成骨生长肽壳聚糖-海藻酸钠微球的制备及体外检测

背景：成骨生长肽体外注射可以刺激外周血和骨髓细胞数增加,增加动物的骨量,加速骨折愈合,但因多肽不稳定性及注射应用不方便,限制了其临床应用。 目的：应用乳化交联法制备成骨生长肽壳聚糖-海藻酸钠缓释微球,并对其粒径、载药、体外释药、理化特性进行检测。 方法：以戊二醛作为交联剂,应用乳化交联法制备具有控制释放功能的负载成骨生长肽壳聚糖-海藻酸钠微球,显微镜及扫描电镜观察微球的形态和粒径;利用酶联免疫吸附实验动态检测成骨生长肽壳聚糖-海藻酸钠微球的载药率、包封率和缓释规律。 结果与结论：乳化交联法制备的壳聚糖-海藻酸钠微球,球形良好,球体表面有较多微孔,具有较高的包封率(>72%)。体外药物释放实验表明,成骨生长肽可以从壳聚糖-海藻酸钠微球中缓慢释放,整个释放过程可达49 d,累积释放率>85%。提示应用乳化交联法制备的负载成骨生长肽壳聚糖-海藻酸钠缓释微球,具有很好的控制释放成骨生长肽的能力。     

磁性壳聚糖微球固定化乳糖酶及其酶学性质

背景:游离酶在使用过程中存在稳定性差、不能重复使用等问题,可利用磁性高分子微球作为结合酶的载体制备固定化酶,使其既保持酶的天然活性并可以重复使用,又为自动化生产管理提供便利的条件.目的:采用磁性壳聚糖微球作为结合酶的载体制备出便于回收、可重复使用、酶活及稳定性较高的固定化乳糖酶.方法:将一定量磁性壳聚糖微球加入磷酸缓冲...     

壳聚糖微球包裹幽门螺杆菌全菌蛋白抗原的制备及体外释放性能

背景：包裹幽门螺杆菌全菌蛋白抗原的研究仍处于探索阶段,有关壳聚糖微球包裹幽门螺杆菌全菌蛋白抗原的制备工艺及体外释放性能的文献甚少。 目的：探讨幽门螺杆菌全菌蛋白抗原壳聚糖微球的制备工艺及体外释放特性。 方法：采用沉淀法制备壳聚糖微球,筛选最佳制备工艺及配比、包裹时间,并在电镜下观察微球的形态和粒径。采用壳聚糖微球包裹幽门螺杆菌全菌蛋白抗原,BCA法测定幽门螺杆菌全菌蛋白抗原微球的包裹率、包裹量及体外释放率。 结果与结论：终体积分数为1%的冰醋酸、硫酸钠为交联剂、pH 5.0、滴加交联剂时不粉碎处理为壳聚糖微球最佳制备工艺,电镜观察显示微球表面光滑、形态圆整,具有良好的分散性,多数微球粒径为1.0-5.0 μm。幽门螺杆菌全菌蛋白抗原微球的包裹率为80.4%,包裹量为16.4%,48 h总释放率为19.4%,幽门螺杆菌全菌蛋白抗原微球整体呈缓慢释放状态。结果证实,实验制备的壳聚糖微球对幽门螺样菌全菌蛋白抗原具有良好的包裹率和包裹量,幽门螺杆菌全菌蛋白抗原微球整体呈缓慢释放状态。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程      

磁性壳聚糖微球的生物相容性

背景：采用壳聚糖对磁性氧化铁纳米颗粒表面进行改性,一方面可改善磁性纳米氧化铁颗粒的团聚性,增加其稳定性,另一方面将来可用于肿瘤热疗及基因治疗。 目的：制备将来可用于肿瘤热疗及基因治疗的磁性壳聚糖微球,评价其生物相容性。 方法：采用改良的化学沉淀法制备磁性氧化铁纳米颗粒。采用超声乳化法将壳聚糖加入到磁性氧化铁纳米颗粒中制备磁性壳聚糖微球,进行以下实验：①MTT实验检测磁性壳聚糖微球浸提液的细胞毒性：分别以1640培养液、聚丙烯酰胺单体溶液、100%,75%,50%,25%的磁性壳聚糖微球浸提液培养L-929细胞。②溶血实验：在兔抗凝血中分别加入磁性壳聚糖微球浸提液、生理盐水及蒸馏水。③微核实验：在昆明小鼠腹腔分别注射含氧化铁磁流体5,3.75,2.5,1.25 g/kg的磁性壳聚糖微球混悬液、环磷酰胺及生理盐水。 结果与结论：磁性壳聚糖微球粒径200-300 nm,分散效果有所提高。不同浓度的磁性壳聚糖微球浸提液对L-929 细胞毒性为1级,属对细胞无毒性范畴。磁性壳聚糖微球浸提液的溶血率为0.69%,小于5%,符合医用材料的溶血实验要求。磁性壳聚糖微球混悬液未导致细胞DNA断裂和非整倍体化,未导致微核产生的遗传毒理作用,材料无致畸或致突变作用,因此磁性壳聚糖微球具有良好的生物相容性。     

重组人卵透明带蛋白及其多克隆抗体对精子-透明带结合的影响

为了探讨毕赤酵母表达的重组人卵透明带ZP3蛋白(recombinant human zona pellucida-3 protein,rhZP3)及其多克隆抗体对小鼠和人精卵结合的影响,采用不同浓度的rhZP3以及空白培养液分别处理小鼠精子,然后再与小鼠卵子进行结合实验,观察经过不同处理的精子对透明带黏附及体外受精率的影响;用rhZP3以及空白培养液分别处理人精子,然后再与人卵子进行结合实验,观察经过不同处理的精子对透明带黏附的影响;用抗rhZP3抗体与阴性血清分别处理小鼠和人卵子,再与精子进行结合实验,观察多克隆抗体对精子粘附以及小鼠体外受精率的影响。实验结果表明,rhZP3和抗rhZP3多克隆抗体既能抑制人的体外精卵结合,也能抑制小鼠体外精卵结合,提示rhZP3具有天然透明带的特性,有发展成避孕疫苗和作为检测透明带抗体检测试剂的可能。     

壳聚糖微球复合丝素基硫酸钙骨水泥的理化特性

背景：脊柱成形和脊柱后凸成形治疗中采用的硫酸钙骨水泥理化性质好,对人体无毒性作用,同时具有降解性能,但单独使用降解较快。 目的：研制具有载药缓释功能的壳聚糖微球丝素基硫酸钙骨水泥。 方法：采用三聚磷酸钠乳化交联法制备壳聚糖微球。采用浓度分别为3%,6%,9%的丝素溶液与CaSO₄•0.5H₂O混合,通过万能力学试验机确定骨水泥力学性能最佳时的丝素浓度,在此浓度下,按壳聚糖微球占CaSO₄•0.5H₂O的质量比分别为0.5%,1%,5%的比例制备壳聚糖微球丝素基硫酸钙骨水泥,测定其抗压强度,并通过X射线多晶衍射仪及傅里叶红外光谱明确达到最佳抗压强度组的骨水泥成分,电镜观察复合骨水泥中壳聚糖微球的形态。 结果与结论：当丝素溶液浓度为6%,壳聚糖微球含量为0.5%时,复合骨水泥的抗压强度最大,为  (39.17±1.96) MPa,此时复合骨水泥的初凝时间为(12.99±1.63) min,终凝时间为(21.55±0.54) min;骨水泥中主要晶相组成为硫酸钙,傅里叶红外光谱结果证实复合骨水泥中含有丝素及壳聚糖;复合骨水泥中的微球表面稍有皱缩,但球形仍然完整,未见明显破坏,可见在制备复合骨水泥的过程中微球能保持稳定而不被破坏。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

胶原-壳聚糖载硫酸长春新碱微球缓释药膜的研究

目的本研究制备载硫酸长春新碱(vincristinesulfate,VCR)微球的胶原-壳聚糖缓释药膜。并考察加入壳聚糖对药膜性质的影响。选定适当的胶原壳聚糖比例制备药膜。方法采用W/O/O溶剂挥发法制备VCR的聚乳酸-羟基乙酸(poly(lactic-co-glycolicacid),PLGA)微球,并对微球性质表征,采用二次冻干法制备载VCR微球的胶原-壳聚糖药膜,对药膜的表面形态、降解性质、热力学性质及释放性质进行表征,并与释放2周后的药膜进行比较。采用高效液相法分析药物含量。结果VCR制成PLGA微球后再制备成药膜,可达到双重缓释的作用,明显减少药物突释,并延缓药物释放。添加了壳聚糖的药膜降解速度明显小于单纯的胶原药膜。在体外释放实验中,微球突释为(27.2±1.2)%,而胶原药膜的突释为(20.4±1.9)%,胶原与壳聚糖比例为9∶1、4∶1、3∶2的药膜突释分别为(20.2±2.1)%、(18.0±1.1)%和(16.3±1.8)%。结论胶原壳聚糖载VCR的缓释药膜能不同程度减少药物的突释,使药物释放更加平稳缓慢,优于单纯的胶原药膜。     

明胶海绵颗粒和壳聚糖α,β-甘油磷酸凝胶微球在肺结核咯血应用中的对比北大核心

背景:文献报道显示,明胶海绵颗粒和壳聚糖/α,β-甘油磷酸凝胶微球在肺结核咯血中具有较高的应用前景。目的:观察明胶海绵颗粒和壳聚糖/α,β-甘油磷酸凝胶微球在肺结核咯血中的应用效果。方法:取40只昆明小鼠,采用雾化吸入感染装置构建肺结核咯血模型,随机分为2组,分别采用明胶海绵颗粒、壳聚糖/α,β-甘油磷酸凝胶微球进行栓塞止血治疗,治疗后15,30 d,观察体质量变化、止血时间、止血有效率、心肌相关指标及肺结核组织病理变化。结果与结论:(1)壳聚糖/α,β-甘油磷酸凝胶微球组治疗后15 d的体质量高于明胶海绵组(P<0.05);(2)壳聚糖/α,β-甘油磷酸凝胶微球组止血有效率高于明胶海绵组(P<0.05),止血时间短于明胶海绵组(P<0.05);(3)两组N端BNP前体、肌酸激酶同工酶及肌酸激酶水平比较差异无显著性意义(P>0.05);(4)壳聚糖/α,β-甘油磷酸凝胶微球组材料周围淋巴细胞浸润,存在坏死病灶,材料上存在大量活性细胞;明胶海绵颗粒组材料上存在少许淋巴细胞,存在坏死病灶;(5)结果表明,将壳聚糖/α,β-甘油磷酸凝胶微球应用于肺结核小鼠动物模型中可获得更好的止血效果。     

重组人卵透明带蛋白3的制备及其免疫学活性分析

目的:纯化制备rhuZP3,并分析其免疫学活性.方法:在含重组质粒pGEX4T-1/ huZP3 的大肠杆菌 BL21中,经IPTG诱导表达出GST-融合蛋白,蛋白经过一系列的纯化,然后SDS-PAGE电泳鉴定蛋白纯度.rhuZP3免疫小鼠,ELISA法检测抗血清对rhZP3的抗体反应.结果:表达出了可溶性融合蛋白,纯化后的rhuZP3纯度达95%.而且它在ELISA 鉴定实验中能被抗rhuZP3抗体识别.结论:通过原核表达系统制备的rhuZP3及其抗体具有免疫学活性.     

人同种异体骨载异烟肼-壳聚糖缓释微球的制备和体内外释药特性☆

背景：壳聚糖微球具有良好的生物相容性及抗菌活性,被广泛地运用于各种药物缓释系统中。 目的：制备人同种异体骨载异烟肼-壳聚糖微球,并分析其体内释药性能。 方法：用喷雾干燥法制备异烟肼-壳聚糖微球,进行体外45 d的药物释放实验。将单独装载异烟肼的异体骨块(对照组)和装载异烟肼-壳聚糖微球的异体骨块(实验组)分别植入家兔两侧髂骨,采用高效液相色谱法检测药物体内释放情况。 结果与结论：异烟肼-壳聚糖微球外观呈圆形、表面光滑、分散良好;平均粒径(3.33±0.9) μm,载药率(16.25±1.24)%。体外药物释放实验显示无突释现象,24 h释放20%左右,45 d释放76%,释放曲线较平缓,释放稳定;数学模型拟合符合Ritger-Peppas模型。实验组异烟肼浓度在前28 d内缓慢升高,其后缓慢下降,持续56 d以上,浓度38.50~155.75 µg/g;对照组异烟肼浓度在1周左右达高峰,为1982.5 µg/g,21 d后骨块周围药物不能测到。说明异烟肼-壳聚糖缓释微球在体内外均可以缓慢平稳释放异烟肼,且持续时间长。提示人同种异体骨载异烟肼-壳聚糖缓释微球复合体可以作为骨结核病灶清除后的一种置入材料,在提供机械支持的同时进行长时间的局部化疗。     

实验性自身免疫性卵巢炎卵巢功能早衰的机理研究

目的：探讨卵透明带免疫对卵巢功能的损伤及作用机理。方法：分别以猪卵透明带抗原和缓冲液通过皮内及两后脚掌多点注射免疫Ｂａｌｂ／ｃ小鼠，每２ｗ免疫１次，连续免疫３次。经３次免疫后，测定外周血雌二醇浓度和血清抗卵透明带抗体滴度，观察卵巢组织病理形态学及免疫组化，分析ＩＬ－１，ＥＧＦ在卵巢中的表达。结果：免疫小鼠外周血抗ＳＰ抗体滴度明显高于对照组，而雌二醇浓度则明显低于对照组，且二者存在显著负相关。     

Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated

Partial zona dissection (PZD) of human oocytes facilitates spermpenetration through mechanically made holes in the zona pellucida.Only 1 of 69 eggs was damaged when sucrose was used to shrinkthe ooplasm during micromanipulation. The fertilization rateof micromanipulated oocytes in 18 couples with male factor infertilitywas 68% (34/50), which compared favourably with inseminationof non-micromanipulated controls (21/45, 47%). PZD was advantageousin oligozoospermic patients, but not in cases of asthenozoospermia,combined semen problems or immunological infertility. Threetwin and two singleton pregnancies resulted following replacementof 23 micromanipulated and eight control embryos in 14 patients.No differences in embryo morphology and development rates werefound between the micromanipulated and control groups. The incidenceof polyspermy in couples with abnormal semen analyses was relativelylow (<20%) possibly due to partial activation of the oocytesfollowing exposure to sucrose. Polyspermy was high (57%) innormozoospermic patients with either immunological infertility(n= 3) or failure of fertilization in previous cycles (n= 4).In the three immunological patients, nine of 11 hyaluronidaseand sucrose-exposed control embryos fertilized and six implanted,possibly indicating that cumulus and corona cells are contributingfactors inhibiting fertilization in such cases.     

人类卵泡糖蛋白3在分子生殖医学上功能的研究

卵泡糖蛋白,为成熟卵细胞的胞外间质组织－透明带（zona pellucida）的主要组成分子,近代研究报告显示：卵泡糖蛋白3（zona pellucida glycoprotein3）在精－卵间的交互作用上担任重要的角色,并被认为是特发性不孕症（idiopathic infertility）的可能主因。为研究卵泡糖蛋白3于精－卵交互作用中,所担当的生物功能与性。本研究应用分子生物和基因工程技术,以制造重组卵泡糖蛋白3,并进一步运用临床细胞分析技术,包括法卵泡试验法（hemizona assay）和精子尖体反应试验法（acrosome reaction assay）,以试验重组糖蛋白之生物活性。实验结果显示：分离之糖蛋白产物呈现抑制（＞40％）精－卵结合,和诱导（＞50％）精子尖体反应的能力,支持人类卵泡糖蛋白具有影响人类精－卵结合,与诱导精子尖体反应的双重功能。     

Genetic disorders in premature ovarian failure

This review presents the genetic disorders associated with premature ovarian failure (POF), obtained by Medline, the Cochrane Library and hand searches of pertinent references of English literature on POF and genetic determinants cited between the year 1966 and February 2002. X monosomy or X deletions and translocations are known to be responsible for POF. Turner's syndrome, as a phenotype associated with complete or partial monosomy X, is linked to ovarian failure. Among heterozygous carriers of the fragile X mutation, POF was noted as an unexpected phenotype in the early 1990s. Autosomal disorders such as mutations of the phosphomannomutase 2 (PMM2) gene, the galactose-1-phosphate uridyltransferase (GALT) gene, the FSH receptor (FSHR) gene, chromosome 3q containing the Blepharophimosis gene and the autoimmune regulator (AIRE) gene, responsible for polyendocrinopathy-candidiasis-ectodermal dystrophy, have been identified in patients with POF. In conclusion, the relationship between genetic disorders and POF is clearly demonstrated in this review. Therefore, in the case of families affected by POF a thorough screening, including cytogenetic analysis, should be performed.     

Antiovarian antibody in premature ovarian failure

Premature ovarian failure is a syndrome consisting of primary or secondary amenorrhoea, hypergonodotropiremia and hypoestrogenemia in women under the age of 40. An autoimmune mechanism was suggested as possible etiology when Vallolton and Forbes in 1966-67 found antibodies to the cytoplasm of rabbit ova in 29 of 232 tested sera. Immune mechanism in the pathogenesis of premature ovarian failure (POF) is suggested by association of autoimmune phenomenon with POF in some cases and demonstration of circulating antibodies to ovary in serum samples from women with POF. The incidence of presence of antiovarian antibody of POF patients has been reported earlier. Evidence of autoimmunity is present in 18-92% of patients with POF. In the present study we have studied 18 cases of POF without any overt manifestation of autoimmune disorder but the antiovarian antibody was detected, with the idea that this autoantibody might be the cause of ovarian dysfunction which is evident in POF. Presence of antiovarian antibody in 16.67% cases with POF in our study that ovarian antibodies may play a role in or reflect an autoimmune process responsible for the development of POF.     

Incipient ovarian failure and premature ovarian failure show the same immunological profile

PROBLEM: Incipient ovarian failure (IOF) is characterized by regular menstrual cycles, infertility and a raised early-follicular FSH in women under 40. IOF might be a precursor or a mitigated form of premature ovarian failure (POF). Disturbances in the immune system may play a role in ovarian failure. METHOD OF STUDY: Autoantibodies and lymphocyte subsets were determined in 63 POF patients, 50 IOF patients, and 27 controls. RESULTS: The prevalence of autoantibodies did not differ between the groups. There was a statistically significant difference in lymphocyte subsets between the control group and the POF group, with the IOF group taking an intermediate position. We found a decrease in percentage of T-suppressor cells with a rise in T-helper/T-suppressor cell ratio, a decrease in natural killer cells, and an increase in B lymphocytes and HLA-DR positive T cells. CONCLUSIONS: These data support the concept that IOF is a mitigated form of POF. The question remains whether these changes are the cause or the consequence of the ovarian failure.     

Preparation and preliminary characterization of concentric multi-walled chitosan microspheres

Chitosan was first converted into micro-droplets by using a high voltage electrostatic field system. The droplets were then dropped into a series of Na(5)P(3)O(10)/NaOH solution mixtures with volume ratio of 17:3, 19:1, 1:0 (pure aqueous Na(5)P(3)O(10)) or 0:1 (pure aqueous NaOH) in order to fabricate chitosan microspheres with different membrane structures. The microspheres exhibit distinct chemical and physical properties, including release behaviors of encapsulated drugs. These chitosan microspheres prepared by this method exhibited good sphericity within the range of (286.6 +/- 15.9) to (356 +/- 9.5) microm in diameters. SEM observations have indicated that the chitosan microspheres exhibited distinct surface structures depending on the post-treatment solutions. The mechanical strength of the chitosan microspheres significantly improved upon treatment with Na(5)P(3)O(10)/NaOH solution at ratio of 17:3 (v/v), as compared with the same but at ratio of 19:1, 1:0 (pure Na(5)P(3)O(10)) and 0:1 (pure NaOH) solutions. In addition, chitosan microspheres with unique multi-walled concentric shell membrane structures were prepared by treating with Na(5)P(3)O(10)/NaOH solution at ratio of 19:1. Release studies were carried out to evaluate the kinetic profiles of two model drugs (5-fluorouracil and cytochrome C) from these prepared chitosan microspheres. When chitosan microspheres treated with Na(5)P(3)O(10)/NaOH ratio at 17:3, the release of cytochrome C was found to be the slowest as compared to those treated by the same Na(5)P(3)O(10)/NaOH solution of other mixing ratios, after a period of 35-day "endurance" test. However, in one case, 5-fluorouracil released quite quickly in a period of 30 min (about 80% completion). The wide range of drug release results might be attributed to the unique and wide range of surface characteristics, porosities, and various structures of chitosan microspheres upon treatment with Na(5)P(3)O(10)/NaOH solutions. These results indicate that, by adjusting the Na(5)P(3)O(10)/NaOH ratios, without extra manipulation on polymer material formulation, one could obtain an additional degree of freedom in drug release profile that permits the simultaneous regulation of morphologies of surface texture and internal structure, mechanical properties, and molecular permeability of the microspheres.     

Biosynthesis and expression of zona pellucida glycoproteins in mammals

The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three glycoproteins (ZPA, ZPB, ZPC), products of the gene families ZPA, ZPB and ZPC that have been found to be highly homologous within mammalian species. Most data on the structure and function of the ZP are obtained from studies in mouse. New data from pig and other domestic animals, however, indicate that the mouse model does not hold for all other species. Whereas in the mouse ZPB is the primary sperm receptor, in the pig ZPA has been shown to possess receptor activity. Contrary to the mouse, where the growing oocyte is the only source of zona glycoproteins, in domestic animals these proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play also a role in granulosa cell differentiation. In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins (oviductins) that become closely associated with the ZP of the ovulated oocyte. Once bound to the ZP, oviductin molecules could act as a protective layer around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains.