RGDyC与PEG共修饰的PAMAM树状大分子载三氧化二砷脑胶质瘤靶向递药系统的制备及体外评价

三氧化二砷(ATO)是中药砒霜的有效成分,已有研究表明其对多种肿瘤具有良好的生长抑制及促凋亡作用,但由于毒性大和透血脑屏障(BBB)难等问题严重限制了其在脑胶质瘤治疗方面的应用。聚酰胺-胺树状大分子(PAMAM)作为一种人工合成的高分子化合物,具有渗透性及稳定性强且生物相容性好等优点,且第5代PAMAM的空间结构更立体,是药物载体的理想选择。该课题采用RGDyC和PEG共同修饰第5代PAMAM,并通过核磁共振氢谱图证明了该纳米载体的成功合成;纳米粒度-电位分析仪和透射电镜图分析显示其平均粒径大约集中在20 nm左右;体外释放实验表明,该递药系统不仅具有缓释效果,同时还有一定的p H敏感特性;细胞结果显示,经RGDyC和PEG共修饰后的PAMAM与未经修饰组相比,细胞毒性显著降低,且该递药系统具有更好的体外跨血脑屏障(BBB)抗肿瘤效果,同时也进一步证明了RGDyC的肿瘤靶向作用。     

叶酸修饰壳聚糖季铵盐载基因纳米复合物的制备及体外转染效果的研究

 目的 合成水溶性阳离子聚合物壳聚糖季铵盐,通过叶酸修饰使其具备主动靶向性,并以此作为基因载体进行研究。方法 利用双功能聚乙二醇将叶酸连接到壳聚糖季铵盐(CSTM)上,以自组装法制备壳聚糖季铵盐-g-聚乙二醇-叶酸（CSTM-g-PEG-FA）/pDNA纳米复合物,并考察其在293T细胞中的转染效率。结果 当壳聚糖季铵盐-g-聚乙二醇-叶酸/pDNA纳米复合物质量比大于等于5时,pDNA能被完全包裹;细胞转染实验中,壳聚糖季铵盐-g-聚乙二醇-叶酸/pDNA纳米复合物的转染效率要明显高于壳聚糖季铵盐/pDNA纳米复合物。结论 带正电荷的壳聚糖季铵盐-g-聚乙二醇-叶酸能够通过静电作用包裹pDNA,叶酸修饰使其具备了一定的主动靶向性。     

整合素受体介导的SiRNA和多柔比星靶向耐药肿瘤的研究

 目的 采用整合素受体介导的靶向载体输送系统实现RNA干扰技术和抗肿瘤药物对耐药肿瘤的联合治疗。方法 分别用静电复合法和硫酸铵梯度法制备载多药耐药基因（MDR1）相关的小干扰RNA（siRNA）的精氨酸-甘氨酸-天冬氨酸三肽（RGD）修饰阳离子脂质体复合物和载多柔比星（DOX）的RGD修饰长循环脂质体，采用激光共聚焦显微镜来观察siRNA和多柔比星的细胞摄取和细胞内分布情况；采用流式细胞术和SRB（磺酰罗丹明B）实验测定多柔比星的细胞累积量和多柔比星对人乳腺癌耐药细胞（MCF7/A）的毒性；采用活体成像技术考察了靶向和非靶向制剂输送siRNA到肿瘤模型裸鼠体内的组织分布情况。结果 所制备的各种脂质体的平均粒径均在200 nm以内；与细胞孵育6 h后，观察发现siRNA和多柔比星各自主要分布在细胞质和细胞核中，与非靶向组相比，靶向脂质体组细胞摄取siRNA更快更多，且有利于siRNA在细胞质中均匀分布；细胞毒实验结果证实，1%摩尔比RGD修饰的阳离子脂质体对siRNA的转染效果最好，流式细胞术实验结果也同样证实RGD修饰脂质体组细胞中多柔比星累积量更高；活体成像结果显示荧光标记的siRNA经靶向脂质体输送后主要集中在肿瘤且比非靶向组蓄积量更高。结论 采用RGD修饰脂质体载运技术可将MDR1 siRNA和多柔比星两种药物同位点输送到肿瘤部位提高靶向性，并有利于改善多药耐药肿瘤的治疗效果。     

通关藤提取物通过调控上皮间充质转化抑制前列腺癌细胞的侵袭及迁移

目的探讨通关藤提取物对前列腺癌细胞增殖、侵袭及迁移的影响,及其对上皮间充质转化（EMT）的作 用机制。方法采用CCK8 法检测通关藤提取物对前列腺癌细胞DU145、PC3、RM-1 和LNCaP 增殖的影响; 采用划痕实验和Transwell 法检测DU145、PC3 细胞迁移和侵袭情况;采用光学显微镜观察细胞形态学变化, 并用Western Blot 法检测EMT 相关核心蛋白E-钙黏蛋白（E-Cadherin）、β-连环蛋白（β-Catenin）、波形蛋白 （Vimentin）的表达。结果通关藤提取物可以明显抑制PC3、DU145、RM-1 和LNCaP 细胞的增殖,并呈现一 定的时间和浓度依赖性,在作用72 h 后,通关藤提取物对PC3、DU145、RM-1 和LNCaP 细胞的半数抑制浓 度（IC50）值分别为53.45、57.22、44.82、50.95 mg·mL-1。与对照组比较,通关藤提取物（30、60 mg·mL-1）可以明 显抑制DU145 和PC3 细胞的迁移及侵袭能力（P < 0.05,P < 0.01,P < 0.001）。通关藤提取物干预后, DU145、PC3 细胞的形态发生了明显改变,与对照组比较,EMT 标志物Vimentin 蛋白表达明显下调（P < 0.05,P < 0.01）。结论通关藤提取物能够抑制前列腺癌细胞的增殖、迁移和侵袭,可能与其下调Vimentin 蛋 白表达,进而抑制EMT 有关。     

反义寡核苷酸/司盘修饰阳离子脂质体复合物的制备和特性

 目的 通过采用司盘系列表面活性剂对阳离子脂质体进行修饰,以提高基因传递效率,寻找高效的基因载体。方法 采用逆相蒸发法制备司盘阳离子脂质体。通过静电作用,得到反义寡核苷酸／阳离子脂质体自组装复合物,并运用电镜、激光 粒度及Zeta电位测定、琼脂糖电泳和荧光光谱等技术手段对司盘阳离子脂质体和自组装复合物进行了物理表征。并在体外 培养细胞系——COS7和RKO进行了摄取实验,对此载体进行生物学评价。结果 体外细胞摄取实验表明,司盘阳离子脂质 体与普通阳离子脂质体相比,可提高细胞对反义寡核苷酸的摄取,司盘40修饰阳离子脂质体效果最为显著。结论 采用表面 活性剂司盘修饰是提高基因传递效率切实可行的方法。表面活性剂司盘修饰阳离子脂质体为颇具发展潜力的非病毒载体。     

聚酰胺-胺结构修饰及在药物传递研究中的应用进展

 目的 综述聚酰胺-胺[poly(amidoamine)，PAMAM]修饰方法及其在药物研究中的应用进展。方法 根据近年来国内外文献资料，对不同官能团结构修饰PAMAM结构的研究进行归纳、分析和总结，并对其修饰后在药物、基因传递研究中的应用进行探讨。结果与结论 采用不同官能团或功能材料对PAMAM的结构进行修饰后，可一定程度的改善PAMAM的生物膜转运特性、降低其毒性、增加其靶向性等，经过不同结构修饰后的PAMAM，作为药物或基因传递系统载体具有很好的应用前景。     

甘草次酸修饰细菌纤维素包载紫杉醇口服胶束的构建与评价

目的 构建一种以肝靶向分子甘草次酸（glycyrrhetinic acid,GA）修饰的细菌纤维素（bacterial cellulose,BC）两亲性纳米载体用于紫杉醇（paclitaxel,PTX）口服给药。方法 以丁二酸酐（succinic anhydride,SA）作为连接臂,将甘草次酸与BC进行偶连,获得GA-BC）,采用红外光谱法、核磁共振氢谱法对合成产物进行结构表征。通过超声法制备GA-BC- PTX载药胶束,再通过粒径、ζ电位、透射电子显微镜及临界胶束浓度的测定进行表征,采用稳定性实验、体外释放实验、MTT、细胞摄取、小鼠活体成像实验及斑马鱼安全性实验对该载药系统进行评价。结果 成功构建GA-BC载体,经过红外光谱、核磁共振氢谱等检测证明偶连成功,GA-BC-PTX载药胶束平均粒径为（292.4±3.7）nm,表面电位为（−16.8±0.9）mV,载药量为（17.89±0.61）%,包封率为（59.81±0.73）%,临界胶束质量浓度为0.063 mg/mL,呈均一球形,制备的载体稳定性较好,体外释放实验表明该载体能在胃肠道环境稳定存在,MTT实验表明GA-BC-PTX胶束对HepG2细胞存在明显抑制作用,并通过共聚焦显微镜观察流式细胞仪分析证明HepG2细胞对GA-BC胶束载尼罗红的摄取明显高于游离的尼罗红。通过对小鼠活体成像证明GA-BC聚合物胶束具有肝靶向作用。通过斑马鱼卵安全性检测证明当GA-BC载体质量浓度小于2 mg/mL时,聚合物载体基本不具有生物毒性。结论 GA-BC载体具有良好的生物安全性和肝靶向作用,制备GA-BC- PTX口服载药胶束可以有效抑制肝肿瘤细胞HepG2的生长,为肝癌的靶向治疗提供新的思路。     

转铁蛋白修饰的姜黄素脂质体的制备及体外抗肿瘤活性

目的 制备转铁蛋白（transferrin,Tf）修饰的姜黄素（curcumin,Cur）脂质体（Tf-Cur-Lip）,并研究其理化性质和体外抗肿瘤活性。方法 采用薄膜分散法制备Tf-Cur-Lip,对其形态、粒径、多分散性指数、包封率、载药量、体外释放等进行表征;以人前列腺癌DU154细胞为模型进行细胞抑制率实验与细胞摄取实验。结果 所制得的Tf-Cur-Lip呈类球形,大小分布均匀,平均粒径为（68.27±0.36）nm,多分散指数为0.194±0.050,平均包封率与载药量分别为（98.13±0.38）%与（2.94±0.38）%。体外释放研究表明,Tf-Cur-Lip相比游离姜黄素具有一定缓释效果。MTT实验结果表明Tf-Cur-Lip对DU145细胞的抑制作用显著高于游离姜黄素,与非靶向姜黄素脂质体（Cur-Lip）相比,转铁蛋白的修饰明显增加了DU145细胞对脂质体的摄取效率。结论 成功制备了Tf-Cur-Lip,该脂质体能够提高肿瘤靶向性,具有一定的抗肿瘤活性。     

聚乙二醇修饰的聚酰胺-胺-甲氨蝶呤分子复合物的制备及体外释药研究

 目的分别制备了聚乙二醇(PEG)修饰的第四代(G4)和第五代(G5)聚酰胺-胺(PAMAM)树枝状大分子-甲氨蝶呤(MTX)复合物,考察其体外释药特性。方法通过酰胺键将功能化PEG与PAMAM表面氨基连接,考察PEG化PAMAM的溶血毒性;制备PAMAM-PEG/MTX复合物,测定最大复合量;考察复合物在不同缓冲溶液及血浆中的体外释药行为及不同储存条件下的稳定性。结果通过PEG化修饰,每分子G4 PAMAM分别连接了11,21和29个PEG分子;每分子G5 PAMAM分别连接了16,30和37个PEG分子。随着PAMAM的PEG化程度提高,其溶血毒性显著减小,复合MTX的量增多。PAMAM-PEG/MTX在10和1mmol·L^-1(pH 7.4)的Tris-HCl缓冲溶液中表现出缓释效果,但加入Nacl后释放加快。放置3个月后,复合物中MTX含量略有下降,药物释放未发生变化。结论与PAMAM相比,PAMAM-PEG的溶血毒性显著降低,并具有一定缓释效果,有望成为一种新型给药载体材料。     

靶向VEGF—hTERT基因siRNA表达载体抑骨肉瘤生长的实验研究

目的：观察靶向VEGF-hTERT基因siRNA表达载体对骨肉瘤细胞增殖的影响和对裸鼠骨肉瘤生长的影响。方法：构RNA干扰腺病毒载体rA-hTERT和rAd-VEGF,单独和联合转染人骨肉瘤细胞系MG-63细胞后实时荧光定量PCR检测hTERT和VEGF基因表达情况,流式细胞仪检测各组细胞凋亡率;人骨肉瘤细胞系MG-63细胞于裸鼠成瘤后使用siRNA表达载体治疗,观察肿瘤的生长状况及组织病理学改变。结果：①转染后48h,rAd-hTERT对hTERT基因mRNA的抑制率约84%,对VEGF基因表达无明显抑制（P〉0.05）;rAd-VEGF对VEGF基因mRNA的抑制率约82%,对hTERT基因表达无明显抑制（P〉0.05）;rAd-hTERT-VEGF干扰组对hTERT基因表达抑制率约87%,VEGF基因表达抑制率约83%,联合干扰与单基因干扰相比,对hTERT-VEGF基因的表达抑制差异无显著性（P〉0.05）。②干扰组转染后l～6d均能促进细胞凋亡,联合干扰细胞凋亡率与单基因干扰细胞凋亡率相比,差异均有显著性（P〈0.05）。裸鼠实验中,基因组肿瘤的生长均受到明显抑制,且联合干扰组与单基因组比较有显著性差异（P〈0.05）。对骨肉瘤的肺转移联合干扰组有显著的抑制作用。结论：靶向VEGF-hTERT基因siRNA表达载体对骨肉瘤细胞系MG-63细胞和裸鼠骨肉瘤的生长及转移均有明显的抑制作用。     

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??OBJECTIVE To identify the possibility of branched polyethyleneimine (BPEI), polyamidoamine dendrimers (PAMAM) and linear polyethylenimine (LPEI) as gene vector for miRNA-15a and miRNA-16-1 gene therapy in prostate cancer. METHODS The diameter and Zeta potential of complex were measured by Zetasizer Nano-ZS9, and its stability was also observed. The efficiency of transfection in vitro was detected by flow cytometer and the positively transfected cells were detected by fluorescence microscope. The inhibition of complex on LNCaP cell was determined by CCK8 assay. The expression of protein BCL-2, CCDN1 and Wnt3a were detected by Western blot method. RESULTS BPEI, PAMAM and LPEI with miRNA formed nano-sized particles. The transfection rate of BPEI/miRNA-CY3 was significantly higher than that of PAMAM/miRNA-CY3 and LPEI/miRNA-CY3 at N/P ratio (P<0.01). The protein level of BCL-2, CCDN1 and Wnt3a in BPEI, PAMAM and LPEI were all lower than the control group in LNCaP cells. CONCLUSION With good stability and transfection rate, BPEI/miRNA, PAMAM/miRNA and LPEI/miRNA can be a promising nano-vector of miRNA transfer system. The complex can inhibit the expression of BCL-2, CCDN1 and Wnt3a gene specifically and efficiently, which may be used for prostate cancer treament.
     

补骨脂酚拮抗AR转录活性抑制雄激素诱导的前列腺癌细胞LNCaP的增殖

[目的]研究补骨脂酚对雄激素受体(AR)的亲和性和转录活性的调控在雄激素依赖的前列腺癌细胞LNCaP中的作用。[方法]用AR竞争性结合试剂盒检测补骨脂酚结合AR的能力;用荧光素酶报告质粒法检测补骨脂酚对睾酮诱导的AR转录活性的影响;用实时定量PCR法检测补骨脂酚对LNCaP细胞中雄激素应答的靶基因——前列腺特异抗原(PSA)表达的影响;用MTT法检测补骨脂酚对睾酮诱导的LNCaP细胞增殖的影响。[结果]补骨脂酚体外结合AR的能力与AR拮抗剂氟他胺相当;睾酮诱导的AR转录活性可以被补骨脂酚阻断;补骨脂酚可以抑制睾酮对LNCaP细胞PSA表达和增殖的上调作用。[结论]补骨脂酚经由AR抑制雄激素依赖的LNCaP细胞的增殖和基因表达,提示:补骨脂酚可以作为潜在的AR拮抗剂,用于雄激素依赖的PCa药物的开发。     

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??OBJECTIVE To study the effect of different graft ratios of PEG on the toxicity in vitro and cellular uptake of PAMAM G5 dendrimers. METHODS Nuclear magnetic resonance (¹H-NMR) and Fourier transform infrared (FT-IR) spectroscopy were used to confirm the structure of PEG-PAMAM G5 dendrimers with four different graft ratios. The particle size and Zeta potential of the nanoparticles were determined by nanoparticle size-Zeta potential analyzer.The toxicity in vitro, cellular uptake, and intracellular localization were tested by hemolysis assay, cytotoxicity assay, cellular uptake test, and laser scanning confocal microscope images, respectively. RESULTS The particle sizes of dendrimers with PEG graft ratios of 7.8%, 14.1%, 20.3%, and 24.2% were (17.05??1.77), (20.77??1.02), (21.68??1.04), and (23.19??0.54)nm, respectively.The Zeta potential decreased from (25.57??1.37) mV of PAMAM G5 to (9.27??0.40) mV of PEG₃₁-PAMAM G5. In addition, the hemolytic toxicity and cytotoxicity of PAMAM G5 dendrimers also markedly decreased especially at high concentrations because of PEG modification. Moreover, the PEG-PAMAM G5 dendrimers with particle diameter of nearly 20 nm not only could be taken in by HBMEC cells, but also accumulated in the cell nucleus.CONCLUSION Modification of PEG can greatly reduce the toxicity of PAMAM G5 dendrimers in vitro, and the higher the degree of modification, the more obvious is the attenuated effect.The PEG-PAMAM G5 dendrimers with particle diameter larger than 20 nm still can be taken in by HBMEC cells and accumulate in the cell nucleus, which provide a foundation for the further research using modified PEG-PAMAM G5 as a basic carrier for genes and nuclear targeting agents in nano medicine.     

载三氧化二砷脑胶质瘤靶向纳米递药系统i RGD/TGN-PEG-PAMAM/ATO的构建及体外研究

目的以树状大分子为载体材料并修饰血脑屏障(blood brain barrier,BBB)靶向短肽TGN和肿瘤靶向短肽i RGD构建脑胶质瘤靶向递药系统(i RGD/TGN-PEG-PAMAM/ATO),旨在解决三氧化二砷(arsenictrioxide,As2O3,ATO)在治疗脑胶质瘤过程中分布缺乏特异性、透BBB难等问题,使其具有更好抗脑胶质瘤作用。方法核磁共振图谱(1H-NMR)、透射电子显微镜(TEM)等考察载体的理化性质;电感耦合等离子发射光谱(ICP)、透析袋法分析其包封率及体外释放情况;通过激光共聚焦及流式细胞仪分析i RGD、TGN对细胞摄取的影响;MTT法考察纳米载体对脑微血管内皮细胞(HBMEC)和脑胶质瘤U87细胞的毒性及BBB模型中递药系统抑制U87细胞生长的情况。结果成功合成了i RGD/TGN-PEG-PAMAM载体,其形态规整,大小均匀,测得其粒径(24.87±0.84)nm,电位(17.26±1.64)m V;该载体对HBMEC和U87细胞均具有较小的毒性;递药系统i RGD/TGN-PEG-PAMAM/ATO的包封率为(71.92±1.17)%,体外释放表明ATO经载体包载后呈现一种缓慢释放趋势,且在酸性条件下更有利于ATO的释放;细胞摄取结果提示iRGD/TGN的修饰有利于U87细胞对递药系统的摄取;体外跨BBB抑制U87细胞生长实验结果表明,i RGD/TGN-PEG-PAMAM/ATO组具有更好的跨BBB抑制U87细胞生长效果。结论 i RGD/TGN-PEG-PAMAM/ATO脑胶质瘤靶向递药系统具有较好的体外跨BBB抑制U87细胞生长的效果,为脑胶质瘤治疗提供了新的策略。     

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

ObjectiveMelittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.MethodsThe melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.ResultsrAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.ConclusionPre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.     

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

ObjectivePlant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.MethodsA miR122 target (122T) sequence was incorporated into the 3′ untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.ResultsThe presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.ConclusionHEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.     

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

OBJECTIVE: Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine(TCM) drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus(r AAV) vectors has not been attempted. METHODS: We synthesized the c DNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged c DNAs were subcloned into a r AAV plasmid vector. The protein expression was confi rmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into r AAV vectors, under the control of a liver cancer-specifi c promoter. The liver tumor growth was then monitored following intratumor administration of the r AAV vectors.RESULTS: The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin(TCS) gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of r AAV vectors containing the TCS gene signifi cantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.CONCLUSION: Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.     

巨噬细胞膜仿生白蛋白纳米体系的构建及其胶质瘤靶向递药性能评价

目的 构建包载WEE1激酶抑制剂adavosertib的巨噬细胞膜仿生白蛋白纳米粒(MM-BSA/Ada),体外评估其作为胶质瘤靶向递药体系的可行性。方法 制备MM-BSA/Ada并筛选最佳膜-核比和最佳药-载比,检测其载体安全性和对C6胶质瘤细胞抗增殖活性,考察其体外细胞摄取、跨血脑屏障转运和跨膜后摄取的能力。结果 MM-BSA/Ada具有良好的稳定性,CCK-8结果初步显示,未载药纳米粒在体外细胞实验中对脑血管内皮细胞呈低毒性;与未包膜纳米粒和游离药物相比,MM-BSA/Ada给药后的体外抗胶质瘤细胞增殖活性(P＜0.001)、胶质瘤细胞摄取量(P＜0.001)、体外血脑屏障透过量(P＜0.01)及跨膜后摄取量(P＜0.001)均显著提高。结论 MM-BSA/Ada有较好的胶质瘤靶向递药性能,有望为胶质瘤提供新的放射增敏策略。     

Blockade of Androgen Markers Using a Novel Betasitosterol,Thioctic Acid and Carnitine‐containing Compound in Prostate and Hair Follicle Cell‐based Assays

Androgenetic alopecia (AGA) affects approximately 70% of men and 40% of women in an age‐dependent manner and is partially mediated by androgen hormones. Benign prostatic hyperplasia (BPH) similarly affects 50% of the male population, rising by 10% each decade. Finasteride inhibits 5‐alpha reductase (5AR) and is used to treat both disorders, despite offering limited clinical benefits accompanied by significant adverse side effects. Building on our previous work demonstrating the efficacy of naturally derived 5AR inhibitors (such as stigmasterol and beta sitosterol), we hypothesize that targeting 5AR as well as inflammatory pathways may yield improved efficacy in AGA and BPH. Here we address these dual pathomechanisms by examining the potency of a novel composition using in vitro assays of representative cell lines for AGA (hair follicle dermal papilla cells) and BPH (LNCaP prostate cells), respectively. Exposure of cells to the novel test composition down‐regulated mRNA expression profiles characteristic of both disease processes, which outperformed finasteride. Changes in mRNA expression were corroborated at the protein level as assessed by western blotting. These studies provide proof of concept that novel, naturally derived compositions simultaneously targeting 5AR and inflammatory mediators may represent a rational approach to treating AGA and BPH. Copyright © 2016 John Wiley & Sons, Ltd.