组织工程纳米心包片的基础实验研究

目的:运用组织工程学方法和纳米技术对牛心包进行处理,研究其免疫原性,以期制备免低疫原性的组织工程补片。方法:采用明胶再基质化、碳化二亚胺/N-羟基琥珀酰亚胺(简称EDC/NHS)交联和高速搅拌复乳溶剂挥发法对牛心包进行相关处理,之后植入大鼠皮下进行观察,通过HE染色对比纳米组、交联组、脱细胞组和新鲜组四组植入前后的组织形态学改变,并对各组心包片进行机械性能、厚度、含水量和钙含量检测;免疫组织化学法检测心包片表面CD4的沉积。结果:肉眼观察到大鼠皮下降解率纳米组明显低于脱细胞组,与交联组相当,脱细胞组降解较快;抗张强度测定结果显示,纳米组抗张强度与交联组(P=0.76)及脱细胞组(P=0.61)相比较差异无统计学意义,但远低于新鲜组(P<0.01);钙含量纳米组远低于新鲜组(P<0.001),与交联组相当;免疫组化结果显示各组心包片表面均有明显的CD4沉积,纳米组的排斥反应远低于新鲜组,与交联组相当。结论:组织工程学方法和纳米技术结合制备的纳米心包片具有低免疫原性,低降解率和抗钙化的优良特性。与交联组相比,纳米微球的嵌合并未明显影响心包片的机械性能和免疫原性,有望成为构建组织工程补片缓释模型的理想载体。     

原花青素处理去细胞牛心包细胞的相容性研究

目的考察原花青素处理去细胞牛心包的细胞相容性。方法采用L929细胞经原花青素处理去细胞牛心包浸提液培养后,噻唑蓝试验（MTT）法测定其相对增值率;将L929细胞与原花青素处理去细胞牛心包直接接触培养,逐日观察细胞生长状态;将L929细胞种植于原花青素处理的去细胞牛心包表面,扫描电镜观察其生长情况。结果原花青素处理去细胞牛心包性质稳定,细胞毒性程度为0～1级。L929细胞与心包材料直接接触培养生长良好,形态学无明显改变。结论原花青素处理去细胞牛心包细胞相容性好。     

应用胶原膜构建组织工程心脏瓣膜的初步研究

目的 :探讨应用胶原膜构建组织工程心脏瓣膜的可行性。方法 :扫描电镜观察材料结构特点。材料兔皮下包埋实验 ,分别于 4,6 ,8,12周观察材料的生物相容性和降解率。培养犬主动脉瓣间质细胞、主动脉壁间质细胞和皮肤成纤维细胞 ,对照其生长曲线、平滑肌α肌动蛋白表达和扫描电镜特点。将犬主动脉壁间质细胞和内皮细胞种植于胶原膜上 ,观察其形态并测定细胞合成前列环素的功能。结果 :胶原膜呈网孔状结构 ,孔径 10 7μm。皮下包埋实验显示材料生物相容性好 ,体内降解时间为 12周。犬主动脉瓣间质细胞和主动脉壁间质细胞平滑肌α肌动蛋白均为部分阳性表达 ,细胞内有大量粗面内质网 ,生长曲线相似。种植实验示细胞在材料表面生长良好 ,并具有合成、分泌前列环素 （84.2 pg/ m L vs0 .4pg/ m L ,P<0 .0 5 ）的功能。结论 :以胶原膜为支架体外构建组织工程心脏瓣膜细胞不仅能在材料表面生长 ,还能合成、分泌血管活性物质 ,是具有“生理功能 的组织工程心脏瓣膜。     

去细胞支架材料构建组织工程心脏瓣膜研究进展

当前所有生物瓣，包括无支架生物瓣共同致命弱点是易钙化、衰坏。提高生物瓣的耐久性是当前研制生物瓣的根本方向。随着组织工程技术的发展，组织工程瓣成为提高生物瓣耐久性新尝试。组织工程心脏瓣膜是应用组织工程学制造出的一种具有细胞活性的新型生物瓣，其原理是利用可吸收的聚合物为支架，再种植细胞包裹瓣叶。因为理想的组织工程瓣膜具有以下特点：组织相容性好、无免疫原性、有活力、耐久性强，并有一定的增长能力和修复能力，所以有着广阔的临床应用前景。如何构建一种良好的组织工程瓣膜，是当前心血管研究领域的热点与前沿。近年来，组织工程瓣，特别是以去细胞基质为支架材料的组织工程瓣的研究取得了一定的进展。     

组织相容性复合体-DMA、DMB基因与1型糖尿病遗传易感相关性的研究

目的　对组织相容性复合体非经典基因 DMA、DMB与 1型糖尿病的易感相关性进行研究。　方法　以 80例 1型糖尿病患者为实验组 ,以 91名健康成年献血员为正常对照组。用酚 氯仿 乙醇沉淀法提取基因组DNA ,然后采用聚合酶链反应和斑点杂交技术对DMA、DMB基因进行分型。　结果　DMA 0 10 3和DMB 0 10 3基因在患者中的频率显著增高 ,DMA 0 10 2和DMB 0 10 1基因在对照组中的频率显著增高。DMA 0 10 1 0 10 2和DMB 0 10 1 0 10 1基因型在对照组中的频率显著增高 (分别为 42 %vs10 .8%;46 %vs7.1%,P <0 .0 1) ,DMB 0 10 3 0 10 3和DMA 0 10 1 0 10 3基因型在患者中的频率显著增高。DMA 0 10 2 DMB 0 10 1二聚体在对照组中的频率显著高于患者组 ( 2 8.6 %vs4.4%,P <0 .0 1) ,DMA 0 10 3 DMB 0 10 2、DMA 0 10 3 DMB 0 10 3和DMA 0 10 3 DMB 0 10 1二聚体在患者中的频率显著增高。　结论　DMA 0 10 3和DMB 0 10 3基因与中国人 1型糖尿病的易感性相关 ,DMA 0 10 2和DMB 0 10 1则与中国人 1型糖尿病的保护性相关。DMB 0 10 3 0 10 3和DMA 0 10 1 0 10 3基因型对 1型糖尿病构成易感性 ,DMA 0 10 1 0 10 2和DMB 0 10 1 0 10 1基因型则对 1型糖尿病构成保护性。DMA 0 10 3 DMB 0 10 2、D     

多株消化道肿瘤细胞在硝酸纤维素膜界面生长的初步研究

目的观察多株消化道肿瘤细胞在硝酸纤维素膜界面上的生长情况,探讨硝酸纤维素膜作为细胞培养载体材料的可行性。方法将大肠癌细胞、胃癌细胞、胰腺癌细胞、肝癌细胞株等肿瘤细胞接种在硝酸纤维素膜界面培养,培养结束后在光学显微镜以及扫描电镜下观察细胞形态,免疫组化法检测肿瘤细胞标志物,对比检测实验组和六孔板对照组培养液上清中两者乳酸脱氢酶漏出率差异,评价硝酸纤维素膜的细胞相容性。结果膜染色透明处理后,在光学显微镜下可以观察到膜表面细胞保持较好的增殖状态和细胞形态;肿瘤细胞主要标志物均呈阳性表达;硝酸纤维素膜及膜上细胞的扫描电镜表征：该膜呈现孔径均一的海绵样空间立体结构,膜表面生长的细胞呈不规则形态且有伪足形成。结论硝酸纤维素膜具有良好的细胞相容性,可以作为一种新的细胞生长载体材料加以应用。     

组织工程软骨的研究现状与进展

随着软骨组织工程学技术的不断发展,构建生物特性与正常关节软骨相似的组织工程软骨已经成为目前组织工程研究的热点之一,并且逐步应用于临床。它的基本思想是综合应用工程学和生命科学的基本原理和方法,采集与靶组织或器官功能相关的活细胞种植于可生物降解的三维支架材料上,通过模拟体内环境的体外培养使附着于支架上的细胞大量增殖,形成有生命的组织,而后将其移人体内,修复组织缺损,替代组织、器官的部分或全部功能,最终与机体组织融为一体,成为自体组织或器官的一部分并发挥其相应的生理功能。组织工程软骨的构建主要包括种子细胞、支架材料、体外培养环境和移植物成血管化四方面因素。     

组织工程心脏瓣膜构建中细胞种植的实验研究

目的 观察不同细胞种植在脱细胞猪主动脉瓣叶支架上的生长特点。方法 将猪主动脉瓣叶行脱细胞处理．并在其上分别种植新生牛主动脉内皮细胞(BAECs)、平滑肌细胞(SMCs)、SMCs和BAECs。结果 猪主动脉瓣叶中的细胞成分能完全去除，种植的BAECs在脱细胞瓣叶表面形成一连续的细胞层．SMCs形成多层连续的细胞层，复合种植细胞则形成多层连续的细胞层；BAECs表面形成一连续的细胞层，其间为多层分布的SMCs，同时内皮细胞分泌前列环素(PGI2)的能力明显增强。结论 不同种类的细胞在脱细胞瓣叶表面均生长良好，并呈现不同的生长方式，SMCs和BAECs复合种植有可能为今后组织工程瓣膜构建中有关细胞种植提供参考。     

雌激素抑制大鼠心室肌细胞L-型钙电流机制的研究

目的 :探讨雌激素抑制大鼠心肌细胞 L -型钙电流 （ICa,L）的可能途径。方法 :通过胶原酶消化得到单个大鼠心室肌细胞 ,膜片钳全细胞电压钳方法记录 ICa,L。结果 :雌激素 （1～ 30μmol/ L ）可剂量依赖性地抑制 ICa,L,雌激素对ICa,L的抑制作用并不能被雌激素受体的阻断剂 Tamoxifen和 ICI 182 780所阻断。细胞外灌流耦联了牛血清白蛋白（BSA）的雌激素 [β- estradiol6 - （o- carboxy- m ethyl） oxime:BSA（EST- BSA） ],不能透过细胞膜 ]也能抑制 ICa,L,但同样浓度的 EST- BSA细胞内灌流对 ICa,L并没有明显的抑制作用。结论 :雌激素可能是通过膜表面上特异的受体介导了其对 L-型钙电流的抑制作用     

纳曲酮缓释剂在家兔体内的组织相容性及安全性研究

将纳曲酮缓释剂置于家兔身体皮下,观察不同时间家兔的体质量、体温变化,以及植入部位伤口的变化和组织病理改变,并以液相色谱-质谱联用仪(LC/MS/MS)测定相应时间家兔体内的血药浓度,评价纳曲酮缓释剂的组织相容性和安全性.结果表明,纳曲酮缓释剂在家兔体内的组织相容性良好,符合一般生物相容性材料所引起的组织反应过程;家兔体内纳曲酮浓度为3.67～34.45ng/ml.纳曲酮缓释剂在家兔体内具有较好的组织相容性和安全性.     

A Double-blind Placebo Controlled Study of Male Alcoholics Given a Subcutaneous Disulfiram Implantation

Twenty-one alcoholics participated in a double blind study to elicit information of the clinical effect of implanted disulfiram. They were randomized to either of two groups, 11 patients to the placebo group and 10 patients to the disulfiram group. Each patient was subjected to seven sessions of intravenous ethanol challenge, once before and six times during the first 20 weeks after implantation. The acute ethanol challenge did not result in any differences between the groups with respect to blood ethanol and acetaldehyde concentrations. No patient showed any clinical signs of disulfiram-ethanol reaction. After a study period of 20 weeks no significant differences were found between the groups with regard to the reduction in average ethanol consumption, days from implantation to the first drink and number of abstinent weeks. Our results do not support the idea that a 1 g disulfiram implant has any pharmacological or clinical action.     

Biomechanical characterization of decellularized and cross-linked bovine pericardium

BACKGROUND AND AIM OF THE STUDY: Although bovine pericardium has been used extensively in cardiothoracic surgery, its degeneration and calcification are important limiting factors in the continued use of this material. The study aims were to decellularize bovine pericardium and to compare the biomechanical properties of fresh and decellularized bovine pericardia to those treated with different concentrations of glutaraldehyde (GA). METHODS: An established protocol for decellularization using sodium dodecyl sulfate was used, and histological analysis performed to validate the adequacy of decellularization. Contact cytotoxicity was used to study the in-vitro biocompatibility of variously treated pericardia. Mechanical testing involved uniaxial testing to failure. Mechanical properties of the fresh and decellularized pericardia (untreated and treated with 0.5% and 0.05% GA) were compared. RESULTS: Histological analysis of decellularized bovine pericardium did not show any remaining cells or cell fragments. The histoarchitecture of the collagen-elastin matrix appeared well preserved. Untreated decellularized pericardium was biocompatible in contact cytotoxicity tests with smooth muscle and fibroblast cells. The GA-treated tissue was cytotoxic. There were no significant differences in the mechanical properties of fresh and decellularized pericardia, but there was an overall tendency for GA-treated pericardia to be stiffer than their untreated counterparts. CONCLUSION: An acellular matrix, cross-linked with a reduced concentration of GA, can be produced using bovine pericardium. This biomaterial has excellent biomechanical properties and, potentially, may be used in the manufacture of heart valves and pericardial patches for clinical application.     

Replacement of right coronary leaflet with bovine pericardium

Bovine pericardium was used to replace the right coronary leaflet to manage aortic insufficiency resulting from ventricular septal defect in 6 patients aged 15 to 34 years. Aortic insufficiency was severe in 5 patients and moderate in one. In all patients, the ventricular septal defect was repaired before aortic valvuloplasty. They were followed up for 5 to 6 months. No mortality was observed. Three patients had relief of aortic insufficiency, 2 had mild residual aortic insufficiency, and one had minor insufficiency not requiring re-operation. Replacement of the right coronary leaflet with bovine pericardium is a promising technique for young patients. The short-term outcomes are encouraging, but longer follow-up is required to assess the durability and function of bovine pericardium in the aortic position.     

Reconstruction of the pericardial sac with glutaraldehyde-preserved bovine pericardium

Complete closure of the pericardial sac after open-heart surgery is thought to be a help in avoiding some postoperative complications and in facilitating reoperation. Since 1977, sixty-six patients who were subjected to open-heart surgery had glutaraldehyde stabilized xenogeneic bovine pericardial patches inserted to achieve closure of their own pericardial sacs. There were no hemodynamic problems related to the implantation of these patches. All 60 surviving patients were clinically well during a follow-up of 145 patient-years. Reoperation was undertaken in three patients 2(1/2), 3, and 7 years after the initial operation. The presence of the pericardial patch greatly facilitated the reopening of the chest because it prevented the formation of adhesions between the patch and the epicardium. Histologically, the implanted pericardium consisted of laminae of collagen fibrils containing evenly dispersed fibrocyte nuclei and parallel runs of fairly wide, separated elastic fibrils. There was no evidence of immune reaction. On the basis of our findings that there was no host reaction or complications after its use, glutaraldehyde stabilized xenogeneic bovine pericardium appears to be the material of choice for patch closure of the pericardium when primary closure alone is not feasible.     

The effect of ethylenediaminetetraacetic acid on calcific degeneration in bovine pericardium

Calcification is the most frequent cause of the clinical failure of bovine pericardium bioprosthetic valves, preventing their widespread application for surgical treatment. The aim of this study was to minimize calcific degeneration in bovine pericardium by using a chelating agent, ethylenediaminetetraacetic acid (EDTA). Freshly excised bovine pericardium was dissected free from adhering fat tissue and cut into 1-cm² pieces that were rinsed in phosphate-buffered saline solution (PBS) and transferred into 4°C PBS containing 1% glutaraldehyde (GA) for initial fixation, then allocated into two groups. Group I received the same treatment in a fresh solution for 5 more days. Group II underwent an additional fixation step in PBS solution (pH 7.4, 37°C) containing 11% EDTA for a period of 48h (30ml/g tissue) and was then transferred into freshly prepared PBS + 1% GA solution at 37°C for another 3 days. To investigate the calcification rate, pericardial patches were inserted into the dorsal pouches of 25 male Wistar rats for 21 days. Calcium levels were measured with an atomic absorption spectrophotometer and examined histo-pathologically. The calcium content of EDTA-treated pericardium (Group II), 21 ± 3.8µg/mg, was significantly lower than that of Group I, 43.3 ± 9.2µg/mg. Assessment of the degree of calcification in the histological sections generally agreed well with the results of the chemical analyses. Calcium deposition in Group I samples were found to be solid mineral depositions, whereas in the Group II pericardial samples, only smaller traces of calcium were found. Calcific degeneration in bovine pericardium can be reduced by using chelates such as EDTA.     

Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic cardiac valves

Bioprosthetic valves fail frequently because of pathological mineralization, a process that begins in cell remnants of the glutaraldehyde (GLUT) fixed tissue. Other pathological cardiovascular calcification and physiological mineralization in skeletal/dental tissues are both largely initiated in cell-derived membranous structures (often called "matrix vesicles"), and the enzyme alkaline phosphatase (AP) likely has an important function in the pathogenesis of mineral nucleation. This study tested the hypothesis that AP might also be present in and contribute to calcification of bioprosthetic valves. AP activity of fresh and GLUT-treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Following 24 hours in 0.6% HEPES-buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained in GLUT-treated tissue relative to that of fresh tissue (Vmax, 24 vs. 45 mumol reaction product/min/mg tissue protein, respectively), although binding was somewhat reduced (Km, 1.9 X 10(3) vs. 1.4 X 10(3) microM substrate, respectively). Enzyme reaction product was demonstrated in both fixed and fresh tissue by light microscopic histochemical studies, confirming the biochemical results. Reaction product was noted along membranes of vascular endothelial cells and interstitial fibroblasts, the sites of early calcific deposits in bioprosthetic valves, by ultrastructural examination of GLUT-treated tissue. We conclude that GLUT-treated bovine pericardium retains much of the hydrolytic activity of AP, an enzyme associated with normal skeletal and pathological cardiovascular and noncardiovascular mineralization, and suggest that further examination of the mechanistic role of this enzyme may stimulate new approaches for slowing or preventing calcification of bioprosthetic tissue.     

诱发大鼠自然感染卡氏肺孢子虫肺炎实验研究

用地塞米松0.3mg～0.6mg肌注大鼠每周两次,连续9周,第10周改为醋酸泼尼松5mg/次,每周两次,肌注7次。激素总量为42.2mg。诱发大鼠重度肺孢子虫性肺炎,第14周死亡。肺印片查见成熟、未成熟及溃破包囊,切片发现典型的病理改变及肺孢子虫体,作为病原与病理诊断的依据。电镜观察包囊有两种形态,一种为园形包囊,另一种为香蕉形包囊。证明新疆地区具有传播与感染该病原的机会与条件,一但免疫功能低下,可诱发人体卡氏肺孢子虫肺炎而死亡。动物实验成功,为本病提供了进一步研究的条件。     

Decellularization of bovine pericardium for tissue-engineering by targeted removal of xenoantigens

BACKGROUND AND AIM OF STUDY: Assessment of decellularization of xenogeneic biological scaffolds for tissue engineering has relied primarily on histological cellularity, though this may not ensure the removal of known xenogeneic antigens such as galactose-alpha1,3-galactose (alpha-gal) and MHC I. METHODS: Bovine pericardium (BP) underwent standard (Std) decellularization consisting of hypotonic lysis and treatment with DNAse/RNAse. In addition to Std decellularization, tissues were treated for 24 h with either 0.5% Triton X-100, 0.5% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), alpha-galactosidase (5 U/ml) or phospholipase (PL) A2 (150 U/ml). Tissues underwent a 96-h washout under gentle agitation at 27 degrees C, and then evaluated by light microscopy for % cellularity, and by immunohistochemistry and Western blot for alpha-gal, bovine MHC I and smooth muscle alpha-actin. RESULTS: Standard treatment of BP resulted in only partial removal histological cellularity and persistence of alpha-gal, MHC I and alpha-actin. Adding SD treatment resulted in apparent acellularity, but persistence of xenogeneic antigens. Only the addition of SDS resulted in complete histological acellularity and removal of xenogeneic antigens. Treatment with alpha-galactosidase selectively removed alpha-gal from BP. CONCLUSION: Histological cellularity is not an adequate end-point for assuring removal of antigenicity from xenogeneic biological scaffolds. However, known xenogeneic antigens can be targeted for removal by novel decellularization treatments such as alpha-galactosidase.