羟基磷灰石/丝素蛋白复合多孔材料的制备及其表征***☆

摘要：以蚕丝丝素蛋白作为羟基磷灰石沉积的模板，制备羟基磷灰石/丝素蛋白复合凝胶，以此为基体，分别以蚕丝短纤维和NaCl颗粒作为增强材料和致孔剂，采用等静压成型法，制备羟基磷灰石/丝素蛋白多孔复合材料。对复合材料结构和力学性能的研究结果表明，材料中含有少量蚕丝短纤维对材料抗弯强度和断裂能力的提高有显著效果，以NaCl颗粒为致孔剂可使材料的平均孔径及孔隙率分别在64~183 µm及55%~75%范围内调节。 关键词：羟基磷灰石；丝素蛋白；多孔材料；复合材料；增强材料     

家蚕后部丝腺丝素蛋白的制备

背景：采用对天然蚕丝进行去丝胶蛋白而获得丝素蛋白的方法,无法完全去除具有显著免疫原性的丝胶蛋白。为此,为了得到完全不含丝胶的丝素蛋白,必须要选择其他方法。 目的：对从家蚕后部丝腺直接提取丝素蛋白的方法进行初步评价,试图为相关研究提供实验依据。 设计、时间及单位：重复测量试验,2008-07/2008-08西南大学生物技术学院,西南大学生命科学学院。 材料：五龄家蚕(Bombyx mori)100头,由西南大学蚕丝生物学实验室提供。 方法：解剖五龄家蚕,取出后部丝腺,在双蒸水中溶解丝素蛋白,过滤,冷冻干燥,研磨,筛分(400目)备用。 主要观察指标：对丝素蛋白粉末进行形态学观察,检测其粒径分布情况。计算丝素蛋白的得率。 结果：通过本方法所提取的丝素蛋白呈乳白色颗粒状,粒径大小介于100~400 μm,平均每头五龄家蚕可制备出0.33 g丝素蛋白。 结论：从家蚕后部丝腺直接提取丝素蛋白的方法简单易行,丝素蛋白得率较高,所制备的丝素蛋白纯度高,是理想的生物组织工程原材料。     

丝素蛋白/壳聚糖三维支架材料的制备方法

背景：很多研究表明丝素蛋白、壳聚糖为天然高分子材料,无毒无味,有良好的生物特性和理化性质。 目的：探讨符合软骨组织工程支架材料要求的丝素蛋白/壳聚糖三维支架材料制备方法。 方法：将丝素蛋白与壳聚糖按质量比分别为3∶1,1∶1,1∶3,0∶1的比例混合制备丝素蛋白-壳聚糖复合材料,通过孔径大小、孔隙率、吸水膨胀率及热水溶失率的测定,寻找丝素蛋白/壳聚糖最佳混合比例。 结果与结论：丝素蛋白/壳聚糖按质量1∶1的比例混合更符合要求：孔径90~280 μm,平均孔径为151.72 μm;孔隙率为(92.72±4.78)%;吸水膨胀率为(141.10±6.87)%;热水溶失率交联后较交联前降低,交联前后比较差异有显著性意义(P < 0.05)。说明丝素蛋白/壳聚糖按1∶1复合支架材料符合软骨组织工程支架材料理化性质的要求,该材料有望作为软骨组织工程研究较理想的支架材料。     

丝素蛋白对可注射磷酸钙骨水泥细胞相容性的影响

背景：已有的研究证实在磷酸钙骨水泥中添加适量的丝素蛋白能增加其抗压强度同时仍保持其可注射性,但制备的丝素蛋白/磷酸钙骨水泥复合材料是否仍具良好的生物相容性尚不确切。 目的：将人骨髓间充质干细胞与丝素蛋白/磷酸钙骨水泥复合材料共培养,评价该材料的细胞相容性。 设计、时间及地点：对比观察体外实验,于2007-09/2008-03在苏州大学附属第一医院骨科实验室完成。 材料：磷酸钙骨水泥由上海瑞邦公司提供。丝素蛋白由苏州大学材料学院提供。 方法：应用密度梯度离心法分离人骨髓间充质干细胞,条件培养基优化分离培养。浸提法制备丝素蛋白/磷酸钙骨水泥复合材料及磷酸钙骨水泥的浸提液。用MTT法测定丝素蛋白/磷酸钙骨水泥及磷酸钙骨水泥浸提液培养人骨髓间充质干细胞的活力,绘制生长曲线、计算第1,3,5,7天的相对增殖率并进行毒性分级;流式细胞仪检测丝素蛋白/磷酸钙骨水泥及磷酸钙骨水泥浸提液培养第4天的人骨髓间充质干细胞的细胞周期及倍体情况;扫描电镜观察丝素蛋白/磷酸钙骨水泥复合材料与人骨髓间充质干细胞共培养后第1,3,5,7天细胞的黏附及增殖情况。 主要观察指标：①细胞毒性分级。②细胞周期。③细胞黏附、增殖情况。 结果：丝素蛋白/磷酸钙骨水泥及磷酸钙骨水泥浸提液培养的人骨髓间充质干细胞具有良好的活力,其毒性分级均为0或1级;流式细胞仪检测见两组细胞均为二倍体细胞,未见异倍体细胞,两组G0/G1期、S期、G2/M期及细胞增殖指数差异无显著性意义（P > 0.05）;扫描电镜见人骨髓间充质干细胞在丝素蛋白/磷酸钙骨水泥复合材料上能黏附、生长,并随时间的推移材料表面的细胞数呈增长趋势。 结论：人骨髓间充质干细胞与丝素蛋白/磷酸钙骨水泥具有良好的体外细胞相容性。     

丝素蛋白作为真皮替代物移植后的组织学变化

背景：目前国外研究者已研发出多种真皮替代物,取得了一定临床效果,但价格非常昂贵,因此研制和开发经济实用的高质量真皮替代物具有重要意义。 目的：了解以柞蚕丝素蛋白材料为支架构建组织工程真皮替代物移植后的组织学变化,并与聚乙烯醇海绵相比较。 方法：取清洁级雄性SD大鼠,背部制作一大小为20 mm×20 mm 的全层皮肤缺损创面,切下皮肤反取成刃厚皮待用,然后随机分为2组,海绵组：创面埋植聚乙烯醇海绵并覆盖自体表皮。丝素组：创面埋植交联柞蚕丝素多孔材料并覆盖自体表皮。分别于术后5,10,15,25 d取材,观察移植物及其周围组织大体标本和组织形态学变化,免疫组织化学染色增殖细胞核抗原和转化生长因子β1的相对定量分析。 结果与结论：移植术后丝素组复合皮片生长良好、全部存活;海绵组移植皮片大部分变黑、结痂,并有脱落。免疫组织化学结果显示丝素组在术后5~25 d 转化生长因子β1和增殖细胞核抗原的表达均显著大于海绵组。结果提示,以丝素蛋白为支架构建的组织工程真皮替代物具有良好的生物相容性,血管化过程快,并能促进皮肤创面的愈合。     

丝素蛋白/羟基磷灰石复合支架对骨髓间充质干细胞增殖分化的支持：优于胶原海绵材料吗？

背景：传统的羟基磷灰石强度低、孔隙度小、骨诱导性和传导性较差。因此,人们采用各种方法制备羟基磷灰石生物复合材料,以改进其性能。 目的：观察丝素蛋白/羟基磷灰石复合材料对骨髓间充质干细胞生物特性的影响,并与常用的胶原海绵相比较。 设计、时间及地点：重复测量观察及多样本观察实验,于2007-01/2008-06在苏州大学附属第一医院骨科实验室完成。 材料：丝素蛋白/羟基磷灰石纳米材料由苏州大学材料学院李明忠教授研制提供,胶原海绵购买于上海其胜公司。 方法：取SD大鼠股骨和胫骨分离提取骨髓间充质干细胞,进行培养,传代。取第3代骨髓间充质干细胞分别接种到丝素蛋白/羟基磷灰石复合材料及三维胶原海绵材料上进行共培养。未加材料,单独培养细胞,作为空白对照组。 主要观察指标：应用倒置显微镜观察细胞生长情况;在培养第2,4,6,8天用MTT法测定细胞增殖情况及测定碱性磷酸酶活性。 结果：①倒置显微镜显示骨髓间充质干细胞能在两种材料上良好地黏附、增殖和生长。②MTT法检测显示两种材料上的细胞增殖明显,碱性磷酸酶活性随培养时间延长而增加,均高于空白对照组。 结论：丝素蛋白/羟基磷灰石复合材料显示出良好的生物相容性,并能促进细胞生长分化,其有望成为胶原组织工程支架材料很好的补充和替代。 关键词：丝素蛋白;羟基磷灰石;组织工程;骨髓间充质干细胞;增殖分化     

膨体聚四氟乙烯人工血管表面肝素固化替代犬下腔静脉的表面抗凝血性能

背景：各种类型人工血管植入机体静脉后,由于血液与人工血管材料表面的不相容性和静脉内血流慢、压力低等原因,极易导致血管腔内血栓形成。目的：观察膨体聚四氟乙烯人工血管表面固化肝素后代替犬下腔静脉的表面抗凝血性能和长期通畅效果。方法：将壳聚糖分子中引入光敏基团后,通过光化学固定至膨体聚四氟乙烯材料表面,在酸性条件下将肝素以离子键形式接枝到壳聚糖上,在膨体聚四氟乙烯人工血管表面形成光滑的肝素层。以固化肝素的膨体聚四氟乙烯人工血管与未处理膨体聚四氟乙烯人工血管间置代替犬下腔静脉,检测其抗凝血性能。结果与结论：固化肝素的膨体聚四氟乙烯人工血管植入后2周、1个月人工血管内壁光滑,仅有少量附壁血栓形成,无充盈缺损,吻合口无狭窄,通畅率达100%;未处理膨体聚四氟乙烯人工血管植入后1周即显示人工血管内附有大量血栓成分,完全堵塞,形成丰富的侧枝。说明固化肝素膨体聚四氟乙烯人工血管是一种理想的下腔静脉替代物。     

In vitro evaluation of a biologic graft surface. Effect of treatment with conventional and low molecular weight (LMW) heparin

Human umbilical vein grafts were treated with either conventional or LMW heparin, followed by exposure to alcohol. The grafts were investigated for their ability to adsorb and inactivate thrombin, and comparison was made with non-heparinized and saline-alcohol treated grafts and grafts supplied with a covalently bonded layer of conventional heparin. In addition, the effect of protamine exposure to heparin-alcohol and LMW heparin-alcohol treated grafts as well as native human umbilical veins (HUV) was studied.

Native HUV and heparin treated graft surfaces adsorbed and inactivated thrombin, whereas non-heparinized and saline-alcohol treated grafts inactivated surface-bound thrombin to only a small degree. Surface-bound LMW heparin exhibited a significantly lower ability to inactivate thrombin as compared with conventional heparin, but LMW heparin-alcohol surfaces were better than non-heparinized ones.

Protamine treatment of “heparinized” surfaces impaired the thrombin inhibiting ability of the heparin-alcohol surface, whereas this property was totally abolished for the LMW heparin-alcohol surface.

The findings indicate that LMW heparin, despite its weaker thrombin inhibiting capacity, may be an alternative to conventional heparin, for “heparinizing” the human umbilical vein graft. Protamine exposure may be potentially harmful for a heparin treated surface, although protamine concentrations used in the present in vitro study may not be reached in vivo. The native HIV was not at all affected by protamine exposure regarding its ability to inactivate thrombin.     

颈动脉粥样硬化性狭窄患者血管内皮生长因子检测及意义

目的探讨颈动脉粥样硬化性狭窄(CAS)患者血管内皮生长因子(VEGF)水平变化。方法用酶联免疫法测定73例颈动脉粥样硬化性狭窄(CAS)患者和40例正常对照组血清中VEGF的水平。结果CAS组VEGF水平均高于正常对照组(P<0.05)。结论颈动脉粥样硬化性狭窄患者的血清VEGF水平明显升高。血清VEGF高水平可能成为CAS出现及以后发展为缺血性脑卒中的预测因素和危险因素之一。     

Quantitative removal of heparin from plasma and other aqueous solutions by affinity adsorption on poly(L-Lysine)-Sepharose 4B

A method is described for removal of heparin from aqueous solutions. Poly(L-lysine) covalently linked to Sepharose 4B has a strong affinity for heparin. Passage of an aqueous heparin solution through a column of poly(L-lysine)-Sepharose 4B (PLLS) results in complete removal of heparin under appropriate experimental conditions. Heparin adsorbed to PLLS then can be eluted quantitatively from the column by addition of 0.1 N NaOH. No significant loss in anticoagulant properties is observed in heparin eluted from PLLS. PLLS can be used to remove heparin from plasma also. Following exposure to plasma containing heparin, PLLS can be washed with solutions of high ionic strength, low pH, or both to remove most nonspecifically adsorbed plasma proteins. Analysis by crossed immunoelectrophoresis of heparin eluted from PLLS showed that the anticoagulant recovered contained less than 1 μg of albumin/ml (0.001% of plasma albumin) but apparently was free of other plasma proteins. Washing of PLLS with solutions of high ionic strength and low pH probably resulted in the loss of certain blood coagulation factors (factors VIII and XII), while the concentration of factor X and antithrombin III did not change appreciably.     

Reliability of an Italian standardized and expanded Brief Psychiatric Rating Scale (BPRS 4.0) in raters with high vs. low clinical experience.

OBJECTIVE: The aims of this study were (i) to assess the inter-rater reliability of the latest Italian expanded 24-item version of the Brief Psychiatric Rating Scale, BPRS version 4.0 and (ii) to assess the feasibility of obtaining reliable BPRS 4.0 ratings by reliability training of clinically less experienced trainees (medical and rehabilitation students). METHOD: A videotape-training procedure was used, and the inter-rater agreement scores of three different groups of raters, namely psychiatrists and psychologists (n=28), psychosocial rehabilitation students (n=27) and medical students (n=54) were calculated and compared. RESULTS: The results indicated that both experienced raters (psychiatrists and psychologists) and inexperienced raters (medical and psychosocial rehabilitation students) were able to achieve high levels of inter-rater reliability. CONCLUSION: Our results are of particular interest in view of the increasing need to draw upon professionals, other than psychiatrists and psychologists, for cost-effective and standardized evaluation of rehabilitation interventions.     

Determination of factor XII in human plasma with arginine proesterase (prekallikrein) I. Preparation and properties of the substrate

An arginine proesterase was purified from human plasma by ion exchange chromatography and was concentrated on hydroxylapatite. Upon activation the preparation yielded 0.96 BAEe esterase units per mg protein. The preparation contained traces of factor XI, but was devoid of factor XII. One major arginine proesterase peak was observed after analytical gel filtration on Sephadex G-100 Superfine and disc electrophoresis at pH 4.3.The proesterase was activated by incubation with kaolin-treated plasma and was identified as plasma prekallikrein. Upon incubation with kaolin-treated plasma the activation proceeded at a constant rate to about 30% consumption of the proesterase. It is concluded that the preparation may serve as a substrate for determination of factor XII in plasma.     

Amelioration of ischemic spinal cord damage by postischemic treatment with propentofylline (HWA 285)

The effect of the xanthine derivative propentofylline (HWA 285) on metabolic and functional recovery in rabbit spinal cord after 20 and 30 min ischemia and 4 days of reperfusion was investigated. Pre-treatment with 20 mg/kg significantly improved recovery of the energy state in the spinal cord, however, without significant functional recovery of hindlimbs. In contrary, post-treatment with HWA 285 recovered the energy state to pre-ischemic value and also significantly improved functional recovery. These findings suggest that the neuroprotective mechanism of HWA 285 in the spinal cord is not associated with inhibition of glutamate release as supposed to operate in the gerbil brain.     

Soluble vascular cell adhesion molecule (VCAM) is associated with treatment effects of Interferon beta-1b in patients with Secondary Progressive Multiple Sclerosis

Abstract Background Subcutaneous IFN-1b (Betaferon^®) is an established immunomodulatory treatment for relapsing remitting MS and active secondary progressive multiple sclerosis (SPMS). It modulates cytokine and adhesion molecule expression but long term in vivo effects of IFN-1b on the immune system are not known in multiple sclerosis. Objective To address the effects of IFN-1b on serum levels for soluble adhesion molecules and cytokine receptors from MS patients. Methods Serial blood samples were obtained from 40 patients of the frequent MRI subgroup (20 patients each from the placebo and the IFN-1b treatment group), participating in the European multi-center clinical trial with IFN-1b for secondary progressive MS, at regular intervals for up to 36 months. Soluble adhesion molecules (sVCAM, sICAM-1, sL-Selectin) as well as TNF-receptor I and II were analysed in the serum of patients by enzyme linked immunosorbent assays (ELISAs). Monthly brain MRI was performed in 34 of these patients (16 patients from the placebo and 18 from the IFN-1b group) during months 1–6 and 19–24 to monitor disease activity as assessed by newly occurring gadolinium (Gd) enhancing lesions. Results An early and significant increase in sVCAM and sTNF-RII serum levels was detected in 16 out of 20 patients (80 %) treated with subcutaneous IFN-1b already at month 1 but was absent in all but one patient during placebo treatment (p<0.01). Raised sVCAM and TNF RII serum levels during months 1–6 inversely correlated with less MRI activity in the 19–24 months treatment interval in the IFNâ-1b treatment group ( p=0.0093 for TNF-RII; p=0.047 for VCAM). Conclusions sVCAM and sTNF RII levels in the serum of SPMS patients are increased during IFN-1b therapy and may at least in part explain some of the treatment effects, like reduced immune cell transmigration.     

The molecular mode of brain mRNA processing damage followed by the suppression of post-transcriptional poly(A) synthesis with cordycepin

Complete suppression of polyadenylation of nuclear precursors of rat brain mRNA by cordycepin leads to degradation of some translatable sequences of both poly(A)(+)- and poly(A)- pre-mRNA localized in 80S hnRNP-particles. This fact has been established by comparative analysis of the data of two-dimensional gel-electrophoretic mapping of translation products synthesized in reticulocytic cell-free system using the exogenous purified templates of rapidly labelling translatable 9-17S hn RNA (pre-mRNA) isolated from brain 80S hn RNP particles of experimental (4 hrs after cordycepin injection) and control (injection of physiological solution) adult healthy male rats. Long contact of brain cells with cordycepin (4 or more hrs) creates conditions for formation of hnRNP-particles devoid of poly(A)+ RNA and poly(A)-binding proteins. These particles differ from "normal" ones by the value of the RNA/protein ratio, and by considerably lower resistance to the action of exogenous ribonucleases and endogenous RNase. Cordycepin does not have a direct effect on biosynthesis of nuclear poly(A)-binding proteins within the duration of the experiment (8 hrs). The phenomena described are discussed.