Insight into the spoilage heterogeneity of meat-borne bacteria isolates with high-producing collagenase

Chilled chicken is inevitably contaminated by microorganisms during slaughtering and processing,resulting in spoilage.Cutting parts of chilled chicken,especially wings,feet,and other skin-on products,are abundant in collagen,which may be the primary target for degradation by spoilage microorganisms.In this work,a total of 17 isolates of spoilage bacteria that could secrete both collagenase and lipase were determined by raw-chicken juice agar(RJA)method,and the results showed that 7 strains of Serratia,Aeromonas,and Pseudomonas could significantly decompose the collagen ingredients.The gelatin zymography showed that Serratia liquefaciens(F5)and(G7)had apparent degradation bands around 50 kDa,and Aeromonas veronii(G8)and Aeromonas salmonicida(H8)had a band around.65 and 95 kDa,respectively.The lipase and collagenase activities were detected isolate-by-isolate,with F5 showing the highest collagenase activity.For spoilage ability on meat in situ,F5 performed strongest in spoilage ability,indicated by the total viable counts,total volatile basic nitrogen content,sensory scores,lipase,and collagenase activity.This study provides a theoretical basis for spoilage heterogeneity of strains with high-producing collagenase in meat.

Long-term assessment of collagenase treatment for Dupuytren’s contracture:A 10-year follow-up study

BACKGROUND Enzymatic fasciotomy with collagenase clostridium histolyticum(CCH)has revolutionized the treatment for Dupuytren’s contracture(DC).Despite its benefits,the long-term outcomes remain unclear.This study presented a comprehensive 10-year follow-up assessment of the enduring effects of CCH on patients with DC.AIM To compare the short-term(12 wk)and long-term(10 years)outcomes on CCH treatment in patients with DC.METHODS A cohort of 45 patients was treated with CCH at the metacarpophalangeal(MCP)joint and the proximal interphalangeal(PIP)joint and underwent systematic reevaluation.The study adhered to multicenter trial protocols,and assessments were conducted at 12 wk,7 years,and 10 years post-surgery.RESULTS Thirty-seven patients completed the 10-year follow-up.At 10 years,patients treated at the PIP joint exhibited a 100%recurrence.However,patients treated at the MCP joint only showed a 50%recurrence.Patient satisfaction varied,with a lower satisfaction reported in PIP joint cases.Recurrence exceeding 20 degrees on the total passive extension deficit was observed,indicating a challenge for sustained efficacy.Significant differences were noted between outcomes at the 7-year and 10-year intervals.CONCLUSION CCH demonstrated sustained efficacy when applied to the MCP joint.However,caution is warranted for CCH treatment at the PIP joint due to a high level of recurrence and low patient satisfaction.Re-intervention is needed within a decade of treatment.

A randomized clinical trial assessing the efficacy of single and multiple intralesional collagenase injections for treating contracted scars

Background:Scar contractions caused by trauma or burns can cause secondary physical dysfunction and disfigurement.Many minimally invasive methods for scar contraction have shown limited applicability and efficacy.This study investigated the feasibility and efficacy of intralesional collagenase injections for scar contraction treatment.Methods:Patients with contracted scars who had limited joint movement and physical disfiguration for>1 year were enrolled in this single-blind,randomized clinical trial from July 2017 to February 2018 at Shanghai Ninth People’s Hospital.Collagenase was injected into the firm-contracted scar(15 U/cm^(2))three times at 4-week intervals in the multiple treatment group and once in the single treatment group,and a placebo injection was performed in the control group.Scar length and skin texture were documented at the 4-and 12-week follow-ups.The safety of the collagenase treatment was also evaluated.Results:The contracted scar was significantly elongated after both single and multiple collagenase treatments.The results showed that,compared to a one-time treatment,repeated injections were more effective at 12 weeks,with an average improvement of 26.83(15.79%).At 12 weeks,78.9% of the patients in the multiple group and 52.9%in the single group achieved significant improvement at 12 weeks.No severe adverse events were observed.Conclusion:Intralesional collagenase injection showed promising results in improving scar contraction and provides an alternative treatment for patients.

Dispase诱发兔眼玻璃体后脱离的效果和安全性评价

目的 采用Dispase在兔眼中诱导完全性PVD形成 ,评价其效果和安全性。 方法 选择健康成年的青紫蓝兔 3 0只 ,随机分成 5组 ,采用玻璃体内注射 ,分别注射Dispase 0 0 0 5U、0 0 12 5U (2组 )、0 0 2 5U、0 0 5U/0 0 5ml,PBS 0 0 5ml注射到另一只眼内作为对照。术前术后眼底镜、裂隙灯和VOLK + 90D随访观察 ,并进行B超和视网膜电图 (ERG)检查 ,最后取眼球对视网膜进行扫描电镜和透射电镜观察。结果 注射Dispase≥ 0 0 12 5U可见玻璃体后脱离 ,部分伴有不同程度的眼底出血 ;B型超声检查见玻璃体混浊和后脱离 ;术后电生理检查 ,0 0 0 5U和 0 0 12 5U组ERG没有变化 ,≥ 0 0 2 5U ,部分出现ERG振幅下降 ;对照组没有以上改变。结论 Dispase 0 0 12 5U可以在兔眼中成功、安全诱发完全性玻璃体后脱离 (PVD) ,更大剂量同样有效 ,但是有一定毒性作用。

Dispase蛋白酶诱导兔眼玻璃体后脱离的实验研究

目的:研究dispase蛋白酶诱导玻璃体后脱离的效果和安全性。方法:24只健康成年的青紫兰兔随机分为A,B,C,D4组,每组6只兔右眼均为实验眼,左眼为对照眼。A～D组实验眼玻璃体腔内分别注射dispase蛋白酶25,50,125和250U/L,对照眼均为眼用平衡盐BSS液0.0001L。注药前后行检眼镜、裂隙灯和VOLK+90D前置镜以及B超和视网膜电图(electroretinogram,ERG)检查,最后取眼球扫描电镜和透射电镜观察。结果:电镜结果A组实验眼无PVD发生;B,C2组实验眼中各有4眼发生完全性PVD,发生率为67%;D组实验眼有5眼发生完全性PVD,发生率为83%;透射电镜观察对照眼及A,B2组实验眼视网膜组织结构正常。C,D组视网膜细胞和细胞器出现水肿和变性。ERG检测A,B2组术后b波振幅与术前比较无明显降低(P>0.05);而C,D2组实验眼术后b波振幅较术前明显降低(P<0.01)。结论:浓度50U/L的dispase蛋白酶可有效地诱导PVD且对视网膜无明显的毒性作用。更高浓度的dispase蛋白酶虽然可迅速有效的诱导完全性PVD,但对视网膜有毒性作用。

Dispase Ⅱ酶对人角膜缘干细胞培养结果的影响

目的 分析Dispase Ⅱ酶对人角膜缘干细胞培养的影响。方法 将110 例人角膜随机分为A、B 2组,在组织块培养过程中分别用和不用Dispase Ⅱ酶处理组织块,观察并分析2种处理方法培养时的细胞生长速度及培养成功率。结果 2 组细胞生长的最早及最晚时间均为(4.00±0.45)d和(11.00±0.27)d,培养成功率分别为93.54%±3.12%和98.51%±1.44%,不使用DispaseⅡ酶处理组比使用DispaseⅡ酶处理组不但培养成功率高,而且从培养至见到细胞生长时间早,统计学上有非常显著的差异(P<0 01,P<0 001)。结论 在人角膜缘干细胞的组织块培养中,不用Dispase Ⅱ酶处理可以得到更快的细胞生长速度,更高的培养成功率。

Dispase在成年鼠骨骼肌干细胞纯化中的价值

目的寻找一种最佳的骨骼肌干细胞分离及纯化方法。方法采用成年雌性SD大鼠,分别采用二步消化法和Dispase消化法分离肌干细胞,然后用差速贴壁技术纯化肌干细胞,骨骼肌肌动蛋白(α-sarcometricactin)免疫细胞化学染色对细胞进行鉴定。结果Dipase组肌干细胞的数量明显多于二步消化法组,且细胞的生长速度也明显较后者快,而衰老速度则明显慢于后者。结论Dipase可明显减轻细胞的损伤,提高肌干细胞的数量和质量,是一种最佳的细胞分离酶。

纤维蛋白溶解酶和Dispase蛋白酶诱导兔眼玻璃体后脱离的实验研究

目的研究纤维蛋白溶解酶和dispase蛋白酶诱导玻璃体后脱离(PVD)的作用。方法24只健康成年的青紫兰兔随机分为4组,右眼均为实验眼,左眼为对照眼。A组实验眼玻璃体腔内注射纤溶酶1U,B组纤溶酶2 U,C组dispase蛋白酶0.0125U和D组dispase蛋白酶0.025U,对照眼均为眼用平衡盐BSS液0.1 ml。注药前后行眼底镜、裂隙灯和VOLK+90D前置镜以及B超和视网膜电图(ERG)检查,最后取眼球进行光镜、扫描电镜和透射电镜观察。结果电镜结果A组2只实验眼后极部发生不完全性PVD,发生率为33.3%;B、C两组实验眼中各有4只眼发生完全性PVD。发生率为66.7%;D组实验眼有5只眼发生完全性PVD,发生率为83.3%。纤溶酶各组实验眼注药后ERG振幅与术前及对照眼比较无显著性差异(P>0.05),光镜和透射电镜观察视网膜组织结构正常,均未发现明显异常改变。而Dispase蛋白酶各组透射电镜观察视网膜细胞和细胞器出现水肿和变性。ERG检测Dispase两组实验眼术后b波振幅较术前明显降低(P<0.01)。结论玻璃体腔注射纤溶酶2U较注射1U更能有效的诱导PVD且对视网膜无明显的毒性作用。而Dispase蛋白酶虽然可迅速有效的诱导PVD,但对视网膜有明显的毒性作用。

利用Dispase高效分离胰岛的实验研究

目的如何获得高质量、数量充足的小鼠胰岛细胞。方法通过比较当前研究中使用的大多数消化酶来分离小鼠胰岛,证实相继使用XI型胶原酶和Dispase比单独使用前者更为有效。结果可获得更多的胰岛细胞(增加至40%)、更短的消化时间(下降25%),并且使用较少的胶原酶(减少40%)。结论这一新改良的方案在小鼠胰岛分离的质量、数量方面都有明显改善。

Dispase Ⅱ和胰酶消化法分离大鼠皮肤角朊细胞的对比研究

引言国内外学者均用酶消化法分离表皮和真皮．消化酶有ＤｉｓｐａｓｅⅡ，胰酶和胶原酶，因胶原酶价钱昂贵，故多数单位不使用它．目前尚无学者对比研究ＤｉｓｐａｓｅⅡ和胰酶，观察其对收集的角朊细胞数、活力、细胞增殖及成纤维细胞（Ｆｂ）污染的影响，故我们希望通过...

Dispase Ⅱ分离皮肤表皮-真皮连接及光镜和扫描电镜样本的制作

ＤｉｓｐａｓｅⅡ分离皮肤表皮－真皮连接及光镜和扫描电镜样本的制作１姜笃银１陈璧２王剑波３史颖辉作者单位：１第四军医大学西京医院烧伤科，西安７１００３２２第四军医大学病理学教研室、３电镜中心，西安７１００３３复合皮（ｃｏｍｐｏｓｉｔｅｓｋｉｎ，ＣＳ）移...

兔眼玻璃体切割术中应用dispase的毒性研究

目的兔眼中央玻璃体切割后注射dispase蛋白酶,观察dispase蛋白酶诱导玻璃体后脱离(PVD)形成的安全浓度。方法新西兰白兔24只,随机分为A、B、C组,每组8只,以右眼为实验眼,左眼为对照眼。兔眼中央玻璃体切割后注射0.5mL dispase,浓度分别为A组0.05μmol/min、B组0.1μmol/min、C组0.2μmol/min;对照组注射0.5mL无菌PBS。用药3个月后光镜和透射电镜观察dispase蛋白酶对视网膜超微结构的影响。结果光镜下可见0.05μmol/min组、0.1μmol/min组、对照组眼视网膜结构完整,排列整齐,未发现异常改变。0.2μmol/min组眼神经节细胞减少。透射电镜下见各实验组动物视网膜内界膜清晰完整,但A组用药3个月后视网膜光感受器内外节结构大致正常,B组、C组实验眼视网膜在用药3个月后有不同程度的光感受器内外节结构紊乱;节细胞水肿,线粒体嵴断裂、空泡变。对照组视网膜超微结构未见明显异常。结论0.05、0.1、0.2μmol/min的dispase均可以诱导兔眼的玻璃体后脱离,随着浓度的增加,毒性增大。

Dispase金属蛋白酶诱导的兔增殖性玻璃体视网膜病变模型

目的建立并评价dispase诱导的增殖性玻璃体视网膜病变(proliferativevitreoretinopathy,PVR)动物模型。方法选择健康成年的青紫蓝兔40只,右眼分别行玻璃体腔注射dispase0.05ml,按注射浓度不同,随机分成4组,A组(0.05U)、B组(0.075U)、C组(0.1U)、D组(0.2U)。左眼分别注射PBS0.05ml作为对照。术后第3小时、第1天、第7天至第70天每周进行眼部检查。检查项目包括裂隙灯、间接眼底镜和眼底照相。在处死动物后,典型的PVR眼进行组织病理学检查及增殖细胞核抗原(proliferatingcellmuclearantigen,PCNA)免疫组织化学染色。结果术后3h所有dispase眼均见视网膜前或玻璃体出血。大约在3周后,dispase眼依次出现视网膜前膜、髓线和血管抬高、固定视网膜皱折以及牵拉性视网膜脱离。组织病理学见视网膜前增殖膜对视网膜的牵拉以及视网膜结构的紊乱。在dispase注药后2周,视网膜节细胞层抗增殖细胞核抗原(PCNA)呈阳性表达,4周时内核层细胞表达阳性,至发生视网膜脱离后视网膜色素上皮开始表达PCNA。对照组未见PVR发生,也未见PCNA的阳性表达。结论Dispase可以在不使用外源性细胞、生长因子和细胞因子的情况下诱导视网膜细胞增殖从而建立PVR模型,操作简单且成功率高。

DispaseⅡ消化法原代培养兔结膜上皮细胞及鉴定

目的研究DispaseⅡ消化法原代培养兔结膜上皮细胞,观察其体外生长特性,并采用免疫组织化学染色鉴定培养的上皮细胞。方法 DispaseⅡ消化法原代培养兔结膜上皮细胞,观察不同时期细胞的生长情况、细胞形态,做广谱角蛋白、MUC5AC免疫荧光染色及PAS染色鉴定;并比较不同部位结膜上皮细胞增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达阳性情况。结果 DispaseⅡ消化法能够获得大量的较纯净的结膜上皮细胞,细胞生长增殖快,能传代4～5代;绝大多数细胞广谱角蛋白免疫荧光染色阳性,少数细胞MUC5AC表达阳性及PAS染色阳性。睑结膜、穹隆部结膜和球结膜细胞PCNA阳性率分别为:(32.14±1.69)%、(27.67±1.20)%、(22.76±1.73)%,兔睑结膜上皮细胞PCNA阳性率较穹隆部及球结膜上皮细胞的PCNA阳性率高,各部位PCNA阳性率间比较差异有统计学意义(P<0.05)。结论 DispaseⅡ消化法是进行体外结膜上皮细胞培养的理想方法,兔眼睑结膜处可能存在结膜干细胞。

Dispase诱导玻璃体后脱离的实验研究

目的 探讨dispase兔眼玻璃体腔内注射诱导玻璃体后脱离 (PVD)的效能及对眼内组织的毒性作用。方法 3 6只兔按观察时间和注入dispase浓度不同分为Ⅰ组 ( 10 μmol/(min·mL)、5 μmol/(min·mL)、1μmol/(min·mL) )和Ⅱ组( 0 2 5 μmol/(min·mL)、0 1μmol/(min·mL)、0 0 5 μmol/(min·mL) )。对照眼注入磷酸盐缓冲液 (PBS)。Ⅰ组注药 3 0min、Ⅱ组 1周内进行PVD和眼内毒性的观察。结果 ( 1)有PVD者Ⅰ组各小组均占 4/5 ;Ⅱ组中 0 2 5 μmol/(min·mL)和 0 1μmol/(min·mL)组各占 5 /5 ,0 0 5 μmol/(min·mL)组占 4/5 ;对照眼均无PVD。两组中实验眼的PVD诱导率与对照眼比较 ,差异均有显著性 (P <0 0 5 )。 ( 2 )两组中较高浓度者可见前房炎性反应、玻璃体混浊或视网膜损害等。在 5 μmol/(min·mL)暴露 3 0min组和 0 2 5 μmol/(min·mL)组电镜下可见视网膜内界膜裸露、感光细胞外段结构紊乱和内段空泡形成。 结论 1μmol/(min·mL) ( 3 0min)或 0 0 5 μmol/(min·mL) ( 1周 )两种应用方式是有效和安全的。

应用DispaseⅡ与淋巴细胞分层液获取高纯度基底细胞

目的 尝试应用DispaseⅡ分离酶获得含有完整基底细胞的表皮 ,再经淋巴细胞分层液收集活性强、生长快的基底细胞。探讨这样的细胞能否加快生长 ,缩短培养周期。方法 取植皮术后所剩中厚皮 ,经Dis paseⅡ处理后 ,用镊子分开表皮与真皮 ,表皮经胰酶进一步消化后 ,把细胞悬液通过淋巴细胞分层液将收集到的细胞作为实验组。以Tr法分离的表皮细胞作为对照组 ,两组间就细胞数、生长速度 ,克隆形成率与成膜时间进行比较。结果 所得数据经统计学处理显示 :实验组细胞的生长速度、克隆形成率与成膜时间比对照组明显加快。两组间有显著差异 (P <0 0 0 1)。结论 应用DispaseⅡ与淋巴细胞分层液能获得纯度高、活性强、增殖快的基底细胞。可以缩短培养周期 ,具有重要的临床意义。

维生素K_3、Dispase联合防治兔增生性玻璃体视网膜病变的实验研究

目的研究Dispase和维生素K3分别单独和联合用药对实验性兔增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的防治作用与毒性。方法40只兔眼,分为对照组、Dispase组、维生素K3组和联合用药组(每组10只眼)。采用眼球穿通伤及玻璃体内注入富含血小板血浆的方法制备外伤性PVR模型后,经睫状体玻璃体腔注入药物,观察比较Dispase、维生素K3及两者联合应用对实验性兔增生性玻璃体视网膜病变的影响。结果用药组平均增生级别均低于对照组,差异有统计学意义(P<0.05);联合用药组平均增生级别低于单独用药组,两者比较,差异有统计学意义(P<0.05);其中,视网膜前的平均增生总细胞数及平均增生成纤维细胞数,维生素K3组和联合用药组少于对照组及Dispase组,比较差异有统计学意义(P<0.05);但前两组比较无差异,后两组亦无差异。视网膜前的平均增生色素上皮细胞数及平均增生神经胶质细胞数,对照组与各用药组两两比较,差异无统计学意义(P>0.05)。电镜结果显示各用药组视网膜超微结构无明显异常。结论维生素K3与Dispase可在一定程度上抑制外伤性PVR的发生、发展,且两者有一定协同作用,对视网膜无毒性损害。

Dispase与RPE细胞联合诱导的小鼠PVR模型的建立

目的用dispase和同种异体视网膜色素上皮(retina pigment epithelium,RPE)细胞联合作用,诱导C57BL/6J小鼠增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的产生,并评价该模型的有效性。方法将32只健康的成年C57BL/6J小鼠随机平均分成4组。分别向右眼玻璃体腔内注射5μL dispase+RPE(A组)、dispase(B组)、RPE(C组)、PBS(D组)。A、B两组dispase的浓度为2.4 U/mL。A、C两组RPE细胞的浓度为2×104/5μL。于注射后3天、1、2、4、6周时,利用裂隙灯显微镜检查眼前节情况,用直接检眼镜检查眼底、记录PVR的分级。结果注射后第3天～6周,A组、B组、C组均不同程度地发生了玻璃体积血,玻璃体内可见弥散色素颗粒、小细胞团,条索及膜结构形成,视网膜皱褶,视网膜脱离等PVR典型征象。A组较B、C两组病变发生时相早、程度深而广泛。D组未见明显的PVR征象发生。注射后第6周结束时,A、B、C 3组视网膜脱离率分别为87.5%、62.5%和75%。结论 Dispase和RPE细胞诱发的小鼠PVR模型,建立方法快速,有效,成功率高,可广泛应用于对PVR病理过程,药物筛选,基因治疗等方面的研究。

A novel arterial pouch model of saccular aneurysm by concomitant elastase and collagenase digestion

Background: An ideal aneurysm model of cerebral aneurysm is of great importance for studying the pathogenesis of the lesion and testing new techniques for diagnosis and treatment. Several models have been created in rabbits and are now widely used in experimental studies; however, every model has certain intrinsic limitations. Here we report the development of a novel saccular aneurysm model in rabbits using an arterial pouch that is subject to in vitro pre-digestion with combined elastase and collagenase. Methods: A segment of right common carotid artery (CCA) was dissected out and treated with elastase (60 U/ml, 20 min) followed by type I collagenase (1 mg/ml, 15 min) in vitro. The graft was anastomosed to an arterial arch built with the left CCA and the remaining right CCA, while the other end of the graft was ligated. The dimension and tissue structure of the pouch were analysed immediately, 2 or 8 weeks after operation. Findings: Ten terminal aneurysms were produced. The gross mor-phology of the aneurysm resembles the human cerebral terminal aneurysms. We have observed the following pathological changes: (1) growth of the aneurysm (mean diameter increased from (2.0±0.1) to (3.2±0.3) mm at 2 weeks, P【0.001, n=7~10); (2) thinning of the aneurysmal wall (the mean wall thickness decreased to 44% at 2 weeks), which was accompanied by significant losses of elastic fibres, collagen and the cellular component; and (3) spontaneous rupture (3 out of 9, one aneurysm ruptured 24 h after operation with the other two at 2 and 4 weeks respectively). Conclusion: This rabbit arterial pouch model mimics human cerebral aneurysms in relation to morphology and histology. In particular, this model exhibited an increased tendency of spontaneous rupture.

Antitumor activity of anti-type IV collagenase monoclonal antibody and its lidamycin conjugate against colon carcinoma

AIM： Type IV collagenase including MMP-2 and -9 plays an important role in cancer cell invasion and metastasis and is an attractive target for rnAb-directed therapy. The irnrnunoreactivity of rnAb 3G11, a rnAb directed against type Ⅳ collagenase in human colorectal carcinomas, was studied by irnrnuno-histochernical （IHC） staining, rnAb 3G11 was conjugated to an antiturnor antibiotic lidarnycin （LDM）. The antiturnor activity of 3G11-LDM conjugate against colon carcinoma was investigated in mice. METHODS： ELISA, gelatin zyrnography, and Western blot assay were used for the biological characterization of rnAb 3G11. The irnrnunoreactivity of rnAb 3G11 with human colorectal carcinomas was detected by IHC staining. The cytotoxicity of LDM and 3G11-LDM conjugate to human colon carcinoma HT-29 cells was examined by clonogenic assay and MTT assay. The therapeutic effect of conjugate 3G11-LDM was evaluated with colon carcinoma 26 in mice. RESULTS： As shown in ELISA, mAb 3Gll reacted specifically with type IV collagenase, while 3G11-LDM conjugate also recognized specifically its respective antigen. In IHC assay, mAb 3G11 showed positive irnrnunoreactivity in most cases of colorectal carcinoma, and negative irnrnunoreactivity in the adjacent non-malignant tissues. By gelatin zyrnography, the inhibition effect of rnAb 3G11 on the secretion activity of type IV collagenase was proved. In terms of IC50 values in MTT assay, the cytotoxicity of LDM to human colon carcinoma HT-29 cells was 10 000-fold more potent than that of rnitornycin C （MMC） and adriarnycin （ADM）. 3G11- LDM conjugate also displayed extremely potent cytotoxicity to human colon carcinoma HT-29 cells with an IC50 value of 5.6× 10^-19 mol/L. 3G11-LDM conjugate at the doses of 0.05 and 0.1 mg/kg inhibited the growth of colon carcinoma 26 in mice by 70.3 and 81.2%, respectively. CONCLUSION： mAb 3G11 is immunoreactive with human colorectal carcinoma and its conjugate with LDM is highly effective against colon carcinoma in mice.