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??OBJECTIVE To establish an interferon-resistant hepatitis B virus cell model and provide experimental basis for further investigating the mechanism of HBV resistance to interferon. METHODS HepG2.2.15 was continuously grown in the presence of 10,30,50 and 70 u??mL^-1 of interferon ??-2b for up to 48 weeks. The HepG2.2.15/IFN??-2b cell model was constructed after 48 weeks of induction. The cells were treated with the best-effect concentration. Then, the levels of HBsAg, HBeAg, and HBV DNA in the supernatant of cell culture medium before and after the treatment were compared. RESULTS After stimulation with low concentrations of IFN??-2b for 12 weeks, the 50 u??mL^-1 group showed significant resistance to the best-effect concentration of IFN??-2b. Compared with the levels before stimulation, the inhibition rate on HBsAg, HBeAg, and HBV DNA decreased by 25.48%, 8.40%, and 15.43%, respectively, suggesting that 50 u??mL^-1 was the best-stimulation concentration. After stimulated with 50 u??mL^-1 IFN??-2b for 12-48 weeks, the results showed that the inhibition rate on HBsAg, HBeAg, and HBV DNA after 36 weeks was the most significant. CONCLUSION Continuous induction with 50 u??mL^-1 IFN??-2b for 36 weeks could most easily induce drug resistance in HepG2.2.15 cells.     

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??Transdermal drug delivery offers a number of advantages including improved patient compliance, sustained release, avoidance of gastric irritation, as well as elimination of pre-systemic first-pass effect. However, the skin barrier function limits the transdermal penetration of macromolecular drugs.Microneedle arrays can remarkably increase the skin permeability for drugs, especially macromolecular drugs, by forming microchannels in the skin. In recent years, the types and usages of microneedle have made great progress. This review focuses on remarking the wound repair and pharmacology evaluation, as well as introduces the microneedle percutaneous drug delivery system.     

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??OBJECTIVE To summarize the pharmacoeconomic studies of adalimumab in the management of Rheumatoid Arthritis, discuss the cost-effectiveness and adverse reaction of combination use of adalimumab and DMARDs, and compare the economic values of TNF-?? inhibitors.METHODS Search Pubmed and MedLINE for pharmacoeconomics studies of adalimumab using in the treatment of RA. All the results are systematically summarized and assessed. RESULTS Seven studies on pharmacoeconomics assessment of adalimumab were selected and discussed. Compared with conventional DMARDs, adalimumab costs more but obviously extends the QALYs of RA patients. Adalimumab also provides significant improvement in symptom severity with less adverse reaction. The efficacy of combination use of adalimumab and MTX is better than adalimumab alone. In terms of cost and effectiveness, adalimumab has advantages over infliximab and is equal to etanercept.CONCLUSION Though adalimumab costs higher for the RA patients, it can significantly improve the quality of life when in the combination treatment with DMARDs, as well as reduce adverse reactions. Compared with other TNF-?? inhibitors, adalimumab shows superiority both in efficacy and cost. Thus, adalimumab is recommended for RA patients that don??t respond to DMARDs.     

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??OBJECTIVE To explore the synthesis of novel phenylalanine dipeptide derivatives and their inhibitory effects on tumor cells. METHODS Starting from L-phenylalanine or L-tyrosine, a series of derivatives were synthesized by reaction with chloroacetyl chloride, followed by condensation with L-phenylalaninol or L-phenylalanine methyl ester hydrochloride and nucleophilic substitution reaction with differently substituted phenol.The cell proliferation inhibiting activities of the derivatives were evaluated by thiazolyl blue tetrazolium bromide(MTT)method.RESULTS Some of the target compounds showed certain inhibitory effect for leukemia cell lines K562 and HEL in vitro.Furthermore, the derivatives 3f and 3q had preferably inhibitory effect on K562 cell line prostate cancer PC3 cells in vitro.CONCLUSION Phenylalanine dipeptide derivatives possess good effect on the leukemia and prostate cancer cells and are worth of further research.     

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??Steroids are natural products with widespread application and attract much attention in the field of chemical industry and medicine. In order to provide reference for further research and development of steroids, this paper mainly reviews the recent advances in the structural modification of steroids at A-ring according to the changes of different structural positions and introduction of functional groups.     

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??OBJECTIVE To establish a high-performance capillary electrophoresis (CE) method for determinating gallic acid in commercial Quanshen pieces of two different colors,amaranth and brownish red. METHODS CE-UV method was used in the following condition: BGE 20 mmol??L^-1 sodium borate solution, voltage 20 kV, detection wavelength 290 nm, sample injection 10 s at 5 kPa, capillary temperature room temperature. RESULTS A quantitative method was successfully developed to determine gallic acid in Quanshen pieces within 6 min. The contents of gallic acid in 7 batches of amaranth colored Quanshen pieces were 2.70% (Hubei province), 12.08% and 7.79% (Shandong province), 2.01%, 5.78%, 6.97%, and 4.22% (Hebei province), respectively. The contents of gallic acid in brownish red colored Quanshen pieces were 1.35% (Hubei province), 1.81% and 3.42% (Shandong province), 1.87%, 3.42%, 0.44%, and 1.52% (Hebei province), respectively. CONCLUSION The method is rapid, economical and simple and can provide a powerful quality control measure for Quanshen pieces. The contents of gallic acid in the amaranth colored Quanshen pieces are higher than those in the brownish red colored pieces.     

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??OBJECTIVE To develop Zaocys dhumnades DNA extraction and detection kit, evaluate the specificity, stability and repeatability of the kit, and investigate the quality of commercial black tip snake products using the kit. METHODS Using the modern DNA fingerprint technology, the pharmacopoeia Zaocys dhumnades DNA detection method was optimized and improved, and Zaocys dhumnades DNA extraction and detection reagent was developed to obtain the tip snake PCR detection kit. The performance parameters of the kit were evaluated. The kit was used on 18 commercial black tip snake products randomly sampled from Beijing, Tianjin, Changchun and Jilin City.The results were compared with those got by using pharmacopoeia method of DNA test. RESULTS Zaocys dhumnades DNA testing kit was still valid after repeated freezing and thawing for 5, 10 and 20 times. Among the 18 commercial black tip snake samples 14 were quality goods, and four were adulterants. Repeated experiments of specificity, stability and repeatability displayed exactly the same results . The test result of the kit method was consistent with the pharmacopoeia method. And the kit method could fulfil the DNA testing task in ordinary laboratories. CONCLUSION Zaocys dhumnades DNA testing kit can identify tip snake products conveniently, accurately and effectively, which can be applied in the rapid detection of Zaocys dhumnades DNA.     

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??OBJECTIVE To understand the condition of antibiotics usage in children with upper respiratory tract infection in China, so as to evaluate the effectiveness of rational drug use policy since the new health care reform promulgated by the state.METHODS Publications were extracted from China National Knowledge Infrastructure(CNKI),Chinese Science and Technology Journal Database(VIP),Wanfang Databases, PubMed, Ebsco, Web of Science and Embase, then Meta-analysis was carried out on them, at the same time, do comparative study on times, regions and categories. RESULTS Thirty three papers were included for Meta-analysis with a total sample of 54 189.Meta-analysis showed that the combined rate of antibiotics usage in children with upper respiratory tract infection in China was 89.2%(95%CI=82.2%-96.2%.Before 2009, the antibiotics use rate was 92.2%,and it turned to be 87.6% after 2009. The antibiotics use rate of eastern,central and western children was 88.3%,88.4% and 93.3%. Tertiary hospitals was 85.2%,secondary hospitals was 92.2% and primary medical institutions(including first level hospitals,community health service centers and township health centers) was 90.2%.CONCLUSION Children's antibiotic use rate of upper upper respiratory tract infection is high in China and the rate is different at different times,regions and institutions. After the health care reform policy was enacted,the use rate of antibiotics fell,this may be related to the implementation of the policy.     

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??OBJECTIVE To investigate the possible mechanism of Panax notoginseng saponins (PNS) protection against cisplatin-induced kidney injury through enhangcing autophagy. METHODS The rats were randomly divided into normal control group, cisplatin model group and cisplatin+PNS group. After exposure to cisplatin for 4 and 8 d, the urinary ??-N-acetyl-??-D-glucosaminidase (NAG), blood urea nitrogen (BUN) and serum creatinine (Scr) were measured by commercial kits, and the pathological change of renal tissue was examined using transmission electron microscopy. Additionally, the expression of LC3, HIF-1??, BNIP3 and Beclin1 were examined by Western blotting. RESULTS PNS decreased the levels of urinary NAG, serum BUN and Scr which were increased by cisplatin (P??0.05). Moreover, PNS significantly increased the expression of LC3, HIF-1??, BNIP3 and Beclin1 (P??0.05), and attenuated the mitochondrial damages of renal cells. Furthermore, the effects of PNS on the mentioned above were more obvious at day 8 than that at day 4 (P??0.05). CONCLUSION PNS may play a protective role against cisplatin-induced kidney injury with certain of time dependence, by enhancing mitochondrial autophagy in rat renal tissue via the HIF-1??/BNIP3 pathway.     

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??OBJECTIVE To evaluate the quality of now available clinical guidelines and consensus statements on type 2 diabetes. Based on this assessment, the comparison of guidelines and consensuses is made to give some suggestions on the METHODS of making these documents. METHODS PubMed, Embase, CNKI, Wanfang, SinoMed and guideline websites were systematically searched. Literatures were screened according to defined criteria for including. AGREE ?? was used to instrument assess the quality of the guidelines and consensuses. RESULTS The result showed that the quality of included guidelines is various, but the overall quality of guidelines is still higher than consensuses. Guidelines got high scores in scope and purpose domain and clarity of presentation domain, but low scores in rigor of development domain and applicability domain. Consensus statements got low scores in all 6 domains. CONCLUSION The main reason for the difference is consensuses have poor methodology. Rapid advice guidelines were suggested as an alternative for consensus statements.     

Production and Characterization of Monoclonal Antibodies Against Primo Vascular System of Rat

BackgroundThe primo vascular system (PVS) has been difficult to detect due to its small diameter and translucent features of the threadlike network. Thus, contrast-enhancing dyes including Alcian blue, Trypan blue and Janus green B had to be used for finding and taking out PVS from rat and mouse.ObjectiveGeneration of monoclonal antibodies (mAbs) against PVS of rat was intended to use as a detector for PVS and a biological tool for functional study of PVS.Materials and methodsPrimo vessel (PV) and Primo node (PN) were isolated from organ surfaces of rat and then their proteins were isolated and injected into mouse as an immunogen. The classical traditional method was applied for production of mAbs against PVS. The various techniques, such as cell fusion, screening of hybridoma, ELISA, Western blotting (WB), immunofluorescence microscopy (IF), and limiting dilution, were used to generate mAbs against PVS.ResultsAmong 16 mAbs generated, 4 representative mAbs were characterized with their specificities in ELISA, WB, and IF. α-rPVS-m1-1 and α-rPVS-m4-6 had strong binding affinities to PVS in both ELISA and WB but did not show specificities in IF at all. On the contrary, α-rPVS-m3-2 and α-rPVS-m3-4 almost did not respond in WB but had strong binding affinities in ELISA and specificities in IF. Two mAbs stained predominantly at extra cellular matrix and cell membrane of PVS of rat in IF, and they were able to discriminate PVS from blood vessel (BV) and lymphatic vessel (LV).Conclusions4 representative mAbs against PVS of rat were characterized by ELISA, WB, and IF. α-rPVS-m3-2 and α-rPVS-m3-4, which had strong specificities in IF, can be used as a tool in discriminating PVS from other similar tissues and in elucidate biological function of PVS.     

苯巴比妥单克隆抗体的研制及竞争ELISA血药浓度监测方法的建立

 目的研制苯巴比妥单克隆抗体(PBmAb),建立PB血药浓度竞争ELISA监测方法。方法将PB改造成对氨基苯巴比妥(pAPB),用重氮化法合成免疫原BSA-pAPB并免疫Balb/c小鼠;细胞融合技术筛选PB mAb杂交瘤细胞株,体内诱生腹水法制备PB mAb;应用PB mAb研制PB血药浓度监测竞争ELISA试剂盒(PB-Kit),并测定其性能。结果BSA-pAPB偶联成功;筛选出4株杂交瘤细胞,应用其中最好的4E6株制备的PB mAb间接ELISA效价为1∶6.4×10⁵,亲和常数(K_a)为2.08×10¹⁰L·moL^-1,50%抑制浓度(IC₅₀)为9.84μg·L^-1,与巴比妥的交叉反应率(CR)为7.9%,与其他化合物无CR;PB-Kit的检测范围为1.0～128μg·L^-1,灵敏度为0.89μg·L^-1,检测限为1μg·L^-1,血清样添加回收率为94.7%,批内和批间变异系数均<10%;PB-Kit与差示分光光度法(DS)的符合率为100%。结论成功研制出高价、敏感、特异的PB mAb,建立了PB血药浓度竞争ELISA监测方法。     

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??OBJECTIVE To develop and validate an acid dissociated bridging ELISA assay for determination of antibodies (ADAs) against a human monoclonal antibody against CD20, Arzerra^TM, in cynomulgus serum, which will be used to reflect the immunogenicity of Arzerra^TM in cynomolgus serum. METHODS Biotin labeled Arzerra^TM and acid treated unknown samples were added into the Arzerra^TM pre-coated 96-well plates, sequentially. Positive controls (ADAs) in unknown samples bridged with coated Arzerra^TM and biotin-labeled Arzerra^TM (Biotin-Drug) to generate ADAs bridged compounds (Drug-ADAs-Biotin-Drug), and coloration was made with enzymatic reaction. RESULTS The sensitivity was 7.4 ng??mL^-1, and the intra-assay and the inter-assay RSDs were less than 17.6% for positive controls. The screening cut point (SCP) for this assay was 1.23 and the confirmatory cut point (CCP) was 42.8%. The assay also showed high drug tolerance, which was 20 fold at 2.0 ??g??mL^-1 ADA level (Drug:ADA=40.0 ??g??mL^-1:2.0 ??g??mL^-1); and no ??Hook effect?? was detected within 3.2-100 000.0 ng??mL^-1. Using this assay to determine ADAs against Arzerra^TM in Cynomolgus serum, ADAs were detected at 21 d post administration. CONCLUSION The bridging ELISA is one of the best assays to determine ADAs against therapeutic monoclonal antibodies, because it shows high sensitivity and good drug tolerance.     

载白藜芦醇的氨基修饰介孔二氧化硅纳米粒的制备及体内体外评价

 目的 本实验拟制备载白藜芦醇的氨基修饰介孔二氧化硅纳米粒(NH₂-MSN-RES),以期提高白藜芦醇(Res)的生物利用度。方法 采用改良的Stober法合成氨基修饰介孔二氧化硅(NH₂-MSN),发明了反复饱和溶液吸附法载入白藜芦醇制备载白藜芦醇的氨基修饰介孔二氧化硅纳米粒,透析袋法考察其体外释药特性,MTT法考察载体对Caco-2的细胞毒性,研究载体对白藜芦醇的跨膜转运能力和在大鼠体内的药动学特性。结果 氨基修饰介孔二氧化硅氨基成功修饰,呈圆整球形,分布均一,对Caco-2细胞无明显的毒性。反复饱和溶液吸附法吸附8次,载白藜芦醇的氨基修饰介孔二氧化硅纳米粒的载药量为(19.26±2.51)%,体外释药呈现明显的缓控释特性,载白藜芦醇的氨基修饰介孔二氧化硅纳米粒对Caco-2细胞单层模型具有良好的跨膜转运能力。药动学参数表明,载白藜芦醇的氨基修饰介孔二氧化硅纳米粒的AUC_0-t是白藜芦醇溶液的2.58倍,并且t_1/2、t_max和ρ_max显著提高,同时载体使白藜芦醇由单室模型变为二室模型。结论 改良的Stober法成功合成氨基修饰介孔二氧化硅,反复饱和溶液吸附法能够有效提高载药量,载白藜芦醇的氨基修饰介孔二氧化硅纳米粒提高了白藜芦醇的生物利用度,氨基修饰介孔二氧化硅是一种有前景的口服给药纳米材料。     

黄芩苷壳聚糖纳米粒的制备及表征

目的：建立小毛茛内酯的含量测定方法,优选猫爪草中小毛茛内酯的提取工艺。方法：采用HPLC测定小毛茛内酯含量,流动相甲醇-乙腈-水（40：40：20）,检测波长254 nm。在单因素试验基础上,以小毛茛内酯质量分数为指标,采用L₉（3⁴）正交试验考察提取时间、料液比、乙醇体积分数及粉碎粒度对提取工艺的影响。结果：最佳提取工艺为粉碎粒度80目,加3倍量70%乙醇提取3次,每次2 h;小毛茛内酯质量分数（1.24±0.22）%。结论：优选的工艺稳定可行且提取率高,为猫瓜草的开发与利用提供参考。     

壳聚糖苦丁茶纳米粒的制备及理化性质考察

目的:制备壳聚糖苦丁茶纳米粒并考察其理化性质和体外释药性能。方法:采用梯度体积分数乙醇提取苦丁茶总黄酮,利用紫外-分光光度法测定总黄酮含量,检测波长510nm。通过离子交联法制备壳聚糖苦丁茶纳米粒,考察其形态、粒子分布、包封率、载药量及体外释放情况。结果:苦丁茶中总黄酮质量分数31.84%。纳米粒呈球形,粒径200~400nm,多分散系数0.137。壳聚糖苦丁茶纳米粒最佳处方为壳聚糖1g·L-1,三聚磷酸钠1g·L-1,苦丁茶总黄酮1g·L-1。体外释放试验显示该纳米粒在前2h有突释效应,释放量45.6%~48.9%,24h总释放量91.9%。结论:壳聚糖纳米粒可作为苦丁茶的优良载体,为新型降脂制剂的开发提供参考。     

壳聚糖修饰的载穿心莲内酯介孔二氧化硅纳米粒的制备及其pH响应性释药性能考察

目的:制备壳聚糖修饰的载穿心莲内酯介孔二氧化硅纳米粒(CS/Ap-MSN),对其进行体外质量评价及p H响应性释药性能考察。方法:根据改良经典stober法一步合成了氨基修饰的介孔二氧化硅纳米粒(NH2-MSN),利用壳聚糖进行偶联修饰,表征其形貌及结构,通过体外释放试验考察CS/Ap-MSN在不同p H条件下的响应性释药性能。结果:CS/Ap-MSN的平均粒径(178.0±3.2)nm,多分散指数0.378±0.117,Zeta电位(34.04±0.22)mV,载药量和包封率分别为(27.8±1.7)%,(63.6±3.2)%,壳聚糖的修饰量6.3%。CS/Ap-MSN在p H 5.0的释放条件下,8 d累计释药量达53.3%;pH 7.4的释放条件下,8 d的累计释药量23.4%。结论:CS/Ap-MSN的体外释药具有p H响应性,累积释放量随p H减小而增大,且具有一定的缓释性能。     

白藜芦醇壳聚糖纳米粒的制备及理化性质

 目的建立白藜芦醇壳聚糖纳米粒的制备方法。方法以三聚磷酸钠作为离子聚合剂,在弱酸性条件下制备白藜芦醇壳聚糖纳米粒;考察pH值、壳聚糖与离子聚合剂浓度、白藜芦醇加入顺序、搅拌时间及探头超声等因素对纳米粒性质的影响,确定最佳制备工艺。结果在透射电子显微镜下,制备的白藜芦醇壳聚糖纳米粒呈球形;平均粒径为38nm,多分散指数为0.13;包封率与载药量分别为(61.6±2.3)%和(13.1±1.1)%;与白藜芦醇壳聚糖溶液相比,白藜芦醇壳聚糖纳米粒显示了明显的缓释效应。结论离子聚合法可用于小粒径壳聚糖纳米粒的制备。     

丹寿汤不同组分配伍对抗心磷脂抗体致复发性流产的定量评价

目的:基于发表的丹寿汤文献数据,采用模型化及模拟化进行再分析,提取更多信息,以期优化配伍方案。方法:原文丹寿汤以正交t值L12设计,1水平为"使用",2水平为"未使用",胚胎吸收率为药效指标。采用正交模拟法结合非线性混合模型结合,定量评价各组分在复方中相对重要程度和相互作用,模型评价采用4种视图进行综合评价。结果:丹寿汤组方中,全方呈现药效相加作用;主要药效学组分重要程度依次为菟丝子(A)、丹参(E)、续断(B)(P<0.05)。理论上不同配伍中,最佳组分配伍为ABCEF,4种视图评价模型表明所建模型预测能力较好。结论:采用正交模拟法结合非线性混合模型定量分析复方配伍数据,可获大量组方定量设计信息。     

靶向抗癌药(As2O3)白蛋白纳米球的研究

目的研究白蛋白纳米球的制备工艺,并检测偶联单克隆抗体白蛋白纳米球对靶细胞的特异性杀伤作用.方法采用高温固化法制备白蛋白纳米球,通过正交设计筛选最佳工艺.制备免疫白蛋白纳米球,观察白蛋白纳米球对急性早幼粒细胞白血病M3亚型白血病细胞的特异性杀伤作用.结果在最佳工艺条件下制备白蛋白纳米球,平均直径为250 nm左右,形态圆整.免疫纳米球(0.3 μmol/mL剂量组作用48 h)对靶细胞(急性早幼粒细胞白血病M3亚型白血病淋巴细胞)杀伤率为(34.12±4.76)%,对正常对照(正常人外周血淋巴细胞)杀伤率仅为(5.37 ±2.21)%.结论免疫纳米球对靶细胞具有特异性杀伤作用,而对非靶细胞的杀伤作用较小.