经导管动脉注入脂质体介导的p53基因治疗肝癌的实验研究

目的 以兔VX2肝癌模型为对象,探讨经导管动脉注入脂质体介导的p53基因治疗肝癌的可行性及转染和表达情况.方法 将pCMV-myc-p53质粒、阳离子脂质体LipofectAMINE以及pCMV-myc-p53和LipofectAMINE的复合体分别注入兔VX2肝癌模型的肿瘤供血动脉,并提取肿瘤组织蛋白,采用蛋白印迹法及免疫组化检测基因转染及其表达.以不同量的pCMV-myc-p53与LipofectAMINE形成的复合体分别注入兔VX2肝癌模型的肿瘤供血动脉内,同法检测基因的转染及其表达.结果 脂质体介导的p53基因经动脉途径成功转染了兔VX2肝癌模型的肿瘤组织并进行表达,其转染效率明显高于单纯基因导入,基因的量与转染效率之间存在量效关系.结论 经动脉途径导入脂质体介导的p53基因治疗肝癌是可行、有效的,具有广阔的应用前景.     

p53基因与肝癌

p53肿瘤抑制基因突变与多数恶性肿瘤的发生发展有关,包括肝细胞癌(hepatocelluar carcinoma,HCC)在内的人类恶性肿瘤中至少有50%发生了p53基因改变。因此,以正常p53基因治疗肿瘤就成了研究热点,随着介入放射学(inter-ventional radiology)向纵深发展,经介入放射方法进行肝癌的基因治疗令人关注。本文介绍了p53基因的结构与功能,其与肝癌的病理联系以及在肝癌治疗中的应用。     

经导管动脉输送转铁蛋白增强p53基因转染效率的研究

目的 研究转铁蛋白(Tf)对p53基因转染效率的影响,并探讨介入技术与Tf联合应用对基因治疗肝癌的双重靶向作用.方法 应用pCMV-myc-p53质粒与阳离子脂质体LipofectAMINE复合物,转染3种肝癌细胞株LM6、Hep3B、YY以及正常肝细胞株L02,以不同浓度Tf(0、10、25、50及100 μg)介导转染,蛋白印迹法检测各细胞株中p53表达情况,并对Tf影响p53转染效率的关系进行分析.再建立兔VX2肝癌动物模型,经介入导管输注Tf-质粒-脂质体复合物,提取肿瘤组织蛋白,蛋白印迹法检测P53表达.结果 4种细胞应用p53-脂质体复合物以及不同浓度Tf转染后48 h的蛋白印迹法检测,发现Tf在10～100 μg可明显增强p53-脂质体的转染效率,且转染效率随Tf浓度增加而增强.动物模型组织提取蛋白检测结果,显示Tf明显增强P53蛋白表达.结论 Tf能增强脂质体-基因转染,介入技术与Tf联合双重靶向对肝癌的基因治疗具有相当的应用前景.     

p53基因治疗肿瘤的研究进展

ｐ5 3被称为“基因组卫士” ,在机体组织细胞的生长发育分化等过程中起重要作用。其主要生物学功能是维持细胞基因组的稳定 ,负调节细胞生长 ,诱导细胞凋亡。研究发现人类恶性肿瘤中至少有 5 0 %发生了ｐ5 3基因改变 ,因此以ｐ5 3基因治疗肿瘤的研究发展非常迅速。最近 ,有学者引入介入技术经肿瘤供血动脉直接灌注重组ｐ5 3,为ｐ5 3基因治疗肿瘤开辟了一条新途径。笔者就肿瘤中ｐ5 3基因突变情况以及ｐ5 3基因治疗的研究进展予以综述。一、ｐ5 3基因及其产物的结构和特性ｐ5 3是一种重要的抑癌基因 ,是目前抑癌基因研究中最令人瞩目者。人类…     

动脉灌注p53基因治疗晚期肝癌的初步临床应用

目的 初步观察动脉灌注p53基因治疗晚期肝癌的疗效.方法 晚期肝癌30例,治疗组14例,对照组16例.根据造影表现决定灌注药物的靶动脉,治疗组在靶动脉注入p53基因,每次用1012VP,加羟基喜树碱20 mg,每周1次,连用3周为1疗程,14例患者分别接受1～4疗程的治疗.对照组给予羟基喜树碱20 mg肝动脉灌注.结果 治疗组生存期最小14 d,最长405 d,平均238.1 d.对照组生存期最小18 d,最长167 d,平均80.7 d.两者相比较,P＜0.05,有显著差异.结论 p53基因治疗晚期肝癌有效.     

碘油联合双基因经肝动脉介入治疗肝癌的实验研究

目的 探讨血管抑素和内皮抑素基因联合超液态碘油对于Wistar大鼠肝癌的抑制效果.方法 将160只Wistar大鼠肝癌模型采用随机表法等分为对照组、碘油组、内皮抑素组、血管抑素组、内皮抑素+血管抑素组、内皮抑素+碘油组、血管抑素+碘油组、内皮抑素+血管抑素+碘油组8组,经相应的治疗后观测肿瘤体积增长率、肺内转移瘤数目、大鼠生存期、微血管密度(MVD)值、细胞凋亡指数(AJ)和血管内皮生长因子(VEGF)的变化,统计学分析采用方差分析和卡方检验.结果 在治疗后第6天,所有治疗组的肿瘤体积生长率均小于对照组,其中双基因与碘油联合应用的抑瘤效果最好,肿瘤的体积增长率最小.治疗后第18天,上述8组肺内转移瘤数目分别为:(37.2±7.2)、(26.2±5.0)、(25.5±4.7)、(26.2±3.9)、(14.9±2.6)、(19.1.4-2.8)、(20.2±2.7)和(6.1±1.2)个,生存时间分别为:(23.3±4.4)、(35.2 4-4.9)、(28.2±3.6)、(29.4±3.4)、(38.3±6.7)、(37.7±5.8)、(36.4±5.5)和(59.8±9.4)d,治疗组肺内转移瘤数目与对照组相比差异均有统计学意义(P值均<0.01);生存时间对照组除与单个基因组比较差异无统计学意义(P值均>0.05)外,与其他各组相比差异均有统计学意义(P值均<0.01).碘油组MVD[(63.4±8.7)条/高倍视野]高于其他治疗组,与对照组[(64.1±11.9)条/高倍视野]比较差异无统计学意义.所有治疗组的AJ均高于对照组[(4.2±1.6)%],其中以双基因联合碘油的治疗组最高[(19.6±2.4)%].VEGF在对照组表达率最高(100%),双基因+碘油组的表达率最低(12.5%).结论 经肝动脉给予双血管生成抑制基因联合碘油对肝肿瘤生长的抑制效果明显好于单纯基因和单纯碘油的作用.     

腺病毒介导人p53抑癌基因对肝癌细胞系HepG2生物学行为的影响

研究腺病毒介导入野生型Ｐ５３抑制肝癌细胞系ＨｅｐＧ２生物学行为的影响。构建含人野生型Ｐ５３抑癌基因的生组腺病组载体，导入肝癌细胞系ＨｅｐＧ２后，分别用光镜，电镜ＴＵＮＥＬ法检测细胞凋亡发生和在裸鼠体内的成瘤性改变。     

腺病毒介导的p53基因治疗肝癌的给药途径研究进展

目前腺病毒介导的p53基因（rAd／p53）治疗肝癌主要的给药方式有选择性肝动脉给药、经门静脉给药、瘤内注射以度静脉给药等。就应用rAd／p53的安全性度不同给药途径进行综述。     

腺病毒介导p53基因转染增加人胃癌细胞的放射敏感性

目的评价腺病毒介导p53基因(Adp53)转染对人胃癌细胞的凋亡效应和放射增敏作用。方法以重组腺病毒介导p53基因感染4种不同p53状况的人胃癌细胞，用免疫组织化学法和Western blot法检测P53蛋白在胃癌细胞中的表达；用细胞集落形成法检测细胞存活率；用TUNEL法检测细胞凋亡。胃癌细胞感染Adp53后照射4Gy，用流式细胞仪检测细胞周期分布和凋亡；胃癌细胞种植肿瘤内注射Adp53后照射6Gy，以肿瘤相对体积增长曲线观察肿瘤抑制情况。结果1：100效靶比(MOI)Adp53产生细胞高转染率，以及p53基因在4种胃癌细胞中均高表达，并产生G2/M期阻滞、凋亡增加和细胞增殖抑制。如果以凋亡评价放射效应，Adp53转染对4Gy照射4种细胞的凋亡率比值为：W细胞3．0，M细胞3．6，neo细胞2．2，823细胞2．5。体内实验结果显示，Adp53对w细胞肿瘤6Gy照射的抑瘤率比值为1．41，而对M细胞肿瘤为1．91。结论腺病毒介导p53基因转染产生细胞凋亡并提高人胃癌细胞的放射敏感性，这种作用不依赖于细胞内在的p53状况。     

p53基因治疗恶性肿瘤的现状与趋势

恶性肿瘤的治疗包括手术、放疗、化疗和生物治疗 ,基因治疗是生物治疗的主要内容 ,该领域的研究正方兴未艾 ,p53基因治疗恶性肿瘤越来越受到人们的重视。本文对p53基因的结构和功能以及该基因治疗恶性肿瘤的现状和趋势作一综述。1　p53基因治疗恶性肿瘤的理论依据　　p53基因是     

CT导引下125I粒子置入治疗兔VX2肝转移癌

目的 探讨CT导引下瘤体内125I粒子置入治疗兔VX2肝转移癌的疗效及其病理改变,为125I粒子临床应用中合理化治疗方案的取得提供理论依据.方法 新西兰兔32只制备成VX2肝转移癌模型,完全随机法分为4组,每组8只,依据术前治疗计划系统(TPS)设计在CT引导下置入不同活度粒子,分别为:37.0、25.9和14.8 m Bq,对照组置入粒子空壳.观察置入后1周、2周、1个月、2个月和3个月各组肿瘤的大小、活性、影像和病理组织形态学的变化.结果 治疗组自然生存期(88±12)d明显高于对照组(22±7)d.治疗3个月后,治疗组平均肿瘤体积分别为:37.0 mBq组(0.06±0.02)cm3、25.9 mBq组(0.21±0.05)cm3、14.8 mBq组(1.74±1.22)cm3,重复测量方差分析及LSD两两比较表明37.0 mBq与25.9 mBq组间差异无统计学意义(t=0.35,P＞0.05),但均低于14.8 mBq组(t值分别为2.59、2.24,P值均＜0.05).治疗后增强MR扫描显示37.0 mBq组强化消失5例,25.9 mBq组强化消失4例,而14.8 mBq组强化消失仅1例.病理检查37.0 mBq组及25.9 mBq组大部分瘤巢缩小至消失,疗效优于14.8 mBq组,但在放射粒子对正常肝组织影响方面37.0 mBq组较其他治疗组严重.结论 短期疗效证明,25.9 mBq的125I粒子可对高增殖活性的肝恶性肿瘤起到良好的控制作用,并且对正常肝组织的损伤程度较小.     

全肝MR灌注成像评价肝细胞癌经导管动脉化疗栓塞短期效果的价值

目的 观察肝细胞癌患者经导管动脉化疗栓塞(TAGE)治疗前后MR PWI表现和灌注值的改变.方法 回顾性分析经穿刺活检病理证实的肝细胞癌患者28例,在TACE术前和术后3～10 d分别进行MR PWI,得出术前及术后负增强积分(NEI)、病灶达峰值时间(TTP)、最大信号下降斜率(MSD)和信号增强比(SER),采用t检验比较TACE术前与术后上述各指标的差异.结果 肝细胞癌瘤区时间信号曲线(TIC)TACE术前呈快速下降,TACE术后趋向平缓;TTP及SER术前分别为(51.2±10.3)s和60.6±36.3,术后分别为(43.7±12.0)s和41.2±27.5,术后较术前降低;NEI值术后为149.6±80.1,术前为108.7±58.9,术后较术前升高,差异均有统计学意义(P＜0.05).MSD值术后较术前降低,但差异无统计学意义(P＞0.05).结论 MR PWI能够敏感地观察到TACE术前后的血流变化,用于评价TACE早期疗效.

Abstract：

Objective To investigate the value of MR perfusion imaging in early detection of findings following arterial chemoembolization of hepatocellular carcinoma Methods Twenty eight consecutive patients with pathologically-confirmed HCC were evaluated. All patients underwent MR perfusion imaging at pre-TACE and 3 to 10 days after TACE. The negative enhancement integral (NEI) ,the time to peak(TTP) ,the maximum slope of decrease (MSD) , the signal enhance ratio (SER) were acquired from MRI software FuncTool 2. 5.36a Version. Statistical analysis using SPSS 14, least significant difference test (t test) were utilized. Results The time intensive curve of tumor was observed to descend rapidly to reach the peak at pre-TACE studies, whereas it descended slowly to reach the peak on post TACE studies. The Value of TTP and SER prior to TACE were(51.2 ± 10. 3) s, 60. 6 ± 36. 3 respectively, and post TACE (43.7 ± 12. 0)s, 41.2 ±27. 5 respectively. The values of TTP and SER post TACE were lower than those prior to TACE (P ＜ 0. 05). The value of NEI prior to TACE was 108.7 ± 58.9, and after TACE 149. 6 ±80. 1 and there was statistically significant difference (P ＜0. 05). The Value of MSD post TACE were lower than those prior to TACE, but there was no statistical significance (P ＞ 0. 05). Conclusion PWI is a very sensitive imaging technique that can be used to monitor early dynamic changes of HCC following TACE.     

放射线联合p53基因及内皮抑素治疗小鼠前列腺癌皮下移植瘤

Objective To test the hypothesis that p53 gene therapy combined with endostatin can enhance tumor response to radiation therapy of RM-1 mouse xenograft prostate cancer and to investigate its mechanism. Methods A mouse prostate cancer model was established. Then mice with xenograft tumor were randomly divided into group A (control), B (radiation), C (radiation and rAdp53), D (radiation and rh-endostatin) and E (radiation and rAdp53 and rh-endostatin). On day 1, rAdp53 was injected intra-tumorously with 1 × 1010 vp per animal to group C and E. From day 1 to 14, rh-endostatin was given 15 mg/kg intraperitoneally daily to group D and E. On day 4 single fraction of 15 Gy was given to tumors in groups B, C, D and E. Normal saline was injected intra-tumorously or intraperitoneaUy accordingly as control. No treatment was done to group A. Tumor volume was measured daily. Samples were collected on Days 5, 10 and 15. Ki67, CD31, p53 and VEGF were detected by means of immunohistochemistry. Results (1) Radiation alone, radiation combined with intra-tumorous injection of Adp53 and/or intraperitoneal injection of rh-endostatin resulted in tumor growth arrest of RM-1 cells in vivo (P = 0.000). Radiation combined with both rAdp53 and rh-endostatin was the most effective treatment (P < 0.05). (2) All the four treatment groups had a decreased expression of mutant type P53 (P = 0.000). The expression of Ki67 in groups B and C were equal (P 0.05) and increasing (P = 0.000), respectively. Group D had a up-down-up curve (P < 0.05), but group E had a up-down one. On day 5 the expresion of VEGF in group E was the lowest (P < 0.05). An increased expression of MVD compared with the control was shown, and MVD in groups C, D and E were always higher than that in the control (P < 0.05). Conclusions The limitation of radiotherapy could be overcome by combination with beth p53 gene therapy and endostatin on the growth of mouse prostate cancer cell. Radiation, rAdp53 and endostatin have their own role but they can be interacted with each other.     

放射线联合p53基因及内皮抑素治疗小鼠前列腺癌皮下移植瘤

Objective To test the hypothesis that p53 gene therapy combined with endostatin can enhance tumor response to radiation therapy of RM-1 mouse xenograft prostate cancer and to investigate its mechanism. Methods A mouse prostate cancer model was established. Then mice with xenograft tumor were randomly divided into group A (control), B (radiation), C (radiation and rAdp53), D (radiation and rh-endostatin) and E (radiation and rAdp53 and rh-endostatin). On day 1, rAdp53 was injected intra-tumorously with 1 × 1010 vp per animal to group C and E. From day 1 to 14, rh-endostatin was given 15 mg/kg intraperitoneally daily to group D and E. On day 4 single fraction of 15 Gy was given to tumors in groups B, C, D and E. Normal saline was injected intra-tumorously or intraperitoneaUy accordingly as control. No treatment was done to group A. Tumor volume was measured daily. Samples were collected on Days 5, 10 and 15. Ki67, CD31, p53 and VEGF were detected by means of immunohistochemistry. Results (1) Radiation alone, radiation combined with intra-tumorous injection of Adp53 and/or intraperitoneal injection of rh-endostatin resulted in tumor growth arrest of RM-1 cells in vivo (P = 0.000). Radiation combined with both rAdp53 and rh-endostatin was the most effective treatment (P < 0.05). (2) All the four treatment groups had a decreased expression of mutant type P53 (P = 0.000). The expression of Ki67 in groups B and C were equal (P 0.05) and increasing (P = 0.000), respectively. Group D had a up-down-up curve (P < 0.05), but group E had a up-down one. On day 5 the expresion of VEGF in group E was the lowest (P < 0.05). An increased expression of MVD compared with the control was shown, and MVD in groups C, D and E were always higher than that in the control (P < 0.05). Conclusions The limitation of radiotherapy could be overcome by combination with beth p53 gene therapy and endostatin on the growth of mouse prostate cancer cell. Radiation, rAdp53 and endostatin have their own role but they can be interacted with each other.     

放射线联合p53基因及内皮抑素治疗小鼠前列腺癌皮下移植瘤

Objective To test the hypothesis that p53 gene therapy combined with endostatin can enhance tumor response to radiation therapy of RM-1 mouse xenograft prostate cancer and to investigate its mechanism. Methods A mouse prostate cancer model was established. Then mice with xenograft tumor were randomly divided into group A (control), B (radiation), C (radiation and rAdp53), D (radiation and rh-endostatin) and E (radiation and rAdp53 and rh-endostatin). On day 1, rAdp53 was injected intra-tumorously with 1 × 1010 vp per animal to group C and E. From day 1 to 14, rh-endostatin was given 15 mg/kg intraperitoneally daily to group D and E. On day 4 single fraction of 15 Gy was given to tumors in groups B, C, D and E. Normal saline was injected intra-tumorously or intraperitoneaUy accordingly as control. No treatment was done to group A. Tumor volume was measured daily. Samples were collected on Days 5, 10 and 15. Ki67, CD31, p53 and VEGF were detected by means of immunohistochemistry. Results (1) Radiation alone, radiation combined with intra-tumorous injection of Adp53 and/or intraperitoneal injection of rh-endostatin resulted in tumor growth arrest of RM-1 cells in vivo (P = 0.000). Radiation combined with both rAdp53 and rh-endostatin was the most effective treatment (P < 0.05). (2) All the four treatment groups had a decreased expression of mutant type P53 (P = 0.000). The expression of Ki67 in groups B and C were equal (P 0.05) and increasing (P = 0.000), respectively. Group D had a up-down-up curve (P < 0.05), but group E had a up-down one. On day 5 the expresion of VEGF in group E was the lowest (P < 0.05). An increased expression of MVD compared with the control was shown, and MVD in groups C, D and E were always higher than that in the control (P < 0.05). Conclusions The limitation of radiotherapy could be overcome by combination with beth p53 gene therapy and endostatin on the growth of mouse prostate cancer cell. Radiation, rAdp53 and endostatin have their own role but they can be interacted with each other.     

肝癌合并胆管癌栓经皮介入引流治疗的临床疗效观察

目的 探讨经皮介入治疗肝癌合并胆管癌栓致梗阻性黄疸的方法和临床价值.方法 回顾性分析16例经病理及影像检查证实为肝癌合并胆管癌栓造成梗阻性黄疸的患者资料,16例均行经皮穿刺造影后置管引流术,根据患者置管后的临床表现,分别采用永久性外引流、内引流定期调整引流管以及覆膜支架植入等治疗方法,术后检测血清总胆红素(TBIL)水平,并采用配对t检验与术前对比,根据TBIL下降情况及临床症状缓解情况来评价治疗的有效性,并在2年的随访期限内观察患者生存期.结果 16例穿刺引流均获得成功,其中永久性外引流2例,内引流并定期调管7例,留置覆膜支架7例.16例经皮介入治疗前总胆红素平均为(261.9±77.2)μmol/L,治疗后2周为(161.2±80.5)μmol/L,差异有统计学意义(t=7.366,P<0.01).16例生存时间为30～391 d,平均生存时间204 d,中位生存时间为200 d.穿刺引流的主要并发症为出血和感染,经止血及抗炎等常规治疗可有效控制.结论 对于肝癌合并胆管癌栓所致的梗阻性黄疸,经皮介入治疗技术成功率高,临床疗效较好.     

人肝细胞肝癌组织中p53蛋白和热休克蛋白70的表达

目的：进行抗人肝癌的肿瘤主动性免疫研究,初步探讨人肝细胞肝癌（HCC）中p53蛋白及热休克蛋白70（HSP70）的表达并分析其间的关系。方法：免疫组织化学Envision法。结果：p53蛋白及HSP70均定位于细胞核或／和细胞质中。HCC中p53蛋白及HSP70阳性率分别为56．60％及49．06％;p53阳性者中88．68％伴有HSP70阳性,而HSP70阳性者中96．23％同时有p53阳性。结论：HCC中p53蛋白及HSP70表达增高,两者表达间有相关性。