4种人肾细胞癌原位移植裸鼠模型建立方法的比较

背景与目的:肾细胞癌是最常见的肾脏恶性肿瘤,起病隐匿,恶性程度高.研究旨在建立成功率高、稳定的人肾细胞癌原位动物造模方法.方法:采用人肾细胞癌细胞(786-0、ACHN)进行细胞悬液原位注射法、皮下成瘤后引瘤原代细胞悬液原位注射法、皮下成瘤后引瘤组织块原位移植肾包膜下法及皮下成瘤后引瘤组织块原位移植肾周筋膜内法建立原位移植裸鼠模型,采用PET/CT、H-E染色、免疫组织化学染色及血清生化检测等手段观察成瘤率及肿瘤生长情况,评估4种方法的建模效果.结果:人肾细胞癌皮下移植瘤裸鼠模型,ACHN组成瘤率(90%)高于786-0组(30%);人肾细胞癌原位移植裸鼠模型,786-0原位注射组、ACHN原位注射组、ACHN皮下移植瘤引瘤后原代细胞原位注射组、ACHN组织块肾包膜下包埋组和ACHN组织块肾周筋膜内包埋组的成瘤率分别为33%、80%、90%、100%和20%,其中以ACHN皮下成瘤后引瘤组织块原位移植肾包膜下法成瘤率最高,影像学及病理组织学检查结果显示符合低分化肾细胞癌.结论:成功建立4种人肾细胞癌原位移植裸鼠模型,其中以ACHN皮下成瘤后引瘤组织块原位移植肾包膜下法成瘤效果最佳,为肾细胞癌发病机制及靶向治疗的进一步研究提供理想的动物模型.     

胱硫醚β合成酶在前列腺癌中的异常表达及其对前列腺癌细胞增殖的调控作用

目的:探讨胱硫醚β合成酶(CBS)在前列腺癌中的差异表达情况,及其对前列腺癌细胞增殖的调控作用。方法:利用GEPIA网站分析来源于TCGA和GTEx数据库的492例前列腺癌组织和152例正常组织中CBS mRNA表达情况。利用ULCAN网站分析来源于TCGA数据库的497例前列腺癌组织和52例正常组织中CBS mRNA的表达情况。用RPIM-1640培养基培养前列腺癌细胞系DU145、PC3、LNCaP和C4-2,免疫印迹实验检测CBS蛋白表达情况。shRNA转染DU145细胞,分为对照shRNA组、CBS shRNA#1组或#2组,克隆形成实验检测细胞克隆形成数量,BrdU标记实验检测细胞增殖能力,免疫印迹实验检测AKT、mTOR、S6K蛋白表达情况,以及AKT S473位点、mTOR S2448位点、S6K T421/S424位点的磷酸化水平。将稳定表达对照shRNA、CBS shRNA#1或#2的DU145细胞注射入小鼠皮下,建立小鼠前列腺癌移植瘤模型,观察肿瘤生长情况。结果:数据库分析结果显示,与正常前列腺组织比较,前列腺癌组织中CBS mRNA的表达水平显著上升。CBS蛋白在...     

米非司酮对子宫内膜癌HHUA细胞裸小鼠移植瘤生长作用的研究

目的探讨米非司酮(RU486)对人子宫内膜癌HHUA细胞裸小鼠移植瘤生长的抑制作用。方法体外培养人子宫内膜癌HHUA细胞,裸小鼠皮下接种HHUA细胞建立裸小鼠移植瘤动物模型,将荷瘤裸小鼠随机分为两组,分别应用米非司酮、精制花生油治疗4周。治疗期间观察各组裸小鼠生长情况、移植瘤体积、增殖率和形态,治疗结束后应用流式细胞术(FCM)检测各组裸小鼠移植瘤组织细胞周期时相的变化和细胞凋亡率;应用免疫组化技术检测基因Bcl-2和Bax蛋白的表达。结果米非司酮组裸小鼠移植瘤体积第3周明显减小,肿瘤增殖率明显降低(P<0.01)。4周治疗结束后米非司酮组裸小鼠移植瘤细胞G0/G1期比例升高(38.17±3.02)%,S期细胞比例(SPF)降低(43.87±4.29)%,与对照组比较差异均有显著性(P<0.05),移植瘤细胞凋亡率为(15.03±2.19)%,明显高于对照组(3.06±1.13)%(P<0.01);治疗组移植瘤细胞Bcl-2蛋白表达明显降低(1.9±0.61),而Bax呈现过度表达(4.0±1.28)(P<0.05)。结论抗孕激素米非司酮(RU486)能显著抑制人子宫内膜癌HHUA细胞裸小鼠移植瘤的生长,其抗肿瘤作用与调控细胞周期时相和诱导细胞凋亡相关。     

裸小鼠人宫颈癌组织模型的建立及其生物学特性初探

目的 建立人宫颈癌HeLa细胞在裸小鼠的体表肿瘤模型,并研究其成瘤性及生物学特性。方法 以不同浓度人宫颈癌HeLa细胞悬液在裸小鼠皮下接种建立人宫颈癌裸小鼠模型,观察荷瘤鼠的成瘤率及肿瘤的一般特性,病理检查其组织学特性,流式细胞术检测其生长、凋亡情况,免疫组织化学法检测肿瘤组织中Survivin,Caspase-3和JAK-STAT3蛋白的表达情况。结果 成瘤率为100%,肿瘤生长以局部浸润为主,未见转移瘤,所有新生肿瘤组织符合人宫颈癌细胞组织特点,绝大多数肿瘤细胞处在增殖分化期,标本宫颈癌组织中有Survivin,Caspase-3和STAT3蛋白的阳性表达。结论 成功建立了裸小鼠人宫颈癌组织模型,该模型较好地模拟了人宫颈癌的生物学特性,为进一步研究针对诱导细胞凋亡治疗的新方法提供了理想的动物模型。初步观察了不同浓度HeLa细胞悬液接种生长的肿瘤组织在生长方面的区别。     

人胃癌裸鼠原位移植瘤模型的建立及其活体荧光成像检测

目的:建立可动态实时监测的人胃癌裸鼠原位移植瘤模型.方法:将稳定表达荧光素酶的人胃癌细胞SGC-7901fLuc+注入裸鼠后肢根部皮下形成皮下移植瘤.待皮下移植瘤瘤块长至直径0.5 cm时,剥取肿瘤组织,应用叠合法将瘤块原位移植于裸鼠胃小弯处,形成原位移植瘤模型.利用小动物活体荧光成像系统,每4日监测移植瘤的进展情况.荷瘤3周后,解剖荧光成像阳性小鼠,利用H-E染色、光镜下观察移植瘤的形态.结果:应用小动物活体荧光成像系统,可在荷瘤裸鼠胃部检测到逐渐增强的荧光信号.荷瘤裸鼠胃部存在肿瘤包块贴附于胃壁,包块直径为0.5 ～1.0 cm,包块周围与相邻组织器官边界清晰,未见粘连,检查腹腔未见转移,未见腹水形成.应用叠合法构建人胃癌裸鼠原位移植瘤模型成功率为95％(19/20).对荷瘤胃组织H-E染色后,可见异常肿瘤细胞,肿瘤包膜完整,符合胃癌组织学特征.结论:成功建立了操作简便、成瘤率高的可动态监测的人胃癌裸鼠原位移植瘤模型,为研究胃癌发生机制、研发抗癌药物提供了理想的实验工具.     

多药耐药细胞系K562/A02裸小鼠皮下移植瘤模型的建立与鉴定

目的建立一种耐药特性稳定的裸小鼠移植瘤动物模型,为进行体内肿瘤耐药机制研究和逆转药物的筛选提供实验模型和实验基础.方法将具有典型MDR表型的K562/A02耐药细胞接种于裸小鼠右侧背侧皮下,而亲本K562细胞接种裸小鼠左侧背侧皮下,接种细胞数为1×107细胞/只,观察肿瘤成瘤情况及生长特性,并比较裸小鼠移植瘤细胞和体外培养细胞对化疗药物的敏感性,同时利用RT-PCR和免疫组织化学染色对裸小鼠K562/A02移植瘤基因表型进行鉴定.结果1)K562/A02裸小鼠移植瘤的成瘤率为100%,大约于第6～9天成瘤(体积＞100mm3),敏感肿瘤生长速度与耐药肿瘤无明显差别;2)裸小鼠K562/A02移植瘤肿瘤细胞和体外培养原代细胞对阿霉素的IC50分别为185.24μg/ml和235.25μg/ml,而K562/A02移植瘤肿瘤的IC50分别为3.07μg/ml和3.26μg/ml;阿霉素治疗组能够明显减慢K562敏感移植瘤的生长,而对K562/A02耐药移植瘤的生长几乎无作用;3)K562/A02裸小鼠移植瘤肿瘤细胞具有mdr1/Pgp表达,而K562裸小鼠移植瘤肿瘤细胞则无mdr1/Pgp表达.结论以K562/A02细胞建立的裸小鼠耐药移植瘤模型,仍保持近似体外的耐药活性和基因表型,为耐药机制和逆转研究提供了良好的体内模型.     

裸小鼠人胃癌原位移植模型的浸润转移特征

目的：建立裸小鼠人胃癌原位移植模型并观察其浸润转移特征。方法：BALB／C-nu／nu裸小鼠12只，以SGC-7901人胃癌细胞株裸鼠皮下移植瘤为材料，通过手术将瘤组织块移植到裸小鼠胃壁，待动物自然灏临死亡时进行系统解剖。结果：原位移植瘤全都成功，且所有动物均出现局部浸润及淋巴结转移，肝脏转移率为66．7％，50“的荷瘤鼠发生腹水，肿瘤晚期与周围脏器发生粘连，荷瘤鼠死于全身衰竭，中位数生存期为18周。结论：裸小鼠人胃癌席位模型的浸润转移特性接近临床病人，是研究人胃癌浸润转移的理想手段。     

雷公藤甲素对人前列腺癌PC-3细胞裸鼠移植瘤模型的作用

目的:探讨雷公藤甲素对前列腺癌PC-3细胞裸鼠移植瘤模型的作用。方法:构建前列腺癌PC-3细胞裸鼠皮下移植瘤模型,分为实验组和对照组,实验组给予雷公藤甲素治疗,0.4mg/(kg.d),皮下注射,连用15天,对照组仅给予含有等量DMSO的生理盐水,检测药物处理后肿瘤的变化,并通过免疫荧光染色的方法检测SUMO特异蛋白酶SENP1的表达。结果:雷公藤甲素能够显著抑制移植瘤的增长,并能抑制SENP1的表达水平。结论:雷公藤甲素可以显著抑制PC-3细胞裸鼠移植瘤的增长,并降低SENP1的表达水平。     

熊果酸对荷人骨肉瘤裸小鼠移植瘤生长的影响

目的:研究熊果酸(ursolic acid,UA)对荷人骨肉瘤裸小鼠移植瘤生长的抑制作用及其机制探讨.方法:采用裸小鼠背部皮下注射接种人骨肉瘤MG-63细胞的方法建立荷人骨肉瘤裸小鼠模型并给予不同剂量熊果酸[5、10、20mg/(kg·d)]和顺铂[2mg/(kg·d)]进行干预治疗,疗程5天.称取瘤重并计算抑瘤率,HE染色观察肿瘤组织形态结构改变.TUNEL染色检测细胞凋亡状况并计算细胞凋亡指数(apoptosis index,AI).免疫组织化学染色(immunohistochemistry,IHC)观察肿瘤组织中caspase-3、Bax、bcl-2蛋白表达并进行半定量分析.结果:较模型组,熊果酸干预组荷人骨肉瘤裸小鼠移植瘤呈现细胞皱缩、片状坏死等病理性改变,凋亡细胞数量明显增多、AI显著升高,瘤体重量显著减轻、抑瘤率显著升高,caspase-3和Bax蛋白表达显著上调,bcl-2蛋白表达显著下调,Bax/bcl-2比值显著升高,熊果酸上述作用均具有一定的剂量依赖性.结论:熊果酸可能通过促进肿瘤细胞凋亡而对荷人骨肉瘤裸小鼠移植瘤生长起到一定的抑制作用,其作用机制可能与熊果酸能够调节凋亡相关蛋白(caspase-3、Bax、bcl-2)并提高Bax/bcl-2比值有关.     

NDV7793对小鼠肺腺癌皮下移植瘤组织CCL21/CCR7表达的影响

摘 要：［目的］研究NDV7793影响小鼠肺腺癌皮下移植瘤CCL21/CCR7表达来抑制小鼠肺腺癌皮下移植瘤转移的机制。［方法］建立小鼠肺腺癌皮下移植瘤模型。用PBS、顺铂和NDV7793给药对移植瘤进行治疗,每两天给移植瘤瘤内注射1次,一共进行5次注射。11d后杀死小鼠取出移植瘤。观察小鼠胸腔有无肿瘤转移,免疫组化检测移植瘤癌旁组织有无肿瘤细胞浸润,流式细胞术检测移植瘤细胞CCR7表达水平,ELISA检测移植瘤CCL21和CCL19。［结果］ PBS阴性实验组免疫组化实验结果显示移植瘤癌旁组织出现明显肿瘤细胞浸润。NDV7793组免疫组化实验结果显示移植瘤癌旁组织未发现有肿瘤细胞浸润。流式细胞术检查发现,NDV7793实验组的CCR7相对于PBS阴性组及顺铂阳性对照组明显降低。ELISA实验显示,NDV7793组CCL21表达水平高于PBS阴性对照组（P=0.049）及顺铂阳性对照组（P=0.046）,差异具有统计学意义。PBS阴性组、NDV7793组及顺铂阳性对照组CCL19水平差异无统计学意义。［结论］ NDV7793在治疗小鼠肺腺癌皮下移植瘤上,可通过上调CCL21和下调CCR7来抑制肺腺癌细胞的浸润和转移。     

Effect of combining anti-epidermal growth factor receptor antibody C225 and radiation on DU145 prostate cancer

The epidermal growth factor receptor (EGFR) network has rich targets for prostate cancer killing. Herein we evaluated the effects of combining the EGFR inhibition and radiation on DU145 prostate cancer. We treated DU145 prostate cancer cells with various doses of anti-EGFR antibody (C225) and gamma-irradiation (RAD). The effects of the treatment on cell viability and growth were assessed with cell counting, XTT and clonogenic assays. In vivo treatment effects were assessed using a subcutaneous tumor xenograft in mice. Cell cycle distribution and progression were assessed with flow cytometry. The apoptotic components of cell death were quantified using Annexin-V binding assays. The results demonstrated that when combined with radiation, C225 augmented the inhibition of cell viability and growth in the DU145 cell line and EGFR inhibition appeared to have some interaction with RAD. C225 inhibited the growth of implanted DU145 tumors and increased the efficacy of radiation treatment. Flow cytometric analysis suggested that mostly necrotic cell death resulted from the EGFR inhibition or irradiation, although there may be some apoptosis. We drew the conclusion that the inhibition of EGFR augments the radiation killing of DU145 prostate cancer via a combination of cytostatic, necrotic and apoptotic mechanisms.     

Comprehensive genotypic analysis of human prostate cancer cell lines and sublines derived from metastases after orthotopic implantation in nude mice

We recently reported on a prostate cancer progression model which was based on repeated orthotopic implantation of human prostate cancer cell lines into athymic nude mice leading to an increase of tumor cell aggressiveness. To assess progression-associated clonal evolution of genotypic changes, we now performed comparative cytogenetic characterization of the original cell lines DU145 and PC3 with derived sublines DU145MN1 and PC3-N. Cell line PC3-125-1L, isolated from a lung metastasis after subcutaneous inoculation of PC3 into nude mice, was included in the study. Whole-genome analysis was performed using spectral karyotyping and comparative genomic hybridization. Fluorescence in situ hybridization was used to assess amplification of selected genes, which are supposed to play a role in prostate cancer progression. Differences in the genetic constitution between parental cell lines and sublines involved gains of genetic material at 2q, 5q, 12p/q, and 18p as well as losses at 6p, 7q, 17p, 18q, and 22q. Loss of 17p in DU145MN1 and high-level amplification of MYC in PC3-125-1L resulted in loss of p53 expression and upregulation of Myc expression, respectively, as was assessed by Western blotting. Thus, the nude mice model is very useful to follow clonal evolution of genetic changes during increase of prostate cancer aggressiveness and possibly to clone genes associated with the progression of prostate cancer.     

PTPRJ通过Src/PI3K/Akt信号通路促进前列腺癌细胞的黏附、迁移和侵袭

目的:探究PTPRJ基因表达对前列腺癌DU145细胞黏附、迁移和侵袭的影响以及可能的调控机制。方法:实时荧光定量PCR、Western blot检测PTPRJ在前列腺肿瘤组织和细胞系中的表达;用携带PTPRJ特异shRNA的重组慢病毒(LV-shPTPRJ)感染沉默PTPRJ表达;MTT检测细胞黏附力,Transwell检测细胞迁移和侵袭;实时荧光定量PCR、Western blot检测信号通路分子mRNA和蛋白表达。结果:与正常前列腺组织和细胞相比,PTPRJ在前列腺肿瘤组织和PC-3、DU145细胞系中表达升高（P＜0.05）;与对照组相比,沉默PTPRJ后前列腺癌DU145细胞黏附、迁移和侵袭能力显著下降（P＜0.01）、信号通路蛋白pY418Src、p-PI3K和p-Akt表达水平均显著降低（P＜0.05）;SC79激活PI3K/Akt可逆转PTPRJ下调对DU145细胞黏附和侵袭的影响;沉默PTPRJ下调裸鼠瘤体组织中pY418Src、p-PI3K和p-Akt表达（P＜0.05）。结论:PTPRJ可能通过激活Src/PI3K/Akt信号通路来促进DU145细胞的黏附、迁移和侵袭,预示PTPRJ可能成为前列腺癌治疗的潜在靶点。     

5-FU节拍化疗策略优化及调节胃癌免疫微环境的体内实验研究

背景与目的：5-氟尿嘧啶（5-fluorouracil,5-FU）是胃癌化疗的骨架药物,传统大剂量5-FU常导致严重不良反应及耐药。低剂量5-FU节拍化疗在不影响疗效的前提下可明显降低药物毒性,但何种给药节拍可达到最佳抗肿瘤作用尚不清楚。本研究旨在探索5-FU的最佳节拍化疗模式,并研究其对胃癌免疫微环境的影响。方法：建立SGC-7901胃癌细胞系的BALB/c裸小鼠皮下移植瘤模型,成瘤后随机分成4组：最大耐受剂量（maximumtolerateddose,MTD）组、每日节拍化疗（daily metronomic chemotherapy,MET-qd）组、隔日节拍化疗（every other day metronomic chemotherapy,MET-qod）组和每周2次节拍化疗（twice-weekly metronomic chemotherapy,MET-biw）组。21 d为1个疗程,共2个疗程。治疗期间观测裸小鼠的一般状况,每周称重并测量瘤体,绘制肿瘤生长曲线。采用流式细胞术检测裸小鼠外周血内皮祖细胞（circulating endothelialprogenitors...     

Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage.

The tumor suppressor gene PTEN/MMAC-1/TEP-1 (referred to hereafter as PTEN) maps to chromosome 10q23 and encodes a dual specificity phosphatase. The PTEN protein negatively regulates cell migration and cell survival and induces a G1 cell cycle block via negative regulation of the phosphatidylinositol 3'-kinase/protein kinase B/Akt signaling pathway. PTEN is frequently mutated or deleted in both prostate cancer cell lines and primary prostate cancers. A murine polyclonal antiserum was raised against a glutathione S-transferase fusion polypeptide of the COOH terninus of PTEN. Archival paraffin tissue sections from 109 cases of resected prostate cancer were immunostained with the antiserum, using DU145 and PC-3 cells as positive and negative controls, respectively. PTEN expression was seen in the secretory cells. Cases were considered positive when granular cytoplasmic staining was seen in all tumor cells, mixed when areas of both positive and negative tumor cell clones were seen, and negative when adjacent benign prostate tissue but not tumor tissue showed positive staining. Seventeen cases (15.6%) of prostate cancer were positive, 70 cases (64.2%) were mixed, and 22 cases (20.2%) were negative. Total absence of PTEN expression correlated with the Gleason score (P = 0.0081) and correlated more significantly with a Gleason score of 7 or higher (P = 0.0004) and with advanced pathological stage (American Joint Committee on Cancer stages T3b and T4; P = 0.0078). Thus, loss of PTEN protein is correlated with pathological markers of poor prognosis in prostate cancer.     

Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype

Proteomic analysis identified differentially expressed proteins between zoledronic acid-resistant and aggressive DU145R80 prostate cancer (PCa) cells and their parental DU145 cells. Ingenuity Pathway Analysis (IPA) showed a strong relationship between the identified proteins within a network associated with cancer and with homogeneous cellular functions prevalently related with regulation of cell organization, movement and consistent with the smaller and reduced cell-cell contact morphology of DU145R80 cells. The identified proteins correlated in publically available human PCa genomic data with increased tumor expression and aggressiveness. DU145R80 exhibit also a clear increase of alpha-v-(αv) integrin, and of urokinase receptor (uPAR), both included within the same network of the identified proteins. Interestingly, the actin-rich structures localized at the cell periphery of DU145R80 cells are rich of Filamin A, one of the identified proteins and uPAR which, in turn, co-localizes with αv-integrin, in podosomes and/or invadopodia. Notably, the invasive feature of DU145R80 may be prevented by blocking anti-αv antibody. Overall, we unveil a signaling network that physically links the interior of the nucleus via the cytoskeleton to the extracellular matrix and that could dictate PCa aggressiveness suggesting novel potential prognostic markers and therapeutic targets for PCa patients.     

Growth-inhibitory effects of luteinizing hormone-releasing hormone (LHRH) agonists on xenografts of the DU 145 human androgen-independent prostate cancer cell line in nude mice

Experiments have been performed to clarify whether LHRH agonists might decrease growth of hormone-unresponsive prostate cancer in vivo. Male nude mice were injected s.c. with the human androgen-independent prostate tumor DU 145 cells; osmotic minipumps releasing the LHRH agonist Zoladex (LHRH-A) for 14 days were simultaneously implanted under the skin. Treatment with LHRH-A induced a significant decrease in tumor growth up to the end of the treatment. In subsequent experiment, minipumps releasing LHRH-A were implanted in nude mice either 7 or 14 days after cell inoculation. When the treatment was started 7 days after inoculation of the cells, tumor growth was significantly decreased up to 28 days; thereafter, tumor volume remained lower than in controls, although not significantly. When LHRH-A was administered beginning 14 days after cell inoculation, tumor growth was not significantly affected at any time interval considered. LHRH-A did not appear to induce apoptosis in DU 145 cells, at least on the basis of the apoptotic index and immunohistochemical staining of the p53 protein. On the other hand, treatment with LHRH-A was accompanied by a significant decrease of the concentration of epidermal growth factor receptors in DU 145 prostate cancer specimens. Our results show that the LHRH agonist used significantly inhibits the growth of DU 145 androgen-independent prostate tumor xenografts in nude mice. Int. J. Cancer 76:506–511, 1998.© 1998 Wiley-Liss, Inc.     

Myeloid cell leukemia-1 is a key molecular target for mithramycin A-induced apoptosis in androgen-independent prostate cancer cells and a tumor xenograft animal model

Mithramycin A (Mith) is a natural polyketide that has been used in multiple areas of research including apoptosis of various cancer cells. Here, we examined the critical role of Mith in apoptosis and its molecular mechanism in DU145 and PC3 prostate cancer cells and tumor xenografts. Mith decreased cell growth and induced apoptosis in DU145 and PC-3 cells. Myeloid cell leukemia-1 (Mcl-1) was over-expressed in both cell lines compared to RWPE1 cells. Mith inhibited Mcl-1 protein expression in both cells, but only altered Mcl-1 mRNA levels in PC-3 cells. We also found that Mith reduced Mcl-1 protein levels through both proteasome-dependent protein degradation and the inhibition of protein synthesis in DU145 cells. Studies using siRNA confirmed that the knockdown of Mcl-1 induced apoptosis. Mith significantly suppressed TPA-induced neoplastic cell transformation through the down-regulation of the Mcl-1 protein in JB6 cells, and suppressed the transforming activity of both cell types. Mith also inhibited tumor growth and Mcl-1 levels, in addition to inducing apoptosis, in athymic nude mice bearing DU145 cell xenografts without affecting five normal organs. Therefore, Mith inhibits cell growth and induces apoptosis by suppressing Mcl-1 in both prostate cancer cells and xenograft tumors, and thus is a potent anticancer drug candidate for prostate cancer.