讨肝脂素（Heplipin)对S37和B16两种肿瘤细胞移植瘤的生长抑制作用。方法：小鼠S37肉瘤和B16黑色素瘤移植瘤在高、中、低剂量（每天分别为3000、1800、1100mg/kg)肝脂素胃饲为治疗组,环磷酰胺（CTX）20mg(kg／天）和生理盐水作为两对照组。比较抑瘤率、癌周边部血管数、总肿瘤面积中坏死区所占比例,结果：肝脂素对S37肉瘤和B16黑色素瘤的抑瘤分别为大剂量：75．1％和47．0％、中剂量：69．7％和32．1％、小剂量：61．7％和13．0％,显示出明显的剂量效关系,与生理盐水对照组相比有显著性差异。在S37肉瘤荷瘤小鼠的实验中,组织学观察发现,肝脂素用药组肿瘤周边部单痊面积血血管数明显少于阴性对照组和CTX组;肝脂素治疗组的肿瘤坏死面积明显大于对照组,结论：肝脂素可明显抑制小鼠S37肉瘤和B16黑色素瘤的生长,其抗肿瘤机制可能与抑制肿瘤生长血管的生成,导肿瘤缺血坏死有关。     

β-榄香烯诱导小鼠黑色素瘤B16细胞凋亡的研究

 目的 研究β-榄香烯（β-E） 诱导小鼠黑色素瘤B16 细胞发生凋亡的作用。方法 应用透射电镜和TdT介导的脱氧核苷酸缺口末端标记（TUNEL） 技术。结果 表明10-40ugmlβ-E作用12-48 小时,可使B16 细胞发生凋亡,且凋亡指数与β-E 的剂量及作用时间有关。结论 诱导肿瘤细胞凋亡的作用是β-E抗癌作用的重要机制之一。     

蛇毒半胱氨酸蛋白酶抑制剂基因转染对小鼠黑色素瘤B16F1细胞基因表达谱的影响

背景与目的:本实验室已证明蛇毒半胱氨酸蛋白酶抑制剂(snake venom cystatin, sv-cystatin)具有抗肿瘤侵袭转移作用.为了进一步阐明其分子机制,本研究利用高通量的基因芯片技术探讨sv-cystatin基因转染对小鼠黑色素瘤细胞(B16F1)基因表达谱的影响.方法:分别将pcrDNA3.1/sv-cystatin及空载体pcDNA3.1转染B16Fl细胞,建立稳定转染的B16Fl/sv-cystatin及B16F1/pcDNA3.1细胞株,提取总mRNA.应用高通量的基因芯片技术检测差异表达基因,半定量RT-PCR法分别验证5个上调和5个下调的差异表达基因.结果:B16F1细胞经转染sv-cystatin后.共检测了1218个基因,其中有45个差异表达基因,21个基因表达上调(Ratio>3),24个基因表达下调(Ratio<0.5),这些基因的功能涉及细胞粘附迁移、细胞免疫调节、增殖分化与凋亡、基因转录和细胞内信号转导等方面.10个差异表达基因的RT-PCR验证结果与基因芯片分析结果相符.结论:Sv-cystatin不仅具有抑制细胞外基质的作用,而且还具有细胞免疫调节、增殖分化与凋亡、基因转录和细胞内信号转导等多种生物学功能.     

IL-2与化疗药物合用对B16黑色素瘤的细胞毒作用

目的研究IL-2与DTIC、DDP不同联合方案对B16黑色素瘤的细胞毒作用,为临床合理应用提供依据.方法采用MTT法检测各实验组的光密度(OD值),计算杀伤率.结果同时加IL-2组及先加IL-2,3小时后加DTIC组对B16抑制率降低,与单药DTIC比较有显著差异(t=3.05,P＜0.05;t=5.51,P＜0.01).同时给DDP和IL-2组或先加IL-2,3小时后再加DDP组对B16抑制率影响不大;与单药DDP比较没有显著差异(t=0.43,P＞0.05;t=0.49,P＞0.05).先加DTIC,3小时后再加IL-2组,与单药DTIC对B16的杀伤率比有所下降,但没有统计学显著差异(t=1.65,P＞0.05).先加DDP,3小时后再加IL-2组与单药DDP对B16的细胞毒作用相比,抑制率有显著提高(t=3.99,P＜0.05).结论IL-2能提高DDP的细胞毒作用,且应在先使用化疗药物的基础上给药.     

小鼠黑色素瘤转移相关蛋白的差异蛋白质组学分析*

目的:分析B16-F10黑色素移植瘤及其肺转移瘤的差异表达蛋白,以筛选黑色素瘤转移相关的分子标志。方法:应用荧光差异凝胶电泳（two-dimensional differential gel electrophoresis ,2D-DIGE）结合基质辅助激光解析电离飞行时间质谱技术（matrix assisted laser desorption ionisation time-of-flight mass spectrometry ,MALDI-TOF-MS）分离鉴定B16-F10黑色素移植瘤及其肺转移瘤的差异表达蛋白,部分差异蛋白经Real-time PCR进行mRNA 表达水平验证。结果:Decyder6.0 软件分析结果显示2D-DIGE图谱分辨率高、重复性好,30个蛋白点在实验组和对照组间存在表达差异（|Ratio| ≥2,P<0.01）,经质谱分析和数据库查询鉴定出9个蛋白在实验组表达上调,包括肌红蛋白（myoglobin,MB）、波形蛋白（vimentin,VIM）、磷酸甘油激酶1（phosphoglycerate kinase 1,PGK 1）、磷酸丙糖异构酶（Triosephosphate isomerase ,TPI 或TIM）、重链结合蛋白（heavy-chain binding protein,BiP）、α- 烯醇化酶（α-enolase 或enolase 1）、β-肌动蛋白（β-actin）、γ-肌动蛋白（γ-actin）、层连蛋白结合蛋白（laminin-binding protein ）,这些蛋白主要参与了细胞骨架构成、糖酵解等生物学过程。Real-time PCR结果显示糖酵解酶PGK 1 及TPI mRNA 表达水平在实验组显著高于对照组（P=0.001,0.003）,变化趋势与蛋白质组学相一致。结论:小鼠黑色素瘤转移过程与多种蛋白的异常表达有关,糖酵解酶PGK 1、TPI 可能参与了黑色素瘤的转移过程。      

表达Angiostatin基因的B16黑色素瘤体内抑制作用

目的:研究表达Angiostatin基因的B16黑色素瘤的体内生长行为.方法:通过改造Plasminogen cDNA得到Angiostatin基因,构建成真核表达载体pAGAGS3后导入B16黑色素瘤细胞使其表达.结果:表达Angiostatin的B16黑色素瘤细胞体外生长速率和软琼脂中克隆形成能力均未改变.但接种小鼠26d后,体内生长抑制率达87%(P<0.01).结论:表达Angiostatin的B16黑色素瘤体内生长受到显著抑制.     

Suramin augments the antitumor and antimetastatic activity of pentoxifylline in B16F10 melanoma

Rapid tumor growth and metastasis are 2 major problems associated with treatment of malignant melanoma. Therefore, drugs that can intervene these processes are of clinical importance. Pentoxifylline (PTX), a methyl xanthine derivative, has been shown to inhibit B16F10 melanoma tumor growth and metastasis. We hypothesized that suramin when combined with PTX enhances its antineoplastic effects, which we have examined using the B16F10 mouse melanoma model. Suramin in simultaneous or sequential combination potentiated the cytotoxic effects of PTX on B16F10 cells. PTX arrested cells in the G0-G1 phase and suramin augmented the effects. Both the drugs inhibited F10 adhesion to laminin, matrigel and collagen type IV and showed enhanced inhibition in combination The combination also demonstrated significantly higher inhibition in cell motility (p = 0.002) and invasion through matrigel (p = 0.005) as compared to the single agents. Suramin synergized with PTX in its effects on secretion of MMP-9 gelatinase. DBA2/J mice implanted with intradermal B16F10 tumor were used as a model to study tumor growth. Animals were intratumorally treated with 50 mg/kg of PTX, 10 mg/kg of suramin and their combinations. Simultaneous administration of the drugs inhibited tumor growth by 5- to 6-folds. Tumor growth was completely blocked in sequential regimen with regression in some cases. The number and size of metastatic nodules on lung was also reduced significantly by the combination treatment. In conclusion, the novel combination of PTX and suramin has synergistic antitumor and antimetastatic activity in B16F10 melanoma and may be a promising approach in treatment of patients suffering from malignant melanoma.     

Multiple human chromosomes carrying tumor-suppressor functions for the mouse melanoma cell line B16-F10, identified by microcell-mediated chromosome transfer

Many tumor-suppressor genes are involved in the development and progression of cellular malignancy. To understand the functional role of tumor-suppressor genes in melanoma and to identify the human chromosome that carries these genes, we transferred individually each normal human chromosome, except for the Y chromosome, into the mouse melanoma cell line B16-F10, by microcell fusion. We examined the tumorigenicity of hybrid cells in nude mice and their in vitro growth properties. The introduction of human chromosomes 1 and 2 elicited a remarkable change in cell morphologic features, and cellular senescence was induced at seven to 10 population doublings. The growth rates of tumors derived from microcell hybrid clones containing introduced human chromosome 5, 7, 9, 10, 11, 13, 14, 15, 16, 19, 20, 21, 22, or X were significantly slower than that of the parental B16-F10 cells, whereas the introduction of other human chromosomes had no effect on the tumorigenicity of these cells. The majority of microcell hybrid clones that exhibited suppressed tumorigenicity also showed a moderate reduction in doubling time compared with B16-F10 cells. Microcell hybrid clones with an introduced human chromosome 5 showed complete suppression of in vitro-transformed phenotypes, including cell growth, saturation density, and colony-forming efficiency in soft agar. Thus, these results indicated the presence of many cell senescence-related genes and putative tumor-suppressor genes for the mouse melanoma cell line B16-F10 and showed in vitro that many tumor-suppressor genes control the phenotypes of transformed cells in the multistep process of neoplastic development.     

Pentoxifylline impedes migration in B16F10 melanoma by modulating Rho GTPase activity and actin organisation

Cancer cell migration is a hallmark of metastatic cascade and compounds that can intervene in this process are clinically important. Pentoxifylline (PTX), a methyl xanthine derivative, inhibits B16F10 melanoma lung homing by inhibiting F10 invasion, MMP secretion and adhesion to matrix components. However, its effect on B16F10 migration remained unexamined, which we investigated in the present study. PTX significantly inhibits F10 migration in scratch wound assay. Elevation in cAMP levels inhibits F10 migration and PTX mediated inhibition of the process was found to be, in part, due to an increase in cellular cAMP levels. PTX induces Protein Kinase A (PKA) activity and PKA inhibitor partly reversed its effects on F10 motility. RhoA and Rac1 GTPases induce B16F10 motility and PTX was found to inhibit migration by affecting these molecules. Stress fibres and lamellipodial protrusions reduced significantly. This was accompanied with inhibition in RhoA and Rac1 membrane localisation. A stark inhibition in RhoA-GTP bound form was also observed. Taken together, the results indicate that PTX, through its phosphodiesterase action, inhibits RhoGTPases and associated actin organisation in B16F10 melanoma, thereby inhibiting cell motility.     

Nitric oxide-mediated regulation of hypoxia-induced B16F10 melanoma metastasis

Tumour hypoxia is associated with resistance to therapy and with increased invasion and metastatic potential. Recent studies in our laboratory have shown that the hypoxic up-regulation of tumour cell invasiveness and chemoresistance is in part due to reduced nitric oxide (NO) signaling. Using B16F10 murine melanoma cells, we demonstrate here that the increased metastatic potential associated with exposure to hypoxia is mediated by a reduction in cGMP-dependent NO-signaling. Pre-incubation of B16F10 cells in hypoxia (1% vs. 20% O(2)) for 12 hr increased lung colonization ability by over 4-fold. This effect of hypoxia on metastasis was inhibited by co-incubation with low concentrations of the NO-mimetic drugs glyceryl trinitrate (GTN) and diethylenetriamine NO adduct (DETA/NO). In a manner similar to hypoxia, pharmacological inhibition of NO synthesis resulted in a significant increase in lung nodule formation, an effect that was prevented by co-incubation with GTN. An important NO-signaling pathway involves the activation of soluble guanylyl cyclase and the consequential generation of cGMP. Culture in the presence of a non-hydrolysable cGMP analogue (8-Br-cGMP) abrogated the hypoxia-induced lung nodule formation, suggesting that the effects of NO on metastasis are mediated via a cGMP-dependent pathway. These findings suggest that a novel mechanism whereby hypoxia regulates metastatic potential involves a downstream inhibition of cGMP-dependent NO signaling.     

Antiproliferative and Antiproteolytic activity of Pentoxifylline in cultures of B16F10 Melanoma cells

Purpose: Pentoxifylline (PTX), a methyl xanthine derivative is widely used as a haemorheological agent in the treatment of peripheral vascular disease. In the present study, we investigated the in vitro effects of PTX on B16F10 melanoma cell proliferation, adhesion and secretion of Matrix metalloproteinases. Methods: The toxic range of PTX was evaluated using MTT test and colony formation assay. The cell cycle study of PTX treated cells was carried out using flow cytometric analysis. Adhesion assay of pretreated melanoma cells was carried out on extracellular matrix (ECM) substrates. The relative levels and activity of matrix metalloprotienase-9 (MMP-9) and MMP-2 were determined by gelatin zymography and western blotting. Results: Pentoxifylline significantly inhibited the in vitro proliferation of B16F10 cells in a concentration dependent manner and displayed an IC₅₀ of 15.2 mM. Non-cytotoxic concentration of 1–3 mM of PTX for an exposure of 24 h demonstrated significant changes in cell morphology. A significant inhibition in G1-S phase transition was observed on PTX treatment. Pretreated F10 cells showed inhibition in adhesion to ECM components and markedly inhibited the secretion of MMP-9 and MMP-2 gelatinases. Conclusion: The results suggest that PTX even at non-toxic pharmacological concentrations acts as an effective antiproliferative agent with significant antiproteolytic and antiadhesive effects.     

Altered characteristics of B16 melanoma cells induced by chemically crosslinking fibronectin to cell surfaces

The loss of fibronectin from tumor cell surfaces has been correlated with an increased incidence of metastases. To determine directly whether cell surface fibronectin influences the metastatic potential of solid tumors, we chemically crosslinked fibronectin to B16 murine melanoma cells using a photosensitive heterobifunctional crosslinking reagent, N-succinimidyl-4-azidophenyl-1,3 dithiopropionate (SADP). Cell attachment to plastic surfaces was increased in cells to which fibronectin was attached; cell growth over a 24-hr period was not significantly affected by the addition of fibronectin. When C57BL/6 mice were injected with fibronectin-crosslinked B16 cells, there was a 63% reduction in the number of pulmonary nodules compared to untreated controls. These results are consistent with the hypothesis that fibronectin enhances the recognition and removal of tumor cells from the circulation, possibly by cells of the reticuloendothelial system.     

三肽化合物酪丝缬肽对小鼠黑色素瘤细胞B16-F10侵袭和转移的抑制作用

背景与目的:三肽化合物酪丝缬肽(tyroservaltide,YSV)对实验性肝癌具有明显抑制作用,本研究观察YSV对小鼠黑色素瘤细胞B16-F10侵袭和转移的抑制作用。方法:MTT法检测YSV对B16-F10的细胞毒作用;利用基质胶Matrigel研究YSV对细胞粘附能力的影响;用Transwell小室侵袭模型研究YSV对肿瘤细胞侵袭能力的改变;经C57/BL6小鼠尾静脉注射B16-F10细胞,建立人工肺转移模型,观察YSV对B16-F10肺转移的影响;免疫组化方法观察YSV对细胞间粘附分子(intercellularadhesionmolecule-1,ICAM-1)在肺组织中表达水平的影响。结果:100μg/mlYSV作用48h对B16-F10细胞的增殖抑制率为24.36%;作用24h对B16-F10细胞在Matrigel胶上的粘附抑制率为36.51%;10μg/mlYSV作用48h对B16-F10细胞的侵袭抑制率为36.53%;640μg·(kg·d)-1YSV抑制B16-F10的肺转移,抑制率为62.21%;YSV组ICAM-1的组织量明显少于生理盐水组。结论:YSV具有抑制B16-F10生长和侵袭转移的作用。     

Paclitaxel Combined with Ticagrelor Inhibits B16F10 and Lewis Lung Carcinoma Cell Metastasis

It is widely accepted that tumor metastasis is the dominant factor leading to cancer-related death. Tumor metastasis is mediated by cell invasion, blood circulation and lymphatic circulation. Paclitaxel, as a common anti-tumor drug and a mitotic inhibitor, promotes microtubule assembly and inhibits microtubule depolymerization. In addition, ticagrelor, an anti-platelet drug, is used to treat acute coronary syndrome. Increasing numbers of studies have reported that platelets can facilitate tumor metastasis. Therefore, inhibiting the effects of platelets can serve as a novel therapeutic strategy for cancer. To explore the effect of anti-tumor and anti-platelet drugs on tumor progression, we chose paclitaxel and ticagrelor. Interestingly, the results demonstrated that paclitaxel and ticagrelor could not only suppress the proliferation, migration and invasion of B16F10 and LLC cells, but they could also prevent tumor metastasis to the lungs. Furthermore, the inhibitory effect of paclitaxel and ticagrelor was more apparent when both drugs were used in combination. Collectively, the current study demonstrated that the combination of paclitaxel and ticagrelor could be considered as a potential anti-tumor therapy approach.     

Comparative protein profiling of B16 mouse melanoma cells susceptible and non-susceptible to alphavirus infection: Effect of the tumor microenvironment

Alphavirus vectors are promising tools for cancer treatment. However, relevant entry mechanisms and interactions with host cells are still not clearly understood. The first step toward a more effective therapy is the identification of novel intracellular alterations that could be associated with cancer aggressiveness and could affect the therapeutic potential of these vectors. In this study, we observed that alphaviruses efficiently infected B16 mouse melanoma tumors/tumor cells in vivo, whereas their transduction efficiency in B16 cells under in vitro conditions was blocked. Therefore, we further aimed to understand the mechanisms pertaining to the differential transduction efficacy of alphaviruses in B16 tumor cells under varying growth conditions. We hypothesized that the tumor microenvironment might alter gene expression in B16 cells, leading to an up-regulation of the expression of virus-binding receptors or factors associated with virus entry and replication. To test our hypothesis, we performed a proteomics analysis of B16 cells cultured in vitro and of B16 cells isolated from tumors, and we identified 277 differentially regulated proteins. A further in-depth analysis to identify the biological and molecular functions of the detected proteins revealed a set of candidate genes that could affect virus infectivity. Importantly, we observed a decrease in the expression of interferon α (IFN-α) in tumor-isolated cells that resulted in the suppression of several IFN-regulated genes, thereby abrogating host cell antiviral defense. Additionally, differences in the expression of genes that regulate cytoskeletal organization caused significant alterations in cell membrane elasticity. Taken together, our findings demonstrated favorable intracellular conditions for alphavirus transduction/replication that occurred during tumor transformation. These results pave the way for optimizing the development of strategies for the application of alphaviral vectors as a potent cancer therapy.