人大肠癌裸小鼠直肠黏膜原位种植癌及转移模型的建立

目的 建立人大肠癌(HICC)组织块裸小鼠直肠黏膜原位种植癌及转移模型.方法 人结肠癌细胞株HCT-116注射接种于裸小鼠直肠黏膜下,获得直肠种植瘤.制备该肿瘤新鲜组织块,原位种植于直肠黏膜面,在2、4、6、8、10、12周取材,进行苏木素一伊红(HE)染色及CEA免疫组织化学检测,观察种植瘤成瘤率,生长情况及转移规律.结果 直肠黏膜组织块法原位种植2周后成瘤率达90%以上.肿瘤在6～12周成指数生长.种植4周见区域淋巴结(LN)部分转移,直肠上动脉LN转移率60%、系膜LN转移率20%;6周见腹主动脉旁LN转移,转移率40%;8周见区域LN均转移充分,分别为100%、80%、80%.10～12周见肝肺转移发生,分别为20%(1/5)、40%(2/5).LN转移发生在肿瘤指数生长之前.HE及CEA免疫组织化学证实LN、肝肺有癌细胞转移浸润.结论 裸小鼠直肠黏膜植块法能成功构建HICC原位模型.此模型操作简便、制模稳定、转移率高,且生物学行为更能模拟临床HICC发生、发展演变过程.     

两种结直肠癌肝转移动物模型建立方法的比较及评价

建立小鼠结直肠癌细胞肝转移动物模型,为研究结直肠癌肝转移机制及筛选评估抗转移药物提供工具。采用60只6～8周龄BALB／c小鼠,选取鼠大肠癌细胞株CT26接种于BALB／c小鼠直肠黏膜下层后,再随机选择30只小鼠行盲肠造口,观察其原位移植瘤体积大小、肝转移癌情况（包括肝脏表面转移癌结节大小、数目和转移率）,成瘤时间及小鼠的生存期。结果显示,两种造模方法均能复制出结直肠癌肝转移动物模型,病理结果证实肝转移肿瘤符合典型的低分化腺癌特征。术后30d两组肝脏转移情况的比较,直肠直接接种组肝转移率（15．7％）和直肠接种盲肠造口组肝转移率（61．5％）,差异有统计学意义（P〈0．05）。术后30d两组淋巴结转移情况比较,直肠直接注射组及直肠接种盲肠造口组淋巴结转移率分别为5．2％和46．2％,差异有明显统计学意义（P〈0．05）。结果表明,两种小鼠大肠癌细胞株肝转移动物模型均能较好模拟结直肠癌体内肝转移过程,直肠接种盲肠造口法的小鼠肝转移模型具有肝脏转移率高,小鼠生存期长,成瘤时间较短等优点。     

巨噬细胞移动抑制因子抗体抑制大肠癌生长与肝转移的研究

目的 观察巨噬细胞移动抑制因子(MIF)抗体对大肠癌生长与肝转移的抑制作用.方法 将CT26细胞接种到BALB/C小鼠项背部获得实体瘤,再对20只BALB/C小鼠行盲肠造疝原位接种瘤块术,术后随机将小鼠分为A、B两组,A组隔天腹腔注射MIF-抗体(400μg/只),B组隔天腹腔注射生理盐水,4周后处死小鼠,观测盲肠原位肿瘤和肝脏情况,并对肝组织进行连续病理切片以观察肝转移情况.ELISA方法检测小鼠血清MIF和MMP-9浓度.结果 MIF-抗体干预的小鼠盲肠原位肿瘤的重量较对照组低[(1.60±0.18)g比(2.11±0.25)g,P<0.01],两组间肝脏重量差异无统计学意义[(0.93±0.07)g比(0.96±0.07)g,P>0.05],但A组大肠癌肝转移率低于B组(1/10比7/10,P<0.05),且A组血清中MIF和MMP-9的浓度也低于B组[(21.15±1.59)ng /L比(35.65±1.34)ng/L,P<0.01;(0.19±0.01)μg/L比(0.28±0.04)μg/L,P<0.01].结论 MIF-抗体腹腔注射不仅抑制大肠癌原位肿瘤的生长,而且降低了大肠癌的肝转移率.     

血管生成拟态在结肠癌肝转移中的表达及分子机制研究

目的 建立结肠癌肝转移动物模型,探讨血管生成拟态在结肠癌肝转移动物模型中的表达规律及相关分子机制.方法 利用改良的盲肠造疝原位接种瘤块法建立BALB/C小鼠结肠癌肝转移模型,随机数字表法将小鼠分为对照组和实验组,每组各16只,CD31/PAS双染法观察血管生成拟态,配对t检验统计分析肝转移灶和原发灶中血管生成拟态表达规律.RT-PCR检测血管生成拟态形成相关基因cox-2、EPHA2基因、Twist基因等的表达,观察cAMP抑制剂对结肠癌肝转移血管生成拟态的影响.结果 BALB/C小鼠结肠癌肝转移模型原发灶和肝转移瘤中均可观察到血管生成拟态的存在,注射8-溴-cAMP后可以抑制结肠癌肝转移的发展进程;实验组血管生成拟态密度显著低于对照组(P＜0.05),实验组血管生成拟态形成相关基因cox-2、E-PHA2、Twist的表达均低于对照组(P＜0.05).结论 小鼠结肠癌肝转移模型中存在血管生成拟态现象,cAMP/PKA特异性抑制剂可以抑制血管生成拟态的形成,cAMP/PKA可能是结肠癌肝转移中血管生成拟态的调节通路.     

经肝内注射肿瘤细胞建立小鼠肝癌实验模型的效果观察

目的 探讨经肝内直接注射肝癌细胞建立小鼠肝癌原位模型的效果。方法 选取Balb/c小鼠来源的ML-3肝癌细胞经肝内直接注射接种至肝脏实质内,与经脾注射肝癌细胞相比较,对其肝内成瘤情况进行观察。结果 肿瘤细胞接种第6天起,经脾脏注射组以及经肝脏注射组小鼠均不同程度出现体重减轻、腹水、黄疸以及腹腔可触及包块。在经脾脏注射组,15只小鼠中仅有3只长出肝脏肿块,其余均腹腔种植转移。在经肝脏直接注射组,15只小鼠中有10只长出肝癌,其中伴腹腔种植转移1只。结论 经肝脏直接注射肿瘤细胞较经脾脏注射法能更有效诱导建立肝癌原位模型。     

C—erbB—2和P53基因产物在实验性人大肠癌裸鼠移植瘤和转…

为研究Ｃ－ｅｒｂＢ－２和Ｐ５３基因产物在实验性大肠癌裸鼠移植瘤和转移瘤中的表达，通过对人大肠癌细胞株ＨＴ２９（低分化腺癌）和Ｌｏｖｏ细胞株（未分化癌）进行ＢＡＬＢ／Ｃ裸鼠脾内接种，分别制成人大肠癌裸鼠移植国肝转移瘤模型。应用快速免疫组化法分别检测裸鼠移植瘤和转移瘤中Ｃ－ｅｒｂＢ－２和Ｐ５３基因产物的表达情况。结果表明：人大肠癌可出现多基因表达产物变化，Ｐ５３蛋白在两咱细胞株接种后的移植瘤和转移瘤中     

结肠癌原位瘤模型方法探讨

目的研究常用结肠癌原位瘤模型方法的优劣,建立稳定的结肠癌原位瘤成瘤模型及肝转移模型。方法采用CT26小鼠结肠癌细胞系直接接种于Balb/c小鼠盲肠浆膜下层,或皮下成瘤后采用组织原位种植接种于小鼠盲肠浆膜下层。分析接种4周后原位成瘤及肝转移发生情况。另外,将CT26细胞分别接种于小鼠的小肠浆膜下和盲肠浆膜下。比较接种后成瘤效果及并发症发生率。结果采用细胞注射和组织种植两种方法的原位成瘤率均为100%(5/5),但细胞注射组原位瘤明显大于组织种植组。细胞注射4周后肝转移率为60%(3/5),而组织种植组没有发生肝转移(P0.01)。小肠原位种植成瘤率为100%(5/5),但早期肠梗阻发生率为80%(4/5),而盲肠注射组早期未见肠梗阻发生。结论就接种类型而言,细胞原位注射较组织原位种植更适合结肠癌肝转移模型;而就注射部位而言,结肠癌肝转移模型应首选盲肠注射,而非小肠注射。     

人胆囊癌裸鼠原位移植瘤模型的建立及初步观察

胆囊癌恶性程度较高，临床预后较差。为了寻找一种能更接近人体内胆囊癌特性的动物模型，我们通过瘤组织块原位接种建立了裸小鼠的胆囊癌原位移植瘤模型，现报告如下。     

裸鼠人肾癌SOI模型的建立及高转移亚株的筛选

目的：建立人肾癌裸鼠转移模型，并分离肺转移亚株。方法；用自建肾癌细胞系RCC-9863建立裸鼠SOI(surgical orthotopic implantation)模型。从裸鼠皮下移植瘤中取组织块，手术包埋于裸鼠左肾实质内。取肺结节，组织块法体外培养。癌细胞行左肾细胞悬液原位接种，重复1次，克隆筛选肺转移亚株。结果；15d左右裸鼠左肾区可触及包块，质硬，有结节。55d裸鼠出现恶液质。SOI模型肿瘤生长快，呈浸润性，肺脏、淋巴结、肝脏可见转移灶。转移株细胞增殖周期短，软琼脂集落形成率高，裸鼠皮下接种后，成瘤潜伏期短，瘤体大，并可以形成广泛肺转移。获得的高转移株命名为MRCC，其IV型胶原酶呈强阳性，增殖细胞核抗原（PCNA）高表达，nm23表达率与RCC-9863差别无显著性意义。结论：肾癌SOI模型的建立并筛选出一株肺脏高转移性细胞亚株，为肾癌高转移性研究提供了有效手段。     

一种新的高转移膀胱癌动物模型的建立

目的　建立一种简便、可靠的小鼠膀胱癌模型。方法　将绿色荧光蛋白 (GFP)表达质粒导入小鼠膀胱癌细胞株BTT T73 9,G418筛选稳定表达GFP的克隆。然后接种于同系小鼠膀胱壁内及后腿皮下 ,荧光显微镜直接观察肿瘤细胞生长、浸润与微转移 ,流式细胞术定量分析转移的肿瘤细胞数 ,并与常规石蜡切片苏木素 伊红 (HE)染色比较。结果　经GFP标记的肿瘤细胞在荧光显微镜下发出绿色荧光 ,膀胱原位接种 (2～ 4)× 10 5后 1周即出现膀胱肿大、血尿 ,以后膀胱肿大加重、出现输尿管阻塞、肾盂积水 ,以及肾、腹腔、肺、肝、脾等远处转移。结论　GFP标记的BTT T73 9膀胱癌细胞和膀胱癌动物模型可十分简便、准确判断膀胱癌肿瘤细胞生长、浸润与微转移等。     

红色荧光蛋白标记的胰腺癌裸鼠原位移植瘤转移模型的建立

目的 建立稳定、高表达红色荧光蛋白(RFP)标记的胰腺癌裸鼠原位移植瘤自发转移模型.方法 将稳定、高表达RFP的人胰腺癌细胞株SW1990-RFP注射至裸鼠皮下,建立人胰腺癌裸鼠皮下移植瘤模型.通过外科手术原位移植裸鼠皮下荧光肿瘤组织小块,建立人胰腺癌裸鼠原位移植瘤自发转移模型.应用整体荧光成像系统评价人胰腺癌裸鼠原位移植瘤模型肿瘤转移及微转移的发生情况.结果 成功建立12只人胰腺癌裸鼠原位移植瘤自发转移模型,建模成功率为100%.应用整体荧光成像系统可以实时监测原发肿瘤的牛长状况以及在肿瘤生长早期即可检测到各脏器微小转移的发生.结论 本实验所建立的RFP标记的胰腺癌裸鼠原位移植瘤自发转移模型稳定、可靠,可以在体外无创性动态观察肿瘤的发生、发展,具较强的灵敏性及特异性.     

Lymph node and pulmonary metastases after transplantation of oral squamous cell carcinoma cell line (HSC-3) into the subcutaneous tissue of nude mouse: detection of metastases by genetic methods using beta-globin and mutant p53 genes.

We attempted to establish an in vivo experimental model of metastasis of oral caner. The HSC-3 cell line, originating from human oral squamous cell carcinoma, was transplanted into nude mice, and metastases to the lymph node and the lung were investigated using genetic analysis. The HSC-3 cells, transplanted subcutaneously into the lateral back of nude mice, and punctured repeatedly after cyst formation, could metastasize to the lymph node and the lung. Genetic analysis using human beta-globin gene demonstrated that the frequency of metastasis to the lymph node and the lung at 12 weeks after the transplantation was 45% and 40%, respectively. Mutant allele-specific amplification of p53 gene could also detect micrometastasis with almost equal sensitivity of polymerase chain reaction with beta-globin gene. Subcutaneous transplantation is easy with excellent reproducibility compared with intravenous or orthotopic transplantation, and we think that this experimental model can be one of standard models of cancer metastasis.     

人肝癌裸鼠NKG2D受体的表达及对NK细胞毒活性的影响

目的 通过研究原发性肝癌中NK细胞的细胞毒活性变化及其活化性受体NKG2D的表达,观察肝脏和脾脏组织病理变化,探讨NK细胞免疫抑制机制,为原发性肝癌的免疫治疗提供理论依据。方法 ①建立人肝癌裸鼠皮下-肝原位移植瘤模型：先用人肝癌细胞株Hep3B接种于裸鼠皮下,形成皮下移植瘤,然后用此移植瘤组织再接种于裸鼠肝内,建立肝原位移植瘤模型（间接肝原位移植瘤模型）;②检测裸鼠原发性肝癌对NK细胞免疫活性的影响：分离裸鼠外周血、肝脏及脾脏组织NK细胞,LDH方法检测NK细胞的细胞毒活性,流式细胞技术检测不同组织NK细胞NKG2D表达百分率,H-E染色观察肝脏移植瘤对肝脏和脾脏淋巴细胞的影响。结果 ①外周血、肝脏及脾脏的NK细胞毒活性及NKG2D受体表达随着肿瘤生长逐渐下降,其中肝脏NK细胞毒活性及NKG2D表达下降明显;②荷瘤裸鼠肝脏癌组织与皮下瘤相同,癌组织异型性与时间呈正相关,脾脏淋巴小结在第4周增生明显,第8周小梁结构增多。结论 原发性肝癌通过下调NKG2D的表达,对NK细胞有免疫抑制作用,这种作用主要发生在肝脏,但对外周血和脾脏也有影响。     

Thrombospondin-1 promotes proliferative healing through stabilization of PDGF

BACKGROUND: Predicting liver metastasis from colon cancer is essential for improving its prognosis. We studied to what extent genetic detection of cancer cells in the resected liver tissue can predict the incidence of macroscopic liver metastasis with a similar mouse model to clinical colorectal cancer that causes a several decade percentage of metachronous hepatic metastases after resection of the primary lesions. MATERIALS AND METHODS: A LS174T human colorectal cancer cell suspension was injected into the spleens of nude mice. One to 10 days after splenic injection, 3 x 3 mm of liver tissue was removed, and a splenectomy was performed. Liver tissue was used for genetic detection and histological examination. Five weeks after splenic injection, the number of macroscopic metastases on the surface of the liver was counted. RESULTS: Eight of the 45 cases were positive for tumor cells in liver tissue genetically, while only 1 was positive for tumor cells histologically. Macroscopic liver metastases were seen 5 weeks after splenic injection in 11 of 37 (29.7%) cases negative for tumor cells genetically and in 8 of 8 (100%) cases positive for tumor cells genetically. Five or more metastases were seen in 3 of 37 (8.1%) cases negative for tumor cells genetically and in 7 of 8 (87.5%) cases positive for tumor cells genetically. CONCLUSIONS: The cases which were positive for tumor cells in liver tissue genetically at the time of splenectomy had more significantly macroscopic liver metastases some weeks later than the cases negative for tumor cells. This study suggests that if micrometastasis was detected genetically, the development of metachronous macroscopic liver metastasis could be predicted.     

胆管癌细胞系QBC939裸鼠肝门部胆管原位种植瘤模型的建立

目的建立胆管癌细胞系QBC939裸鼠肝门部胆管原位种植瘤模型并观察淋巴引流情况。方法应用我们前期建立的肝门部胆管癌细胞系QBC939,行裸鼠背部皮下接种,建立高转移特性胆管癌裸鼠皮下种植瘤模型,然后将高转移性种植瘤组织接种于裸鼠肝门部胆管与门静脉间组织间隙紧贴胆管处,4周及6周后行种植瘤瘤组织解剖学和病理学检查,并观察淋巴管引流情况。结果模型建立过程中,胆管癌细胞系QBC939细胞裸鼠皮下接种成瘤率为100%(10/10);原位种植4周后,肝门部胆管原位种植瘤成瘤率为100%(10/10),原位种植6周后,合并发生肝脏转移及腹腔淋巴结转移为80%(8/10)。结论成功建立了胆管癌细胞系QBC939裸鼠肝门部胆管原位种植瘤模型。     

三七总皂甙对大鼠移植肝缺血再灌注损伤的保护作用

摘要：目的:探讨三七总皂甙预处理对大鼠移植肝缺血再灌注损伤的保护作用。方法:建立大鼠原位肝移植模型，然后分为三七预处理组（P组）、对照组（N组）和假手术组（S组）3组，取血检查ALT，AST；用免疫组化法检测caspase-3和TNF-α的表达，TUNEL法检测肝细胞凋亡，行肝组织病理检查。结果:鼠肝移植模型N组的血ALT，AST数值、肝细胞中caspase-3和TNF-α的表达及肝细胞的凋亡均明显高于P组，差异均有显著意义（P<0.05）。结论:肝细胞凋亡加重移植肝缺血再灌注损伤，三七总皂甙预处理对大鼠肝移植缺血再灌注损伤有保护作用。     

Oral Administration of Uracil-Tegafur (UFT) Inhibits Liver Micrometastasis of Human Colon Cancer in an Orthotopic Nude Mouse Model and Its Early Detection System

Purpose To evaluate the effect of UFT (an oral antineoplastic drug combining uracil and tegafur) as an adjuvant chemotherapy.Methods We examined whether UFT inhibits micrometastasis of the liver from colon cancer implanted into the cecum of nude mice in an orthotopic model. Moreover, we studied whether our early detection system using a polymerase chain reaction (PCR) of the human -globin gene would be useful in this model.Results The administration of 20mg/kg UFT p.o., which is a relatively small dose compared with 65mg/kg of the maximum tolerated dose of this drug in mice, inhibited liver metastasis completely when started immediately after a cecectomy (micrometastasis present at this time), but did not inhibit liver metastasis significantly when started at 4 weeks after a cecectomy (gross tumor present at this time). There were no severe toxicities at this dose. In our PCR study, all livers in 10 mice to which therapy was given immediately after a cecectomy and without liver metastasis showed no PCR-amplified fragment, while 7 of 10 livers in the nontreatment group in which gross liver metastases were not observed demonstrated this fragment.Conclusions These findings indicate that UFT is useful for either adjuvant chemotherapy or the inhibition of micrometastasis, and our system to detect micrometastasis by examining the human -globin gene is useful for the early evaluation of the efficacy of these drugs.     

Ischemia-reperfusion of small liver remnant promotes liver tumor growth and metastases--activation of cell invasion and migration pathways.

Elucidating the mechanism of liver tumor growth and metastasis after hepatic ischemia-reperfusion (I/R) injury of a small liver remnant will lay the foundation for the development of therapeutic strategies to target small liver remnant injury, and will reduce the likelihood of tumor recurrence after major hepatectomy or liver transplantation for liver cancer patients. In the current study, we aimed to investigate the effect of hepatic I/R injury of a small liver remnant on liver tumor development and metastases, and to explore the precise molecular mechanisms. A rat liver tumor model that underwent partial hepatic I/R injury with or without major hepatectomy was investigated. Liver tumor growth and metastases were compared among the groups with different surgical stress. An orthotopic liver tumor nude mice model was used to further confirm the invasiveness of the tumor cells from the above rat liver tumor model. Significant tumor growth and intrahepatic metastasis (5 of 6 vs. 0 of 6, P=0.015), and lung metastasis (5 of 6 vs. 0 of 6, P=0.015) were found in rats undergoing I/R and major hepatectomy compared with the control group, and was accompanied by upregulation of mRNA levels for Cdc42, ROCK (Rho kinase), and vascular endothelial growth factor, as well as activation of hepatic stellate cells. Most of the nude mice implanted with liver tumor from rats under I/R injury and major hepatectomy developed intrahepatic and lung metastases. In conclusion, hepatic I/R injury of a small liver remnant exacerbated liver tumor growth and metastasis by marked activation of cell adhesion, invasion, and angiogenesis pathways.     

High-malignancy orthotopic nude mouse model of human prostate cancer LNCaP.

BACKGROUND: An animal model of human prostate cancer LNCaP demonstrating high rates of spontaneous metastasis from the orthotopic site after tumor implantation would be very valuable for mechanistic and drug discovery studies. We previously developed microsurgical techniques to implant histologically intact tumor tissues orthotopically in nude mice in order to develop high metastatic mouse models of human cancer. METHODS: Intact tissue of the androgen-dependent human prostate cancer cell line, LNCaP, was implanted on the ventral lateral lobes of the prostate gland by surgical orthotopic implantation (SOI) in a series of 20 nude mice. Mice were autopsied, and histopathological examination of primary tumors and relevant organs was done to identify and quantitate micrometastasis. RESULTS: Eighteen of 20 animals transplanted with LNCaP by SOI had tumor growth. Mean primary tumor weight in the prostate was 9.24 g at time of necropsy. Sixty-one percent of the transplanted animals had lymph node metastasis. Forty-four percent had lung metastasis. Mean survival time was 72 days, indicating a high degree of malignancy of the tumor. CONCLUSIONS: The extensive and widespread lung metastasis as well as lymph node metastasis following orthotopic implantation of LNCaP in nude mice and the short survival time provide a high-malignancy nude model of the LNCaP human prostate tumor.     

Technic for orthotopic hepatic transplantation in rats using cuffs for 3 vascular anastomoses

A technique for orthotopic hepatic transplantation in the rat uses polyethylene cuffs to re-establish vascular and biliary continuity. Introduction of the suprahepatic cuff into the intrahepatic vena cava of the receiver rat, after partial hepatectomy, reduces the duration of vascular clamping and improves survival of grafted rats (long-term survival: 70%). This technique is simple to reproduce after several weeks of training and can allow research teams to study immunologic mechanisms of tolerance or rejection of liver allografts in the pure strain rat.