磁性siRNA-CCMN沉默Survivin基因对人膀胱癌细胞增殖和凋亡作用的实验研究
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摘要
背景与目的：在我国,膀胱癌是泌尿系统最常见的恶性肿瘤,它直接威胁患者的生命。目前,膀胱癌的治疗仍以手术为主,辅以化疗、放疗、免疫治疗等多种方法联合治疗模式,但是,对于浸润性和转移性膀胱癌,以上治疗方式的远期疗效不尽人意,大多数患者最终死于肿瘤的浸润和转移。所以,探索膀胱癌诊断、治疗的新措施、新途径,提高治疗膀胱癌患者的总体生存率,是泌尿外科学者一直关注、研究的主要焦点。在当代,分子生物学得到日新月异的迅速发展,研究人员不断相继发现、克隆了一些与膀胱癌相关的原癌基因、抑癌基因和抗凋亡基因等,这就为探讨膀胱癌的发病机理、研究膀胱癌新的诊断和治疗方法提供了新的靶点,使基因治疗成为治愈膀胱癌的新希望。
Survivin基因属于凋亡抑制蛋白(IAPs)家族新成员,其分子量最小,结构相对简单、独特,与其他IAPs家族成员存在结构上的差别。已有研究表明：Survivin基因具有抑制细胞凋亡,调节细胞周期的作用,还有参与新生血管形成等作用。Survivin基因被认为是迄今为止所发现的所有凋亡抑制因子中抗凋亡作用最强的,它抑制细胞凋亡的作用远远强大于IAPs家族中的其他因子。据文献报道,Survivin基因对膀胱肿瘤细胞的发生发展起促进作用,并且可能与肿瘤的转移和预后相关。由于Survivin基因在大多数恶性肿瘤组织中选择性显著表达,具有强大的抑制肿瘤细胞凋亡的作用,从理论上说,抑制Survivin基因的表达,足以诱导肿瘤细胞明显的自发性凋亡,这是其他凋亡抑制基因所不具备的特点。
RNA干扰(RNAi)技术是最近十年来发展最快的特异性基因阻断技术,它是将具有特定分子结构特点的、长度约19~25nt的双链小RNA (dsRNA),也叫做小干扰RNA (siRNA),通过一定的方式导入细胞内,然后siRNA特异性的降解与其序列具有同源性的宿主细胞的mRNA,因为降解发生在基因的转录后阶段,因此也叫转录后水平的目的基因沉默(PTGS)。因为RNA干扰技术可使靶基因稳定沉默,而不影响其他正常基因的表达,并且具有特异性、高效性、简易性的特点,因此,RNA干扰技术在用于恶性肿瘤的基因治疗方面前景广阔。
恶性肿瘤的基因靶向治疗可以包含两大方面：首先是靶向目的基因的选择；二是基因靶向治疗方法和策略的选择。利用与Survivin基因的mRNA序列互补的特异性siRNA作用于有凋亡抵抗的肿瘤细胞,阻断Survivin基因的表达,为调控肿瘤细胞凋亡提供了新的治疗手段。目前,研究表明,靶向Survivin基因的siRNA干扰可以明显抑制肿瘤细胞的生长和增殖,诱导其凋亡。文献报道,靶向Survivin基因的siRNA质粒能够明显诱导肿瘤细胞凋亡,抑制肿瘤细胞的增殖。但是,目前缺乏能够驱动目的基因高效稳定表达、安全无害、靶向性高、简便的新型非病毒型基因导入载体系统。
随着现代纳米技术和医用高分子材料的发展,以磁性纳米粒子作为基因载体的研究引起广泛的关注。磁导向靶向治疗是一种最新的抗肿瘤基因靶向治疗方法,它是通过具有良好生物相容性和低毒副作用特性的磁性纳米粒子载体携带目的基因治疗分子,在一定强度的外加磁场的作用下,到达病灶靶部位而肿瘤细胞被消灭。羧甲基壳聚糖磁性纳米颗粒(CCMN)是国内、外目前医学领域纳米材料研究领域的最新成果,CCMN在外加磁场的作用下,可以引导所负载的物质在体内做定向移动、定位浓聚,特异选择性定位,发挥主动的靶向作用；因此协同CCMN磁性纳米颗粒的磁导向作用,可完成对靶器官、靶细胞甚至到细胞内靶结构的选择性导入。目前,国内外尚未见羧甲基壳聚糖磁性纳米颗粒(CCMN)系统协同外磁场用于肿瘤基因靶向治疗的报道,鉴于目前肿瘤基因靶向治疗中存在的诸多问题和提高靶向治疗水平的迫切需要,开展此项研究具有广阔的前景和很大的临床应用价值。
本研究采用特异性高的靶向survivin基因的siRNA重组质粒(siRNA-survivin)作为负载基因治疗分子,以磁性CCMN粒子作为基因载体,并在外磁场的协同作用下转染siRNA-survivin入人膀胱癌BIU-87细胞,观察siRNA对Survivin基因的沉默作用,以及Survivin基因沉默对膀胱癌细胞增殖和凋亡的影响,探讨磁性siRNA-CCMN纳米粒子在膀胱癌基因靶向治疗中的作用以及CCMN作为新型非病毒型基因导入载体系统的可能性。
材料与方法：
1.主要材料
1.1人膀胱癌细胞株BIU-87细胞,为贴壁细胞。
1.2主要仪器设备：超净工作台,台式低温高速离心机,恒温C02培养箱,超低温冰箱,可调式脉冲电磁场发生器频率50Hz：H-600型透射电镜,Zeta粒度分析仪,振动样品磁信号处理仪,Spectra-30型原子吸收光谱仪,ELX-800酶标仪,PCR仪,GS-800凝胶成像分析仪,DU-800紫外分光光度计。
1.3主要试剂：RPMI-1640培养干粉,OPTI-MEM无血清培养基,羧甲基壳聚糖,聚丙烯酰胺葡聚糖凝胶,多聚赖氨酸(PLL),四甲基偶氮唑盐(MTT),去内毒素质粒提取试剂盒E.I.N.A.TM, RT-PCR试剂盒(TaKaRa RNA PCR kit(AMV)Ver3.0),总RNA提取试剂盒,Trizol试剂盒,DNA原位末端标记(TUNEL)检测试剂盒,Western Blotting和ECL发光试剂盒。
2.方法
2.1Survivin-siRNA干扰序列设计、合成
根据Reynolds制定的siRNA设计原则,确定目标序列：ACGAGCCAGACTTGGCCCAGT.使用美国Invitrogen公司提供的在线si RNA设计软件,根据选定的干扰目标序列设计出相应的shRNA DNA表达模板。经BLAST同源性分析后,进行shRNA的DNA模板合成及其与质粒载体(pSilencer1.0-U6)的连接、鉴定,并转化到大肠杆菌DH5a菌株,命名为pSilencer1.0-siRNA-survivin。
2.2重组质粒的扩增、提取
将已转化入重组质粒的DH5a菌株,接种于含0.05g/L卡那霉素的LB固体培养板上,置于37℃,5%CO2恒温培养箱中培养过夜。挑取培养板上直径lmm左右的3个单菌落,溶解于5m1含0.05g/L卡那霉素的LB液体培养基中,将液体培养基在摇床上以>225rpm的速度振荡,置于37℃,5%CO2恒温培养箱中培养过夜。次日可得到已活化扩增的菌液,用中量去内毒素质粒提取试剂盒,按操作说明提取质粒。提取的质粒用DU-800紫外分光光度计检测质粒DNA浓度和纯度。
2.3CCMN粒子的制备：采用化学共沉淀法制备CCMN粒子,将1.5g的羧甲基壳聚糖、0.234g的FeCl3·6H20、0.086g的FeCl2·4H2O-起溶于3ml水中,一边用磁力搅拌一边滴加一定量的氨水,并将温度迅速升到60℃,反应30min,然后调节pH值到中性,再将聚集物和大粒子低速离心15min去除后,再使用Sephacryl S-300HR凝胶柱分离并收集凝胶过柱首峰,随后进行透析充分,冷冻真空干燥即得CCMN。
2.4siRNA-CCMN粒子的构建及理化性质检测：在pH=7时,将PLL、CCMN与pSilencer1.0-siRNA-survivin三者在无血清的RPMI-1640培养液中按1：1：2的质量比混匀振荡,4℃下放置1h,即可形成siRNA-CCMN,分别用H-600型透射电镜、粒度分析仪、振动样品磁信号处理仪和Spectra-30型原子吸收光谱仪分别检测其直径、有效粒径、分散度、比饱和磁化强度、矫顽力及铁离子含量等。
2.5BIU-87细胞培养及转染：将细胞按每孔1×105个的数量,加入含有10%小牛血清的RPMI-1640培养液的6孔板内培养。待细胞生长处于对数期时,用无血清培养液洗涤2次,每孔内滴加含2μg siRNA-survivin的siRNA-CCMN粒子。转染6h后加入含10%胎牛血清的RPMI-1640培养液中继续培养48h后进行后续操作,同时用荧光显微镜检测细胞转染效率。本实验设立正常对照组,无磁场作用的siRNA-CCMN组和有磁场作用的siRNA-CCMN组(siRNA-CCMN+磁场)(以下分组和处理均同)。磁场作用组：脉冲电磁场强度为10mT,按常规方法进行干预。转染48～72h后收集细胞样品进行后续操作。
2.6MTT法检测对BIU-87细胞增殖的抑制作用
上述3组均设置5个复孔,并设空白对照组。将单细胞悬液接种于96孔培养板上,分别于转染后24、48、72h,采用MTT法对BIU-87细胞的增殖抑制率进行检测。取处于对数生长期的BIU-87细胞,用不含血清和抗生素的RPMI-1640培养液制成单细胞悬液,调整浓度至1×104个/孔,将ELX-800酶标仪调于490nm波长处,测定各孔的吸光度值(A),然后计算各组细胞增殖的抑制率(IR)：IR=(1-A实验组/A空白对照组)×100%。
2.7半定量RT-PCR法检测Survivin基因的mRNA表达水平
转染后48h,常规消化、收集处于对数生长期生长良好的各组培养细胞5×104个,按Trizol试剂说明书操作提取总RNA。再用DU-800紫外分光光度计测定总RNA的浓度和纯度。随后按照RT-PCR反应试剂盒的说明书进行逆转录(RT)反应合成cDNA,然后进行PCR反应。PCR反应结束后,对反应产物于4℃进行琼脂糖凝胶电泳检测。标本用GS-800型凝胶成像分析仪进行扫描,再用Doiphin1D软件进行半定量分析。用Survivin产物的吸光度值(A)与内参照β-actin产物的吸光度值(A)的比值表示Survivin基因mRNA的表达强度。Survivin基因的mRNA表达抑制率=(1—实验组表达强度-空白对照组表达强度)×100%。
2.8TUNEL法检测BIU-87细胞凋亡
转染后48h,分别消化、收集各组细胞1×106个,制备细胞爬片,用PBS洗涤3次。然后参照TUNEL试剂盒说明书进行操作,结果判断：凋亡细胞的细胞核在显微镜下观察,呈棕褐色颗粒状着色。在显微镜下随机选择5个视野(×400),每个视野计数100个细胞,并计数凋亡细胞占细胞总数的百分比,即为凋亡指数(AI)。
2.9Western-Blotting法检测Survivin基因的蛋白表达强度
转染后72h,分别消化收集各组细胞约2×106个于1.5ml离心管中,加入冰上预冷1×PBS1m1,洗涤2次,离心lmin。弃去上清,加入5倍体积的细胞蛋白裂解液,振荡混匀,冰浴30min。14000rpm,4℃离心15min。吸取上清至一预冷的EP管中,加入2×SDS上样缓冲液按1：4混匀,100℃加热5min,冷却,高速离心lmin。将变性后的总蛋白100μg经10%SDS-PAGE凝胶电泳。再进行电转膜,将蛋白转移到硝酸纤维素膜上。将硝酸纤维素膜置于含有封闭缓冲液的平皿中2h。一抗用TBST1:400(0.2μg/μl)稀释,装入密封袋中,将硝酸纤维素膜放入其中,4℃平缓摇动过夜。再将二抗用TBST1:1000稀释,装入密封袋中。取适当量ECL试剂盒中的A液、B液等体积混合lmin,硝酸纤维素膜蛋白面朝下与此混合液充分接触5min。将硝酸纤维素膜沥干,放在包有保鲜膜的薄板上,用GS-800凝胶成像分析仪将膜照相,保存图像,Quantity One软件分析条带像素值,蛋白相对表达量=待测蛋白的光密度值/内参的光密度值。
2.10统计学处理
本研究实验数据采用SPSS10.0软件进行统计学处理。各组相关数据均以x±s来表示,采用单因素方差分析和X2检验进行组间数据分析比较,p<0.05为差异有统计学意义。
结果：
1.重组质粒siRNA-DNA的浓度和纯度
将扩增、提取得到的质粒siRNA-DNA通过紫外分光光度计测定其浓度是1.208±0.0045μ g/μl,纯度是1.890±0.034。所提取的重组质粒siRNA-DNA的OD260/OD290比值在1.8～1.9之间,表明重组质粒DNA较纯,另外重组质粒siRNA-DNA的含量在0.8～1.3μg/μl之间,达到细胞转染实验的要求。
2. siRNA-CCMN粒子的理化性质：在电镜下观察,siRNA-CCMN为圆形,其直径为10nm-12nm,分散度为0.34,有效粒径为94.0nm,比饱和磁化强度为0.18emu/g、剩磁、矫顽力分别为0.11emu/g、2.42,铁离子含量为340μmol/。
3. siRNA-CCMN转染效率
siRNA-CCMN转染BIU-87细胞4～6h后,在荧光显微镜下,可见较多细胞带有绿色荧光,siRNA-CCMN+磁场组转染效率为65%左右。
4. siRNA-CCMN对BIU-87细胞增殖的抑制作用
MTT法检测各组BIU-87细胞转染后24h、48h、72h的增殖抑制率(%)分别是：正常对照组：1.23%,1.66%,1.61%；siRNA-CCMN组：20.54%,27.66%,24.65%;siRNA-CCMN+磁场组：33.62%,44.98%,36.57%。
MTT法检测的结果表明,siRNA-CCMN转染BIU-87细胞后,siRNA-CCMN+磁场组的BIU-87细胞增殖水平均显著低于siRNA-CCMN组和正常对照组(P<0.05)。siRNA-CCMN组和正常对照组之间的BIU-87细胞增殖水平也存在显著性差异(P<0.05)。
5. siRNA-CCMN对BIU-87细胞Survivin基因mRNA表达水平的影响
5.1总RNA提取结果
通过核酸蛋白检测仪所检测的各组细胞提取的总RNA的浓度(μg/μl)及纯度(OD260/OD280)分别是：正常对照组：0.599,1.912,siRNA-CCMN组：0.663,1.902,siRNA-CCMN+磁场组：0.625,1.895。各组的OD260/OD280的比值在1.8～2之间,表明所提取的总RNA纯度较高。
5.2细胞瞬时转染48h后Survivin基因mRNA表达水平
(1)各组细胞瞬时转染48h后,用半定量RT-PCR检测细胞Survivin mRNA表达情况的凝胶电泳图,未见其他非特异性条带的出现。第1、2、3泳道分别是siRNA-CCMN组,正常对照组,siRNA-CCMN+磁场组的Survivin及其对应的β-actin扩增条带。各组在311bp区域内的内参照β-actin扩增条带的亮度相等,说明表达水平无明显差别：而在587bp处的Survivin扩增条带,siRNA-CCMN+磁场组条带亮度明显弱于其他两组,说明表达水平有明显差别。
(2)各组细胞瞬时转染48h后,用半定量TR-PCR检测细胞Survivin基因mRNA表达的相对水平结果：正常对照组：0.773,siRNA-CCMN组：0.545,抑制率是32.62%,siRNA-CCMN+磁场组：0.379,抑制率是50.97%。结果表明,siRNA-CCMN+磁场组BIU-87细胞的Survivin mRNA表达水平分别与正常对照组、siRNA-CCMN组比较,均明显下调,差异有统计学意义(P<0.05),其抑制率达50.97%。而siRNA-CCMN组和正常对照组BIU-87细胞的Survivin mRNA表达水平之间也有显著性差异(P<0.05),其抑制率达32.62%。
6. siRNA-CCMN对BIU-87细胞凋亡率的影响
各组BIU-87细胞的凋亡指数(AI)分别是：正常对照组：2.84,siRNA-CCMN组：13.46,siRNA-CCMN+磁场组：28.40。结果表明,siRNA-CCMN+磁场组BIU-87细胞的凋亡率明显高于正常对照组和siRNA-CCMN组,差异有统计学意义(P<0.05); siRNA-CCMN组也显著高于正常对照组(P<0.05)。
7. siRNA-CCMN对BIU-87细胞Survivin蛋白表达的影响
7.1Western Blotting法检测各组细胞Survivin蛋白的条带图,如图所不,siRNA-CCMN+磁场组条带灰度明显变浅,弱于正常对照组和siRNA-CCMN组,提示Survivin的蛋白表达被抑制。
7.2Western Blotting法检测各组细胞Survivin蛋白的表达强度,结果分别是：正常对照组：0.874, siRNA-CCMN组：0.475,siRNA-CCMN+磁场组：0.253。结果表明,siRNA-CCMN+磁场组BIU-87细胞的Survivin蛋白的表达强度明显低于正常对照组和siRNA-CCMN组,差异有统计学意义(P<0.05)。siRNA-CCMN组也显著低于正常对照组(P<0.05)。
结论：
1.本研究采用靶向survivin基因的特异性siRNA重组质粒pSilencer1.0-siRNA-survivin作为负载基因治疗分子,以CCMN粒子作为基因转染载体,在外磁场的协同作用下转染siRNA-survivin入人膀胱癌BIU-87细胞,观察siRNA-CCMN对Survivin基因的沉默作用,以及Survivin基因沉默对膀胱癌细胞增殖和凋亡的影响,探讨磁性siRNA-CCMN纳米粒子在膀胱癌基因靶向治疗中的作用以及CCMN作为新型非病毒型基因导入载体的可能性。
2.本研究用化学共沉淀法成功制备了羧甲基壳聚糖氧化铁磁性纳米颗粒(CCMN),化学共沉淀法制备简单,反应条件容易控制,制备出来的CCMN具有良好的生物相容性和生物可溶解性、特殊的纳米效应、超顺磁性、无毒无副作用,在溶液中稳定性好,具有较好的具有较好的单分散性。化学修饰后的羧甲基壳聚糖磁性纳米颗粒(CCMN)上的高分子羧甲基壳聚糖上的活性基团可通过经典的氧化还原反应与pSilencer1.0-siRNA-survivin稳定结合形成siRNA-CCMN, siRNA-CCMN在细胞摄粒作用下可以转染入细胞内,而且效率较高,外磁场可以显著加强转染效率。与现有的载体相比,羧甲基壳聚糖磁性纳米颗粒具有安全、特异,可行性好,这为其作为新型基因和药物纳米载体的研究提供了实验依据,为肿瘤的磁导向靶向治疗奠定实验基础。
3.本实验结果显示,靶向Survivin的siRNA-CCMN成功转染了人膀胱癌细胞株BIU-87细胞后,能够显著下调BIU-87细胞中Survivin基因mRNA转录和蛋白表达水平,明显诱导BIU-87细胞的凋亡,抑制细胞的增殖,而且在恒定外磁场协同作用下,siRNA-CCMN的作用显著加强。说明siRNA-CCMN可以特异、高效地转染膀胱癌BIU-87细胞,并且能有效的发挥对Survivin基因的RNA干扰作用,为膀胱癌的磁导向基因靶向治疗提供了一种可能的新途径与实验依据。
Background&objective:Bladder cancer is a common urologic cancer, which directly threaten to the life of patients. At present, the main treatment of bladder cancer is still surgery, and the auxiliary methods are chemotherapy、actinotherapy、 immunotherapy and a variety of methods with combination treatment mode. However, to invasive and metastatic bladder cancer, the prostecdtive efficacy of treatment methods above is less than satisfactory and most patients eventually die of infiltrating and metastasis of the tumor.
Survivin gene belongs to the new member of IAPs family, whose molecular weight is the smallest and structure is relatively simple and unique. It is very different from another members in IAPs family in the structure. Literature reports, Survivin has positive function to the occurance and development of cell carcinoma of bladder and is possibly related to metastasis and prognosis of the tumor. Survivin can be an important parameter to filtration、early diagnosis and prognostic ability of bladder cancer. Because Survivin in most cancer tissues with survivin gene has significant express of selectivity, strong function of inhibitor of apoptosis and inhibitor to survivin expression enough to induce the spontaneous apoptosis of cancer cell. This is the characteristic that another apoptosis inhibiting genes not have.
RNAi technique is an agene disruption technique that develops rapidly in recent years, which thus leads to gene silencing of level of purpose (PTGS) through making the dsRNA with the regular structure characteristic and having a length of19-25nt, which is siRNA, import into host cells and on the level of specificity degradation to the mRNA of host cells that have homology with it. The technique of RNAi can make target genes stable and silent, not impact the expression of normal genes, and have the characteristics of simplification、specificity and high efficiency. Consequently, the RNAi technique is used in gene treatment for cancer with broad prospects.
Anti-tumor gene therapy by the way of magnetic targeting was a new type tumor therapy system, in which the magnetic nanoparticles with satisfactory biocompatibility and low-toxicity side effect can carry genetic therapeutic molecules and initiatively reach locally advanced tumors, then kill the tumor cells under external magnetic fields. In the previous experiments, we found that iron oxide magnetic nanoparticles (CMN) has some satisfactory properties, such as superparamagnetism, biocompatibility, special nanometer effect and stability, etc. It not only can conjugate with the green fluorescent protein plasmid DNA, but also shows good magnetic targeting effect under external magnetic fields which was controled.
The gene treatment of cancer contains two areas:one is the choice of target gene; the other is method and strategy of gene treatment. Using the specificity for complementary sequence to mRNA of Survivin genes, siRNA function in tumor cell with apoptotic resistance and block the Survivin gene's expression, which offers a new treatment method to control tumor cell apoptosis. In the present study, siRNA recombinant plasmid which specific targeted survivin (siRNA-survivin) was used as a loaded gene therapeutic molecule. The effects of magnetic siRNA-CCMN nanoparticles transfection in combination with external magnetic fieldson silencing survivin gene expression of human bladder cancer cells and inducing human bladder cancer cells apoptosis were evaluated, In which CCMN produced by us was used as a gene carrier, respectively in vivo.
Materials and Methods:
1. The main materials
1.1People bladder cancer cell line of BIU-87cells, which is adherent cell.
1.2The main instrument and equipment:Bechtop, Constant temperature CO2incubator, Desktop low temperature high speed centrifuge, H-600TEM, Zeta particle size analysis instrument, Vibrating Sample Magnetometer Signal Processorultra, low temperature refrigerator, ELX-800eliasa, PCRAmplifier, GS-800gel imaging analyzer, DU-800ultraviolet spectrophotometer.
1.3The main reagent:RPMI-1640training powder, Sephacryl S-300HR, Polylysine (PLL), carboxymethyl chitosan, OPTI-MEM serum-free medium, Methyl thiazolyl tetrazolium (MTT), E.I.N.A.TM kit to endotoxin plasmid for extracting RT-PCR kit (TaKaRa RNA PCR kit(AMV)Ver3.0), Trizol Kit, DNA in situ end labeling (TUNE) detection kit, Western Blotting and ECL CLIA kit.
2. Methods
2.1Designing and synthetizing the interference sequences of Survivin-siRNA According to Reynolds's designing principles of siRNA, make sure the targeted sequences of interference:ACGA GCCAGACTTGGCCCAGT. Use the online designing software offered by Invitrogen Company to design the DNA expression models of the homologous shRNA based on the selected interference target sequence. Through the homology analysis of BLAST, all the work of the synthesis of shRNA DNA template, connection with pSilencerl.0-U6and determination, and metaplasia to escherichia coli DH5a bacterial strain are completed by Wuhan Jing Sai. These recombined plasmids are named after pSilencer1.0-siRNA-survivin.
2.2Amplification and extraction of recombinant plasmid
Inoculating Escherichia coli DH5a strain already transformed into recombinant plasmid in LB Solid culture plat containing0.05g/L kanamycin, puting in37℃5%CO2constant temperature incubator to train overnight. Selecting3almost lmm single colonies to dissolve into5ml LB liquid medium containing0.05g/L kanamycin, make liquid medium oscillate on the table concentrator at a speed larger than225rpm, put in37℃,5%CO2constant temperature incubator to train overnight. On the next day, we can get inocula which is already activation of amplification, use E.I.N.A.TM kit to endotoxin plasmid for extracting plasmid, extracting plasmid based on operation instructions. The concentration and purity of the extracted plasmid DNA can be test through DU-800ultraviolet spectrophotometer.
2.3Preparation of CCMN
CCMN was produced by the methods of chemical coprecipitation method. Taking carboxymethyl chitosan (1.5g), FeCl3·6H2O (0.234g) and FeCl2-4H2O (0.086g) in3ml of water dissolved. When Magnetic stirring, droping with a certain amount of ammonia. The temperature quickly rose to60℃, reaction time was30min, adjusting pH value to the neutral. Large particles will was removaled after aggregate15minafter low speed centrifugation. Then using the Sephacryl S-300HR gel column to separate and collect the first peak of the gel column. After full dialysis, CCMN was made through frozen vacuum drying.
2.4Construction and characters detection of siRNA-CCMN conjugates
At pH=7, PLL, CCMN and pSilencerl.0-siRNA-survivin were mixed and stirred according to the quality control rate (1:1:2) in the culture solution of RPMI-1640in free of serum. The siRNA-CCMN conjugates compound formed after the mixture reacted in the dark at4℃for1hour. Using H-600TEM, Zeta particle size analysis instrument and Vibrating Sample Magnetometer Signal Processor to detect the diameter, effective diameter, polydispersity and saturation magnetization of siRNA-DMN conjugates, respectively. The ferri ion (Fe) content of conjugates was detected by Spectra-30atomic absorption photometer.
2.5Cells culture and transfection
BIU-87cells were seeded into6-well microplate at a density of1×105/well and cultured in RPMI-1640with10%fetal bovine serum in a humidified chamber at37℃and5%CO2. When the cells grow to logarithm growth phase, which were washed by pure RPMI-1640. The siRNA-survivin was transferred into BIU-87cells by CCMN when the siRNA-CCMN containing2μg of siRNA-survivin recombinant plasmid was added into each well. After6hours, cells were cultured in RPMI-1640with10%fetal bovine serum again for48hours. The subsequent procedure was in progress. The study has three groups:The control group, siRNA-CCMN group, siRNA-CCMN+PEMFs group. The following grouping and process were done in the above same manner. The intensity of PEMFs was10mT. The treating methods were described according to the documents provided.
2.6Inhibitory effect of siRNA-CCMN on BIU-87cell proliferation by MTT method test
In3groups as the above same, every group having5ventral orifices. Conduct cell transfection, having steps as above. Separately after transfection in24、48、72h, use MTT method to test inhibition rate of cells. Use ELX-800microplate reader at490nm wavelength to test the absorbance value of separated hole.(A), compute inhibition rate of cell proliferation (IR):IR=(A blank control group-A experiment group)/A blank control group×100%.
2.7Semiquantitative RT-PCR testing expression level of Survivin mRNA
Inoculating cells on96hole culture plate.48h after transfection, digest and collect5×104well grown cells in every group, and extract overall RNA according to Trizol reagent instruction. Use DU-800ultraviolet spectrophotometer to test concentration and purity of overall RNA. According to directions of RT-PCR kit TaKaRa RNA PCR kit(AMV)Ver3.0, reverse transcrip-tion (RT) reaction to synthesize cDNA. Then conduct PCR reaction. After the PCR reaction has finished, conduct agarose gel electrophoresis test to reaction product at4℃. Use GS-800gel imaging analyzer to conduct specimen scanning, and use Doiphin1D software to conduct semi-quantitative analysis. The expression intensity of survivin gene mRNA is expressed through optical density of survivin products (A) and optical density of internal controlδβ-actin products ratio. Inhibition rate of survivin gene mRNA expression=(1-survivin expression intensity of experimental group/survivin expression intensity of blank control group)×100%.
2.8TUNEL method testing BIU-87cell apoptosis
48h after transfection, collect1×106cells, prepare cell climb pieces and use PBS to wash them three times. Then according to directions of TUNEL kit, conduct to operate that. Judgment of result:Observed under a microscope, if nucleus is brown granular coloring, that is apoptosis. Under a microscope, select random five field of vision (×400), every of which is courted100cells, and court the percentage that overall apoptosis makes up. It is apoptosis index (AI)。
2.9Western Blotting method testing the expression intensity of Survivin protein
72h after transfection, digest and collect almost2×106cells separately every group in1.5ml centrifuge tube, put into Ice precooling forl×PBS lml, wash twice and centrifuge1min. Abandon to supernatant, putting into cell protein cracking fluid of5times in the volume, oscillation blending and ice-bath with30min.14000rpm,4℃centrifugalization of15min. Absorb supernatant into a precooling EP tube, add to2×SDS loading buffer, blending based on1:4, heat at100℃for5min, cool that, and centrifuge at high speed for1min. Make total protein100μg after degeneration to be gel electrophoresis through10%SDS-PAGE. Then conduct to transfer tank for electrophoresis to transfer protein to the nitrocellulose membrane. Thus, put the nitrocellulose membrane in a plate having block buffers5%of skim milk for2h. Use TBST1:400(0.2μg/μl) to dilute primary antibodies,put that into a hermetic bag, place nitrocellulose membrane into the bag and shake gently overnight at4℃. Then use TBST1:1000to dilutesecond antibodies, put that into a hermetic bag. Select Standard A and standard B with optimum in ECL kit to mix with equal volume for1min, and make nitrocellulose membrane protein face down to contact mixed liquorfully for5min. Use GS-800gel imaging analyzer to take photo for the firm, save the image and use Quantity One software to analysis banding pixels, showing the relative amount of protein expression=optical density value of target protein/internal control optical density value.
2.10Statistics processing
Use SPSS10.0software to make experimental data to be statistics processing. Data of every group is expressed based on x±S, and one-way anova and X2are used to analysis among groups. If p<0.05, differences among groups has statistical significance.
Results:
1. Concentration and purity of recombinant plasmid DNA
Measure the concentration and purity of plasmid DNA get by amplification and extraction through ultraviolet spectrophotometer. The results are1.208±0.0045μ,g/μl and1.89±0.034。The result shows:the OD260/OD280ratio of each recombinant plasmid DNA is all between1.8and1.9, each recombinant plasmid DNA is showed to be pure. In addition, computing DNA of each recombinant plasmid in0.8～1.3μg/μl is also meet the requirement of cell transfection experiment.
2. Character of siRNA-DMN
TEM image of siRNA-CCMN showed that its shape was round. The nanoparticles joining with each other and had an average particle size of about10～12nm. The effective diameter and polydispersity of siRNA-CCMN were94.0nm and0.34, respectively. The saturation magnetization was0.18emu/g, the remanent magnetization and coercive field determination were0.11emu/g and2.42, respectively. The atomic absorption photometer detected ferriion (Fe) concentration was340μmol/L.
3. The transfection efficiency of siRNA-CCMN
After siRNA-CCMN has transfected BIU-87cells for4-6h, under fluorescence microscope, we can see more cells having green fluorescence. Transfecting efficiency is about60%in SiRNA-DMN+PEMFs group, and the control group cell without transfecting does not have green fluorescence.
4. The inhibiting effect of siRNA-CCMN on BIU-87cell proliferation
The inhibited proliferation rate of every group BIU-87cells detected with MTT method after transfected24h,48h,72h were:normal control group:1.23%,1.66%,1.61%; siRNA-CCMN group:20.54%,27.66%,24.65%; siRNA-CCMN+magnetic field group:33.62%,44.98%,36.57%,respectively. The results showed that: BIU-87cell proliferation level of siRNA-CCMN+PEMFs group was significantly lower than that of siRNA-CCMN group and normal control group (P<0.05). There were significant differences in BIU-87cell proliferation levels between siRNA-CCMN group and normal control group (P<0.05).
5. The influence of siRNA-CCMN on expression level of Survivin mRNA in BIU-87cell
5.1The results of total RNA extraction
The concentration and purity of total RNA extracted in every group of cell through nucleic acid protein detector are:normal control group:0.599,1.912, siRNA-CCMN group:0.663,1.902,0.625,1.895:siRNA-CCMN+PEMFs group. Each of the OD260/OD280ratio was between1.8-2, showed that the total RNA with higher purity of the extracted.
5.2Survivin mRNA expression level after cell transient transfection for48h
(1) After transient transfection of every group of cell for48h, gel electrophoresis results for using semiquantitative RT-PCR to test cell Survivin mRNA expression level is shown in figure.As shown in figure, we cannot see additional nonspecific banding appearing. The first, second and third lane are separately siRNA-CCMN, control group、siRNA-CCMN+PEMFs group, withp-actin amplification banding it refers to. Every group's internal control β-actin amplification banding's luminance in the area of587bp is equal, with expression level of no significant difference; while for the Survivin amplification banding at311bp, the banding luminance of siRNA-CCMN+PEMFs group is significantly weaker than another two groups. After transient transfection of every group of cell for48h, using semiquantitative TR-PCR to test cell Survivin mRNA expression relative level are:Normal control group:0.773, siRNA-CCMN:0.545, inhibition rate was32.62%, the siRNA-CCMN+PEMFs group:0.379, the inhibition rate is50.97%. The results show that, the expression level of Survivin mRNA in siRNA-CCMN+PEMFs group was significantly reduced than normal control group, and siRNA-CCMN group, the difference was statistically significant (P<0.05), the inhibition rate was50.97%. There was also a significant difference between siRNA-CCMN group and normal control group, the expression level (P<0.05), the inhibition rate was32.62%.
6. The influence of siRNA on BIU-87cell apoptotic rate
Every group's apoptosis index value (AI) is:normal control group:2.84, siRNA-CCMN:13.46, siRNA-CCMN+PEMFs:28.40. The results show that the apoptotic rate of BIU-87cells in the siRNA-CCMN+PEMFs group was significantly higher than that in normal control group and siRNA-CCMN group, the difference was statistically significant (P<0.05); siRNA-CCMN group was significantly higher than that in the normal control group (P<0.05).
7. The influence of siRNA-CCMN on BIU-87cell's Survivin protein expression
7.1Histogram of Western Blotting method testing Survivin protein expression is shown, the banding gray of siRNA-CCMN+PEMFs group distinctly becomes shallow, which is weaker than control group and siRNA-CCMN group. It prompts that Survivin's protein expression is restrained.
7.2Western Blotting method testing Survivin protein's expression strength. The result are:Normal control group:0.874, siRNA-CCMN:0.475, siRNA-CCMN+PEMFs:0.253. The results show that, the intensity of Survivin protein expression in BIU-87cells in siRNA-CCMN+PEMFs group is significantly lower than that of normal control group and siRNA-CCMN group, the difference was statistically significant (P<0.05). The siRNA-CCMN group were significantly lower than that of normal control group (P<0.05)..
Conclusion:
1. This study used siRNA recombinant plasmid which specificly targeting survivin (siRNA-survivin) as a loaded gene therapeutic molecule in the present study. CCMN produced by us was used as a gene carrier, respectively in vivo. The effection of magnetic siRNA-CCMN which was transfected in BIU-87cells on silencing survivin gene expression of human bladder cancer cells and inducing human bladder cancer cells apoptosis in combination with external magnetic fields were evaluated.
2. This study used the method of chemical coprecipitation to instruct CCMN. The method of chemical coprecipitation was simple and the preparation conditions were easily controlled. The CCMN had satisfactory compatibility, degradation and non-immunoantigenicity properties and possessed good hydrophilic property, superparamagnetism, especial nanometer effect and better single dispersion. The active groups of high polymer of CCMN could conjugate with bioactive molecules, such as plasmid by chemical modification. The compounds could enter intra-cellular by the cell taking in and succeed in transferring the GFP gene into human bladder cancer cells BIU-87. The CCMN after chemical modification could conjugate stably with pSilencerl.0-siRNA-survivin by oxidation-reduction reaction. The siRNA-CCMN conjugates could effective inhibite proliferation of BIU-87cells and induce apoptosis. The effects of combination with external magnetic fields on killing BIU-87cells were enhaced. Compared with existing carriers, CCMN have safety, specificity and feasibility which provided the experimental data for the feasibility of CCMN as gene drug nanometer carrier and established the foundation for the magnetic targeted treatment of tumor.
3. The experimental results show, siRNA-CCMN targeting Survivin after it successfully transfected with human bladder cancer cell line BIU-87cells can significantly down-regulated mRNA transcription and protein expression level of Survivin gene mRNA, induced BIU-87cell apoptosis, inhibited cell proliferation. With a constant external magnetic field, the effects of siRNA-CCMN were strengthened. It provides a possible new way and experimental basis for magnetic targeting gene target to bladder cancer treatment.
Survivin基因属于凋亡抑制蛋白(IAPs)家族新成员,其分子量最小,结构相对简单、独特,与其他IAPs家族成员存在结构上的差别。已有研究表明：Survivin基因具有抑制细胞凋亡,调节细胞周期的作用,还有参与新生血管形成等作用。Survivin基因被认为是迄今为止所发现的所有凋亡抑制因子中抗凋亡作用最强的,它抑制细胞凋亡的作用远远强大于IAPs家族中的其他因子。据文献报道,Survivin基因对膀胱肿瘤细胞的发生发展起促进作用,并且可能与肿瘤的转移和预后相关。由于Survivin基因在大多数恶性肿瘤组织中选择性显著表达,具有强大的抑制肿瘤细胞凋亡的作用,从理论上说,抑制Survivin基因的表达,足以诱导肿瘤细胞明显的自发性凋亡,这是其他凋亡抑制基因所不具备的特点。
RNA干扰(RNAi)技术是最近十年来发展最快的特异性基因阻断技术,它是将具有特定分子结构特点的、长度约19~25nt的双链小RNA (dsRNA),也叫做小干扰RNA (siRNA),通过一定的方式导入细胞内,然后siRNA特异性的降解与其序列具有同源性的宿主细胞的mRNA,因为降解发生在基因的转录后阶段,因此也叫转录后水平的目的基因沉默(PTGS)。因为RNA干扰技术可使靶基因稳定沉默,而不影响其他正常基因的表达,并且具有特异性、高效性、简易性的特点,因此,RNA干扰技术在用于恶性肿瘤的基因治疗方面前景广阔。
恶性肿瘤的基因靶向治疗可以包含两大方面：首先是靶向目的基因的选择；二是基因靶向治疗方法和策略的选择。利用与Survivin基因的mRNA序列互补的特异性siRNA作用于有凋亡抵抗的肿瘤细胞,阻断Survivin基因的表达,为调控肿瘤细胞凋亡提供了新的治疗手段。目前,研究表明,靶向Survivin基因的siRNA干扰可以明显抑制肿瘤细胞的生长和增殖,诱导其凋亡。文献报道,靶向Survivin基因的siRNA质粒能够明显诱导肿瘤细胞凋亡,抑制肿瘤细胞的增殖。但是,目前缺乏能够驱动目的基因高效稳定表达、安全无害、靶向性高、简便的新型非病毒型基因导入载体系统。
随着现代纳米技术和医用高分子材料的发展,以磁性纳米粒子作为基因载体的研究引起广泛的关注。磁导向靶向治疗是一种最新的抗肿瘤基因靶向治疗方法,它是通过具有良好生物相容性和低毒副作用特性的磁性纳米粒子载体携带目的基因治疗分子,在一定强度的外加磁场的作用下,到达病灶靶部位而肿瘤细胞被消灭。羧甲基壳聚糖磁性纳米颗粒(CCMN)是国内、外目前医学领域纳米材料研究领域的最新成果,CCMN在外加磁场的作用下,可以引导所负载的物质在体内做定向移动、定位浓聚,特异选择性定位,发挥主动的靶向作用；因此协同CCMN磁性纳米颗粒的磁导向作用,可完成对靶器官、靶细胞甚至到细胞内靶结构的选择性导入。目前,国内外尚未见羧甲基壳聚糖磁性纳米颗粒(CCMN)系统协同外磁场用于肿瘤基因靶向治疗的报道,鉴于目前肿瘤基因靶向治疗中存在的诸多问题和提高靶向治疗水平的迫切需要,开展此项研究具有广阔的前景和很大的临床应用价值。
本研究采用特异性高的靶向survivin基因的siRNA重组质粒(siRNA-survivin)作为负载基因治疗分子,以磁性CCMN粒子作为基因载体,并在外磁场的协同作用下转染siRNA-survivin入人膀胱癌BIU-87细胞,观察siRNA对Survivin基因的沉默作用,以及Survivin基因沉默对膀胱癌细胞增殖和凋亡的影响,探讨磁性siRNA-CCMN纳米粒子在膀胱癌基因靶向治疗中的作用以及CCMN作为新型非病毒型基因导入载体系统的可能性。
材料与方法：
1.主要材料
1.1人膀胱癌细胞株BIU-87细胞,为贴壁细胞。
1.2主要仪器设备：超净工作台,台式低温高速离心机,恒温C02培养箱,超低温冰箱,可调式脉冲电磁场发生器频率50Hz：H-600型透射电镜,Zeta粒度分析仪,振动样品磁信号处理仪,Spectra-30型原子吸收光谱仪,ELX-800酶标仪,PCR仪,GS-800凝胶成像分析仪,DU-800紫外分光光度计。
1.3主要试剂：RPMI-1640培养干粉,OPTI-MEM无血清培养基,羧甲基壳聚糖,聚丙烯酰胺葡聚糖凝胶,多聚赖氨酸(PLL),四甲基偶氮唑盐(MTT),去内毒素质粒提取试剂盒E.I.N.A.TM, RT-PCR试剂盒(TaKaRa RNA PCR kit(AMV)Ver3.0),总RNA提取试剂盒,Trizol试剂盒,DNA原位末端标记(TUNEL)检测试剂盒,Western Blotting和ECL发光试剂盒。
2.方法
2.1Survivin-siRNA干扰序列设计、合成
根据Reynolds制定的siRNA设计原则,确定目标序列：ACGAGCCAGACTTGGCCCAGT.使用美国Invitrogen公司提供的在线si RNA设计软件,根据选定的干扰目标序列设计出相应的shRNA DNA表达模板。经BLAST同源性分析后,进行shRNA的DNA模板合成及其与质粒载体(pSilencer1.0-U6)的连接、鉴定,并转化到大肠杆菌DH5a菌株,命名为pSilencer1.0-siRNA-survivin。
2.2重组质粒的扩增、提取
将已转化入重组质粒的DH5a菌株,接种于含0.05g/L卡那霉素的LB固体培养板上,置于37℃,5%CO2恒温培养箱中培养过夜。挑取培养板上直径lmm左右的3个单菌落,溶解于5m1含0.05g/L卡那霉素的LB液体培养基中,将液体培养基在摇床上以>225rpm的速度振荡,置于37℃,5%CO2恒温培养箱中培养过夜。次日可得到已活化扩增的菌液,用中量去内毒素质粒提取试剂盒,按操作说明提取质粒。提取的质粒用DU-800紫外分光光度计检测质粒DNA浓度和纯度。
2.3CCMN粒子的制备：采用化学共沉淀法制备CCMN粒子,将1.5g的羧甲基壳聚糖、0.234g的FeCl3·6H20、0.086g的FeCl2·4H2O-起溶于3ml水中,一边用磁力搅拌一边滴加一定量的氨水,并将温度迅速升到60℃,反应30min,然后调节pH值到中性,再将聚集物和大粒子低速离心15min去除后,再使用Sephacryl S-300HR凝胶柱分离并收集凝胶过柱首峰,随后进行透析充分,冷冻真空干燥即得CCMN。
2.4siRNA-CCMN粒子的构建及理化性质检测：在pH=7时,将PLL、CCMN与pSilencer1.0-siRNA-survivin三者在无血清的RPMI-1640培养液中按1：1：2的质量比混匀振荡,4℃下放置1h,即可形成siRNA-CCMN,分别用H-600型透射电镜、粒度分析仪、振动样品磁信号处理仪和Spectra-30型原子吸收光谱仪分别检测其直径、有效粒径、分散度、比饱和磁化强度、矫顽力及铁离子含量等。
2.5BIU-87细胞培养及转染：将细胞按每孔1×105个的数量,加入含有10%小牛血清的RPMI-1640培养液的6孔板内培养。待细胞生长处于对数期时,用无血清培养液洗涤2次,每孔内滴加含2μg siRNA-survivin的siRNA-CCMN粒子。转染6h后加入含10%胎牛血清的RPMI-1640培养液中继续培养48h后进行后续操作,同时用荧光显微镜检测细胞转染效率。本实验设立正常对照组,无磁场作用的siRNA-CCMN组和有磁场作用的siRNA-CCMN组(siRNA-CCMN+磁场)(以下分组和处理均同)。磁场作用组：脉冲电磁场强度为10mT,按常规方法进行干预。转染48～72h后收集细胞样品进行后续操作。
2.6MTT法检测对BIU-87细胞增殖的抑制作用
上述3组均设置5个复孔,并设空白对照组。将单细胞悬液接种于96孔培养板上,分别于转染后24、48、72h,采用MTT法对BIU-87细胞的增殖抑制率进行检测。取处于对数生长期的BIU-87细胞,用不含血清和抗生素的RPMI-1640培养液制成单细胞悬液,调整浓度至1×104个/孔,将ELX-800酶标仪调于490nm波长处,测定各孔的吸光度值(A),然后计算各组细胞增殖的抑制率(IR)：IR=(1-A实验组/A空白对照组)×100%。
2.7半定量RT-PCR法检测Survivin基因的mRNA表达水平
转染后48h,常规消化、收集处于对数生长期生长良好的各组培养细胞5×104个,按Trizol试剂说明书操作提取总RNA。再用DU-800紫外分光光度计测定总RNA的浓度和纯度。随后按照RT-PCR反应试剂盒的说明书进行逆转录(RT)反应合成cDNA,然后进行PCR反应。PCR反应结束后,对反应产物于4℃进行琼脂糖凝胶电泳检测。标本用GS-800型凝胶成像分析仪进行扫描,再用Doiphin1D软件进行半定量分析。用Survivin产物的吸光度值(A)与内参照β-actin产物的吸光度值(A)的比值表示Survivin基因mRNA的表达强度。Survivin基因的mRNA表达抑制率=(1—实验组表达强度-空白对照组表达强度)×100%。
2.8TUNEL法检测BIU-87细胞凋亡
转染后48h,分别消化、收集各组细胞1×106个,制备细胞爬片,用PBS洗涤3次。然后参照TUNEL试剂盒说明书进行操作,结果判断：凋亡细胞的细胞核在显微镜下观察,呈棕褐色颗粒状着色。在显微镜下随机选择5个视野(×400),每个视野计数100个细胞,并计数凋亡细胞占细胞总数的百分比,即为凋亡指数(AI)。
2.9Western-Blotting法检测Survivin基因的蛋白表达强度
转染后72h,分别消化收集各组细胞约2×106个于1.5ml离心管中,加入冰上预冷1×PBS1m1,洗涤2次,离心lmin。弃去上清,加入5倍体积的细胞蛋白裂解液,振荡混匀,冰浴30min。14000rpm,4℃离心15min。吸取上清至一预冷的EP管中,加入2×SDS上样缓冲液按1：4混匀,100℃加热5min,冷却,高速离心lmin。将变性后的总蛋白100μg经10%SDS-PAGE凝胶电泳。再进行电转膜,将蛋白转移到硝酸纤维素膜上。将硝酸纤维素膜置于含有封闭缓冲液的平皿中2h。一抗用TBST1:400(0.2μg/μl)稀释,装入密封袋中,将硝酸纤维素膜放入其中,4℃平缓摇动过夜。再将二抗用TBST1:1000稀释,装入密封袋中。取适当量ECL试剂盒中的A液、B液等体积混合lmin,硝酸纤维素膜蛋白面朝下与此混合液充分接触5min。将硝酸纤维素膜沥干,放在包有保鲜膜的薄板上,用GS-800凝胶成像分析仪将膜照相,保存图像,Quantity One软件分析条带像素值,蛋白相对表达量=待测蛋白的光密度值/内参的光密度值。
2.10统计学处理
本研究实验数据采用SPSS10.0软件进行统计学处理。各组相关数据均以x±s来表示,采用单因素方差分析和X2检验进行组间数据分析比较,p<0.05为差异有统计学意义。
结果：
1.重组质粒siRNA-DNA的浓度和纯度
将扩增、提取得到的质粒siRNA-DNA通过紫外分光光度计测定其浓度是1.208±0.0045μ g/μl,纯度是1.890±0.034。所提取的重组质粒siRNA-DNA的OD260/OD290比值在1.8～1.9之间,表明重组质粒DNA较纯,另外重组质粒siRNA-DNA的含量在0.8～1.3μg/μl之间,达到细胞转染实验的要求。
2. siRNA-CCMN粒子的理化性质：在电镜下观察,siRNA-CCMN为圆形,其直径为10nm-12nm,分散度为0.34,有效粒径为94.0nm,比饱和磁化强度为0.18emu/g、剩磁、矫顽力分别为0.11emu/g、2.42,铁离子含量为340μmol/。
3. siRNA-CCMN转染效率
siRNA-CCMN转染BIU-87细胞4～6h后,在荧光显微镜下,可见较多细胞带有绿色荧光,siRNA-CCMN+磁场组转染效率为65%左右。
4. siRNA-CCMN对BIU-87细胞增殖的抑制作用
MTT法检测各组BIU-87细胞转染后24h、48h、72h的增殖抑制率(%)分别是：正常对照组：1.23%,1.66%,1.61%；siRNA-CCMN组：20.54%,27.66%,24.65%;siRNA-CCMN+磁场组：33.62%,44.98%,36.57%。
MTT法检测的结果表明,siRNA-CCMN转染BIU-87细胞后,siRNA-CCMN+磁场组的BIU-87细胞增殖水平均显著低于siRNA-CCMN组和正常对照组(P<0.05)。siRNA-CCMN组和正常对照组之间的BIU-87细胞增殖水平也存在显著性差异(P<0.05)。
5. siRNA-CCMN对BIU-87细胞Survivin基因mRNA表达水平的影响
5.1总RNA提取结果
通过核酸蛋白检测仪所检测的各组细胞提取的总RNA的浓度(μg/μl)及纯度(OD260/OD280)分别是：正常对照组：0.599,1.912,siRNA-CCMN组：0.663,1.902,siRNA-CCMN+磁场组：0.625,1.895。各组的OD260/OD280的比值在1.8～2之间,表明所提取的总RNA纯度较高。
5.2细胞瞬时转染48h后Survivin基因mRNA表达水平
(1)各组细胞瞬时转染48h后,用半定量RT-PCR检测细胞Survivin mRNA表达情况的凝胶电泳图,未见其他非特异性条带的出现。第1、2、3泳道分别是siRNA-CCMN组,正常对照组,siRNA-CCMN+磁场组的Survivin及其对应的β-actin扩增条带。各组在311bp区域内的内参照β-actin扩增条带的亮度相等,说明表达水平无明显差别：而在587bp处的Survivin扩增条带,siRNA-CCMN+磁场组条带亮度明显弱于其他两组,说明表达水平有明显差别。
(2)各组细胞瞬时转染48h后,用半定量TR-PCR检测细胞Survivin基因mRNA表达的相对水平结果：正常对照组：0.773,siRNA-CCMN组：0.545,抑制率是32.62%,siRNA-CCMN+磁场组：0.379,抑制率是50.97%。结果表明,siRNA-CCMN+磁场组BIU-87细胞的Survivin mRNA表达水平分别与正常对照组、siRNA-CCMN组比较,均明显下调,差异有统计学意义(P<0.05),其抑制率达50.97%。而siRNA-CCMN组和正常对照组BIU-87细胞的Survivin mRNA表达水平之间也有显著性差异(P<0.05),其抑制率达32.62%。
6. siRNA-CCMN对BIU-87细胞凋亡率的影响
各组BIU-87细胞的凋亡指数(AI)分别是：正常对照组：2.84,siRNA-CCMN组：13.46,siRNA-CCMN+磁场组：28.40。结果表明,siRNA-CCMN+磁场组BIU-87细胞的凋亡率明显高于正常对照组和siRNA-CCMN组,差异有统计学意义(P<0.05); siRNA-CCMN组也显著高于正常对照组(P<0.05)。
7. siRNA-CCMN对BIU-87细胞Survivin蛋白表达的影响
7.1Western Blotting法检测各组细胞Survivin蛋白的条带图,如图所不,siRNA-CCMN+磁场组条带灰度明显变浅,弱于正常对照组和siRNA-CCMN组,提示Survivin的蛋白表达被抑制。
7.2Western Blotting法检测各组细胞Survivin蛋白的表达强度,结果分别是：正常对照组：0.874, siRNA-CCMN组：0.475,siRNA-CCMN+磁场组：0.253。结果表明,siRNA-CCMN+磁场组BIU-87细胞的Survivin蛋白的表达强度明显低于正常对照组和siRNA-CCMN组,差异有统计学意义(P<0.05)。siRNA-CCMN组也显著低于正常对照组(P<0.05)。
结论：
1.本研究采用靶向survivin基因的特异性siRNA重组质粒pSilencer1.0-siRNA-survivin作为负载基因治疗分子,以CCMN粒子作为基因转染载体,在外磁场的协同作用下转染siRNA-survivin入人膀胱癌BIU-87细胞,观察siRNA-CCMN对Survivin基因的沉默作用,以及Survivin基因沉默对膀胱癌细胞增殖和凋亡的影响,探讨磁性siRNA-CCMN纳米粒子在膀胱癌基因靶向治疗中的作用以及CCMN作为新型非病毒型基因导入载体的可能性。
2.本研究用化学共沉淀法成功制备了羧甲基壳聚糖氧化铁磁性纳米颗粒(CCMN),化学共沉淀法制备简单,反应条件容易控制,制备出来的CCMN具有良好的生物相容性和生物可溶解性、特殊的纳米效应、超顺磁性、无毒无副作用,在溶液中稳定性好,具有较好的具有较好的单分散性。化学修饰后的羧甲基壳聚糖磁性纳米颗粒(CCMN)上的高分子羧甲基壳聚糖上的活性基团可通过经典的氧化还原反应与pSilencer1.0-siRNA-survivin稳定结合形成siRNA-CCMN, siRNA-CCMN在细胞摄粒作用下可以转染入细胞内,而且效率较高,外磁场可以显著加强转染效率。与现有的载体相比,羧甲基壳聚糖磁性纳米颗粒具有安全、特异,可行性好,这为其作为新型基因和药物纳米载体的研究提供了实验依据,为肿瘤的磁导向靶向治疗奠定实验基础。
3.本实验结果显示,靶向Survivin的siRNA-CCMN成功转染了人膀胱癌细胞株BIU-87细胞后,能够显著下调BIU-87细胞中Survivin基因mRNA转录和蛋白表达水平,明显诱导BIU-87细胞的凋亡,抑制细胞的增殖,而且在恒定外磁场协同作用下,siRNA-CCMN的作用显著加强。说明siRNA-CCMN可以特异、高效地转染膀胱癌BIU-87细胞,并且能有效的发挥对Survivin基因的RNA干扰作用,为膀胱癌的磁导向基因靶向治疗提供了一种可能的新途径与实验依据。
Background&objective:Bladder cancer is a common urologic cancer, which directly threaten to the life of patients. At present, the main treatment of bladder cancer is still surgery, and the auxiliary methods are chemotherapy、actinotherapy、 immunotherapy and a variety of methods with combination treatment mode. However, to invasive and metastatic bladder cancer, the prostecdtive efficacy of treatment methods above is less than satisfactory and most patients eventually die of infiltrating and metastasis of the tumor.
Survivin gene belongs to the new member of IAPs family, whose molecular weight is the smallest and structure is relatively simple and unique. It is very different from another members in IAPs family in the structure. Literature reports, Survivin has positive function to the occurance and development of cell carcinoma of bladder and is possibly related to metastasis and prognosis of the tumor. Survivin can be an important parameter to filtration、early diagnosis and prognostic ability of bladder cancer. Because Survivin in most cancer tissues with survivin gene has significant express of selectivity, strong function of inhibitor of apoptosis and inhibitor to survivin expression enough to induce the spontaneous apoptosis of cancer cell. This is the characteristic that another apoptosis inhibiting genes not have.
RNAi technique is an agene disruption technique that develops rapidly in recent years, which thus leads to gene silencing of level of purpose (PTGS) through making the dsRNA with the regular structure characteristic and having a length of19-25nt, which is siRNA, import into host cells and on the level of specificity degradation to the mRNA of host cells that have homology with it. The technique of RNAi can make target genes stable and silent, not impact the expression of normal genes, and have the characteristics of simplification、specificity and high efficiency. Consequently, the RNAi technique is used in gene treatment for cancer with broad prospects.
Anti-tumor gene therapy by the way of magnetic targeting was a new type tumor therapy system, in which the magnetic nanoparticles with satisfactory biocompatibility and low-toxicity side effect can carry genetic therapeutic molecules and initiatively reach locally advanced tumors, then kill the tumor cells under external magnetic fields. In the previous experiments, we found that iron oxide magnetic nanoparticles (CMN) has some satisfactory properties, such as superparamagnetism, biocompatibility, special nanometer effect and stability, etc. It not only can conjugate with the green fluorescent protein plasmid DNA, but also shows good magnetic targeting effect under external magnetic fields which was controled.
The gene treatment of cancer contains two areas:one is the choice of target gene; the other is method and strategy of gene treatment. Using the specificity for complementary sequence to mRNA of Survivin genes, siRNA function in tumor cell with apoptotic resistance and block the Survivin gene's expression, which offers a new treatment method to control tumor cell apoptosis. In the present study, siRNA recombinant plasmid which specific targeted survivin (siRNA-survivin) was used as a loaded gene therapeutic molecule. The effects of magnetic siRNA-CCMN nanoparticles transfection in combination with external magnetic fieldson silencing survivin gene expression of human bladder cancer cells and inducing human bladder cancer cells apoptosis were evaluated, In which CCMN produced by us was used as a gene carrier, respectively in vivo.
Materials and Methods:
1. The main materials
1.1People bladder cancer cell line of BIU-87cells, which is adherent cell.
1.2The main instrument and equipment:Bechtop, Constant temperature CO2incubator, Desktop low temperature high speed centrifuge, H-600TEM, Zeta particle size analysis instrument, Vibrating Sample Magnetometer Signal Processorultra, low temperature refrigerator, ELX-800eliasa, PCRAmplifier, GS-800gel imaging analyzer, DU-800ultraviolet spectrophotometer.
1.3The main reagent:RPMI-1640training powder, Sephacryl S-300HR, Polylysine (PLL), carboxymethyl chitosan, OPTI-MEM serum-free medium, Methyl thiazolyl tetrazolium (MTT), E.I.N.A.TM kit to endotoxin plasmid for extracting RT-PCR kit (TaKaRa RNA PCR kit(AMV)Ver3.0), Trizol Kit, DNA in situ end labeling (TUNE) detection kit, Western Blotting and ECL CLIA kit.
2. Methods
2.1Designing and synthetizing the interference sequences of Survivin-siRNA According to Reynolds's designing principles of siRNA, make sure the targeted sequences of interference:ACGA GCCAGACTTGGCCCAGT. Use the online designing software offered by Invitrogen Company to design the DNA expression models of the homologous shRNA based on the selected interference target sequence. Through the homology analysis of BLAST, all the work of the synthesis of shRNA DNA template, connection with pSilencerl.0-U6and determination, and metaplasia to escherichia coli DH5a bacterial strain are completed by Wuhan Jing Sai. These recombined plasmids are named after pSilencer1.0-siRNA-survivin.
2.2Amplification and extraction of recombinant plasmid
Inoculating Escherichia coli DH5a strain already transformed into recombinant plasmid in LB Solid culture plat containing0.05g/L kanamycin, puting in37℃5%CO2constant temperature incubator to train overnight. Selecting3almost lmm single colonies to dissolve into5ml LB liquid medium containing0.05g/L kanamycin, make liquid medium oscillate on the table concentrator at a speed larger than225rpm, put in37℃,5%CO2constant temperature incubator to train overnight. On the next day, we can get inocula which is already activation of amplification, use E.I.N.A.TM kit to endotoxin plasmid for extracting plasmid, extracting plasmid based on operation instructions. The concentration and purity of the extracted plasmid DNA can be test through DU-800ultraviolet spectrophotometer.
2.3Preparation of CCMN
CCMN was produced by the methods of chemical coprecipitation method. Taking carboxymethyl chitosan (1.5g), FeCl3·6H2O (0.234g) and FeCl2-4H2O (0.086g) in3ml of water dissolved. When Magnetic stirring, droping with a certain amount of ammonia. The temperature quickly rose to60℃, reaction time was30min, adjusting pH value to the neutral. Large particles will was removaled after aggregate15minafter low speed centrifugation. Then using the Sephacryl S-300HR gel column to separate and collect the first peak of the gel column. After full dialysis, CCMN was made through frozen vacuum drying.
2.4Construction and characters detection of siRNA-CCMN conjugates
At pH=7, PLL, CCMN and pSilencerl.0-siRNA-survivin were mixed and stirred according to the quality control rate (1:1:2) in the culture solution of RPMI-1640in free of serum. The siRNA-CCMN conjugates compound formed after the mixture reacted in the dark at4℃for1hour. Using H-600TEM, Zeta particle size analysis instrument and Vibrating Sample Magnetometer Signal Processor to detect the diameter, effective diameter, polydispersity and saturation magnetization of siRNA-DMN conjugates, respectively. The ferri ion (Fe) content of conjugates was detected by Spectra-30atomic absorption photometer.
2.5Cells culture and transfection
BIU-87cells were seeded into6-well microplate at a density of1×105/well and cultured in RPMI-1640with10%fetal bovine serum in a humidified chamber at37℃and5%CO2. When the cells grow to logarithm growth phase, which were washed by pure RPMI-1640. The siRNA-survivin was transferred into BIU-87cells by CCMN when the siRNA-CCMN containing2μg of siRNA-survivin recombinant plasmid was added into each well. After6hours, cells were cultured in RPMI-1640with10%fetal bovine serum again for48hours. The subsequent procedure was in progress. The study has three groups:The control group, siRNA-CCMN group, siRNA-CCMN+PEMFs group. The following grouping and process were done in the above same manner. The intensity of PEMFs was10mT. The treating methods were described according to the documents provided.
2.6Inhibitory effect of siRNA-CCMN on BIU-87cell proliferation by MTT method test
In3groups as the above same, every group having5ventral orifices. Conduct cell transfection, having steps as above. Separately after transfection in24、48、72h, use MTT method to test inhibition rate of cells. Use ELX-800microplate reader at490nm wavelength to test the absorbance value of separated hole.(A), compute inhibition rate of cell proliferation (IR):IR=(A blank control group-A experiment group)/A blank control group×100%.
2.7Semiquantitative RT-PCR testing expression level of Survivin mRNA
Inoculating cells on96hole culture plate.48h after transfection, digest and collect5×104well grown cells in every group, and extract overall RNA according to Trizol reagent instruction. Use DU-800ultraviolet spectrophotometer to test concentration and purity of overall RNA. According to directions of RT-PCR kit TaKaRa RNA PCR kit(AMV)Ver3.0, reverse transcrip-tion (RT) reaction to synthesize cDNA. Then conduct PCR reaction. After the PCR reaction has finished, conduct agarose gel electrophoresis test to reaction product at4℃. Use GS-800gel imaging analyzer to conduct specimen scanning, and use Doiphin1D software to conduct semi-quantitative analysis. The expression intensity of survivin gene mRNA is expressed through optical density of survivin products (A) and optical density of internal controlδβ-actin products ratio. Inhibition rate of survivin gene mRNA expression=(1-survivin expression intensity of experimental group/survivin expression intensity of blank control group)×100%.
2.8TUNEL method testing BIU-87cell apoptosis
48h after transfection, collect1×106cells, prepare cell climb pieces and use PBS to wash them three times. Then according to directions of TUNEL kit, conduct to operate that. Judgment of result:Observed under a microscope, if nucleus is brown granular coloring, that is apoptosis. Under a microscope, select random five field of vision (×400), every of which is courted100cells, and court the percentage that overall apoptosis makes up. It is apoptosis index (AI)。
2.9Western Blotting method testing the expression intensity of Survivin protein
72h after transfection, digest and collect almost2×106cells separately every group in1.5ml centrifuge tube, put into Ice precooling forl×PBS lml, wash twice and centrifuge1min. Abandon to supernatant, putting into cell protein cracking fluid of5times in the volume, oscillation blending and ice-bath with30min.14000rpm,4℃centrifugalization of15min. Absorb supernatant into a precooling EP tube, add to2×SDS loading buffer, blending based on1:4, heat at100℃for5min, cool that, and centrifuge at high speed for1min. Make total protein100μg after degeneration to be gel electrophoresis through10%SDS-PAGE. Then conduct to transfer tank for electrophoresis to transfer protein to the nitrocellulose membrane. Thus, put the nitrocellulose membrane in a plate having block buffers5%of skim milk for2h. Use TBST1:400(0.2μg/μl) to dilute primary antibodies,put that into a hermetic bag, place nitrocellulose membrane into the bag and shake gently overnight at4℃. Then use TBST1:1000to dilutesecond antibodies, put that into a hermetic bag. Select Standard A and standard B with optimum in ECL kit to mix with equal volume for1min, and make nitrocellulose membrane protein face down to contact mixed liquorfully for5min. Use GS-800gel imaging analyzer to take photo for the firm, save the image and use Quantity One software to analysis banding pixels, showing the relative amount of protein expression=optical density value of target protein/internal control optical density value.
2.10Statistics processing
Use SPSS10.0software to make experimental data to be statistics processing. Data of every group is expressed based on x±S, and one-way anova and X2are used to analysis among groups. If p<0.05, differences among groups has statistical significance.
Results:
1. Concentration and purity of recombinant plasmid DNA
Measure the concentration and purity of plasmid DNA get by amplification and extraction through ultraviolet spectrophotometer. The results are1.208±0.0045μ,g/μl and1.89±0.034。The result shows:the OD260/OD280ratio of each recombinant plasmid DNA is all between1.8and1.9, each recombinant plasmid DNA is showed to be pure. In addition, computing DNA of each recombinant plasmid in0.8～1.3μg/μl is also meet the requirement of cell transfection experiment.
2. Character of siRNA-DMN
TEM image of siRNA-CCMN showed that its shape was round. The nanoparticles joining with each other and had an average particle size of about10～12nm. The effective diameter and polydispersity of siRNA-CCMN were94.0nm and0.34, respectively. The saturation magnetization was0.18emu/g, the remanent magnetization and coercive field determination were0.11emu/g and2.42, respectively. The atomic absorption photometer detected ferriion (Fe) concentration was340μmol/L.
3. The transfection efficiency of siRNA-CCMN
After siRNA-CCMN has transfected BIU-87cells for4-6h, under fluorescence microscope, we can see more cells having green fluorescence. Transfecting efficiency is about60%in SiRNA-DMN+PEMFs group, and the control group cell without transfecting does not have green fluorescence.
4. The inhibiting effect of siRNA-CCMN on BIU-87cell proliferation
The inhibited proliferation rate of every group BIU-87cells detected with MTT method after transfected24h,48h,72h were:normal control group:1.23%,1.66%,1.61%; siRNA-CCMN group:20.54%,27.66%,24.65%; siRNA-CCMN+magnetic field group:33.62%,44.98%,36.57%,respectively. The results showed that: BIU-87cell proliferation level of siRNA-CCMN+PEMFs group was significantly lower than that of siRNA-CCMN group and normal control group (P<0.05). There were significant differences in BIU-87cell proliferation levels between siRNA-CCMN group and normal control group (P<0.05).
5. The influence of siRNA-CCMN on expression level of Survivin mRNA in BIU-87cell
5.1The results of total RNA extraction
The concentration and purity of total RNA extracted in every group of cell through nucleic acid protein detector are:normal control group:0.599,1.912, siRNA-CCMN group:0.663,1.902,0.625,1.895:siRNA-CCMN+PEMFs group. Each of the OD260/OD280ratio was between1.8-2, showed that the total RNA with higher purity of the extracted.
5.2Survivin mRNA expression level after cell transient transfection for48h
(1) After transient transfection of every group of cell for48h, gel electrophoresis results for using semiquantitative RT-PCR to test cell Survivin mRNA expression level is shown in figure.As shown in figure, we cannot see additional nonspecific banding appearing. The first, second and third lane are separately siRNA-CCMN, control group、siRNA-CCMN+PEMFs group, withp-actin amplification banding it refers to. Every group's internal control β-actin amplification banding's luminance in the area of587bp is equal, with expression level of no significant difference; while for the Survivin amplification banding at311bp, the banding luminance of siRNA-CCMN+PEMFs group is significantly weaker than another two groups. After transient transfection of every group of cell for48h, using semiquantitative TR-PCR to test cell Survivin mRNA expression relative level are:Normal control group:0.773, siRNA-CCMN:0.545, inhibition rate was32.62%, the siRNA-CCMN+PEMFs group:0.379, the inhibition rate is50.97%. The results show that, the expression level of Survivin mRNA in siRNA-CCMN+PEMFs group was significantly reduced than normal control group, and siRNA-CCMN group, the difference was statistically significant (P<0.05), the inhibition rate was50.97%. There was also a significant difference between siRNA-CCMN group and normal control group, the expression level (P<0.05), the inhibition rate was32.62%.
6. The influence of siRNA on BIU-87cell apoptotic rate
Every group's apoptosis index value (AI) is:normal control group:2.84, siRNA-CCMN:13.46, siRNA-CCMN+PEMFs:28.40. The results show that the apoptotic rate of BIU-87cells in the siRNA-CCMN+PEMFs group was significantly higher than that in normal control group and siRNA-CCMN group, the difference was statistically significant (P<0.05); siRNA-CCMN group was significantly higher than that in the normal control group (P<0.05).
7. The influence of siRNA-CCMN on BIU-87cell's Survivin protein expression
7.1Histogram of Western Blotting method testing Survivin protein expression is shown, the banding gray of siRNA-CCMN+PEMFs group distinctly becomes shallow, which is weaker than control group and siRNA-CCMN group. It prompts that Survivin's protein expression is restrained.
7.2Western Blotting method testing Survivin protein's expression strength. The result are:Normal control group:0.874, siRNA-CCMN:0.475, siRNA-CCMN+PEMFs:0.253. The results show that, the intensity of Survivin protein expression in BIU-87cells in siRNA-CCMN+PEMFs group is significantly lower than that of normal control group and siRNA-CCMN group, the difference was statistically significant (P<0.05). The siRNA-CCMN group were significantly lower than that of normal control group (P<0.05)..
Conclusion:
1. This study used siRNA recombinant plasmid which specificly targeting survivin (siRNA-survivin) as a loaded gene therapeutic molecule in the present study. CCMN produced by us was used as a gene carrier, respectively in vivo. The effection of magnetic siRNA-CCMN which was transfected in BIU-87cells on silencing survivin gene expression of human bladder cancer cells and inducing human bladder cancer cells apoptosis in combination with external magnetic fields were evaluated.
2. This study used the method of chemical coprecipitation to instruct CCMN. The method of chemical coprecipitation was simple and the preparation conditions were easily controlled. The CCMN had satisfactory compatibility, degradation and non-immunoantigenicity properties and possessed good hydrophilic property, superparamagnetism, especial nanometer effect and better single dispersion. The active groups of high polymer of CCMN could conjugate with bioactive molecules, such as plasmid by chemical modification. The compounds could enter intra-cellular by the cell taking in and succeed in transferring the GFP gene into human bladder cancer cells BIU-87. The CCMN after chemical modification could conjugate stably with pSilencerl.0-siRNA-survivin by oxidation-reduction reaction. The siRNA-CCMN conjugates could effective inhibite proliferation of BIU-87cells and induce apoptosis. The effects of combination with external magnetic fields on killing BIU-87cells were enhaced. Compared with existing carriers, CCMN have safety, specificity and feasibility which provided the experimental data for the feasibility of CCMN as gene drug nanometer carrier and established the foundation for the magnetic targeted treatment of tumor.
3. The experimental results show, siRNA-CCMN targeting Survivin after it successfully transfected with human bladder cancer cell line BIU-87cells can significantly down-regulated mRNA transcription and protein expression level of Survivin gene mRNA, induced BIU-87cell apoptosis, inhibited cell proliferation. With a constant external magnetic field, the effects of siRNA-CCMN were strengthened. It provides a possible new way and experimental basis for magnetic targeting gene target to bladder cancer treatment.
引文
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[1]Suzuki A, Ito T, Kawano H, et al. Survivin initiates procaspase-3/p21 complex formation as a result of interaction with CDK4 to resist Fas-mediated cell death. Oncogene,2000,19:1346-1353.
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[12]Blanc-Brude OP, Yu J, Smosa H, et al. Inhibitor of apoptosis protein surviving regulates vascular injury. Nat Med,2002,8 (9): 987-994.
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