木瓜蛋白酶对凝血功能的抑制作用及机制的体外研究

目的：观察木瓜蛋白酶体外对凝血功能的抑制作用,并探讨其可能机制。方法：将不同剂量木瓜蛋白酶分别与贫血小板血浆（PPP）和富血小板血浆（PRP）作用,分为生理盐水组、10U/L组和20U/L组,分别以血凝仪测定PPP和PRP的凝血酶原时间（PT）和活化部分凝血活酶时间（APTT）,以血气分析仪和血凝仪分别测定PPP的Ca2＋浓度和凝血因子V、VII、VIII、IX、X和XI活性（FV：C、FVII：C、FVIII：C、FIX：C、FX：C和FXI：C）;将新鲜全血与前述3种浓度木瓜蛋白酶作用,采用硅化管法测定全血凝血时间（CT）。同时测定0、20、40、60和80U/L木瓜蛋白酶PPP的PT和APTT值。结果：10 U/L和20U/L木瓜蛋白酶组PPP和PRP的PT和APTT值、全血CT值分别显著高于生理盐水组和10U/L木瓜酶组（P〈0.01）,FV：C和FVIII：C水平分别显著低于生理盐水组和10U/L木瓜酶组（P〈0.05）;三组PPP与PRP之间PT和APTT值、各组间Ca2＋浓度以及其余凝血因子活性差异均无显统计学意义（P〉0.05）。PPP的PT和APTT值均与木瓜蛋白酶剂量呈显著正相关（r=0.995和0.991,P〈0.01）。结论：木瓜蛋白酶可通过抑制凝血因子V和VIII活性,从而对凝血功能有剂量依赖性的抑制作用,具有抗凝的功效。     

高原鼢鼠肉木瓜蛋白酶酶解物对小鼠免疫功能的影响

目的研究高原鼢鼠肉蛋白的木瓜蛋白酶酶解物生理活性功能,对酶解物进行了免疫活性实验。方法通过MTT法和中性红比色法测定其对小鼠脾淋巴细胞增殖、腹腔巨噬细胞吞噬功能、网状内皮系统吞噬功能、NK细胞杀伤毒性,鼠抗体生成脾细胞数(IgM-PFC)的影响。结果酶解物高、中剂量与对照相比有显著差异,而其中以高剂量组的活性最明显,能够显著提高小鼠淋巴细胞的增殖能力;激活小鼠网状内皮系统,增强吞噬功能;增强NK细胞活性;增加小鼠脾脏的抗体生成细胞数。结论高原鼢鼠肉蛋白的木瓜蛋白酶酶解物能明显提高正常小鼠细胞免疫、体液免疫及NK细胞杀伤毒性的能力。     

Thiirancarboxamides as inhibitors of papain

Derivatives of the thiirancarboxylic acid building-block containing a peptide bond were synthesised and screened against the model cysteine protease papain. The most active of the series showed a second-order rate constant of inactivation comparable to that of the parent compound. The insertion of a peptide moiety seems to compensate the lack of a free carboxylate interacting with the histidinium ion at the enzyme's active site.     

微波辐射在血型血清学试验中的应用

目的 为了缩短疑难配血、新生儿溶血病母婴血型不合抗体检测及Rh血型不完全抗体鉴定试验时间。方法 应用家和微波炉：①做间接抗球蛋白试验：检测Rh血型IgG抗-D抗体效价，试用微波辐射二档3min加一档5min致敏；②木瓜蛋白酶试验，采用低强度的微波辐射一档6～8min做试验，通过木瓜蛋白酶介质破坏红细胞上唾液酸的作用，检测Rh血型IgG抗-D抗体效价。结果 ①间接抗球蛋白试验采用微波辐射效价与37℃水浴相同，但前者积分高出10min左右，较常规的抗球蛋白法，致敏细胞阶段可缩短时间50min。②木瓜蛋白酶试验6～8min，效价高出1个滴度，积发高出14分。试验可缩短时间12min。结论 该方法操作简单，节约时间，可重复性好。我们用微波辐射法鉴定Rh(D)抗原200人，结果与37℃木瓜蛋白酶法一致。     

甘蔗渣纤维素固定化木瓜蛋白酶及其应用研究

匀浆状甘蔗渣纤维素用NaOH活化后，顺次经过高碘酸钠、尿素、甲醛处理，成为高反应性固定化酶载体，可用于木瓜黄蛋白酶的固定化，其酶蛋白的结合量与甲醛处理时间、酶固定时间、溶液酶浓度等有关，甲醛处理最适时间为14h，酶固定8h后，载体结合酶量趋向稳定；溶液酶最佳浓度范围为1－2．0mg/ml粗酶，固定化酶活力回收达34．5％，半衰期35天，固定化酶和溶液酶的Km值（底物酪蛋白，w/v,%)分别为0．12％和0．26％；固定化酶和溶液酶的最适PH分别为PH8．0和PH8．5，二者的最适温度均为60－70℃。固定化酶和溶液酶一样有底物抑制现象，固定化酶在6mol/L脲中处理6h活力趋向稳定，其活力为原有活力的46．75，用固定化酶处理啤酒，啤酒浊度下降了2－9．25倍，蛋白质含量下降了78．8％，冷藏120天，处理啤酒无冷混浊现象发生，同时啤酒原有风味和其它理化指标保持不变。     

木瓜蛋白酶水解对β-酪蛋白膜的形态和黏弹性的影响

目的：评价木瓜蛋白酶水解对牛β-酪蛋白（β-CN）膜表面形态和黏弹性的影响。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膜能量消散因子（△D）和频率（△F）和黏弹性变化。β-CN膜水解前后的表面形态变化通过原子力显微镜（atomic force microscopy,AFM）表征。结果：伴随着酶浓度提高,残余的β-CN膜的黏弹性增加（P＜0．05）。AFM图像显示水解后残余的β-CN膜的厚度和粗糙度均降低。结论：木瓜蛋白酶水解β-酪蛋白β-CN膜的反应具有浓度依赖性特征;水解反应使膜变得较薄而光滑,且更加紧凑和较硬。     

Randomized controlled clinical trial of long-term chemo-mechanical caries removal using PapacarieTM gel

Objectives

Compare the effectiveness of Papacarie^TM gel for the chemo-mechanical removal of carious lesions on primary teeth to conventional caries removal with a low-speed bur with regard to execution time, clinical aspects and radiographic findings.

Material and Methods

A randomized controlled clinical trial with a split-mouth design was carried out. The sample was composed of 20 children aged four to seven years, in whom 40 deciduous teeth were randomly divided into two groups: chemo-mechanical caries removal with Papacarie^TM and removal of carious dentin with a low-speed bur. Each child underwent both procedures and served as his/her own control. Restorations were performed with glass ionomer cement. The time required to perform the procedure was also analyzed. The patients underwent longitudinal clinical and radiographic follow-up of the restorations.

Results

No statistically significant difference between groups was found regarding the time required to perform the procedures and the radiographic follow up. Statistically significant differences between groups were found in the clinical evaluation at 6 and 18 months after treatment.

Conclusion

Papacarie^TM is as effective as the traditional method for the removal of carious dentin on deciduous teeth, but offers the advantages of the preservation of sound dental tissue as well as the avoidance of sharp rotary instruments and local anesthesia.     

从RPMI8866细胞制备CD23分子最佳条件及提取方法的研究

建立从高表达CD23分子的RPMI8866细胞株分离制备CD23分子的两种方法:自然裂解法和木瓜蛋白酶水解法。比较了此两法的优劣,探讨了CD23回收量的影响因素,经Western blot-ting技术鉴定出制备品中18、37、42、45和86KDa的CD23片段。结果表明,上述方法简便、可靠、易行,且回收量较高。可供研究及分离其它膜分子参考。     

High frequency jet ventilation in experimental pulmonary emphysema

The effects of high frequency jet ventilation (HFJV, f=2 Hz and 8 Hz, I:E=0.43, FiO₂=0.4) were studied and compared with intermittent positive pressure ventilation (IPPV, f=10–14 breaths/min, V_T=15 ml/kg, I:E=0.5, FiO₂=0.4) in 8 dogs before and after induction of panlobular emphysema (PLE). PLE increased alveolar-arterial PO₂ difference (P_A-aO₂) during all modes of ventilation, whereas PaCO₂ did not change significantly. In both periods of the study, HFJV_8Hz was less effective in terms of CO₂-elimination and oxygenation. In the control-period, functional residual capacity (FRC) was 937±212 ml. The increase during HFJV (HFJV_2Hz:1156±508 ml, HFJV_8Hz:1153±433 ml) did not reach significance (P=0.09). Closing volume (CV) increased from 1.5±4.3% of vital capacity (%VC) (IPPV) to 6.3±7.1%VC (HFJV_2Hz) and 10.8±9.8% VC (HFJV_8Hz), respectively. In the PLE-period, FRC and CV increased significantly to 1107±207 ml and 14.1±7.0% VC respectively during IPPV (P<0.05). Application of HFJV neither increased FRC (HFJV_2Hz: 1153±433 ml, HFJV_8Hz: 1005±344 nor CV 14.8±6.0% VC and 13.9±8.1% VC, respectively). It is concluded that HFJV induces no alveolar overdistension in dogs with emphysematous lungs.     

Single breath N2 washout in papain-induced pulmonary emphysema

Single breath nitrogen washout tests were analyzed in dogs (n=8) with healthy lungs and after development of emphysema. The animals were in the supine position and studied during anaesthesia and mechanical ventilation (F_iO₂=0.4, F_iN₂=0.6). During controlled expiration with constant flow (V_E=0.15 1/s) onset of phase IV of the alveolar plateau was related to airway closure of dependent lung regions (closing volume CV). In the control state, CV accounted for 6.2±1.5% VC, and closing capacity (CC) was lower than functional residual capacity (FRC). Likewise, gas exchange was normal in all animals (PaO₂=24.7±3.32 kPa, PaCO₂=5.18±0.53 kPa, P_A-aO₂=2.6±0.3 kPa). Panlobular emphysema (PLE) was induced by inhalation of papain (100 mg/kg). After three weeks development of PLE was documented by messurements of lung volumes (functional residual capacity (FRC), expired vital capacity (EVC), total lung capacity (TLC), residual volume (RV)), pulmonary mechanics (dynamic and static compliance (C_dyn, C_stat), mean airway resistance (R_aw)), gas exchange (PaO₂, PaCO₂, P_A-aO₂), and by radiomorphological analysis. In the PLE-group, FRC and RV (p0.05), and C_stat (p0.01) were significantly elevated. CV increased to 16.2±2.7% VC (p0.01) and CC exceeded FRC by 80 ml, indicating that tidal volume breathing took place within the range of closing volume. Oxygenation was significantly impaired (PaO₂=18.6±3.72 kPa, P_A-aO₂-6.5±1.1 kPa, p0.05), but not CO₂-elimination. Pathological analysis by radiomorphological means showed dissiminate parenchymal lesions compatible with emphysema of grade II severity located predominantly in subpleural areas. In dogs with papain-induced PLE, premature closure of dependent airways is enhanced, which is due to structural changes and a loss of elastic recoil in the lungs.