肝癌细胞不同制悬方法建立裸鼠移植瘤模型的比较

目的比较肝癌细胞不同制悬方法建立裸鼠皮下瘤及原位瘤模型,优选建模方法。方法将肝癌细胞分别采用PBS缓冲液(PBS组)、无血清DMEM溶液(DMEM组)和含10%血清的DMEM溶液(血清DMEM组)3种液体制悬后注入裸鼠皮下建立肝癌裸鼠皮下瘤模型,比较皮下瘤的体积、瘤重及成瘤率;将肝癌细胞分别采用PBS缓冲液(PBS组)、无血清DMEM溶液(DMEM组)和含10%血清的DMEM溶液(血清DMEM组)3种液体制悬后注入裸鼠肝脏建立肝癌裸鼠原位瘤模型,比较原位瘤的成瘤率。结果3组裸鼠皮下瘤模型的皮下瘤体积及瘤重相比,差异无统计学意义(P0.05),但是PBS组及DMEM组的成瘤率(90%和100%)明显高于血清DMEM组(40%),差异有统计学意义(P0.05);裸鼠原位瘤模型中PBS组的成瘤率(90%)明显高于DMEM组(40%)及血清DMEM组(20%),差异有统计学意义(P0.05)。结论采用PBS缓冲液或无血清DMEM溶液对肝癌细胞稀释制悬可以有效建立裸鼠皮下瘤模型,采用PBS缓冲液对肝癌细胞稀释制悬可以有效建立裸鼠原位瘤模型。     

抗端粒酶核酶抑制肝癌细胞裸鼠移植瘤生长的研究

设计针对端粒酶RNA组分的特异性核酶，研究该核酶对肝癌细胞裸鼠移植瘤生长的影响。构建含针对端粒酶RNA组分锤头状核酶基因的重组真核表达质粒pBBS212-RZ，以及建立人肝癌细胞株HepG2裸鼠移植瘤模型。将不同剂量的pBBS212-RZ用脂质体包裹后进行瘤体内多点注射，对照组注射生理盐水或空质粒载体pBBS212。连续注射2l天后，测量移植瘤体积和重量，检测瘤组织端粒酶活性。抗端粒酶特异性核酶使瘤组织端粒酶活性下降(抑制率为72％)，明显地抑制了肝癌细胞裸鼠移植瘤生长(抑制率为68％)，且存在剂量依赖性。抗端粒酶特异性核酶作为一种有效的端粒酶抑制剂，可抑制肝癌细胞的生长，有望在肝癌基因治疗中发挥作用。     

人肝癌裸鼠移植模型的研究进展

肝癌的基础与临床研究迫切需要能真正模拟肝癌在人体内自然生长、侵袭及转移全部过程的动物模型.目前,人肝癌裸鼠移植模型是人体外最接近人类肝癌的整体实验模型,并且造模时间短,成功率高,按移植部位可以分为皮下移植、原位移植、腹腔移植以及转移模型.影响裸鼠移植模型建立的因素主要有人肝癌细胞或外科标本的特性、移植部位及移植癌细胞数量、裸鼠的品系和周龄以及生长环境和其他因素的影响.     

荷瘤裸鼠膀胱癌原位模型的建立

目的通过细胞移植法建立模拟人的膀胱原位癌荷瘤裸鼠模型，为临床膀胱癌的防治提供新的思路。方法将人膀胱移行细胞癌株T24原位移植到裸鼠膀胱内后定期用磁共振成像（MRI）检测，并进行解剖，组织病理学和免疫细胞学检查。结果裸鼠膀胱内的癌发生率为94．0％（47／50），组织病理学检查：Ⅰ级23例，Ⅱ级15例，Ⅲ级9例，免疫细胞学证实该模型具有人膀胱移行细胞癌株T24的特性。结论模拟人的荷瘤裸鼠膀胱癌原位模型符合临床上膀胱肿瘤向膀胱内生长的过程，具有很强的实用性。     

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.     

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.     

超声内镜引导下钬激光对裸鼠胰腺原位种植瘤消融效果的实验研究

目的：本研究的主要目的是探索超声内镜引导下钬激光对裸鼠胰腺原位种植瘤消融治疗的有效性,为超声内镜引导下钬激光对肿瘤的消融治疗应用于临床提供实验基础。方法：选取4-6周龄的雄性balb/c裸鼠20只,将胰腺癌细胞株SW1990接种至裸鼠胰腺,构建裸鼠原位种植瘤模型,随机分配裸鼠成2组,每组10只,实验组给予超声内镜引导下钬激光的消融治疗,对照组不给予任何治疗。于治疗后第7天,第14天和第28天予超声内镜下测量实验组及对照组裸鼠肿瘤的大小,观察裸鼠活动、食欲等变化。解剖裸鼠,取出肿瘤,行HE染色显微镜下观察胰腺肿瘤组织的病理变化。结果：所有裸鼠均可耐受超声内镜引导下钬激光的消融治疗,实验组裸鼠术后无明显不适反应,精神状态、活动量及食欲较好,对照组裸鼠精神状态逐渐恶化,身材极度消瘦,消融后28天实验组裸鼠肿瘤体积缩小,对照组裸鼠肿瘤增大,两组肿瘤体积的变化差异具有统计学意义,钬激光的治疗疗效大约持续28天左右。HE染色可见消融部位呈凝固性坏死。结论：超声内镜引导下钬激光对裸鼠原位种植瘤的消融治疗是有效的,治疗疗效大约持续28天,主要引起肿瘤组织的凝固性坏死。     

胰腺癌循环微小核糖核酸载体的研究

目的 寻找胰腺癌循环微小核糖核酸（miRNA）的载体.方法 常规培养、传代胰腺癌细胞SW1990、BxPC3,采集6例胰腺癌患者及6例健康体检者（对照组）的血清.应用梯度离心方法获取血清和细胞培养上清中的微囊泡（MV）.采用蛋白质印迹法检测RNA诱导沉默复合体的核心元件Ago2蛋白和MV的标志性蛋白CD63,采用荧光定量PCR方法检测MV包裹的和游离的miRNA.结果 胰腺癌细胞株培养上清的MV中含有Ago2、CD63蛋白.胰腺癌组、对照组的血清及MV中均存在miR-20a、miR-21、miR-24、miR-25、miR-191、miR-483-5p,但以MV包裹的量较多,且胰腺癌组MV包裹的miRNA量与对照组不完全一致,其中miR-20a、miR-24、miR-191分别为对照组的（2.93±0.29）、（2.73±0.46）、（2.39±0.51）倍,差异均有统计学意义（F值分别为75.97、25.80、12.94,P＜0.05或＜0.01）.结论 MV是胰腺癌循环miRNA的主要载体.     

人胰腺癌细胞株PANC1裸鼠原位模型的建立及磁共振监测

目的 建立一种稳定的人胰腺癌裸小鼠原位移植瘤模型,并探讨无创的磁共振(MRI)对其监测的应用价值.方法 人胰腺癌细胞株PANC1体外常规传代后建立裸鼠皮下种植瘤模型,将皮下种植瘤制成细胞悬液,植入20只BALB/C-nn裸鼠的胰腺包膜下构建人胰腺癌原位移植模型;通过MRI检查监测模型的成瘤率、成瘤时间、肿瘤生长速度、肿瘤形态、信号变化等特征,于第7周末取肿瘤组织行病理学检查.结果 接种后15 d,7只(35%)荷瘤鼠在MRI上显示成瘤,至接种后27 d,成瘤率为100%.肿瘤信号与邻近组织相比,90%(18/20)病灶T1WI呈均匀稍低信号,10%(2/20)呈等信号;75%(15/20)在T2WI呈均匀高信号.移植后2、3、4、5、6、7周经MRI测量的移植瘤体积分别为(12.6±2.4)mm3、(94.3±11.2)mm3、(175.9±82.5)mm3、(395.8±126.6)mm3、(1290.2±167.2)mm3、(1583.4±87.4)mm3.病理学检查确诊为胰腺低分化腺癌,并保持原发瘤的生物学特征.结论 人胰腺癌细胞株PANC1移植瘤制成的细胞悬液种植裸小鼠胰腺包膜下制备胰腺癌模型符合人胰腺癌的特征,且易于无创监测,为临床研究提供了一个有效、稳定的体内实验体系.     

裸鼠前列腺原位肿瘤骨转移模型的建立

目的制备裸鼠原位前列腺癌骨转移模型。方法选取8周龄雄性BALB/c裸鼠10只,下腹部切口,显露膀胱和前列腺,在其前列腺左右腹侧叶包膜下种入预先制备好的小瘤块,建立原位肿瘤模型。术后3 d随机分为对照组和单核细胞趋化蛋白-1(MCP-1)组,每组5只。对照组无特殊处置;MCP-1组隔日给予尾静脉注射MCP-1 1μg,3 w后行核素骨扫描检查确定是否出现骨转移及肿瘤骨转移部位。取原位肿瘤及骨组织,HE染色后显微镜下观察细胞形态。结果裸鼠原位前列腺癌模型全部建立成功,MCP-1组均出现骨转移,对照组未见骨转移病灶。结论裸鼠原位前列腺癌骨转移模型模拟了人类前列腺癌骨转移的发生过程。     

Microencapsulated tumor assay: evaluation of the nude mouse model of pancreatic cancer

AIM: To establish a more stable and accurate nude mouse model of pancreatic cancer using cancer cell microencapsulation.METHODS: The assay is based on microencapsulation technology, wherein human tumor cells are encapsulated in small microcapsules (approximately 420 μm in diameter) constructed of semipermeable membranes. We implemented two kinds of subcutaneous implantation models in nude mice using the injection of single tumor cells and encapsulated pancreatic tumor cells. The size of subcutaneously implanted tumors was observed on a weekly basis using two methods, and growth curves were generated from these data. The growth and metastasis of orthotopically injected single tumor cells and encapsulated pancreatic tumor cells were evaluated at four and eight weeks postimplantation by positron emission tomography-computed tomography scan and necropsy. The pancreatic tumor samples obtained from each method were then sent for pathological examination. We evaluated differences in the rates of tumor incidence and the presence of metastasis and variations in tumor volume and tumor weight in the cancer microcapsules vs single-cell suspensions.RESULTS: Sequential in vitro observations of the microcapsules showed that the cancer cells in microcapsules proliferated well and formed spheroids at days 4 to 6. Further in vitro culture resulted in bursting of the membrane of the microcapsules and cells deviated outward and continued to grow in flasks. The optimum injection time was found to be 5 d after tumor encapsulation. In the subcutaneous implantation model, there were no significant differences in terms of tumor volume between the encapsulated pancreatic tumor cells and cells alone and rate of tumor incidence. There was a significant difference in the rate of successful implantation between the cancer cell microencapsulation group and the single tumor-cell suspension group (100% vs 71.43%, respectively, P = 0.0489) in the orthotropic implantation model. The former method displayed an obvious advantage in tumor mass (4th wk: 0.0461 ± 0.0399 vs 0.0313 ± 0.021, t = -0.81, P = 0.4379; 8th wk: 0.1284 ± 0.0284 vs 0.0943 ± 0.0571, t = -2.28, respectively, P = 0.0457) compared with the latter in the orthotopic implantation model.CONCLUSION: Encapsulation of pancreatic tumor cells is a reliable method for establishing a pancreatic tumor animal model.     

Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

AIM： To investigate anti-tumor activities and apoptosisregulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.
METHODS： BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups （n = 15）. Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg （BF1）, 1 mg/kg （BF2） and 0.5 mg/kg （BF3） for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues.
RESULTS： The tumor volumes in each group of bufalin were reduced significantly （35.21±12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P 〈 0.01, respectively）, and the survival times were prolonged in group BF1-2 （31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P 〈 0.05, respectively）, and necrosis was mainly in severe or moderate degree in group BF1-2. No morphological changes were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA     

Metastatic models of human liver cancer in nude mice orthotopically constructed by using histologically intact patient specimens

In this study of orthotopic implantation of histologically intact surgical specimens, the authors constructed metastatic models of human hepatocellular carcinoma (HCC) in nude mice. Histologically intact human liver cancer specimens, derived from patients, were implanted directly into the liver of nude mice, and their orthotopic growth and metastases were observed. The transplantability and metastatic rate of two specimen groups (primary and metastatic lesions) were analysed. -Fetoprotein (AFP) was also determined in transplanted tumours by an immunohistochemical method. Orthotopic growth was observed in 14 of 30 transplanted specimens and formation of metastases in 7 cases, which exhibited the variety of clinical behaviours seen in patients with HCC. These behaviours included local growth, regional invasion, spontaneous intrahepatic, lymph node and lung metastasis and peritoneal seeding. In two groups the growth rate of metastatic lesions following implantation was clearly higher than that of primary tumours. Chromosome analysis from locally growing tumours confirmed their morphologically human origin. An immunohistochemical study showed that implanted tumours originating from AFP-positive specimens maintained AFP expression. These results indicated that the animal models should prove valuable for developing new treatment modalities and studying the mechanism of metastasis of human HCC.Abbreviation HCC hepatocellular carcinoma - AFP -fetoprotein     

胰腺神经内分泌肿瘤伴肝转移的治疗

胰腺神经内分泌肿瘤（pNET）是所有胰腺肿瘤中较少见的一类肿瘤,近些年发病率呈逐渐增加趋势。肝脏是其远处转移的常见器官。对于分化良好、没有肝外转移、可切除的患者,手术仍是首选的治疗方法。对于不可切除的肝转移患者联合肝动脉栓塞化疗、射频消融、化疗和分子靶向药物等多种治疗方法可以延长患者的存活时间。而对于此类患者的肝移植指征则需要根据个体化情况严格掌握。     

Generation and characterization of a transgenic mouse model for pancreatic cancer

AIM: To generate a SV40Tag transgenic tumor animal model and to study the mechanism underlying tumorigenesis. METHODS: A mammary gland expression vector containing SV40Tag DNA was generated. Transgene fragments were microinjeted into fertilized eggs of FVB mice. The genetically manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. PCR and Northern blot analysis were used for genotype analysis of F1 and F2 mice. Transgene expression was detected by RT-PCR and immunohistochemistry. RESULTS: SV40Tag gene was detected in two lines of transgenic mice. One of them delivered the transgene to Fl and a tumor was found in the pancreas of these mice. RT-PCR and immunohistochemistry showed that SV40Tag gene was expressed in the tumor. Pathological characterization of the transgenic mice demonstrated that the tumor belonged to pancreatic cystic neoplasm. CONCLUSION: SV40Tag transgenic mouse model can be successfully established. The transgenic mice develop a pancreatic tumor, which can be used for investigation of the molecular mechanism of tumorigenesis in vivo.     

Anticancer activity of genistein on implanted tumor of human SG7901 cells in nude mice

AIM： To investigate genistein-induced apoptosis of implanted tumors of SG7901 cells in nude mice, and the relationship between this apoptosis and expression of Bcl-2 and Bax. METHODS： Establishing a transplanted tumor model by injecting human SG7901 cells into subcutaneous tissue of nude mice. Genistein （0.5, 1 and 1.5 mg/kg） was directly injected adjacent to the tumor, six times at 2-d intervals. Then, changes in tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphological alterations by transmission electron microscopy （TEN）, measured the apoptotic rate by the TUNEL staining method, and detected the expression of apoptosisregulated gene Bcl-2 and bax by immunohistochemical staining and RT-PCR. RESULTS： Genistein 0.5, 1 and 1.5 mg/kg significantly inhibited carcinoma growth when it was injected near the tumor by 10.8%, 29.9% and 39.6%, respectively. Genistein induced implanted tumor cells to undergo apoptosis, with apoptotic characteristics seen by TEM. The apoptosis index was increased progressively with increasing genistein dose （28.9% ± 1.2%, 33.8% ±1.6% and 37.7% ±1.2%）. The positive rate of Bcl-2 protein was decreased progressively （11.9%± 0.9%, 5.9%± 0.7% and 4.2% ±0.6%）, and the positive rate of bax protein was increased progressively （0.9% ±1.7%, 24.9% ±0.8% and 29.6% ± 1.7%） by immunohistochemical staining, with increasing dose of genistein. The density of Bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR. CONCLUSION： Genistein was able to induce apoptosisof transplanted tumor cells. This apoptosis may be mediated by down-regulation of the apoptosis-regulated gene Bcl-2 and up-regulation of apoptosis-regulated gene bax.     

遗传印记基因PEG 10转基因小鼠的建立及其皮下移植瘤生长和转移的变化

目的 建立PEG10转基因小鼠模型,研究PEG10对小鼠皮下移植瘤的生长和转移的影响. 方法 PCR阳性转基因鼠经RT-PCR、Western blot鉴定后,皮下注射H22细胞,连续测量肿瘤体积.12 d后取肿瘤和肝脏组织进行HE染色,肝脏组织进行SP染色,检测PEG10蛋白的表达.计量资料采用独立样本的f检验.结果 阳性首建鼠的肝脏中枪测到目的 基因和蛋白的表达.转皋因小鼠皮卜瘤的体积(4.08、4.23 cm3)及质量(6.89、6.48 g)均显著高于野生昔小鼠(1.61 cm3及1.63 g,P<0.05),均向周围组织侵袭并出现肝转移,肝脏中检测到PEG10蛋白;野生型小鼠的肿瘤组织有包膜,未出现肝转移. 结论构建的PEG10转基因小鼠模型可促进皮卜移植瘤的生长、侵袭和转移.     

Detection of circulating tumor cells in patients with pancreatic cancer: a preliminary result

Background/Purpose

It has been reported that circulating tumor cells (CTCs) can be used to predict survival in metastatic breast cancer. In this preliminary study, we examined the level of CTCs in pancreatic cancer (PC) patients to elucidate whether we could predict survival in PC.

Methods

The eligible subjects, at Tokyo Medical University Hospital, were 26 patients with PC, 11 with chronic pancreatitis, and 10 healthy volunteers. Three PC patients underwent surgery, 18 patients (who were stage IV) were treated with gemcitabine (GEM), and 5 patients received best supportive care (BSC).

Results

The CTC count was 1/7.5 ml blood or higher (defined as positive) in 11 of the 26 patients (42%; mean, 16.9/7.5 ml blood; range, 1-105/7.5 ml blood). Gemcitabine was administered to 6 of the 11 CTC-positive patients (3.8 courses on average). The treatment was continued for more than three courses in 2 patients, in both of whom the CTC count was only 1/7.5 ml blood. Operation was performed in 1 of the 11 CTC-positive patients. The remaining 4 patients of the 11 CTC-positive patients received only BSC. CTC was negative in 15 patients with PC (stage II, 1; stage III, 1; stage IVa, 7; and stage IVb, 6) and in the subjects with benign conditions. The median survival times (MSTs) of the CTC-positive and-negative patients were 110.5 and 375.8 days (P < 0.001). When the analysis was limited to the 14 stage-IVb patients, the MSTs of the CTC-positive and-negative patients were 52.5 and 308.3 days (P < 0.01).

Conclusions

The present study demonstrated that the detection of CTCs in peripheral blood may be useful to predict prognosis in patients with PC.