原位注射法建立人胰腺癌SW1990裸鼠种植瘤模型及高频内镜超声探头对其的监测

目的 建立裸小鼠人胰腺癌原位种植瘤模型,探索监测种植瘤生长的方法.方法 将对数生长期的人胰腺癌细胞株SW1990制备成细胞悬液,原位注射于Balb/c-nu裸小鼠胰腺尾部包膜下,利用高频内镜超声(EUS)探头体表观察肿瘤结节的生长及声像图像.结果 20只裸小鼠均接种成功,1只裸鼠于接种后25 d时死亡.接种后14 d,EUS检查的瘤体大小为(8.09±2.61)mm3,肿瘤结节呈均质低回声,边界清楚,周边有包膜及声晕,形态规则,30%的肿瘤结节周边可见低速环绕彩色血流信号;接种后28 d,瘤体增大至(12.40±3.51)mm3,70%的肿瘤结节呈不规则形,部分为分叶状,肿块呈低回声,不均质,未见液化坏死区,70%的肿瘤结节周边可见低速环绕彩色血流信号.结论 原位注射法是建立裸小鼠人胰腺癌原位种植瘤模型较理想的方法,操作简便,成瘤率高;高频内镜超声显像是可靠的监测胰腺原位种植瘤的手段.     

人胰腺癌原位移植模型MRI增强扫描特征及病理对照研究

目的 探讨人胰腺癌原位移植瘤模型的MRI影像学表现及其病理基础.方法 应用3.0T磁共振成像仪及临床型乳腺线圈对30只胰腺癌裸鼠原位模型行冠状位及横断位TSE-T1 WI/T2 WI平扫,经腹腔注射钆喷酸葡胺(Gd-DTPA)行连续动态增强扫描,测量平扫和增强扫描各时相肿瘤信号强度,计算强化率,分析MR图像特征并与病理对照.结果 30只裸鼠荷瘤接种成功率为100%,组织学检查符合胰腺低分化腺癌.与邻近组织信号相比,90%(27/30)肿瘤T1 WI呈均匀稍低信号,10%(3/30)信号欠均匀;80%(24/30)肿瘤T2 WI呈不均匀高信号、内见斑片状更高或等信号区,20%(6/30)呈均匀等高信号.平扫肿瘤信号强度为228.35±11.71,增强扫描后1.5、3、6,9、12 min的肿瘤信号强度分别为258.20±11.17、301.75±17.09、358.65±25.13、480.05±19.01、558.35±40.49,均明显高于平扫(P值均<0.01);各时相强化率分别为0.13±0.04、0.35±0.11、0.56±0.10、1.10±0.10、1.45±0.18,各时相间差异均有统计学意义(P值均<0.01).MR强化明显区为供血丰富的肿瘤生长活跃区域,中央无强化区为坏死组织和(或)肿瘤细胞致密且毛细血管较少区域.结论 经腹腔注射对比剂后可获得清晰的移植瘤MRI图像,与病理检查结果具有很好的一致性.     

三种胰腺癌细胞株Mesothelin的表达及成瘤速度的比较

目的 比较3种人胰腺癌细胞株在体外和裸鼠体内Mesothelin的表达及裸鼠皮下种植瘤生长速度的差异.方法 培养人胰腺癌细胞株SW1990、BxPC3、PANC1,取对数生长期分别注射于裸鼠左腋窝皮下,每周测量裸鼠皮下种植瘤的长、短径,计算体积,SW1990、BxPC3组裸鼠观察3周,PANC1组裸鼠观察5周;用蛋白质印迹法分别检测3种细胞及裸鼠皮下种植瘤组织中的Mesothelin表达,用免疫组化染色检测3种裸鼠皮下种植瘤组织中Mesothelin表达.结果 人胰腺癌细胞株体外Mesothelin表达的强弱顺序是BxPC3＞ PANC1＞ SW1990,体内种植瘤组织表达的强弱顺序是SW1990＞ BxPC3＞ PANC1.3种人胰腺癌细胞种植于裸鼠皮下的成瘤率均为100％,各组肿瘤的生长速度顺序为SW1990＞ BxPC3＞ PANC1.结论 不同人胰腺癌细胞株Mesothelin的表达量不同,裸鼠皮下种植瘤的生长速度亦不同,两者无相关性.     

多时间节点荷人胰腺癌SW1990细胞裸鼠模型的建立及其MRI表现

目的 研究裸鼠荷载多时间节点人胰腺癌细胞的能力,分析其MRI表现及皮下移植瘤MRI检出率.方法 将人胰腺癌SW1990细胞悬液在第1天、第8天、第15天分别注射于6只裸鼠左腋窝、右腋窝、右腹股沟皮下,制成分别荷3、2、1枚瘤裸鼠各2只.3周后行MRI平扫加增强扫描.然后剥取皮下肿块行病理学检查.结果 实验期间受试裸鼠全部存活,12枚肿瘤均可见,肿瘤体积与生长时间呈正相关.MRI平扫检出9枚肿瘤,增强扫描又增加检出1枚肿瘤,2枚不能检出.未检出的肿块位于右腹股沟皮下,生长时间最短,长径＜5mm.裸鼠皮下种植瘤具有类似人胰腺癌的MRI信号,有出血、坏死灶,但所有肿瘤边缘清晰,具“假包膜”征.光镜下12枚肿块均可见类似人胰腺癌的细胞.结论 裸鼠可以分期皮下种植3枚人胰腺癌细胞肿瘤,并且至少存活3周,常规3.0T MRI尚不能完全检出生长时间1周、长径＜5mm的早期肿瘤.     

携带人Endostatin基因的腺病毒治疗胰腺癌移植瘤

目的 观察携带人Endostatin基因的重组腺病毒载体Ad-hEnd对裸鼠胰腺癌移植瘤的治疗作用.方法 将胰腺癌细胞株SW1990细胞皮下注射建立裸鼠移植瘤模型,随机分为Ad-hEnd组、报告基因LacZ重组腺病毒组(Ad-LacZ组)和对照组,每组8只.重组腺病毒200 μl瘤内注射,隔日1次,共4次.观察移植瘤的生长情况,免疫组化染色检测血管内皮生长因子(VEGF)的表达和微血管密度(MVD),原位缺口末端标记法(TUNEL)检测肿瘤细胞凋亡.结果 移植瘤成瘤率100%.治疗后4周,Ad-hEnd组、Ad-LacZ组和对照组移植瘤体积分别为(921.9±279.7)mm3、(2804.4±553.5)mm3和(3040.6±487.6)mm3;瘤重分别为(1.19±0.18)g、(2.38±0.42)g和(2.41±0.47)g;VEGF表达阳性率分别为(36.3±7.1)%、(81.2±6.6)%和(79.4±6.2)%;MVD分别为12±4、27±5和25±6;细胞凋亡率分别为(31.2±5.4)%、(9.4±4.9)%和(8.5±3.7)%.与Ad-LacZ组和对照组比较,Ad-hEnd组以上各项指标均有显著性差异(P＜0.01);Ad-LacZ组和对照组之间的差异无统计学意义.结论 重组腺病毒介导的hEndostatin基因可抑制胰腺癌的生长和血管生成,促进肿瘤细胞凋亡,可用于胰腺癌抗血管生成的基因治疗.     

胰腺癌伴神经浸润动物模型的建立

目的 建立胰腺癌伴神经浸润的动物模型.方法 32只裸鼠分为4组,每组8只.将人胰腺癌细胞SW1990、CAPAN-2、PANC1分别注射于裸鼠的坐骨神经周围,以不做任何处理的裸鼠作为对照组.观察术后裸鼠体质量变化、成瘤情况、成瘤时间、造模成功率等指标.结果 对照组裸鼠体质量增加,各癌细胞注射组裸鼠成瘤后体质量明显下降,甚至呈现恶液质,成瘤侧肢体活动受限.CAPAN-2注射组及SW1990注射组的8只裸鼠最终均成瘤,PANC1注射组只有5只成瘤.以肿瘤最长径长至约0.8 cm为时间截点,CAPAN-2注射组、PANC1注射组、SW1990注射组裸鼠的成瘤时间分别为（49.8±5.0）、（56.6±2.4）、（25.4±3.0）d.SW1990注射组成瘤时间最短,PANC1注射组成瘤时间最长,3组间成瘤时间的差异具有统计学意义（F=73.51,P＜0.01）.CAPAN-2注射组、PANC1注射组、SW1990注射组裸鼠神经浸润造模成功率分别为87.5％ （7/8）、20.0％（1/5）、50.0％ （4/8）.结论 成功建立了胰腺癌伴神经浸润动物模型,但不同的胰腺癌细胞系建立的动物模型具有不同的特点.     

磷酸化转录激活因子5在胰腺癌细胞的表达及生长激素的干预作用

目的 检测磷酸化转录激活因子5(pSTAT5)在7株胰腺癌细胞株中的表达,观察生长激素(GH)处理SW1990细胞及其移植瘤后pSTAT5表达的变化,探讨GH的分子作用机制.方法 体外培养人胰腺癌细胞株SW1990、Cap-1、Colo、Mia、Aspc、P3、PANC1,Western blotting检测各株细胞的pSTAT5表达;收集指数生长期的SW1990细胞接种于BALB/C裸鼠,成瘤后随机分为GH组(瘤内注入GH 4 mg·kg-1·d-1,连续2周)和对照组(NS组),最后一次注射GH后1、2、24 h分批处死裸鼠,Western blotting检测SW1990细胞和移植瘤pSTAT5蛋白表达的变化.结果 所有胰腺癌细胞(SW1990、Cap-1、Colo、Mia、Aspc、P3、PANC1)均有pSTAT5表达.GH(50 ng/ml)刺激后5 min,SW1990细胞pSTAT5的表达量为0.57±0.05,较刺激前显著增高,10 min达到0.64±0.04,15 min很快下降至0.39±0.03,但直至1 h仍高于对照组(0.33±0.02对0.25±0.06),2 h后表达量为0.26±0.03,回落到基础水平.移植瘤pSTAT5表达无明显变化.结论 GH可迅速上调SW1990细胞的pSTAT5表达,但维持时间较短,而对胰腺癌移植瘤pSTAT5的表达无显著影响.     

RPL31-siRNA对人胰腺癌BxPC-3细胞裸鼠皮下移植瘤生长的抑制作用

目的:研究siRNA沉默糖体蛋白L31(ribosomal protein L31,RPL31)对人胰腺癌BxPC-3细胞裸鼠皮下移植瘤的抑制作用,探讨RPL31基因在胰腺癌中可能的作用机制.方法:设计合成靶向人RPL31基因的siRNA及阴性对照siRNA;建立人胰腺癌BxPC-3细胞的Balb/c裸鼠皮下移植瘤模型,按照移植瘤体积随机化分为3组:溶剂对照组、阴性对照组及RPL31-siRNA干预组;使用RNAi-Mate转染试剂将RPL31-siRNA进行移植瘤瘤内注射,观察各组裸鼠体内瘤体生长速度,绘制肿瘤生长曲线;采用实时定量PCR及Western blot的方法检测移植瘤组织RPL31基因的表达;免疫组织化学法(immunohistochemistry,IHC)检测裸鼠皮下移植瘤Ki-67及CD31的表达;原位末端标记技术(TUNEL)检测移植瘤组织的细胞凋亡.结果:与溶剂对照组和阴性对照组相比,RPL31-siRNA注射组裸鼠皮下移植瘤生长缓慢,移植瘤体积明显缩小,差异具有统计学意义(P<0.05);移植瘤组织中RPL31基因的mRNA水平和蛋白水平表达下调;另外,RPL31-siRNA注射组裸鼠移植瘤中Ki-67表达下调(55.78%±4.63%、51.37%±5.05%vs11.08%±1.31%),CD31的表达下调(56.53%±6.03%、44.84%±5.24%vs9.67%±1.39%);RPL31-siRNA注射组裸鼠移植瘤细胞凋亡增加(2.92%±0.54%、3.85%±0.87%vs39.58%±4.02%).结论:RPL31-siRNA可以下调胰腺癌BxPC-3细胞裸鼠皮下移植瘤中RPL31基因的表达,抑制移植瘤的生长,并且还可以抑制移植瘤中Ki-67及CD31的表达,诱导移植瘤细胞的凋亡.以RPL31为靶点的基因治疗有潜在临床应用前景.     

鸟苷酸环化酶C基因沉默对人胃癌裸鼠皮下种植瘤的影响

目的:探讨鸟苷酸环化酶C(GC-C)基因沉默对人胃癌裸鼠皮下种植瘤的影响及其机制.方法:将SGC-7901细胞(SGC-7901组)、空质粒转染的细胞(PRNA组)、GC-C基因稳定沉默的细胞(GC-C-shRNA组)悬液先在裸鼠皮下接种成瘤,再取瘤组织块分别接种到裸鼠皮下,建立3种胃癌裸鼠皮下种植瘤模型.观察裸鼠一般情况、肿瘤的成瘤率、生长速度,描绘肿瘤的生长曲线,计算肿瘤的抑瘤率;采用实时荧光定量PCR(RFQ-PCR),Western blot和免疫组织化学法检测GC-C和趋化因子受体4(CXCR4)在各组移植瘤组织中的表达.结果:与SGC-7901组和PRNA组比较,GC-C-shRNA组裸鼠移植瘤生长速度明显减慢、体积明显变小(抑制率分别为33.7%、33.2%),肿瘤异型性、坏死程度明显降低,差异具有统计学意义(均P<0.05),且移植瘤组织中GC-C和CXCR4 mRNA和蛋白均明显下调(GC-CmRNA:7.47±1.70 vs 11.18±0.60,11.28±0.85;GC-C蛋白:0.52±0.15 vs 1.04±0.19,1.03±0.24;CXCR4蛋白:0.67±0.13 vs 1.02±0.21,1.03±0.23,均P<0.05).结论:GC-C基因稳定沉默在体内能保持稳定,且能抑制裸鼠移植瘤生长,该过程可能与CXCR4下调有关.     

PUMA基因转染对胰腺癌细胞生长的影响

目的 探讨PUMA基因转染抑制胰腺肿瘤生长的体内外效果.方法 利用脂质体转染法将表达PUMA的质粒转染导入胰腺癌细胞株PC-3中,G418筛选出阳性克隆,Western和RT-PCR法检测PUMA转染后PC-3 PUMA的表达,流式细胞仪检测转染后细胞凋亡率;分别将转染PUMA的PC-3细胞(实验组)和未转染的PC-3细胞移植到裸鼠体内,比较裸鼠移植肿瘤的大小和重量以及PUMA表达.结果 PUMA表达质粒转染的PC-3细胞(PC-3/PUMA)稳定表达PUMA,其细胞凋亡率为(5.50 ± 0.90)%,明显高于未转染组的(1.073 ± 0.248)%和空载体转染组的(1.08 ± 0.35)%(P ＜0.05);裸鼠接种4周后PC-3/PUMA细胞成瘤率为70%,PC-3细胞和空载体PC-3细胞成瘤率为100%(P ＞ 0.05),PC-3/PUMA细胞形成的肿瘤体积比PC-3细胞和空载体PC-3细胞明显减小(P ＜0.05),并且形成的肿瘤组织中PUMA高表达.结论 胰腺癌细胞中缺失PUMA基因表达,PUMA转染胰腺癌细胞后表达PUMA,并能促进细胞凋亡.     

康莱特注射液与光动力疗法联合治疗大鼠胰腺移植瘤

目的 探讨康莱特注射液与光动力疗法(PDT)联合应用对裸鼠胰腺癌移植瘤生长的抑制作用.方法 将人胰腺癌细胞株SW1990移植至裸鼠皮下制备皮下移植瘤模型.60只荷瘤鼠按随机数字法分为6组:对照组,不予任何治疗;康莱特1组,激光照射前一天开始从尾静脉注射康莱特注射液1.25 g/kg体重,连续10 d;康莱特2组,同上法注射康莱特注射液2.5 g/kg体重;光动力(PDT)组,腹腔内注射Photosan 2 mg/kg体重,48 h后予激光照射;联合1组,治疗方法采用康莱特1组+PDT组;联合2组,治疗方法为康莱特2组+PDT组.每组10只.每周2次测量肿瘤大小.PDT后14 d处死动物,取瘤块称重,计箅抑瘤率.结果 对照组、康莱特1组、康莱特2组、PDT组、联合1组和联合2组治疗后14 d的瘤体积分别为(550.08±52.46)mm3、(519.71±46.44)mm3、(405.29±38.67)mm3、(199.27±37.37)mm3、(107.47±14.13)mm3和(75.58±12.53)mm3;瘤重分别为(0.82±0.08)g、(0.77±0.06)g、(0.61±0.06)g、(0.41±0.05)g、(0.28±0.04)g和(0.16±0.04)g.2个联合组的肿瘤体积和瘤重明显小于其他各组(P＜0.05);联合1组抑瘤率从PDT组的50%上升到65.9%,联合2组上升到80.5%.结论 康莱特与PDT联合应用可明显抑制移植瘤体积,有协同增效作用.     

Orthotopic transplantation model of human gastrointestinal cancer and detection of micrometastases

AIM To establish a relevant animal model ofhuman gastrointestinal cancer,which can beused for repetitive investigations,so as toimprove our understanding and management ofcarcinogenesis and cancer metastasis.METHODS Intact tissues of human colorectaland pancreatic cancers were transplanted innude mice.The biological characteristics of theoriginal and the corresponding transplantedtumors were investigated by HE staining,PASstaining and immunostaining.The metastases inthe livers and lungs of nude mice wereinvestigated by immunostaining withbiotinylated mab KL-1 and by RT-PCR using CK20specific primers.RESULTS There were totally 9 of 16 surgicalspecimens growing in nude mice subcutaneouslyand/or orthotopically(4 of 6 colorectal and 5 of10 pancreatic cancer).Tumor cell content of thespecimens and freezing of tissue specimens areimportant factors influencing the growth oftransplanted tumor.In the group of fresh tumortissues with greater than 50% tumor cell content,the success rate of the transplantationwas 100%(3 cases of pancreatic cancer and 3cases of colorectal cancer).The orthotopicallytransplanted tumors resemble the original tumormorphologically and biologically,including TAAexpression such as CEA byimmunohistochemistry,and CEA level in theserum of mice.Ki-67 labeling index and theexpression of TAA especially K-ras,17-1A andRA-96,are associated with the potential of tumorgrowth in nude mice.Micrometastases in thelungs and livers of tumor bearing mice can bedetected by immunostaining with biotinylatedmab KL-1 and CK20-specific RT-PCR.CONCLUSION An orthotopic transplantationmodel for human colon and pancreatic cancer innude mice has been set up.We have alsoestablished sensitive detection methods withCK-immunohistochemistry and CK20-RT-PCR tostudy xenotransplanted human cancer and itsmetastatic cancer cells in the liver and lung ofnude mice.This study may be helpful inunderstanding the mechanism of cancermetastasis and in developing new diagnosticmethods and therapeutic strategies formetastases including micrometastases.     

Effects of recombinant human canstatin protein in the treatment of pancreatic cancer

INTRODUCTION The prognosis of patients with pancreatic cancer is poor, with or without treatment. The American National Cancer Institute (NCI) reported in its SEER Cancer Statistics Review that there are approximately 27 000 new cases of pancreatic cancer…     

Study of orthotopic transplantation model of human gastrointestinal cancerand detection of micrometastases

AIM To establish a relevant animal model of human gastrointestinal cancer, which can be used forrepetitive investigations and may improve our understanding of carcinogenesis and cancer metastasis.METHODS Intact tissue of human colorectal and pancreatic cancers was transplanted in nude mice. Thebiological characteristics of the original and corresponding transplanted tumors were investigated by HEstaining, PAS staining and immunostaining. The metastases in livers and lungs of the nude mice wereinvestigated by immunostaining with biotinylated mab KL-1 and by RT-PCR using CK20 specific primers.RESULTS Nine of 16 surgical specimens grew in the nude mice subcutaneously and/or orthotopically (4 of6 colorectal and 5 of 10 pancreatic cancer). Tumor cell content of the specimens and freezing of tissuespecimens are important factors influencing the growth of transplanted tumor. In the group of fresh tumortissues with greater than 50% tumor cell content, transplantation rate was 100% (3 cases of pancreatic cancerand 3 cases of colorectal cancer). The orthotopically transplanted tumors resembled the original tumormorphologically and biologically, including TAA expression such as CEA by immunohistochemistry, andCEA level in the serum of mice. Ki-67 labeling index and the expression of TAA especially K-ras, 17-1A and RA-96, were associated with the potential of tumor growth in nude mice. Micrometastases in the lungs andlivers of tumor bearing mice could be detected by immunostaining with biotinylated mab KL-1 and CK20-sepcific RT-PCR.CONCLUSION An orthotopic transplantation model for human colon and pancreatic cancer in nude micehas been established. The sensitive detection methods with CK-immunohistochemistry and CK20-RT-PCRwere also established to study xenotransplanted human cancer and its metastatic cancer cells in the liver andlung of nude mice. This study may be helpful in understanding the mechanism of cancer metastasis and indeveloping new diagnostic methods and therapeutic strategies for metastases.Acknowledgement The authors thank Dr. J. Luettges, Department of Pathology; Kiel University, for investigating thepathological characterics of the specimens; Dr. N. Zawazawa, Institute of Immunology, Kiel University, for the quantitativemeasurement of serum of CEA.     

光动力疗法对胰腺癌移植瘤的疗效及其时-效关系

目的 探讨光动力疗法(PDT)对人胰腺癌裸鼠移植瘤的疗效及其时-效关系.方法 将人胰腺癌细胞SW1990皮下种植后形成的瘤块剪成1 mm3组织块再移植至36只裸鼠皮下,待皮下移植瘤直径达0.8～1 cm时按随机分组法分为3组:荷瘤对照组、光敏剂组(腹腔内注射Photosan 2 mg/kg体重)、光动力组(腹腔内注射Photosan 2 mg/kg体重,48 h后予激光照射),每组12只.每周2次测量肿瘤大小,3周后取瘤称瘤重,计算瘤体积及抑瘤率.结果 PDT组治疗后第6天瘤体积为(0.24±0.15)cm3,显著小于荷瘤对照组的(0.43±0.18)cm3和光敏剂组的(0.39±0.15)cm3(P＜0.05),并随着时间延长,差异更加显著.但治疗后15 d起,PDT组移植瘤体积又开始增大.治疗21 d后,PDT组瘤重为(0.69±0.23)g,显著小于荷瘤对照组的(1.65 ±0.21)g和光敏剂组的(1.62±0.12)g(P＜0.05).PDT组抑瘤率为58.18%,显著高于光敏剂组的1.80%(P＜0.05).结论 PDT对胰腺癌具有显著的抑制作用,起效快,但单用疗效维持时间不长.     

Magnetic resonance imaging to measure therapeutic response using an orthotopic model of human pancreatic cancer

Pancreatic cancer is one of the most incurable and lethal human cancers in the United States. To facilitate development of novel therapeutic agents, we previously established an orthotopic pancreatic tumor model that closely mimics the natural biological behavior of human pancreatic cancer. In this study, magnetic resonance imaging (MRI) techniques were developed to detect tumor formation noninvasively and monitor serially tumor growth kinetics in this orthotopic model used for experimental drug testing. By using an optimized T2-weighted imaging method, we were able to distinguish human pancreas cancer from normal mouse pancreas. Orthotopic tumor formation was detected as early as day 1 after tumor cell implantation with a tumor volume as small as 12 mm3. Mice with evidence of tumor were separated into four treatment groups: control, auristatin-PE, gemcitabine, and their combination. After treatment, the mice were imaged at least three times before termination of the experiment. Comparison between MRI tumor volume measurements and tumor weights made at biopsy resulted in a correlation coefficient of 0.98. The tumor growth curves constructed from serial magnetic resonance imaging (MRI) measurements clearly showed tumor growth inhibition in treated mice compared with the control group. As expected, the group treated with the combination had the highest response rate compared with either auristatin-PE or gemcitabine alone, and the data were statistically highly significant (p < 0.004). From these results, we conclude that noninvasive MRI can be used to monitor serially therapeutic response in this orthotopic human pancreatic tumor model and can be used in the future to evaluate novel antitumor agents before human studies.