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.     

应用阳离子脂质体介导内皮抑素和(或)p53基因治疗Lewis肺癌小鼠的实验研究

目的 观察SA阳离子脂质体介导内皮抑素基因和（或）p53基因对Lewis肺癌小鼠移植瘤生长、转移的抑制作用。方法 取Lewis肺癌细胞悬液建立C57BL/6j小黑鼠肺癌动物模型后,选择40只随机分成5组,每组8只,分别为空白对照组、实验对照组、p53治疗组、pEnd治疗组、pEnd＋p53联合治疗组。小鼠成瘤后,瘤内注射SA阳离子脂质体介导的内皮抑素基因和（或）p53基因,每周2次,共6周。观察瘤体大小变化、小鼠营养状况、生存期等。结果 各治疗组均能抑制肿瘤生长及肺内转移,与对照组比较有统计学意义（P〈0.01）,小鼠活动能力、饮食、对外界刺激反应能力等均优于对照组。结论 应用SA阳离子脂质体介导内皮抑素基因和（或）p53基因瘤内注射可有效地抑制Lewis肺癌移植瘤的生长、转移,小鼠生存期明显延长于对照组。内皮抑素基因与p53基因联合未发现协同作用。     

Targeted IL-24 gene therapy inhibits cancer recurrence after liver tumor resection by inducing tumor cell apoptosis in nude mice

BACKGROUND:Interleukin-24(IL-24)is a novel candi-date tumor suppressor that induces tumor cell apoptosis experimentally in a variety of human malignant cells including liver cancer cells.The present study was conducted to investigate the potential effect of recombinant adeno-associated virus(rAAV)-mediated IL-24 gene therapy on tumor recurrence and metastasis by inducing tumor cell apoptosis in a hepatocellular carcinoma(HCC)model in nude mice.METHODS:We established a recurrent and metastatic HCC model in n...     

Retrovirus—mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

AIM: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice. METHODS: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin. The inhibitory effect of endostatin expressed by transfected SMMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted. RESULTS: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P<0.01). In vitro experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P<0.001). The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6+/-1.1, much fewer than that of 22.6+/-4.5 from SMMC-pLncx cells (P<0.01). CONCLUSION: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.     

ATRA inhibits experimental liver metastasis of gastric cancer cells in nude mice

AIM To study the effects of ATRA on experimental liver metastasis of gastric cancer cells.METHODS MGc80-3 and SGC-7901 cells were injectied into spleen subcapsule of nude mice, who weresubsequently administrated with ATRA every other day. Food-intake and body weight of mice were measuredweekly. After six weeks, the nude mice were executed, tumors in spleen and liver were examinedpathologically, microtumor vessel density (MVD) was accounted by immunohistochemical method and serumCEA was measured by radioimmunoassay.RESULTS Nude mice administrated with ATRA, the growth of spleen tumor and its metastatic ability toliver were inhibited, the metastatic rate was decreased by 33.3% (MGc80-3) and 50.0% (SGC-7901). SpleenMVD and liver MVD were reduced by 28.6% and 22.9% (MGc80-3), 23.7% and 37.6% (SGC-7901),respectively. The serum CEA was lowered by 43.4% (MGc80-3).CONCLUSION ATRA can effectively inhibit the experimental liver metastasis of gastric cancer cells,which is relavant with the decrease of MVD and CEA.     

分泌型血管内皮抑素重组腺病毒的构建及其在基因治疗载体肝干细胞中的表达

肝干细胞具有自我更新、高度增殖和多向分化潜能,研究表明肝干细胞为良好的治疗载体细胞,广泛应用于生物型人工肝、肝细胞移植以及先天性代谢性肝病的基因治疗,而近年其在肝癌治疗中的潜力也备受关注.     

重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

介入化疗联合放疗对胃癌肝转移患者免疫调控作用及疗效的影响

目的:探讨介入化疗联合放疗对胃癌肝转移患者的免疫调控作用及其疗效.方法:胃癌肝转移患者33例分为2组,对照组(n=16)采用经肝动脉EAP栓塞化疗方案,治疗组(n=17)经肝动脉EAP栓塞化疗方案药物灌注1次后,再行常规分割外放疗(TD2000-3500cGy).两组患者在治疗后分别检测免疫调节因子IL-2及TNF-α、IFN-g的水平,并观察其近期疗效.结果:治疗后治疗组及对照组患者外周血中IL-2及TNF-α、IFN-g的水平分别为54.7±7.2,43.1±4.0,26.6±2.7ng/L和34.6±5.2,27.9±3.3,11.5±1.7ng/L,两组数据有显著性差异(P<0.01).治疗组的近期有效率为94.4%,0.5及1a生存率均为88.2%.对照组的近期有效率为62.5%,0.5及1a生存率为81.3%和43.8%,治疗组的近期有效率及1a生存率显著高于对照组(P<0.01).结论:经肝动脉EAP栓塞化疗方案联合放疗治疗胃癌肝转移患者,可显著提高患者的近期有效率及1a生存率,上调免疫调节因子IL-2及TNF-α、IFN-g的水平.     

肝癌的综合治疗及肿瘤抗血管联合疗法

肝癌死亡率极高,发病隐匿,且85%合并肝硬化不能耐受手术.肝动脉化疗栓塞术(trans-catheter arterial chemoembolization,TACE)是不能手术切除肝癌的首选治疗方法之一,但由于门静脉血供的残存,单纯TACE难以使肿瘤细胞完全坏死,术后癌细胞残留是复发的根源.放疗亦作为治疗肝癌的常规手段,但其促进肿瘤血管新生,因而促使大肝癌放疗抗拒和放疗后复发、转移.而抗血管治疗能够抑制肿瘤血管形成,降低血管通透性,提高内皮细胞对放射的敏感性及促进瘤细胞凋亡和减少肿瘤内的乏氧细胞数,从而抵抗放射抗拒.综上,与单一的手术、化疗、放疗及抗血管治疗相比,肝癌的综合治疗能提高患者生存率,改善预后.治疗策略的制定需要掌握不同肿瘤的生物学特性,权衡各种治疗方案的利弊.     

5-氟脲嘧啶腹腔化疗预防裸鼠人结肠癌细胞肝转移研究

目的探索预防大肠癌根治术后肝转移局部区域性辅助化疗新途径.方法利用裸鼠人结肠癌细胞肝转移模型观察术后早期大剂量大容积5-氟脲嘧啶腹腔化疗预防裸鼠经睥接种的人结肠癌细胞肝转移的疗效.结果术后早期5-氟脲嘧啶40 mg/生理盐水40 ml/kg,1次/d,连续2 d 的腹腔化疗可使裸鼠肝转移发生率降低40%,平均每只裸鼠肝转移瘤数目减少50.89%,平均每只裸鼠生存时间延长48.21%.结论腹腔化疗是一个预防大肠癌根治术后肝转移有效的辅助化疗新途径.     

p21基因转染人肝癌SMMC-7721 细胞对化疗药物敏感性的研究

目的探讨p21基因转染人肝癌SMMC-7721细胞（7721细胞）对化疗药物的敏感性，深入研究p21基因对肝癌细胞的治疗作用。方法应用磷酸钙介导p21基因转染人肝癌7721细胞；通过G418筛选稳定表达细胞株并扩大培养；采用MTT法检测肿瘤细胞增殖速度；流式细胞术检测细胞周期变化。结果经过3～4w G418筛选得到稳定表达的细胞株，p21基因稳定转染并联合化疗药物应用可明显抑制7721细胞的体外增殖，细胞周期明显改变，细胞从G1期到G2期发生阻滞。结论提示转染外源p21基因联合化疗药物可使7721细胞周期阻滞于G1期，并可抑制肿瘤细胞增殖。     

Egr-内皮抑素基因联合放射治疗裸鼠膀胱癌模型的实验研究

目的 探讨Egr-内皮抑素（Egr-Endostatin，Egr-Endo）基因联合放射治疗抑制裸鼠膀胱癌移植瘤生长的效应及作用机理。方法 裸鼠皮下注射入膀胱癌T24细胞建立移植瘤模型，特肿瘤长径生长到约4mm后随机分为4组（对照组、Egr-Endo组、5 Gy辐照组、Egr-Endo＋Gy辐照组），每组6只；，定期观察各组裸鼠移植瘤体积变化；瘤细胞移植45d后（辐照后15d）处死各组荷瘤鼠，测量瘤重、检测NK细胞毒活性和腹腔巨噬细跑TNF-α分泌活性，免疫组化法检测肿瘤组织微血管密度（MVD）。结果 瘤细胞移植45d后（辐照后15d）Egr-Endo＋5Gy辐照组移植瘤体积、重量明显低于对照组、Egr-Endo组、5Gy辐照组，差异有显著性；辐照后15d Egr-Endo＋5Gr辐照组NK细胞毒活性、腹腔巨噬细胞TNF-α分泌活性及肿瘸组织MVD均明显高于对照组，差异有显著性。结论Egr-Endo基因联合放射治疗对膀胱癌生长及血管生成有协同抑制作用。     

腺病毒介导生长抑素2型受体对裸鼠人胰腺癌移植瘤抑制作用的研究

目的构建表达生长抑素2型受体(SSTR2)的重组腺病毒,探讨其对裸鼠人胰腺癌移植瘤生长的影响及机制。方法2004-06-01—2005-12-10,在暨南大学生物工程学系采用位点特异性重组方法构建和包装携带人生长抑素2型受体和报告基因LacZ的重组腺病毒Ad-SSTR2和Ad-LacZ。建立裸鼠人胰腺癌移植瘤模型,分别于瘤内注射生理盐水(阴性对照组)、Ad-LacZ(报告基因对照组)和Ad-SSTR2(实验组),观察肿瘤大小和重量变化。通过逆转录-聚合酶链反应(RT-PCR)和免疫印迹试验(Western blot)鉴定SSTR2在裸鼠肿瘤组织中的表达,Western blot测定信号传导通路蛋白ERK2和ras的表达情况。结果获得滴度分别为6.0×1012pfu/L和6.5×1012pfu/L的重组腺病毒Ad-SSTR2和Ad-LacZ。Ad-SSTR2转染裸鼠胰腺癌移植瘤后SSTR2 mRNA和蛋白都得到有效表达,实验组对移植瘤生长具有明显的抑制作用,抑制率为48.2%。与阴性对照组和报告基因对照组相比,实验组ERK2和ras蛋白的表达量明显减少(P<0.01)。结论腺病毒介导的生长抑素2型受体对裸鼠人胰腺癌移植瘤的生长具有抑制作用,其作用机制可能与信号传导通路蛋白ERK2和ras的表达下调有关,提示其有可能成为胰腺癌基因治疗的一种有效方法。     

人胰岛素基因转染鼠成纤维细胞致糖尿病大鼠血糖下降效应的研究

目的研究人胰岛素基因表达质粒转染鼠成纤维细胞（Ｌｔｋ－）后对糖尿病大鼠血糖的影响。方法重组的人胰岛素基因表达质粒通过脂质体法转染鼠成纤维细胞（Ｌｔｋ－），经Ｇ４１８选择性筛选获得一株较高胰岛素表达水平的细胞株（Ｌｔｋ－／ＰＲＩ１２），大量扩增，直接注射至糖尿病大鼠腹腔内，并与转染空载体的对照Ｌｔｋ－细胞比较，观察在糖尿病大鼠体内的胰岛素表达及对血糖等变化的影响。结果空载体转染的对照细胞与未转染组相似，无胰岛素表达，植入糖尿病大鼠腹腔后，血糖仍持续稳定在较高水平，体重进行性下降；而接受胰岛素表达载体细胞的糖尿病大鼠，在观察的２１天内血Ｃ肽持续表达，血糖明显下降，体重逐渐恢复。结论人胰岛素基因能成功转染非胰岛β细胞并表达目的基因使血糖水平下降。本研究为进一步开展糖尿病基因治疗研究奠定了基础。     

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。     

Phase tissue intercellular adhesion molecule-1 expression in nude mice human liver cancer metastasis model

Ｐｈａｓｅｔｉｓｓｕｅｉｎｔｅｒｃｅｌｕｌａｒａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅ１ｅｘｐｒｅｓｓｉｏｎｉｎｎｕｄｅｍｉｃｅｈｕｍａｎｌｉｖｅｒｃａｎｃｅｒｍｅｔａｓｔａｓｉｓｍｏｄｅｌＳＵＮＪｉｎｇＪｉｎｇ,ＺＨＯＵＸｉｎＤａ,ＬＩＵＹｉｎＫ...     

Phase tissue intercellular adhesion molecule-1 expression in nude mice human liver cancer metastasis model

AIM To study the phase cancer tissue intercellular adhesion molecule-1 (ICAM-1) expression of human cancer metastasis model in nude mice, and to analyze the relationship between ICAM-1 expression and the metastasis and recurrence of hepatocellular cancinoma (HCC).METHODS HCC tissues from liver cancer metastasis model in nude mice (LCI-D20) was orthotopically implanted, and ICAM-1 expression in HCC tissues at different growing time were detected by immunodot blot. Tumor size, intrahepatic and extrahepatic metastasis foci were observed by naked eyes and under light microscope.RESULTS ICAM-1 was positively correlated to the tumor growing time (r=0.88, P＜0.01) and tumor size r=0.5, P＜0.05). It was higher in metastatic HCC than in nonmetastatic HCC (8.24±0.95 vs 3.03±0.51, P＜0.01). ICAM-1 content in cancer tissues increased suddenly after metastasis occurred and then maintained in a high level. ICAM-1 was also higher in multimetastasis group than in monometastasis group (10.05±1.17 vs 5.48±0.49, P＜0.05).CONCLUSION Tissue ICAM-1 could predict not only the metastasis of human liver cancer metastasis model in nude mice early and sensitively, but also the metastasis degree. So tissue ICAM-1 may be a potential index indicating the status of metastasis of HCC patients.