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目的　观察电穿孔技术结合博莱霉素化疗对结肠癌裸鼠移植瘤的抗肿瘤效果 ,探讨其免疫效应机制 ,为其临床应用提供实验依据。方法　 2 0 0 2年 11月至 2 0 0 3年 6月 ,用人结肠癌Lovo细胞系建立结肠癌裸鼠移植瘤模型 ,随机分为电穿孔化疗组 (B E )、单纯化疗组 (B E - )、单纯电击组 (B -E )和阴性对照组 (B -E - ) ,动态观察肿瘤大小 ,7d后处死并取出皮下肿瘤称重 ,观察其组织学改变 ,并用LDH法检测小鼠脾NK、LAK杀伤活性。结果　B E 组移植瘤较其他各组明显缩小 (P <0 0 1) ;B E 组完全缓解 1例 ,部分缓解 7例 ,B -E 和B E -组内各有 1例部分缓解 ,而B -E -组中无一例有效 ,B E 组有效率与其余各组的差异有统计学意义 (P <0 0 1) ;组织学观察也显示B E 组的移植瘤内大片肿瘤细胞坏死、血管损伤和炎细胞浸润 ;各组小鼠的NK和LAK细胞的杀伤活性差异无显著意义 (P >0 0 5 )。结论　电穿孔化疗可显著增强结肠癌裸鼠移植瘤对化疗的敏感性和疗效 ,可能主要与T细胞介导的肿瘤特异性免疫机制有关 ,可成为结肠癌治疗的一种新方法。     

蟾毒灵对裸鼠结肠癌原位移植瘤凋亡基因Caspase-3表达的影响

目的:探讨蟾毒灵治疗裸鼠结肠癌原位移植瘤的作用及机制。方法 :应用不同剂量蟾毒灵(0.5、1.0、1.5 mg/kg)处理人结肠癌细胞株HCT-116建立的裸鼠原位移植瘤模型,分别用生理盐水(NS)(0.2 mL/只)、5-FU(25 mg/kg)做阴性(NS组)和阳性对照(5-FU组),测量移植瘤的体积,观察蟾毒灵对移植瘤的肿瘤抑制率、裸鼠生存期的影响;HE染色观察移植瘤的坏死程度;TUNEL标记法检测肿瘤细胞的凋亡指数;荧光定量PCR方法检测基因Caspase-3 mRNA的表达,免疫组化法和Western印迹法检测Caspase-3蛋白的表达。结果:蟾毒灵各剂量组和5-FU组裸鼠原位移植瘤体积明显小于NS组(P<0.05);蟾毒灵各组移植瘤的凋亡指数与NS组相比明显增加(P<0.05)。结论:蟾毒灵能够抑制裸鼠人结肠癌原位移植瘤的生长,促进肿瘤细胞凋亡,其机制可能与上调基因Caspase-3表达有关。     

缺氧诱导因子1α表达对裸鼠肝癌皮下移植瘤生长的促进作用

目的 探讨缺氧诱导因子1α表达对肝癌移植瘤生长的影响.方法 建立强力霉素诱导缺氧诱导因子1α表达的裸鼠肝癌HepG2 Tet-on-HIF-1α 细胞皮下移植瘤模型;荷瘤裸鼠口服强力霉素(Dox)后,观察上调缺氧诱导因子1α对皮下移植瘤生长的影响.结果 荷瘤裸鼠口服强力霉素可以上调裸鼠皮下移植瘤中缺氧诱导因子1α mRNA和蛋白表达水平;肿瘤体积Dox(+)组vs.Dox(-)组为(513.545±276.229)mm 3 vs.(166.506±110.142)mm 3 (P<0.05),肿瘤重量(1.251±0.438)g vs.(0.640±0.296)g(P<0.05),肿瘤生长速度Dox(+)组明显超过Dox(-)组,肿瘤内面积坏死率明显小于Dox(-)组(31.360%±2.728%)vs.(36.640±3.804%)(P<0.05);同Dox(-)组相比,Dox(+)组裸鼠体重下降更为明显(P<0.01).两组荷瘤鼠均无肝、肺转移发生.结论 强力霉素可以诱导裸鼠肝癌HepG2 Tet-on-HIF-1α 细胞皮下移植瘤模型中缺氧诱导因子1α的表达,促进肿瘤的生长.     

结肠皮下易位结肠癌荧光原位移植模型的建立

目的 建立结肠皮下易位结肠癌荧光原位移植模型并对其肿瘤生物学进行评价.方法 选取10只BALB/C nu/nu裸鼠,将其盲肠转移至皮下,并将携带荧光的肿瘤移植在盲肠处;对5只荷瘤裸鼠予以20 mg/kg的氟尿嘧啶(5-FU)腹腔注射(处理组),另5只作为对照.观察成瘤裸鼠肿瘤生长情况、肠系膜淋巴结及肝脏转移情况.结果 10只裸鼠模型均成功构建,肿瘤移植后荧光灯下最早发现肿瘤时间为3～9(4.7±1.3)d.5-FU处理组肿瘤生长速度明显低于对照组,差异有统计学意义(P＜0.05).处理组肠系膜淋巴结转移1例,对照组淋巴结转移1例,腹膜转移1例;原位肿瘤、淋巴结和腹膜转移肿瘤病理检查均为低分化癌.结论 结肠皮下易位结肠癌荧光原位移植模型有建立方便、成功率高、肿瘤易早期观察、可反复取材等优点,是一种实用的结肠癌新模型.     

结肠皮下易位结肠癌荧光原位移植模型的建立

目的 建立结肠皮下易位结肠癌荧光原位移植模型并对其肿瘤生物学进行评价.方法 选取10只BALB/C nu/nu裸鼠,将其盲肠转移至皮下,并将携带荧光的肿瘤移植在盲肠处;对5只荷瘤裸鼠予以20 mg/kg的氟尿嘧啶(5-FU)腹腔注射(处理组),另5只作为对照.观察成瘤裸鼠肿瘤生长情况、肠系膜淋巴结及肝脏转移情况.结果 10只裸鼠模型均成功构建,肿瘤移植后荧光灯下最早发现肿瘤时间为3～9(4.7±1.3)d.5-FU处理组肿瘤生长速度明显低于对照组,差异有统计学意义(P＜0.05).处理组肠系膜淋巴结转移1例,对照组淋巴结转移1例,腹膜转移1例;原位肿瘤、淋巴结和腹膜转移肿瘤病理检查均为低分化癌.结论 结肠皮下易位结肠癌荧光原位移植模型有建立方便、成功率高、肿瘤易早期观察、可反复取材等优点,是一种实用的结肠癌新模型.     

多西他赛联合照射对人胃癌裸鼠移植瘤生物效应的实验研究

目的:观察多西他赛对人胃癌裸鼠移植瘤的放射增敏作用和多西他赛联合放射对肿瘤细胞凋亡的影响。方法:建立人AGS胃癌裸鼠移植瘤动物模型28只,随机分为4组:对照组、单纯照射组、单纯多西他赛组、多西他赛 照射组。测量各组移植瘤体积,计算各组抑瘤率,评价多西他赛的放射增敏效果,并对标本进行光镜观察及原位末段标记(TUNEL)检测。结果:28只裸鼠的成瘤率为100%,实验期间无死亡,比较各组移植瘤体积差异有统计学意义(P<0.01)。各组抑瘤率分别为单纯照射组36.4%,单纯多西他赛组44.2%,多西他赛 照射组86.9%。多西他赛对放射治疗的增敏值(enhancement factor,EF)值为1.5。多西他赛 照射组的平均凋亡指数(AI)为(15.6±2.3)%,与其它各组比较差异有统计学意义(P<0.01)。结论:多西他赛对人AGS胃癌裸鼠移植瘤有良好的放射增敏作用,多西他赛联合放疗能更有效地诱导肿瘤细胞凋亡。     

低分子肝素对种植性乳腺癌生长和转移的抑制作用

目的 观察低分子肝素在种植性乳腺癌动物模型中对肿瘤生长和转移的抑制作用.方法 制作120例MCF-7乳腺癌细胞移植瘤裸鼠模型,将其随机分为低分子肝素组、生理盐水组、化疗组、内分泌治疗组,每组30只;观察裸鼠一般情况及移植瘤的牛长变化,28 d后统一脱颈椎处死裸鼠,切取移植瘤标本进行常规镜检及应用免疫组织化学检测瘤组织内血管内皮生长因子(VEGF)和微血管密度(MVD)的变化.结果 实验4周后,低分子肝素组、化疗组、内分泌治疗组不仅在移植瘤体积、胸壁浸润及淋巴转移等方面明显优于生理盐水组,其VEGF、MVD的表达亦低于生理盐水组,差异有统计学意义(P<0.01);其中尤以低分子肝素组中VEGF、MVD的表达更低,但与化疗组、内分泌治疗组间对比差异并无统计学意义(P>0.01).结论 低分子肝素在裸鼠MCF-7移植瘤模型中可以通过抑制肿瘤血管形成而抑制移植瘤的生长及转移.     

TK/GCV、CD/5-Fc双自杀基因系统治疗结肠癌的实验研究

目的观察胸苷激酶/丙氧鸟苷(TK/GCV)、胞嘧啶脱氨酶/5-氟胞嘧啶(CD/5-Fc)双自杀基因系统对结肠癌的治疗效果,并结合研究结果进行作用机理的分析。方法采用培养细胞移植法,将人结肠癌细胞系SW480接种于裸鼠背部皮下,建立裸鼠人结肠癌移植瘤模型。将32只裸鼠随机分为4组:空白对照组,TK/GCV治疗组,CD/5-Fc治疗组,TK/GCV CD/5-Fc联合治疗组,每组8只。TK/GCV CD/5-Fc采用逆转录病毒介导,采用瘤体内直接注射法,同时腹腔注射GCV、5-Fc治疗,观察各组小鼠的生存状况及肿瘤体积、瘤重、肿瘤生长抑制率、常规病理、生存期等指标,比较观察各治疗组的治疗效果及对荷瘤裸鼠存活的影响。结果各治疗组裸鼠人结肠癌移植瘤的生长均受到显著抑制,治疗后各组体积数增加(P<0.05),治疗组裸鼠存活期显著延长,TK/GCV CD/5-Fc联合治疗组效果最好,疗效q值=1.675>1.15,即TK/GCV、CD/5-Fc有交互协同作用。结论TK/GCV与CD/5-Fc对人结肠癌SW480细胞有明显抑制作用,TK/GCV、CD/5-Fc双自杀基因系统效果更强。     

RhoC-siRNA 对裸鼠结肠癌移植瘤的生长抑制作用

目的 探讨小干扰RNA(siRNA)载体介导抑制RhoC表达对人结肠癌裸鼠移植瘤的抑制作用.方法 将人结肠癌细胞HT-29接种于裸鼠背部皮下,成功建立裸鼠移植瘤模型;成瘤后将RhoC-siRNA注射入裸鼠移植瘤瘤体,观察RhoC-siRNA对裸鼠移植瘤生长的影响,Western blot检测瘤体中RhoC、基质金属蛋白(MMP-2)和血管内皮生长因子(VEGF)蛋白表达.结果 RhoC-siRNA对结肠癌裸鼠移植瘤的生长有明显抑制作用,瘤体体积重量明显减轻; RhoC-siRNA治疗后瘤体内VEGF和MMP-2蛋白表达明显下降.结论 通过抑制裸鼠结肠癌移植瘤中RhoC的表达,可以抑制肿瘤的生长并下调VEGF和MMP-2的表达水平.     

PPIB蛋白对结肠癌成瘤的影响和低氧机制

目的:研究肽酰脯氨基顺反异构酶B(peptidyl-prolyl cis-trans isomerase,PPIB)蛋白对结肠癌细胞成瘤的影响,探讨结肠癌低氧发病的机制。方法:应用RNAi技术,构建下调PPIB蛋白表达的稳转结肠癌细胞株,移植到BALB/c裸鼠皮下,检测PPIB蛋白下调组与对照组的成瘤差异。TUNEL法检测两组移植瘤的细胞凋亡水平。低氧培养结肠癌细胞株,Western印迹法检测PPIB的表达水平与低氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)的关系。结果:下调PPIB蛋白表达后,在裸鼠皮下成瘤的能力显著减弱。SW480-si PPIB下调组的移植瘤体积显著小于SW480-NC对照组(P<0.01)。TUNEL检测显示下调组的癌细胞凋亡比例显著高于对照组(P<0.001)。在低氧环境下培养后,结肠癌细胞的PPIB蛋白表达水平随着低氧条件下HIF-1α的上调而升高(P<0.05)。结论:PPIB蛋白在结肠癌中因为低氧环境而表达升高,且在升高后通过抑制癌细胞的凋亡而发挥促进结肠癌形成和进展的作用,是一个极具临床应用价值的防治结肠癌的分子靶点。     

电穿击化疗治疗异位移植裸小鼠人结直肠肿瘤肝转移

目的 :用电穿击化疗对异位移植裸小鼠人结直肠肿瘤肝转移模型进行治疗 ,探讨其疗效和机制。方法 :将 4 0只皮下人结肠癌LoVo细胞系肿瘤的裸鼠随机分成 1 0区组 ,每区组 4只 ,随后将每区组的小鼠分至电穿击化疗组、化疗组、电击组、阴性对照组 ,并给予相应的治疗。 1w后处死小鼠 ,观察瘤重及病理改变情况。结果 :电穿击化疗组小鼠的肿瘤显著减小 ,其中有一只小鼠的肿瘤完全消失 ,平均瘤重为 1 8.3mg ,与其余各组平均瘤重 (化疗组 36 .9mg、电击组 37.9mg、阴性对照组 4 2 .7mg)相比在统计学上差异有显著性 ,P<0 .0 1。光镜下可见电穿击化疗组的肿瘤组织有大量片状坏死、炎性细胞浸润、血管的扭曲及内皮的脱落和坏死 ,而化疗组和电击组则仅见少量炎性细胞的浸润和单个细胞的坏死。结论 :电穿击化疗可以显著增强肿瘤对化疗的敏感性 ,为人结直肠肿瘤肝转移的治疗提供了一种新的途径     

电脉冲化疗治疗人前列腺癌裸鼠移植瘤

目的　探讨平阳霉素（ＰＹＭ） 与电脉冲（ＥＰ） 相结合的电脉冲化疗对人前列腺癌细胞系ＰＣ３ｍ 裸鼠皮下移植瘤的作用。方法　将左右侧颈背部荷有ＰＣ３ｍ 肿瘤的裸鼠随机分为５ 组：按左右不同分别给予如下治疗：（１）Ｐ－ Ｅ－ ｜Ｐ＋ Ｅ－ ,（２）Ｐ－ Ｅ－ ｜Ｐ－ Ｅ＋ ,（３）Ｐ－ Ｅ－ ,｜Ｐ＋ Ｅ＋ ,（４）Ｐ＋ Ｅ－｜Ｐ＋ Ｅ＋ ,（５）Ｐ－ Ｅ＋ ｜Ｐ＋ Ｅ＋ 。Ｐ－ Ｅ－ 代表未治疗,Ｐ＋ Ｅ－ 代表肿瘤内注射平阳霉素,Ｐ－ Ｅ＋ 代表单给电脉冲,Ｐ＋ Ｅ＋ 代表电脉冲化疗,即肿瘤内注射平阳霉素后给予电脉冲。结果　亚治疗量的平阳霉素肿瘤内注射后给予电脉冲,１００％ 有效,其中６２．５ ％ 部分缓解、３７．５％ 完全缓解,即Ｐ＋ Ｅ＋ 有效,而其他组合无效。结论　平阳霉素介导的电脉冲为前列腺肿瘤及其他肿瘤的局部治疗提供了新思路。     

In vitro chemosensitivity tests on human tumor xenografts by clonogenic assay: the combined use of mitomycin C with alpha-interferon or gamma-interferon

Using the human tumor clonogenic assay technique, the effects of Mitomycin C plus either alpha-interferon or gamma-interferon were studied against five human tumor xenografts serially transplanted into nude mice (three gastric and two colon cancers). When the survival fraction found with the drug combination was smaller than the multiplication of survival fractions with either drug alone, the combined effect was defined as synergism. Similarly, antagonistic effect was defined when the survival fraction of drug combination was larger than the larger one observed in either interferon or Mitomycin C alone. Four out of five human tumor xenografts (three gastric and one colon cancers) showed synergistic effects in combination of alpha-interferon with Mitomycin C. Though two gastric cancer xenografts exhibited synergistic effects in combination of gamma-interferon with Mitomycin C, antagonistic effects, which was not found in combination of alpha-interferon with Mitomycin C, were observed in one gastric cancer and one colon cancer xenografts.     

Electrochemotherapy: aspects of preclinical development and early clinical experience

OBJECTIVE: To develop an optimized, reproducible system of electrochemotherapy, and to investigate its clinical application in patients with cutaneous or subcutaneous recurrences of inoperable or progressive disease recalcitrant to current anticancer treatments. BACKGROUND: Electrochemotherapy is the application of electric pulses to tumor tissue, rendering the cell membranes permeable to otherwise impermeant or poorly permeant anticancer drugs. This facilitates a potent local cytotoxic effect. STUDY DESIGN: The optimal parameters for electrical pulses and bleomycin concentration were obtained in vitro and then applied to tumors derived from 4 histologically distinct human cancer cell lines (7860, PC3, OE19, MCF-7) established in athymic nude mice. Comparison was made with tumors that received bleomycin alone, electric pulses alone, and untreated controls. The optimized electrochemotherapy was then applied to patients with cutaneous or subcutaneous tumors, of any histologic type, recurrent or metastatic and unresponsive to standard chemotherapy and/or radiotherapy regimens. Tumors were assessed at monthly intervals to determine response to the treatment. RESULTS: In vivo: Using the optimal parameters ascertained in vitro, all tumors treated by electrochemotherapy with bleomycin (n = 24) had significantly regressed (P < 0.001, all 4 lines) compared with control tumors (n = 72). Twelve tumors completely regressed (50%) following a single application, with 12 partial regressions (50%). Clinical: In 30 patients (111 tumors), none of the treated tumors progressed. Sixty percent of tumors (66 of 111) showed complete regression, 22% (24 of 111) partial response, and 18% (21 of 111) no change. Electrochemotherapy was more effective in smaller tumors (<3 cm), 71% (64 of 90) showing complete regression, 20% (18 of 90) partial response, and 9% (8 of 90) no change. CONCLUSIONS: Electrochemotherapy parameters optimized in vitro are applicable in vivo. This treatment is effective in athymic nude mice for all histologic types indicating a nonimmunologic mode of action. In clinical application, electrochemotherapy is an effective, safe, and reproducible therapy. Patients with cutaneous or subcutaneous tumors previously refractory to surgical intervention, systemic chemotherapy, and/or radiotherapy responded successfully irrespective of histologic type.     

PTEN真核表达质粒对人膀胱癌裸鼠移植瘤的抑制效应

目的:探讨PTEN真核表达质粒对人膀胱癌裸鼠移植瘤的抑制作用。方法:将人膀胱癌细胞株BIU-87裸鼠背部皮下接种,成瘤后于瘤内多点注射重组真核表达质粒pBp-PTEN,设pBp空质粒和生理盐水为对照,观察肿瘤生长情况、PTEN表达的变化。结果:pBp-PTEN治疗组肿瘤变小,PTEN表达阳性率提高,与对照组比较,差异有统计学意义(P<0.05)。结论:PTEN真核表达质粒pBp-PTEN瘤内注射对人膀胱癌裸鼠移植瘤有一定的抑制作用。     

Possible Involvement of Antitumor Immunity in the Eradication of Colon 26 Induced by Low-Voltage Electrochemotherapy with Bleomycin

Purpose: The antitumor efficiency of electrochemotherapy using chemotherapeutic agents and high-voltage electric pulse has been reported. This study was done to define the precise nature of the involvement of antitumor immunity in the regression of tumor nodules in electrochemotherapy, and to evaluate the effectiveness of using low-voltage electroporation. Methods: Balb/c mice and Balb/c nu/nu nude mice were inoculated subcutaneously with Colon 26 cells or Meth A cells. Electrochemotherapy using bleomycin and low-voltage electroporation (CUY21) was performed as a treatment against tumor nodules. Results: Colon 26 tumors were eradicated in the mice given an intratumor (i.t.) injection of 500 μg bleomycin followed by treatment with electric fields ranging from 50 to 150 V/cm, with complete response rates ranging from 80% to 100%. The mice rejected inoculations of rechallenged Colon 26 cells, but not Meth A cells. In the Balb/c nu/nu nude mice, complete regression of the tumor was not seen after electrochemotherapy under the same therapeutic conditions that resulted in almost complete cure in the Balb/c mice. Conclusion: Our results suggest that the generation of T-cell-dependent, tumor-specific protective immunity might be involved in the process of tumor nodule regression in low-voltage electrochemotherapy. Received: February 14, 2002 / Accepted: July 2, 2002 Reprint requests to: Y. Gunji     

Characterization of HCT116 human colon cancer cells in an orthotopic model

BACKGROUND: Colorectal cancer metastases result in a significant number of cancer related deaths. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. In the absence of any transgenic models of colon cancer metastases, an in vivo model system that fulfills the rate limiting steps of metastasis (local invasion and distant colony formation) is needed. The purpose of this study was to characterize the behavior of a human colon cancer cell line, HCT116 in an orthotopic model. MATERIALS AND METHODS: HCT116 cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into 32 other male BALB/c nude mice using microsurgical techniques. Animals were serially imaged and euthanized at 6-8 weeks post-implantation. Tissues were procured and processed for hematoxylin and eosin analysis. RESULTS: All 32 animals demonstrated primary tumor growth, invasion and peritoneal spread. Liver metastases were identified in 15/32 (47%), and lung metastases were confirmed in 13/32 (41%). In total, 19/32 (59%) animals demonstrated distant metastatic colony formation. CONCLUSIONS: This orthotopic model of colon cancer fulfills the rate limiting steps of local invasion and distant colony formation in the process of metastases. HCT116 human colon cancer cell line in this in vivo model system provides a tool to dissect the molecular mechanism involved in the metastatic cascade.     

Treatment with soybean-derived Bowman Birk inhibitor increases serum prostate-specific antigen concentration while suppressing growth of human prostate cancer xenografts in nude mice.

BACKGROUND: Bowman Birk inhibitor (BBI) is an anticarcinogenic serine protease inhibitor that may inhibit the protease activity of prostate-specific antigen (PSA) and the growth of human prostate cancer xenografts in nude mice. METHODS: Human prostate cancer xenografts were established by implanting LNCaP cells into the prostate glands of NCRNU-M athymic nude mice. The animals with established tumors were maintained on a control diet or diets supplemented with 1% BBI or 1%, 2%, or 3% BBI concentrate (BBIC) for 6 weeks. The serum PSA concentrations were determined before and after the BBI or BBIC treatment period. The final tumor loads were determined at autopsy. RESULTS: Treatment with BBI or BBIC decreased the final tumor load and increased the tumor doubling time and PSA density in the nude mice bearing human prostate cancer xenografts. CONCLUSIONS: BBI and/or BBIC could be useful for prostate cancer treatment.     

Electrochemotherapy significantly inhibits the growth of colon 26 tumors in mice

Electrochemotherapy is a novel antitumor treatment involving the systemic administration of bleomycin followed by the delivery of electrical pulses to the tumor. The present study investigates the effects of electrochemotherapy on the growth of colon 26 cells inoculated subcutaneously into the backs of BALB/c mice. The mice were divided into the following four experimental groups: 20 that received no further treatment after the inoculation of colon 26 cells (control group); 20 that received 500 μg of bleomycin intraperitoneally 7 and 9 days after the inoculation (BLM group); 20 that received electric pulses to the tumor 7 and 9 days after the inoculation (EP group); and 30 that received electrochemotherapy 7 and 9 days after the inoculation (ECT group). During 28 days of observation, no deaths due to tumor progression occurred in the ECT group, but there were 18 in the control group, 11 in the BLM group, and 18 in the EP group. While weight loss was observed in all groups, it was most remarkable in the control group. Tumor growth was significantly inhibited in the ECT group, compared to the other experimental groups (P<0.01). The results of this study demonstrated that electrochemotherapy significantly inhibited the growth of colon 26 tumors in mice, without causing any remarkable adverse effects.