纳米纤维介导的紫杉醇/阿霉素序贯联合治疗SHg-44胶质瘤

背景：纳米纤维技术可同时担载多种药物,避开血脑屏障限制实现脑胶质瘤的序贯联合化疗。 目的：用乳液电纺法制备同时担载紫杉醇和阿霉素的聚乙二醇-聚乳酸共聚物纳米纤维并实现两种药物的序贯释放,探讨纳米纤维介导的紫杉醇和阿霉素序贯联合治疗SHg-44胶质瘤的效果及机制。 方法:实验分为4组,1640培养液对照组,1%阿霉素组,1%紫杉醇组,5%(阿霉素+紫杉醇)组。采用高效液相色谱法测定紫杉醇和阿霉素的体外释放情况。四甲基偶氮唑盐法检测纳米纤维介导的紫杉醇和阿霉素序贯治疗对SHg-44胶质瘤细胞的增殖抑制率;流式细胞仪检测法检测紫杉醇和阿霉素序贯治疗对SHg-44胶质瘤细胞的凋亡诱导作用。 结果与结论：纳米纤维介导的紫杉醇和阿霉素序贯治疗对SHg-44胶质瘤细胞具有明显的生长抑制及促凋亡作用,且作用效果好于单独药物应用。提示聚乙二醇-聚乳酸纳米纤维作为一种药物载体能提高紫杉醇和阿霉素对SHg-44胶质瘤细胞增殖抑制和诱导凋亡作用。     

升压联合化疗对大鼠脑胶质瘤中白介素-1β-转化酶活性变化的影响

目的:通过升压联合化疗方法,观察大鼠脑胶质瘤组织中白介素-1β-转化酶(ICE)活性的变化,说明凋亡基因ICE在胶质瘤治疗中所起的作用。方法:利用体外胶质瘤细胞培养,采用立体定位注射接种法,复制动物模型,通过变压化疗治疗大鼠脑胶质瘤,治疗前后作比较,观察肿瘤大小,大鼠生存期,脑血流及瘤组织ICE活性的变化。结果:升压联合化疗后,肿瘤局部脑血流和药物浓度均增加,大鼠生存期明显延长。联合化疗升压组瘤组织ICE活性高于未升压组,联合化疗组高于单化组及对照组,肿瘤体积变小。结论:升压联合化疗使大鼠脑胶质瘤组织ICE活性增加,从而促进肿瘤细胞凋亡.     

生物可降解高分子材料—聚酸酐

聚酸酐是80年代初美国麻省理工学院Langer [1]等发现的一类新型合成生物可降解高分子材料,由于其具有良好的生物相容性、表面溶蚀(surface erosion)降解性、降解速度可调及易加工性等优异性能[2,3],很快在医学前沿领域得到应用[4]。作为一类新型药物控释材料,前后历经近二十年的系统研究,于1996年获FDA批准应用于复发恶性脑胶质瘤的术后辅助化疗[5]。     

阿霉素性心力衰竭模型的氧化应激和凋亡机制

目的:观察阿霉素(ADR)复制慢性心力衰竭模型的心功能、氧化应激和心肌细胞凋亡发生情况。方法:TBA法测定丙二醛(MDA)含量、邻苯三酚法测定超氧化物歧化酶(SOD)活性动态变化,TUNEL法检测细胞凋亡,免疫组化检测P53蛋白含量,RT-PCR法检测p53mRNA表达。结果:ADR引起大鼠心功能显著降低的同时MDA含量显著增加,SOD活性显著下降,心肌细胞发生凋亡,心肌组织p53mRNA表达增加,P53蛋白合成明显增多。结论:阿霉素性心力衰竭模型有显著的氧化应激反应和细胞凋亡发生,p53基因在其凋亡发生机制中有一定作用。而且阿霉素性心力衰竭中心肌细胞凋亡的发生与氧化应激有密切关系。     

TNF-α在热疗降低胶质瘤侵袭性过程中的作用

 目的 探讨肿瘤坏死因子-α(TNF-α)在热疗抑制肿瘤侵袭性过程中的作用。方法 热处理大鼠恶性胶质瘤细胞(C6细胞)和胶质瘤大鼠后,放射免疫法监测培养液和脑胶质瘤组织内TNF-α的浓度;免疫组化法检测经热疗/ TNF-α/生理盐水处理过的胶质瘤组织内增殖细胞核抗原(PCNA)蛋白的表达。利用Transwell构建肿瘤侵袭模型,通过结晶紫染色法检测肿瘤侵袭性。电镜观察C6恶性胶质瘤大鼠肿瘤血管内皮细胞的凋亡。结果 热疗可增加C6细胞培养液和胶质瘤大鼠肿瘤组织内的TNF-α含量及降低胶质瘤侵袭性,均于热疗后120min时达高峰(P＜0.01)。热疗与TNF-α单独作用于胶质瘤大鼠后,均可引起胶质瘤大鼠肿瘤血管内皮细胞的凋亡。且TNF-α引起内皮细胞的凋亡水平与热处理后C6细胞培养液中TNF-α含量一致。结论 热疗可能是通过增加TNF-α引起肿瘤血管内皮细胞凋亡而抑制了肿瘤侵袭性。     

海藻酸盐控释微球的制备及其体外释药特性

研制白蛋白海藻酸钠(BSA-海藻酸钙微球)控释微球,并对其体外释药特性等进行考察,为应力控释VEGF促进组织工程骨血管化提供理论依据。以海藻酸钠为载体,采用W/O乳化-离子交联法制备BSA-海藻酸钙微球;检测粒径大小、外观、包封率等理化特性;考察微球的体外释药特性。微球球形圆整,分散性好,平均粒径为230±60μm,载药量达80.3μg/mg,包封率为61%;微球的体外释药速率平稳,周期达2周余。海藻酸钠可以作为蛋白、多肽类药物的可生物降解辅料;乳化离子交联法的制备工艺简便,有利于蛋白、多肽类药物结构和功能的稳定性并有效延长其作用时间。     

仙台病毒 Tianjin 株缺陷干扰颗粒体内外诱导大鼠脑胶质瘤细胞凋亡的研究

目的探讨仙台病毒Tianjin株缺损干扰颗粒（ defective interfering particles ,DI颗粒）体内外诱导大鼠脑胶质瘤细胞C6凋亡的作用。方法将不同滴度仙台病毒Tianjin株DI颗粒分别与大鼠脑胶质瘤细胞C6作用不同时间,以培养基作为阴性对照、完整病毒作为阳性对照,通过DNA片段琼脂糖凝胶电泳、TUNEL染色、AnnexinⅤ-FITC/PI标记流式细胞仪分析等检测细胞凋亡情况。建立大鼠皮下胶质瘤模型,通过测量肿瘤大小观察DI颗粒抑瘤作用,病理切片HE染色观察肿瘤组织病理变化,TUNEL法检测肿瘤组织细胞凋亡情况。结果 C6细胞在体外经DI颗粒诱导后, DNA片段琼脂糖凝胶电泳呈阶梯状;流式细胞仪及TUNEL检测显示,DI颗粒组与完整病毒组细胞凋亡率明显增高,且呈时间-剂量依赖型。动物实验结果显示,DI颗粒和完整病毒均可明显抑制肿瘤生长;肿瘤组织病理切片HE染色显示DI颗粒组和完整病毒组瘤结节内瘤细胞较少;TUNEL原位细胞凋亡检测显示DI颗粒组和完整病毒组凋亡细胞明显增加,以上结果与阴性对照组比较差异均有统计学意义（P＜0．01）。结论仙台病毒Tianjin株DI颗粒在体内外均能引起大鼠脑胶质瘤C6细胞凋亡,且呈时间-剂量依赖型,提示DI颗粒有辅助治疗脑胶质瘤的可能性。     

PLGA/O-CMC载药纳米微粒的体外降解及释药行为研究

本研究以聚乳酸-乙醇酸共聚物（PLGA）和自行制备的O-羧甲基壳聚糖（O-CMC）为原料，以5-氟尿嘧啶（5-FU）为抗癌药物模型，采用自身设计的改良复乳法制备了载药纳米微粒。微粒平均粒径为98.5nm，粒径分布指数为0.192，粒子表面∈电位为61．48eV，载药率高达18.9％。然后用SEM动态监测载药纳米粒子降解过程中表面形貌的变化，并连续追踪粒子降解过程中的质量损失和降解介质的pH变化。载药纳米粒子在PBS中的释药行为研究表明，（1）前12h的释药动力学符合Huguchi方程，具有一级释放特性；（2）在20d内的释药动力学符合零级释放特性。细胞凋亡实验结果表明载药纳米粒子对TJ905脑胶质瘤细胞增殖有明显的抑制作用。     

脑胶质瘤间质内化疗联合用药的敏感性研究

目的 观察卡铂（CBP）、威猛（Vm-26）、甲氨喋呤（MTX）及尼莫地平（NIM）联合应用对脑腔质瘤的抑制作用，探讨脑间质内联合用药与服质瘤细胞敏感性的关系，为临床脑胶质瘤间质内化疗提供有效的化疗依据。方法 40倒脑胶质瘤标母体外细胞培养，采用噻唑兰（MTT）比色法测定体外用药对脑肢质瘤的增殖抑制作用。光镜和电镜下观察药物作用的细胞形态学改变，并采用流式细胞术（FCM）测定药物在脑腔质瘤细胞周期中的分布厦细胞凋亡情况，比较脑胶质瘤细胞对CBP、Vm-26、MTX及NIM单独使用、联合使用的敏感性。结果CBP、Vm-26、MTX、NIM联合用药对肿瘤细胞的抑制率可连06．64％；明显高于CBP＋NIM（69.03％）、Vm-26＋NIM（71.53％）、MTX＋NIM（52.75％）及CBP＋Vm-26＋MTX（78．59％）的抑制效果（P〈0．01），同时CBP、Vm-26、MTX的浓度降低为单独用药的1／10～I／100；光镜和电镜检查发现，在药物作用下肢质瘤细胞形态发生明显改变；FCM检查结果表明，MTX＋CBP＋Vm-26＋NIM联合应用能显著诱导细胞发生凋亡，且主要作用于G2期、S期细胞。结论 联合应用CBP、Vm～26、MTX及NIM对肢质瘤细胞的敏感性明显优于单用药，具有抑制率高、用药浓度低的特点。药物对胶质瘤细胞各个周期均有杀伤力，以G2期和S细胞为主。     

残瘤腔间质内化疗治疗脑胶质瘤的临床研究

目的 探讨术中残瘤腔安置Ommaya贮液囊对脑胶质瘤患者术后间质内化疗(interstitial chemotherapty)的疗效。方法 在脑胶质瘤手术中尽可能切除肿瘤，于残瘤腔安置Ommaya贮液囊，术后定期行经贮液囊内注射化疗药VM-26。结果 手术切除后间质内化疗能提高患者的生存率和生存质量。结论 瘤腔间质内化疗方法简捷、费用低、全身毒副作用小，能有效改善患者的生存质量，是一种值得推广的方法。     

Scheduled eating increases dopamine release in the nucleus accumbens of food-deprived rats as assessed with on-line brain dialysis

This study examined the effect of scheduled eating on the in vivo release of dopamine (DA) in the nucleus accumbens of rats that were maintained on a food deprivation schedule. DA release was measured by means of a fully automated on-line brain dialysis system. The initiation of eating increased the release of DA, which remained elevated during the entire eating period. Termination of eating caused a gradual decrease of the release of DA to basal values. Increased motor activities did not change the release of DA. These results indicate a link between eating and DA release and demonstrate the suitability of on-line brain dialysis for behavioural experiments.     

The targeted delivery of anticancer drugs to brain glioma by PEGylated oxidized multi-walled carbon nanotubes modified with angiopep-2

In this study, a dual-targeting drug delivery system based on PEGylated oxidized multi-walled carbon nanotubes (O-MWNTs) modified with angiopep-2 (O-MWNTs-PEG-ANG) was successfully developed for treatment of brain glioma. O-MWNTs can not only distribute in brains but also accumulate in tumors, and have ultrahigh surface area with remarkably high loading anticancer drug of doxorubicin (DOX), which was selected as drug carrier. Angiopep-2 can specifically combine to the low-density lipoprotein receptor-related protein (LRP) receptor overexpressed on the blood-brain barrier (BBB) and glioma cells, which was selected as targeting ligand. The cooperative dual-targeting to brain glioma by O-MWNTs-PEG-ANG was evaluated by intracellular tracking in vitro and fluorescence imaging in vivo, which demonstrated that the combination of O-MWNTs-PEG and angiopep-2 constituted an ideal dual-targeting drug delivery system. The anti-glioma effect of DOX-loaded O-MWNTs-PEG-ANG (DOX-O-MWNTs-PEG-ANG) was assessed by C6 cytotoxicity and median survival time of glioma bearing mice, which showed a better anti-glioma effect than DOX. The biological safety of O-MWNTs-PEG-ANG was evaluated by BCEC and C6 cytotoxicity, hematology analysis and CD68 immunohistochemical analysis, which proved O-MWNTs-PEG-ANG was good biocompatibility and low toxicity. The biological safety of DOX-O-MWNTs-PEG-ANG was evaluated by histopathological analysis, which suggested a lower cardiac toxicity than DOX. In conclusion, O-MWNTs-PEG-ANG is a promising dual-targeting carrier to deliver DOX for the treatment of brain tumor.     

Paclitaxel delivery from PLGA foams for controlled release in post-surgical chemotherapy against glioblastoma multiforme

Paclitaxel loaded biodegradable poly-(dl-lactic-co-glycolic) acid (PLGA) foams with microporous matrix were fabricated by a novel pressure quenching approach to provide a sustained paclitaxel release. The foams with micropores provided increased surface area to volume ratio and were also implantable for post-surgical chemotherapy applications. The two formulations 5% (w/w) paclitaxel loaded PLGA 85:15 foam (F1) and 10% (w/w) paclitaxel loaded PLGA 50:50 foam (F2), were evaluated in vitro and in vivo. Both the foams were found to provide a paclitaxel release beyond a month in vitro with a near zero-order kinetics and with minimum burst release. Furthermore, apoptosis of C6 glioma cells in vitro demonstrated the benefits of sustained paclitaxel release by the foams in comparison to acute Taxol^® exposure. Both the foams exhibited continuous paclitaxel release in an in vivo (subcutaneous) environment up to a month which correlated well with the in vitro release profiles. Bio-distribution results in the rat brain showed paclitaxel penetration at therapeutic levels up to 3 mm into the tissue from the site of foam implantation. Hence these foams could be employed as potential implants for post-surgical chemotherapy against malignant glioma.     

Study and evaluation of mechanisms of dual targeting drug delivery system with tumor microenvironment assays compared with normal assays

A dual targeting delivery system was developed to completely conquer the two barriers that glioma treatment faces: the blood–brain barrier (BBB) and the brain–glioma barrier. Recently, a system comprising AS1411 aptamer (for glioma targeting) and TGN peptide (for BBB targeting) modified nanoparticles (AsTNPs) was developed, which can effectively target brain glioma and improve the survival of glioma-bearing mice. However, the in vitro models currently used are far too different from the in vivo tumor microenvironment that the glioma targeting delivery system actually faces. In this study, the pharmacology mechanisms of AsTNPs were explored in several models that imitated the tumor microenvironment. AsTNPs can be selectively taken up by endothelial and glioma cells, effectively penetrating the BBB and brain–glioma barriers to reach glioma cells and display their anti-glioma effect. The cell monolayers, tumor spheroids and coculture systems were more suitable in vitro models for the pharmacology evaluation of targeted drug delivery systems.     

An in vivo microdialysis study of light/dark-modulation of vitreal dopamine release in zebrafish

Dopamine (DA) is an important neuromodulator in the visual system. The release of DA in the retina largely depends on environmental lighting conditions. Most previous studies have assessed the effect of illumination on retinal DA or its metabolites using homogenates or in vitro preparations. This study was designed to investigate the effect of transitions between lighting conditions--from dark to steady or flickering light and vice versa--on retinal DA release in zebrafish using in vivo microdialysis. The transition from dark to flickering light increased DA release, whereas the transition from flickering light to dark decreased it. This latter effect depended on time of day within the light period, e.g., it was strongest in the late afternoon. When using steady light, none of these effects were seen. Our study also demonstrates that in vivo microdialysis can successfully be applied to the investigation of retinal DA release in zebrafish.     

Carbachol effect on carotid body dopamine in vitro release in response to hypoxia in adult and pup rabbit

Dopamine (DA) release from the adult carotid body (CB) is dependent, in part, upon CB cholinergic receptor stimulation. The aim of the present study was to determine the role of cholinergic stimulation on DA release from rabbit pup CB with reference to adult's. CBs sampled from adult (n = 52) and 10-day-old (n = 49) rabbits were incubated in vitro for 1 h in a surviving medium bubbled with either 100 or 8% O2 in N2, without (control) or in the presence of the cholinergic agonist carbachol 1 microM. In adults, DA released (DAr) in the medium was significantly larger with 1 microM carbachol compared with control in either 100 or 8% O(2) (P < 0.01). In pups, carbachol 1 microM had no effect in 100% O2 but significantly increased DAr compared with control in 8% O2 (P < 0.01). The data suggest that cholinergic mechanisms regulating DAr are not fully expressed in pup rabbit CBs, in contrast with adults and thus, exhibit maturation-related functional differences.     

Dopamine release in the nucleus accumbens of freely moving rats determined by on-line dialysis: effects of apomorphine and the neuroleptic-like peptide desenkephalin-gamma-endorphin

This study examined the effects of apomorphine, sulpiride, desenkephalin-gamma-endorphin (DE gamma E) and a combination of DE gamma E with apomorphine on the release of dopamine (DA) and its main metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens of freely moving rats. A fully automated on-line brain dialysis system was used. A small dose of s.c. administered apomorphine induced a decrease in the output of DA and DOPAC. Sulpiride, infused into the nucleus accumbens, induced a 2-fold increase in the output of DA, DOPAC and HVA. DE gamma E hardly modified either the basal release of DA, DOPAC and HVA or the apomorphine-induced attenuation of the release of DA and DOPAC. These results indicate a dissociation between the behavioural effects of DE gamma E and its effect on the release of DA in vivo.     

Ascorbic acid does not modulate stimulated dopamine release: In vivo voltammetric data in the rat

Electrical stimulation of the nigrostriatal pathway released dopamine (DA) in the striatum of the anaesthetized rat. The level of DA released by 10-s stimulus trains was measured by high-speed cyclic voltammetry. Metoclopramide (10 mg/kg) increased DA release by 20%. Apomorphine (1.76 mg/kg) caused a 40% decrease in release which was blocked by metoclopramide. Ascorbate (1.76 g/kg) had no effect on stimulated DA release. Furthermore, pretreatment of rats with ascorbate trebled the striatal extracellular ascorbate level, but failed to modify the effects of metoclopramide and apomorphine on DA release. We conclude that ascorbate has no effect on the presynaptic autoreceptors that modulate striatal DA release in vivo.     

升压联合化疗对大鼠脑胶质瘤形态学变化的影响

目的:通过体外细胞化疗和体内对大鼠胶质瘤升压联合化疗后, 观察体外细胞形态变化和体内肿瘤抑制作用, 观察变压联合化疗对胶质瘤的治疗效果。方法:采用胶质瘤体外培养、化疗。体内采用脑定位注射接种法, 复制大鼠脑胶质瘤动物模型。体内进行联合升压化疗。结果: 体外培养瘤细胞, 使用化疗药后, 可见细胞体积增大、胞质呈颗粒样变、胞内容物外溢等形态学变化;体内化疗后肿瘤体积变小, 瘤内可见坏死区等病理变化;升压化疗组肿瘤内血流增加、药物浓度增加;动物生存期延长。 结论:升压联合化疗对大鼠脑胶质瘤具有明显抑制作用, 可使动物生存期延长。     

Intracranial MEMS based temozolomide delivery in a 9L rat gliosarcoma model

Primary malignant brain tumors (BT) are the most common and aggressive malignant brain tumor. Treatment of BTs is a daunting task with median survival just at 21 months. Methods of localized delivery have achieved success in treating BT by circumventing the blood brain barrier and achieving high concentrations of therapeutic within the tumor. The capabilities of localized delivery can be enhanced by utilizing mirco-electro-mechanical systems (MEMS) technology to deliver drugs with precise temporal control over release kinetics. An intracranial MEMS based device was developed to deliver the clinically utilized chemotherapeutic temozolomide (TMZ) in a rodent glioma model. The device is a liquid crystalline polymer reservoir, capped by a MEMS microchip. The microchip contains three nitride membranes that can be independently ruptured at any point during or after implantation. The kinetics of TMZ release were validated and quantified in?vitro. The safety of implanting the device intracranially was confirmed with preliminary in?vivo studies. The impact of TMZ release kinetics was investigated by conducting in?vivo studies that compared the effects of drug release rates and timing on animal survival. TMZ delivered from the device was effective at prolonging animal survival in a 9L rodent glioma model. Immunohistological analysis confirmed that TMZ was released in a viable, cytotoxic form. The results from the in?vivo efficacy studies indicate that early, rapid delivery of TMZ from the device results in the most prolonged animal survival. The ability to actively control the rate and timing of drug(s) release holds tremendous potential for the treatment of BTs and related diseases.