CS/HACC/GP温敏水凝胶复合MSCs的实验研究

目的制备壳聚糖(CS)/壳聚糖季铵盐(HACC)/甘油磷酸钠(GP)温敏性水凝胶,检测其成胶时间、超微结构、渗透压、生物安全性等性质,将其和骨髓间充质干细胞(MSCs)复合培养,观察MSCs的生长情况。方法制备壳聚糖季铵盐,将壳聚糖/壳聚糖季铵盐/甘油磷酸钠按一定配比制备温敏性水凝胶,观察其成胶情况、内部超微结构,测量孔隙率、孔径大小,评估其降解情况,进行生物安全性检测。将MSCs与支架复合培养,倒置显微镜观察凝胶表面细胞生长情况,扫描电镜观察凝胶内部细胞的生长、增殖情况。结果成功制备HACC,水凝胶37℃时10分钟可成胶,生物安全性好,渗透压为290~310 mmol/kg,水凝胶无细胞毒性,其浸提液对小鼠体重增加无影响。扫描电镜见内部结构多空疏松的三维网状结构,孔径50~100um,孔隙率≥85%,降解时间约12周。MSCs在水凝胶中能正常生长、增殖。结论本实验证明CS/HACC/GP温敏水凝胶孔隙率、孔径大小合适,无细胞毒性,适合MSCs生长、增殖,具有较大的潜力成为神经组织工程中的细胞支架。     

壳聚糖水凝胶与星形胶质细胞体外生物相容性评价的实验研究

研究壳聚糖水凝胶材料与星形胶质细胞的体外生物相容性,初步探讨壳聚糖水凝胶作为神经组织工程支架材料的可行性.利用氯化壳聚糖、β-甘油磷酸钠和羟乙基纤雏素制备壳聚糖水凝胶,MTT法评价其细胞毒性;体外培养鉴定新生Wistar大鼠脑皮层星形胶质细胞;壳聚糖水凝胶与星形胶质细胞体外共培养,观察星形胶质细胞在材料上的生长;MTT...     

负载肝细胞生长因子水凝胶对心肌梗死组织区域的作用

背景：研究证实肝细胞生长因子可显著减轻糖尿病大鼠心肌梗死后心肌细胞凋亡、改善心功能,但是以往的研究多是静脉注射或是基因转染的给药方式,利用药物缓释系统给药的研究不多见。目的：以水凝胶作为肝细胞生长因子的载体,有效注射至心肌梗死部位,观察其改善心功能的效果。方法：制备甘油磷酸钠/壳聚糖/海藻酸钠/肝细胞生长因子水凝胶,检测该水凝胶对心肌细胞的相容性与体外生长因子释放特征。取3月龄雄性SD大鼠54只,结扎冠状动脉左前降分支复制心肌梗死模型,采用随机数字表法分3组处理：未治疗组于心肌梗死边缘分多点注射PBS,无生长因子水凝胶组于心肌梗死边缘分多点注射甘油磷酸钠/壳聚糖/海藻酸钠水凝胶,载生长因子水凝胶组心肌梗死边缘分多点注射甘油磷酸钠/壳聚糖/海藻酸钠/肝细胞生长因子水凝胶,每组18只,于设定时间点进行相关检测。结果与结论：(1)活死染色与CCK-8实验结果显示,甘油磷酸钠/壳聚糖/海藻酸钠/肝细胞生长因子水凝胶具有良好的细胞相容性,可促进心肌细胞的增殖与存活;甘油磷酸钠/壳聚糖/海藻酸钠/肝细胞生长因子水凝胶可持续释放肝细胞生长因子达42 d以上;(2)造模后第7,14,35天,与未治疗组...     

壳聚糖水凝胶与星形胶质细胞体外生物相容性评价的实验研究

研究壳聚糖水凝胶材料与星形胶质细胞的体外生物相容性,初步探讨壳聚糖水凝胶作为神经组织工程支架材料的可行性。利用氯化壳聚糖、β-甘油磷酸钠和羟乙基纤维素制备壳聚糖水凝胶,MTT法评价其细胞毒性;体外培养鉴定新生Wistar大鼠脑皮层星形胶质细胞;壳聚糖水凝胶与星形胶质细胞体外共培养,观察星形胶质细胞在材料上的生长;MTT法检测接种后1、35、、7d的细胞增殖度。体外成功制备壳聚糖水凝胶,该材料无细胞毒性;体外分离、培养得到状态良好的星形胶质细胞;体外星形胶质细胞在材料上培养呈星形,生长良好,有分支状突起形成;MTT结果表明,材料-细胞共培养组中的细胞增殖度明显高于单纯细胞组（P〈0.001）。壳聚糖水凝胶与星形胶质细胞在体外具有良好的生物相容性,有望作为神经组织工程支架材料。     

骨髓间充质干细胞复合温敏水凝胶支架的制备

背景：壳聚糖及其衍生物制备的支架对细胞迁移和神经轴突再生有重要作用。壳聚糖及其衍生物的组织相容性好,易使干细胞在其表面附着生长,在神经组织工程具有较为广阔的应用前景。 目的：制备适宜骨髓间充质干细胞生长的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,观察骨髓间充质干细胞在细胞支架中的生长情况。 方法：将壳聚糖进行季铵盐化改性处理,通过傅里叶变换红外光谱分析谱检测确定其生成。实验以壳聚糖与壳聚糖季铵盐配比为8∶1成功制备出较为稳定的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性温敏水凝胶细胞支架,观察成胶情况,并进行生物安全性检测。 结果与结论：实验在傅里叶变换红外光图谱上发现了季铵基基团的特征峰。细胞毒性实验显示,水凝胶浸提液干预的大鼠骨髓间充质干细胞无毒性。急性全身毒性实验显示,浸提液对大鼠体质量增加无明显影响,支架生物安全性较好。扫描电镜观察显示,骨髓间充质干细胞在细胞支架中能正常的生长和增殖。结果证实,实验成功制备了壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,适合骨髓间充质干细胞生长和增殖。     

导电弹性水凝胶促进C2C12细胞增殖的实验研究

目的　本文主要探讨一种多巴胺激活的导电冷冻水凝胶(Dopamine-based polypyrrole -methacrylated gelatin cryogel, Ppy-GelMA)对成肌细胞活性的影响。 方法　通过扫描电镜了解材料结构、细胞粘附生长情况,并通过活死染色、CCK-8试验、Ki-67免疫荧光染色明确此水凝胶对C2C12细胞的影响。 结果　扫描电镜结果显示此水凝胶具有均一连通的多孔结构, Ppy-GelMA导电水凝胶上的成肌细胞具有同向排列性;活死染色显示此水凝胶对C2C12成肌细胞有良好的细胞相容性;CCK-8检测显示Ppy-GelMA导电水凝胶上的细胞活力显著高于GelMA水凝胶上的细胞活力; Ki67免疫染色结果显示Ppy-GelMA导电水凝胶相比GelMA水凝胶拥有更高的Ki67阳性细胞率。 结论　Ppy-GelMA导电水凝胶可促进成肌细胞增殖。     

可注射性纳米粒/凝胶复合物缓释SDF-1α体系促进颅骨再生的研究

目的 采用壳聚糖(CS)为主要原料制备可缓释基质细胞衍生因子-1α(SDF-1α)的纳米粒,将其与壳聚糖/β-甘油磷酸钠(CS/β-GP)水凝胶复合,通过持续释放SDF-1α诱导大鼠颅骨再生修复情况.方法 分别制备SDF-1α/壳聚糖/羧甲基壳聚糖纳米粒(SDF-1α/CS/CMCSNPs)和CS/β-GP水凝胶,将S...     

季铵盐壳聚糖三维支架复合GNDF载间充质干细胞向神经样细胞分化

背景：壳聚糖类水凝胶因其良好的生物相容性、可降解性及对药物的缓释作用,作为支架材料近年来在组织损伤修复领域逐渐成为研究热点。 目的：探索大鼠骨髓间充质干细胞在季铵盐壳聚糖温敏凝胶支架上生长、向神经样细胞定向分化的可行性,为治疗神经系统损伤寻找理想的组织工程材料。 方法：季铵盐壳聚糖与β-甘油磷酸钠复合制成温敏凝胶,扫描电镜观察凝胶的三维结构,MTT法评价凝胶浸提液对骨髓间充质干细胞活力的影响;将牛血清白蛋白加载于凝胶支架,紫外光谱吸收法分析凝胶支架对牛血清白蛋白的缓释效果。接种大鼠骨髓间充质干细胞于凝胶支架,扫描电镜观察在支架缓释胶质细胞源性神经营养因子作用下,骨髓间充质干细胞的生长、分化情况,免疫荧光技术检测神经元烯醇化酶的表达。 结果与结论：季铵盐化壳聚糖与甘油磷酸钠复合所得凝胶支架,其多孔性特点明显,有温敏特性,对蛋白的缓释效果良好,承载大鼠骨髓间充质干细胞后,对其增殖无明显不利影响。在凝胶支架缓释的胶质细胞源性神经营养因子作用下,骨髓间充质干细胞呈现神经样细胞形态,表达神经元特异性标记物神经元烯醇化酶。说明季铵盐壳聚糖温敏凝胶对胶质细胞源性神经营养因子的缓释效果良好,其凝胶支架具有多孔径、良好生物相容性特点,可承载大鼠骨髓间充质干细胞体外生长和向神经元定向分化。     

缓释抗菌微球复合型组织工程材料水凝胶的制备及性能评价

背景：氧化羟丙基甲基纤维素-透明质酸钠水凝胶体系具有良好的力学性能、生物相容性与降解性,可作为组织工程支架对受损组织起保护支撑作用,也可作为药物载体实现局部缓释作用。目的：制备缓释抗菌微球复合型组织工程材料水凝胶,研究其理化性能、生物学性能、骨诱导性能和抗菌性能。方法：以聚乳酸乙醇酸共聚物、壳聚糖和透明质酸钠为材料,通过乳液法制备出具有多层结构的多孔微球载体,并对酸万古霉素进行负载,制备出抗菌缓释微球;以氧化羟丙基羧甲基纤维素和胺化透明质酸钠为基质制备成可注射水凝胶,并将不同质量浓度的抗菌缓释微球(0.1,0.2,0.3 g/m L)添加到水凝胶中,制备出载抗菌缓释微球的可注射水凝胶,对其理化性能(成胶时间测定、溶胀性能、降解性能)、生物学性能(细胞毒性、细胞溶血性)及成骨性能(碱性磷酸酶活性及诱导成骨基因RUN-x2、骨形态发生蛋白2、骨钙蛋白、Ⅰ型胶原蛋白表达水平)、抗菌性能进行检测及评价。结果与结论：(1)3种载抗菌缓释微球水凝胶具有良好的成胶性能与较小的溶胀比;体外降解扫描电镜结果显示,水凝胶具有稳定的自身降解性能;(2)细胞毒性实验显示,当抗菌缓释微球添加量≤0.2 g/m ...     

壳多糖-胶原凝胶构建组织工程口腔黏膜固有层

背景:有研究探讨了以胶原/壳聚糖聚合物为支架材料构建组织工程人工皮肤的可行性。目的:拟探讨以壳多糖-胶原凝胶支架材料与口腔黏膜成纤维细胞在体外构建人工固有层的可行性。方法:口腔黏膜成纤维细胞取自成年鼠颊部、牙龈或腭部。以2×DMEM培养液、胎牛血清、胶原溶液、壳多糖等或2×DMEM培养液、胎牛血清、胶原溶液等按一定比例配置成凝胶溶液,将大鼠口腔黏膜成纤维细胞与上述凝胶溶液迅速混合制成口腔黏膜人工固有层,动态观察细胞生长状况和凝胶收缩情况。结果与结论:胶原-口腔黏膜成纤维细胞复合物、壳多糖-胶原-口腔黏膜成纤维细胞复合物体外培养1d,口腔黏膜成纤维细胞在支架材料中生长良好,细胞呈长梭形,排列无一定方向。随时间推移,口腔黏膜成纤维细胞逐渐增多,凝胶颜色变浅,厚度变薄,分别于2,3d后发生收缩。结果证实,口腔黏膜成纤维细胞可在壳多糖-胶原凝胶形成的三维空间结构生长并分泌基质,形成类似黏膜固有层的致密结缔组织。壳多糖-胶原凝胶是构建组织工程化口腔黏膜固有层的合适材料。     

Effect of radiographic contrast media on complement components C3 and C4: generation of C3b-like C3 and C4b-like C4

Various water soluble iodinated radiographic contrast media (RCM) have been studied for their effect on complement components C3 and C4, purified and in serum. Hepatotropic RCM, and at higher concentration also some nephrotropic RCM, were found to exert a direct effect on purified C3 and C4. RCM treated human C3 and C4 are characterized by (a) loss of haemolytic function, (b) retention of activity in the formation of fluid phase C3 convertases and (c) an antigenic relationship to activated C3 and C4 (C3b and C4b, respectively). This direct alteration of C3 and C4 can probably also occur in serum since loss of haemolytic function is observed at similar RCM concentrations after incubation of serum and of purified components. It is concluded that RCM treated C3 and C4 represent altered forms of these components that resemble C4b and C3b in activity and conformation (C3b-like C3 and C4b-like C4). The alteration is probably caused by binding of RCM, exerting a mild denaturing effect. C3b-like C3 is a potential activator of the alternative pathway, and both C3b-like C3 and C4b-like C4 are known to be cleaved by serum inactivators. A possible pathological significance of the generation of C3b-like C3, C4b-like C4 and their split products remains to be evaluated.     

Synthesis of C3, C5, C6, C7, C8, and C9 by Human Fibroblasts

We investigated the ability of human fibroblasts to produce the components of the final common pathway (C3-C9) of complement in vitro by co-culturing an alternative complement activator (agarose beads) with the cells. The test system involved incubation of beads with anti-complement antibodies followed by radioactive-labelled anti-Ig detection antibodies. Subsequently, the beads were examined in a radioimmunoassay. Our results indicate that human fibroblasts produce C3, C5, C6, C7, C8, and C9. A neoepitope selectively expressed on activated C9 was detected, indicating assembly of the terminal complement complex and thus formation of a functional terminal complement pathway by the fibroblasts.     

Complement component C6 and C7 haplotypes associated with deficiencies of C6

Both complete C6-deficiency (C6*Q0) and subtotal C6-deficiency (C6*SD) have been described as simple recessive traits and C6*SD has been described in combination with subtotal deficiency of the C7 coded at an adjacent locus. The trace of C6 protein found in both C6*SD traits is phenotypically indistinguishable, being smaller than normal C6 and having different isoelectric properties. A defect has been found in the C6 gene which plausibly explains the C6*SD phenotype, and this defect is also common to both C6*SD traits. We present data from seven DNA markers of the C6 and C7 genes which show that although at least four haplotypes are associated with C6*Q0, most South African C6*Q0 patients carry a common defective haplotype. The most common haplotype associated with C6*Q0 has been observed only once among unaffected haplotypes of relatives. In one family, the cases of C6*SD share a complete haplotype with both cases of combined deficiency and are probably heterozygous for this condition and complete deficiency of C6. In another family, the C6*SD is on a slightly different haplotype and C7 is normally expressed. Thus, the C6 defect is not sufficient on its own to explain the C7 deficiency in the combined deficient haplotype. The haplotype associated with the combined deficiency is found not only in normal control subjects, but also in one case of complete C6 deficiency. In this case the molecular defect seen in combined or C6*SD cases is absent.     

Electrophoresis of guinea-pig complement components (C3, C5, C6, C7, C8 and C9) on polyacrylamide gel

The electrophoretic mobilities of C3, C5, C6, C7, C8 and C9 of guinea-pig complement were studied using polyacrylamide (PAA) gel as a supporting medium. The haemolytic activity of each complement component was readily revealed as a discrete haemolytic band in blood agar containing intermediate red blood cells and appropriate reagents which was overlayed on the PAA gel plate after electrophoresis. By this method, the relative mobilities of C3, C5, C6, C7, C8 and C9 were determined as 0.19, 0.40, 0.17, 0.27, 0.31 and 0.57 respectively. An additional, distinct, haemolytic C5 band was detected when native guinea-pig serum was run. The relative mobility of the haemolytic activity which was not detected when purified C5 was electrophoresed, was 0.25.     

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.     

Use of C6- and C7-deficient human sera in quantitative hemolytic assays for C6 and C7.

Quantitative hemolytic assays for C6 and C7 using as R reagents sera from patients deficient in these components are described. The assays gave linear results. Normal range for serum C6 was found to be 21,400--41,700 C6 hemolytic units/ml; for serum C7 the normal range was 5540--9860 C7 hemolytic units/ml.     

Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

Monocytes cultured under serum-free conditions secreted protein which bound covalently and non-covalently to agarose beads, an activator of the alternative pathway of complement. There was a significantly binding of monoclonal anti-C3c antibodies, polyclonal anti-C5, anti-C6, anti-C7, anti-C8, and anti-C9 antibodies, and of a monoclonal antibody against a neoantigen of polymerized C9 to agarose beads incubated with the monocytes for 24, 48, 72 or 96 h. From these results, we conclude that monocytes produce C5, C6, C7, C8 and C9 that assemble as the terminal complement complex on the surface of the agarose beads. Activation by agarose of the alternative pathway with generation of particle bound C3 and C5 convertases is a prerequisite for the subsequent formation of the terminal complement complex. Whether SC5b-9 or the membrane attack of complement (C5b-9) is formed on the beads will be examined.