可溶性固定化木瓜蛋白酶的制备及性质研究

目的 制备一种可溶性固定化木瓜蛋白酶(PP)并研究其部分酶学性质.方法 将PP偶联在羧甲基纤维素醋酸琥珀酸酯(AS-L)上,制备得到水溶性固定化PP,并测定游离和水溶性固定化PP最适作用pH、最适作用温度、Km值、热稳定性、酸碱稳定性、低温稳定性和回收稳定性.结果 最适作用pH分别为6.0、5.0,最适作用温度分别为60℃、70℃,Km值分别为2.53、3.07 mg·ml-1;可溶性固定化PP在60℃条件下,保温12 h,仍有62%的活性;在4℃条件下,30 d后活性保留达90%以上;在pH6时稳定性最好,pH4～7范围内也较稳定(相对活性＞80%).结论 可溶性固定化PP能够在大于pH5.5的溶液中完全溶解,最适作用pH范围变窄,最适作用温度和Km值升高;热和酸碱的稳定性均明显增强.     

改良木瓜酶介质输血相容性试验及临床应用

本文介绍一种筛选免疫性抗体的改良木瓜酶介质输血相容性试验。此法与传统的木瓜酶试验及低离子强度抗球蛋白试验同时检测免疫性Ｒｈ抗体（抗－Ｄ，抗－Ｃ抗－Ｆ，抗－ｃ的凝集反应强度（平均效价积分值），其结果没有显著性差异（Ｐ均）０．２０）。本法灵敏、快速、简便，也不需特殊设备，能与盐水法输血相容性试验同步进行，常规开展十分方便，临床应用效果较好，有一定推广价值     

肺泡壁细胞凋亡参与木瓜蛋白酶所致大鼠肺气肿的形成

目的：观察木瓜蛋白酶所致的肺气肿大鼠肺泡壁细胞凋亡，探讨细胞凋亡在肺气肿发病中的作用。方法：大鼠随机分为3组：正常对照组、木瓜蛋白酶组、木瓜蛋白酶联合［60Co］处理组。观察肺组织病理学变化，TUNEL法检测大鼠肺泡壁凋亡细胞，免疫组化法检测肺泡壁细胞PCNA、Bax和SP-C蛋白的表达。TUNEL与SP-C免疫荧光法鉴定凋亡细胞中的II型肺泡上皮细胞。结果： 木瓜蛋白酶组、木瓜蛋白酶联合［60Co］处理组大鼠出现明显肺气肿。木瓜蛋白酶联合［60Co］处理组平均内衬间隔，平均肺泡面积和单位面积肺泡数与木瓜蛋白酶组比较有显著差异。木瓜蛋白酶组，木瓜蛋白酶联合［60Co］处理组大鼠肺泡壁细胞的AI、PI以及Bax染色阳性的细胞百分比显著高于正常对照组；而SP-C染色阳性的细胞百分比显著低于正常对照组。木瓜蛋白酶联合［60Co］处理组大鼠的AI、PI、Bax染色阳性的细胞百分比显著高于木瓜蛋白酶组；而SP-C染色阳性的细胞百分比显著低于木瓜蛋白酶组。部分TUNEL阳性细胞表达II型肺泡上皮细胞标志SP-C。结论：大鼠肺泡壁细胞特别是II型肺泡上皮细胞凋亡参与肺气肿的形成，Bax蛋白的表达上调可能导致肺气肿大鼠肺泡壁细胞的凋亡。     

血型不规则抗体检测的方法学评价

目的寻找简便、快速、敏感的用于红细胞不规则抗体筛选的试验方法。方法收集6例临床疑难病例样本和1份IgG型试剂,采用盐水法、木瓜酶法、凝聚胺法、抗人球蛋白法、微柱凝胶法平行检测患者血清中不规则抗体的效价,比较5种方法的敏感性、特异性。结果盐水法仅检出IgM类不规则抗体;木瓜酶法、凝聚胺法、抗人球蛋白法、微柱凝胶法在检测IgG类抗体效价时差异不大,木瓜酶法的敏感性略低,微柱凝胶法对弱阳性IgG抗体检测的敏感性最高,但4种方法间差异无统计学意义(P>0.05),相关性较好(r>0.99)。结论盐水法不能检出IgG抗体;木瓜酶法检测IgG抗体具有一定的局限性;凝聚胺法简便、快速,但为手工操作,不易标准化;抗人球蛋白法需要反复洗涤红细胞,费时,不便于开展大规模筛查;微柱凝胶法操作简便迅速、敏感性高、重复性好、易于标准化,适于临床输血前和孕妇产前大批量抗体筛选,是检测IgG抗体最为敏感的方法。     

玻璃酸钠对关节腔内注入木瓜蛋白酶所致兔膝关节退行性改变的改善作用

采用在兔膝关节内注入木瓜蛋白酶造成的膝关节退行性改变模型,考察关节腔内长期多次注射玻璃酸钠对这种退行性改变的抑制和改善作用。结果表明:玻璃酸钠对已造成退行性改变的关节软骨有减轻和改善其病变程度的作用。     

A conserved poxvirus NlpC/P60 superfamily protein contributes to vaccinia virus virulence in mice but not to replication in cell culture

Of the vaccinia virus genes that are conserved in all sequenced poxviruses, each one except for VACWR084 (G6R) has been at least partially characterized. The poxvirus protein encoded by G6R belongs to the NlpC/P60 superfamily, which consists of proteins with a papain-like fold and known or predicted protease, amidase or acyltransferase activity. The G6 protein was synthesized late in infection and localized to the interior of virions, primarily between the membrane and core. Unlike other conserved poxvirus genes, G6R was not required for virus propagation and spread in a variety of cells. Nevertheless, G6R null mutants caused less severe disease in mice than the parent or revertant virus. Moreover, mutation of the predicted catalytic cysteine led to the same level of attenuation as a null mutant, suggesting that the G6 protein has enzymatic activity that is important in vivo. Conservation of G6R amongst poxviruses and the disparity between its role in vitro and in vivo imply that the protein is involved in an aspect of the virus-host interaction that is common to vertebrates and insects.     

用木瓜蛋白酶在两相系统中合成二肽甜味剂Aspartame

研究了多种因素对木瓜蛋白酶在水－有机溶剂两相反应体系中合成二肽甜味剂Ａｓｐａｒｔａｍｅ能力的影响，结果表明，在本实验条件下，水与乙酸乙酯的比例为６：１００～７：１００之间为宜；反应最适ｐＨ为５．２；最适温度３７℃；反应时间为９ｈ；酶用量为１００～１２０ｍｇ／ｍｍｏｌ底物；Ｌ－ＰｈｅＯＭｅ与ＣＢＺ－Ｌ－Ａｓｐ的摩尔比为１：１。     

Efficacy of Papacarie® in reduction of residual bacteria in deciduous teeth: a randomized,controlled clinical trial

OBJECTIVES:

The aim of the present study was to analyze the efficacy of Papacarie® gel compared with the traditional method (low-speed bur) in reducing the counts of total bacteria, Lactobacillus, total Streptococcus and Streptococcus mutans group.

METHODS:

A randomized, controlled clinical trial with a split-mouth design was performed. The sample comprised 40 deciduous teeth in 20 children (10 males and 10 females) aged four to seven years. The teeth were randomly allocated to two groups: G1, or chemomechanical caries removal with Papacarie Duo®, and G2, or the removal of carious dentin tissue with a low-speed bur. Infected dentin was collected prior to the procedure, and the remaining dentin was collected immediately following the removal of the carious tissue. Initial and final counts of bacterial colonies were performed to determine whether there was a reduction in the number of colony-forming units (CFUs) of each microorganism studied. ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01811420","term_id":"NCT01811420"}}NCT01811420.

RESULTS:

Reductions were found in the numbers of total bacteria, total Streptococcus and Streptococcus mutans group following either of the caries removal methods (p<0.05). A reduction was also noted in the number of Lactobacillus CFUs; however, this difference did not achieve statistical significance (p>0.05).

CONCLUSION:

Papacarie® is an excellent option for the minimally invasive removal of carious tissue, achieving significant reductions in total bacteria, total Streptococcus and S. mutans with the same effectiveness as the traditional caries removal method.     

关节腔内注射不同浓度木瓜蛋白酶建立小鼠膝骨关节炎模型的研究

目的探讨关节腔内注射不同浓度木瓜蛋白酶建立小鼠膝骨关节炎模型的可行性。方法健康10周龄雄性昆明小鼠80只,体重(45±3)g,随机分为2%木瓜蛋白酶组、4%木瓜蛋白酶组、8%木瓜蛋白酶组和对照组。实验第1、3、5天分组行右膝关节腔内注射2%、4%、8%(w/v)木瓜蛋白酶和0.3mol/LL-半胱氨酸的混合溶液0.25 m L/kg或等量生理盐水。记录小鼠一般情况及右膝关节宽度。所有小鼠首次注射1周后处死,取关节行常规HE染色及甲苯胺蓝染色,镜下观察并评分。结果造模组右膝关节出现肿胀、功能障碍;右膝关节软骨染色变浅、软骨层变薄、基质分解破坏等病理学改变,其严重程度与木瓜蛋白酶浓度呈正相关;对照组无上述改变。结论膝关节腔内注射2%、4%、8%(w/v)木瓜蛋白酶和0.3mol/L L-半胱氨酸的混合溶液0.25 m L/kg可建立小鼠不同严重程度的膝骨关节炎,周期短、成功率高。     

木瓜酶促进大鼠血管内皮细胞成血管的功能研究

目的　通过体外鼠细胞模型实验,检测木瓜酶在体外培养环境下对血管内皮细胞成血管向分化的促进功能。方法　采用P3代大鼠血管内皮细胞,将木瓜酶溶解于完全培养基内,按照10 μg/ml、50 μg/ml、100 μg/ml浓度配置含木瓜酶的完全培养基,与空白对照组共同培养0、7、14 d,通过逆转录聚合酶链反应（qRT-PCR）法检测成血管分化相关基因VEGF（血管内皮生长因子）、CD31（血小板-内皮细胞粘附分子）的表达水平,通过CCK-8法检测不同组细胞的增殖活力。统计学方法为方差分析。结果　经14 d培养,添加木瓜酶的细胞VEGF、CD31表达水平及增殖能力显著高于空白对照组,木瓜酶浓度达到50 μg/ml时VEGF表达水平达到最高,为空白对照组的3.28倍,CD31表达水平随着木瓜酶浓度升高而升高,最高达到空白对照组的3.66倍。结论　适宜浓度的木瓜酶能够在体外促进内皮细胞的成血管分化及增殖,表明木瓜酶对血管组织形成具有一定的促进作用。