慢性粒细胞白血病相关抗原HLA-A2.1限制性CTL表位的生物信息学分析

目的:预测慢性粒细胞白血病BCR/ABL融合基因HLA-A2.1限制性细胞毒性T细胞表位.方法:运用BIMAS、SYFPEITHI、Predep、Immune Epitope Database和Analysis Resource(IEDB)软件预测BCR/ABL融合基因可能的HLA-A2.1限制性CTL表位.结果:结合BIMAS、SYFPEITHI、Predep和IEDB预测BCR/ABL融合基因的HLA-A2.1限制性CTL表位,共6条.结论:综合运用多个预测软件可提高预测效率获得目的表位肽,为下一步基础实验打下基础.     

千里光碱脂质体对黑色素瘤B16细胞周期和超微结构的影响

目的观察千里光碱脂质体对黑色素瘤B16细胞周期和超微结构的影响。方法采用流式细胞仪和电子显微镜观察黑色素瘤B16细胞周期和超微结构的变化。结果千里光碱可以阻滞细胞周期,抑制肿瘤细胞DNA合成,促进细胞凋亡,改善黑色素瘤细胞的超微结构。结论千里光碱可能是通过阻滞细胞周期,促进细胞凋亡来改变肿瘤细胞超微结构,而发挥抑制肿瘤的作用。     

小鼠黑色素瘤B16F10-Red细胞株的建立与生物学特性分析

目的:构建表达香菇珊瑚红色荧光蛋白(discosomasp red fluorescent protein,DsRed)的小鼠黑色素瘤B16F10-Red细胞株,并检测其生物学特性.方法:用GenEscortTMⅡ转染试剂将pDsRed质粒导入小鼠黑色素瘤B16F10细胞,G418加压培养联合极限稀释法建立稳定、高水平表达DsRed的单克隆细胞系.FCM检测B16F10和B16F10-Red细胞的细胞周期.比较B16F10-Red和B16F10细胞的克隆球形成能力和小鼠体内致瘤能力.结果:稳定表达DsRed的小鼠黑色素瘤B16F10-Red细胞株基本保持了其亲代细胞的特征,能在C57BL/6小鼠腹部皮下形成肿瘤并继续生长和转移.结论:B16F10-Red细胞株构建成功,其移植瘤模型成瘤率和转移情况同B16F10肿瘤相比无明显差别.     

原癌基因Bmi-1 B细胞抗原表位及 HLA I类限制性CTL表位的生物信息学分析

目的:预测原癌基因Bmi-1(B-cell-specific Moloney murine leukemia virus insertion site 1)的B细胞抗原表位及HLA I类限制性细胞毒性T细胞（cytotoxic T cell）表位。方法:采用Ellipro(http://tools.immuneepitope.org/ellipro/)预测Bmi-1蛋白中可能存在的线性B细胞表位和构象B细胞表位；采用ProtParam、NetCTL等软件和方法预测Bmi-1中可能存在的HLA I类限制性CTL表位。结果:Bmi-1蛋白中含有6个线性B细胞表位和8个构象B细胞表位；结合HLA结合力、蛋白酶体切割效率和TAP转运效率，经过NetCTL程序预测表明:针对不同的HLA I类分子，原癌基因Bmi-1中都存在多个HLA I类分子的限制性CTL表位。结论:综合运用多个程序预测了原癌基因Bmi-1中的B细胞抗原表位及HLA I类分子的限制性CTL表位，为下一步开展针对Bmi-1的免疫靶向治疗等研究打下了基础。     

mGM-CSF转导的小鼠黑色素瘤细胞融合巨噬细胞的抗肿瘤作用研究

目的 比较小鼠黑色素瘤细胞B16致瘤性与转导mGM-CSF基因的小鼠黑色素瘤细胞B16致瘤性的差别,同时研究转导该基因阳性的B16肿瘤细胞与同源小鼠巨噬细胞融合后其致瘤性变化,初步探讨巨噬细胞在肿瘤生成中的作用.方法 本实验以FuGENE6介导,将融合基因Lxpsp-mGM-CSF转导逆转录病毒包装细胞PA317,以含...     

苦参碱衍生物M19对于肿瘤细胞转移的抑制作用

目的:探讨苦参碱衍生物M19对于肝细胞癌和黑色素瘤细胞转移的抑制作用。方法:肝癌细胞Hep3B、MHCC-LM3和黑色素瘤细胞B16F10经M19处理后,检测肿瘤细胞体外迁移能力。建立C57BL/6小鼠黑色素瘤B16F10和裸鼠肝细胞癌MHCC-LM3转移模型,经M19体内给药后,观察肿瘤细胞肝、肺转移情况。结果:M19能浓度依赖性地抑制肝癌细胞和黑色素瘤细胞迁移。M19体内给药使B16F10细胞在小鼠肺的转移减少,且还能抑制MHCC-LM3细胞在裸鼠肺和肝的转移。结论:M19能在体内外抑制肝细胞癌和黑色素瘤细胞的转移。     

敲除PIP5K1α抑制黑色素瘤细胞B16F10增殖、迁移及侵袭的机制探讨

目的:探讨敲除PIP5K1α对黑色素瘤细胞B16F10增殖、迁移和侵袭的作用及机制。方法:采用CRISPR-Cas9技术在B16F10细胞中敲除PIP5K1α;采用CCK-8法检测细胞的增殖活性;Wound healing检测细胞迁移;Transwell检测细胞的侵袭能力;Elisa实验检测PIP2的合成;Western blot检测细胞蛋白表达变化。结果:CCK-8实验表明:敲除PIP5K1α可明显抑制B16F10细胞的增殖。敲除PIP5K1α明显抑制了B16F10细胞的迁移和侵袭能力。在B16F10细胞中,敲除PIP5K1α后,总AKT的蛋白表达不变,p-AKT、Cyclin A2及Cyclin D1的蛋白表达下降。结论:PIP5K1α可调节黑色素瘤B16F10细胞中p-AKT、Cyclin A2和Cyclin D1蛋白的表达,敲除PIP5K1α表达可抑制细胞增殖、迁移和侵袭能力。     

没食子酸诱导ROS积蓄介导黑色素瘤B16-F10细胞内源性凋亡及周期阻滞北大核心

目的:探究没食子酸诱导活性氧(reactive oxygen species,ROS)积蓄介导黑色素瘤B16-F10细胞凋亡及周期阻滞的作用机制。方法:以梯度浓度的没食子酸作用于黑色素瘤B16-F10细胞,采用MTT法检测没食子酸对细胞生长的影响,平板克隆技术检测细胞克隆形成率,Transwell实验检测细胞迁移及侵袭能力,ROS Assay Kit检测B16-F10细胞内ROS水平,线粒体膜电位检测试剂盒检测细胞膜电位的变化,Hoechst 33258荧光染色法进行细胞形态学检测,流式细胞术检测细胞凋亡及细胞周期阻滞水平,Western blot检测细胞内相关蛋白水平的变化。结果:结果显示,没食子酸明显抑制B16-F10细胞的生长,且具有浓度依赖性。没食子酸作用后,B16-F10细胞增殖、迁移及侵袭能力明显下降,细胞内ROS水平明显升高,线粒体膜电位明显下降;细胞数减少,细胞凋亡率增加,G_(0)/G_(1)期细胞数明显增多;细胞内凋亡相关蛋白Bax、Cytochrome C、Caspase-9以及Caspase-3表达量增加,而Bcl-2表达量减少;周期相关蛋白Chk2、p53、p21表达量明显增加,CyclinE1、CDK2表达量减少。结论:没食子酸作用于黑色素瘤B16-F10细胞后,通过诱导ROS积蓄,引起B16-F10细胞内源性凋亡及周期阻滞。     

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.     

B7-1基因转染小鼠EL-4淋巴瘤诱导带瘤宿主抗瘤效应的体内研究

本文研究了IL-2、IL-4、IL-6基因转染后B16黑色素瘤细胞表面MHCⅠ类抗原及ICAM-1的表达水平,并探讨了其在CTL诱导过程中的作用.结果表明,IL-2、IL-4、IL-6基因转染的B16黑色素瘤细胞表面MHCⅠ类抗原及ICAM-1表达均高于野生型B16黑色素瘤细胞及转染对照质粒的B16黑色素瘤细胞.体内接种后,小鼠脾脏CTL活性明显增强,CTL培养体系中IFN-γ、TNF-α的分泌水平也增高.在CTL诱导体系中加入抗ICAM-1单抗可以抑制CTL的活化,加入抗MHCⅠ类分子单抗后可使CTL的活化完全阻断.这提示细胞因子基因转染可能通过使肿瘤细胞表面MHCⅠ类抗原、ICAM-1的表达增加,从而增强瘤苗的免疫原性.     

Opposite effects of TPA on G1/S transition and on cell size in the low metastatic B16F1 with respect to high metastatic BL6 murine melanoma cells.

Phorbol esters, known activators of c- and n-protein kinase C (PKC) isoforms, play a pivotal role in tumor promotion. In order to better understand the relationships between PKC activation, the metastatic potential and the proliferative capacity, we have analyzed the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment on the proliferative as well as on the cell cycle distribution and on the cell size of low and high metastatic murine B16F1 and B16-BL6 (BL6) melanoma cells, respectively. TPA-treated B16F1 cells showed an increased proliferative capacity up to 72 h, the cytofluorimetric analysis revealing an increased number of cells in the S + G2-M phase of the cell cycle. In contrast, TPA-treated BL6 cells reached a plateau at 48 h and showed an increased cell volume in the G1 and S phases of the cell cycle, with a reduction in the percentage of cells in the S + G2-M phase. Taken together, our results indicate opposite effects of TPA treatment in murine melanoma cells of different metastatic potential. TPA could cause a block in the G1 phase of the cell cycle with enhanced cell volume in high metastatic BL6 cells. The same treatment, on the contrary, induced an increased entry into the cell cycle of low metastatic B16F1 cells, suggesting a relationship between cell proliferation and the metastatic potential of B16 murine melanoma cells. Moreover, under the present conditions, classical PKC isoforms were inactivated, suggesting the involvement of the TPA-dependent novel PKCs.     

Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors

Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8(+) T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8(+) T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4(+) T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)-derived 8 mer peptide, OVA(257-264), and 16mer peptide, OVA(265-280), were used as CD8(+) and CD4(+) T cell epitopes, respectively. Vaccination with hsc70-OVA(257-264) generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA(265-280) to the amino-terminus of hsc70-OVA(257-264) (OVA(265-280)-hsc70-OVA(257-264)) enhanced the generation of the OVA(257-264)-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA(257-264). Our results suggest that fusion of both CD4(+) and CD8(+) T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8(+) T cell epitope alone.     

基因枪介导的neu基因DNA疫苗抗肿瘤作用的实验研究

目的 探讨DNA疫苗pWRG-neu的皮内免疫,对高表达neu基因的小鼠移植瘤生长和转移的抑制作用。方法 向小鼠黑色素瘤B16F10细胞系转染pcDNA-neu,用有限稀释法筛选一株高表达neu基因的细胞株B16F10-neu。在基因枪介导下,向C57BL／6小鼠导入DNA疫苗pWRG-neu,通过观察免疫动物的生存期,评价DNA疫苗的抗肿瘤作用。分离免疫动物脾细胞,经自体淋巴细胞混合培养实验,分析DNA疫苗体内免疫后机体的CTL应答。结果 筛选到一株高表达neu基因的B16F10-neu细胞株,转基因过程和外源基因的表达没有改变细胞系的增殖特性。用基因枪轰击,进行DNA疫苗pWRG-neu皮内免疫,对小鼠黑色素瘤B16F10-neu进行预防、治疗和抗转移的实验研究,结果表明,DNA疫苗的免疫能够明显推迟移植瘤的生长,延长小鼠生存期,获得明显的抗肿瘤效果。DNA疫苗免疫后可诱导小鼠脾淋巴细胞CTL活性。结论 基因枪介导的DNA疫苗pWRG-neu经皮内免疫,能够有效的诱导机体的细胞免疫应答,预防和治疗小鼠移植瘤的发生,并有一定的预防肿瘤肺转移的作用。     

Migration and metalloproteinases determine the invasive potential of mouse melanoma cells,but not melanin and telomerase

The biological characteristic cell locomotion and invasion, melanin content, metalloproteinases and telomerase activity were studied in a parental mouse melanoma cell line B16 and two descendents B16BL6 and B16F10. The invasive potential of melanoma cells was assayed in a transwell cell culture chamber. Melanin content was determined by the absorbance value at 470 nm per 10⁶ cells. Tumor cells migration within the 3-D collagen matrix was microscopically recorded with a time-lapse video recorder and analyzed by computer-assisted cell tracking. Gelatin zymography was adopted to assay the metalloproteinases secretion. A polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) was used for measuring telomerase activity. The results demonstrated that B16BL6 and B16F10 cells were highly invasive compared to B16 cells, but the melanin content of B16F10 was very low. B16F10 and B16BL6 were hypermotile and secreted much more metalloproteinases than B16. No differences were observed in telomerase activity among the three melanoma cell lines. Invasion of mouse melanoma was closely correlated to tumor cell migration and secretion of metalloproteinases. Melanin content and telomerase activity were phenotypically not related to invasiveness in these three mouse melanoma cell lines.     

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.     

Transcutaneous immunization in mice: induction of T-helper and cytotoxic T lymphocyte responses and protection against human papillomavirus-induced tumors

Previous reports have shown that transcutaneous immunization (TCI) with proteins or peptides in combination with adjuvants efficiently induces specific cellular and humoral immune responses. However, depending on the kind of skin pretreatment, induction of cellular immune responses was restricted to generation of either specific cytotoxic T lymphocytes (CTLs) or T-helper (Th) cells. In this study, we induced antigen-specific CTL responses together with the appropriate Th responses by TCI of C57BL/6 mice. We applied ovalbumin protein or an ovalbumin-derived fusion peptide containing a CTL and Th epitope together with a combination of cholera toxin (CT) and CpG oligodeoxynucleotide (CpG) onto cold wax-depilated and hydrated bare skin. TCI with the ovalbumin fusion peptide induced more robust CTL and Th responses than that with ovalbumin protein. The fusion peptide in combination with the nontoxic CT derivative CTA1-D2D1 and CpG induced an antigen-specific CTL response, albeit less efficiently than in combination with complete CT. Further, we compared the potency of HPV-16 E7 oncoprotein-derived peptides containing single (CTL) or multiple (CTL + Th + B cell) epitopes to induce effective CTL responses. Strong E7-specific CTL responses were detected only after TCI with the E7 multiepitope peptide. This peptide was also shown to protect mice against tumor growth after challenge with HPV-16 E7-positive tumor cells. TCI with E7 protein and CT/CpG led to formation of an E7-specific humoral immune response.     

Guanosine Potentiates the Antiproliferative Effect of Cytosine-β-D-Arabinofuranoside in Melanoma Cell Lines

Guanosine is shown to potentiate markedly the antiproliferative effect of cytosine-β-D-arabinoside (ara-C) on B16 F10 mouse and SKMEL-28 human melanoma cell lines. Several metabolic consequences of the synergistic interaction between ara-C and guanosine on cell growth were determined in BI6 F10 mouse melanoma cells. Treatment of the cells with guanosine for 24hr resulted in an increase in the percentage of cells in the S phase of the cell cycle, a threefold increase in intracellular GTP concentration, and an increase in the incorporation of ara-C into acid-insoluble material and phosphorylated metabolites. These findings suggest that guanosine potentiates the growth-inhibitory effect of ara-C in B16 F10 melanoma cells by increasing the intracellular concentration of its active metabolites.