胰腺癌神经转移原位移植瘤动物模型的构建及其生物学特性研究

目的建立人类胰腺癌细胞系MIA PaCa—Ⅱ裸鼠胰腺原位移植瘤模型,观察神经侵袭情况,并研究其生物学特性。方法将MIA PaCa—Ⅱ细胞接种于裸鼠背部皮下和胰腺被膜下,建立皮下和原位移植瘤模型,分别于4、6、8周处死裸鼠行原位移植瘤病理学检查,HE和银染观察神经侵袭情况,对K—ras、C—erbB2、环氧合酶-2（COX-2）、前列腺干细胞抗原（PSCA）、p53、DPC4行SABC免疫组织化学染色。结果皮下移植瘤接种成瘤率为100％（10／10）,呈局限性生长,无脏器和神经转移。原位移植4、6、8周时成瘤率均为100％（10／10）,神经转移率分别为50％（5／10）、80％（8／10）和60％（6／10）,并可见多个脏器转移。MIA PaCa—Ⅱ细胞、皮下及原位移植瘤细胞对K—ras、C—erbB2、COX-2、PSCA呈阳性表达,且高于裸鼠正常胰腺细胞,而p53、DPC4表达则低于正常胰腺组织细胞。结论成功建立神经侵袭原位移植瘤动物模型,研究嗜神经转移以6周为宜。K—ras、C—erbB2、COX-2、PSCA高表达与p53、DPC4低表达可能参与了胰腺癌的发生。     

裸鼠动物模型中胰腺癌神经侵袭的研究

  目的   探讨在裸鼠动物模型中胰腺癌神经侵袭发生情况。   方法  人Mia PaCa-2和Capan-2胰腺癌细胞株培养,裸鼠皮下胰腺癌细胞注射成瘤后,将瘤块取出切碎后裸鼠胰腺原位移植建立裸鼠原位胰腺癌动物模型,于术后4、6、8周分批处死裸鼠,采用大体标本观察和病理切片HE染色的方法观察模型中肿瘤大小、转移情况和胰腺癌神经侵袭的情况。   结果  裸鼠胰腺原位植瘤术后成瘤率100%,术后4周Mia PaCa-2组3例（50.00%）发生腹膜后神经侵犯,Capan-2组2例（33.33%）发生腹膜后神经侵犯;原位植瘤术后6周,Mia PaCa-2组5例（83.33%）发生腹膜后神经侵犯,Capan-2组4例（66.67%）发生腹膜后神经侵犯;原位植瘤术后8周,Mia PaCa-2组8例（100.00%）发生腹膜后神经侵犯,Capan-2组7例（100.00%）发生腹膜后神经侵犯。腹膜后神经侵犯组与其他组比较差异均有统计学意义（P<0.01）。   结论  神经侵袭是胰腺癌发展过程中的一个早期、独立的事件,在胰腺癌发生的早期,无其他转移或很少时,已经发生了腹膜后神经侵犯。      

神经菌毛素在胰腺癌组织及MIA PaCa-Ⅱ细胞系中的表达

目的探讨神经菌毛素（Neuropilin-1，NRP-1）在胰腺导管癌组织及MIA PaCa-Ⅱ胰腺癌细胞系中的表达及意义。方法运用免疫组化和RT-PCR法分别检测在正常胰腺组织、癌旁组织、胰腺癌组织及MIA PaCa-Ⅱ细胞系中Neuropilin-1蛋白及mRNA表达水平。结果蛋白水平：可见正常胰腺组织无表达，癌旁组织轻度表达，而胰腺癌组织及MIA PaCa-Ⅱ胰腺癌细胞中高水平表达。神经组织也可表达Neuropilin-1 mRNA水平见正常胰腺组织呈微量表达，癌旁组织中度表达，而胰腺癌组织及MIA PaCa-Ⅱ胰腺癌细胞中呈高水平表达。结论Neuropilin-1可能与参与了胰腺癌的发生发展，在胰腺癌神经转移中可能起着重要作用。     

人骨肉瘤原位移植模型的建立及生物学特征

目的 用人骨肉瘤细胞系HOS-98建立人骨肉瘤裸鼠胫骨原位移植模型,以探讨宿主器官微环境对人骨肉瘤细胞侵袭及转移等生物学行为的影响。方法 将人骨肉瘤细胞系HOS-98接种于裸鼠皮下,形成移植瘤,用传代移植瘤组织作为移植材料,进行胫骨原位移植及皮下移植。分别于移植后4周和8周处死小鼠,进行病理形态学检查,并对两种方法在成瘤率、生长方式及侵袭、转移等生物学行为比较。结果 两种移植方式在成瘤率及形态学上无明显不同,胫骨原位移植的潜伏期较短,并且生长快于皮下移植方式。皮下移植瘤呈局限性膨胀生长,有不完整的纤维包膜,瘤内类骨基质较少见,未见肺转移,观察8周时无明显消瘦;而胫骨原位移植瘤侵袭周围组织,可见发生肺转移,8周明显消瘦。原位移植的裸鼠血清ALP水平高于皮下移植者。原位移植的X线检查有明显的类似于人的骨性反应。结论 用人骨肿瘤细胞系HOS-98皮下接种的移植瘤作为移植材料是建立肿瘤异位移植的可行途径,裸鼠胫骨微环境较皮下组织更适合于人骨肉瘤的侵袭及转移表达,裸鼠胫骨原位移植模型的恶性生物学行为更接近临床骨肉瘤患者的体内侵袭及转移实际,该原位移植模型为今后的实验研究提供了更加接近患者实际的实验模型。     

人类胃癌裸鼠淋巴结转移模型的建立

 目的　研究胃癌淋巴结转移动物模型的建立方法。方法　人类胃癌低分化细胞系SGC-7901体外培养、传代并扩增后，收集细胞行皮下种植成瘤，鼠间传代至第6代，以皮下肿瘤组织块原位种植于裸鼠胃壁建立动物模型。种植后第9周处死裸鼠，观察原位种植瘤生长、淋巴结转移及其他脏器转移情况，测定荷瘤裸鼠血清癌胚抗原（CEA）值。结果　原位移植瘤种植成功率100 ％，胃周淋巴结转移率93.3 ％，移植瘤可发生局部浸润及远处脏器转移，荷瘤裸鼠CEA值明显高于正常裸鼠（P＜0.01）。结论　应用SGC-7901细胞系可成功建立胃癌的淋巴结转移动物模型。     

人胃癌组织块裸鼠原位移植/转移模型的建立

 目的　用肿瘤组织块原位移植 ,建立人胃癌裸小鼠原位移植 /转移模型。方法　以人胃低分化腺癌细胞系接种于裸小鼠皮下 ,形成稳定传代的皮下移植瘤 ,再取该肿瘤组织块原位移植于裸鼠胃壁 ,观察移植肿瘤的生长状况、移植成功率和自发转移的发生率。结果　原位移植成功率 (成瘤率 )为 1 0 0 %、局部淋巴结转移率 1 0 0 %、远处淋巴结转移率 90 %、肝转移发生率为 75%。荷瘤鼠的中位生存期为 1 4周 ,晚期出现消瘦和全身衰竭。结论　该裸小鼠原位移植 /转移模型的生物学行为与人胃癌自然生长和转移过程相似 ,可作为一种有价值的工具用于胃癌转移机理和抗转移实验治疗的研究。     

采用OB胶粘贴法建立人胃癌裸鼠原位种植转移模型

背景与目的建立理想的动物肿瘤移植模型是进行肿瘤防治研究的重要前提,目前公认的胃癌造模的方法为采用完整组织块的“皮下移植法”、“胃囊法”,但传统的造模方法目前尚存在操作复杂、成活率低等缺点,本研究拟采用“OB胶粘贴法”更简便地建立人胃癌裸鼠原位移植模型。方法以反复接种传代于裸鼠皮下的SGC鄄7901人胃癌细胞株建立的移植瘤组织块为材料,将其用生物吻合“OB胶”原位粘贴于裸鼠胃壁,并与传统“胃囊法”、“皮下移植法”比较观察移植肿瘤的生长情况、移植成功率和自发转移的发生率。结果“OB胶粘贴法”组原位移植成功率为100%,局部淋巴结转移率为100%,肺转移发生率为62.5%,肝转移发生率为87.5%,腹膜转移发生率为87.5%,较之胃囊法(分别为100%、100%、50.0%、50.0%和33.3%),除腹膜转移率升高外,远处脏器转移差异无显著性,但成活率高。皮下移植组未发生局部浸润及远处转移。结论“OB胶粘贴法"能更简便地建立人胃癌高转移模型,重现临床转移过程。     

裸鼠人胃癌原位移植模型的建立

目的：建立裸鼠人胃癌原位移植模型。方法：将ＳＧＣ－７９０１人胃癌细胞株细胞接种至裸鼠皮下，建立皮下模型并反复传代，再以第八代移植瘤组织为材料，通过剖腹手术将其移植到裸鼠胃壁，待动物自然死亡后尸检。结果：该模型原位瘤沿胃壁呈浸润性生长，潜伏期为３￣４周，以后迅速增长并与腹膜粘连，约１２周以后逐渐消瘦，生存期为１６￣２４周，原位成瘤率及局部浸润率均为１００％，部分运动（２／６）出现肝脏转移。结论：裸鼠     

裸鼠人胃腺癌SGC-7901原位移植模型的构建及其生物学特性

目的 :建立裸鼠人胃癌原位移植模型。方法 :以反复接种传代于裸鼠皮下的SGC 790 1人胃癌细胞株建立的移植瘤组织块为材料 ,将其原位移植于裸鼠胃壁 ,观察移植肿瘤的生长情况、移植成功率和自发转移的发生率。结果 :原位移植成功率及局部浸润率为 8 8,局部淋巴结转移率为 8 8,肺转移发生率为 5 8,肝转移发生率为 7 8,腹膜转移发生率为 7 8。结论 :裸鼠人胃癌原位移植模型的生物学行为与人胃癌自然生长和转移过程相似 ,可作为肿瘤转移机制及其抗转移治疗实验研究的理想模型     

人类卵巢癌原位移植自发转移裸小鼠模型的构建

 目的　用人类卵巢癌组织块原位移植构建与人类卵巢癌最相似的裸鼠原位移植自发转移模型。方法　将人类卵巢癌高转移细胞系8910PM在裸鼠皮下注射成瘤,然后将瘤组织运用显微外科的方法植入裸鼠卵巢包膜内,原位移植成功后继续在鼠间卵巢包膜内传代,并运用透射电镜进一步验证肿瘤形成和转移效果。结果　裸鼠皮下与卵巢部位成瘤率均为100 %（分别为2／2和16／16）,原位移植瘤转移率为50 %。卵巢包膜间传代,第2代、第4代分别出现卵巢癌的肝转移,其他部位尚未见转移灶。最早成瘤时间1周,最早转移出现时间2周。在连续传代过程中移植瘤仍保持原癌组织病理形态特点及人类肿瘤染色体的特点。结论　成功构建了人类卵巢癌原位移植自发转移裸鼠模型,为卵巢癌的转移机制及治疗研究提供了一个理想的动物模型。     

Establishing orthotopic transplanted models of human pancreatic cancer in nude mice and study on their biological properties]

Two cell lines of human pancreatic cancer have been established, which can be successfully transplanted into pancreas of nude mice, the first of this kind of cell lines in China. Fresh specimens human of pancreatic cancer taken surgically were transplanted in the pancreas of pure line BALB/C-nu/nu nude mice. The transplanted tumours grew and reproduced successfully, and were named PINMP-1 and PINMP-2, respectively. So far, 9 generations of PINMP-1 and 6 generations of PINMP-2 were obtained. Their biological properties, ways of invasion and metastasis and morphological characteristics under light and electron microscope were studied. The results showed a 95%-100% transplanting success rate, with the success rate of transplanting from tissues revivified from the liquid nitrogen preservation being 100%. Both of the lines could produce large amount of CEA, and chromosome analysis confirmed that they had retained a karyotype of the human cancer cells. In nude mice transplanted with the tumours, metastasis could be found in the lymph nodes, lungs and livers. Metastasis via lymphatic channels and blood vessels were also demonstrated. The pathological and ultrastructural examination confirmed that the transplanted tumours had identical characteristics as their donor tumours. The transplanted cells grew independently in the pancreas of the nude mice, making a better model for study on tumour invasion and metastasis than subcutaneously transplanted tumours. This indicated that the microenvironment in the transplantation site had certain influence on the biological behavior of the transplanted tumours. The models could be used in the study on the invasion, metastasis and experimental therapy of pancreatic carcinomas.     

Inhibitory effect of 4-methylesculetin on hyaluronan synthesis slows the development of human pancreatic cancer in vitro and in nude mice

We report the inhibitory effect of 4-methylesculetin (ME), a 4-methylumbelliferone derivative, on hyaluronan (HA) synthesis by pancreatic cancer cells, and its resulting anticancer action. First, HA in cell culture was analyzed using competitive inhibition with hyaluronic acid-binding protein (HABP) to study HA synthesis by the human pancreatic cancer cell line KP1-NK, and cell-surface HA was visualized using a particle-exclusion assay to study the synthesis of extracellular matrix HA. We also analyzed the inhibitory effect of ME on cell adhesion and invasion, which play a role in the invasion, growth and metastasis of human pancreatic cancer. Furthermore, we examined HA in human pancreatic cancer cells transplanted into the hypodermis of nude mice to study the inhibitory effect of ME on HA synthesis. Moreover, pancreatic cancer cells were also transplanted into the abdomen of nude mice to study whether ME would have the potential to prolong the survival of patients with end-stage pancreatic cancer. ME at 10 muM did not inhibit the growth of human pancreatic cancer cells, but inhibited HA synthesis in cell culture by 40%, adhesion by 44% and invasion by 40%. ME inhibited the proliferation of subcutaneous tumors and HA synthesis (by 50%) of pancreatic cancer transplanted into the hypodermis of nude mice. ME also prolonged the survival time of nude mice bearing abdominally transplanted pancreatic cancer cells. ME inhibited pancreatic cancer growth and metastasis by inhibition of HA synthesis. These results suggest that ME may prolong the survival time of patients with end-stage pancreatic cancer.     

Establishment of perineural invasion models and analysis of gene expression revealed an invariant chain (CD74) as a possible molecule involved in perineural invasion in pancreatic cancer.

PURPOSE: Perineural invasion causes frequent local recurrence even after resection and a poor prognosis for pancreatic cancer. We established perineural invasion models and analyzed the molecular mechanism of perineural invasion in pancreatic cancer. EXPERIMENTAL DESIGN: Seven pancreatic cancer cell lines with or without human peripheral nerves were s.c. implanted in nonobese diabetes/severe combined immunodeficient mice. We compared expression profiles among high and low perineural invasion cell lines by using an oligonucleotide microarray. We examined up-regulation of the invariant chain (CD74) in high perineural invasion cell lines in mRNA and protein levels and surgical cases immunohistochemically. RESULTS: Four of seven pancreatic cancer cell lines (CaPan1, CaPan2, CFPAC, and MPanc96) showed perineural invasion to s.c. transplanted human peripheral nerves. Moreover, CaPan1 and CaPan2 (high perineural invasion group) also resulted in a high frequency of perineural invasion to mouse s.c. peripheral nerves, whereas three pancreatic cancer cell lines HPAFII, AsPC1, and Panc1 (low perineural invasion group) did not show perineural invasion to either human or mouse nerves. We identified 37 up-regulated genes and 12 down-regulated genes in the high perineural invasion group compared with the low perineural invasion group. Among them, CD74 was up-regulated in the high perineural invasion group in mRNA and protein levels. Furthermore, immunohistochemical expression of CD74 in clinical cases revealed its significant overexpression in pancreatic cancer with perineural invasion (P < 0.008). CONCLUSIONS: This is the first report of perineural invasion models using human pancreatic cancer cell lines. In combination with gene expression profiling, it was indicated that CD74 could be a candidate molecule involved in perineural invasion. These models provide new approaches for study of perineural invasion in pancreatic cancer.     

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。     

高表达MUC1的人食管癌裸鼠移植瘤模型的建立

背景与目的:Mucin-1(MUC1)粘蛋白是一种肿瘤相关抗原,是肿瘤免疫治疗良好的分子靶点之一.本研究建立高表达MUCl的人食管癌裸鼠皮下移植瘤模型,为研究以MUC1为靶点的食管癌的生物治疗提供体内模型.方法:以体外培养的高表达MUC1的人食管癌细胞株EC-109接种于4～5周龄的BALB/c裸小鼠皮下,观察移植瘤生长情况,对移植瘤进行病理组织学检查,采用免疫组化方法检测移植瘤细胞增殖细胞核抗原(proliferating cell nuclear antigens,PCNA),采用流式细胞仪检测移植瘤细胞的细胞周期及MUC1的表达.结果:裸鼠皮下移植瘤成瘤率为86.0%,移植瘤具有与人恶性肿瘤相似的组织学和生物学特点,其平均PCNA标记指数为(63.5 3.6)%、S期细胞百分比(S-phase fraction,SPF)平均值为(37.6±3.7)%、MUC1的平均表达率为97.5%.结论:高表达MUC1的人食管癌裸鼠皮下移植瘤模型具有恶性肿瘤的生物学特性,可用于研究人食管癌的生物学特点,同时为以MUC1为靶点的食管癌的免疫治疗研究提供了良好的体内模型.     

康莱特注射液合并健择对移植于裸鼠的人胰腺癌的疗效初步研究

目的:探讨康莱特注射液合并健择对移植于裸鼠的人胰腺癌的作用。方法:24只裸鼠皮下接种人胰腺癌细胞PANC-1,10d后随机分成4组,每组6只裸鼠。对照组给予生理盐水;康莱特组自接种第10天起连续给药10次,给药剂量为5·0g/kg,静脉推注;健择组在接种第12天给药1次,给药剂量为60mg/kg;合并给药组剂量及给药时间同单独给药组。停药后1周脱颈处死动物,解剖裸鼠,称体质量、瘤质量,测量肿块大小并计算抑瘤率。结果:单独注射康莱特及健择组的抑瘤率分别为12·24%和13·26%;合并用药组抑瘤率为40·11%,明显高于单独用药组。结论:康莱特注射液与健择对移植于裸鼠的人胰腺癌的抑制作用具有协同效应。     

Nerve growth factor and enhancement of proliferation,invasion, and tumorigenicity of pancreatic cancer cells

Nerve growth factor (NGF) exerts both stimulatory and inhibitory effects on neuronal and certain non-neuronal tumors. In pancreatic cancer NGF is overexpressed, and this overexpression is associated with increased perineural invasion. NGF has the potential to stimulate the growth of some pancreatic cancer cell lines, and this effect is mediated by the phosphorylation of tyrosine kinase receptor A and mitogen-activated protein kinase activation; it is dependent on the expression levels of tyrosine kinase receptor A and p75 receptors. To determine whether cancer cell-derived NGF can participate in the regulation of pancreatic cancer cell proliferation, PANC-1 human pancreatic cancer cells were stably transfected with a full-length human beta-NGF expression vector. In vitro and in vivo growth characteristics were analyzed by proliferation assays and invasion assays and in a nude mouse tumor model. Stable transfection of NGF in PANC-1 cells resulted in enhanced anchorage-dependent growth, with a decrease in doubling times of up to 50%, and in an approximately twofold increase in anchorage-independent cell growth and cell invasion. Furthermore, stably transfected PANC-1 cells showed enhanced tumorigenicity in nude mice. These results suggest that NGF has the capacity to act in a paracrine and/or an autocrine manner in pancreatic cancer and that it enhances cancer cell growth and invasion in vivo, thereby contributing to the aggressiveness and poor prognosis of this disease.