壳聚糖在眼科临床中的应用

甲壳素是一种来源于动物的天然多糖 ,学名为 1,4 2 乙酰胺基 α 脱氧 β D萄聚糖 ,普遍存在于虾、蟹等低等动物及昆虫等节肢动物的外壳中 ,也存在于真菌和藻类的细胞壁中 ,资源丰富 ,分布广泛。壳聚糖是甲壳素脱去部分乙酰基后的产物。甲壳素和壳聚糖的制备方法比较简单 ,虾或蟹壳经清洗后浸酸脱钙 ,再用 10 %的碱液脱除蛋白即得到甲壳素 ,如继续用浓碱去乙酰基则得到壳聚糖。甲壳素的发现已有 10 0多年的历史 ,近年来由于其在生物技术、医药、生物医学工程等众多领域具有极大的潜在应用价值和广阔的发展前景而引起人们的重视。在医学领…     

毒鼠强气相色谱测定方法的探讨

毒鼠强又名"四二四"、"没命鼠",是一种剧毒灭鼠剂,对人的半数致死量(LD50)为100μg/kg,它的结构特殊(见图1),化学名称:四次甲基二砜四胺.毒鼠强为白色粉末,熔点250～254℃,255～260℃分解.微溶于丙酮,易溶于苯,不溶于甲醇和乙醇,稍溶于水,在稀酸、稀碱(0.1moL/L)中稳定[1].     

兔骨髓基质干细胞在不同脱钙程度骨基质材料上的增殖

背景:脱钙骨基质具有天然网状孔隙结构,有较好的可塑性和生物相容性,但目前报道对制备脱钙骨基质所用的脱钙时间各异.目的:比较不同脱钙时间的脱钙骨基质对兔骨髓基质干细胞增殖的影响,为组织工程软骨筛选最佳支架材料.方法:取兔髂骨制成条块状,经反复脱脂后,分别脱钙处理6,12,24 h,乙醇浸泡2 d制得脱钙骨基质,置于24孔培养板中.取浓度为5×10~9L~(-1)的第3代兔骨髓基质干细胞悬液40 μL,接种于上述培养板不同脱钙时间的预湿材料上.扫描电镜下观察脱钙骨基质形态及其孔隙率、孔径,MTT法测定骨髓基质干细胞在脱钙骨基质上的增殖情况,扫描电镜下观察骨髓基质干细胞在脱钙骨基质上的黏附情况.结果与结论:脱钙骨基质呈天然的高密度孔隙网架结构,骨小梁、小梁间隙和骨内管腔系统同时存在,脱钙6,12,24 h的脱钙骨基质其孔隙率、孔径大小无明显差异(P>0.05).与脱钙12,24 h的脱钙骨基质比较,骨髓基质干细胞与脱钙6 h的脱钙骨基质复合更为紧密,细胞相容性最好.扫描电镜下,脱钙6 h的脱钙骨基质上生长的骨髓基质干细胞培养8 d后增殖稳定,适合移植,且细胞数量明显多于脱钙12,24 h的脱钙骨基质(P<0.01).     

壳聚糖纳米混悬剂的研制和表征

背景:实验于2006-07/2007-06在浙江省医学科学院生物工程研究所完成.壳聚糖(脱乙酰度为87.85%,黏度为195 mPa·s,水分7.13%,灰分0.82%,批号:050621160)由浙江金壳生物化学有限公司提供.运用化学酸解、60Co γ射线辐照、高压、超声波及稳定剂等综合理化手段制备壳聚糖纳米粒子.透射电镜观察制备的壳聚糖纳米粒子为类圆球形;激光动态光散射仪检测壳聚糖纳米粒子平均粒径25.3 nm,占总粒子数99.1%,粒径分布图形较窄,表明颗粒大小均匀.实验结果表明运用以60Co γ射线辐照为主要手段进行壳聚糖纳米混悬剂的制备,是一种简便、快速、无污染、重复性好的方法,具备可行性.     

介绍一种改良的Neutral EDTA脱钙液

脱钙是制作骨组织切片的重要环节,尤其是要进行免疫组织化学染色的骨组织,如何达到骨组织既充分脱钙,又保护骨组织的抗原不受破坏的目的,我们参考有关文献[1,2],配制了一种改良的Neutral脱钙液,用于骨组织脱钙,并进行相应的免疫组化染色。该方法既不损害组织结构也不影响染色效果,简化了步骤,保证了质量。1材料与方法1·1材料24例来自大段同种异体骨愈合实验研究之兔骨。改良的Neutral脱钙液配制方法:将乙烯二胺四乙酸二钠(EDTA-2Na)250 g,溶于1 750 ml蒸溜水中,此时溶液呈灰白色浑浊状,不停摇晃使溶液彻底溶解。加入氢氧化钠25 g,继续摇…     

甲酸混合酸脱钙液在兔股骨头制片中的应用

目的观察甲酸混合酸脱钙液对股骨头组织的制片效果。方法采用成年新西兰大白兔10只,切断股骨头圆韧带,取股骨上1/3段置于甲酸混合酸脱钙液中脱钙1周,随即常规脱水、透明、石蜡包埋,制备5μm厚纵切片,行HE染色。结果所有组织切片厚薄均匀,未见脱片现象,股骨头完整性较好,各层次分明,骨小梁结构完整,软骨细胞、骨细胞细胞形态清晰,着色鲜艳。结论选用甲酸混合酸脱钙液制片股骨头组织具有脱钙时间相对较短、操作简单、组织结构完整、染色效果较好的特点。     

药用茶叶提取物的制备工艺研究

目的:以天然绿茶为原料,采用现代化的制备工艺研制出药用茶叶提取物,运用于医药领域。方法:对药用茶叶提取物生产技术及其装备进行了优化和改进,包括茶叶选择、提取、过滤、碱沉、酸溶、萃取、浓缩、干燥、粉碎、包装等各工序的关键点。结果:选用在浙江东白山春夏两季采收的茶叶为原料,提取罐温度控制在80℃,提取30 min,工序包括氢氧化钙碱沉,柠檬酸酸溶,乙酸乙酯萃取,减压浓缩及真空干燥,并用高效液相法对茶叶提取物的含量进行检测。结论:该制备工艺适用于工业化生产,能够批量生产出药用级的茶叶提取物。     

用于肾结石检查的尿标本的收集和处理

草酸钙肾结石患者的诊断常须测定24小时尿的钙、磷、尿酸和(或)草酸盐。尿中钙、草酸盐和尿酸的排泄对这些病人特别重要,约30%患者的尿钙排泄和约25%患者的尿酸排泄高,这些生化值可指示治疗。钙、草酸盐和尿酸的溶解性完全不同。钙和草酸盐溶于酸,而尿酸溶于碱。因此收集标本时应注意它们溶解度的差异。作者研究了pH对准确分析尿中钙、草酸盐、尿酸、镁、磷和肌酐的影响。实验表明,不恰当的pH可异致某些分析结果无效,将留取标本的pH改变并加热,可使单一尿标本获得正确的测定结果。     

犬同种异体脱钙骨基质复合细胞片层的体外培养与观察(英文)

背景:如何构建具有类似天然骨结构组织工程骨的问题是组织工程学发展的一个难题.细胞片层技术的出现,为其提供了新的途径.目的:观察细胞片层与脱钙骨基质的生物相容性及其在脱钙骨基质上的生长情况.设计、时间及地点:体外观察性实验,于2009-06/2009-09在青岛大学医学院附属医院中心实验室完成.材料:利用温度感应培养基制备犬骨髓基质细胞片层;脱脂、脱钙、脱非胶原蛋白方法制备犬脱钙骨基质.方法:将温度感应技术制备刮取的细胞片层铺盖于脱钙骨基质表面,用含体积分数为10%胎牛血清及成骨诱导剂的DMEM培养液浸没培养.主要观察指标:扫描电镜下观察脱钙骨基质的结构及细胞片层在脱钙骨基质上的附着、生长情况.计算脱钙骨基质孔隙率和孔径大小.结果:扫描电镜下观察可见脱钙骨基质呈三维立体网孔结构.材料孔隙率约为.75%.平均孔隙直径为(250.11±98.89)μm,成正态性分布.扫描电镜下观察细胞片层在脱钙骨基质上的附着、生长状况良好,增殖迅速.结论:细胞片层与脱钙骨基质有较好的生物相容性,脱钙骨基质/细胞片层复合体能够应用于组织工程骨,为构建类似骨结构组织工程骨起到促进作用.     

环孢素A影响脱钙骨基质诱导成骨的组织学观察

背景:脱钙骨可用于成骨细胞的支架材料,机体组织对脱钙骨有一定的免疫排斥反应.目的:观察环孢素A影响脱钙骨诱导成骨的组织学变化,探讨脱钙骨成为理想的骨移植材料的可行性.设计、时间及地点:随机对照动物实验,于2007-03/2008 04在大连医科大学附属_二院实验中心完成(国家分子生物学三级实验室).材料:用16只新西兰大白兔,分为4组,同种脱钙骨对照组和实验组,异种脱钙骨实验组和对照组,每组4只.环孢素注射液为北京诺华制药有限公司产品.方法:制备脱钙骨,取白兔的脱钙骨命名为同种脱钙骨,取自犬的脱钙骨命名为异种脱钙骨.于兔的脊旁肌肉内植入相应脱钙骨制备物,每侧2处.实验组每人肌注含环孢素A的实验液2 mg/kg,对照组肌注对照液2 mg/kg,共4周.植入物标本进行苏木精-伊红染色,光镜观察.主要观察指标:各组脱钙骨组织学观察结果.结果:同种脱钙骨在所有动物中均引起了骨形成.环孢素A并没有改变同种脱钙骨诱导成骨的形态学.异种脱钙骨对照组在第4周时没有骨或软骨细胞生成.异种脱钙骨环孢素A实验组在第4周时有软骨、骨形成.结论:环孢素A并没有改变同种脱钙骨诱导成骨的形态学.免疫反应抑制了异种脱钙骨的成骨,环孢素A可抵消这种抑制作用,环孢素A可提高异种脱钙骨治疗骨不连或骨缺损的成功率.     

Decolorization of malachite green and crystal violet by waterborne pathogenic mycobacteria

Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium marinum, and Mycobacterium chelonae tolerate high concentrations of the dyes malachite green and crystal violet. Cells of strains of those species decolorized (reduced) both malachite green and crystal violet. Because decolorized malachite green lacked antimicrobial activity, the resistance of these mycobacteria could be due, in part, to their ability to decolorize the dyes. Small amounts of malachite green and its reduced, decolorized product were detected in the lipid fraction of M. avium strain A5 cells grown in the presence of malachite green, suggesting that a minor component of resistance could be due to sequestering the dyes in the extensive mycobacterial cell surface lipid. The membrane fraction of M. avium strain A5 had at least a fivefold-higher specific decolorization rate than did the crude extract, suggesting that the decolorization activity is membrane associated. The malachite green-decolorizing activity of the membrane fraction of M. avium strain A5 was abolished by either boiling or proteinase exposure, suggesting that the decolorizing activity was due to a protein. Decolorization activity of membrane fractions was stimulated by ferrous ion and inhibited by dinitrophenol and metyrapone.     

LmCDA1 organizes the cuticle by chitin deacetylation in Locusta migratoria

Cells produce an extracellular matrix (ECM) with a stereotypic organization that is important for tissue function. The insect cuticle is a layered ECM that mainly consists of the polysaccharide chitin and associated proteins adopting a quasi‐crystalline structure. Our understanding of the molecular mechanisms deployed during construction of the highly ordered protein–chitin ECM so far is limited. In this study, we report on the role of the chitin deacetylase 1 (LmCDA1) in the organization of the protein–chitin ECM in the migratory locust Locusta migratoria, and LmCDA1 localizes predominantly to the apical tier of the protein–chitin ECM, but it is also found in lower regions. Reduction of LmCDA1 function correlates with lower amounts of chitin and impedes conversion of chitin to chitosan by deacetylation. Establishment of the quasi‐crystalline architecture of the protein–chitin ECM is, however, independent of LmCDA1 activity, but it is dependent on another chitin deacetylase, LmCDA2, which has no detectable effects on chitin deacetylation and, as shown previously, no influence on chitin content. Our data reveal that LmCDA1 and LmCDA2 act in parallel and independently from each other in defining the dimensions of the cuticle. Both enzymes are non‐uniformly distributed within the protein–chitin matrix, suggesting a site‐autonomous function.     

Design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole.

It was the purpose of this study to design and evaluate a new bioadhesive vaginal drug delivery system for clotrimazole. Chitosan, a cationic biopolymer derived by deacetylation of chitin, was modified by the introduction of thioglycolic acid (TGA). The modification was achieved by utilising a carbodiimide to link the carboxylic acid moieties of TGA covalently to the primary amino groups of chitosan. The amount of added carbodiimide was thereby varied, resulting in chitosan-TGA conjugates A and B with 160 microM (=micromol) and 280 microM thiol groups per gram polymer, respectively. In order to characterise the new polymers the water uptake, the disintegration behaviour, the bioadhesive properties utilising the rotating cylinder method, as well as the release of clotrimazole from tablets based on these derivatives were studied. The water uptake and cohesive properties of vaginal tablets consisting of these new conjugates could be significantly (p<0.05) improved. By adding clotrimazole the disintegration time of the conjugates was prolonged 1.6-fold for conjugate A and even 100-fold for conjugate B. Furthermore, the adhesion on vaginal mucosal tissue could be significantly improved. The addition of clotrimazole had also an impact on the adhesion time of chitosan-TGA conjugate B, which remained 26-times longer on vaginal mucosa than the corresponding unmodified polymer. The immobilisation of thiol groups guarantees a controlled drug release. Results of this study demonstrate that these new chitosan-TGA conjugates are very promising vehicles for the vaginal application of clotrimazole in treatment of mycotic infections.     

经鼻置气管插管患者改进护理方法的效果分析

目的通过呼吸道护理改进,达到湿化气道、稀化痰液、解除支气管痉挛、改善通气功能的目的。方法持续气道滴入无菌蒸馏水,患者出现痰鸣音时及时吸痰,雾化吸入改为2次/h,每次15min,雾化液为蒸馏水加潘诺、盐酸氨溴索。通过痰液情况评估,比较改进技术与传统气道护理技术的区别。结果相对于传统气道护理组,经过改进气道护理技术护理的患者,气道痰液的黏稠程度都明显降低。结论改进的气道护理技术有利于保持气管插管患者气道的清洁程度,降低因痰液堵塞造成的并发症。     

不同制剂二氧化氯杀菌效果观察

目的观察不同制剂稳定性二氧化氯消毒液杀菌效果。方法采用悬液定量杀菌方法,对不同活化剂配制的二氧化氯消毒液杀菌效果进行了实验室检测。结果采用柠檬酸和盐酸作为活化剂,以亚氯酸钠作为二氧化氯原液,将二者同时投入到不同倍数水中进行活化。随着原液稀释倍数的增加,经柠檬酸和盐酸活化的二氧化氯对金黄色葡萄球菌、白色念珠菌和枯草杆菌黑色变种芽孢的杀灭效果均呈下降趋势。结论将活化剂与亚氯酸钠同时投入到水中,随着水量的递增,其杀菌效果下降;不能将活化与稀释两个过程同步进行,必须先活化后再稀释。     

Studies on the chitin/chitosan binding properties of six cuticular proteins analogous to peritrophin 3 from Bombyx mori

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated‐chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3‐A1, BmCPAP3‐A2, BmCPAP3‐B, BmCPAP3‐C, BmCPAP3‐D1 and BmCPAP3‐D2) were cloned and expressed in Escherichia coli and purified using metal‐chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3‐D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3‐D1 was similar to BmCPAP3‐A1 and BmCPAP3‐C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin‐binding protein, BmCPAP3‐D1, which exhibits high binding affinity to deacetylated chitin.     

改良复乳法制备神经生长因子微球及其体外释药性能

背景:已往研究表明,神经生长因子微球对脊髓损伤的修复有促进作用.然而,传统复乳法制备微球过程中诸多因素会严重影响生物大分子药物的生物活性,如何改善制备工艺提高微球的缓释性能至关重要.目的:课题提出改良复乳法制备神经生长因子微球的方法,并考察和验证其一般性质和体外释药特征.设计、时间及地点:随机对照细胞学实验,于2008-05/2009-05在温州医学院药学院及温州医学院附属第二医院骨科分子生物学实验室完成.材料:重组人神经生长因子(美国R&D公司),乳酸/羟基乙酸共聚物(聚乳酸/聚羟基乙酸75/25,Mw=20 000,黏度=0.025 L/g)(山东省医疗器械研究所);聚乙烯醇(日本可乐丽公司);聚乙二醇400(德国AppliChem公司).方法:以乳酸/羟基乙酸共聚物为载体材料,对传统复乳溶剂挥发法进行改进,采用全循环一体机装置制备神经生长因子缓释微球,对缓释微球的物理表观性质进行检测,然后通过EUSA+Kit法和PC12细胞共培养法来检测微球中神经生长因子的活性.主要观察指标:以微球形态、粒径、包封率等为评价指标,并通过体外释放率和PC12细胞活性考察其体外释药性质.结果:改良法和传统法制备的微球粒径分别为(22.61±3.94)μm和(21.32±4.82)μm,包封率分别为(92.08±4.39)%和(89.17±3.74)%.改良组的神经生长因子微球可以持续分泌有生物活性的神经生长因子长达7周,累积释放率为85.7%,而传统组的微球只能释放4周,累积释放率为60 8%,且第4周时释放的神经生长因子活性明显低于改良组,差异有显著性意义(P<0.01).结论:改良法制备的神经生长因子微球粒径适宜、包封率高,较传统复乳溶剂挥法制备的微球有更好的缓释性能.     

甲壳素类神经导管修复周围神经的研究与进展

目的:概述近年来甲壳素类材料制备神经导管修复周围神经损伤的研究进展.资料来源:应用计算机检索Medline和Springerlink 2000-01/2009-08有关神经导管材料修复周围神经损伤方面的文献,检索词"nerve conduit,peripheral nerve injury",限定文献语言种类为"English";同时检索中国期刊全文数据库、重庆维普数据库2000-01/2009-08相关文献,检索词"神经导管,周围神经损伤",限定文章语言种类为中文.资料选择:纳入与修复周围神经损伤有关的非生物降解材料、生物降解材料和生物衍生材料的文献.对资料进行初审,选取符合甲壳素类神经导管材料修复周围神经损伤要求的有关文章.排除标准相关度不大和重复性文章.结局评价指标:神经组织工程;甲壳素类神经导管材料;神经导管制备.结果:①随着医学材料的进展,天然或人工合成材料的神经导管用于桥接神经缺损的组织工程支架材料,具有引导和促进神经再生作用.生物可降解材料中甲壳素类神经导管可在合理的时间段内降解,有可控的生物亲和性、降解性能、多孔性和机械性能.②在导管结构、复合其他生物可降解材料、表面修饰、添加种子细胞及神经生长因子等方面进行实验研究.③通过改变再生室的空间结构和微环境,从而加快神经生长速度,促进神经功能的良好恢复.并对材料表面修饰及制备方法加以改进,使得导管适应神经再生.结论:随着生物学技术和其他相关技术的发展,甲壳素类神经导管材料在周围神经组织工程中的应用必将得到不断的展现.     

Highly efficient biodegradation of reactive blue 19 under the activation of tea residue by a newly screened mixed bacterial flora DDMY2

In this study, a newly screened mixed bacterial flora DDMY2 had high decolorization capacity for anthraquinone dye reactive blue 19 (RB19) and the decolorization efficiency of 300 mg L⁻¹ RB19 could reach up to 98% within 48 h in the presence of tea residue. Results indicated that RB19 could be efficiently decolorized by flora DDMY2 in wide ranges of pH values (5.0–9.0), temperatures (30–40 °C) and initial dye concentrations (50–500 mg L⁻¹) under the activation of tea residue. Concentration of tea residue had been proved to significantly impact the decolorization performance. UV-vis spectrophotometry, Fourier transform infrared spectrometry and liquid chromatography/time-of-flight/mass spectrometry analysis showed three identified degradation products and the possible degradation pathway of RB19 was speculated. High-throughput sequencing analysis revealed the community structures of bacterial flora before and after domestication by tea residue. Based on the result, it was inferred that unclassified_o_Pseudomonadales, Brevibacillus, Stenotrophomonas and Bordetella activated by tea residue were responsible for the excellent decolorization performance. Results of this research deepen our understanding of the biodegradation process of anthraquinone dyes by bacterial flora and broaden the knowledge of utilizing tea residue as a bioactivator in biological treatment.

Tea residue promoted the decolorization of RB19 by activating flora DDMY2, revealing corresponding degradation pathways and functional genera in DDMY2.     

中药复方对急性髓细胞性白血病干细胞Flt3和N-ras基因表达的影响

背景:在白血病患者体内存在一群白血病干细胞,这些恶性干细胞是白血病细胞存在和不断增殖的根源,针对干预白血病干细胞的特异性靶向治疗药物已成为近年来研究热点.目的:探讨中药复方对急性髓细胞性白血病干细胞Flt3和N-ras基因表达的影响.设计、时间及地点:随机区组实验,于2007-09/2008-12在山东中医药大学附属医院完成.对象:2006-2007年山东中医药大学附属医院、山东大学齐鲁医院血液科就诊的急性髓细胞性白血病初治患者50例,FAB分型M15例,M215例,M49例,M521例.方法:原方:黄芪、白花蛇舌草、小蓟、太子参、半枝莲、蒲公英、生地、黄精、女贞子、旱莲草、天冬、麦冬、白术、茯苓、甘草.扶正方:黄芪、太子参、生地、黄精、女贞子、天冬、麦冬、白术、茯苓、甘草.祛邪方:白花蛇舌草、小蓟、半枝莲、蒲公英、旱莲草.制剂:上述3组组方药物由山东中医药大学附属医院中药制剂实验室(国家三级重要制剂实验室)配制成1 g/mL的药液,无菌试验阴性后过滤除菌备用.常规无菌采集急性髓细胞性白血病M1,M2,M4,M5型患者骨髓各5 mL,稀释后加入淋巴细胞分离液,联合应用免疫磁珠分选系统和流式细胞仪分离纯化白血病干细胞.调整细胞浓度至2×108L-1,均分成4组:对照组不加药物,实验组分别加入原方、扶正方、祛邪方的对应中药制剂,终浓度为100 mg/L,继续培养48 h后进行指标检测.主要观察指标:RT-PCR法检测Flt3、N-ras基因的表达.结果:与对照组比较,原方组、扶正方组、祛邪方组Flt3表达阳性率均明显降低(P<0.05),但原方组降低幅度明显大于扶正方组、祛邪方组(P<0.05);扶正方组与祛邪方组Flt3表达阳性率比较无明显差异(P>0.05).4组N-ras表达阳性率与Flt3表达情况基本一致.结论:Flt3和N-ras基因在白血病干细胞中存在过度表达,中药原方及其拆方后的扶正方、祛邪方均能够降低白血病干细胞中Flt3和N-ras基因的表达水平.