Angiostatin基因真核表达载体的构建及对B16黑色素瘤体内生长抑制作用

Angiostatin是一种新发现的对肿瘤生长有特异抑制作用的抗血管生成因子,实验已证实其对多种肿瘤有明显的抑制作用。本文报告构建了含Angiostation基因的真核表达载体pAG3,通过建立荷瘤小鼠模型来研究Angiostation对人黑色素瘤B16的原位生长,植入及与化疗药物DTIC的联合作用等来探讨Angiostation裸DNA肌肉注射的体内抗瘤效应。实验结果表明Angiostation可明显抑制C57荷瘤小鼠的肿瘤生长;人黑色素瘤B16细胞植入前5天肌肉注射pAG3能显著阻止下C57小鼠新肿瘤的形成;但在pAG3与DTIC联合化疗实验中,两者未表现出明显的增强效应。本实验为拓展非病毒介导的Angiostation抗血管生成基因治疗途径奠定了基础。     

Angiostatin基因真核表达载体的构建及对B16黑色素瘤体内生长抑制作用

Angiostatin是一种新发现的对肿瘤生长有特异抑制作用的抗血管生成因子,实验已证实其对多种肿瘤有明显的抑制作用.本文报告构建了含Angiostatin基因的真核表达载体pAG3,通过建立荷瘤小鼠模型来研究Angio-statin对人黑色素瘤B16的原位生长,植入及与化疗药物DTIC的联合作用等来探讨Angiostatin裸DNA肌肉注射的体内抗瘤效应.实验结果表明Angiostatin可明显抑制C57荷瘤小鼠的肿瘤生长;人黑色素瘤B16细胞植入前5天肌肉注射pAG3能显著阻止正常C57小鼠新肿瘤的形成;但在pAG3与DTIC联合化疗实验中,两者未表现出明显的增强效应.本实验为拓展非病毒介导的Angiostatin抗血管生成基因治疗途径奠定了基础.     

抗原负载DCs诱导的CIK细胞对B16黑色素瘤抑制作用的形态学观察

目的:探讨抗原负载树突状细胞(dentritic cells,DCs)诱导的CIK(cytokine induced killer)细胞对B16黑色素瘤的抑瘤作用。方法:分离、培养DC和CIK细胞,取部分DC进行肿瘤抗原负载,将其与CIK细胞按1:10的比例共培养3d,即为抗原负载的DC-CIK。建立B16黑色素瘤小鼠模型,分别于瘤周围皮下注射经Brdu标记的CIK、DC-CIK、抗原负载DC-CIK。按注射细胞进行分组,测量注射前后各组小鼠的瘤体积,计算抑瘤率,比较其抑瘤作用。应用免疫组化方法和透射电镜观察抗原负载DC-CIK细胞在皮肤中的分布及杀伤肿瘤细胞的形态学表现。结果:抗原负载DC诱导的CIK(细胞组抑瘤率(86.57%)高于CIK细胞组(33.34%,P<0.05)和DC-CIK细胞组(61.08%,P<0.05);光镜下抗原负载DC-CIK细胞主要分布在皮下组织,癌组织周围,特别是癌巢周边。透射电镜下抗原负载DC-CIK细胞体积大,核有切迹,细胞质内细胞器丰富,粗面内质网扩张。细胞表面有突起,与肿瘤细胞密切接触。大量肿瘤细胞凋亡、坏死。结论:CIK细胞经抗原负载DC诱导后抑瘤作用明显强于单纯CIK细胞和DC-CIK细胞。     

重组FN多肽CH50抑制黑色素瘤B16细胞体内转移的研究

目的研究重组纤黏连蛋白（FN）多肽CH50对小鼠黑色素瘤B16细胞体内转移的影响,以探讨CH50多肽抑制肿瘤转移的可能分子机制。方法体外培养黑色素瘤B16细胞,用荧光染料CFSE标记,接种脾脏后24h取脾、肝、肺做冰冻切片,观察肿瘤细胞在3种组织中的侵袭情况。从脾脏接种B16细胞,建立体内肿瘤转移动物模型,采用基于流体动力学的体内基因转染方法于小鼠体内表达CH50多肽,RT-PCR检测CH50mRNA在肝组织的表达,Western印迹检测CH50多肽的表达。通过比较原位肿瘤结节及转移结节在数量、大小、分布上的差异及检测原位肿瘤组织中MMP-2、MMP-9表达差异,观察CH50多肽的治疗效果。结果注射24h后即可在脾脏形成荧光结节。pCH510质粒通过尾静脉注射后,可在肝组织中检测到CH50mRNA及CH50多肽的表达。从脾脏接种B16细胞后第14天可在脾脏形成原发肿瘤,至第35天肝脏表面已形成转移瘤结节,成功建立了体内器官问（脾转肝）肿瘤转移动物模型。体内转染表达CH50多肽能抑制肿瘤生长、侵袭和转移,抑制原位肿瘤结节中MMP-2、MMP-9的表达。结论CH50多肽可以通过对MMP-2、MMP-9蛋白表达的抑制作用来抑制黑色素瘤B16细胞的成瘤能力和体内侵袭、转移能力。     

单基因和双基因联合治疗对小鼠B16-F10黑色素瘤生长抑制作用研究

目的研究阳离子脂质体介导mGM-csf和mFlt-kdr3基因治疗以及2个基因联合治疗对小鼠B16-F10黑色素瘤肺转移以及实体瘤的生长抑制作用。方法通过尾静脉注射法和皮下注射法分别将10⁵个以及10⁶个对数生长期黑色素瘤细胞注入BALB/c小鼠体内,构建小鼠肺转移模型和腋下实体瘤模型。将2组模型小鼠分别分成5组:mFlt-kdr3治疗组、mGM-csf治疗组、联合治疗组(1次mFlt-kdr3,2次mGM-csf)、H1299脂质体质粒对照组和生理盐水对照组。肺转移模型小鼠建模3周后,尾静脉给药治疗,每次给药80μL,每次间隔1 d,共3次。完成后3 d,解剖取出小鼠肺组织并对肿瘤灶进行计数、苏木精-伊红染色。实体瘤模型小鼠在建模1周后开始瘤体穿刺给药,每次给药25μL,每次间隔1 d,共9次,每次给药前用游标卡尺测量瘤体的长、短径。结果肺转移治疗实验中,治疗组平均肿瘤个数显著少于对照组(P<0.05),且联合治疗组肿瘤个数最少,并且肺部结构完整,基本未见明显的肿瘤灶,可以看到明显的肺泡结构。实体瘤模型实验中,治疗组瘤体平均体积显著小...     

免疫毒素Luffin B-Ng76对人黑色素瘤细胞的体外抑制作用

用ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６Ｂ凝胶亲和层析法从丝瓜籽中分离纯化了单链致核糖体失活蛋白（ｒｉｂｏｓｏｍｅｉｎａｃ－ｔｉｖａｔｉｎｇｐｒｏｔｅｉｎ,ＲＩＰ）——ｌｕｆｉｎＢ。并将ｌｕｆｉｎＢ与抗人黑色素瘤细胞单抗Ｎｇ７６制成了免疫毒素,命名为ｌｕｆｉｎＢ－Ｎｇ７６,它对体外培养的黑色素瘤细胞Ｍ２１有很强的抑制作用,ＩＣ５０为２．５×１０－１１ｍｏｌ／Ｌ,毒性比游离的ｌｕｆｉｎＢ提高４０００倍,而它对非靶的ＨｅＬａ细胞的毒性较Ｍ２１细胞低１２００倍。结果提示ｌｕｆｉｎＢ用于制备免疫毒素具有良好的应用前景。     

小鼠正常黑色素细胞与黑色素瘤细胞(B16)mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径,主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrinβ1和Integrinβ5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响

为研究黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响,以及初步研究其作用机制,采用磺酰罗丹明B(SRB)法测黄泥螺提取物对B16细胞的生长抑制作用,得到48 h后半数抑制浓度(IC50)为68.56 ug/ml.当黄泥螺提取物浓度为70ug/ml时,台盼蓝排斥试验显示有部分细胞死亡.经Hoechest 33258染色并用荧光显微镜观察,发现培养的细胞中出现凋亡小体,细胞核发生固缩;DNA琼脂糖凝胶电泳呈现出DNA大片段;用流式细胞仪进一步检测表明,细胞生长周期发生变化并检测到凋亡峰.结果表明,体外培养的B16细胞经过黄泥螺提取物处理后,B16细胞增殖受到抑制,细胞周期被阻滞,B16细胞受到诱导后存在凋亡.因此,黄泥螺提取物对小鼠黑色素瘤细胞B16生长有明显的影响.     

小鼠正常黑素细胞与黑色素瘤细胞（B16）mRNA表达差异分析

黑色素瘤是一种高侵袭性的恶性皮肤肿瘤,转移率高、预后差。研究黑素瘤细胞生物学特性对黑素瘤的治疗和控制具有重要的意义。本研究以C57BL/6J小鼠的正常黑色素细胞及B16黑色素瘤细胞为研究对象,采用二代测序技术分析两种细胞间的转录组表达差异,筛选差异基因,为后续黑色素瘤的形成机制研究提供理论依据。采用差异倍数及错误率分析测序数据,鉴定出1 436个新的mRNA和4 086个差异表达的已知mRNA。GO数据库和KEGG数据库分析显示,差异表达的mRNAs参与了149个调控途径, 主要集中在疾病调控、细胞周期调节和环境信息调控方面。qRT-PCR及Western印迹检测发现,调节细胞增殖、迁移的Pdgf-B、Integrin-β1和Integrin-β5以及调节黑色素颗粒增加的Mitf、Tyr、Tyrp1和Tyrp2在B16细胞中的表达量显著高于在正常黑色素细胞中的表达。本研究获得的差异基因为后续黑色素瘤的研究提供了新的候选基因。     

静磁场对小鼠黑色素瘤细胞B16生长和氧化应激的影响

目的:利用小鼠黑色素瘤细胞B16,研究静磁场对肿瘤细胞生长和氧化应激的影响,探讨氧化应激介导静磁场影响肿瘤细胞生长的机制,为磁场在肿瘤疾病的治疗中的应用提供理论依据。方法:采用MTT法测定磁场对B16细胞活力的影响;利用流式细胞仪测定静磁场暴露对B16细胞周期分布的影响;利用生物化学方法测定磁场暴露对细胞氧化防御系统相关蛋白酶活性的影响。结果:24 h内50 m T-200 m T静磁场暴露可以抑制B16生长,但超过24 h的磁场暴露可以促进B16生长;100 m T和200 m T静磁场暴露对B16的细胞周期分布没有影响;B16暴露于100 m T和200 m T静磁场48 h,GST活性和GSH/GSSG水平表现为先上升后下降,SOD活性和T-AOC水平先下降后上升,CAT活性没有受到影响。结论:50 m T-200 m T静磁场可以抑制小鼠黑色素瘤细胞B16的生长,诱导肿瘤细胞产生氧化应激。     

豹蛙酶研究进展

豹蛙酶是一种抗肿瘤药物,属于核糖核酸酶A超家族,它的核糖核酸酶活性低,细胞毒性较强,在体内外对多种肿瘤具有显著杀伤作用,是目前全球正在重点研究的100种新药之一。豹蛙酶用于恶性间皮瘤的治疗目前处于Ⅲ期临床研究阶段,用于非小细胞肺癌和其他固体肿瘤的治疗也处于临床Ⅰ期或Ⅱ期研究阶段。豹蛙酶通过多种机制导致肿瘤细胞凋亡,抗病毒作用是其进一步研究开发的重点。     

STAT3 Knockdown in B16 Melanoma by siRNA Lipopolyplexes Induces Bystander Immune Response In Vitro and In Vivo

Persistent activation of STAT3 plays a major role in cancer progression and immune escape. Therefore, targeting STAT3 in tumors is essential to enhance/reactivate antitumor immune response. In our previous studies, we demonstrated the efficacy of stearic acid-modified polyethylenimine (PEI-StA) in promoting small interfering RNA (siRNA) silencing of STAT3 in B16.F10 melanoma in vitro and in vivo. In the current study, we examine the immunologic impact of this intervention. Toward this goal, the infiltration and activation of lymphocytes and dendritic cells (DCs) in the tumor mass were assessed using flow cytometry. Moreover, the levels of IFN-γ, IL-12, and TNF-α in homogenized tumor supernatants were determined. Moreover, mixed lymphocytes reaction using splenocytes of tumor-bearing mice was used to assess DC functionality on siRNA/lipopolyplexes intervention. Our results demonstrated up to an approximately fivefold induction in the infiltration of CD3(+) cells in tumor mass on STAT3 knockdown with high levels of CD4(+), CD8(+), and NKT cells. Consistently, DC infiltration in tumor milieu increased up to approximately fourfold. Those DCs were activated, in an otherwise suppressive microenvironment, as evidenced by a high expression of costimulatory molecules CD86 and CD40. ELISA analysis revealed a significant increase in IFN-γ, IL-12, and TNF-α. Moreover, mixed lymphocytes reaction demonstrated alloreactivity of these DCs as assessed by high T-cell proliferation and IL-2 production. Our results suggest a bystander immune response after local STAT3 silencing by siRNA. This strategy could be beneficial as an adjuvant therapy along with current cancer vaccine formulations.     

豹蛙核酸酶与斑蝥酸钠对肺腺癌细胞增殖的协同抑制作用

豹蛙核酸酶(onconase,Onc)是从美洲北方豹蛙卵母细胞中提取的一种核糖核酸酶,对许多肿瘤细胞都具有杀伤作用。斑蝥素(cantharidin)是存在于芫青科昆虫斑蝥体内的一种天然防御性毒素,斑蝥酸钠(sodium cantharidate,SCA)是斑蝥素半合成衍生物。鉴于Onc与SCA对非小细胞肺癌都具有杀伤作用,采用MTT法测定Onc与SCA单独与联合作用于两株肺腺癌细胞的IC50值,运用联合作用指数(combination index,CI)和等效线分析评价两者联合作用的效果。结果表明,Onc与SCA联合作用时,CI值均小于0.7,等效线分析图显示,代表Onc与SCA联合作用的点均位于加成线下方,Onc与SCA对肺腺癌SPC-A-1、A549细胞株增殖的抑制作用具有协同效应。用流式细胞仪进行的凋亡细胞检测结果也支持上述"Onc/SCA联合使用具有协同抗癌作用"的结论。     

CRMP-2 Is Involved in Axon Growth Inhibition Induced by RGMa In Vitro and In Vivo

Repulsive guidance molecule-a (RGMa) is associated with axon growth inhibition in different central nervous system (CNS) injuries, but its signaling pathways remain unclear. We examined the involvement of collapsin response mediator protein-2 (CRMP-2), a common downstream target of Rho-kinase and GSK-3β, in vitro by culturing neonatal rat primary cortical neurons with RGMa protein, Rho-kinase inhibitor (Y-27632), and GSK-3β inhibitor. We examined CRMP-2 in vivo by suppressing RGMa expression using recombinant adenovirus (rAd-shRGMa) in a rat MCAO/reperfusion model. RGMa induced neurite retraction and CRMP-2 phosphorylation in vitro, which were reversed by either Rho-kinase or GSK-3β inhibitors. After MCAO/reperfusion in rats, pCRMP-2 protein was greatly increased in the ischemic cortex, axons were damaged severely, Neurofilament-200 (NF-200) expression was significantly decreased, and neurological deficits were significant, which were all improved by down-regulating RGMa. We concluded RGMa inhibits axon growth by phosphorylating CRMP-2 via both Rho-kinase and GSK-3β signaling pathways.     

Inhibition of Sphingolipid Synthesis by Cycloserine In Vitro and In Vivo

Abstract: d - and l -cycloserine were shown to be irreversible inhibitors of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase, in a study using bacterial and brain microsomal enzymes, l-Cycloserine was shown to be 100 times more inhibitory than the d-isomer for the brain microsomal enzyme in vitro. In vivo , l -cycloserine caused a 70% inhibition of brain microsomal enzyme. Following one injection, enzyme activity recovered 80% of normal after 16 hours. Daily dosages of l -cycloserine on a regimen of intraper-itoneal injection for 7 days caused a significant reduction in total brain ganglioside and cerebroside plus sulfatide levels.     

蛇毒金属蛋白酶抑制剂抗小鼠黑色素瘤肺转移

探讨蛇毒金属蛋白酶抑制剂BJ46a抗肿瘤侵袭转移作用。以BJ46a为目的基因,选择杆状病毒表达系统生产重组BJ46a蛋白,ProBondTM亲和层析纯化后处理黑色素瘤细胞B16,经尾静脉接种C57BL/6小鼠,建立B16细胞实验性肺转移模型,20天后以颈椎脱臼法处死小鼠,观察肺组织病理学变化。结果表明：不同浓度重组BJ46a蛋白处理组肺部转移瘤数分别为1.1±0.83、0.9±0.7,明显低于对照组（6.3±3.00,P<0.001）和空白对照组（10.7±5.73,P<0.001）,光镜下对照组肿瘤病理学征象明显,而重组BJ46a蛋白处理组瘤结节小。研究结果首次证明了重组BJ46a蛋白能抑制小鼠黑色素瘤细胞的侵袭转移,为其作为抗肿瘤侵袭转移药物的进一步研制和开发应用提供理论依据和前提条件。探讨蛇毒金属蛋白酶抑制剂BJ46a抗肿瘤侵袭转移作用。以BJ46a为目的基因,选择杆状病毒表达系统生产重组BJ46a蛋白,ProBond™ 亲和层析纯化后处理黑色素瘤细胞B16,经尾静脉接种C57BL/6小鼠,建立B16细胞实验性肺转移模型,20天后以颈椎脱臼法处死小鼠,观察肺组织病理学变化。结果表明：不同浓度重组BJ46a蛋白处理组肺部转移瘤数分别为1.1±0.83、0.9±0.7,明显低于对照组（6.3±3.00,P<0.001）和空白对照组（10.7±5.73,P<0.001）,光镜下对照组肿瘤病理学征象明显,而重组BJ46a蛋白处理组瘤结节小。本研究结果首次证明了重组BJ46a蛋白能抑制小鼠黑色素瘤细胞的侵袭转移,为其作为抗肿瘤侵袭转移药物的进一步研制和开发应用提供理论依据和前提条件。     

FAK Inhibition Decreases Hepatoblastoma Survival Both In Vitro and In Vivo

Hepatoblastoma is the most frequently diagnosed liver tumor of childhood, and children with advanced, metastatic or relapsed disease have a disease-free survival rate under 50%. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult hepatocellular carcinoma, leading us to hypothesize that FAK would be present in hepatoblastoma and would impact its cellular survival. In the current study, we showed that FAK was present and phosphorylated in human hepatoblastoma tumor specimens. We also examined the effects of FAK inhibition upon hepatoblastoma cells using a number of parallel approaches to block FAK including RNAi and small molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion, and migration and increased apoptosis. Further, small molecule inhibition of FAK led to decreased tumor growth in a nude mouse xenograft model of hepatoblastoma. The findings from this study will help to further our understanding of the regulation of hepatoblastoma tumorigenesis and may provide desperately needed novel therapeutic strategies and targets for aggressive, recurrent, or metastatic hepatoblastomas.