【作者】 曹辉；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 肿瘤学， 2011， 博士

【摘要】 原发性肝癌是恶性度高,预后差的恶性肿瘤,被称为“癌中之王”,随着美国[1]及我国[2]肝癌发病率的增加,肝癌已跃居世界癌症死因第四位[3]。全球范围内,原发性肝癌占所有癌的5.4%[4],每年约有610,000人死于肝癌[3]。由于其恶性度高、进展快、容易复发,将近80%的患者在诊断后一年内死亡[5]。肝细胞癌总体疗效不理想,部分原因是由于肿瘤细胞对放、化疗的抵抗[6]。肿瘤干细胞(cancer stem cells,CSCs)理论的提出为抗肿瘤治疗提供了新的思路,肿瘤细胞群体中绝大多数肿瘤细胞是已经分化成熟了、没有增殖能力的细胞,常规的放、化疗只是杀灭了这部分细胞,而具有增殖和成瘤能力的肿瘤干细胞存活了下来。这部分细胞则成为肿瘤复发、治疗抵抗的根源[10]。因此,探索肿瘤干细胞在肿瘤发生发展过程中的作用,开发靶向肿瘤干细胞的治疗手段,通过不同手段杀灭或者抑制肿瘤干细胞,有望成为肿瘤治疗中新的研究领域。有报道指出,肝癌细胞株中存在功能性的肝癌干细胞(liver cancer stem cells,LCSCs)。有效地清除肝癌干细胞对于获得理想的抗肝癌疗效是十分必要的。人类CD133基因在多种肿瘤干细胞中表达,是多种肿瘤干细胞(包括LCSCs)的新型标志物。分子靶向治疗可以有针对性地作用于有特异性分子标记的细胞群体。肿瘤干细胞作为肿瘤内部一个亚群细胞是否可选择分子靶向治疗作为一种针对性的治疗手段?已有一些研究针对肿瘤干细胞和非成瘤细胞群体基因表达差异性进行基因靶向治疗的探索。从基因水平调控细胞的基因表达过程理应成为治疗肿瘤的理想手段。腺病毒是目前在肿瘤基因治疗中应用相当广泛的载体之一,增强其对外源基因的转导和转录靶向性,可以大大提高治疗效果。利用肿瘤特异性启动子(Tumor specific promoter, TSP)在转录水平对外源基因的调控实现其在肿瘤细胞中的特异表达。因此,从某种程度上说,启动子的转录靶向性评价在肿瘤的基因治疗中不仅具有重要价值,而且也是决定其能否最终走向临床试验的必要研究。Survivin是一种细胞凋亡抑制因子,组织分布特征具有明显的细胞选择性,仅在胚胎细胞和多种肿瘤细胞中表达,而在成人正常组织中基本不表达。黄爱龙等[7]证实在肝癌的靶向性基因治疗中,Survivin启动子具有较强的转录活性。Jin XD等[8]研究证实HepG2肝癌细胞中,Survivin的表达在低剂量率照射的条件下随着剂量的增加而增高。Nandi S等[9]表明,在脑胶质瘤细胞U118MG中,2Gy剂量照射后survivin的mRNA水平比照射前上调了10倍。本研究探讨Survivin作为一种有效的放射诱导的肿瘤特异性启动子在肝癌干细胞中的转录活性。目的通过MACS分选技术分离肝癌干细胞,探讨其生物学特性。构建Survivin启动子调控的重组腺病毒载体,判定其在肝癌干细胞中的启动子活性,观察其是否具有辐射诱导性,确定其在肝癌干细胞中的靶向性,从而为肝癌干细胞的临床靶向治疗提供实验依据。方法1、以肝癌HepG2为研究对象,通过MACS分选技术分离纯化肝癌干细胞。并通过流式细胞术检测分选前后CD133的表达率变化。2、无血清培养技术富集肝癌干细胞,并通过血清诱导肝癌干细胞的分化。3、通过流式细胞术,分析低剂量辐射后肝癌细胞中肝癌干细胞百分比的变化。4、体外克隆形成实验、增殖实验和体内成瘤实验分别判定肿瘤干细胞的生物学特性。5、RT-PCR方法分别检测辐射对Survivin mRNA表达水平的影响。6、构建Survivin启动子介导的含萤光素酶报告基因的pGL3B载体,通过萤光素酶报告基因检测试剂盒,体外分析初步判定其在肝癌干细胞中的转录活性。7、低剂量辐射对Survivin启动子活性的影响。8、利用基因重组技术构建Survivin启动子介导的含萤光素酶报告基因的腺病毒载体,通过萤光素酶报告基因检测试剂盒,体外分析判定其在肝癌干细胞中的转录靶向性。9、观察低剂量辐射对重组腺病毒的诱导作用。结果1、分选前,流式细胞术测试CD133~+细胞的百分比为1.54±0.26%,分选后,CD133~+细胞的百分率为89.58±0.72%,统计学分析表明分选前后有显著差异(p<0.05)。免疫磁珠分选技术是纯化HepG2细胞系中的CD133~+细胞的理想方法之一,分选纯度高,且不影响细胞的活性。2、CD133~+肝癌细胞分选后,能在含有生长因子的无血清培养条件下逐渐形成细胞球,且随着时间的延长,细胞球日益增多且致密。用含血清的培养液诱导,24小时即贴壁分化。3、未照射组,CD133~+细胞的百分比为1.24±0.36%,低剂量(2Gy)照射肝癌HepG2细胞后,所得细胞中CD133~+细胞的百分比为3.91±0.21%,p<0.05,两者具有明显的统计学差异,提示低剂量(2Gy)照射可能使CD133~+细胞增多。4、与CD133-肿瘤细胞相比,CD133~+肿瘤细胞在体外有较强的克隆形成能力和增殖能力,在NOD/SCID小鼠体内中有较强的成瘤能力。5、辐射诱导HepG2细胞Survivin mRNA表达。6、成功构建了真核表达质粒pGL3B-SUR-PRO,质粒转染肝癌干细胞,通过萤光素酶活性分析,结果表明其在肝癌干细胞中具有转录活性。7、低剂量辐射(2Gy)处理后,增加了质粒在肝癌干细胞中的转录活性。8、成功构建了重组腺病毒pAd-Sur-luc,感染肝癌干细胞,通过萤光素酶活性分析,表明与肝癌细胞相比,重组腺病毒在肝癌干细胞中的转录活性更强。9、与未照射组相比,低剂量辐射具有诱导重组腺病毒在肝癌干细胞转录活性的作用。结论免疫磁珠分选技术是纯化HepG2细胞系中的CD133~+细胞的理想方法之一,分选纯度高,且不影响细胞的活性。分选的肝癌干细胞能在无血清培养基中成球生长,在有血清培养基中能贴壁分化。Survivin启动子介导的重组腺病毒在肝癌细胞及肝癌干细胞中均具有较强的转录活性,并且,低剂量辐射能够诱导重组腺病毒对肝癌干细胞的靶向杀伤作用。目的和意义:通过复习文献,对TACE联合HIFU与单纯TACE治疗中晚期肝癌的疗效进行循证医学研究,综合分析得到一个对疗效较为普遍性的结论,用于指导临床。材料和方法:检索国内外有关数据库,查找符合纳入标准的临床对照试验(截至到2010年6月)。采用Meta分析方法,对国内外公开发表的有关TACE联合HIFU与单纯TACE治疗中晚期肝癌疗效的临床随机对照试验观察随访研究文献进行综合分析,对纳入文献进行质量评价。根据获得的文献数据,分别对两种疗法引起的总有效率及临床受益率及患者的0.5, 1, 2, 3年生存情况进行分析。应用Stata10.0软件中统计分析相应的研究指标。当各项研究间存在异质性时,用固定效应模型表达,反之用随机效应模型表示。如试验结果存在异质性,进行敏感性分析及亚组分析。结果:9项独立的临床随机对照试验研究共计736例病例进入了本次meta分析,与单纯TACE组相比,TACE联合HIFU治疗组能明显提高总有效率CR+PR (OR=2.43; 95% CI 1.55–3.82; p=0.000)和临床受益率CR+PR+SD (OR=2.75; 95% CI 1.83–4.13; p=0.000),同时改善患者的0.5年生存率(OR=5.95; 95% CI 3.43-1033; P=0.000), 1年生存率(OR=3.25; 95% CI 2.31-4.59; P=0.000), 2年生存率(OR=3.34; 95% CI 1.93-5.76; P=0.000)和3年生存率(OR=2.97; 95% CI 1.77-4.98; P=0.000)。敏感性分析证实这些结果可信且不受发表偏倚的影响。结论:TACE联合HIFU治疗肝癌的近、远期疗效优于单纯TACE治疗。由于结果基本来自单个研究,纳入研究质量不高,因此使用这些结论必须谨慎,该结果不能作为推荐临床应用的证据。更多还原

【Abstract】 Primary liver cancer is a malignant tumor of high malignancy and poor prognosis, known as the "the King of cancer ". Hepatocellular carcinoma (HCC) is the fourth leading cause of death from cancer in the world, with an increasing incidence in the United States and the China. Globally, HCC accounts for approximately 5.4% of all cancers and causes 610,000 deaths per year. Most patients with HCCs are diagnosed either at an intermediate to advanced stage or at the point with poor liver function, and few meaningful therapeutic options are available. With a poor prognosis and limited systemic treatment options, approximately 80% of patients die within a year of diagnosis. Overall efficacy of HCC is not satisfactory, partly due to the tumor cells to radiotherapy and chemotherapy resistance.The theory of Cancer stem cells (CSCs) provides a new way for the anti-tumor therapy. The majority of tumor cell populations have differentiated and matured, which is no proliferation ability. Conventional radiotherapy and chemotherapy is to kill off this part of the cells, so CSCs of proliferation and tumor formation ability survived. This part of CSCs becomes the root of cancer recurrence and treatment resistance. Therefore, exploring the role of CSCs in the occurrence and development of tumor, developing targeting treatment of CSCs, and killing or inhibiting tumor stem cells through different means, are expected to become the new field of study. It is reported that the presence of functional liver cell lines of liver cancer stem cells (LCSCs). Effective way to remove stem cells is very necessary for liver cancer to get a good anti-liver cancer. Human CD133 gene,which is expressed in a variety of cancer stem cells, is a new marker of cancer stem cells (including LCSCs).Molecular targeted cancer therapy (MTCT) is the "personalized" or "individualized" approaches toward cancer which targets the particular molecular or genetic changes. CSCs represent a distinct subpopulation of cancer cells of integral importance. May molecular targeted therapy choose as a treatment for CSCs? Some research explored that the differences of gene expression in CSCs and non-tumor cell population. The regulation of gene expression should be an ideal means of treating cancer.Adenovirus, a wide range of carriers in cancer gene therapy, can enhance the foreign gene transduction and transcriptional targeting and greatly improve the therapeutic effect. However, to date, there have been two limitations to the clinical application of these agents, i.e. poor viral infectivity and tumor specificity. To bypass these problems, a new generation of vectors is being developed, which selectively target tumor cells and which allow for increased viral replication. This can be achieved by use of tumor-specific promoters (TSP). Therefore, to some extent, the evaluation of transcription activity of TSP in tumor is not only of great value in targeted gene therapy, but also is a significant therapeutic end point in human clinical trails.Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in transformed cell lines and in all the most common human cancers and undetectable in terminally differentiated adult tissues. kuang ea al. reveals that the survivin promoter possesses a high transcriptional activity in all three established HCC cell lines and may serve as a useful tool for transcriptional targeting gene therapy of HCCs.Jin XD et al. found that low-LET X-rays induced dose-dependent increases in survivin expression. Nandi S showed that the survivin gene was up-regulated to f10-fold in U118MG, indicating a strong radiation-inducible promoter. This study is to reveal the activity of the survivin gene promoter in liver cancer stem cells and evaluate the possible application of this promoter as a radioinducible promoter in tumor gene therapy.ObjectiveTo isolate and characterization of CD133 positive cells from of liver stem cells by MACS sorting and investigate its biological characteristics. To Construct Survivin promoter-mediated recombinant adenovirus vector, determine the promoter activity in liver stem cells, evaluate the adenovirus with respect to radioinducible properties, test the capacity of our novel adenovirus to effectively target CD133~+ liver cancer stem cells and provide experimental basis for targeted therapy.Methods1. Isolation and characterization of CD133~+ LCSCs from the HepG2 liver cancer by MACS sorting. The percentage of CD133~+ LCSCs was evalating by flow cytometry.2. To observe the growth of the isolated CD133~+ LCSCs in serum-free medium and in medium containing 10% FBS.3. To examine the impact of radiation on HepG2, we evaluated the percentage of CD133~+ LCSCs before and after radiation exposure by FACS.4. To determine whether CD133~+ cells are more tumorigenic than their CD133- counterparts in vitro and vivo, we compared their proliferative, clonal ability and tumorigenicity using proliferation, colony formation assay and tumor development experiments, respectively.5. To examine the specific expression of possible candidate for promoter-inducible elements, we evaluated the level of Survivin mRNA expression after different dose radiation exposure by RT-PCR.6. The genomic fragment encoding the Survivin promoter was isolated cloned into the KpnI/HindIII sites of the luciferase reporter plasmid pGL3-Basic. We examined the luciferase activity in the CD133~+ cells.7. We examined the promoter activity in the CD133~+ HepG2 cells in response to low-dose radiation. 8. Construction of Survivin promoter-mediated recombinant adenovirus vector with luciferase reporter pAd-sur-luc by recombinant DNA technology. We examined the luciferase activity in the CD133~+ cells to see whether our pAd-sur-luc could preferentially target CD133~+ cells.9. We examined the promoter activity in the CD133~+ cells in response to pAd-sur-luc in conjunction with radiation.Results1. The percentage of CD133~+ LCSCs was 1.54±0.26% and 89.58±0.72% before and after sorting, respectively(p < 0.05). MACS technology is an ideal method for purify and separate CD133~+ cells from the HepG2 cell line, with high purity and no influence of cell activity.2. The isolated CD133~+ LCSCs generate suspension spheres which have certain stem properties in serum-free medium, and increased as days passed by. But the differentiation were induced in medium containing 10% FBS in 24h. So CD133~+ cell subpopulation has stronger ability to differentiate in vitro.3. The percentage of CD133~+ LCSCs was 3.91±0.21% and 1.24±0.36% after 2Gy XRT or not, respectively(p<0.05), suggesting that radiation induce the increase of CD133~+ cells.4. CD133~+ cells isolated from the HepG2 cell line possess higher proliferative and clonogenic potential in vitro and higher tumorigenicity ability in vivo than CD133- cells. 5. The mRNA levels of Survivin were up-regulated on exposure to radiation, especially in 2Gy radiation, indicating that survivin promoter is a strong low-dose radiation-inducible promoter.6. The recombinant pGL3B-SUR-PRO was successfully generated and sequenced. The constructed vector was transfected into CD133~+ HepG2 cells and the result showed that pGL3B-SUR-PRO has activity in CD133~+ HepG2 cells.7. pGL3B-SUR-PRO showed an increase activity in response to low-dose radiation.8. The recombinant pAd-Sur-luc was successfully constructed with improved two-step homologous recombination protocol, which drives the expression of a reporter luciferase gene and sequenced. The constructed vector was transfected into CD133~+ HepG2 cells and the result showed that the CD133~+ enriched cells are targeted by pAd-Sur-luc in vitro.9. Low-dose radiation enhances survivin-mediated virotherapy against liver cancer stem cells.ConclusionsMACS technology is an ideal method for purify and separate CD133~+ cells from the HepG2 cell line, with high purity and has no influence of cell activity. CD133~+ cell subpopulation has stronger ability to differentiate, higher proliferative and clonogenic potential in vitro and higher tumorigenicity ability in vivo. Low-dose radiation enhances survivin-mediated virotherapy against liver cancer stem cells. Objective: To evaluate the therapeutic effects of Transcatheter arterial chemoembolization (TACE) plus High Intensity Focused Ultrasound (HIFU) for unresectable hepatocellular carcinoma (UHCC).Methods: We performed a systematic review and meta-analysis of Chinese literature by searching the Chinese Biomedical Database, Wei-Pu database and Wan-Fang database and Chinese scientific Journals database (up to June 2010). Randomized controlled trials (RCTs) and non-randomized studies comparing TACE plus HIFU with TACE alone in treating UHCC were identified using a pre-defined search strategy. Outcomes such as tumor response and survival between the two operations were compared by using the methods provided by the Cochrane Handbook for Systematic Reviews of Interventions. According to the test of heterogeneity, a fixed-effect model or a random effect model was used and the odds ratio (OR) was the principal measure of effects.Results: Nine controlled trials with a total of 736 patients compared the effect of TACE plus HIFU with TACE alone in treating UHCC. TACE plus HIFU Significantly improved the 0.5 year survival (OR=5.95; 95% CI 3.43-1033; P=0.000), 1 year survival (OR=3.25; 95% CI 2.31-4.59; P=0.000), 2 year survival (OR=3.34; 95% CI 1.93-5.76; P=0.000), 3 year survival (OR=2.97; 95% CI 1.77-4.98; P=0.000) and tumor response [CR+PR (OR=2.43; 95% CI 1.55–3.82; p=0.000); CR+PR+SD (OR=2.75; 95% CI 1.83–4.13; p=0.000)] of patients. The findings were stable in sensitivity analyses and were not affected by publication bias.Conclusions: Transcatheter arterial chemoembolization (TACE) plus High Intensity Focused Ultrasound (HIFU) was more therapeutically beneficial. However, considering the strength of the evidence, additional randomized controlled trials are needed before combined therapy can be recommended routinely.更多还原