非缝合式无支架异种主动脉生物瓣膜的研制

目的: 研制新型非缝合式无支架异种主动脉生物瓣膜,在体外对其功能进行初步研究。方法: 将猪主动脉瓣缝合到NiTi形状记忆合金支架上,在离体猪心脏内置入新型瓣膜,肉眼及彩超下观测瓣膜闭合、冠状动脉血流及瓣周漏情况。通过TH-1200型人工心脏瓣膜脉动流测试系统测定25#非缝合式无支架异种主动脉生物瓣膜平均跨瓣压差、返流百分比、瓣膜开口面积。结果: 新型瓣膜能较简单的植入离体猪心脏内。且不同压力（80～250mmHg）下,瓣膜闭合良好,无明显瓣周漏,冠状动脉血流良好;心脏彩超检测瓣周结构与主动脉壁之间可见无回声间隙,无明显的瓣周漏。体外脉动实验提示新型瓣膜与机械瓣膜相比跨瓣压差小,返流少,开口面积大。结论: 该型瓣膜制备相对简单,具有血流动力学良好,跨瓣压小,无需抗凝等优点,可简化手术操作,降低手术难度。     

Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties

Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris. In high-density fermentations, engineered P. pastoris yielded > 3 g of the protein per litre of culture. Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines.     

异种脱钙骨基质,异种骨基质胶加释放系统移植的组织学观察

Ｗｉｓｔａｒ大鼠２４只分４组，分别将４种复合异种骨植入大鼠股四头肌内，定期取材制片，光镜及ＴＥＭ－１０ＳＸ透射电镜观察。结果：２ｄ巨噬细胞等密集成团，细胞器发达；４ｄ间充质细胞伪足伸入异种脱钙骨基质（Ｄｅｍｉｎｅｒａｌｉｚｅｄ ｘｅｎｏｇｅｎｅｉｃ ｂｏｎｅ ｍａｔｒｉｘ，ＸＤＢＭ），异种骨基质胶（Ｘｅｎｏｇｅｎｅｉｃ ｂｏｎｅ ｍａｔｒｉｘ ｇｅｌａｔｉｎ，ＸＢ－ＭＧ）隙内；８ｄ间充质细胞增多，     

T淋巴细胞在异种鼠肝移植免疫排斥反应中的作用

目的 探讨Ｔ淋巴细胞是否参与了异种肝移植免疫排斥反应。方法 建立仓建到大鼠原全肝移植模型,用免疫组织化学染色方法检测异种肝移植免疫排斥反应中Ｔ淋巴细胞浸润情况及其Ｆａｓ抗原配体（Ｆａｓ－Ｌ）的表达,并用普通组织学及原位末端标记方法检测移植肝中细胞凋亡。结果 异种肝移植免疫排斥过程中,移植肝中Ｔ淋巴细胞,包括ＣＤ４、ＣＤ８亚型大量浸润,术后４天开始表达Ｆａｓ－Ｌ,随着排斥反应的加重,Ｆａｓ－Ｌ表达越     

Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity.
2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity.
3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells.
4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.

     

协调性异种肝移植免疫病理学的实验研究

观察协调性异种肝移植免疫病理变化,探讨其免疫排斥反应机制。方法：建立仓鼠到大鼠原位肝移植模,运用组织学方法检测异种／肝移植受体脾脏、淋巴结的组织学的改变;采用免疫组织化学的方法检测移植肝大鼠抗体的沉着、淋巴细胞的浸润及穿孔素、Ｆａｓ－Ｌ的表达;原位末端标记方法,检测植肝细胞凋亡。     

经hCTLA4-Ig基因转染的猪胰岛细胞在小鼠体内的存活与功能状况研究

目的研究将人细胞毒T淋巴细胞相关抗原4免疫球蛋白（hCTLA4-Ig）基因转染至猪胰岛细胞后，再将其移植至小鼠体内的存活及功能状况。方法采用腺相关病毒载体（AAV）介导hCTLA4-Ig基因体外转染新生猪胰岛细胞（NIPs），再将转基因的NIPs移植至构建了人免疫系统的SCID糖尿病小鼠左肾被膜下。逆转录聚合酶链（RT-PCR）反应和免疫荧光染色法检测转染后hCTLA4-Ig基因的表达状况；观察小鼠移植转基因NIPs后的生存时间；移植物免疫组织化学分析及酶联免疫（ELISA）法测定受者血清中细胞因子的水平。结果NIPs经转染后，可检测到hCTLA4-Ig基因及其蛋白表达，且葡萄糖刺激胰岛素释放试验与未转染的对照组比较，差异无统计学意义（P〉0．05）；糖尿病小鼠经转基因的NIPs移植后，生存时间及血糖维持正常的平均时间分别为（72．5±30．6）d和（59．1±24．0）d，较未转染的对照组（26．9±6．9）d和（12．7±3．3）d显著延长（P〈0．01）；免疫组织化学染色检测移植后不同时间（15、30、90d）的移植物组织，可见转基因细胞移植部位有完整的胰岛细胞，而对照组胰岛细胞破坏、消失，周围可见较多的炎性细胞浸润；且转基因细胞移植的小鼠血清白细胞介素2（IL-2）、γ干扰素（IFN-γ）、肿瘤坏死因子α（TNF-α）的水平明显低于对照组（P〈0．05）。结论AAv介导hCTLA4-Ig基因体外转染猪胰岛细胞后再移植至受者体内，可提高受者的免疫耐受水平，延长胰岛细胞在异种受者体内的存活时间，而胰岛细胞的内分泌功能不受明显影响。     

经肝动脉肝内移植异种胰岛细胞治疗糖尿病的实验研究

目的 探讨经肝动脉异种肝内植入新生儿猪胰岛细胞的可行性，以及移植物生理功能和产生副反应的特点。方法 化学药物诱导法制作５只犬的糖尿模型以后，将分离，纯化后的新生猪胰岛细胞肝动脉植入糖尿病动物模型的肝内，测量移植前后胰岛纱用量，血糖以及血清Ｃ肽水平和肝功能的变化，并对肝，肾进行病理检查。结果 移植以后血清Ｃ肽明显增高，对葡萄糖刺激试验反应敏感（Ｐ＝０．０１２２）血糖逐步恢复正常（Ｐ＝０．００００１５