微孔高分子PLGA涂层疏水性及血小板粘附行为的研究

利用水蒸气辅助自组装方法在心血管支架用316L不锈钢表面上制备出了规则排列的微孔丙交酯乙交酯共聚物(PLGA)薄膜.实验研究表明,大气湿度和聚合物溶液浓度对孔径大小和分布有较大影响.接触角检测结果表明,微孔结构改变了PLGA涂层的亲疏水性质,呈现出疏水特性,而致密涂层及不锈钢基体表面则为亲水性.血小板粘附实验显示血小板在孔径小于3～5μm微孔涂层的表面几乎不粘附;在孔径大于5μm微孔涂层表面有微量粘附;而在致密涂层和不锈钢表面则发生粘附、聚集,甚至产生伪足.这一研究结果证明:通过水蒸气辅助自组装法制备的PLGA微孔涂层具有较强的抗血小板粘附的能力,有助于提高金属血管支架表面的血液相容性.     

基于电穿孔和孔径变化方程的球形细胞微孔特性研究

为进一步探究电穿孔理论机制,通过COMSOL建立单细胞电穿孔二维轴对称模型,该模型同时纳入表征微孔密度的电穿孔渐进方程和表征微孔动态演变过程的孔径变化方程,且模型的轴对称性使穿孔面积的计算更为准确,从而得到微孔的时空分布特性,并在此基础上探讨场强和脉宽对该特性的影响。结果表明:脉宽100μs、场强2kV/cm的脉冲作用下,产生微孔7862个,穿孔面积达细胞表面积的6.3%,电穿孔各参量的时空分布规律与文献结果一致,从而可验证所建模型的有效性;在1～5kV/cm范围内增大脉冲场强,微孔数与场强成正比,孔径则与场强成反比,孔面积与细胞面积之比从1.3%增至12.9%;对两组能量相同的纳秒脉冲和微秒脉冲进行比较,发现脉冲结束时前者产生的微孔数是后者的353.1倍,而在细胞膜上最靠近电极的点,后者的孔径是前者的19.3倍,说明纳秒脉冲有利于微孔数增加,而微秒脉冲有利于孔径扩大。仿真结果表明,微孔特性决定电穿孔的发生和发展过程,微孔特性的精确计算是阐释电穿孔效应的关键所在。     

羟基磷灰石与骨界面抗张强度测试及扫描电镜观察

本文测试了孔隙率为１４％，孔径小于２μｍ的纯羟基磷灰石（ＨＡ）与兔胫骨皮质骨之间界面的抗张强度，２周时为０．７２ＭＰａ；４、８、１６周之间强度无明显差异平均为１．５ＭＰａ。扫描电镜观察表明，２周时拉伸断裂位于ＨＡ－骨界面，４周以后多在ＨＡ晶体颗粒之间。ＨＡ与骨生物适应性好，矿化组织可以直接沉着于ＨＡ表面或ＨＡ内部，近界面处的ＨＡ发生了一定的生物降解反应，且骨矿化与ＨＡ的降解可能达到某种平衡。     

CEA时间分辨荧光免疫分析

以ＣＥＡ单克隆抗体Ｃ５０包被微孔板,异硫氰酸苄基二乙烯三胺四乙酸络合Ｅｕ＾３＋标记Ｃ１７单抗。采用平衡法建立ＣＥＡ时间分辨荧光免疫分析,数据采用Ｌｏｇ＝Ｌｏｇｉｔ函数数据自理程序处理。方法的批内和批间ＣＶ分别为２．９７％和１．６０％,平均回收率为１０１．９８％,灵敏度为０．２０μｇ／Ｌ,可测范围为２．３９～５０８．９μｇ／Ｌ,ＥＤ５０为６０．９１μｇ／Ｌ。本方法与ＡＦＰ和ＣＡ１２５和ＣＡ１５３无交     

微孔化羊脱细胞真皮基质复合人脐带间充质干细胞的实验研究

目的观察人脐带间充质干细胞(hUCMSC)在微孔化羊脱细胞真皮基质(ADM)中的生长特性,探索制作hUCMSC复合微孔化羊ADM生物敷料的可行性。方法制作羊ADM,并对其微孔化处理。体外分离培养hUCMSC。将hUCMSC分别种植于羊ADM和微孔化羊ADM上,观察hUCMSC的生长增殖情况。结果羊ADM颜色呈瓷白色,苏木精-伊红HE染色显微镜下未见细胞成分、血管及皮肤附件残留无细胞碎片。微孔化羊ADM颜色呈白色,苏木精-伊红HE染色显微镜下可见较为均匀的孔径。将hUCMSC分别种植于羊ADM和微孔羊ADM培养6 d后显微镜下观察,hUCMSC在羊ADM表面贴壁生长,hUCMSC在微孔化羊ADM的表面及内部孔径里呈梭形贴壁生长。结论 hUCMSC能够沿着微孔在羊ADM生长,微孔化羊ADM能够为hUCMSC提供一个良好的生物三维载体。     

味感受器模型薄膜的电阻和电容特性

味感受器模型薄膜的电阻和电容特性［日］／小岛洋一郎…／／ＢＭＥ．—１９９４，８（１２）．—４１我们研究了微孔ＤＯＰＨ膜的电特性。该薄膜是由细孔上吸附有合成类脂物的微孔过滤物组成的，由压力刺激产生的薄膜阻抗的振荡现象与部分机械感受器薄膜相仿，然而，很难...     

肾衰竭及恶性肿瘤等患者血清中sIL－2R水平

本文报道用双抗体夹心ＥＬＩＳＡ检测多种疾病患者血清ｓＩＬ－２Ｒ水平。认为用市售试剂盒应选用合适的微孔板并确定相应的合适抗体稀释度。结果显示，肾衰竭患者和未经治疗的恶性肿瘤患者的ｓＩＬ－２Ｒ水平显著高于正常对照，数例何杰金氏淋巴瘤患者血清ｓＩＬ－２Ｒ尤为增高。上述结果与文献报道结果接近。本文支持肾功能衰竭致使ｓＩＬ－２Ｒ不能及时降解清除是该病患者血清ｓＩＬ－２Ｒ高水平的主要原因，而在恶性肿瘤患者的免疫抑制状态中高水平的ｓＩＬ－２Ｒ起重要作用。     

测定红细胞变形性的微孔滤膜及其孔径

在微孔过滤法测定红细胞变形性的实验中,微孔滤膜是最关键的部分。我们从表观特性和过滤特性两个方面对目前使用的不同类型的滤膜进行了比较,发现具有规则一致几何特性的镍膜,对物理、化学,尤其是病理因素引起的红细胞变形性改变最敏感。以往普遍认为,3μm孔径的滤膜对红细胞平均体积的改变最敏感,对红细胞内粘度的改变不敏感,而5μm孔径的滤膜则相反。我们通过对使用3μm和5μm滤膜的实验结果的分析,纠正了这一片面的观点,认为较小的滤膜孔径对红细胞变形性的测定更合适。     

肾衰竭及恶性肿瘤等患者血清中sIL—2R水平

本文报道用双抗体夹心ＥＬＩＳＡ检测多种疾病患者血清ｓＩＬ－２Ｒ水平。认为用市售试剂盒应选用合适的微孔板并研究相应原合适抗体稀释度。结果显示，肾衰竭患者和未经治疗的恶性肿瘤患者的ｓＩＬ－２Ｒ水平显著高于正常对照，数例何杰金氏淋巴瘤患者血清ｓＩＬ－２Ｒ尤为增高。上述结果与文献报道结果接近。本文支持肾功能衰竭致使ｓＩＬ－２Ｒ不能除解清除是该病患者血清ｓＩＬ－２Ｒ高水平的主要原因，而在恶性肿瘤患者的免疫抑     

胰腺癌相关抗原PCA_2－Ag的酶联免疫分析

应用建立的单克隆抗体ＰＣＡ_２的ＥＬＩＳＡ结合抑制法对５０名正常人，３１例经手术病理证实的胰腺癌，１７例急性胰腺炎和８例慢性胰腺炎进行检测。结果表明，单抗ＰＣＡ_２所对应的抗原（ＰＣＡ_２－Ａｇ）在胰腺癌患者血清中的平均含量显著高于正常对照组（Ｐ＜０．００１）。ＰＣＡ_２－Ａｇ诊断胰腺癌的灵敏度为７７．４％，特异性为９５．７％。ＰＣＡ_２－Ａｇ与ＣＡ１９－９、ＣＥＡ联合检测可提高胰腺癌的正确诊断率。ＰＣＡ_２－Ａｇ的检测对胰腺癌具有一定的诊断价值。     

APA微胶囊膜厚的理论计算与实验研究

以静电脉冲技术成功地制备了海藻酸-聚-L-赖氨酸-海藻酸（APA）生物微胶囊，结合元素分析方法，推导出膜厚的理论计算公式，通过扫描电子显微镜（SEM）和光学显微镜测定了囊膜厚度，实验结果表明膜厚的理论计算和实验测定值一致，APA微胶囊膜厚约为7-10μm。     

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。     

永生化下颌骨髁突软骨细胞的微囊化研究

为了探讨微囊包裹软骨细胞在软骨组织工程中的适用性 ,根据气流切割原理采用海藻酸钠 -多聚赖氨酸 -海藻酸钠 ( APA)对永生化下颌骨髁突软骨细胞 ( Im mortalized mandibular condylar chondrocyte,IMCC)进行微囊包裹。用倒置显微镜观察、台盼蓝染色、细胞记数、HE染色、免疫组化等方法检测微囊的大小、细胞的生长及微囊内组织的软骨特性等情况。研究发现 ,IMCC可在微囊内存活 ,活细胞率 >80 % ,微囊直径平均 779μm。细胞数量随着培养时间的延长逐渐增多 ,约 2 0 d左右达到平台期 ,细胞在囊内呈簇样生长 ,高表达软骨特异的蛋白多糖和 型胶原。提示 IMCC可在微囊内形成类软骨组织样结构 ,微囊技术适用于包裹软骨细胞     

Histological techniques for preservation of alginate bead structural integrity using glycolmethacrylate

Abstract

The applications of alginate as a drug delivery vehicle and 3D cell culture scaffold have become increasingly popular in the fields of biomaterials and tissue engineering. Although histological analysis of intact alginate scaffold and cells is a critical aspect of investigations, the existing methods of histological processing often lead to distortions in the scaffold shape, resulting in poor image quality and misrepresentation of the cellular environment. For this reason, the use of glycol methacrylate (GMA) has been explored as an embedding material of alginate scaffolds for histological analysis of embedded cells. The results of the present study demonstrated that soaking alginate scaffolds in a barium chloride solution prior to fixation in neutral buffered formalin (NBF) was a critical step for maintenance of the structure. By slowly infiltrating the hydrogel matrix with GMA using a commercially available embedding kit, the techniques developed in the present study allowed preservation of alginate bead structural integrity and successful staining of the embedded cells.     

Culture and Growth Characteristics of Chondrocytes Encapsulated in Alginate Beads

Methodology is described for the culture of avian and mammalian chondrocytes in ionotrophically gelled “semisolid” and “hollow” alginate beads. Chondrocytes grown in “semi-solid” gels exhibited a spherical shape as opposed to a fibroblastic morphology observed in monolayer culture. In the “semi-solid” beads, the cells grew as small clumps and as large aggregates. The aggregates were round or elliptical in appearance and surrounded by a dense Alcian Blue positive halo. Preliminary studies with collagen and chitosan matrixes encapsulated in “hollow” beads suggest that cell growth and morphology are profoundly influenced by the composition of the cellular environment. Chondrocyte structure and function in the “semi-solid” and “hollow” beads were partially characterized by light microscopy, histochemical and biochemical means. The encapsulation methodology is readily applicable for the culture of chondrocytes in single beads, in multiwell dishes, or mass culture.     

Development of alginate gel dressing

Alginate-based wound dressing materials have been widely used to promote wound healing and to reduce blood loss from wounds. However, recently a few drawbacks of well-established commercial alginate dressings have been reported. Therefore, we tried to develop a new alginate dressing to reduce the drawbacks. First, four new dressings with different calcium content were prepared, and the cytotoxicity of these four materials, and Kaltostat and Sorbsan, was tested in vitro by culture of fibroblasts with their extracts. Second, full-thickness wounds in pigs were used for the evaluation of wound healing in vivo. Finally, a newly developed alginate dressing was used clinically for treatment of split-thickness skin graft donor sites. The extract medium from ALG3, ALG4, Kaltostat, and Sorbsan induced a significant inhibitory effect on proliferation of fibroblasts. As for wound closure rate, the ALG2-covered wounds had the smallest wound area on day 15. Histologically, foreign-body reaction was least in ALG2-treated wounds. In a clinical study, the main drawback of ALG2 was leakage of wound exudate due to dissolution of the dressing material. However, the transparency of moistened ALG2 allowed easy evaluation of the wound, and after healing it was easy to remove ALG2 from the wound without injury to the reepithelialized skin because ALG2 was relatively nonadherent to the wound.     

新型mucA基因突变的铜绿假单胞菌生物被膜的形成和藻酸盐相关基因表达的研究

目的 确定黏液型铜绿假单胞菌PA17的mum基因突变位点，研究藻酸盐合成相关基因在其生物被膜形成过程中的表达，并观察PAl7生物被膜形成过程和形态。方法 PCR方法扩增铜绿假单胞菌PA17的mueA基因全长并测序；改良平板培养法建立PA17的生物被膜模型，半定量RT-PCR测定生物被膜形成24h、3d．6d时藻酸盐合成相关基因,algD、algU和algR的表达，并进行统计学分析；扫描电镜观察不同时间点的生物被膜形态。结果 PA17的mucA基因第166～333位核苷酸片段缺失，第342位A→G；其藻酸盐相关基因algD和algU均在生物被膜形成过程的第6天表达水平最高，algR在24h表达最高，单因素方差分析显示，上述基因在生物被膜形成过程不同时间点表达的差异有统计学意义；PA17于第6天形成成熟生物被膜，形态为薄膜状。结论 PA17是一株含新型mucA突变基因的黏液型铜绿假单胞菌，其藻酸盐相关基因在生物被膜形成的不同时间点的表达差异具有统计学意义，其生物被膜形态为薄膜状。     

Modulation of systemic cytokine levels by implantation of alginate encapsulated cells

The availability of cell lines that are transfected with IL-4, IL-5 and IFN-γ cytokine genes permits the prolonged in vivo delivery of functional cytokines in relatively large doses for the modulation of specific immune responses. Often the transfected cells are xenogeneic or allogeneic to the experimental animal and have to be encapsulated in such a way that no cellular response by the host will be induced. Alginate has proven to be a simple matrix for encapsulating cells under mild conditions suitable for in vivo implantation. Encapsulated cells express the transfected IL-4 gene for at least 14 days after in vivo implantation and were shown to be functional during that period by modulating ongoing IgE responses. The application of adherent growing transfected cells permits dose-response titrations and provides an easy method for local and systemic cytokine delivery. Alternatively, hybridoma cells can be encapsulated and the secreted antibody monitored in the serum. It was found that no host immune response was triggered by alginate encapsulated cells. The efficiency of treatment by encapsulated cells was shown to be equivalent to that of injecting purified antibodies.     

几丁糖/海藻酸敷料止血性能的实验研究

目的研究一种新型敷料：几丁糖／海藻酸敷料（本项目组自行研制，已申请国家专利）的止血性能。方法取新西兰兔4只，在背部两侧对称性剪5个直径1cm的圆型创口，分别与创面大小相当的几丁糖／海藻酸敷料和明胶海绵止血，观察与创面的粘附情况，记录出血时间：止血停止后，将几丁糖／海藻酸敷料和明胶海绵放入预先配制好的氰化高铁血红蛋白检测试剂中仔细清洗，用分光光度计在540nm波长处光度比色，测出的Hb光度吸收值表示出血量。结果几丁糖／海藻酸敷料与创面粘附较好，几丁糖／海藻酸敷料、明胶海绵组的出血时间分别为87.7±19.1妙、170.7±22.6妙，Hb光度吸收值分别为1.131±0.44、1.733±0.733，经统计学分析，两组数据都有显著性差异（P〈0.01），几丁糖／海藻酸敷料组明显优于明胶海绵组。结论几丁糖／海藻酸敷料具有较好的止血性能。     

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r² = 0.47, p < 0.001) and GAG content (r² = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r² = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.