八聚精氨酸修饰的载紫杉醇脂质体的制备工艺研究

目的 研究八聚精氨酸(R8)修饰的载紫杉醇脂质体的制备工艺.方法 采用有机相反应法,将R8修饰到脂质体表面,并以薄膜分散-探头超声法制备载紫杉醇脂质体,以细胞摄取、粒径、PDI、包封率为主要指标对磷脂/胆固醇、药脂比、水化液种类、R8密度、DSPE-PEG2000密度进行筛选,进一步优化处方.结果 最优处方为磷脂-胆固醇2∶1、药脂比1∶40、水化液为PBS(pH6.5)、R8密度5％、DSPE-PEG2000密度3％,所制脂质体的粒径为156 ±2 nm,PDI＝ 0.25,包封率为80.87％±8.9％.结论 成功制备了八聚精氨酸(R8)修饰的载紫杉醇脂质体.     

穿膜肽TAT和脑肿瘤靶向肽T7双修饰脂质体的制备和体外靶向性评价

本文旨在制备T7肽和穿膜肽TAT双修饰的脂质体(T7 and TAT dual modified liposomes,T7-TAT-LIP)用于血脑屏障和脑肿瘤细胞双级靶向药物递送。研究以CFPE为荧光探针,T7修饰的PEG-DSPE、TAT修饰的PEG-DSPE、卵磷脂、PEG-DSPE和胆固醇为材料,采用成膜水化法制备脂质体,对T7浓度、TAT浓度、连接T7和TAT的PEG长度进行优化,表征其粒径、zeta电位、形态和稳定性。以b End.3细胞和C6细胞为模型,考察T7-TAT-LIP的细胞摄取能力,表征其穿过血脑屏障和脑肿瘤细胞靶向能力。结果表明,T7用量为脂质的6%、修饰T7所用PEG链长为2000、TAT用量为脂质的0.5%、修饰TAT所用PEG链长为1000时所得到的双修饰脂质体被C6细胞摄取能力最强。优化后T7-TAT-LIP粒径为118 nm,zeta电位为-6.32 m V,透射电镜下形态圆整。脂质体在PBS中较为稳定,37℃放置24 h,浊度和粒径无明显变化;4~8℃放置1个月,粒径和PDI无明显变化。在不同时间点,b End.3和C6细胞摄取T7-TAT-LIP的强度均高于单配体修饰脂质体,且随着孵育时间提高,摄取浓度逐渐提高。这些结果说明,双修饰脂质体具有血脑屏障和脑肿瘤细胞双级靶向能力,且效果优于单配体修饰脂质体。     

RGD修饰载紫杉醇脂质体用于抗脑胶质瘤的体外研究

目的制备RGD修饰载紫杉醇脂质体并考察其体外抗脑胶质瘤治疗效果。方法采用薄膜分散法制备RGD修饰载紫杉醇脂质体（RGD—LP-PTX）,考察其理化性质。用鼠源性脑胶质瘤c6细胞考察肿瘤细胞对脂质体的摄取效率。MTT实验考察脂质体对肿瘤细胞的增殖抑制率。结果制备得到的RGD—LP粒径在120nm左右,PD,〈0.3。C6细胞对RGD．LP的摄取能力显著〉LP;RGD-LP-PTX对C6细胞的生长的抑制作用显著强于LP。结论经过RGD修饰后,脂质体具有良好的脑胶质瘤细胞靶向性,且能够显著增强栽药脂质体对脑胶质瘤细胞的增殖抑制作用。     

RGD修饰的多柔比星脂质体的制备及荷瘤动物活体成像研究

目的制备精氨酸-甘氨酸-天冬氨酸环状三肽(arginine-glycin-aspartic acid,RGD)序列修饰的多柔比星(doxorubicin,DOX)脂质体(RGD-DOX-LP),考察其粒径、zeta电位、包封率,并探究其肿瘤细胞摄取率及在荷瘤裸鼠中的靶向分布情况。方法采用薄膜分散法制备RGD-DOX-LP及多柔比星脂质体(DOX-LP)。荧光分光光度法研究SUP-M2细胞对RGD-DOX-LP,DOX-LP及DOX溶液的摄取效率。以Cyp790为染料,采用近红外小动物活体成像仪研究比较Cyp靶向脂质体(Cyp-RGDDOXLP)与Cyp常规脂质体(Cyp-DOX-LP)在荷瘤裸鼠体内的分布情况。结果 RGD-DOX-LP粒径为(117±1.8)nm,zeta电位为(-12.46±0.86)mv。细胞摄取实验结果显示,SUP-M2细胞对RGDDOXLP的摄取率是DOX-LP的2.8倍,差异有统计学意义(P<0.05)。在荷瘤裸鼠中,Cyp-RGDDOXLP组在肿瘤部位的荧光强度显著高于Cyp-DOX-LP组,荧光持续时间也显著延长。结论 RGD修饰载多柔比星的脂质体能够高效、靶向性地富集于肿瘤组织,是一种有效的抗肿瘤给药系统。     

PCM和TAT双修饰脂质体的制备及心肌靶向性的初步评价

目的 研究心肌特异性靶向肽PCM和穿膜肽TAT双修饰的载荧光探针香豆素-6脂质体的制备工艺,并初步考察其心肌靶向性.方法 采用薄膜分散-超声法,以PCM和TAT为靶头,制备以大豆磷脂、胆固醇、DSPE-mPEG2000为载体材料的载香豆素-6的双修饰脂质体;优化香豆素-6用量、靶头连接方法及靶头用量,以形态、粒径分布、电位、包封率及体外稳定性对脂质体进行表征;考察心肌细胞H9C2对双修饰脂质体的摄取能力,表征其心肌靶向性.结果 PCM和TAT通过插入法连接,当PCM的用量为脂质的3％、TAT的用量为脂质的1％、香豆素-6的用量为20 μg时,所制得的双修饰脂质体的形态圆整,粒径分布为115.7 ±2.91 nm,Zeta电位为-13.1 ±1.81 mV,包封率为83.2％±3.1％,具有良好的体外稳定性,双修饰脂质体的心肌细胞摄取率明显高于未修饰和单修饰的脂质体.结论 双修饰脂质体的制备工艺简单,PCM和TAT双修饰可提高脂质体的心肌细胞靶向性.     

pH敏感多肽修饰载紫杉醇脂质体的制备及评价

目的制备p H敏感多肽修饰载紫杉醇脂质体(p HS-LP-PTX),并评价其体外性质。方法采用薄膜分散法制备PHS-LP-PTX,MTT实验考察脂质体对MCF-7细胞和Hep G2细胞的毒性,通过细胞摄取实验考察脂质体与肿瘤细胞的结合能力。结果所制备的p HS-LP-PTX在p H6.4时平均粒径为125.5±13.4 nm,Zeta电位为10.47±2.53 m V,24 h内具有良好的血清稳定性。MTT结果显示:p H6.4时,p HS-LP-PTX的细胞毒性优于各对照组。体外细胞摄取实验表明:p H6.4时,MCF-7细胞和Hep G2细胞对p HS-LP-PTX的摄取效率优于p H7.4时和普通脂质体。结论紫杉醇脂质体经过p H敏感多肽修饰后,能增强脂质体在酸性环境下的细胞穿透能力,是一种良好的p H敏感型肿瘤靶向给药系统。     

RVG29修饰脂质体对脑胶质瘤靶向性的初步研究

目的 研究一种新型配体RVG29修饰脂质体后,对体外脑胶质瘤的靶向性.方法 采用有机相合成法制备DSPE-PEG2000-RVG29 PPP(D-RVG29)材料,按照薄膜分散法制备脂质体,通过C6细胞和Hela细胞的细胞摄取考察D-RVG29修饰脂质体后,对体外脑胶质瘤的靶向性.结果 在脂质体处方中加入DSPE-PEG2000-OME(D-OME)可使D-RVG29修饰脂质体具有更好的粒径和分布范围,C6细胞对D-RVG29修饰脂质体的摄取强于未修饰D-RVG29的脂质体,Hela细胞对两种脂质体的摄取强度无明显区别.结论 D-OME可以提高D-RVG29修饰脂质体的稳定性,D-RVG29修饰脂质体具有体外脑胶质瘤细胞的靶向性.     

两种RGD肽修饰的新型阿霉素脂质体的体外表征

目的作为配体,肽对于多种受体显示出良好的靶向性,例如在肿瘤表面过度表达的整合素家族受体。本文主要研究和表征分别用精氨酸-甘氨酸-天冬氨酸(RGD)三肽和甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(GRGDS)五肽修饰的载药脂质体。方法分别采用RGD和GRGDS对包载阿霉素的立体稳定脂质体(SSL-doxorubicin)进行修饰,以制备RGD-SSL-doxorubicin和GRGDS-SSL-doxorubicin。在体外表征试验中,测定了各种脂质体的包封率、粒径、Zeta电位和释放率,采用SRB试验研究了各脂质体对卵巢癌细胞的细胞毒作用,并应用流式细胞仪和共聚焦显微镜考察了肿瘤细胞对各脂质体包封的阿霉素的摄取情况。结果所有脂质体的包封率均在95%以上,采用RGD或GRGDS进行的修饰并未影响长循环脂质体的包封率。各种脂质体的平均粒径在105.7±3.5nm和130.5±3.0nm之间,Zeta电位在–3.3±0.3和–6.1±0.3mV之间,在模仿体内环境的释放介质(含胎牛血清)中,12小时内约有2/5的阿霉素从脂质体中释放。与游离阿霉素相比,修饰后的脂质体对肿瘤细胞的抑制率略有下降;在研究对阿霉素摄取的流式细胞试验和共聚焦试验中,也有类似现象出现。将各种脂质体分别加入肿瘤细胞后,阿霉素主要分布于SKOV-3的细胞核。结论本研究成功制备了两种分别用精氨酸-甘氨酸-天冬氨酸(RGD)三肽和甘氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸(GRGDS)五肽修饰的阿霉素脂质体。体外表征结果显示,该修饰不会显著改变立体稳定脂质体的性质。     

基因miRNA-34a靶向纳米复合物抗前列腺癌细胞增殖的作用研究

目的 通过构建具有前列腺癌细胞特异性靶向作用的基因载体适配体——聚乙二醇-聚酰胺-胺(APT-PEG-PAMAM),携带具有抗前列腺癌增殖作用的基因miRNA-34a,考察载体系统的转染效率及其对前列腺癌细胞的抑制作用。 方法 运用核磁共振(NMR)鉴定APT-PEG-PAMAM基因载体的结构;利用粒径电位仪对APT-PEG-PAMAM/miRNA纳米复合物进行表征;利用基因转染实验考察APT-PEG-PAMAM/miRNA纳米复合物在前列腺癌细胞(PC3和LNCaP)上的表达效果;利用CCK-8细胞增殖抑制实验考察APT-PEG-PAMAM/miRNA-34a对前列腺癌细胞的抑制作用。 结果 通过结构鉴定确定APT-PEG-PAMAM合成成功。定性、定量转染效率实验证明,经APT进一步修饰后,对LNCaP细胞转染效率显著增加,证明APT的靶向作用。CCK-8细胞增殖实验证明,APT-PEG-PAMAM/miRNA-34a对前列腺癌细胞具有抑制作用。 结论 APT-PEG-PAMAM/miRNA-34a有望成为今后靶向治疗前列腺癌的基因药物。     

壳聚糖与细胞穿透肽共修饰的纳米粒制备及抗结肠癌的体外细胞学评价

目的:为了提高5-氟尿嘧啶(5-FU)抗结肠癌的口服疗效,构建了一种由N,N,N-三甲基-N-硬脂酰壳聚糖(TSCS)、腺苷和细胞穿透肽(TAT)共修饰的聚乳酸-羟基乙酸共聚物(PLGA)纳米粒,并对其进行相关评价。方法:合成TSCS、硬脂酰腺苷与5-FU磷脂复合物,制备了腺苷穿透肽共修饰纳米粒(AD-CPP-NPs),并对其进行粒径、包封率、体外释放等药剂学和细胞学评价。结果:纳米粒外观为类球形,分布均一。测得纳米粒粒径约为177 nm, Zeta电位为38.2 mV,包封率为56.6%。细胞实验表明,与HT-29细胞共孵育4 h后,与表面未修饰的PLGA纳米粒相比,共修饰纳米粒摄取能力相对聚乙烯醇纳米粒提高16.42倍。结论:共修饰纳米粒能主动靶向结肠癌细胞、增强细胞摄取效率、增加5-FU在癌细胞内的蓄积。     

Affective and Anxiety Disorders and Alcohol and Drug Dependence:

Depression and anxiety frequently coexist in patients with substance use disorders. This clinically-oriented article examiens the relationship between these conditions and emphasizes data showing that substances of abuse can cause signs and symptoms of both depression and anxiety. These substance-related syndromes appear to have a different course and prognosis than uncomplicated, independent anxiety and major depressive disorders, and clinicians should consider the role of alcohol and other drugs in all patients presenting with these complaints. The authors will also outline an approach for diagnosing and managing patients with the combination of a substance use and depressive or anxiety disorder.     

Modelling and simulation of variability and uncertainty in toxicokinetics and pharmacokinetics

Two important methodological issues within the framework of the variability and uncertainty analysis of toxicokinetic and pharmacokinetic systems are discussed: (i) modelling and simulation of the existing physiologic variability in a population; and (ii) modelling and simulation of variability and uncertainty when there is insufficient or not well defined (e.g. small sample, semiquantitative, qualitative and vague) information available. Physiologically based pharmacokinetic models are especially suited for separating and characterising the physiologic variability from the overall variability and uncertainty in the system. Monte Carlo sampling should draw from multivariate distributions, which reflect all levels of existing dependencies in the intact organism. The population characteristics should be taken into account. A fuzzy simulation approach is proposed to model variability and uncertainty when there is semiquantitative, qualitative and vague information about the model parameters and their statistical distributions cannot be defined reliably.     

Synthesis and anti-nociceptive and anti-inflammatory effects of gaultherin and its analogs

The synthesis of gaultherin (1) and its analogs was carried out to provide 11 glycosides under phase-transfer catalytic conditions. The activities of all synthesized compounds were evaluated by nitric oxide production inhibitory assay in vitro. Methyl 2-O-(4-O-β-d-galactopyranosyl)-β-d-glucopyranosylbenzoate (5f) showed significantly anti-nociceptive and anti-inflammatory effects by the evaluation in vivo. Structure–activity relationships within these compounds were discussed.     

Antiinfective and encrustation-inhibiting materials--myth and facts

Catheters, urethral and ureteral stents and other urological implants are frequently affected by encrustration and infection due to their permanent contact with urine. Indwelling urinary catheters provide a haven for microorganisms and thus require extensive monitoring. Several surface modification techniques have been proposed to improve the performance of devices including the immobilization of biomolecules, the incorporation of hydrophilic grafts to reduce protein adsorption, the creation of hydrophobic surfaces, the creation of microdomains to regulate cellular and protein adhesion, new polymers and antimicrobial coatings. Physico-chemical explanation to elucidate the mechanism of such encrustation or infection inhibiting materials is still not available. Our series of experiments showed a marked decrease of silver-activity in biological fluids which corresponds with the controversial clinical results obtained with silver coated urinary catheters. Rifampicin/minocycline coated catheters had very low activity against Gram-negative rods, enterococci and Candida spp., the main causing organisms of urinary catheter infection. Surface engineered materials and antimicrobial drug delivery systems will be the next generation of sophisticated urinary catheters and stents, if both efficacy as well as efficiency has been proved clinically.     

成骨细胞的功能与损伤和骨质疏松的防治

骨质疏松是一种全身性骨骼疾病,导致骨折风险增加。成人的骨量通过破骨细胞的骨吸收和成骨细胞的骨形成作用来维持动态平衡,治疗骨质疏松症的理想策略是抑制破骨细胞的骨吸收和/或增强成骨细胞的骨形成功能。目前针对保护成骨细胞及增强其功能的骨质疏松疗法相对较少。因此,本文针对成骨细胞相关功能蛋白、各种细胞损伤机制（内质网应激、氧化应激、机械过载、微小RNA和长链非编码RNA的影响等）及骨质疏松的治疗与预防作一综述,以期为针对增强成骨细胞功能的骨质疏松治疗策略提供新思路。     

Self-stimulation and amphetamine: Tolerance to d and l isomers and cross tolerance to cocaine and methylphenidate

The effects of the d and l isomers of amphetamine on self-stimulation responding were tested following acute and chronic administration. Tolerance and post-drug depression of responding occurred in tests with both isomers, indicating no role for p-hydroxynorephedrine (PHN) which is one of the metabolites of d-amphetamine. In the second experiment, d-amphetamine, methylphenidate and cocaine all produced quantitatively and qualitatively similar effects on self-stimulation responding following acute administration. Following chronic administration of d-amphetamine, animals showed tolerance to all three drugs, indicating cross-tolerance among them. These data are consistent with an hypothesis that tolerance and post-drug depression following chronic amphetamine treatment are the result of decreases in postsynaptic receptor sensitivity, which would lead to a decreased effectiveness of all three drugs, regardless of their pre-synaptic mechanisms.     

益生菌与肿瘤防治

益生菌广泛存在于自然界中,通过维持宿主体内菌群平衡、影响肠屏障功能和调节免疫应答等作用,提高宿主健康水平,被公认为"肠道健康卫士".一些益生菌可以增强机体的免疫功能,抑制致癌物质,影响肿瘤细胞的基因表达,对肿瘤具有拮抗作用.大量研究表明,益生菌在未来的肿瘤防治中有很好的应用和发展前景.     

Interaction of estrone and estradiol with DNA and protein of liver and kidney in rat and hamster in vivo and in vitro

[6,7-³H] Estrone (E) and [6,7-³H]estradiol-17 (E₂) have been synthesized by reduction of 6-dehydroestrone and 6-dehydroestradiol with tritium gas. Tritiated E and E₂ were administered by oral gavage to female rats and to male and female hamsters on a dose level of about 300 g/kg (54 mCi/kg). After 8 h, the liver was excised from the rats; liver and kidneys were taken from the hamsters. DNA was purified either directly from an organ homogenate or via chromatin. The radioactivity in the DNA was expressed in the units of the Covalent Binding Index, CBI = (mol chemical bound per mol DNA-P)/(mmol chemical administered per kg b.w.). Rat liver DNA isolated via chromatin exhibited the very low values of 0.08 and 0.09 for E and E₂, respectively. The respective figures in hamster liver were 0.08 and 0.11 in females and 0.21 and 0.18 in the males. DNA isolated from the kidney revealed a detectable radioactivity only in the female, with values of 0.03 and 0.05 for E and E₂, respectively. The values for male hamster kidney were < 0.01 for both hormones. The minute radioactivity detectable in the DNA samples does not represent covalent binding to DNA, however, as indicated by two sets of control experiments. (A) Analysis by HPLC of the nucleosides prepared by enzyme digest of liver DNA isolated directly or via chromatin did not reveal any consistent peak which could have been attributed to a nucleoside-steroid adduct. (B) All DNA radioactivity could be due to protein contaminations, because the specific activity of chromatin protein was determined to be more than 3,000 times higher than of DNA. The high affinity of the hormone to protein was also demonstrated by in vitro incubations, where it could be shown that the specific activity of DNA and protein was essentially proportional to the concentration of radiolabelled hormone in the organ homogenate, regardless of whether the animal was treated or whether the hormone was added in vitro to the homogenate.Carcinogens acting by covalent DNA binding can be classified according to potency on the basis of the Covalent Binding Index. Values of 10³–10⁴ have been found for potent, 10² for moderate, and 1–10 for weak carcinogens. Since estrone is moderately carcinogenic for the kidney of the male hamster, a CBI of about 100 would be expected. The actually measured limit of detection of 0.01 places covalent DNA binding among the highly unlikely mechanisms of action. Similar considerations can be made for the liver where any true covalent DNA binding must be below a level of 0.01. It is concluded that an observable tumor induction by estrone or estradiol is unlikely to be due to DNA binding.Paper presented at the Satellite Symposium of the European Society of Toxicology, Rome, March 29, 1983     

Isolation and characterization of glycosylated and non-glycosylated prolactins from alligator and crocodile

Two molecular forms of prolactin (PRL). glycosylated and non-glycosylated, were isolated from pituitary glands of two reptiles, alligator and crocodile. The reptilian PRLs were extracted under alkaline conditions from the precipitate obtained after pituitaries were first extracted with 0.25 m sucrose, 1 mM NH₄HCO₃, pH 6.3. Purification was performed by ion exchange chromatography on DE-52, gel filtration on Sephadex G-75 superfine, and reversed phase high performance liquid chromatography. Two forms of both alligator and crocodile PRL, designated PRLI and PRLII, with molecular weights of 26000 and 24000 were isolated. Alligator and crocodile PRLI and PRLII were stained specifically in immunoblots with anti-sea turtle PRL and anti-ostrich PRL. Sequence analysis revealed that both forms of alligator and crocodile PRLs consisted of 199 amino acid residues with a glycosylation consensus sequence (Asn-Ala-Ser) at position 60 in alligator and crocodile PRLs with a molecular weight of 26000 (PRLI). In contrast, Thr was substituted for Asn at position 60 in the PRLs with a molecular weight of 24000 (PRLII). The sequences of alligator PRLs differed from crocodile PRLs only in position 134: Val for alligator PRLs and He for crocodile PRLs. There is a high degree of structural conservation between the reptilian PRLs isolated in this study and avian PRL; each showed 92% sequence identity with chicken PRL and 89% with turkey PRL.