兔肺气肿模型的建立

目的：应用木瓜蛋白酶雾化吸入和静脉注射法来复制兔肺气肿模型。方法：兔耳缘静脉注入8％木瓜蛋白酶和让兔雾化吸入5％木瓜蛋白酶液制作模型。观测其动脉血气及肺功能并对肺组织进行病理学观察。结果：与正常组相比模型组兔动脉血pH值、PaO2浓度降低，PaCO2浓度升高；潮气量下降，呼吸总阻力、中心阻力、总气道阻力升高；病理组织切片光镜下见肺泡壁变薄、断裂，肺泡腔扩张、融合，典型肺气肿表现。结论：木瓜蛋白酶雾化吸入和静脉注射能成功复制出兔肺气肿模型。     

Effectiveness of papain gel in venous ulcer treatment: randomized clinical trial

OBJECTIVE:

to assess the effectiveness of 2% papain gel compared to 2% carboxymethyl cellulose in the treatment of chronic venous ulcer patients.

METHOD:

randomized controlled clinical trial with 12-week follow-up. The sample consisted of 18 volunteers and 28 venous ulcers. In the trial group, 2% papain gel was used and, in the control group, 2% carboxymethyl cellulose gel.

RESULTS:

the trial group showed a significant reduction in the lesion area, especially between the fifth and twelfth week of treatment, with two healed ulcers and a considerable increase in the amount of epithelial tissue in the wound bed.

CONCLUSION:

2% papain gel demonstrated greater effectiveness in the reduction of the lesion area, but was similar to 2% carboxymethyl cellulose gel regarding the reduction in the amount of exudate and devitalized tissue. Multicenter research is suggested to evidence the effectiveness of 2% papain gel in the healing of venous ulcers. UTN number: U1111-1157-2998     

一种改进的大鼠皮层神经元原代培养方法及其性质鉴定

目的：对原有大鼠皮层神经元原代培养方法进行改进, 以获得数量更多、纯度更高、体外生长时间更长的神经细胞进行相关实验研究。方法: 使用3种不同成分的液体对培养器皿进行序贯预处理, 分离16~17 d胎龄的Wistar大鼠胎鼠皮层, 采用木瓜蛋白酶化学消化与机械吹打相结合的方法制备单细胞悬液, 经细胞计数后按照不同实验目的进行梯度密度接种。细胞接种当日4~6 h将含有血清的接种培养液换为添加B27的Neurobasal无血清培养基, 培养第3天(DIV3)用终浓度10 μmol/L的阿糖胞苷(cytosine arabinoside, Ara-C)处理24 h抑制胶质细胞增殖, 以后每周半量换液1次。用倒置相差显微镜观察细胞形态, 利用神经元特异性标志物微管相关蛋白2(microtubule associated protein 2, MAP2)与细胞核双标记法鉴定培养神经细胞的纯度, 并以免疫荧光法检测突触前、后标记物以评估突触形成情况。 结果: 与单纯多聚赖氨酸预处理培养器皿, 胰蛋白酶化学消化法分离细胞及含血清培养基培养的原方法相比, 方法改进以后收获的细胞产量明显增加, 且消化过程对细胞损伤小, 接种后细胞分散均匀, 杂质少, 纯度高, 树突、突触发育正常, 可长期存活。 结论: 该方法简便可行, 结果稳定,用该方法培养的大鼠皮层神经元可作为神经元体外培养的良好实验模型。     

木瓜蛋白酶水解对β-酪蛋白膜的润湿性和构象的影响

目的：评价木瓜蛋白酶水解对牛β-酪蛋白（β-CN）膜的润湿性和构象的影响。方法：将β-CN通过自组装固定于芯片表面,采用消散因子石英晶体微天平（quartz crystal microbalance with dissipation,QCM-D）测量β-CN膜频率（ΔF）变化。β-CN膜水解前后的润湿性和构象变化分别通过接触角测量仪和掠角红外光谱（GA-FTIR spectra）仪测量。结果：水解后β-CN膜接触角变小,伴随酶的浓度从0.5～12.5mg/L增加,β-CN膜由疏水性向亲水性转变。FTIR测量表明β-CN膜水解后的构象发生了改变。除α-螺旋结构外,β-CN膜含有的二级结构（%）均变小,表明水解反应首先发生与β-CN分子的疏水性C端。疏水性反应和氢键结合是水解反应的主要驱动力。结论：木瓜蛋白酶水解β-酪蛋白β-CN膜导致了润湿性和构象的改变,其生物学意义有待于进一步研究。     

Melatonin Attenuates the Progression of Osteoarthritis in Rats by Inhibiting Inflammation and Related Oxidative Stress on the Surface of Knee Cartilage

ObjectiveTo investigate the correlation between melatonin and osteoarthritis (OA) in rats. To explore the relevant mechanisms in the occurrence and development of osteoarthritis in rats, and to further understand the disease of osteoarthritis.MethodsForty healthy 6‐month‐old male SD rats were randomly divided into two groups: sham and drug intervention groups. Pre‐OA modeling, enzyme‐linked immunosorbent assay was employed to detect the levels of IL‐1β, IL‐6, COX‐2, and melatonin in the serum of the rats in each group. For OA modeling, we administered an injection of papain into the knee cavity of all rats. The levels of IL‐1β, IL‐6, and COX‐2 in the serum of rats in each group were detected 2 weeks after the modeling. Additionally, 2 weeks after the modeling, the rats in the drug intervention group were intraperitoneally injected with melatonin antagonists. The rats in the sham group were intraperitoneally injected with normal saline for 2 weeks. The levels of IL‐1β, IL‐6, and COX‐2 in the serum of each group were measured at the second, third, and fourth weeks after the drug intervention, and the levels of melatonin in the serum were measured at the second week after the drug intervention. Finally, the rats were euthanized by cervical dislocation, and pathological sections were collected from the knee joint to observe the pathological tissue changes under a microscope, and Mankin score was determined. The independent samples t‐test method was used for analysis.ResultsThe imaging examination after the drug intervention showed that the modeling of knee osteoarthritis in rats was successful. In the pathological findings, HE staining showed a legible cartilage structure of each layer, with cartilage proliferation and partial cartilage tearing to the radial layer. The tide line was intact; toluidine blue staining revealed more obvious changes. The differences among the mean values of IL‐6, IL‐1β, and COX‐2 measured in each period were statistically significant (t = 5.50, p < 0.05). The measured mean values of IL‐6, IL‐1β, and COX‐2 revealed statistically significant differences among the groups (t = 2.01, p < 0.05). The intergroup comparison of the Mankin scores in each period showed statistically significant differences.ConclusionMelatonin may inhibit inflammation and associated oxidative stress on the surface of knee cartilage. It may be related to the repair and regeneration of articular surface cartilage during the development of OA in the rat knee joint.     

木瓜蛋白酶水解β-酪蛋白膜的动力学研究

目的：采用消散因子石英晶体微天平（QCM-D）原位、实时和动态监测木瓜蛋白酶水解β-酪蛋白生物膜的动力学过程,比较不同浓度酶的水解效率。方法：用β-酪蛋白在QCM-D上建立体外蛋白膜模型,采用不同浓度的木瓜蛋白酶水解膜,观察频率和厚度的改变,运用Boltzmann方程对实验数据进行拟合,单因素方差分析和SNK-q检验比较水解参数B、Δh、V50和C。结果：随着酶浓度增大,膜的厚度Δh减薄,ΔF上升增多,水解参数B增大,C减小,V50缩短（P〈0.05）。结论：在一定浓度范围内水解蛋白生物膜时,酶的水解效率随浓度增大而提高。     

酶解法制备可口革囊星虫抗菌物质的研究

目的 确定可口革囊星虫体壁中抗菌物质的最佳酶解工艺。方法 以可口革囊星虫体壁为原料，利用筛选出的木瓜蛋白酶酶解可口革囊星虫体壁蛋白，获得含可口革囊星虫体壁的酶解产物，并用抑菌圈法比较不同条件下酶解产物对大肠埃希菌活性的影响。在单因素试验的基础上，应用四因素三水平正交设计分析法，探讨酶解温度、pH值、酶解时间、加酶量及底物浓度对酶解产物抑菌活性的影响。结论 木瓜蛋白酶最适合水解可口革囊星虫体壁制备抗菌物质，其最佳工艺条件为：底物浓度为4g/L、加酶量为4000U/g、温度为55℃、pH值为8.0的条件下酶解4h，获得平均抑菌圈大小为(15.78±0.5)mm。     

一种高效便捷的家兔肺气肿模型建立方法

目的应用猪胰蛋白酶气管注入同时联合木瓜蛋白酶静脉注射法来建立家兔肺气肿模型。方法将家兔随机分为模型组及对照组。模型组兔耳缘静脉注入8%木瓜蛋白酶同时5%猪胰蛋白酶气管注入制作模型。对照组:家兔经环甲膜穿刺一次性注入生理盐水1 ml/kg。再经家兔耳缘静脉一次性注入生理盐水1 ml/kg。观测实验动物动脉血气及肺功能,并对肺组织进行病理学观察。结果与正常对照组相比,模型组兔动脉血Pa CO2升高;而p H值、Pa O2、Sp O2降低;潮气量下降,呼吸总阻力、中心阻力、总气道阻力升高;病理组织切片光镜下见肺泡壁变薄、断裂,肺泡腔扩张、融合,典型肺气肿表现。结论应用猪胰蛋白酶气管注入同时联合木瓜蛋白酶静脉注射法能成功建立兔肺气肿模型。该方法快捷可靠,适用于各种肺气肿动物研究实验。     

木瓜酶致大鼠软骨基质糖胺多糖代谢改变

给大鼠一次腹腔注射1.5％粗制木瓜酶溶液(按150mg/kg体重计量),可明显引起大鼠软骨基质可复性损伤。注射后6h,实验组大鼠软骨硫酸软骨素(CH·S)含量便开始下降,至24h仅为对照组含量的一半,同时软骨CH·S有机硫含量明显减少。实验组大鼠尿液糖胺多糖(GAG)排泄量子注射后12h开始升高,24h达到最高峰,为对照组排泄量的3倍,GAG排泄量的增加主要成分为CH·S。上述代谢损伤至72h可完全恢复正常。实验证明,木瓜酶对软骨CH·S分子量无明显影响。     

汞和金属离子及多硫化物对木瓜蛋白酶活性的抑制作用

目的半胱氨酸蛋白酶参与了很多动植物生理过程和病理过程,是Hg2＋及其化合物作用的潜在靶点。木瓜蛋白酶是半胱氨酸蛋白酶家族中最具代表性、研究最为广泛深入的一种蛋白酶,本文以木瓜蛋白酶为模型, 研究了汞等金属离子及其化合物对半胱氨酸蛋白酶活性的抑制作用。结果 Hg2＋对木瓜蛋白酶活性的抑制能力很强（Ki=2×10^-7 mol·L^-1）。过量的还原型谷胱甘肽和半胱氨酸能够恢复木瓜蛋白酶的活性。多种金属离子（Cu^2＋、Ag^＋、Au^3＋、Zn^2＋、Cd^2＋、Fe^3＋、Mn^2＋、Pb^2＋、Yb^3＋）对木瓜蛋白酶的活性具有抑制能力,而且抑制能力与金属离子自身的软硬度具有良好的相关性。研究结果表明 Hg2＋ 可能作用于木瓜蛋白酶的活性中心。此外,本文首次研究了汞多硫化物复合物对木瓜蛋白酶活性的抑制作用,发现其表观抑制常数（Ki= 7×10^-6 mol·L^-1）与柳硫汞（Ki=2.7×10^-6mol·L^-1）十分接近,而且其caco-2细胞单层的表观渗透能力（1.9×10^-5cm/s） 也较好。结论汞多硫化物复合物可能通过与半胱氨酸蛋白酶或者其它含巯基蛋白分子作用,发挥生物学活性。本文的研究工作可能有助于阐明汞化合物（ 特别是朱砂等不溶性汞化合物） 的毒理及药理作用的机制。