人结肠癌裸鼠肝转移模型的建立

目的 建立人结肠癌裸鼠肝转移模型,用于肿瘤转移防治的实验研究。方法 30只裸鼠随机分为两组,分别用肝脏注射法和脾脏注射法建立结肠癌肝转移模型, 术后待裸鼠濒临死亡或自然死亡时,观察其生存天数,解剖裸鼠,观察腹腔内转移情况和肝脏成瘤的情况。结果 两组裸鼠的肝脏成瘤率均为100%,生存期无明显差异。肝脏注射组裸鼠均于注射部位出现单一的巨大瘤结节,脾脏注射组裸鼠肝脏表面及切面见多发散在的转移瘤结节。两组的肝脏瘤组织细胞形态学相似,符合低分化腺癌的特征。结论 经脾注射法建立裸鼠结肠癌肝转移模型比较符合结肠癌体内肝转移过程的规律,是一种简便有效的、重复性好的方法。     

三氧化二砷抑制我卵巢癌裸鼠腹腔转移瘤的形成及其机制的初步研究

背景与目的:三氧化二砷(arsenictrioxide,As2O3)已成功地应用于急性早幼粒细胞白血病的临床化疗,并已用于治疗肝癌、结肠癌、胃癌等的实验研究。本研究探讨As2O3对人卵巢癌细胞株3AO细胞裸鼠腹腔种植转移的影响及其作用机制。方法:采用四甲基偶氮唑蓝(methylthiazolyltetrazolium,MTT)法检测不同浓度As2O3作用48h对人卵巢癌细胞株3AO细胞的生长抑制率,并与顺铂(cisplatin,cDDP)进行比较。取4×106个3AO细胞接种于裸鼠腹腔,96h后随机分为5组,分别腹腔内注射生理盐水、3mg/kg顺铂及0.8、1.2、2.4mg/kgAs2O3,观察3AO细胞在各组裸鼠的成瘤率、裸鼠的死亡率及生存期;采用流式细胞术观察As2O3作用后3AO细胞中Fas、FasL、nm23基因表达的变化。结果:As2O3能明显抑制3AO细胞的生长,抑制作用呈浓度依赖性,与顺铂相比差异有显著性(P<0.05);As2O3能明显降低3AO细胞的裸鼠成瘤率及荷瘤鼠的死亡率,延长荷瘤鼠的生存期,与顺铂相比差异有显著性(P<0.05);As2O3使裸鼠腹腔种植瘤Fas基因及nm23基因的表达水平升高(P<0.05),而对FasL基因的表达无影响(P>0.05)。结论:As2O3对人卵巢癌裸鼠腹腔种植具有明显的抑制作用,其机制可能与Fas基因及nm23基因的过度表达有关。     

三氧化二砷抑制人卵巢癌裸鼠腹腔转移瘤的形成及其机制的初步研究

背景与目的:三氧化二砷(arsenictrioxide,As2O3)已成功地应用于急性早幼粒细胞白血病的临床化疗,并已用于治疗肝癌、结肠癌、胃癌等的实验研究.本研究探讨As2O3对人卵巢癌细胞株3AO细胞裸鼠腹腔种植转移的影响及其作用机制.方法:采用四甲基偶氮唑蓝(methylthiazolyltetrazolium,MTT)法检测不同浓度As2O3作用48h对人卵巢癌细胞株3AO细胞的生长抑制率,并与顺铂(cisplatin,cDDP)进行比较.取4×106个3AO细胞接种于裸鼠腹腔,96h后随机分为5组,分别腹腔内注射生理盐水、3mg/kg顺铂及0.8、1.2、2.4mg/kgAs2O3,观察3AO细胞在各组裸鼠的成瘤率、裸鼠的死亡率及生存期;采用流式细胞术观察As2O3作用后3AO细胞中Fas、FasL、nm23基因表达的变化.结果:As2O3能明显抑制3AO细胞的生长,抑制作用呈浓度依赖性,与顺铂相比差异有显著性(P<0.05);As2O3能明显降低3AO细胞的裸鼠成瘤率及荷瘤鼠的死亡率,延长荷瘤鼠的生存期,与顺铂相比差异有显著性(P<0.05);As2O3使裸鼠腹腔种植瘤Fas基因及nm23基因的表达水平升高(P<0.05),而对FasL基因的表达无影响(P>0.05).结论:As2O3对人卵巢癌裸鼠腹腔种植具有明显的抑制作用,其机制可能与Fas基因及nm23基因的过度表达有关.     

康莱特对人结肠癌裸鼠种植瘤肝转移的影响

目的:探讨中药康莱特对人结肠癌裸鼠种植瘤肝转移的抑制作用。方法:将结肠癌LoVo细胞注入裸鼠脾脏,建立结肠癌肝转移模型。将成功制模的裸鼠随机分为4组:对照组,CTX组,康莱特组及CTX+康莱特组。治疗45天后,处死裸鼠,计算肝转移结节数。采用ELISA法测定肿瘤组织上清液骨桥蛋白(OPN)的浓度。结果:在肝转移结节数目上,与对照组相比其余3组肝转移结节个数均降低且有统计学意义。在组织骨桥蛋白浓度上,其余3组组织骨桥蛋白浓度降低且有统计学意义。与CTX组相比,CTX+康莱特组组织骨桥蛋白浓度降低且有统计学意义,但CTX组与康莱特组二者无统计学差异P=0.176。结论:康莱特可以抑制裸鼠结肠癌的肝转移,康莱特联合CTX具有协同作用。     

三氧化二砷和5-氟脲嘧啶对人结肠癌裸鼠移植瘤端粒酶及其催化亚单位的影响

目的：研究三氧化二砷(As2O3)注射液、5-氟脲嘧啶(5-Fu)注射液单用和两药联合使用埘人结肠癌裸鼠移植瘤端粒酶活性及其催化亚单位表达的影响：方法：建立人类结肠癌裸鼠移植瘤模型,随机分为4组：牛理盐水组;As2O3组(2．5mg／kg);As2O3(2．5mg／kg) 5-Fu组(20mg／kg);5-Fu组(20mg／kg)。采用腹腔注射给药。利用银染-端粒重复扩增(TRAP)法测定人结肠癌裸鼠移植瘤端粒酶活性、RT—PCR法检测端粒酶催化亚基(human telomerase reverse transeriptases,hTERT)的表达：结果：生理盐水组和5-Fu组的端粒酶活性及其催化亚单位表达不受影响．As2O3单药组和联合用药组均明显抑制端粒酶活性及其催化亚单位表达。结论：As2O3单用以及联合5-Fu对于人结肠癌裸鼠移植瘤细胞的端粒酶活性及其催化亚单位的表达均具有明显的抑制作用。     

康莱特对人结肠癌裸鼠种植瘤肝转移的影响

目的：探讨中药康莱特对人结肠癌裸鼠种植瘤肝转移的抑制作用。方法：将结肠癌LoVo细胞注入裸鼠脾脏,建立结肠癌肝转移模型。将成功制模的裸鼠随机分为4组：对照组,CTX组,康莱特组及CTX＋康莱特组。治疗45天后,处死裸鼠,计算肝转移结节数。采用ELISA法测定肿瘤组织上清液骨桥蛋白（OPN）的浓度。结果：在肝转移结节数目上,与对照组相比其余3组肝转移结节个数均降低且有统计学意义。在组织骨桥蛋白浓度上,其余3组组织骨桥蛋白浓度降低且有统计学意义。与CTX组相比,CTX＋康莱特组组织骨桥蛋白浓度降低且有统计学意义,但CTX组与康莱特组二者无统计学差异P=0.176。结论：康莱特可以抑制裸鼠结肠癌的肝转移,康莱特联合CTX具有协同作用。     

三氧化二砷抗裸鼠人结肠癌移植瘤作用及其机制的研究

目的:探讨三氧化二砷(As2O3)抗人结肠腺癌裸鼠移植瘤作用、作用机制以及药物毒性.方法:建立人结肠癌裸鼠移植瘤模型,随机分为4组,即生理盐水组、5-氟脲嘧啶(5-FU)组、低剂量As2O3组和高剂量As2O3组,比较各组的抑瘤作用和裸鼠一般状态的变化,并对标本分别行光镜和电镜观察、原位末端标记(TUNEL)、免疫组化和血常规检测.结果:As2O3能够明显抑制结肠癌裸鼠移植瘤的增长,并能诱导癌细胞凋亡,调控相关基因表达;未见As2O3引起明显肝、肾和造血系统损害.结论:As2O3对人结肠腺癌细胞裸鼠移植瘤具有显著的抗癌作用,此作用可能与诱导癌细胞凋亡密切相关,且受到多种有关基因的调控;但是对裸鼠的肝、肾和造血系统无明显的毒性.     

人参皂苷Rg3联合三氧化二砷抑制乳腺癌移植瘤生长及其新生血管形成的研究

目的探讨人参皂苷Rg3(简称Rg3)联合三氧化二砷(As2O3)对裸鼠人乳腺癌移植瘤模型中肿瘤生长及新生血管密度的影响。方法右前肢腋窝皮下移植人乳腺癌MCF-7细胞株的雌性裸鼠28只,随机分成4组:①联合用药组即Rg3(12mg/kg.d)+As2O3(45μg/kg.d);②Rg3组(12mg/kg.d);③三氧化二砷组(45μg/kg.d);④对照组(0.5m l/d)。自肿瘤接种第5天开始,Rg3隔日灌胃,共25次;三氧化二砷腹腔内注射,每日1次,共35次,期间观察裸鼠的生存质量。停药1周后,脱颈处死裸鼠,检测肿瘤的重量,并计数肿瘤内微血管密度(MVD)。结果 Rg3组、三氧化二砷组、联合用药组对裸鼠人乳腺癌移植肿瘤生长的抑制作用明显,联合用药组抑制肿瘤生长的作用比单一用药组更强,联合用药组MVD明显低于三氧化二砷组及对照组。结论 Rg3与三氧化二砷联合应用具有协同作用,可明显抑制乳腺癌新生血管形成,降低乳腺癌组织内的MVD,进而抑制裸鼠乳腺癌移植瘤的生长,同时降低三氧化二砷的毒副反应。     

三氧化二砷抑制小鼠H22肝癌移植瘤血管生长的实验研究

目的：探讨三氧化二砷（As2O3）对小鼠H22肝癌移植瘤组织血管生成的抑制作用。方法：建立小鼠H22肝癌移植瘤模型，腹腔应用As2O3治疗后，通过瘤体体积及瘤重的比较，病理观察血管分布，VEGF及bFGF免疫组化检测，反映As2O3对小鼠H22肝癌组织血管生成的抑制作用。结果：As2O3高剂量及低剂量组均有效地抑制荷瘤鼠皮下肿瘤的体积及瘤重，瘤重抑瘤率分别为39．32％、44．89％，病理观察发现As2O3治疗组的肿瘤血供较对照组明显减少，免疫组化检测结果为As2O3治疗组肿瘤细胞的VEGF阳性细胞数低于对照组VEGF阳性细胞数，As2O3治疗组bFGF阳性表达率明显低于对照组（P〈0．01），有显著差异。结论：As2O3对小鼠肝癌移植瘤血管生成具有明显的抑制作用，具有治疗肝癌的潜在价值。     

三氧化二砷联合5-氟尿嘧啶抗裸鼠人结肠癌移植瘤的作用及其机制的研究

目的探讨三氧化二砷（As2O3）联合5-氟尿嘧啶（5-FU）对裸鼠人结肠癌移植瘤的抗肿瘤作用、作用机制以及药物毒性.方法建立裸鼠人结肠癌模型,随机分为生理盐水组、As2O3组、5-FU组和As2O3联合5-FU组,腹腔分别注射生理盐水、As2O3、5-FU和As2O3 ＋5-FU,观察各组抑瘤作用和裸鼠一般状态的变化,并对体内标本行光镜、电镜、原位末端标记（TUNEL）、免疫组化和血常规检测.结果与单药作用相比,As2O3联合5-FU可显著抑制结肠癌移植瘤的增长,CDI值〈1,并显著增强诱导瘤细胞凋亡作用、调控凋亡相关基因（Bcl-2、Bax、Fas和FasL）表达.联合用药并未增加裸鼠肝、肾和造血系统的毒性.结论 As2O3与5-FU联合应用对结肠癌移植瘤有协同抗肿瘤作用,其机制可能与增强诱导癌细胞凋亡、细胞周期特异性药物与细胞周期调控剂联合增效以及调控凋亡相关基因表达有关;同时联合用药对于荷瘤裸鼠的肝、肾和造血系统无明显毒性.     

An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.

Whether liver metastases from colon cancer are capable of metastasizing to other sites is an important question in surgical oncology. To answer this question, we have developed a highly metastatic orthotopic transplant model of a liver metastasis from a human colon cancer patient in nude mice that targets the liver and lymph nodes. The metastatic human tumor was transplanted in athymic nude mice by surgical orthotopic implantation (SOI) of a liver metastasis from a colon cancer patient. The human colon tumor was then subsequently implanted in the colon by SOI or, in an additional series of nude mice, in the liver by surgical hepatic implantation (SHI). The mice were then explored over time for lymph node involvement beginning 10 days after implantation. After SOI, 100% of the animals had liver metastasis within 10 days, and subsequently, 19 days after SOI, all lymph nodes draining the liver were involved with metastasis without any retroperitoneal or lung tissue involvement. After SHI, all sites of lymphatic drainage of the liver, including portal, celiac, and mediastinal lymph nodes, were massively involved by metastasis in 100% of the animals as early as 10 days after tumor implantation on the liver. The results of this study demonstrate that liver metastases from colon cancer are capable of remetastasizing to other sites. This study thus suggests that in colon cancer patients with liver metastasis, mediastinal, celiac, and portal lymph node metastases originate from the liver metastasis and not, as previously thought, from primary colon cancer.     

Antitumor effect of the angiogenesis inhibitor TNP-470 on human digestive organ malignancy

The antitumor and antimetastatic effects of TNP-470, an angiogenesis inhibitor, on human gastrointestinal tumors xenotransplanted into nude mice were investigated. When two gastric cancer (MT-2 and MT-5) and two colon cancer (TK-4 and TK-13) xenografts are transplanted orthotopically into nude mice, liver metastasis develops 6 weeks after transplantation. TNP-470 30 mg/kg had a significant inhibitory effect on primary tumor growth of gastric cancers when given on alternate days from 7 days after transplantation. However, when given from 10 days or 14 days after transplantation, no inhibitory effect on the growth of any tumor xenograft was observed. In contrast, liver metastasis of each xenograft type was inhibited significantly by TNP-470. The effect of TNP-470 on prognosis was investigated using a hepatic metastatic model of rat hepatoma. Although all untreated rats that received AH-130 cell implants died within one month of massive hepatic metastasis, >50% of rats treated with TNP-470 survived for 4 months. The number of apoptotic cells in hepatic metastatic foci was significantly increased by TNP-470 administration. These results suggest that TNP-470 may provide a new approach to the treatment of digestive organ malignancies.     

Contribution of angiogenesis to the progression of colon cancer: possible inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and hepatic metastasis

The contribution of angiogenesis to tumor growth and hepatic metastasis of colorectal cancer was investigated by means of immunohistochemical study and in vitro and in vivo experiments. Colorectal cancer specimens from 30 patients with hepatic metastasis and 39 patients without hepatic metastasis were studied by staining with antibodies against factor VIII-related antigen. Microvessel count in patients with liver metastasis was significantly higher than in those without liver metastasis (p<0.005). The effect of TNP-470 was evaluated with in vitro and in vivo experiments using human colon cancer cell line, LM and the highly hepatic metastasis cell line, LM-H5. The effect of TNP-470 on the proliferation of the cancer cells and human umbilical vein endothelial cells (HUVECs) was examined. TNP-470 inhibited more sensitively the proliferation of HUVECs than cancer cells in vitro. IC50 was approximately 3 pg/ml in HUVECs and approximately 2 microg/ml in cancer cells. The effect of TNP-470 on the growth of xenografts and liver metastases by LM-H5 in nude mice was examined. TNP-470 (30 mg/kg) was administered by subcutaneous injection every third day for 4 weeks. TNP-470 inhibited both the growth of xenograft and the hepatic metastasis. The number of metastatic foci in the liver was 78.2+/-30.1 in the control group and 20.6+/-16.5 in the treated group. These results suggest that TNP-470 is a potent agent to inhibit tumor growth and hepatic metastasis of colon cancer.     

TSU-68 (SU6668) inhibits local tumor growth and liver metastasis of human colon cancer xenografts via anti-angiogenesis

A number of receptor tyrosine kinases (RTKs) are involved in angiogenesis. TSU-68 (SU-6668) was developed as an inhibitor of RTKs involved in VEGF, bFGF and PDGF signaling, which then inhibits endothelial cell proliferation. We investigated the antitumor effects of TSU-68 against human colon cancer xenografts in male SCID mice and its anti-angiogenic activity using a dorsal air-sac (DAS) assay. TSU-68 was administered orally at a dose of 200 mg/kg twice daily. Mice bearing human colon carcinoma, HT-29, or WiDr xenografts were treated for 16 days. To determine the effect on hepatic metastasis, cell suspensions of HT-29 or WAV-I were injected into the spleen of mice on day 0, and mice treated for 28 days starting from day 1. For the DAS assay, HT-29, WiDr or WAV-I cells suspended in PBS at 2 x 10(7) cells/Millipore chamber were implanted subcutaneously into SCID mice, which were then treated from day 0 to 5, On day 6, the anti-angiogenic effects were assessed. Results indicated that TSU-68 significantly inhibited the growth of subcutaneous tumors. In the hepatic metastasis model, liver weights of the TSU-68-treated group were significantly reduced, compared to those of control mice. In the DAS assay, the angiogenic indices of the treated groups were significantly decreased for HT-29, WiDr and WAV-I tumors, with T/C ratios of 13.4, 50 and 35.3%, respectively. As TSU-68 significantly inhibited tumor growth and liver metastasis formation of human colon cancer xenografts, probably through anti-angiogenic activity, this agent may be useful for the treatment of colon cancer.     

肝纤维化对结肠癌肝转移的影响及TIMP-1、CTGF在其过程中的机制研究

目的：通过建立小鼠肝纤维化模型及肝纤维化基础上的结肠癌肝转移模型,阐明肝纤维化对肝转移癌发生的影响,探讨TIMP-1、CTGF在肝纤维化抑制结肠癌肝转移中的作用.方法：BALB/c小鼠76只,随机分为A、B两组,A组为正常对照组,B组采用CCl4灌胃法构建肝纤维化模型.病理切片证实肝纤维化模型制作成功后,将B组随机分为B1和B2两组,A组同时随机分为A1和A2两组,每组均为19只.取A2和B2组,将培养好的小鼠结肠癌CT26细胞应用脾脏种植切除脾脏法构建结肠癌肝转移模型.2周后处死各组动物,比较A2和B2组小鼠肝转移率,并采用免疫组化方法对TIMP-1、CTGF在各组肝脏中的表达进行分析.结果：动物模型构建成功,病理切片证实肝纤维化及肝转移癌的发生.A2组肝转移发生率为82.4％（14/17）,B2组肝转移发生率为25％ （4/16）,差异有统计学意义（P＜0.05）.TIMP-1、CTGF在B1、B2组肝脏的表达与A1、A2组相比均升高,差异有统计学意义（P＜0.05）.结论：肝纤维化对肝转移癌的发生有抑制作用.TIMP-1、CTGF可能在肝纤维化抑制结肠癌肝转移中发挥作用.     

高脂饮食对结肠癌肝脏转移的影响及作用机制

目的 探讨高脂饮食对结肠癌肝脏转移的影响及其作用机制。方法 30只NSI第三代免疫缺陷裸鼠脾脏种植结肠癌细胞DLD1构建移植瘤裸鼠模型,分别喂养普通饲料（对照组）、高脂饲料（高脂饮食组）和高脂饲料并腹腔注射CXCR4拮抗剂AMD3100（高脂饮食+AMD3100组）,每组10只。12周后颈椎脱臼处死,称量各组裸鼠体重、肝脏重量,统计肝脏中肿瘤转移灶数目,并采用Western blot和RT-qPCR检测肿瘤组织中SDF-1、CXCR4的蛋白和mRNA表达水平。结果 12周后,高脂饮食组裸鼠体重、脂肪重量、肝脏重量及血清瘦素浓度高于对照组和高脂饮食+AMD3100组（P<0.05）。对照组、高脂饮食组和高脂饮食+AMD3100组裸鼠发生肝脏转移的比例分别为30.0%、80.0%和40.0%。高脂饮食组裸鼠的肿瘤体积及肝脏转移灶数量均明显高于对照组［（3.83±0.42） mm³  vs （1.00±0.15） mm³,P<0.001;（4.33±0.58） 个 vs （1.33±0.58） 个,P=0.002］和高脂饮食+AMD3100组 ［（3.83±0.42） mm³  vs （1.96±0.15） mm³,P<0.001;（4.33±0.58） 个 vs （2.33±0.58） 个,P=0.002］。Western blot和RT-qPCR检测结果显示,高脂饮食组结肠癌组织中的SDF-1、CXCR4的蛋白和mRNA表达水平均较对照组和高脂饮食+AMD3100组明显上调（均P<0.05）。结论 高脂饮食可通过促进结肠癌癌组织中SDF-1和CXCR4的表达水平而增强结肠癌的肝脏定向转移。     

三氧化二砷-泊洛沙姆４０７缓释剂瘤内注射治疗裸鼠人肝癌移植瘤的实验研究

目的：观察三氧化二砷-泊洛沙姆４０７（Ａｓ_２Ｏ_３-Ｐ４０７）缓释剂对裸鼠皮下人肝癌移植瘤的有效性。方法：以人肝癌ＨｅｐＧ２细胞为来源建立裸鼠皮下移植瘤模型,实验共分３组：Ａｓ_２Ｏ_３-Ｐ４０７缓释剂治疗组、Ａｓ_２Ｏ_３治疗组和生理盐水（ＮＳ）对照组,于造模成功后６～８天行瘤体注射,每４天１次,共４次。比较各组抗癌疗效,观察裸鼠体重、肿瘤体积变化、抑瘤率、肿瘤组织病理及骨髓涂片以及裸鼠的存活情况、生存质量。结果：Ａｓ２Ｏ３ Ｐ４０７缓释剂治疗组肿瘤生长受到显著抑制,肿瘤体积较Ａｓ_２Ｏ_３组小（Ｐ＜０.０５）,瘤重分别为（０.２５７±０.０８０）ｇ和（０.６４１±０.１０９）ｇ,差异有统计学意义（Ｐ＜０.０５）;两组与ＮＳ对照组瘤重（１.０９４±０.１４７）ｇ相比,差异有统计学意义（Ｐ＜０.０５）。治疗后Ａｓ_２Ｏ_３-Ｐ４０７缓释剂组裸鼠体重大于Ａｓ_２Ｏ_３组和ＮＳ对照组,差异有统计学意义（Ｐ＜０.０５）。各组均未见骨髓抑制现象。结论：Ａｓ_２Ｏ_３-Ｐ４０７缓释剂瘤内注射,使用安全,可维持局部有效药物浓度、延长作用时间,疗效优于Ａｓ_２Ｏ_３注射液。     

结肠癌转移相关基因1对结肠癌细胞肝转移的影响

目的:研究结肠癌转移相关基因1(metastasis-associated in colon cancer1,MACC1)的表达对结肠癌肝转移的影响。方法:将融合绿色荧光蛋白(green fluorescent proteins,GFP)的慢病毒表达载体分别感染具有不同转移潜能的人结肠癌细胞株SW1116和HCT116。给予BALB/c-nu/nu裸鼠脾脏注射感染后的结肠癌细胞,构建结肠癌肝转移模型。注射后35d,在荧光解剖显微镜下观察肝脏内结肠癌细胞的转移情况,计算肝转移率。应用蛋白质印迹法检测SW1116和HCT116细胞中MACC1蛋白的表达水平,评估MACC1的表达水平与肝转移率的关系。结果:SW1116细胞的肝转移率明显高于HCT116细胞。MACC1蛋白在HCT116细胞中呈低表达,而在SW1116细胞中呈高表达,差异有统计学意义(P<0.01)。结论:MACC1蛋白的表达水平可能与结肠癌的肝转移能力呈正相关。