促红细胞生成素对达卡巴嗪和顺铂抗小鼠黑色素瘤活性的调节作用

目的：研究促红细胞生成素（erythropoietin,EPO）对荷黑色素瘤小鼠肿瘤生长的影响及其对达卡巴嗪（dacarbazine,DTIC）和顺铂（cisplatin,DDP）抗肿瘤活性的调节作用。方法：将荷黑色素瘤B16细胞的C57BL/6小鼠,分别用EPO、DTIC和DDP以及EPO分别联合DTIC、DDP药物处理,以盐水处理组为对照。观察肿瘤在小鼠体内的生长情况。部分小鼠于停药次日摘除眼球取血,分析血液中红细胞（RBC）、血红蛋白（Hb）、红细胞压积（Hct）和白细胞（WBC）水平。并颈椎脱臼处死小鼠,剥离瘤体,分别称量瘤质量。其余小鼠用于生存期观察。结果：EPO处理组小鼠的肿瘤体积和质量与盐水处理组相比差异无统计学意义（P〉0.05）;DTIC联合EPO处理组小鼠的肿瘤体积和质量与DTIC处理组相比差异无统计学意义（P〉0.05）;DDP联合EPO处理组小鼠的肿瘤体积与DDP处理组相比明显缩小（P〈0.05）。应用EPO处理组的小鼠外周血RBC、Hb和Hct水平明显高于未应用EPO组（P〈0.05）。结论：EPO对黑色素瘤细胞在小鼠体内的生长没有明显影响;但EPO能调节黑色素瘤细胞对DTIC、DDP的敏感性,且该调节作用与化疗药的类型有关。     

东亚钳蝎毒素ITX对C57BL/6小鼠Lewis肺癌组织生长的影响

目的 观察东亚钳蝎毒素ITX对C57BL/6小鼠Lewis肺癌(LLC)组织生长的影响.方法 32只C57BL/6小鼠随机分为空白组、模型组、阳性对照rIL-2组和ITX组.将LLC细胞接种至模型组、阳性对照rIL-2组和ITX组C57BL/6小鼠的有前腋下.各组按不同的处理因素连续干预16 d.实验期间观察各组小鼠的一般状态,监测瘤重和瘤体积的变化.末次给药24 h后处死小鼠,剥取肿瘤,摘取双肺叶,在光学显微镜下观察肿瘤和肺脏的炎性细胞浸润情况.结果 与模型组相比,ITX组和rIL-2组小鼠的生存质量有一定程度的下降,但差异无统计学意义(P>0.05);各组肿瘤体积比较差异无统计学意义(P>0.05);肿瘤问质和肺泡间隔的炎细胞数增多(P<0.05).随着时间延长,模型组、ITX组和rIL-2组肿瘤体积均有明显增长(P<0.05).结论 ITX能够通过加强肿瘤微环境中以炎症为代表的非特异免疫促进C57BL/6小鼠LLC的生长.     

静脉注射特异性激活的巨噬细胞抑制癌的肺部转移

对 B-16黑色素瘤在体内致敏的异种淋巴细胞和 B-16肿瘤细胞一起在体外培养,将其上清与带有 B-16肿瘤的 G57BL/6小鼠的巨噬细胞在体外培养,经培养的巨噬细胞静脉注入接种过 B-16肿瘤的G57BL/6小鼠,抑制了肿瘤在肺部的转移。用2毫升 thioglycollate 腹腔注入进行性生长的 B-16黑色素瘤小鼠,4～5天     

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。     

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。     

组胺对C57BL/6小鼠B16黑色素瘤转移行为的影响

背景与目的:肿瘤生物治疗中微血管壁通透性是大分子抗体偶联药物进入瘤体的主要屏障.组胺可通过增加微血管壁通透性和组织液的生成促进抗体偶联物在肿瘤局部的富集.本研究观察组胺所致微血管壁通透性的增加和组织液的生成增多是否会增加肿瘤血行及/或淋巴的转移.方法:采用瘤细胞悬液接种法将小鼠黑色素瘤细胞B16注入小鼠左上肢腋部皮下,建立C57BL/6小鼠荷瘤模型.实验组接种第6天起,背部皮下注射300 mg/kg组胺,隔日一次,共5次,对照组注射等体积生理盐水.组织化学方法检测肿瘤细胞在各脏器的转移情况.分别用Student t-test和四格表资料确切概率法分析组胺对肿瘤细胞增殖和转移的作用.结果:接种成瘤率100%.300 mg/kg组胺隔日一次皮下注射能明显抑制肿瘤的生长,实验组与对照组瘤重分别为(5.26 1.55)g和(6.96±1.31)g,二者间差异有统计学意义(P＜0.01).实验组与对照组淋巴结转移率分别为33.3%和75.0%,血行转移率分别为25.0%和75.0%,差异均有统计学意义(P＜0.05).结论:组胺能够抑制C57BL/6小鼠B16黑色素瘤的血行和淋巴转移,该抑制作用部分与其抑制瘤细胞的增殖有关.     

顺铂预处理化疗通过免疫调节增强CIK细胞对B16恶性黑色素瘤的抑制作用

目的 观察顺铂( Cisplatin,DDP)预处理化疗联合CIK细胞对B16恶性黑色素瘤的抑制作用,探讨DDP预处理化疗增强细胞因子诱导的杀伤细胞(cytokine - induced killer cells,CIK cells)抑瘤作用的潜在机制.方法 建立C57BL/6小鼠B16黑色素瘤模型,测量肿瘤体积,绘制...     

改造的人乳头瘤病毒16型E6/E7融合蛋白疫苗抗肺癌作用的实验研究

目的 研究经改造的HPV16 E6／E7蛋白质疫苗(HPV16 mE6Δ／mE7)对表达HPV16 E6和E7的小鼠肺癌细胞的体内生长抑制作用，为肺癌免疫治疗提供新线索。方法 预防性实验：对C57BL／6小鼠先进行免疫，然后接种表达HPV16E6和E7癌蛋白的TC-1肿瘤细胞；第33天，对未出瘤小鼠进行第二次大剂量肿瘤细胞攻击。治疗性实验：C57BL／6小鼠先接种TC-1细胞，然后进行两次免疫治疗，再对末出瘤小鼠进行第二次大剂量TC-1细胞攻击实验。计算各组肿瘤发生率、肿瘤体积。采用MTT方法检测淋巴细胞增殖反应。结果预防性实验：在100天的观察时间内，免疫组小鼠可以有效抵抗第一、二次肿瘤细胞攻击，所有小鼠均未见肿瘤发生；而PBS组及单纯佐剂组小鼠在第一次肿瘤细胞接种后6～12天内均有肿瘤形成，30天内因肿瘤负荷出现衰竭。在治疗性实验中，免疫组、PBS组、单纯佐剂组小鼠成瘤率分别为20％、90％、60％；第二次攻击实验中，免疫组仍有87．5％的小鼠无肿瘤生长，而其他两组动物在4～6天内均有肿瘤形成。MTT实验表明经mE6Δ／mE7免疫的小鼠脾淋巴细胞增殖反应明显强于对照组。结论 经改造的HPV16型mE6Δ／mE7蛋白质疫苗对于实验动物肺癌有较好的抑制作用，为HPV16相关肺癌的免疫治疗提供了实验数据。     

小鼠放射性肺纤维化模型的建立与检测北大核心CSCD

目的构建C57BL/6小鼠放射性肺纤维化动物模型,并从功能学、影像学及病理学等方面检测鉴定及分析。方法C57BL/6雄性小鼠30只随机分为对照组、16 Gy照射组、20 Gy照射组。照射组小鼠接受单次16 Gy或20 Gy胸部X线照射,于照射后3个月及6个月行功能学、影像学与病理学检测。结果在照射后6个月时,小鼠胸背部被毛变白脱落,气道阻力显著增加,影像学检查显示肺内广泛的斑片影、实变影,三维重建提示小鼠肺扭曲形变、体积显著缩小,病理学证实出现了广泛的肺纤维化。结论照射小鼠胸部6个月后出现显著肺纤维化,成功构建C57BL/6小鼠放射性肺纤维化动物模型。     

β-胡萝卜素增强小鼠对同基因肿瘤的免疫应答

给接种 B_(16)黑色素瘤的 C57BL/6小鼠行β-胡萝卜素灌胃,每日10mg/kg。20天后,取小鼠脾细胞和B_1~6黑色素瘤细胞混合注入健康 C57BL/6小鼠皮下,观察肿瘤生长情况并检测脾细胞增殖活性和腹腔巨噬细胞的细胞毒活性。实验组和对照组小鼠肿瘤大小无显著性差异,但实验组肺部瘤转移灶数明显低于对照组;实验组脾细胞增殖活性和巨噬细胞细胞毒活性明显高于对照组。提示β-胡萝卜素可增强小鼠的抗肿瘤免疫功能。     

B16 murine melanoma and aging: slower growth and longer survival in old mice

The growth characteristics and colonization potential of a transplantable melanoma administered to young (3 mo) and old (24 mo) C57BL/6 mice were investigated. After sc injection of B16-F10 melanoma cells, tumor growth was slower, and final tumor volume was less in the older mice. Furthermore, after iv injection of B16-F1 melanoma cells, the number of pulmonary colonies was also less, and the survival was greater in the older mice. These findings indicate an age advantage in this experimental tumor model that may be attributed to either physical or immunologic factors.     

Translational strategies exploiting TNF-alpha that sensitize tumors to radiation therapy

TNFerade is a radioinducible adenoviral vector expressing tumor necrosis factor-alpha (TNF-alpha) (Ad.Egr-TNF) currently in a phase III trial for inoperable pancreatic cancer. We studied B16-F1 melanoma tumors in TNF receptor wild-type (C57BL/6) and deficient (TNFR1,2-/- and TNFR1-/-) mice. Ad.Egr-TNF+IR inhibited tumor growth compared with IR in C57BL/6 but not in receptor-deficient mice. Tumors resistant to TNF-alpha were also sensitive to Ad.Egr-TNF+IR in C57BL/6 mice. Ad.Egr-TNF+IR produced an increase in tumor-associated endothelial cell apoptosis not observed in receptor-deficient animals. Also, B16-F1 tumors in mice with germline deletions of TNFR1,2, TNFR1 or TNF-alpha, or in mice receiving anti-TNF-alpha exhibited radiosensitivity. These results show that tumor-associated endothelium is the principal target for Ad.Egr-TNF radiosensitization and implicate TNF-alpha signaling in tumor radiosensitivity.     

Inhibitory effect of Cordyceps sinensis on experimental hepatic metastasis of melanoma by suppressing tumor cell invasion

We investigated the anti-metastatic activity of a water extract of Cordyceps sinensis (WECS) using a model of mice injected with B16-F0 mouse melanoma cells into the spleen. WECS administered intraperitoneally reduced the number of metastatic surface nodules of B16-F0 cells in the liver of C57BL/6Cr mice in a dose-dependent manner, and significantly prolonged their survival. To identify the mechanism of the anti-metastatic effect of WECS, we examined its effects on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. As a result, WECS reduced HGF-accelerated B16-F0 cell invasion in a concentration-dependent manner. These findings suggest that WECS exerts an anti-metastatic action, in part by inhibiting the HGF-accelerated tumor invasiveness of mouse melanoma cells.     

Effect of retinoic acid on tumor-mediated immunologic alterations in mice bearing a variant of the B16 melanoma

This investigation examined the effect of retinoic acid on tumor progression and immunological status of mice bearing the B16-F10 melanoma (previously selected for high lung-colonizing capacity). Tumor cells were implanted s.c. in syngeneic C57BL/6 mice, half of which were treated with beta-all trans retinoic acid (RA). Although RA failed to exhibit direct toxicity on this variant at the concentration used, the immunologic aberrations induced by the tumors were diminished by i.p. RA administration (at 45 micrograms twice/week for 3 weeks). In mice bearing B16-F10 tumors, tumor burdens were decreased from 2.9% of body weight to 1.6%. The mitogenic responses of splenic lymphocytes to concanavalin A (ConA) were increased in tumor-bearing mice following this RA treatment. The presence of these tumor cells decreased the absolute number of CD4- and CD8-positive splenic lymphocytes. Following RA treatment, the CD8-positive population was increased in tumor-bearing mice, while the CD4+ population was not significantly altered. Since previous studies indicated that plasma membrane fragments (or vesicles) could alter lymphocyte distributions and proliferative capacities, the in vitro shedding of membrane fragments from B16-F10 tumor cells was assayed and observed to be decreased after continuous treatment of cultures with 10(-6) M RA for 21 days. Membrane shedding from B16-F10 cells was inhibited by 48.5% following RA treatment. Based on these in vivo and in vitro results, we suggest that RA treatment may diminish tumor growth by decreasing tumor-induced immunosuppressive events.     

Suppression of tumor metastasis by blockade of transforming growth factor beta signaling in bone marrow cells through a retroviral-mediated gene therapy in mice

Transforming growth factor B (TGF-beta) is a potent immunosuppressive cytokine that is frequently associated with mechanisms of tumor escape from immunosurveillance. We report that transplantation of murine bone marrow (BM) expressing a dominant-negative TGF-beta type II receptor (TbetaRIIDN) leads to the generation of mature leukocytes capable of a potent antitumor response in vivo. Hematopoietic precursors in murine BM from donor mice were rendered insensitive to TGF-beta via retroviral expression of the TbetaRIIDN construct and were transplanted in C57BL/6 mice before tumor challenge. After i.v. administration of 5 x 10(5) B16-F10 murine melanoma cells into TbetaRIIDN-BM transplanted recipients, survival of challenged mice at 45 days was 70% (7 of 10) versus 0% (0 of 10) for vector-control treated mice, and surviving TbetaRIIDN-BM mice showed a virtual absence of metastatic lesions in the lung. We also investigated the utility of the TGF-beta-targeted approach in a mouse metastatic model of prostate cancer, TRAMP-C2. Treatment of male C57BL/6 mice with TbetaRIIDN-BM resulted in the survival of 80% (4 of 5) of recipients versus 0% (0 of 5) in green fluorescent protein-BM recipients or wild-type controls. Cytolytic T-cell assays indicate that a specific T-cell response against B16-F10 cells was generated in the TbetaRIIDN-BM-treated mice, suggesting that a gene therapy approach to inducing TGF-beta insensitivity in transplanted BM cells may be a potent anticancer therapy.     

Triflavin, an Arg-Gly-Asp-containing antiplatelet peptide inhibits cell-substratum adhesion and melanoma cell-induced lung colonization.

Triflavin, an Arg-Gly-Asp (RGD) containing peptide purified from Trimeresurus flavoviridis snake venom, inhibits human platelet aggregation by blocking fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex. In this study, we show that triflavin (1-30 micrograms/mouse) inhibits B16-F10 melanoma cell-induced lung colonization in C57BL/6 mice in a dose-dependent manner. In vitro, triflavin dose-dependently inhibits adhesion of B16-F10 melanoma cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, vitronectin, and collagen type I). Triflavin is approximately 600-800 times more potent than GRGDS at inhibiting cell adhesion. In addition, triflavin dose-dependently inhibits B16-F10 cell-induced platelet aggregation. These results imply that the inhibitory effect of triflavin on the adhesion of tumor cells to ECMs (e.g., fibronectin, vitronectin and collagen type I) and/or tumor cell-induced platelet aggregation may be partially responsible for its antimetastatic activity in C57BL/6 mice.     

Anti-tumor activity of N-trimethyl chitosan-encapsulated camptothecin in a mouse melanoma model

Background

Camptothecin (CPT) has recently attracted increasing attention as a promising anticancer agent for a variety of tumors. But the clinical application is largely hampered by its extreme water insolubility and unpredictable side effect. It is essential to establish an efficient and safe protocol for the administration of CPT versus melanoma.

Methods

Camptothecin was encapsulated with N-trimethyl chitosan (CPT-TMC) through microprecipitation and sonication. Its inhibition effect on B16-F10 cell proliferation and induction of apoptosis was evaluated by MTT assay and flow cytometric analysis in vitro. The anti-tumor activity of CPT-TMC was evaluated in C57BL/6 mice bearing B16-F10 melanoma. Tumor volume, tumor weight and survival time were recorded. Assessment of apoptotic cells within tumor tissue was performed by TUNEL assay. Antiangiogenesis and antiproliferation effects of CPT-TMC in vivo were conducted via CD31 and PCNA immunohistochemistry, respectively.

Results

CPT-TMC efficiently inhibited B16-F10 cells proliferation and increased apoptosis in vitro. Experiment group showed significant inhibition compared with free CPT-treated group (81.3% vs. 56.9%) in the growth of B16-F10 melanoma xenografts and prolonged the survival time of the treated mice (P < 0.05). Decreased cell proliferation, increased tumor apoptosis as well as a reduction in angiogenesis were observed.

Conclusions

Our data suggest that N-trimethyl chitosan-encapsulated camptothecin is superior to free CPT by overcoming its insolubility and finally raises the potential of its application in melanoma therapy.     

Aerosol delivery of PEI-p53 complexes inhibits B16-F10 lung metastases through regulation of angiogenesis

Inhibition of pulmonary metastases poses a difficult clinical challenge for current therapeutic regimens. We have developed an aerosol system utilizing a cationic polymer, polyethyleneimine (PEI), for topical gene delivery to the lungs as a novel approach for treatment of lung cancer. Using a B16-F10 murine melanoma model in C57BL/6 mice, we previously demonstrated that aerosol delivery of PEI-p53 DNA resulted in highly significant reductions in the tumor burden (P < .001) in treated animals, and also lead to about 50% increase in the mean length of survival of the mice-bearing B16-F10 lung tumors. The mechanisms of this antitumor effect of p53 are investigated in this report. Here, we demonstrate that the p53 transfection leads to an up-regulation of the antiangiogenic factor thrombospondin-1 (TSP-1) in the lung tissue and the serum of the mice. Furthermore, there is a down-regulation of vascular endothelial growth factor (VEGF) in the lung tissue and serum of the B16-F10 tumor-bearing mice treated with PEI-p53 DNA complexes, compared with untreated tumor-bearing animals. In addition, staining for von Willebrand factor (vWF), a marker for the angiogenic blood vessels, revealed that p53 treatment leads to a decrease in the angiogenic phenotype of the B16-F10 tumors. Immunohistochemistry for transgene expression reveals that the PEI-p53 aerosol complexes transfect mainly the epithelial cells lining the airways, with diffuse transfection in the alveolar lining cells, as well as, the tumor foci in the lung tissue. There was also some evidence of apoptosis in the lung tumor foci of animals treated with p53. The data suggest that aerosol delivery of PEI-p53 complexes leads to inhibition of B16-F10 lung metastases, in part by suppression of angiogenesis.     

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。