RNAi沉默SDF-1基因对人胃癌裸鼠原位移植瘤生物学行为的影响及机制

目的:探讨RNAi技术沉默基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)对人胃癌裸鼠原位移植瘤生物学行为的影响及可能机制。方法:利用RNAi-SDF-1细胞及对照组细胞建立人胃癌裸鼠皮下移植瘤,Western Blot检测裸鼠皮下移植瘤SDF-1蛋白表达情况。利用裸鼠皮下移植瘤建立其原位移植瘤模型,8周后处死裸鼠解剖尸体,检测原位移植瘤生长、凋亡及远处脏器转移情况。Western Blot检测SDF-1沉默对通路蛋白Akt、p-Akt、NF-κB、p-NF-κB以及侵袭转移相关基因E-cadherin、MMP-7表达的影响。结果:成功建立RNAi-SDF-1裸鼠原位移植瘤模型。与空白及阴性对照组比较,RNAi-SDF-1组裸鼠原位移植瘤生长缓慢,体积与质量均明显降低,差异有统计学意义(P0.01)。流式细胞术结果显示,RNAi-SDF-1组肿瘤细胞凋亡率明显高于空白及阴性对照组(P0.01)。尸体解剖结果显示,RNAi-SDF-1组肿瘤腹腔淋巴结及肝脏转移率明显低于空白及阴性对照组(P0.05)。Western Blot结果显示,与空白及阴性对照组比较,RNAi-SDF-1组肿瘤组织中Akt、p-Akt、NF-κB、p-NF-κB、MMP-7表达水平明显降低,E-cadherin表达水平明显升高(P0.01)。结论:RNAi-SDF-1能够有效抑制人胃癌SGC7901细胞裸鼠原位移植瘤的生长,诱导肿瘤细胞凋亡,抑制其腹腔淋巴结、肝脏转移,其机制可能与抑制PI3K-Akt、NF-κB信号通路及MMP-7的表达并上调E-cadherin的表达相关。     

GFP标记的肺癌细胞生长和转移的在体荧光成像

目的:利用稳定表达GFP的人高转移肺巨细胞癌细胞株95D,在体观察肿瘤的生长和淋巴结转移情况.方法:利用逆转录病毒感染人高转移肺巨细胞癌细胞株95D,经过G418筛选获得稳定表达GFP的细胞株.接种裸鼠皮下,成瘤后使用Nikon公司SMZ1000 P-FLA型荧光体视显微镜观察皮下原发肿瘤病灶及转移淋巴结.结果:使用逆转录病毒感染,可以获得稳定表达高强度GFP的肺癌细胞株,且生物学行为无明显改变.接种至裸鼠皮下后,2周左右成瘤,皮下肿瘤原发病灶在荧光体视镜下可清晰观察到肿瘤侵犯范围及周边血管生长情况.切开皮肤,可以观察到表达的GFP的转移淋巴结.结论:GFP标记人高转移肺巨细胞癌细胞株95D,建立裸鼠移植瘤模型,能更方便的观察肿瘤的生长、侵润和转移,为研究肿瘤的生长转移机制打下基础.     

基于临床肿瘤标本的胃癌转移模型建立

目的建立基于临床肿瘤标本的胃癌转移模型,为胃癌的转移研究提供个体化动物模型。方法将胃癌新鲜的手术标本移植到裸鼠皮下,建立胃癌患者异种移植(patient-derived xenograft,PDX)模型。进一步通过手术将皮下瘤组织原位移植到裸鼠胃部肌层,连续观察裸鼠的体征状态,通过近红外荧光活体成像技术检测肿瘤转移的发生。解剖荷瘤小鼠,将肺部转移灶进一步移植裸鼠皮下获得实体瘤。HE染色观察原发瘤与转移瘤的结构特征,(short tandem repeat) STR分析原发瘤和转移瘤的遗传特性。PCR-Array分析转移瘤和原发瘤中转移相关基因的表达。结果成功建立胃癌PDX模型,移植瘤组织结构与患者保持基本一致;通过胃部原位移植发现编号C19751的小鼠发生肺和肝的转移。其中肺转移灶皮下移植后获得了实体瘤,STR分析显示原发瘤保持了与肺转移瘤一致的遗传特征。PCR-Array结果显示,与原发瘤相比,转移瘤中CXCL12,IGF1和MMP2基因表达均显著上调。结论利用临床肿瘤标本成功建立胃癌转移模型,为胃癌转移研究提供了良好的个体化模型。     

人胰腺癌裸鼠异种移植瘤模型的生物发光成像和超声成像比较

目的利用荧光素酶基因标记的人胰腺癌细胞株Capan-2建立胰腺癌裸鼠移植模型,评价生物发光和小动物超声成像在移植瘤模型建立中的作用。方法将表达荧光素酶基因的真核表达载体转入人胰腺癌细胞Capan-2,将1×106人胰腺癌细胞悬液分别接种于裸鼠胰腺和右后肢皮下,使其成瘤。生物发光成像和小动物超声成像系统观察肿瘤的生长情况。结果肿瘤细胞原位移植成功率为75%,皮下移植成功率为100%。生物发光成像系统在肿瘤细胞原位接种第7天,可以观察到肿瘤发光;小动物超声成像系统在肿瘤细胞皮下接种第7天,可以测量肿瘤的大小,但在肿瘤细胞原位接种的第7天不能测量肿瘤的大小。另外肿瘤细胞在裸鼠皮下生长的速度比原位生长速度快3倍左右。结论生物发光成像系统更适用于肿瘤早期监测,为深入研究胰腺癌的发生发展、侵袭转移机制提供理想工具。     

裸鼠体内高转移人肺癌模型的筛选

将人肺巨细胞癌ＰＬＡ－８０１Ｄ裸鼠皮下移植瘤作为瘤源,建立了ＰＬＡ－８０１Ｄ－ＡＳ裸鼠腹水鼠模型,其转移率及转移程度明显增高;以ＰＬＡ－８０１Ｄ－ＡＳ腹水瘤模型的肺转移灶细胞进行裸鼠皮下连续传代,建立了肺转移率及程度高于ＰＬＡ－８０１Ｄ的ＰＬＡ－８０１ＤＬ皮下移植瘤株。本文从肿瘤的异质性以及肿瘤与宿主相互作用的角度讨论了其肺转移提高的原因。     

活体荧光成像对裸鼠肝癌细胞系MHCC97-H原位移植模型的动态量化分析

目的:利用生物自发光的裸鼠肝癌原位移植模型,以活体荧光成像技术对肝癌的生长和转移情况进行动态、量化分析.方法:将稳定转染了荧光素酶(luciferase)基因的人肝癌细胞株MHCC97-H-LUC细胞,移植至裸鼠肝脏包膜下,每周利用活体荧光成像系统对裸鼠体内移植瘤的生长部位和范围进行成像,测量肿瘤细胞生物发光量,动态观察肝癌细胞在裸鼠体内的肿瘤数量、生长速度和转移情况.结果:建立可稳定表达荧光素酶的人肝癌细胞株MHCC97-H-LUC并用于进行生物自发光的裸鼠原位移植模型;利用活体荧光成像系统对裸鼠体内的移植瘤成像,见发光部位由肝脏向腹腔扩散,发光量随时间呈指数级增长;病理学观察证实肿瘤细胞长.结论:利用活体荧光成像技术的动态量化分析可灵敏、准确地监测裸鼠肝癌原位移植模型中肿瘤细胞的生长及转移情况,为肿瘤发生、生长、转移机制及对抗肿瘤生长和转移的体内研究提供了科学的量化手段.     

MMTV-Wnt-1转基因小鼠作为高发乳腺癌动物模型的观察

目的　观察MMTV Wnt 1转基因小鼠的乳腺癌发病情况及病理学变化规律。方法　观察MMTV Wnt 1转基因小鼠肿瘤发生情况 ,并采用原位移植将瘤组织置于裸鼠皮下 ,通过组织病理学切片来观察MMTV Wnt 1阳性转基因小鼠和移植鼠的病理学变化。结果　MMTV Wnt 1转基因小鼠最早从 7周龄开始出现乳腺瘤 ,发瘤鼠剖检可见脾、肝有不同程度的肿大 ,其他器官无明显病变 ;病理组织学检查发现发瘤鼠各脏器有不同程度的病变 ,但未出现肿瘤转移。将瘤组织移植裸鼠后 ,肿瘤可在裸鼠皮下生长 ,移植肿瘤病理学形态与原发瘤一致 ,未出现转移。结论　实验结果验证MMTV Wnt 1转基因小鼠可稳定自发乳腺肿瘤 ,可作为研究乳腺癌的良好的动物模型     

人卵巢癌裸鼠移植实体瘤模型的建立

目的建立人卵巢癌裸鼠移植实体瘤模型。方法将1例人卵巢癌组织移植裸鼠,建立人卵巢癌裸鼠原代移植实体瘤模型的基础上,再将实体瘤皮下移植、实体瘤原位移植、实体瘤细胞移植裸鼠。观察裸鼠实体瘤生长和转移情况,称量其体重、子宫卵巢重、瘤重及瘤的大小,并作病理、电镜、染色体检查。结果成功地建立人卵巢癌裸鼠移植实体瘤模型,并已传至第18代,传代移植成功率100％,组织学和超微结构形态均证明该实体瘤保留了原人卵巢癌特征,有人卵巢癌染色体特征,并出现肝、脾转移。结论本研究建立人卵巢癌裸鼠移植实体瘤模型与人相似,通过18代传代和实验观察方法稳定可靠。为人卵巢癌的研究提供了理想的动物模型。     

人肝细胞肝癌MHCC97H裸鼠移植模型的建立及索拉菲尼的药效研究

索拉菲尼是靶向血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)、B-Raf原癌基因(B-Raf proto-oncogene)等多种酪氨酸激酶的抑制剂,能有效延长晚期肝细胞肝癌(简称肝癌)患者的生存时间。该研究利用人肝癌细胞MHCC97H成功建立了裸鼠皮下异位移植模型以及原位移植模型,并评估了索拉菲尼对MHCC97H移植瘤的治疗作用。结果表明,MHCC97H可以在裸鼠皮下形成异位移植瘤,每天灌胃30 mg/kg索拉菲尼可显著抑制肿瘤生长。同时,MHCC97H也可以在裸鼠肝脏形成原位移植瘤。每天灌胃30 mg/kg索拉菲尼可以显著抑制裸鼠的血清甲胎蛋白(alpha fetoprotein,AFP)水平及原位瘤生长。综上所述,MHCC97H是构建皮下异位移植以及原位移植模型的一个理想肝癌细胞系,灌胃索拉菲尼在这两种移植瘤模型中都表现出显著的肿瘤抑制效果。     

miRNA-194 在裸鼠体内对骨肉瘤增殖和转移的影响

目的:通过使用慢病毒技术,建立肺转移裸鼠模型,观察miRNA-194对于骨肉瘤增殖和转移的影响,为研究miRNA-194在骨肉瘤中的作用及进一步的治疗提供理论依据。方法:使用慢病毒技术对骨肉瘤细胞系U2-OS进行转染后分组,然后将转染后的各组细胞注入裸鼠体内建立肺转移模型。5周后将裸鼠处死,观察和比较原位及肺部肿瘤的大小。结果:1)mi RNA-194下调组裸鼠的原位肿瘤的体积(3920±860 mm~3)和重量(2.15±0.32 g)明显的大于其余各组(P0.05);2)miRNA-194上调组的肺部情况(36.7±12.4个)明显的优于其余各组(P0.05);3)病理学检测证实其确实为原位骨肉瘤及肺部转移病灶。结论:miR-194可以明显的在裸鼠体内抑制骨肉瘤的增殖及肺部的转移,从而显示mi R-194在动物水平在骨肉瘤中呈现明显的抑癌基因的作用,为接下来关于miRNA-194的各项实验奠定了良好的实验基础。     

Nuclear architecture supports integration of physiological regulatory signals for transcription of cell growth and tissue-specific genes during osteoblast differentiation

Metastatic model of human tumor xenografts have been developed using orthotopic transplantation of histologically intact tissue (onplantation) of lung, stomach, colon, pancreatic, prostate and bladder carcinomas. These models represent the entire process of the metastasis, consisting of local tumor growth, vascular and lymphatic invasion at the local site, flow in the vessels and lymphatic, extravasation at the metastatic organs, and seeding and growth at relevant metastatic sites. Orthotopically transplanted human small-cell lung carcinoma displayed a different chemosensitivity pattern compared with the subcutaneous transplanted model, suggesting different pharmacodynamics between the orthotopic lung and the ectopic subcutaneous sites. The intact-tissue orthotopic-onplantation model seems to be useful to study the mechanism of metastasis for discovery of antimetastatic agents and for the patient tumors and for this treatment design.     

Feasibility and limits of an orthotopic human colon cancer model in nude mice

We sought to develop an accurate colorectal cancer model in nude mice with stable local growth, tumor cell dissemination, and reproducible metastatic capacity. To this end, we orthotopically transplanted histologically intact human colorectal cancer tissue from 10 human patients into nude mice. After successful local tumor growth, tumor tissues were retransplanted as many as 9 times in serial passage. All specimens were transplanted using microsurgical techniques. Histologic, immunohistochemical, and polymerase chain reaction techniques were used to determine tumor growth rates and kinetics, development of regional lymph node and distant hepatic metastases, and the induction of minimal residual disease (MRD). Stable local tumor growth rates with variable growth kinetics were detected in 73.4% of all mice. The lymph node and hepatic metastasis rates were low, at 18.4% and 4.9%, respectively. MRD, as reflected by CK20 positivity of the bone marrow in animals with lymph node and hepatic metastases, was present in 22.2%. The orthotopic colorectal cancer model described here is feasible for the induction of reproducible local tumor growth but is limited by variable growth kinetics and the low rate of lymph node and hepatic metastases. Cytokeratin-positive cells indicative of MRD could be detected in the bone marrow of approximately 25% of the nude mice with metastases. The observed induction of MRD after orthotopic implantation of intact human colon cancer in animals with lymph node and hepatic metastases might be improved if established colon cancer cell lines were used.     

Establishment of a Patient-Derived Orthotopic Xenograft (PDOX) Model of HER-2-Positive Cervical Cancer Expressing the Clinical Metastatic Pattern

Squamous cell carcinoma of the cervix, highly prevalent in the developing world, is often metastatic and treatment resistant with no standard treatment protocol. Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). Unlike subcutaneous transplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most importantly, the metastasis pattern correlates to the patient. In the present report, we describe the development of a PDOX model of HER-2-positive cervical cancer. Metastasis after SOI in nude mice included peritoneal dissemination, liver metastasis, lung metastasis as well as lymph node metastasis reflecting the metastatic pattern in the donor patient. Metastasis was detected in 4 of 6 nude mice with primary tumors. Primary tumors and metastases in the nude mice had histological structures similar to the original tumor and were stained by an anti-HER-2 antibody in the same pattern as the patient’s cancer. The metastatic pattern, histology and HER-2 tumor expression of the patient were thus preserved in the PDOX model. In contrast, subcutaneous transplantation of the patient’s cervical tumors resulted in primary growth but not metastasis.     

Combined bioluminescence and fluorescence imaging visualizing orthotopic lung adenocarcinoma xenograft in vivo

Lung adenocarcinoma is the most common type of lung cancer. A close monitor of in vivo tumor development may help to better understand the pathogenesis and pathological processes of this disease. A bimodal imaging strategy has been developed, which is a very important tool to investigate the growth and metastasis of lung adenocarcinoma. In the present study, we used a combined labeling strategy in p53RE-luc-A549 cells via transfecting the reporter gene EGFP. In order to unambiguously identify the growth and metastasis of transfected A549 tumor cells, we established and observed subcutaneous and orthotopic xenografts in nude mice by in vivo bioluminescence and fluorescence imaging, which was verified by our post-mortem histological analysis. In vivo bioluminescence signal was observed for the progression of both subcutaneous and orthotopic xenografts in EGFP-p53RE-luc-A549 cells; in vivo fluorescence was only observed for the growth of subcutaneous xenograft of EGFP-p53RE-luc-A549 cells. Moreover, EGFP-p53RE-luc-A549 cells allow for the improved identification of implanted cells within host tissue during histological analysis. In conclusion, we presented a combined labeling strategy for bimodal A549 cell imaging which leads to improved detection of cellular grafts.     

Antisense Oligonucleotide against hTERT (Cantide) Inhibits Tumor Growth in an Orthotopic Primary Hepatic Lymphoma Mouse Model

Background

Human xenograft models, resulting from orthotopic transplantation (implantation into the anatomically correct site) of histologically intact tissue into animals, are important for investigating local tumor growth, vascular and lymphatic invasion at the primary tumor site and metastasis.

Methodology/Principal Findings

We used surgical orthotopic transplantation to establish a nude mouse model of primary hepatic lymphoma (PHL), HLBL-0102. We performed orthotopic transfer of the HLBL-0102 tumor for 42 generations and characterized the tumor cells. The maintenance of PHL characteristics were supported by immunohistochemical and cytogenetic analysis. We also report the antitumor effect of Cantide, an antisense phosphorothioate oligonucleotide against hTERT, on the growth of HLBL-0102 tumors. We showed a significant, dose-dependent inhibition of tumor weight and serum LDH activity in the orthotopically transplanted animals by Cantide. Importantly, survival was prolonged in Cantide-treated HLBL-0102 tumor-bearing mice when compared to mock-treated mice.

Conclusions/Significance

Our study provided the basis for the development of a clinical trial protocol to treat PHL.     

优化胶粘贴法建立裸鼠胃癌原位种植模型

目的通过对两种胶粘贴法建立的胃癌原位种植动物模型的比较研究,为探讨胃癌的发病机制和实验治疗提供理想的动物模型。方法用OB胶和FS生物蛋白胶法分别建立胃癌原位种植动物模型,观察和比较两种方法所建立的模型肿瘤生长状况、转移情况和形态学变化。结果FS生物蛋白胶组未出现肿瘤大片坏死,腹水形成率为85.7%,幽门梗阻发生率为57.1%;而OB胶组肿瘤大片坏死发生率为100%,腹水形成率为14.3%,未出现幽门梗阻。FS生物蛋白胶组有三例出现了肺和脑转移。结论FS生物蛋白胶法建立的裸鼠胃癌原位种植动物模型能更好的模拟人胃癌患者的临床过程,为研究人胃癌转移机制和实验治疗提供理想的动物模型。     

人肝癌原位移植转移模型特性实验研究

目的　建立人肝癌细胞系HCC-9724(简称H)淋巴结转移模型,研究肿瘤转移机理。方法　采用裸鼠肝脏原位移植法,接种肿瘤细胞,取其淋巴结转移灶反复肝内接种,连续传三代后,观察其转移特性,采用SABC法测定淋巴结中nm23和Ⅳ型胶原酶表达。结果　裸鼠原位接种50d,肝内长出约1.7cm×6.0cm大小的肿瘤,呈分叶状,质地较软,周围血供丰富,瘤组织与邻近脏器粘连,有明显的浸润和转移,经裸鼠三次筛选后肿瘤潜伏期短(15d),瘤体大,形成广泛的肠系膜淋巴结转移,淋巴结中Ⅳ型胶原酶表达呈强阳性;而nm23呈弱阳性。结论　采用裸鼠肝原位移植法,反复筛选,获得了人肝癌淋巴结高转移模型。     

人肝癌裸小鼠常位移植实验研究

采用硫贲妥钠（３０ｍｇ／ｋｇ、３０％乙醇配制）麻醉和蘸上立止血（２５０ｋＩＵ／ｍｌ）的明胶海棉止血措施确保了人肝癌裸小鼠常位移植术能安全、可靠地进行。本中心建立的两株人肝癌裸小鼠移植瘤株（ＨＨＣ４、ＨＨＣ１５）已成功地移植于裸小鼠（ＳＰＦ级）肝脏内,移植瘤生长良好、传代稳定和持续分泌ＡＦＰ。荷瘤（ＨＨＣ１５）３～６周裸小鼠血清ＡＦＰ含量与瘤体积的增加呈正相关。常位移植前（７ｄ）裸小鼠皮下注射０．１ｍｌ０．５％ＣＣ１（Ｖ／Ｖ。橄榄油配制）能明显提高ＨＨＣ４常位移植的成功率（Ｘ２检验、Ｐ＜０．０１）;在微量ＣＣｌ４作用下,裸小鼠肝细胞受损伤,发生肝硬化,在此基础上所移植和生长的人肝癌常位移植瘤与大多数人肝癌病变发生的肝脏病理生理学特点相似,本模型的建立更适用于抗肝癌药物的模拟治疗和筛选。