siRNA干扰鸟氨酸脱羧酶抗酶抑制因子-1的表达抑制黑素瘤细胞增殖

目的:研究siRNA干扰鸟氨酸脱羧酶抗酶抑制因子-1(ornithine decarboxylase antizyme inhibitor-1,OAZI-1)的表达对小鼠黑素瘤B16-F1细胞增殖的影响。方法:构建OAZI-1特异性siRNA表达质粒psilencer2.1-U6/OAZI-1及对照psilenc-er2.1-U6/scrambled质粒,脂质体转染法将质粒转染入B16-F1细胞,Western blotting和定量PCR检测B16-F1细胞中OAZI-1的表达。MTT法和流式细胞术检测psilencer2.1-U6/OAZI-1对B16-F1细胞增殖和细胞周期的影响;MTT法检测干扰OAZI-1表达后B16-F1细胞对抗肿瘤药物紫杉醇(docetaxel)的敏感性。结果:成功获得稳定转染psilencer2.1-U6/OAZI-1质粒的B16-F1细胞(B16/OAZI-1细胞),B16/OAZI-1细胞中OAZI-1 mRNA和蛋白表达分别为阴性对照B16/scrambled细胞的25.0%和18.9%。干扰OAZI-1的表达抑制B16-F1细胞的增殖,G0/G1期细胞比例增加[(57.0±0.8)%vs(63.5±0.7)%,P<0.01],而S期和G2/M期细胞比例减少[(31.5±0.7)%vs(27.5±0.3)%,P<0.05;(11.5±0.3)%vs(9.1±0.6)%,P<0.01]。干扰OAZI-1的表达能降低B16-F1细胞对紫杉醇的敏感性。结论:siRNA干扰OAZI-1的表达能抑制黑素瘤B16-F1细胞的增殖,并降低其对紫杉醇的敏感性。     

多胺类似物BENS对黑素瘤B16-F1细胞生长的影响

目的:探讨多胺类似物N1,N11-bis(ethyl)norspermine(BENS)对黑素瘤B16-F1细胞生长及凋亡的影响.方法:BENS处理B16-F1细胞,MTT法检测细胞存活率,FCM法检测细胞凋亡率,荧光染色法检测线粒体膜电位的变化,DNA片段化检测细胞凋亡,Western印迹法检测凋亡相关蛋白的含量.结果:多胺类似物BENS能显著抑制B16-F1细胞增殖,抑制率呈药物浓度依赖性.BENS可诱导DNA片段化,导致FCM分析中出现典型的亚凋亡峰,同时伴有细胞线粒体膜电位降低、细胞色素C由线粒体向细胞质释放和Bax由细胞质向线粒体转移等凋亡相关性改变.结论:多胺类似物BENS抑制黑素瘤B16-F1细胞生长,并能诱导该细胞凋亡,具有潜在的临床应用价值.     

人核糖核酸酶抑制因子对小鼠B16黑素瘤的基因治疗研究

目的　观察人核糖核酸酶抑制因子(RI)逆转录病毒真核表达载体(p L NCX- ri)对小鼠B16黑素瘤生长的抑制作用。方法　将转染细胞PA317- p L NCX和PA317- p L NCX- ri分别置于DMEM培养基上,经G4 18筛选培养。用Sepharose CL- 4 B凝胶过滤层析纯化病毒颗粒。经G4 18筛选培养,在倒置相差显微镜下计数克隆细胞数。用RT- PCR方法和Western blotting方法检测被感染的NIH3T3细胞及B16黑素瘤组织中RI基因的表达。给接种B16黑素瘤细胞的C5 7BL/ 6 J小鼠分别皮下注射纯化的p L NCX- ri病毒和空载体病毒,观察C5 7BL/ 6 J小鼠肿瘤的生长状况,并对肿瘤组织称重。镜下观察肿瘤组织切片的血管密度。结果　RI在感染了p L NCX- ri病毒的NIH3T3细胞和B16黑素瘤组织中得到有效表达。p L NCX- ri病毒治疗组的瘤组织重量(0 .6 0±0 .2 6 ) g明显低于p L NCX病毒对照组(1.84±1.6 4 ) g(P<0 .0 1)和生理盐水对照组(1.77±1.13) g(P<0 .0 1)。p L NCX病毒对照组(1.84±1.6 4 ) g和生理盐水对照组(1.77±1.13) g无显著性差异(P>0 .0 5 )。与p L NCX病毒对照组和生理盐水对照组相比,p L NCX- ri病毒治疗组肿瘤组织的血管密度明显减少,而两对照组间无明显差异。结论　人核糖核酸酶抑制因子(RI)对小鼠B16黑素瘤的生长有明显的抑制作用     

重组腺相关病毒载体介导人血管抑素K1-5基因预防和治疗B16黑素瘤的实验研究

目的:研究重组腺相关病毒载体介导人血管抑素K1-5基因(rAAV-AG)对B16F0恶性黑素瘤细胞生长的影响。方法:观察rAAV-AG单次肌肉注射在预防肿瘤生长、血行转移及治疗肿瘤方面的不同作用;Western blot法定性检测血管抑素表达情况;免疫组化法检测肿瘤组织中的微血管密度(MVD);TUNEL法检测肿瘤凋亡细胞数。结果:rAAV-AG单次肌肉注射后第2周至实验结束时(~70 d)小鼠体内均有血管抑素表达;预防肿瘤生长抑瘤率为97%(P<0.01);预防肝、肺转移抑瘤率分别为37.5%(P<0.05)和47.7%(P<0.01);低、中、高剂量组治疗肿瘤抑瘤率分别为44%、61%和65%,小鼠中位生存时间分别为58 d6、6 d和68 d,与生理盐水组比较,低剂量组无显著性差异(P=0.06),中、高剂量组均有显著性差异(P<0.01);基因治疗各组肿瘤MVD减少,凋亡细胞数增加,与生理盐水组比较,统计结果与各组抑瘤率一致。结论:rAAV-AG单次肌肉注射能有效预防和治疗B16恶性黑素瘤的生长及血行转移,延长荷瘤小鼠的生存期;在一定剂量范围内其抑瘤效应呈剂量依赖性;抑瘤作用可能是通过抑制肿瘤血管生成、诱导肿瘤细胞凋亡来实现的。     

人参皂苷Rg3对小鼠B16黑素瘤细胞侵袭、转移及MMP-9表达的影响

目的:观察人参皂苷Rg3(简称Rg3)对小鼠黑素瘤细胞B16的肺转移、侵袭能力及基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达的影响,探讨其抗肿瘤转移的作用机制。方法:体内建立小鼠自发肺转移和实体瘤模型,观察腹腔注射不同剂量的Rg3(对照组给予0.9%氯化钠溶液)后,肺部肿瘤转移灶的数目,并检测实体瘤中MMP-9蛋白的表达情况。采用Boyden小室侵袭实验及免疫细胞化学染色法检测Rg3对肿瘤细胞体处侵袭能力及MMP-9表达的影响。结果:采用不同剂量的Rg3(0.3、1.0和3.0mg/kg)治疗后,小鼠肺部转移灶的数目均较少,肿瘤组织中MMP-9的表达水平降低,与对照组比较差异有统计学意义(P<0.05)。在体外,2.5和5.0μg/mLRg3治疗组中侵袭穿过人工基膜的B16细胞数目明显少于对照组(P<0.01),且5.0μg/mLRg3可抑制肿瘤细胞中MMP-9的表达。结论:Rg3能够抑制小鼠黑素瘤细胞的肺转移,其抗肿瘤转移作用可能是通过降低肿瘤细胞中MMP-9的表达水平及细胞侵袭能力来实现的。     

木鳖子醇提物抑制小鼠黑素瘤B16细胞体内外侵袭转移的实验研究

目的:观察木鳖子醇提物(ethanol extract from cochinchina momordica seed,CMSEE)对小鼠黑素瘤细胞B16及其荷瘤小鼠移植瘤侵袭转移的影响,初步探讨其作用机制.方法:通过划痕实验和体外迁移实验检测不同质量浓度CMSEE(20、25、30μg/mL)作用24和48 h后,B16细胞侵袭和转移能力的改变;分别用RT-PCR和Western印迹法检测不同质量浓度CMSEE(20、25、30μg/mL)作用24 h以及25μg/mL CMSEE作用不同时间(0、6、12、24、36、48 h)对B16细胞中基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9 mRNA及其蛋白以及基质金属蛋白酶组织抑制因子(tissue inhibitor of metalloproteinase,TIMP)-1、TIMP-2蛋白表达的影响.以B16细胞建立小鼠荷瘤模型,用10 mg/kg CMSEE灌胃治疗荷瘤小鼠,HE染色观察各组小鼠的肿瘤、肝、脾组织的病理变化.结果:CMSEE(20～30 μg/mL)可明显抑制B16细胞的侵袭和转移能力,可使B16细胞划痕的直径明显变宽(P＜0.05),穿过Transwell小室膜的细胞数显著减少(P＜0.05),并呈浓度依赖性;CMSEE(20～30μg/mL)可明显下调B16细胞中MMP-2、MMP-9 mRNA及蛋白的表达(P＜0.05),并有浓度依赖性;而CMSEE对B16细胞中TIMP-1和TIMP-2蛋白的表达无明显影响(P＞0.05).CMSEE可有效抑制小鼠体内黑素瘤的侵袭和转移.结论:CMSEE在体内外均能明显抑制小鼠B16细胞的侵袭和转移能力,其作用机制可能与其抑制MMP-2和MMP-9的表达有关.     

木鳖子醇提物对黑素瘤B16细胞增殖的抑制及其可能机制

目的：观察木鳖子醇提物(ethanol extract from cochinchina momordica seed, CMSEE)对黑素瘤B16细胞增殖的影响,初步探讨其作用机制。方法：MTT法和克隆集落形成实验检测CMSEE对小鼠黑素瘤B16细胞生长的影响,倒置相差显微镜和瑞氏吉姆萨染色观察细胞形态学变化,流式细胞技术（FCM）检测B16细胞周期分布和凋亡率的变化,比色法检测CMSEE对B16细胞黑素生成和酪氨酸酶活性的影响,RTPCR法检测CMSEE对B16细胞中Cmyc、〖STBX〗P38和Tyr〖STBZ〗基因表达的影响。结果：CMSEE（10～100 mg/L）明显抑制B16细胞的增殖(P<0.05, P<0.01),呈质量浓度和时间依赖性。10～40 mg/L CMSEE处理后,B16细胞呈现典型的细胞分化形态,细胞周期阻滞于G0/G1期,黑素产生和酪氨酸酶活性增加(P<0.01),Cmyc基因表达下调,〖STBX〗P38和Tyr〖STBZ〗基因表达上调;100 mg/L CMSEE处理后的B16细胞呈现凋亡形态变化\[凋亡率达(37.2±329)%\],黑素生成减少(P<0.05),酪氨酸酶活性降低(P<0.01)。结论：CMSEE体外对B16细胞增殖有明显的抑制作用,其机制与其低剂量诱导分化和高剂量诱导凋亡有关。     

恶性肿瘤细胞及组织中凋亡抑制蛋白livin的表达及其临床意义

目的：探讨凋亡抑制蛋白livin的两种异构体livinα、和livinβ在多种肿瘤细胞系及肿瘤组织中的表达及其与各类肿瘤临床病理的关系。方法：采用RT-PCR法检测HeLa、SBC-2等12种肿瘤细胞和50例不同肿瘤组织及20例癌旁组织中livinα和livinβ的mRNA的表达水平。结果：12个细胞系中8个细胞系的livin mRNA表达为阳性，分别为HeLa、SPCA-1、SBC-2、PC14、MKN45、LOVO、HHCC、A549，其中A549为弱表达，且livinα的表达强于livinβ；HL-7702、Hep-2、7721、K562四种细胞中livin表达为阴性；50例肿瘤组织中livin mRNA表达阳性率为64．0％，而在癌旁组织中为2％。Livin mRNA的表达与肿瘤患者的肿瘤分化程度、淋巴结转移及TNM分期均无明显相关性。结论：Livin在肿瘤细胞系及肿瘤组织中表达的研究，为进一步研究livin与肿瘤的发生、发展的关系，及其以livin为靶位的肿瘤基因治疗研究提供了有力依据。     

人颊癌细胞信号转导抑制因子1沉默对细胞增殖及化疗药物敏感性的影响

目的:探讨人颊癌细胞中细胞因子信号转导抑制因子1（SOCS1）沉默与细胞增殖及化疗药物敏感性影响的关系。方法:Western blot、PCR及定量PCR验证SOCS1干扰序列沉默人颊癌细胞系BcaCD885中SOCS1的表达。MTT法检测颊癌细胞对化疗药物敏感性的变化。细胞计数法观察细胞的增殖速度。流式细胞术检测细胞周期的变化。结果:将SOCS1干扰序列转染BcaCD885细胞后,SOCS1 mRNA及蛋白表达水平均明显下降。SOCS1表达抑制后,转染72h后,BcaCD885细胞数量较对照组显著减少（P＜0.05）。化疗药物卡铂和紫杉醇对BcaCD885细胞的半数抑制浓度（IC50）均显著降低（P＜0.01）。SOCS1表达抑制后BcaCD885细胞G0/G1期细胞比例明显升高,而S期和G2/M期细胞比例明显降低,与对照组比较,差异均具有统计学意义（P＜0.05）。结论:SOCS1基因沉默后,人颊癌细胞株BcaCD885的增殖能力下降,并增强对化疗药物的敏感性。     

EGFL6基因沉默对人黑素瘤细胞增殖及侵袭的影响

目的 :通过特异性小干扰RNA(small interference RNA,si RNA)抑制人黑素瘤A375细胞中表皮生长因子样结构域蛋白6(epidermal growth factorlike-domain 6,EGFL 6)基因表达,探讨EGFL 6基因沉默对人黑素瘤细胞增殖及侵袭的影响。方法:设计合成靶向EGFL 6基因的siRNA(命名为EGFL6-siRNA),通过脂质体转染法将该siRNA转染入黑素瘤A375细胞,然后采用实时荧光定量PCR法和蛋白质印迹法分别检测黑素瘤A375细胞中EGFL6 mRNA及蛋白的表达水平,细胞计数试剂盒-8(cell counting kit-8,CCK-8)法和Transwell实验分别检测A375细胞增殖和侵袭能力的变化。结果:在成功转染EGFL6-siRNA 48 h后,A375细胞中EGFL6 mRNA及蛋白表达水平均明显下降(P值均<0.01)。EGFL6-siRNA转染组A375细胞在转染后24~72 h各时间点的细胞增殖能力均受到明显抑制(P值均<0.01),且细胞侵袭能力显著降低(P<0.01)。结论:特异性siRNA能够有效地沉默人黑素瘤A375细胞中EGFL 6基因的表达,并抑制肿瘤细胞的增殖和侵袭。     

lncRNA CDKN2B-AS1对黑色素瘤B16-F10细胞恶性生物学行为的影响

目的：探讨长链非编码RNA CDKN2B反义RNA1（long non-coding RNA CDKN2B-antisense RNA 1, lncRNA CDKN2B-AS1）通过靶向miR-7-5p影响黑色素瘤B16-F10细胞的恶性生物学行为的影响。方法：选用黑色素瘤B16-F10细胞,构建shRNA CD‐KN2B-AS1载体并转染到B16-F10细胞,实验分为对照组、sh-CDKN2B-AS1 组、miR-7-5p mimics 组、miR-7-5p inhibitor组。用RT-PCR检测转染后B16-F10细胞中CDKN2B-AS1表达水平,用克隆形成实验和MTT法检测克隆形成数及细胞的增殖能力,用划痕愈合实验和Transwell实验检测细胞的迁移和侵袭能力。用荧光素酶报告基因实验检证CDKN2B-AS1和miR-7-5p相互间靶向关系。用RT-PCR和Western blotting检测转染miR-7-5p mimics、inhibitor后B16-F10细胞中miR-7-5p和Ki67、cleaved caspase-3、上皮钙黏蛋白（E-cadherin）、神经钙黏蛋白（N-cadherin）、Twist1蛋白的表达水平。结果：与对照组比较,sh-CDKN2B-AS1组 B16-F10细胞中CDKN2B-AS1表达水平显著下调（P<0.01）,细胞的增殖、迁移及侵袭能力显著下降（均P<0.01）。荧光素酶报告基因实验证明 CDKN2B-AS1 与 miR-7-5p 存在靶向作用关系。sh-CDKN2B-AS1 组与 miR-7-5p mimics 组细胞中 miR-7-5p 和cleaved caspase-3、E-cadherin水平均明显上调（均P<0.05）,Ki67、N-cadherin、Twist1水平明显下调（均P<0.05）。结论：CDKN2BAS1通过靶向miR-7-5p促进黑色素瘤发展,干扰CDKN2B-AS1可抑制黑色素瘤B16-F10细胞的恶性生物学行为。     

Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A

Immunotherapy based on the administration of the mycobacterium bacillus Calmette-Guerin has been successfully used in the treatment of in situ transitional cell bladder cancer, and may be applicable to the treatment of cutaneous malignant melanoma. Antigen 85A (Ag85A) and heat shock protein 65 kDa (hsp65) are major secreted proteins of Mycobacterium species and potent stimulators of cell-mediated immunity. This study evaluated the ability of Ag85A and hsp65 gene transfection to limit tumor growth by B16-F10 mouse melanoma cells. Immunoblotting confirmed protein expression and secretion by B16-F10 cells that were transiently transfected with plasmid DNA containing the Ag85A or hsp65 gene. Groups of syngeneic C57BL/6 mice were injected subcutaneously with 1x10(5) untransfected B16-F10 cells or B16-F10 cells transiently transfected with either empty vector or vector containing the Ag85A or hsp65 gene. Ag85A-expressing B16-F10 cells exhibited a dramatic 76% reduction (p<0.05, Mann-Whitney U test) in tumor weight in comparison to empty vector controls at 14 days post-inoculation. In contrast, hsp65-transfected B16-F10 cells did not show any change in tumorigenicity. Decreased tumorigenicity by Ag85A-transfected B16-F10 cells was not due to a reduced ability of Ag85A-transfected B16-F10 cells to proliferate since both mock- and Ag85A-transfected B16-F10 cells showed increased in vitro proliferation in comparison to untransfected cells. Hematoxylin and eosin staining revealed that Ag85A-transfected B16-F10 tumors contained an inflammatory leukocyte infiltrate that was not present in hsp65-transfected tumors. Reduced tumor progression by Ag85A-transfected B16-F10 melanoma cells suggests that immunotherapy based on the transient induction of Ag85A expression may be an effective approach for the treatment of cutaneous malignant melanoma.     

Identification of B16-F1 melanoma autocrine motility-like factor receptor

B16-F1 melanoma cells express augmented glycosylation of a Mr 78,000 (gp78) cell surface glycoprotein in response to cell shape modulation which is correlated to an increased metastatic ability in vivo and motility in vitro. A monoclonal antibody (mAb) directed against gp78 was used to study its surface distribution and possible function in cell locomotion. On motile cells, gp78 is localized by immunofluorescence to the leading lamella as well as to the trailing edge, suggesting shuffling of gp78 during cell migration. When bound to the cells the mAb induced locomotory activity similar to the effect of the cells' autocrine motility-like factor (AMLF). The enhanced motility induced by either anti-gp78 mAb or autocrine motility factor (AMF) were both inhibited by pertussis toxin, indicating that the 3F3A mAb induces cell kinesis via the same pertussis toxin-sensitive G protein pathway as has been described for other motility factors. The binding of anti-gp78 mAb to its ligand was inhibited (10-fold) by preincubation with B16-F1 AMLF containing conditioned media. Based on such functional properties, it was concluded that gp78 behaves as an AMF receptor of the B16-F1 melanoma cell.     

Modulation of the lung colonization of B16-F1 melanoma cells by citrus pectin.

CONTEXT: Studies have shown that the galactoside-containing simple sugars and anti-galactoside-binding lectin antibodies may affect experimental tumor cell metastasis. However, the limited number of reagents used thus far necessitate further observations. PURPOSE: Natural citrus pectin (CP) and pH-modified CP (MCP), rich in galactose residues, were used to study the involvement of carbohydrates containing galactoside residues in cellular interaction in vitro and in lung colonization in vivo of B16-F1 melanoma cells. METHODS: B16-F1 melanoma cells were incubated with various concentrations of CP and MCP. Their ability to form homotypic aggregation in vitro and tumor lung colonization in vivo in 8-week-old female C57BL/6 mice was then analyzed. RESULTS: The CP binds to the surface of B16-F1 melanoma cells; this binding can be inhibited by lactose at a concentration of 0.15 M. Intravenous injection of the murine B16-F1 melanoma cells with the natural CP resulted in a significant increase (up to threefold) in the appearance of tumor colonies in the lung and in increased homotypic aggregation properties of the cells, while injection of MCP significantly decreased B16-F1 experimental metastasis (greater than 90%). CONCLUSIONS: Tumor galactoside-binding proteins mediate cellular recognition by linking oligosaccharides with terminal D-galactoside residues on adjacent cells. Successful interference with such a process with MCP may lead to a reduced ability to form tumor cell emboli and metastasis. IMPLICATIONS: These findings imply that the galactose-containing carbohydrate side chains of CP might mimic or compete with the natural ligand(s) of the tumor galactoside-binding protein (gal-lectin) and thus affect cellular interactions relevant for metastasis.     

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.     

Neural crest cell migration of mouse B16-F1 melanoma cells transplanted into the chick embryo is inhibited by the BMP-antagonist noggin

Melanoma cells are derived from the neural crest and characterized by high migratory potential and invasive growth. To test the analogies between malignant and embryonic cell migration, in previous studies we transplanted melanoma cells and non-transformed mouse neural stem cells into the neural crest compartment of the chick embryo. Human and mouse melanoma cells spontaneously migrated along the neural crest pathways while emigration of neural stem cells was dependent on pre-treatment with BMP-2 (bone morphogenetic protein-2). In the embryo neural crest cell migration is induced by BMP and inhibited by its antagonist noggin. We tested whether the spontaneous neural crest cell migration of melanoma cells was dependent on their endogenously expressed BMP and could be inhibited by noggin. Mouse B16-F1 melanoma cells transfected with GFP-VASP (vasodilator-stimulated phosphoprotein) were cultured as aggregates and treated with BMP-2 or noggin. Untreated and treated aggregates were transplanted into the neural tube of the E2 chick embryo. Untreated and BMP-2-treated melanoma cells emigrated from the neural tube along with the chick host neural crest cells. Noggin-treated aggregates showed no emigration. We conclude that spontaneous emigration of melanoma cells depends on their constitutive overexpression of BMP, and that noggin efficiently suppresses the emigration of melanoma cells in the embryonic micro-environment, thus rendering noggin a promising agent for the inhibition of melanoma cell migration in vivo.     

Anti-tumor effect of adenovirus-mediated gene transfer of pigment epithelium-derived factor on mouse B16-F10 melanoma

Background

Angiogenesis plays an important role in tumor growth, invasion, and eventually metastasis. Antiangiogenic strategies have been proven to be a promising approach for clinical therapy for a variety of tumors. As a potent inhibitor of tumor angiogenesis, pigment epithelium-derived factor (PEDF) has recently been studied and used as an anticancer agent in several tumor models.

Methods

A recombined adenovirus carrying PEDF gene (Ad-PEDF) was prepared, and its expression by infected cells and in treated animals was confirmed with Western blotting and ELISA, respectively. Its activity for inhibiting human umbilical vein endothelial cell (HUVEC) proliferation was tested using the MTT assay. C57BL/6 mice bearing B16-F10 melanoma were treated with i.v. administration of 5 × 10⁸ IU/mouse Ad-PEDF, or 5 × 10⁸ IU/mouse Ad-Null, or normal saline (NS), every 3 days for a total of 4 times. Tumor volume and survival time were recorded. TUNEL, CD31 and H&E stainings of tumor tissue were conducted to examine apoptosis, microvessel density and histological morphology changes. Antiangiogenesis was determined by the alginate-encapsulated tumor cell assay.

Results

The recombinant PEDF adenovirus is able to transfer the PEDF gene to infected cells and successfully produce secretory PEDF protein, which exhibits potent inhibitory effects on HUVEC proliferation. Through inhibiting angiogenesis, reducing MVD and increasing apoptosis, Ad-PEDF treatment reduced tumor volume and prolonged survival times of mouse bearing B16-F10 melanoma.

Conclusion

Our data indicate that Ad-PEDF may provide an effective approach to inhibit mouse B16-F10 melanoma growth.     

Oxidative burst of neutrophils against melanoma B16-F10

Intensive oxidative burst was determined by chemiluminescence of peripheral blood neutrophils of mice that were intramuscularly injected with melanoma B16-F10 and/or subcutaneously with Sephadex G-200. The neutrophils from papula developed at the site of Sephadex injection were cytotoxic for the B16-F10 cells in vitro. However, survival of Sephadex injected tumour-bearing mice was lower than of control animals bearing B16-F10, while their tumours grew faster and were less necrotic. Thus, it is likely that injection of Sephadex distracted the neutrophils from the tumour allowing faster progression of the tumour, indicating that neutrophils may have an important role in the host defence against malignant cells in the early stage of tumour development.