RNA干扰技术及其在肝癌治疗中的应用

RNA干扰(RNAi)是指生物体内由双链RNA(dsRNA)介导同源mRNA的特异性降解,从而导致基因沉默的现象.随着研究的不断深入,RNAi在肝癌治疗中的应用也取得了积极的进展,显示着巨大的潜力,本文就此予以综述.     

小干扰RNA在临床治疗中的应用

RNA沉默现象是真核细胞生物一种重要的基因调节机制。利用小干扰RNA影响基因表达的RNA干扰技术在基因功能研究及基因治疗中得到了很大发展。目前已有数种针对不同疾病的siRNA药物进入了临床试验阶段。本文重点介绍了针对呼吸道合胞病毒的小干扰RNA——ALN—RSV01的临床试验进展情况。将小干扰RNA作为分子药物治疗疾病具有广阔的发展空间。但在临床试验及应用中需要以生物医学中的无害、有利、尊重．公正原则进行合理的风险一收益评估．以期其能更好地发挥作用。     

小干扰RNA在肝病中的应用进展

近年来,RNA干扰(RNAi)技术已成为分子生物学研究领域的新热点,已广泛应用于许多研究领域,尤其是在肝病治疗方面,取得了一些突破性进展。它不仅能够发挥抗肝炎病毒作用,还能够调节细胞增殖、凋亡、抑制致病基因的表达、影响细胞的信号转导等方面的作用,可能成为肝病治疗有效的潜在手段。     

小干扰RNA技术与骨质疏松症治疗

以骨量减少、骨微结构破坏为主要特征的骨折疏松症及其所致骨折严重影响人均寿命及生活质量，目前的治疗方法因多种原因并不能很好地解决相关问题。RNA干扰是一种序列特异的转录后基因沉默技术，通过小干扰RNA技术可以特异性调控蛋白表达，达到靶向治疗目的。该文就小干扰RNA技术在骨质疏松症治疗方面的研究作一综述。     

化学修饰小干扰RNA研究进展

RNA干扰(RNA interference,RNAi)是指转录双链的小干扰RNA(small interfering RNA,siRNA)能够高效、特异地抑制同源基因的表达。近年来,RNAi以其快速、简便、特异、高效等优势被广泛应用于基因功能研究,肿瘤治疗和抗病毒感染等领域。而采用胆固醇、锁核酸、二硝基苯酚等化学基团对双链小干扰RNA(siRNA)的正义或反义链进行化学修饰,可增强siRNA的稳定性,提高其干扰活性,克服siRNA传统载体(病毒、质粒等)对机体潜在的风险性,避免脱靶、利于被细胞摄取等,在基础研究和临床治疗方面具有非常广阔的应用前景。     

小干扰RNA与口腔癌研究进展综述

口腔癌是口腔外科常见恶性肿瘤,近年来的研究显示其发生、发展、侵袭转移和预后与体内小干扰RNA表达水平密切相关。因此本文通过对小干扰RNA产生机制、与肿瘤的发生、与口腔癌前病变、与口腔癌及与口腔癌的治疗等方面综述如下。     

小干扰RNA抑制肝癌HepG2细胞VEGF表达的研究

目的 观察小干扰RNA(small interference RNA, siRNA)对肝癌HepG2细胞血管内皮生长因子(VEGF)表达的抑制作用.方法 将VEGF siRNA经LipofectamineTM 2000脂质体转染肝癌HepG2细胞后,实时荧光定量PCR和Western blot方法分别检测VEGF mRNA和蛋白的表达,ELISA检测细胞培养上清液中VEGF蛋白浓度. 结果细胞培养6 h后siRNA的转染效率为(90.4±2.9)%,转染后肝癌HepG2细胞中VEGF mRNA和蛋白的表达明显减少,细胞培养上清液中VEGF蛋白表达下降. 结论运用RNA干扰技术,可以有效地干扰肝癌HepG2细胞VEGF的表达并能降低VEGF的生成.     

RNA干扰技术与肝细胞癌基因治疗

肝细胞癌已被证实为一种基因病,相关基因的异常表达引起细胞生物学特性改变,导致细胞永生化和癌变,因此,深入探讨肝细胞癌发病的分子机制以寻求新的诊治方法是提高疗效的关键.RNA干扰作为一种高效特异性抑制基因表达的新技术,广泛应用于研究基因功能和基因治疗,本文就近年来RNA干扰技术应用于肝细胞癌基因治疗领域的研究进展予以综述...     

甲胎蛋白增强子调控的小干扰RNA质粒载体的构建

目的 构建AFP增强子调控的小干扰RNA质粒载体.方法 从HePG2细胞中提取AFP基因.采用定向克隆的方法,构建质粒pGenesil-AFP.pGenesil-AFP质粒DNA扩增、提取后转化到DH5感受态细胞.针对目的 基因Survivin的序列,构建pGenesil-AFP-shRNA.结果 pGen-esil-AFP-shRNA质粒经XbaI和Hind Ⅲ双酶切鉴定,获得一856 bp大小和4506 bp大小的片段,通过基因测序分析,证明质粒载体构建正确,用紫外分光光度计测得质粒的浓度为1.3 g/L,A值为1.94,说明制备的质粒纯度较高.结论 成功构建AFP增强子调控的Survivin shRNA融合质粒.     

小干扰RNA对小鼠淋巴细胞cbl-b基因表达的抑制作用

目的 设计合成及筛选自身免疫关键基因--cbl-b (Casitas B-cell lineage lymphoma-b,cbl-b)基因小分子干扰RNA(siRNA).方法 应用RNA设计软件,模拟cbl-b小鼠cbl-b mRNA二级结构,设计并合成针对cbl-b mRNA的4对21核苷酸(nt)siRNA,96孔板转染小鼠淋巴细胞,以空白及转染非特异siRNA(与cbl-b mRNA无同源性的21nt siRNA)作为对照,应用蛋白免疫印迹(Western blot)方法 检测小鼠淋巴细胞cbl-b蛋白表达.结果 cbl-b基因siRNA工作浓度为100nmol/L时转染小鼠淋巴细胞转染率最高,可达(87.48±1.94)%,平均荧光强度最强,可达33.09±1.77.与对照相比,转染cbl-b siRNA的小鼠淋巴细胞蛋白表达明显下调,以siRNA-4最显著,抑制率达85%,转染非特异性siRNA的小鼠淋巴细胞cbl-b蛋白表达水平无明显变化.结论 得到cbl-b siRNA转染小鼠淋巴细胞最佳转染条件,成功筛选能高效抑制cbl-b蛋白表达的siRNA-4,其有效抑制时间约为48h,有望通过沉默cbl-b基因直接活化淋巴细胞,增强机体主动免疫杀伤肿瘤细胞.

Abstract：

Objective To design, synthesize screen small interfering RNA (siRNA) targeting to Casitas B-cell lineage lymphoma-b (cbl-b).Methods Four pairs of 21 nucleotide siRNAs directed to Cbl-b mRNA were designed and synthesized by utilizing RNA design software to simulate secondary structure of cbl-b mRNA in mice. These siRNAs were respectively transfected into lymphocytes in 96 shadows mask by oligofectamine package, and untreated and unspecific siRNA-transfected lymphocytes served as controls. The expression of cbl-b protein was detected by Western blotting.Results When the work concentration of siRNA was 100 nmol/L, transfection efficiency of lymphocytes was highest, up to (87.48±1.94)% and the mean fluorescence intensity was strongest, up to 33.09±1.77. Compared with bland controls, the expression of cbl-b protein level was markedly down-regulated in siRNA-transfected lymphocytes. The inhibitory rate of the siRNA of the target-4 was highest, up to 85%. The expression of cbl-b protein in unspecific siRNA-transfected lymphocytes had no significant changes.Conclusion siRNA-4, which can highly effectively inhibit protein expression of cbl-b gene, was screened successfully, and its inhibition effect can maintain near 48 h. It is hopeful that the cbl-b siRNA will activate lymphocytes directly by cbl-b gene silencing, and kill tumor by activate immunization.     

Surgical therapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) accounts for most primary malignancies of the liver. The most important risk factor is liver cirrhosis. HCC can be traced by the tumor marker alpha-fetoprotein. Patients with a known liver cirrhosis should regularly be screened, including sonography and alpha-fetoprotein evaluation. Surgical therapy - either partial liver resection or liver transplantation - is the only treatment that can potentially achieve long-term survival. The presence of liver cirrhosis is likely to induce postoperative liver insufficiency and is associated with higher local tumor recurrence rate. Patients without liver cirrhosis or Child-Pugh A patients with tumors smaller than 5 cm may be considered as the ideal target group for resection. For more advanced stages of cirrhosis and tumors of less than 5 cm up to 7 cm in size, liver transplantation offers a better prognosis. Long waiting time for a suitable organ negatively influences liver transplantation outcome. Living donor liver transplantation is a novel therapeutic option that improves posttransplant survival and extends the indication for transplantation in hepatocellular carcinoma.     

Multimodal therapy concepts in hepatocellular carcinoma

HCC is the most severe complication of liver cirrhosis. For the majority of patients the chances of cure are still limited. The recent experience in liver transplantation for HCC in the authors unit and in other centers allows the definition of a subgroup of patients, with small tumors (up to 3.0 cm, or 5 cm if solitary), no more than 3 nodules, and the absence of portal vein tumor thrombus. In these patients, liver transplantation offers a disease-free survival that is better than after liver resection, and similar to the survival of liver transplantation for benign liver disease. Patients with contraindications to transplantation, patients in whom a long waiting-time before transplantation is anticipated, and patients in countries with limited access to transplantation can be treated with a palliative intent (because of de novo tumors) by liver resection. Depending on liver function and local expertise, percutaneous ethanol injection (PEI) can be equally effective for small HCC. Other forms of treatment currently being evaluated, such as radio-frequency, thermal destruction or cyro-therapy, offer advantages similar to PEI without the need for multiple sessions. Further progress will probably come from a wider use of screening to detect a larger proportion of treatable lesions, and from strategies to prevent carcinogenesis in the cirrhotic liver, and possibly from innovative treatments such as gene therapy to alter the tumor biology.     

Microwave coagulation therapy for hepatocellular carcinoma

The efficacy and safety of microwave coagulation therapy (MCT) in patients with hepatocellular carcinoma (HCC) and impaired hepatic reserve were studied. Preoperative background factors, postoperative results, and prognostic factors were compared in 51 patients who underwent hepatic resection (HR group) and 38 patients who underwent microwave coagulation therapy (MCT group). Before surgery, measures of hepatic function, including level of albumin (P = 0.0072), prothrombin time (P < 0.0001), hepaplastin test (P = 0.0088), and the radioactivity of technetium-99m galactosyl-human serum albumin 15 min in the liver after injection divided by that in both liver and heart (P < 0.0001) were significantly lower in the MCT group than in the HR group. The indocyanine green dye retention rate at 15 min was significantly greater (P < 0.0001) in the MCT group than in the HR group, and a significant difference was noted in Child-Pugh grade between the groups (P < 0.0001). Operative time (P = 0.0014) and blood loss during surgery (P = 0.0005) were significantly lower in the MCT group than in the HR group. In contrast, no significant differences were recognized between the groups in the changes in postoperative liver function, or in the rates of morbidity, mortality, local recurrence, and survival. Moreover, the type of treatment (HR or MCT) was not a prognostic factor. The results indicate that MCT can be used safely as an alternative to hepatic resection in patients with poor liver function without reducing the efficacy of local control. Received for publication on Jan. 17, 2000; accepted on April 1, 2000     

siRNA对肝癌细胞中特异性异常表达FGFR3基因的抑制效应研究

目的设计和筛选有效抑制纤维母细胞生长因子受体3基因（FGFR3）表达的siRNA序列,构建相应表达载体,研究其对肝癌细胞HepG2生物学特性的影响。方法根据FGFR3序列,设计3条siRNA序列及1条对照序列：以pRNAT-U 6．1／Neo为载体,并以载体中同时构建的绿色荧光蛋白（GFP）基因编码序列为转染效率内参照;采用脂质体法将pRNAT-U 6．1／Neo-siRNA转染肝癌细胞HepG2;以实时荧光定量PCR（Real time PCR）检测FGFR3的mRNA表达变化,Western印迹法检测FGFR3蛋白质表达变化;[^3H]胸腺嘧啶脱氧核苷掺入法（^3H-Tdr）和细胞克隆形成法分析DNA合成变化及细胞生长能力。结果所设计合成的3条siRNA序列中均能抑制FGFR3的表达,对mRNA表达抑制率约为74．04％～87．25％,在蛋白质水平表达抑制率为19．65％～90．40％。转染siRNA后,HepG2的细胞克隆形成能力和DNA合成能力明显受到抑制。结论siRNA能够有效抑制肝癌细胞FGFR3的表达,并继而抑制肝癌细胞的DNA合成和生长,能够为肝癌基因表达调控研究提供新的靶基因。     

肝癌的分子靶向治疗进展

随着对肝癌分子机制的深入研究,分子靶向治疗成为近年来肝癌治疗的研究热点,一些分子靶向治疗药物的疗效获得了认可.经查阅国内、外近年有关肝癌的分子靶向治疗的文献,本文就肝癌的分子靶向治疗进展进行综述.     

干扰素在肝细胞癌治疗中的应用及其作用机制的研究进展

干扰素（Interferons,IFNs）是一种具有抗病毒、抗细胞增殖和免疫调节作用的细胞因子,在临床上已广泛应用于抗病毒和抗肿瘤治疗。INFs已被认为是对慢性病毒性肝炎最有效的抗病毒药之一,近年来,大量研究显示IFNs可以预防慢性病毒性肝炎患者中肝细胞癌（hepatocellular carcinoma,HCC）的发生,以及具有预防HCC术后复发和抗HCC侵袭和转移的作用。     

肝细胞癌转化治疗的研究进展

随着治疗方式的多元化和多学科联合诊断与治疗的规范化,综合运用经肝动脉插管治疗、放射治疗、靶向治疗等方法使中晚期肝细胞癌实现降期,或通过门静脉栓塞、联合肝脏分隔和门静脉结扎使剩余肝脏快速增生,以及常规行抗病毒和保肝治疗,让不同比例的中晚期或剩余肝脏储备不足的肝细胞癌能够成功转化为可切除肝细胞癌。这将成为提高肝细胞癌诊断水平的重要措施和肝细胞癌临床研究的热点。因此,笔者对肝细胞癌转化治疗的内涵、相应策略和进展进行深入阐述。     

甲胎蛋白的生理功能及其在肝癌分子治疗中的应用前景

甲胎蛋白长时间以来一直作为肝细胞癌的重要分子标记用于临床诊断、治疗及预后判断。但多年以来,临床医师对甲胎蛋白生理功能的认识远不如对其临床意义的了解。本文概述了甲胎蛋白的分子结构、生物学特性及其生理功能,包括其在临床诊断方面的应用、运输功能、免疫抵制、增生调控和凋亡调控。并对其在肝癌分子治疗方面的应用前景研究作一综述和展...     

巨噬细胞移动抑制因子小干扰RNA对肝癌细胞表达血管内皮生长因子的抑制效应

目的 观察巨噬细胞移动抑制因子(MIF)小干扰RNA(siRNA)对肝癌细胞表达血管内皮生长因子(VEGF)的影响.方法 Western blot检测MIF和VEGF在肝癌和癌旁组织的表达情况;人工化学合成MIF siRNA和对照siRNA,将其转染PLC、HepG2肝癌细胞株,定量聚合酶链反应(PCR)和Western blot检测MIF和VEGF mRNA及其蛋白的表达.结果 MIF、VEGF mRNA和蛋白在肝癌组织中高表达.MIF siRNA(50、100 nmol/L)转染肝癌细胞株后,PLC细胞MIF和VEGFmRNA水平分别下调(78.8±7.2)%、(90.4±2.9)%和(60.6±2.6)%、(79.8±1.2)%;HepG2细胞MIF和VEGF mRNA水平分别下调(74.3±8.9)%、(88.4±4.6)%和(65.6±4.6)%、(80.7 ±2.2)%.MIF蛋白分别下调(57.3±3.4)%、(78.7±1.2)%和(54.8±6.8)%、(77.9±2.3)%,并呈剂量依赖关系;伴随MIF mRNA和蛋白的表达下调VEGF蛋白分别下调(52.6±12.9)%、(87.4±2.3)%和(52.4±11.1)%、(68.5±2.8)%,亦呈剂量依赖关系,与对照组比较差异有统计学意义(P<0.01),两干预组间的差异也有统计学意义(P<0.05).结论 MIF siRNA能特异性阻断PLC及HepG2细胞MIF mRNA和MIF蛋白的表达,并且同时有效下调VEGF mRNA及其蛋白的表达,MIF可能通过调控VEGF基因和蛋白的表达,参与肿瘤血管的生成和促进肿瘤细胞的转移.