叶酸修饰的聚乙烯亚胺聚合物载PD-L1 siRNA体外靶向胃癌细胞的研究

目的:构建高效的siRNA纳米载体靶向SGC-7901胃癌细胞,并下调胃癌表达的程序性死亡配体1(PD-L1)。方法:检测叶酸(FA)-PEG-SS-PEI-SPION纳米载体与siRNA复合后的粒径、电位等表征;体外实验检验siRNA的结合能力、复合物细胞毒性、细胞摄入能力及转染效率;磁共振(MR)成像检测示踪能力;检验胃癌细胞PD-L1下调效应及共培养T细胞的细胞因子水平。结果:N/P比值为10时,FA-PEG-SS-PEI-SPION完全复合siRNA,形成电位为(9.14±0.80)m V、粒径为(116.7±2.5)nm的多聚复合物。靶向组的转染率为(95.06±0.44)%,与非靶向组的(93.87±1.05)%相当;平均荧光强度为1 892.67±81.51,高于非靶向组的1 324.33±186.58(P0.05)。普鲁士蓝染色和激光共聚焦显微镜成像证实了复合物的细胞摄入。体外MR成像验证了聚合物的MR造影成像能力。靶向组PD-L1的mRNA最低相对表达量为9.07%±0.79%,Western blot显示PD-L1的表达显著降低。共培养实验显示IFN-γ和TNF-α的分泌水平增加,IL-10的分泌水平降低(P0.05)。结论:本研究构建了FA-PEG-SS-PEI-SPION纳米载体,并证明了其体外靶向细胞及载siRNA下调PD-L1表达的能力和MR示踪的能力,是一种高效和安全的靶向治疗纳米载体。     

miR-155特异性siRNA增强阿糖胞苷诱导的Burkitt淋巴瘤Raji细胞凋亡

 目的：研究miR-155特异性siRNA增强阿糖胞苷（Ara-C）诱导的Burkitt淋巴瘤Raji细胞凋亡及其机制。方法：Ara-C与miR-155 siRNA单独或者联合干预Raji细胞增殖。qRT- PCR检测脂质体转染siRNA后miR-155的表达;CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡;Western blotting法检测caspase-3的表达。结果：qRT-PCR显示,转染miR-155 siRNA后,Raji细胞的miR-155表达明显下降（P<0.05）,Ara-C或miR-155 siRNA单独处理细胞后均显示出增殖抑制作用,而两者联合使用的增殖抑制作用更显著（P<0.05）。各组细胞进行药物干预48 h后行流式细胞术,结果显示Ara-C或miR-155 siRNA单独处理细胞凋亡率分别为（16.5±0.3）%和（14.6±0.3）%,与对照组［（3.6±0.4）%］比较差别有统计学意义（P＜0.05),Ara-C+miR-155 siRNA组的凋亡率［（38.4±1.4）%］分别与Ara-C和miR-155 siRNA单独处理组比较,差别有统计学意义（P＜0.05）。Ara-C+miR-155 siRNA组与Ara-C组及miR-155 siRNA组相比,caspase-3的水平明显增高。结论：miR-155特异性siRNA具有增强阿糖胞苷对Raji细胞增殖抑制及诱导Raji细胞凋亡的作用,其机制与caspase-3凋亡途径有关。     

内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用

 目的：探讨内质网应激在Bim介导缺氧致心肌细胞凋亡中的作用。方法：在体外原代培养出生1~3 d大鼠心肌细胞,并用抗α-横纹肌肌动蛋白免疫组化法进行鉴定。设计并化学合成3对靶向bim的siRNA,用脂质体法将siRNA转染心肌细胞,筛选沉默效率最高的siRNA。实验分组：（1）空白对照组;（2）缺氧组;（3）缺氧+脂质体组;(4)缺氧+阴性对照siRNA组;(5)缺氧+Bim-siRNA组。MTT法观察细胞活性;流式细胞术检测细胞凋亡率及细胞内钙离子浓度变化情况;Western blotting检测内质网应激标志分子caspase-12和三磷酸肌醇（IP3）的表达情况。结果：免疫组化鉴定证实大鼠心肌细胞原代培养成功。在荧光显微镜下,转染了阴性对照siRNA组的细胞中观察到绿色荧光,即转染成功;Western blotting 结果显示,Bim-siRNA转染均能有效降低Bim蛋白的表达,其中第2对沉默效率最高,达到86.73%。缺氧损伤导致心肌细胞活性明显下降（P<005）,转染Bim-siRNA后细胞活性较阴性对照组升高。缺氧细胞凋亡率较对照组明显增加（P<0.01）,细胞内钙离子浓度明显增高,而沉默bim的表达能降低细胞凋亡率和细胞内钙离子浓度。缺氧导致内质网应激标志分子caspase-12和IP3表达较空白对照组明显上调（均P<005）,而抑制Bim表达后caspase-12和IP3表达明显降低。结论：沉默bim的表达能有效抑制缺氧导致心肌细胞凋亡的作用,内质网应激标志分子caspase-12和IP3可能参与了Bim介导缺氧致心肌细胞凋亡的过程。这有望为临床心肌缺血缺氧损伤的治疗提供新思路。     

敲低膜联蛋白A7对人肝癌HepG2细胞Bcl-2和Bax表达的影响

目的 探讨敲低膜联蛋白A7表达对人肝癌细胞系HepG2细胞凋亡及凋亡相关蛋白Bcl-2和Bax表达的影响。方法 将HepG2细胞接种于6孔板,分为3组：siRNA干扰组、阴性对照组和空白对照组,其中干扰组只转染靶向膜联蛋白A7的siRNA,阴性对照组只转染阴性对照siRNA,空白对照组只加转染试剂。通过Western blotting鉴定膜联蛋白A7在转染后48h可被最大程度的抑制,于是在转染后48h采用流式细胞术检测各组细胞凋亡率,通过免疫组织化学、Western blotting和RT PCR检测Bcl-2和Bax蛋白及mRNA的表达。 结果 与阴性对照组和空白对照组相比,siRNA干扰组细胞凋亡率显著增高（P<0.05）,Bcl-2蛋白和mRNA的表达均显著降低（P<0.05）,而Bax蛋白和mRNA均未发生显著变化（P>0.05）。结论 敲低膜联蛋白A7可促进HepG2细胞的凋亡,降低Bcl-2和Bax的比值。     

RICTOR对类风湿关节炎成纤维样滑膜细胞活力的影响

 目的：通过siRNA介导的RNA干扰技术沉默类风湿关节炎（RA）成纤维样滑膜细胞（FLS）mTORC2的特异组成蛋白RICTOR的表达,观察其对细胞活力的影响。方法：组织块法培养RA-FLS。应用阳离子脂质体转染的方法,把化学合成的特异性RICTOR siRNA转染RA-FLS,并以转染非特异性siRNA作为阴性对照。利用荧光定量PCR法分析转染24 h后细胞RICTOR mRNA表达水平的变化;Western blotting法分析转染48和72 h后细胞RICTOR蛋白表达水平的变化;以噻唑蓝（MTT）比色法检测转染成纤维样滑膜细胞不同时间（24、48和72 h）RICTOR siRNA对细胞活力的影响。结果：荧光定量PCR结果显示特异性RICTOR siRNA转染组与对照组相比,细胞中RICTOR的mRNA表达水平显著下调,24 h干扰效率达78.3%±63.71%（P<0.01）。Western blotting结果显示与对照组相比,RICTOR siRNA转染组48 h和72 h后RICTOR蛋白表达水平明显降低,沉默效率分别为92.48%±6.14%和98.57%±1.40%（均P<0.01）。MTT结果显示,早期（24和48 h）RICTOR siRNA转染组与阴性对照组细胞存活率比较无显著差异;72 h后,RICTOR siRNA转染组与阴性对照相比,细胞活力明显降低,抑制率为90.14%±1.90%（P<0.01）。结论：转染特异性RICTOR siRNA可降低RA-FLS的活力,提示mTORC2可能与RA-FLS的生长有关。     

慢病毒介导的caspase-3沉默对大鼠骨髓间充质干细胞增殖和凋亡的影响

 目的：探讨沉默caspase-3对大鼠骨髓间充质干细胞（MSCs）增殖、细胞周期和凋亡的影响。方法：构建靶向caspase-3的shRNA重组慢病毒并转染MSCs,通过real-time PCR和Western blotting 在mRNA及蛋白水平鉴定转染结果。采用MTS法检测细胞增殖,流式细胞术检测细胞周期。Real-time PCR检测bcl-2和bax mRNA的表达。Hoechst荧光染色法检测细胞的凋亡情况。结果：Real-time PCR和Western blotting结果均表明成功建立稳定转染shRNA-caspase-3的大鼠MSCs细胞株。沉默caspase-3使MSCs的增殖率明显提高（P＜0.05）,且S期细胞百分比明显增多,为（52.66±0.30）%。沉默caspase-3后bcl-2 mRNA表达上调,bax mRNA表达下调,bcl-2/bax比值升高（P＜0.05）。转染组的细胞凋亡率为（15.01±1.73）%,低于空载体组的（25.67±3.05）%和空白对照组的（23.67±1.16）%（P＜0.05）。结论: 沉默caspase-3能调控MSCs的细胞周期,促进细胞增殖,减少细胞凋亡。     

PKM2 影响鼻咽癌细胞增殖凋亡及机制研究

目的：探讨PKM2对鼻咽癌细胞增殖凋亡的影响。方法：鼻咽癌细胞CNE-1转染PKM2小干扰RNA（PKM2-siRNA1和PKM2-siRNA2）和阴性对照（siRNA control）,荧光定量PCR和Western blot检测细胞中PKM2水平,筛选干扰效果好的PKM2 siRNA2继续研究。噻唑蓝（MTT）检测细胞增殖,细胞克隆试验检测细胞克隆形成能力,流式细胞术检测细胞凋亡,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)法检测活性氧（ROS）水平,Western blot检测p38丝裂原活化蛋白激酶（p38MAPK）、磷酸化的p38MAPK（p-p38MAPK）、C-myc、β-连环蛋白（β-catenin）、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）蛋白水平。结果：细胞转染PKM2 siRNA1和PKM2 siRNA2后PKM2 mRNA和蛋白水平与没有转染的细胞相比均明显下降,并且转染PKM2 siRNA2后细胞中PKM2水平下降更多,而转染siRNA control的细胞中PKM2水平与没有转染的细胞相比没有明显变化。下调PKM2表达后的细胞凋亡率由（9.36±1.04）%升高至（48.42±5.28）%,细胞克隆形成率从（75.48±8.25）%降低至（46.15±3.47）%,细胞OD值从（0.86±0.11）下降至（0.52±0.04）,细胞中ROS水平升高,细胞中p-p38MAPK、Cleaved Caspase-3蛋白水平也明显升高,细胞中C-myc、β-catenin水平明显下降。结论：PKM2表达下调抑制鼻咽癌细胞生长,促进鼻咽癌细胞凋亡,作用机制可能与p38MAPK和Wnt/β-catenin信号通路有关。     

膜联蛋白A7低表达对人肝癌HepG2细胞增殖的影响

目的 探讨膜联蛋白A7低表达对人肝癌HepG2细胞的增殖产生的影响。方法 采用Western blotting法鉴定siRNA可有效抑制膜联蛋白A7表达,然后用脂质体转染法将siRNA转染入HepG2细胞,将细胞分为siRNA干扰组、阴性对照组和空白对照组,在转染后24h、48h、72h进行细胞计数以绘制细胞增殖曲线,转染后48h进行MTT实验以检测细胞增殖活力;免疫组织化学法检测cyclinD1和Ki67的表达,Western blotting法检测cyclinD1的表达情况。结果 转染了靶向膜联蛋白A7的siRNA后48h的HepG2细胞,细胞计数和MTT实验可见siRNA干扰组细胞增殖活力较阴性对照组和空白对照组显著降低（P<0.05）;cyclinD1和Ki67蛋白表达均为siRNA干扰组细胞表达显著低于两对照组（P<0.05）。 结论 膜联蛋白A7低表达可能对HepG2细胞的增殖具有一定的抑制作用。     

ClC-3氯离子通道对高分化鼻咽癌细胞突起形成的影响

目的：探讨ClC-3氯离子通道在高分化鼻咽癌CNE-1细胞突起形成中的作用。方法：采用免疫荧光技术（Alexa Fluor 488标记抗体）检测CNE-1细胞中ClC-3蛋白的分布；MQAE荧光探针检测细胞内氯离子浓度；吸附式单通道膜片钳技术记录氯离子单通道活动；采用FAM标记的siRNA转染细胞，实验分为对照组、阴性对照（NC） siRNA组和ClC-3 siRNA组；激光共聚焦显微镜观察转染后绿色荧光情况，流式细胞术检测转染效率，Western blot法测ClC-3蛋白的表达，倒置显微镜下观察CNE-1细胞突起形成情况。结果：ClC-3蛋白主要分布于细胞膜和细胞质，且在细胞膜突起形成部位明显聚集；细胞突起处MQAE荧光弱，氯离子浓度较高，而突起根部荧光强，氯离子浓度较低；突起根部记录到翻转电位为4.06 mV的单通道电流，在-120 mV钳制电压下，该通道电导约为78.4pS; siRNA转染细胞48 h后，ClC-3 siRNA组细胞ClC-3蛋白表达量显著降低（P<0.01），且ClC-3 siRNA组细胞无片状伪足伸出，而对照组和NC siRNA组细胞伸出明显的片状伪...     

聚乙烯亚胺-聚乙二醇-siRNA纳米复合物的制备和理化性质的研究

目的 探索聚乙烯亚胺-聚乙二醇(PEI-PEG)-siRNA纳米复合物的制备方法及其理化性质,以提高siRNA的细胞转染率.方法 设计合成了PEI-PEG共聚物基因载体,使其与针对细胞表面受体CD44v6的siRNA形成纳米复合物.通过粒径与电位测定、凝胶阻滞电泳、扫描电镜、流式细胞仪测定等方法,观察不同N/P比的纳米复合物的复合效果、表面形态和大小、基因转染率等.结果 电镜下纳米复合物呈近球形、大小较一致、分散良好的纳米颗粒.N/P=5、10时复合物粒径分别为(174.6±1.2)nm,(267.7±1.8)nm.当N/P超过10时纳米复合物粒径减小,zeta电位为正值且增大.此时,siRNA被PEI-PEG完全复合,产生一种荧光淬灭作用.流式细胞仪结果表明纳米复合物的基因转染率随着N/P的增加而增大.当N/P比为30时,其转染率为(75.6±9.2)%.结论 PEI-PEG是一种有潜力的阳离子基因载体,它的制备为下一步体外实验及动物实验提供了条件.     

Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons

Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25 kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons.     

Bone site-specific delivery of siRNA

Small interfering RNAs（siRNA） have enormous potential as therapeutics to target and treat various bone disorders such as osteoporosis and cancer bone metastases.However,effective and specific delivery of siRNA therapeutics to bone and bone-specific cells in vivo is very challenging.To realize the full therapeutic potential of siRNA in treating bone disorders,a safe and efficient,tissue- and cell-specific delivery system must be developed.This review focuses on recent advances in bone site-specific delivery of siRNA at the tissue or cellular level.Bone-targeted nanoparticulate siRNA carriers and various bone-targeted moieties such as bisphosphonates,oligopeptides（Asp）₈ and（AspSerSer）₆,and aptamers are highlighted.Incorporation of these bone-seeking targeting moieties into siRNA carriers allows for recognition of different sub-tissue functional domains of bone and also specific cell types residing in bone tissue.It also provides a means for bone-formation surface-,bone-resorption surface-,or osteoblastspecific targeting and transportation of siRNA therapeutics.The discussion mainly focuses on systemic and local bone-specific delivery of siRNA in osteoporosis and bone metastasis preclinical models.     

透明质酸偶联壳聚糖微球对非小细胞肺癌的靶向性作用

背景：部分肿瘤细胞表面的CD44受体表达上调,如大肠癌、非小细胞肺癌,提示透明质酸与CD44受体在肿瘤的生长与扩散有着一定的关系。 目的：观察透明质酸偶联壳聚糖纳米微球对非小细胞肺癌的靶向性。 方法：采用离子交联法制备负载多西紫杉醇的透明质酸偶联壳聚糖微球(DTX-HACTNPs),电镜观察其形态学特征,激光粒度分析仪测定粒径大小及分布。FITC标记微球,作用于CD44+人非小细胞肺癌细胞株(A549),荧光显微镜观察。MTT法检测载药微球的体外细胞毒性。 结果与结论：DTX-HACTNPs形态规则,粒径分布较为均匀,平均粒径为(228.0±2.6) nm。DTX-HACTNPs对A549细胞的杀伤力高于非透明质酸偶联载药微球,但仍低于普通注射用DTX,3者的半数抑制浓度(IC50)分别为(15.06±0.94),(25.73±3.37),(5.35±0.61) mg/L(F=73.871,P=0.000)。提示HACTNPs通过受体途径主动靶向性结合于非小细胞肺癌细胞,可提高化疗药对肿瘤细胞的选择性杀伤力。     

Targeted delivery of siRNA

Therapeutic application of siRNA requires delivery to the correct intracellular location, to interact with the RNAi machinery within the target cell, within the target tissue responsible for the pathology. Each of these levels of targeting poses a significant barrier. To overcome these barriers several strategies have been developed, such as chemical modifications of siRNA, viral nucleic acid delivery systems, and nonviral nucleic acid delivery systems. Here, we discuss progress that has been made to improve targeted delivery of siRNA in vivo for each of these strategies.     

Poly(beta-amino ester) as a carrier for si/shRNA delivery in lung cancer cells

Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNA interference (RNAi) studies. In the present study, we evaluated biodegradable poly(beta-amino ester) (PAE) carrier composed of low molecular weight polyethylenimine and poly(ethylene glycol) for si/shRNA delivery in lung cancer cells. PAE carrier successfully delivered EGFP (enhanced green fluorescence protein) siRNA (siGFP) and silenced EGFP expression. The silencing achieved with PAE carrier was found to be nearly 1.5 times superior and safer than standard PEI25K. Also, our PAE carrier exhibited superior Akt1 shRNA delivery (shAkt) and thereby silenced oncoprotein Akt1 efficiently. PAE-shAkt mediated Akt1 knock-down hindered cancer cell growth in Akt1 specific manner. Superior shAkt delivery and low cytotoxicity of PAE carrier promoted Akt1 knock-down specific apoptosis, while low delivery efficiency and high cytotoxicity of PEI25K carrier mainly exhibited undesirable necrosis. Moreover, basic cancer properties like cell proliferation, malignancy and metastasis were reduced more efficiently using PAE-shAkt system. These findings demonstrated the potential of PAE as an alternative to PEI25K in si/shRNA-based RNAi studies.     

RNA干扰沉默增殖诱导配体基因对结肠癌细胞周期的影响

目的 探讨沉默增殖诱导配体(a proliferation-inducing ligand,APRIL)基因对人结肠癌SW480细胞增殖及细胞周期的影响.方法 将APRIL基因的小干扰RNA质粒载体(siRNA-APRIL)转染结肠癌SW480细胞株,以非特异性序列载体转染组(nontargeting control)及未转染组(nontransfected control)作为对照.Real-time PCR和Western blot评价APRIL沉默效率;CCK-8(cell counting kit-8)法检测细胞增殖情况;流式细胞术检测细胞周期变化;RT-PCR检测细胞周期调控基因p21及p27的表达.结果 与非特异性序列载体转染组及未转染组相比,siRNA-APRIL显著抑制APRIL mRNA及蛋白的表达(P＜0.05);siRNA-APRIL转染SW480细胞48 h、72 h和96 h后细胞增殖能力明显下降(P＜0.05);转染48 h,siRNA-APRIL组G0/G1期细胞比例增高,S期及G2/M期细胞比例减少,细胞凋亡的数量增加,同时p21及p27 mRNA的表达上调(P＜0.05);而上述指标两对照组之间比较差异无统计学意义(P＞0.05).结论 siRNA-APRIL能特异性抑制结肠癌SW480细胞APRIL的表达,并抑制细胞增殖,使细胞周期出现G0/G1期阻滞,其机制可能与上调p21和p27的表达有关.     

Genetically modified Tomato aspermy virus 2b protein as a tumor-targeting siRNA delivery carrier

In nature, there exist a wide range of dsRNA-binding proteins that have different binding modes for small interfering RNA (siRNA) as well as structural differences, and some of these proteins have potential as effective siRNA delivery carriers. In order to deliver siRNA into cancer cells, a dsRNA-binding 2b protein derived from Tomato aspermy virus was genetically modified by fusing the integrin-targeting RGD peptide to its C-terminus, and biosynthesized. The resulting 2b-RGD protein possesses distinct characteristics favorable for biomedical applications of siRNA: (i) high affinity for siRNA, (ii) siRNA protection against RNases in serum, (iii) low cytotoxicity compared to the polycationic polymers often employed in conventional siRNA carriers, (iv) specific binding to integrins on cancer cells, and the ability to pass through the cell membrane via endocytosis, and (v) the ability to facilitate cytosolic release of siRNA. Here, we demonstrate that the 2b-RGD/siRNA complexes have great potential as a tumor-targeting siRNA delivery carrier and suggest their possible therapeutic applications for cancer treatment.