肿瘤细胞在活体小鼠肝脏微循环中动态变化的可视性研究

目的：在小鼠活体内动态观察肿瘤细胞在肝脏微循环中的运动、分布及凋亡。方法：用Calcein AM荧光标记B16F1黑色素瘤细胞,将其注射入C57BL/6小鼠的肠系膜静脉,在荧光倒置显微镜下动态观察肿瘤细胞在肝脏微循环中的运动及分布,测量并计算肿瘤细胞在终末门静脉中的运动速度。注射肿瘤细胞8小时后,切取肝脏,用凋亡标记荧光素对肝组织切片进行原位DNA末端标记,在荧光显微镜下计算凋亡细胞百分比。结果：B16F1黑色素瘤细胞进入肝脏微循环中后,90%的细胞通过机械性嵌顿停留在肝窦中,10%的肿瘤细胞通过黏附停留在终末门静脉中,B16F1细胞在肝脏终末门静脉中的运动速度为648±53μm/s。在注射肿瘤细胞8小时后,在肝窦中约有30%的肿瘤细胞发生凋亡,在终末门静脉中约有10%的肿瘤细胞发生凋亡。结论：利用荧光标记的肿瘤细胞及荧光倒置显微镜为探讨肿瘤肝转移机制提供了一个良好的活体观察方法。     

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。     

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.     

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.     

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.     

小鼠黑色素瘤某些生物学特性与其侵袭潜能的相关性研究

目的 研究具有相同起源而转移能力不同的小鼠黑色素瘤细胞系（B16、B16F10、B16BL6）的某些生物学特性与其侵袭潜能的相关性。方法 用重组基质膜实验考察细胞的侵袭能力;用分光光度法测定黑色素瘤细胞的黑色素含量;利用细胞运动记录分析系统研究细胞在三维胶原基质中的运动状态;用明胶底物酶谱法分析细胞分泌Ⅳ型胶原酶的能力;用端粒重复扩增（TRAP）－PCR法测定细胞的端粒酶活性。结果 B16B6力B16F10具有较高的侵袭能力,其运动能力及分泌Ⅳ型胶原酶的能力亦较强,但B16F10的黑色素含量却较低;端粒酶活性在三者之间无明显差异。结论在所研究的小鼠黑色素瘤不同亚系的声东击西 些生物学特性中,侵袭能力与细胞运动能力、分泌Ⅳ型胶原酶的能力之间有较好的相关性,而与黑色素含量及端粒酶活性无直接相关性。     

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。      

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。     

增强型绿色荧光蛋白与MAGE-n共表达载体的构建及表达

目的构建增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)与人黑色素瘤抗原-n(melanomaantigenn,MAGE-n)真核共表达载体,并在小鼠黑色素瘤B16细胞中进行表达。方法采用酶切法从pLXSN-MAGE-n中得到MAGE-n基因片段,克隆至真核表达载体pIRES2-EGFP中,构建真核共表达质粒pIRES2-EGFP-MAGE-n。用脂质体法转染小鼠黑色素瘤B16细胞,G418筛选阳性克隆,荧光显微镜检测阳性克隆中EGFP的表达,RT-PCR、Westernblot和免疫组化分别检测阳性克隆中MAGE-nmRNA和蛋白的表达水平。结果从pLXSN-MAGE-n重组质粒中酶切获得一条约950bp的片段,成功构建了真核共表达载体pIRES2-EGFP-MAGE-n,转染B16细胞后筛选得到阳性克隆,可见到EGFP的表达,产生明亮的绿色荧光,RT-PCR检测到MAGE-nmRNA的表达,Westernblot和免疫组化检测到MAGE-n蛋白的表达。结论成功构建了共表达质粒pIRES2-EGFP-MAGE-n,获得了稳定共表达EGFP和MAGE-n的小鼠黑色素瘤B16细胞系,为进一步研究MAGE-n在肿瘤免疫治疗中的作用奠定了基础。     

p16基因重组质粒的构建及其对人肺癌细胞的抑制作用

表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,发现当上清仅占6.25%时即可对野生型B16F10细胞发挥明显的杀伤作用,提示AdCD/5FC介导的旁观者效应可能是通过5FC经CD酶代谢产生的毒性产物扩散而实现的.本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠经注射AdCD并连续10天给予5FC治疗后,与PBS、对照病毒AdLacZ/5FC治疗小鼠比较,小鼠肿瘤生长明显受到抑制,小鼠存活期明显延长.     

Vascular endothelial growth factor is an in vivo survival factor for tumor endothelium in a murine model of colorectal carcinoma liver metastases

BACKGROUND: Recent studies have suggested that vascular endothelial growth factor (VEGF), in addition to its proangiogenic properties, also functions as a survival factor for endothelial cells. The authors hypothesized that inhibition of VEGF activity by blockade of VEGF receptor-2 (R-2) function prevents angiogenesis and decreases tumor growth in colon carcinoma liver metastases. METHODS: Spleens of mice were injected with human colon carcinoma cells producing liver metastases. After 7 days of tumor growth, groups of mice received either antibody to VEGFR-2 (DC101) or phosphate-buffered saline (control). In a follow-up experiment, a similar treatment regimen was followed except that mice were sacrificed at 1-week intervals to assess the time course of endothelial cell and tumor cell apoptosis. RESULTS: After 21 days of therapy, the authors observed a significant decrease in vessel counts in liver metastases from human colon carcinoma in nude mice after therapy with VEGFR-2 antibody. Tumor cell apoptosis was increased significantly in the tumors of mice receiving DC101. Temporal studies with immunofluorescent double staining for the microvasculature and apoptotic cells revealed an increase in endothelial cell apoptosis that preceded an increase in tumor cell apoptosis. In vitro, treatment of human umbilical vein endothelial cells with antibody to VEGFR-2 produced a > 2.5-fold increase in endothelial cell apoptosis. CONCLUSIONS: Therapy targeting the VEGFR-2 inhibited tumor growth in a murine model of colon carcinoma liver metastasis. Surprisingly, this therapy did not only inhibit angiogenesis but also led to endothelial cell death. These findings suggest that VEGF, via VEGFR-2 signaling, functions as a survival factor for tumor endothelial cells in liver metastases from colon carcinoma.     

Sialyl Lewis-X抗原在大肠癌肝转移中的作用研究及临床意义

目的探讨SialylLewis-X(SLeX)抗原在大肠癌肝转移中的作用及临床意义。方法采用大肠癌Lovo、HT29细胞与人脐静脉血管内皮及内皮细胞的粘附试验,分别用扫描电镜及透射电镜观察Lovo、HT29细胞与脐静脉血管内皮及内皮细胞的粘附性及SLeX单抗封闭大肠癌细胞后,这种粘附性的改变;采用实验性裸鼠大肠癌肝转移模型分别观察SLeX单抗封闭Lovo、HT29细胞前后对实验性裸鼠大肠癌肝转移的影响。结果高表达SLeX抗原的Lovo细胞与脐静脉血管内皮的粘附性较低表达SLeX抗原的HT29细胞强,Lovo细胞与脐静脉血管内皮细胞的连接方式与HT29细胞明显不同;高表达SLeX抗原的Lovo细胞引起裸鼠肝转移率高于低表达SLeX抗原的HT29细胞。结论大肠癌细胞表面SLeX抗原在大肠癌细胞与脐静脉血管内皮细胞的粘附及实验性裸鼠肝转移中起重要作用,SLeX单抗能有效地抑制肿瘤细胞与脐静脉血管内皮细胞的粘附,并能降低实验性裸鼠肝转移的形成。     

B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis

The formation of liver metastases involves interactions between intravascular cancer cells and the hepatic microvasculature. Here we provide evidence that the arrest of intravascular B16F1 melanoma cells in the liver induces a rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. B16F1 melanoma cells (5 x 10(5)) labeled with fluorescent microspheres were injected into the portal circulation of C57BL/6 mice. The production of NO in vivo was detected by electron paramagnetic resonance spectroscopy ex vivo using an exogenous NO-trapping agent. A burst of NO was observed in liver samples examined immediately after tumor cell injection. The relative electron paramagnetic resonance signal intensity was 667 +/- 143 units in mice injected with tumor cells versus 28 +/- 5 units after saline injection (P < 0.001). Two-thirds of cells arrested in the sinusoids compared with the terminal portal venules (TPVs). By double labeling of B16F1 cells with fluorescent microspheres and a TdT-mediated UTP end labeling assay, we determined that the melanoma cells underwent apoptosis from 4-24 h after arrest. The mean rate of apoptosis was 2-fold greater in the sinusoids than in the TPVs at 4, 8, and 24 h after injection (P < 0.05-0.01). Apoptotic cells accounted for 15.9 +/- 0.8% of tumor cells located in the sinusoids and 7.1 +/- 0.9% of tumor cells in the TPVs. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester completely blocked the NO burst (P < 0.001) and inhibited the apoptosis of B16F1 cells in the sinusoids by 77%. However, the rate of tumor cell apoptosis in the TPVs was not changed. There were 5-fold more metastatic nodules in the livers of N(G)-nitro-L-arginine methyl ester-treated mice (P < 0.05). The inactive enantiomer N(G)-nitro-D-arginine methyl ester had no effect on the initial NO burst or on apoptosis of tumor cells in vivo. Both annexin V phosphatidylserine plasma membrane labeling and DNA end labeling of apoptotic cells were demonstrated after a 5-min exposure (a time equivalent to the initial transient NO induction in vivo) of B16F1 cells to a NO donor in vitro. These results identify the existence of a natural defense mechanism against cancer metastasis whereby the arrest of tumor cells in the liver induces endogenous NO release, leading to sinusoidal tumor cell killing and reduced hepatic metastasis formation.     

Regulation of B16F1 melanoma cell metastasis by inducible functions of the hepatic microvasculature

We have previously shown that circulating intravascular cells generally arrest by mechanical restriction in the hepatic sinusoids, causing rapid release of nitric oxide (NO) which is cytotoxic to these cells and inhibits their growth into metastatic tumours. Here, we present evidence that these NO-dependent cytotoxic mechanisms are susceptible to upregulation by lipopolysaccharide (LPS). Five x 10(5) fluorescently labelled melanoma cells were injected into the mesenteric vein of C57BL/6 mice to effect their localisation in the hepatic microvasculature. Test mice were then given 1 mg/kg LPS intraperitoneally (i.p.) to activate the microvascular cells. By electron paramagnetic resonance (EPR) spectroscopy, the expression of NO in the liver was significantly increased by 8 h in the LPS-treated mice. The non-selective NO synthase inhibitor L-NAME inhibited the induction of NO by LPS, while its inactive enantiomer D-NAME had no significant effect. Using immunohistochemistry (IHC), iNOS-positive microvascular cells were detected in the terminal portal venule (TPV) region of the liver 8 h after LPS stimulation. LPS treatment also increased the retention of melanoma cells in the liver between 8 and 24 h, especially in the TPV region. Eight hours after cell injection, local expression of VCAM-1 and ICAM-1 was detected by double-label immunohistochemistry at the sites of tumour cell arrest. Expression of these adhesion molecules was enhanced in mice treated with LPS. Using flow cytometry, 98% of the B16F1 melanoma cells expressed VLA-4, the counter receptor of VCAM-1, and approximately 1.5% expressed LFA-1, the counter receptor of ICAM-1. LPS did not significantly alter the expression of either counter receptor on melanoma cells in vitro or in vivo. By DNA end-labelling, the rates of melanoma cell apoptosis were significantly increased from 8 to 24 h in the TPV region (but not in the sinusoids) of LPS-treated mice. Fourteen days after tumour cell injection, the LPS-treated mice had a significantly smaller hepatic metastatic tumour burden than the control mice. These data suggest that LPS can inhibit the metastasis of melanoma cells in the liver by inducing the expression of NO and adhesion molecules by the hepatic endothelium. The induction of iNOS and the inducible cytotoxic effect of LPS appear to be primarily located within the TPV region of the liver acinus.     

重组人血管内皮抑素联合顺铂治疗小鼠肺转移癌实验研究

目的:探讨重组人血管内皮抑素(恩度)联合顺铂抑制小鼠肺转移癌的作用和机制。方法:取H22肝癌细胞株腹水瘤反复从小鼠尾静脉注入,建立高肺转移小鼠模型,随机分为4组:生理盐水、顺铂、恩度和顺铂与恩度联合治疗组,10只/组。从尾静脉接种后第7天腹腔内给药,1次/d,连用14 d,第21天处死,解剖出小鼠肺脏,称质量,计数肺转移结节数,并对肺转移瘤组织进行免疫组化染色,检测血管内皮生长因子(VEGF)表达,计数微血管密度。结果:生理盐水组、恩度组、顺铂组和联合治疗组肺转移率分别为100%、70%、60%和40%,肺转移结节数分别为(28.6±10.2)、(20.7±5.6)、(14.3±6.4)和(8.9±3.8)个,微血管密度计数为(31.4±3.1)、(24.6±5.9)、(21.2±4.7)和(15.2±3.1),各治疗组结果与生理盐水组比较差异均有统计学意义(P<0.05),联合组与顺铂组或恩度组比较也具有差异(P<0.05)。生理盐水组肺转移瘤组织的VEGF表达明显高于各治疗组(P<0.05)。结论:恩度单用或联合顺铂均能显著抑制H22肝癌肺转移组织的血管生成,降低了肺转移。     

Vaccination with viable human umbilical vein endothelial cells prevents metastatic tumors by attack on tumor vasculature with both cellular and humoral immunity.

PURPOSE: Because tumor endothelium is rarely targeted by immunity but is critically important for tumor growth, the immunity against tumor endothelium is to be developed as a novel antitumor strategy. EXPERIMENTAL DESIGN: First, viable human umbilical vein endothelial cells (HUVEC) were immunized to C57BL/6 and BALB/c mice to evoke specific CTLs as well as antibodies against tumor endothelium. Lewis lung carcinoma or myeloma cells were subsequently inoculated to evaluate the effect on tumor growth by vaccination. Second, the effect on tumor metastasis by vaccination was studied using tumor-resected mice receiving HUVEC immunization 3 days after excision. Third, the immune sera and T lymphocytes from HUVEC-immunized mice were transferred to tumor-bearing mice and added to cultured HUVECs to investigate their antiproliferative effect. RESULTS: Viable HUVEC immunization showed potent antitumor effects in Lewis lung carcinoma and myeloma tumor models. Both immune sera and CTL inhibited tumor growth and specifically suppressed proliferation of HUVECs. Particularly, tumors entirely disappeared on day 90 after tumor inoculation in four of six tumor-bearing mice receiving CTL therapy. In a metastatic tumor model, we found that the HUVEC vaccination prolonged life span from 30.9 to 41.5 days after tumor resection compared with PBS-treated mice without apparent side effects. CONCLUSIONS: Vaccination with viable HUVECs evoked both humoral and cellular immunity against tumor microvasculature, and therefore significantly inhibited tumor growth and prolonged life span of tumor-resected mice. This may provide with a novel treatment for metastatic tumors. Moreover, we have established a convenient method to evoke specific CTL against tumor angiogenesis.     

基于共培养技术的肿瘤微血管细胞模型的建立

目的建立可研究肿瘤微血管的分子学特性及结构特性的细胞模型,应用于药物筛选及靶向药物输送的研究.方法通过TRANSWELL共培养肿瘤细胞MCF-7和内皮细胞HUVEC的方法,建立肿瘤微血管细胞模型,检测了该模型下的细胞形态、结构、及通透性的变化.结果在肿瘤细胞共生条件下的内皮细胞层结构疏松,微环境呈明显酸性,而且细胞间隙增大,对100纳米左右粒子的通透性有显著提高.结论这一细胞模型的建立将有助于肿瘤血管的分子、结构特性以及相关药物的筛选和输送研究.     

Inhibition of hepatocarcinoma by systemic delivery of Apoptin gene via the hepatic asialoglycoprotein receptor

Specificity is a prerequisite for systemic gene therapy of hepatocarcinoma. In vitro, the tumor-specific viral death effector Apoptin selectively induces apoptosis in malignant hepatic cells. Intratumoral treatment of xenografted subcutaneous hepatomas with Apoptin results in tumor regression. Here, we report a systemic delivery vehicle containing the Apoptin gene linked to asialoglycoprotein (Asor), which targets asialoglycoprotein receptor (ASGPR) present only on the surface of hepatocytes. In vitro, the protein-DNA complex Asor-Apoptin induced apoptosis in HepG2 hepatocarcinoma cells but not in normal L-02 hepatocytes. Non-hepatocyte-derived tumorigenic human A549 cells lacking the membrane ASGPR were not affected by Asor-Apoptin. In vivo systemic delivery of Asor-Apoptin via the tail vein into mice bearing in situ hepatocarcinoma resulted in specific and efficient distribution of Apoptin in both hepatocarcinoma cells and normal hepatocytes. Five days after injection of Asor-Apoptin, the in situ hepatocarcinomas showed significant signs of regression, whereas the surrounding normal hepatocytes did not. Systemically delivered Asor-LacZ expressing non-apoptotic LacZ gene did not inhibit tumor growth. Our data reveal that systemic delivery of Asor-Apoptin specifically induces apoptosis in malignant hepatocytes and thus constitutes a powerful and safe therapeutics against hepatocarcinomas.