黑色素瘤B16细胞热休克蛋白—抗原肽复合物的体内外抑？…

目的：黑色素瘤Ｂ１６细胞胞浆ＨＳＰ－抗原肽复合物（ＨＡＣ）的制备、免疫原性的诱导及其抑瘤作用的研究。方法：采用Ｔｒｉｓ－ＨＣＩ提取和Ｓｅｐｈａｃｒｙｌ　Ｓ－２００凝胶过滤制备Ｂ１６细胞ＨＡＣ,通过Ｃ５７ＢＬ／６Ｊ小鼠体内诱导特异性ＣＴＬ,再经体内、体外实验检测其抑癌作用。结果：凝胶过滤获得的含６０～９７ｋＤ蛋白的４１,４７和５３管的ＨＡＣ可以降低肿瘤发生率、延长肿瘤出现时间及降低小鼠死亡率。结论：Ｂ１６细胞胞浆中的６０～９７ｋＤ的ＨＡＣ具有免疫原性及抑瘤作用,为制备肿瘤疫苗提供了实验依据。     

黑色素瘤B16细胞热休克蛋白 抗原肽复合物的体内外抑瘤效应 *

黑色素瘤B16细胞胞浆HSP-抗原肽复合物（HAC）的制备、免疫原性的诱导及其抑瘤作用的研究。方法:采用Tris-HCl提取和Sephacryl S-200凝胶过滤制备B16细胞HAC,通过C57BL/6J小鼠体内诱导特异性CTL,再经体内、体外实验检测其抑瘤作用。结果:凝胶过滤获得的含60～97 kD蛋白的41,47和53管的HAC可以降低肿瘤发生率、延长肿瘤出现时间及降低小鼠死亡率。结论: B16 细胞胞浆中的60～97 kD的HAC具有免疫原性及抑瘤作用,为制备肿瘤疫苗提供了实验依据。     

TNF-α诱导小鼠B16黑色素瘤细胞凋亡及其对小鼠B16细胞移植瘤生长的抑制作用

目的:探讨TNF-α对小鼠B16黑色素瘤(MM)细胞凋亡的诱导及其对小鼠B16移植瘤生长的抑制作用.方法:用不同浓度的TNF-α直接作用于培养的小鼠B16黑色素瘤细胞,36h后,采用流式细胞仪(FCM)及原位末端标记法(TUNEL法)检测B16细胞的凋亡率.动物实验观察TNF-α小量瘤体内注射对小鼠B16移植瘤生长的抑制作用.结果:在1000、3000、5000及10000U/mL TNF-α作用下,B16细胞的凋亡指数均明显高于空白对照组(P＜0.01),随着TNF-α浓度增加,B16凋亡细胞数呈增加趋势.动物实验结果显示,TNF-α治疗3周后,治疗组荷瘤小鼠MM移植瘤的体积和重量明显低于对照组(P＜0.01).结论:TNF-α能够诱导小鼠B16黑色素瘤细胞发生凋亡,且小剂量TNF-α瘤体内注射能显著抑制小鼠移植性MM的生长.     

Raw264.7细胞蛋白和PD-L1抗体在小鼠黑色素瘤(B16)中的抑瘤效果评价

目的探讨小鼠来源白血病毒诱导的肿瘤细胞(Raw264.7)蛋白和程序性死亡-配体(PD-L1)在C57BL/6小鼠黑色素瘤(B16)中的抑瘤效果。方法采用6~8周龄清洁级C57BL/6小鼠30只(雌雄各半),平均分为3组。各组小鼠分别于皮下接种B16细胞(2×10⁵个/只),待小鼠肿瘤平均体积达到100 mm³时,给予治疗。group A:腹腔注射Raw264.7细胞蛋白(50μg/只),每周1次,共2次。group B:腹腔注射PD-L1抗体(600μg/只,本课题组生产纯化),每3天1次,共4次。group C:相同时间点给予等体积生理盐水对照。各组小鼠给药后,测量肿瘤大小,末次测量后,各组小鼠无菌取脾脏,应用流式细胞术分析CD3/4/8⁺T细胞群比例。结果各组荷瘤小鼠给予相应治疗后,组C小鼠肿瘤体积增长迅速,末次测量平均体积达到4004 mm³;组B小鼠增长次之,末次平均体积达到1637 mm³;组A小鼠体积增长最慢,末次平均体积仅为882 mm³(P<0.01)。对小鼠脾脏免疫细胞分析显示,组B小鼠中CD3⁺CD8⁺T cell比例明显上调,平均达到77.65%,而CD3⁺CD4⁺T比例明显下调,仅为11.86%(P<0.01);而组A和组C两组小鼠CD3⁺CD4⁺T和CD3⁺CD8⁺T没有明显区别(P>0.05)。结论制备的RAW264.7细胞灭活蛋白和PD-L1抗体对于B16荷瘤小鼠均有抑瘤作用,且前者效果更为明显。     

抑瘤素M联合达卡巴嗪抑制黑色素瘤细胞B16的增殖

  目的  通过体内外实验探讨抑瘤素M（OSM）联合达卡巴嗪（DTIC）对小鼠黑色素瘤细胞B16的抑制作用。  方法  在体外分别采用MTT法和FCM法检测达卡巴嗪单药以及联合OSM对黑色素瘤B16细胞增殖和凋亡的影响;Hochest染色法检测达卡巴嗪单药以及联合OSM对黑色素瘤B16细胞的细胞核形态学变化,研究OSM联合DTIC对黑色素瘤B16细胞的抑制作用;体内实验将黑色素瘤细胞B16接种于小鼠,观察OSM、DTIC及DTIC联合OSM治疗对小鼠的成瘤性的影响。  结果  体外实验中OSM、DTIC或DTIC联合OSM对黑色素瘤B16细胞的增殖抑制率分别为11.2±2.3%,25.3±4.6%和32.5±3.8%,差异具有统计学意义（P < 0.05）,诱导黑色素瘤B16细胞的凋亡率分别为1.32±0.42%,10.64±2.13%和15.86±2.76%,差异具有统计学意义（P < 0.05）。在形态学上,DTIC联合OSM可明显引起细胞核破碎增加;在体内实验中,DTIC相对于对照组能明显抑制小鼠黑色素瘤的生长,DTIC联合OSM能增加DTIC的抑瘤作用。  结论  OSM联合DTIC体外可以明显抑制黑色素瘤B16细胞增殖,诱导其凋亡,为黑色素瘤的治疗提供了一种新的可能性方案。      

苯乙酸钠对小鼠B16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。     

乙双吗啉对B16黑色素瘤细胞作用的形态学研究

乙双吗啉(Bimolane,AT-1727)是我所研制的新药。临床上用于治疗银屑病和眼葡萄膜炎。动物实验观察到它有抑制肿瘤生长的作用。临床上也已试用于治疗恶性肿瘤。但其作用机理还不太清楚。本文应用显微缩时电影和电子显微镜观察此药对体外培养的B_(16)黑色素瘤细胞的作用。方法和材料乙双吗啉由本所合成室提供,白色粉剂,用少量DMSO溶解,无菌生理盐水稀释,置4℃冰箱避光保存。 B_(16)黑色素瘤细胞由日本引进原代瘤源,经体内外交叉传代10次以上获肺100％高转移株,以1×10~5/ml细胞数接种于玻璃培养瓶中,含10％小牛血清,100 IU/ml青霉素,100μg/ml链霉素的RPMI-1640     

MAGE-1与人HSP70融合基因真核表达载体的构建及在小鼠黑色素瘤细胞内的表达

目的:构建MAGE-1(melanomaantigen1)与人HSP70(heatshockprotein70)融合基因的真核表达载体PIRES2-EGFP-MAGE-1-HSP70,并观察在小鼠黑色素瘤细胞内的表达,为肿瘤DNA疫苗研究奠定基础。方法:HSP70基因经酶切后连入真核表达载体PIRES2-EGFP,再通过PCR方法扩增MAGE-1基因,测序后插入PIRES2-EGFP-HSP70中HSP70基因的5'端,构建了MAGE-1与人HSP70融合基因表达载体PIRES2-EGFP-MAGE-1-HSP70。结果:扩增了MAGE-1基因,测序结果表明与GenBank公布的序列一致,成功地构建了PIRES2-EGFP-MAGE-1-HSP70真核表达载体。结论:成功地构建MAGE-1与人HSP70融合基因的真核表达载体PIRES2-EGFP-MAGE-1-HSP70,并在小鼠黑色素瘤细胞系稳定表达,为进一步DNA肿瘤疫苗研究提供了实验基础。     

5-FC/CD 自杀基因疗法结合热休克蛋白-多肽复合物瘤苗治疗小鼠黑色素瘤的研究

 目的　研究 5 -氟尿嘧啶 /胞嘧啶脱氨酶 (5 FC/ CD)基因疗法与热休克蛋白 -多肽复合物 (HSP- PC)瘤苗免疫疗法联合抗肿瘤效果。方法　将携带 CD基因的重组腺病毒注射到小鼠黑色素瘤体内 ,腹腔注射 5 - FC,同时皮下接种 HSP70 - PC。结果　经联合治疗后 ,70 %荷瘤小鼠肿瘤体积缩小、消退 ,小鼠存活期延长 ,肿瘤组织明显坏死 ,炎症细胞、CD+4 及 CD+8T细胞浸润明显。结论5 - FC/ CD基因疗法结合 HSP- PC瘤苗免疫疗法抗小鼠黑色素瘤作用显著 ,具有临床应用前景。     

肾腺癌中T细胞亚群和HSP96肿瘤抗原肽复合物关系的探讨

目的：探讨肾腺癌无出血坏死者和肾腺癌有出血坏死与行肾动脉栓塞术者T细胞亚群的分布及意义、两组肾腺癌患者肿瘤组织中热休克蛋白（HSP96）的分布有无差异及意义。通过提取出肾腺癌患者癌组织中的HSP96肽复合物找到肿瘤相关的抗原肽。方法：1）选取肾腺癌无出血坏死患者30例,肾腺癌有出血坏死者17例, 行肾动脉栓塞术患者13例。应用两步法免疫组织化学染色观察其T亚群的分布及HSP96的含量和分布。2） 通过经典的HSP96-抗原肽复合物提取方法提取出热休克蛋白肽复合物并进行质谱分析。结果：1）两组比较CD3+T细胞、CD4+T细胞、CD8+T细胞及CD4+/CD8+的比值有统计学差异（P<0.05）。2）两组比较肾腺癌中含有的HSP96没有统计学意义（P>0.05）。3）经LC-ESI-MS-MS测得其中两个主要肽段的序列分别为LVPLE?GWGGNVM和PPVYYVPYVVL,其分子量分别为1 270.68和1 307.70。结论：术前行动脉栓塞术和肾腺癌有出血坏死者因为肿瘤细胞的坏死而使其细胞内具有抗原性的肽段释放出来,从而提高了效应T细胞在肿瘤局部的数量, 对肿瘤组织的杀伤及抑制肿瘤细胞的扩散具有重要的意义。根据gp96结合肽的特点推测其可能为肿瘤抗原肽, 有待进一步鉴定。     

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).     

Effect of medroxyprogesterone and an aorta-derived cell growth inhibitor on B16 melanoma in mice

An aorta-derived inhibitor of endothelial cell and tumor cell growth and medroxyprogesterone, which depresses collagenase expression in vivo, were tested alone and in combination against B16-F10 melanoma in C57BL/6 mice in such doses that either agent alone had little effect. Together, these agents retarded growth of subcutaneously transplanted tumor cells and reduced the number and size of pulmonary tumors after iv tumor cell injection. Of the treatments used, only the aortic factor administered alone prolonged life in mice with pulmonary tumors.     

Effects of sodium cyanate in mice bearing B16 melanoma

Summary Sodium cyanate injected IP at a dose level of 200 or 250 mg/kg caused a 90% or greater inhibition of the incorporation of [³H]thymidine into DNA of B16 melanoma transplanted SC in mice. Despite the inhibitory effect of sodium cyanate on precursor incorporation into DNA, no significant effect on host survival was observed when sodium cyanate was administered as a single agent in the diet, in drinking water, or by IP injection to mice that had received IP transplants of B16 melanoma. The action of melphalan and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in prolonging the survival time of melanoma-bearing mice was not enhanced by combined treatment with sodium cyanate. However, combined injections of sodium cyanate and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) increased the survival of tumor-bearing mice significantly more than injections of BCNU alone at a lower dose than the maximum tolerated one. These data and other studies suggest that B16 melanoma may be less responsive to the action of sodium cyanate than are murine leukemic cells or rat hepatomas.The work described in this paper was supported by NIH grant CA-35315 and a research award from the Veterans Administration     

Novobiocin-induced anti-proliferative and differentiating effects in melanoma B16.

The antibiotic drug novobiocin was evaluated for its anti-tumour properties in B16 melanoma cells. Novobiocin is shown to inhibit melanoma B16 cell proliferation. The anti-proliferative effect was gradually reversible upon removal of novobiocin from the culture medium. Growth inhibition by novobiocin was accompanied by phenotypic alterations, that included morphological changes, lipid accumulation and marked increases in the activities of NADPH cytochrome c reductase and gamma glutamyl transpeptidase. In vivo administration of repeated i.p. doses of novobiocin, to mice implanted with B16 melanoma cells resulted in growth retardation. The combined treatment of the B16 melanoma cells with novobiocin and other chemical inducers of differentiation was examined in a cell growth assay. Novobiocin and sodium butyrate inhibited cell growth in a near additive manner, while combination of novobiocin with the GTP-depleting agents, tiazofurin or mycophenolic acid resulted in a synergistic decrease in cell growth. Our results support the contention further that novobiocin and other differentiating agents might be of potential value in melanoma therapy.     

Size dependence of cell cycle parameters for lung metastases from B16 melanoma

Data presented show that the cell cycle time of the lung metastases from a B16 melanoma tumor growing in the foot pad of C57 BL/6J mice increases as the size of the metastases increases. This increased cell phase durations of the small metastases being closer to those of the primary tumor. The growth fractions of the primary tumor and of the lung metastases of different sizes are analyzed.     

B16 melanoma in C57BL-6J mice: kinetics and effects of heterologous serum

B16 melanoma was studied in syngeneic C57BL/6J mice as a therapeutic model of melanoma. The irregular behavior of tumor after trochar passage was eliminated by serial intraperitoneal passage, but abundant melanin production was maintained. Mean viability of dispersed suspensions was 56%. Median survival time after injection of 10⁷ cells intraperitoneally was 11-16 days (average 13.20). In vivo doubling time was estimated at 3.24 days. Tenfold increase time was approximately 10.75 days. Preliminary studies with rabbit antiserum demonstrated possible enhancement after incubation with tumor in vitro, and a possible therapeutic effect after administration in vivo.     

Properties of mouse melanoma antigen and its secretion mechanism from the cell surface

We analyzed the biochemical properties and biological significance of the melanoma antigen secreted in the culture supernatants of B16 melanoma cells. The 80 kilodalton (kd) molecule bearing the epitopes of mouse melanoma antigen was found to associate noncovalently with an 18 kd moiety in the culture supernatants as well as on the cell surface. Tunicamycin treatment of B16 cells did not affect the expression of the 69 kd nonglycosylated form of the 80 kd molecule but did abolish the association between the two molecules on the cell surface. We could not detect this antigen as a soluble form when the N-linked glycosylation was inhibited. Therefore, the glycosylation of the 80 kd molecule is essential for the formation of the 80 kd/18 kd complex and also for the secretion. Moreover, the affinity-purified melanoma antigen from the supernatants could induce anti-melanoma suppressor cells which block the generation of cytotoxic T lymphocytes against melanoma cells. Thus, the 80 kd glycoprotein as a soluble melanoma antigen performed a pivotal function in the escape mechanisms of melanoma cells from the host immune surveillance system.     

Effect of melatonin on B16 melanoma growth in athymic mice

The effect of melatonin on the growth of B16 mice melanoma was examined. Male and female BALB/c athymic mice, inoculated with 7 X 10(4) melanoma cells, were given drinking water containing melatonin (5 micrograms/g body weight/day) and 0.5% ethanol. Compared to control animals the melatonin treated male and female athymic mice had significantly smaller tumors on Day 40. The weights of the testes, the ovaries, and the adrenal glands of melatonin treated mice were significantly reduced compared to control animals. These data indicate that melatonin p.o. significantly inhibited the growth of B16 mouse melanoma and that the antitumor effect of melatonin was associated with a significant decrease in gonadal and adrenal weights.