Morphological and Cytogenetic Characterization and N-myc Oncogene Analysis of a Newly Established Neuroblastoma Cell Line

A permanent cell line established from a xenograft of neuroblastoma which occurred in a 5 year old girl was investigated for its morphological and biological characteristics. The cultured cells were tumorigenic in nude mice. Microscopically, each tumor consisted of small round to polygonal cells with irregular nuclei and prominent nucleoli, corresponding to the features of the primary and xenografted tumor cells. Electron microscopic examination revealed that both the transplanted tumor cells and the cultured cells contained scanty microtubules and dense-core neurosecretory granules. Chromosome analysis of this cell line showed monosomy for chromosomes 1,10,19 and X, and structural rearrangements involving chromosomes 8, 17 and 20, in addition to numerous double minutes. The N- myc oncogene was found to be amplified 40 to 80 fold in the transplanted and cultured tumor cells, as well as in the primary tumor cells. In situ hybridization with a digoxigenin labeled uridine-triphosphate N- myc RNA probe detected abundant mRNA in the tumor cells. This neuroblastoma line may become a valuable in vitro experimental model system for studies aimed at better characterization of neuroblastoma. Acta Pathol Jpn 41: 507 515, 1991.     

β-Endorphin alters the course of central nervous system disease induced by a temperature-sensitive vesicular stomatitis virus in reconstitued nude mice

A 100 plaque forming unit (pfu) dose of a temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), tsG31 KS5, engendered a slowly progressive paralytic central nervous system (CNS) disease that killed all BALB/c nude mice within 28 days. Reconstitution of nude mice with 10⁷ syngeneic splenocytes 24 h before intracerebral inoculation with tsG31 KS5 VSV, however, protected 92% of the animals from death. When these reconstituted animals were injected intracerebroventricularly with 14 pmol of β-endorphin 24 h after reconstitution with splenocytes and 24 h befor inoculation with tsG31 KS5 VSV, only 72% of the animals survived. Furthermore, whereas 40% of the afflicted reconstituted nude mice given intracerebroventricular injections of sterile water were able to recover from the symptoms of disease, those surviving animals which received β-endorphin were unable to do so. A single intravenous injection of 14 pmol β-endorphin, or repeated postinfection administration of 28 pmol of β-endorphin intravenously into nude mice reconstituted with syngeneic splenocytes, which were pretreated with β-endorphin, did not alter the course of CNS disease induced by tsG31 KS5 VSV. The effect induced by intracerebroventricular injection of β-endorphin as antagonized by naloxone, but not by the neuropeptide fragment β-endorphin-(1–27). A simultaneous intracerebroventricular injection of reconstituted nude mice with 1220 pmol of naloxone and 14 pmol of β-endorphin resulted in a 89% survival rate, and 33% of the afflicted animals were able to overcome the symptoms of the disease induced by tsG31 KS5 VSV. Intracerebroventricular injection of reconstituted nude mice with 330 pmol of β-endorphin-(1–27) and 14 pmol of β-endorphin resulted in a 72% survival rate and the surviving animals were unable to improved appreciably the clinical status of their disease. Injection of reconstituted nude mice with either 1220 pmol of naloxone or 330 pmol of β-endorphin-(1–27) alone did not alter the course of the CNS disease in any way. A single intracerebroventricular injection of 29 pmol of another psychoactive peptide, [Des-Tyr]-endorphin, 24 h after reconstitution of nude mic with splenocytes and 24 h prior to infection with virus, resulted in 74% survival; and 39% of the afflicted animals were able to recover from the clinical symptoms.     

2-氯乙基-3-肌氨酸酰胺-1-亚硝脲对荷人脑胶质瘤动物模型的疗效分析

目的　探讨新型亚硝脲类抗癌药２氯乙基３肌氨酸酰胺１亚硝脲（ Ｓａｒ Ｃ Ｎ Ｕ） 在动物体内抗人脑胶质瘤的效果。方法　腹腔给 Ｓａｒ Ｃ Ｎ Ｕ 和 Ｂ Ｃ Ｎ Ｕ、 Ｖ Ｍ２６ ，观察位于皮下和颅内移植瘤的生长情况、宿主的体重变化。结果　 Ｓａｒ Ｃ Ｎ Ｕ 与对照组相比能明显抑制皮下移植瘤的生长和延长宿主的生存期。结论　 Ｓａｒ Ｃ Ｎ Ｕ 比目前临床上常用的 Ｂ Ｃ Ｎ Ｕ 和 Ｖ Ｍ２６ 具有更强的抑制人脑胶质瘤生长的作用。     

导入FasL基因对荷肝癌裸鼠肿瘤增殖影响的实验研究

目的　对 PA 317/ Fas L - p L XSN在裸小鼠体内抗肿瘤作用及机理作进一步探讨。方法　制备 PA317/ p KXSN -Fas L +病毒上清及转 Fas L CBRH7919细胞株。 6 0只实验动物 ,随机分成 4组。收集处于对数生长期的 CBRH 7919肝癌细胞 ,A、C、D三组每只裸小鼠接种肝癌细胞于腋窝皮下。 A组 (对照组 ) :接种 5天后 ,在腋窝部肿瘤内注射生理盐水 ;B组 :接种Fas L - CBRH7919制备模型 ;C组 :实验方法同 A组 ,在腋窝部肿瘤内注射 PA 317/ p L XSN- Fas L +病毒上清 ;D组 :实验方法同 C组 ,在腋窝部肿瘤内注射 PA 317/ p L XSN空载体。肿瘤细胞接种后测量、计算肿瘤的体积 ;间接免疫荧光流式细胞仪(flowcytom eter,FCM)检测各组肿瘤组织 Fas L的表达情况。采用 PI- Annexin V方法 ,用 FCM检则各组肿瘤细胞凋亡情况。结果　观察时间为 2 0天 ,B、C组肿瘤生长受到明显抑制 ,但并未完全消退 ,且与对照组有显著差异 ;D组肿瘤生长与对照组无显著差异。 B、C组肿瘤组织中 Fas L表达显著高于 A、D组 (分别为 72 .3% ,6 8.6 % vs7.2 % ,11.3% ,P<0 .0 1) ;与此对应 ,B、C组肿瘤组织中细胞凋亡显著高于 A、D组 (分别为 30 .5 % ,31.3% vs 12 .1% ,19.4 ,P<0 .0 1)结论　采用逆转录病毒载体将Fas L基因导入裸小鼠肝癌组织 ,     

重组人可溶性凋亡配体相关蛋白联合顺铂抑制肺癌裸鼠移植瘤的生长

目的:探讨重组人可溶性凋亡配体相关蛋白(rhTRAIL)和顺铂联用对抑制人肺腺癌细胞系A549裸鼠移植瘤的生长有无协同增效作用。方法:将24只荷人A549肺腺癌BALB/CA-nu裸鼠随机分成4组,(1)阴性对照组;(2)rhTRAIL组(1μg/mL,隔日1次×8次);(3)顺铂组(1.5mg/kg,每周2次×4次);(4)联合用药组(rhTRAIL1μg/mL,隔日1次×8次 顺铂1.5mg/kg,每周2次×4次)。分别测量瘤重、瘤体积,计算肿瘤生长指数和抑瘤率,并观察各组移植瘤组织的毒副作用和病理形态结构。结果:阴性对照组肿瘤生长指数为7.63±2.01,而rhTRAIL组和顺铂组分别为4.15±1.04和4.37±0.93,联合用药组则减低至1.69±0.37。rhTRAIL组、顺铂组的抑瘤率分别为36.8%和30.3%,联合用药组则增高达69.5%。rhTRAIL组、顺铂组和阴性对照组比较,差异具有显著性(P<0.05);联合用药组和其他3组比较差异均有显著性(P<0.01)。各组裸鼠的毒副作用轻微,治疗前后体重无明显变化。结论:rhTRAIL、顺铂均可抑制A549裸鼠移植瘤的生长,rhTRAIL和顺铂联用具有协同增效作用。rhTRAIL有望成为一种重要的生物制剂应用于肺癌的多学科综合治疗。     

Fusion of <Emphasis Type="Italic">Gaussia</Emphasis> Luciferase to an Engineered Anti-carcinoembryonic Antigen (CEA) Antibody for <Emphasis Type="Italic">In Vivo</Emphasis> Optical Imaging

The bioluminescent protein Gaussia luciferase (GLuc) was fused to an anti-carcinoembryonic antigen (CEA) antibody fragment, the diabody, for in vivo optical tumor imaging. A 15-amino acid N-terminal truncation (GLΔ15) resulted in a brighter protein. Fusions of the anti-CEA diabody to full-length GLuc and GLΔ15 retained high affinity for the antigen, emitted light, and exhibited excellent enzymatic stability. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-GLΔ15 to CEA-positive xenografts, with a tumor/background ratio of 3.8 ± 0.4 at four hours after tail-vein injection, compared to antigen-negative tumors at 1.3 ± 0.1 (p = 0.001). MicroPET imaging using ¹²⁴I-diabody-GLΔ15 demonstrated specific uptake in the CEA-positive tumor (2.6% ID [injected dose]/g) compared to the CEA-negative tumor (0.4% ID/g) at 21 hours. Although further optimization of this fusion protein may be needed to improve in vivo performance, the diabody-GLΔ15 is a promising optical imaging probe for tumor detection in vivo.     

HCV体内感染裸鼠模型的建立

目的:探讨利用裸鼠建立感染丙型肝炎病毒(HCV)体内动物模型的可能性。方法:将体外感染了HCV的人胎肝细胞(HFH)移植到裸鼠脾脏。移植术后3个月,取脾作病理学检查,以评估感染了HCV的HFH是否还存活。并且通过透射电镜检查,以评估移植入裸鼠脾脏的HFH胞浆中是否仍有HCV。同时,取裸鼠的外周血液作液相RT-PCR检查,以明确外周血液中HCV RNA是否为阳性。结果:移植术后3个月,移植入裸鼠脾脏内的HFH依然存活,经检查移植入裸鼠脾脏中的HFH胞浆内仍然存在HCV样颗粒。然而,体内有HCV感染的裸鼠外周血液仅70％HCV RNA为阳性。结论:裸鼠可作为“活试管”,支持体外感染了HCV的HFH在其脾脏内较长期生存。但仅使用外周血液HCV RNA检查这一个指标来判断裸鼠体内是否有HCV感染是不够的。     

顺铂结合CIK细胞对裸鼠人胃癌模型体内的抗肿瘤作用

目的:探讨顺铂、CIK细胞以及顺铂联合CIK对裸鼠人胃癌移植瘤生长的抑制作用,为临床上联合应用顺铂和CIK细胞治疗胃癌提供实验依据.方法:应用淋巴细胞分离液分离外周血单个核细胞,给予多种细胞因子(rhIFN-g、CD3mcAb、rh1L-1、rhlL-2),诱导生成CIK细胞;培养人胃癌细胞株SGC-7901,接种至80只裸鼠右腋皮下,10 d后取移植瘤直径基本一致的裸鼠随机分4组:NS对照组、顺铂组、CIK细胞组和顺铂+CIK细胞组,每组16只.连续5 d在接种肿瘤细胞部位处给予相应注射治疗,观察其对胃癌移植瘤模型的抗肿瘤疗效.结果:与NS对照组相比,顺铂组、CIK细胞组、顺铂+CIK细胞组裸鼠人胃癌移植瘤模型治疗后肿块质量均减轻,生存期均明显延长,尤以顺铂+CIK组效果显著(P<0.01).裸鼠体内实验表明,顺铂+CIK细胞联合应用能够显著抑制胃癌细胞的生长,其抑瘤率可达57.8%,明显高于NS对照组(P<0.01).免疫功能检测显示红细胞C3b反应受体明显升高,红细胞免疫复合物受体明显降低(P<0.01).结论:顺铂联合CIK细胞对人胃癌移植瘤生长的抑制作用大于单独应用顺铂或CIK细胞.     

采用在体分时取样法研究2种咪喹莫特乳膏的裸鼠皮肤药动学行为

目的:采用裸鼠在体分时取样法比较国产和进口2种咪喹莫特乳膏的皮肤药动学行为。方法:分别在裸鼠相同面积的皮肤上涂抹2种乳膏各约0.03g(n=32)后,于0.25、0.5、1、2、4、6、8、24h取皮制备匀浆,采用高效液相色谱法测定皮肤中的药物浓度,比较2种制剂的药动学参数并计算偏差以对该方法进行评价。结果:国产和进口乳膏的咪喹莫特cmax分别为(82.80±8.13)、(76.46±13.08)μg·g-1,tmax分别为(6.0±0.0)、(5.0±1.2)h,t1/2分别为(25.28±4.27)、(23.30±7.42)h,AUC0～24h分别为(992.27±226.55)、(1037.02±175.93)μg·h·g-1,二者比较均无统计学差异(P>0.05)。2种制剂cmax、tmax、AUC0～24h的RSD分别为9.8%、17.1%,0、24.0%,22.8%、17.0%。结论:2种乳膏的裸鼠皮肤药动学行为无显著性差异;主要药动学参数的RSD偏差较小,表明在体给药分时取样法能较好地反映药物在裸鼠体内的经皮代谢过程,适用于临床前外用药物的皮肤药动学筛选与评价研究。     

Growth-promoting effect of bisphenol A on neuroblastoma in vitro and in vivo

Purpose

To investigate the effect and mechanism of bisphenol A (BPA), one of the main environmental endocrine disruptors, on the proliferation of human neuroblastoma cells.

Methods

In vitro, cultured SK-N-SH cells were treated with 17β-estradiol (E₂; 1 ng/mL), BPA (2 μg/mL) with or without estrogen receptor antagonist ICI182,780 (10⁻⁶ mol/L). Viable cell number, DNA proliferation index, and expression of cyclin-dependent kinase 4 and cyclin D1 were assessed by absorbance reading, flow cytometry, and western blotting, respectively. In vivo, ovariectomized nude mice bearing SK-N-SH tumors were administered by gavage with E₂ (500 μg/kg per day, n = 11), BPA (200 mg/kg per day, n = 10), or vehicle (n = 9) for 18 days. Mice body weight, tumor volume and weight were examined every 3 days. Tumor microvessel density, proliferating cell nuclear antigen and vascular endothelial growth factor expression were evaluated by immunohistochemical staining or western blotting.

Results

In vitro, the BPA group had 20% higher number of viable cells, 70% higher proliferation index (both P < .01), and higher expression of cyclin-dependent kinase 4 and cyclin D1 than the nontreated group. In vivo, the BPA group had over 50% higher gross tumor volume, tumor weight, microvessel density, proliferating cell nuclear antigen (P < .05 or .01), and higher vascular endothelial growth factor protein expression than the mock control group. Both in vitro and in vivo BPA effects were comparable with those by E₂. ICI182,780 effectively abolished the promoting effect for both.

Conclusions

Bisphenol A can promote the growth of neuroblastoma to a level similar to that of E₂. Estrogen receptor-dependent pathway and angiogenesis may contribute to the underlying mechanisms.