复方苯佐卡因凝胶的局部毒性研究

目的评价复方苯佐卡因凝胶的局部刺激性和致敏性。方法依照国家食品药品监督管理局《化学药物刺激性、过敏性和溶血性研究技术指导原则》（以下简称指导原则）,采用大鼠口腔黏膜刺激性实验法、兔眼刺激性实验法以及豚鼠皮肤致敏实验法。结果复方苯佐卡因凝胶给大鼠口腔正常黏膜连续涂药3次,相当于3g·kg-1,均无刺激和毒性反应;一次性按每只0．1g给兔眼结膜涂药,亦未出现刺激性反应;给每只豚鼠皮肤0．2g致敏接触和激发接触,均无过敏反应发生。结论复方苯佐卡因凝胶对口腔黏膜无刺激性,对皮肤也没有致敏性。     

Application of sodium alginate microspheres in ischemic stroke modeling in miniature pigs

The miniature pig is an optimal animal model for studying nervous system disease because of its physiologic and pathologic features. However, the rete mirabile composed of arteries and veins at the skull base limits their application as a model of ischemic stroke by middle cerebral artery occlusion. The present study investigated the possibility of establishing an ischemic stroke model in the miniature pig by blocking the skull base retia with sodium alginate microspheres. Three Bama miniature pigs were used. Using the monitor of C-arm X-ray machine, sodium alginate microspheres (100-300 μm), a novel embolic material, were injected through the femoral artery, aortic arch, common carotid artery, ascending pharyngeal artery and the retia. Results were evaluated using carotid arteriography, MRI, behavior observation and histology. The unilateral rete mirabile was completely blocked, resulting in disturbance in blood supply to the basal ganglia, astasia of the right hind limb and salivation. MRI and hematoxylin-eosin staining showed an evident infarction focus in the basal ganglia. These findings indicate that sodium alginate microspheres are a suitable embolic material for blocking the skull base retia in miniature pigs to establish an ischemic stroke models.     

蛋白多糖在脱细胞猪肺动脉带瓣管道中抗钙化的作用

目的:证实去除细胞外基质蛋白多糖对提高脱细胞猪肺动脉带瓣管道抗钙化性能的作用,为研制组织工程化肺动脉带瓣管道做准备。方法:实验分为3组,即A组:为新鲜猪肺动脉带瓣管道组织,B组:用胰蛋白酶+Triton X-100处理的脱细胞猪肺动脉带瓣管道组织和C组:在B组处理的基础上再经透明质酸酶消化,去除细胞外蛋白多糖基质成分的猪肺动脉带瓣管道组织,每组4份(n=4)。方法:实验室:将3组样本分别进行HE染色后,用光镜和扫描电镜观察肺动脉管壁及瓣膜组织的变化。采用盐酸胍抽提结合阿利新蓝染色法测定蛋白多糖的含量。同时将3组样本包埋于大鼠皮下,于6周后取出标本进行Van Kosaa银染色法(钙盐染色)和原子吸收分光光度计法分别定性、定量分析组织的钙化程度。结果:光镜和电镜检查结果显示,猪肺动脉管壁和瓣膜组织的细胞可以较完整的去除,纤维网架结构可以完整保持。蛋白多糖含量的测定显示,与A、B组相比,C组细胞外基质蛋白多糖的含量显著下降(P<0.05)。大鼠皮下包埋实验显示,与A、B两组相比,C组的钙化反应更少,管壁组织钙的含量显著下降(P<0.05)。结论:采用胰蛋白酶+Triton X-100脱细胞方法可以达到去除细胞的目的。通过大鼠皮下包埋实验证明,采用透明质酸酶消化减少细胞外基质蛋白多糖的含量可以进一步减少脱细胞组织的钙化反应,为组织工程肺动脉带瓣管道的构建提供较为理想的脱细胞基质材料。     

猪精子细胞中胰岛素诱导产生的一氧化氮自由基促进精子获能

近期,胰岛素被证实具有诱导猪精子获能的能力。在不同的哺乳动物中,精子获能被认为与一氧化氮信号通路相关;因此,本研究使用荧光激活细胞分选技术探究经胰岛素处理的猪精子中一氧化氮水平。在同一样本中,用金霉素染色、蛋白质酪氨酸磷酸化模式和顶体状态来评估精子获能。结果显示经胰岛素处理的精子内一氧化氮水平及三种评价精子获能的指标均有显著提高;相反,用一氧化氮合成酶抑制剂（N-硝基-L-精氨酸甲酯）或抗胰岛素受体β抗体预处理的精子中胰岛素相关的作用全被逆转。这些结果表明胰岛素具有增强猪精子细胞内一氧化氮浓聚的能力并且提示胰岛素可能通过一氧化氮促进精子获能。     

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 4: pre‐clinical efficacy and complication data required to justify a clinical trial

In 2009, the International Xenotransplantation Association (IXA) published a consensus document that provided guidelines and “recommendations” (not regulations) for those contemplating clinical trials of porcine islet transplantation. These guidelines included the IXA's opinion on what constituted “rigorous pre‐clinical studies using the most relevant animal models” and were based on “non‐human primate testing.” We now report our discussion following a careful review of the 2009 guidelines as they relate to pre‐clinical testing. In summary, we do not believe there is a need to greatly modify the conclusions and recommendations of the original consensus document. Pre‐clinical studies should be sufficiently rigorous to provide optimism that a clinical trial is likely to be safe and has a realistic chance of success, but need not be so demanding that success might only be achieved by very prolonged experimentation, as this would not be in the interests of patients whose quality of life might benefit immensely from a successful islet xenotransplant. We believe these guidelines will be of benefit to both investigators planning a clinical trial and to institutions and regulatory authorities considering a proposal for a clinical trial. In addition, we suggest consideration should be given to establishing an IXA Clinical Trial Advisory Committee that would be available to advise (but not regulate) researchers considering initiating a clinical trial of xenotransplantation.     

Cartilage degeneration and excessive subchondral bone formation in spontaneous osteoarthritis involves altered TGF‐β signaling

Transforming growth factor‐β (TGF‐β) has been demonstrated as a potential therapeutic target in osteoarthritis. However, beneficial effects of TGF‐β supplement and inhibition have both been reported, suggesting characterization of the spatiotemporal distribution of TGF‐β during the whole time course of osteoarthritis is important. To investigate the activity of TGF‐β in osteoarthritis progression, we collected knee joints from Dunkin–Hartley (DH) guinea pigs at 3, 6, 9, and 12‐month old (n = 8), which develop spontaneous osteoarthritis in a manner extraordinarily similar to humans. Via histology and micro‐computed tomography (CT) analysis, we found that the joints exhibited gradual cartilage degeneration, subchondral plate sclerosis, and elevated bone remodeling during aging. The degenerating cartilage showed a progressive switch of the expression of phosphorylated Smad2/3 to Smad1/5/8, suggesting dual roles of TGF‐β/Smad signaling during chondrocyte terminal differentiation in osteoarthritis progression. In subchondral bone, we found that the locations and age‐related changes of osterix⁺ osteoprogenitors were in parallel with active TGF‐β, which implied the excessive osteogenesis may link to the activity of TGF‐β. Our study, therefore, suggests an association of cartilage degeneration and excessive bone remodeling with altered TGF‐β signaling in osteoarthritis progression of DH guinea pigs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:763–770, 2016.     

软骨－软骨膜复合体重建鼓膜的组织学变化

目的：探讨软骨－软骨膜复合体修补鼓膜穿孔后其软骨厚度及软骨膜内血管密度的变化。方法45只雄性豚鼠随机分为A、B、C三组,每组15只,每只豚鼠分别于左侧耳制作鼓膜穿孔模型后立即采用同侧耳甲腔软骨－软骨膜复合体植入修补鼓膜,植入前先测量并记录植入复合体的厚度,分别观察术后2周（A组）、6周（B组）及12周（C组）植入复合体的厚度、软骨膜内血管密度;另取B组5只豚鼠的右耳（对照组）行鼓膜穿孔造模,不行鼓膜修补术。结果 A、B、C三组豚鼠左耳鼓膜穿孔平均愈合时间为3．8±0．84天,对照组鼓膜穿孔自愈时间为7．2±0．84天;A、B、C三组软骨－软骨膜复合体植入前厚度分别为0．199±0．023、0．198±0．017、0．193±0．021mm ,三组间差异无统计学意义（ P＞0．05）,而植入后2周（A组）植入体厚度为0．227±0．024mm ,较植入前增厚,植入后6周（B组）植入体厚度为0．191±0．016mm ,较植入前无变化（ P＞0．05）,但较植入后2周（A组）变薄（ P＝0．016）,植入后12周的C组植入体厚度为0．169±0．017mm ,较植入前明显变薄（ P＜0．05）,且较植入后2周（A组）、6周（B组）厚度变薄（P＝0．00）;植入后第2、6周,软骨膜单位面积中血管数量分别为13．28±2．49、7．71±2．49个,第2、6周时重建鼓膜的组织学结构为鳞状上皮层、纤维－软骨膜层、软骨层、粘膜层,第12周时为鳞状上皮层、软骨层及粘膜层。结论耳廓软骨－软骨膜复合体重建鼓膜的愈合时间短于鼓膜自行愈合的时间;植入后复合体的厚度较植入前先增厚再变薄,软骨膜层血管数量表现为先增加后减少;重建鼓膜最终组织学分层与正常鼓膜组织学结构相似。