微生物胶原酶

某些微生物可以分泌微生物胶原酶。微生物胶原酶具有和脊椎动物胶原酶相似的生物学作用 ,可以降解细胞外基质的主要成分—胶原 ,其活性依赖于Ca2 + 等二价金属阳离子。但微生物胶原酶又具有许多不同于脊椎动物胶原酶的生物学特性。它不仅可以降解天然不溶性胶原纤维 ,也可以降解变性的可溶性胶原蛋白、人工合成的胶原酶底物以及其它细胞外基质糖蛋白。越来越多的研究表明 ,微生物胶原酶在一些微生物致病过程中起重要作用 ,是某些微生物的重要毒力因子。而且微生物胶原酶也被越来越广泛地应用于生命科学基础研究和临床某些疾病治疗 ,具有极为广阔的应用前景。     

产胶原酶的蜡样芽胞杆菌发酵条件优化及酶的分离纯化

【目的】优化蜡样芽胞杆菌R75E菌株产胶原酶的条件,并通过蛋白分离纯化技术获得高纯度胶原酶。【方法】利用单因素及正交试验优化蜡样芽胞杆菌R75E产胶原酶的发酵条件及发酵培养基,将发酵液离心除菌后得到粗酶液,对其依次通过硫酸铵分级沉淀、Butyl FF疏水层析及SuperdexTM 200凝胶过滤层析等方法对目标胶原酶进行分离纯化,利用SDS-PAGE电泳检测其纯度。【结果】优化后发酵条件为培养温度41°C、接种量6%、培养时间36 h,优化后发酵培养基为葡萄糖10 g/L、蛋白胨5 g/L、起始p H 7.0,粗酶液酶活力较优化前提高了2.9倍;将该粗酶液经过一系列纯化后得到纯度超过90%的胶原酶产物,其纯化倍数和回收率分别为18.4和1.1%。【结论】获得蜡样芽胞杆菌R75E的最佳产酶条件,并对胶原酶分离纯化的方法进行了探索,为微生物胶原酶的开发应用奠定基础。     

蜡样芽孢杆菌ColR75E胶原酶的表达、纯化及酶学性质研究

目的:为了进一步了解蜡样芽孢杆菌R75E胶原酶colR75E的特性,在大肠杆菌原核表达系统中表达colR75E胶原酶,并对表达的重组胶原酶进行纯化以及酶学性质研究。方法:以蜡样芽孢杆菌R75E基因组DNA为模板采用PCR法获得胶原酶colR75E基因,构建pET28a/colR75E重组质粒,并在大肠杆菌BL21(DE3)中进行诱导表达,利用Ni-NTA纯化的方式获得高纯度ColR75E胶原酶,利用胶原酶谱、胶原酶活力测定、Ⅰ型胶原蛋白降解产物电泳检测等方式对ColR75E胶原酶的酶学特性进行分析。结果:通过Ni-NTA纯化获得的蛋白经胶原酶谱、Ⅰ型胶原蛋白降解产物的检测时均表现出胶原蛋白的降解能力。在标准条件下测得其比活力为3.62 U/mg,Km为25.55μmol/L(2.93 mg/ml),Vmax为5.71μmol/(mg·min)。ColR75E胶原酶的最适反应温度为45℃,最佳反应p H为8.0。此外,ColR75E胶原酶对于不高于50℃的温度以及pH6.0~8.0的酸碱度范围具有良好的稳定性。ColR75E胶原酶可被Ca2+激活、但被Zn2+、Pb2+、Fe2+、Mn2+等金属离子抑制,抑制能力依次为Pb2+Zn2+Fe2+Mn2+。ColR75E胶原酶对EDTA和EGTA的高敏感性进一步证实了该胶原酶为一种金属蛋白酶。结论:利用大肠杆菌原核表达系统获得高纯度高活性的蜡样芽孢杆菌胶原酶是可行的,为该胶原酶广泛应用于医疗、食品等工业领域中奠定了理论基础。     

单独及混合胶原酶消化小鼠乳腺癌移植瘤组织获取细胞活性比较

[目的]比较单独及混合胶原酶消化小鼠乳腺癌移植瘤组织后获取的细胞活性。[方法]用4T1细胞建立小鼠乳腺癌动物模型,待成瘤后取乳腺癌肿瘤组织。比较4种消化酶(Ⅰ型胶原酶、Ⅳ型胶原酶、Ⅰ+Ⅳ型混合胶原酶和商用胶原酶),两种消化方法:单酶或多酶单次消化30 min;单酶或多酶分次消化30 min。用碘化丙啶(PI)染色各组消化后的细胞悬液,流式细胞仪检测细胞活率。[结果]不同酶单次消化后的细胞活率分别为:Ⅰ型胶原酶为55. 5±7. 2%、Ⅳ型胶原酶为26. 9±7. 6%、Ⅰ+Ⅳ型混合胶原酶为43. 5±10%、商用胶原酶为30. 4±10. 6%;在分次消化后分别为:Ⅰ型胶原酶为70. 6±4. 1%、Ⅳ型胶原酶为65. 5±17. 2%,Ⅰ+Ⅳ型混合胶原酶为78. 3±2. 2%,商用胶原酶为48. 5±11. 6%。[结论]使用Ⅰ+Ⅳ型混合胶原酶分次消化得到的乳腺癌移植瘤细胞活性最高,并为小鼠乳腺癌移植瘤组织消化提供了一种有效的新方法。     

海洋微生物酶的开发

海洋酶（marizyme）制剂的开发将成为酶制剂工业一个较活跃的领域,应用前景广泛。海洋微生物是获取海洋酶的重要来源。美国一家公司已获得500种不同的海洋微生物分离物,成为筛选酶和药物的好材料,得到10种海洋生物酶如蛋白酶、琼脂糖酶和胶原酶等。海洋极端环境如海底火山口附近微生物产生的耐热酶有着开发潜力。美国研究人员从这种特殊环境火山口壁（105—113℃）找到嗜极微生物（exeemophiles）,如神rolobusfUI）larii的细菌能在这种高温环境中生长繁殖,在90℃以下则不能生长;还有一种在150℃下能生存的产甲烷大古单细胞生物如Met…     

蜡样芽孢杆菌胶原酶基因colR75E在毕赤酵母中的重组表达

【目的】利用毕赤酵母真核表达系统表达蜡样芽孢杆菌胶原酶Col R75E,寻找一种安全、稳定的方式体外制备具有高活性的胶原酶。【方法】以蜡样芽孢杆菌R75E基因组DNA为模板,采用PCR法扩增胶原酶col R75E基因,构建p PICZαA/col R75E重组质粒,将该质粒线性化后电转化至毕赤酵母X-33菌株,诱导其表达并对表达条件进行优化。将表达后的酵母发酵液上清通过硫酸铵沉淀、脱盐处理及亲和层析纯化步骤获得高纯度重组Col R75E胶原酶。利用胶原酶活力测定、SDS-PAGE电泳、胶原酶谱、I型胶原蛋白及不同底物蛋白降解产物电泳等方法对重组胶原酶Col R75E的活性及底物特异性进行分析。【结果】毕赤酵母中最佳表达重组胶原酶Col R75E的条件为p H 6.0,甲醇终浓度为2.5%,诱导时间72 h,诱导后的蛋白经SDS-PAGE、胶原酶谱以及I型胶原蛋白降解产物电泳分析发现,毕赤酵母中表达的重组胶原酶分子量符合预期,蛋白纯度超过95%,具有较好的胶原蛋白水解活性并测得其比活力为4.977 U/mg。该酶对I型胶原蛋白表现出较好的专一性,但是对牛血清白蛋白、酪蛋白及溶菌酶蛋白没有水解活性。【结论】利用毕赤酵母真核表达系统能够获得高活性的蜡样芽孢杆菌胶原酶Col R75E,为该胶原酶广泛应用于医疗、食品等工业领域奠定了理论和方法基础。     

小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

Ⅳ型胶原酶人基因工程抗体的研制

肿瘤组织内Ⅳ型胶原酶表达和活性升高与肿瘤转移密切相关 .抑制Ⅳ型胶原酶活性的物质能够有效地抑制或降低肿瘤的转移 .因此Ⅳ型胶原酶已成为恶性肿瘤辅助诊断和控制癌转移的标志分子之一 .利用噬菌体抗体展示技术 ,在人抗体库研究的基础上 ,成功地研制出一种Ⅳ型胶原酶特异人源单链抗体hCo4.该抗体主要是由抗体轻链可变区和重链可变区组成 ,分子量约为 2 7ku .ELISA和免疫印迹均证明hCo4能够与Ⅳ型胶原酶特异结合 ,是目前所报道的Ⅳ型胶原酶特异抗体中分子量最小的人基因工程抗体 .该抗体的亲和力与从分泌抗Ⅳ型胶原酶单抗的杂交瘤细胞制备的鼠源单链抗体亲和力相同 .它有可能成为一种研究Ⅳ型胶原酶与肿瘤转移相互关系的工具以及用于肿瘤免疫诊断和治疗 .     

酶消化对肿瘤浸润淋巴细胞活力影响的研究

本文系统地研究了酶消化(胶原酶Ⅳ、胶原酶Ⅱ,透明质酸酶和胰蛋白酶)对肿瘤浸润淋巴细胞(TIL)活力、增殖力等影响,并用单个酶消化法与Rosenberg法比较。结果提示:胶原酶Ⅳ或胶原酶Ⅱ消化肿瘤组织块时对TIL细胞活力、增殖力损伤小;透明质酸酶与胰蛋白酶消化对TIL细胞活力,增殖力损伤大。在37℃,1h消化组与4℃,24h消化组对同一来源肿瘤组织酶消化配对比较中,结果提示同一酶4℃,24h消化比37℃1h消化对TIL细胞增殖力损伤小,与Rosenberg方法比较中也发现单一胶原酶消化比三酶联合消化影响小。配对各实验组TIL细胞~3H-TdR掺入率分析中,也反映了类似变化。本文经4℃,24h胶原酶Ⅳ消化后培养的TIL细胞在60天和75天仍对自身靶细胞有杀伤力。     

不同二步酶灌注法分离大鼠肝星状细胞的比较研究

目的:在现有二步酶灌注法分离大鼠肝星状细胞(hepatic stellate cells,HSC)的基础上,探索更加高效的分离HSC方法。方法:分别采用链酶蛋白酶+胶原酶循环灌注、链酶蛋白酶非循环灌注+胶原酶循环灌注以及胶原酶单独循环灌注法分离大鼠HSC,比较三种方法的细胞获得率、活性和纯度差异。应用0.4%台盼蓝染色判断活性,结蛋白(desmin)、波形蛋白(vimentin)细胞免疫荧光方法鉴定纯度。结果:链酶蛋白酶非循环灌注+胶原酶循环灌注法细胞获得率高于另两种方法,细胞活力高于链酶蛋白酶循环灌注+胶原酶循环灌注法,三组得到的细胞纯度均高于90%且无显著差异。结论:在三种二步酶灌注方法中,链酶蛋白酶非循环灌注+胶原酶循环灌注法能显著提高HSC获得率,且对细胞活力影响小,不降低细胞纯度,是一种高效的分离方法,有利于HSC相关肝脏疾病的生物学研究。     

Collagenase synthesis by cloned rabbit pulp cells--molecular and immunological identity of pulp cell and fibroblast enzyme.

Cultures of cloned rabbit pulp (RP) cells without stimulation produced collagenase of a concentration as high as reference rabbit skin fibroblast cultures which were stimulated with phorbol myristate acetate (PMA, 100 ng/ml). The RP cell collagenase was compared with reference fibroblast collagenase in Western blot analysis using monoclonal antibodies prepared against RP cell collagenase and a polyclonal antibody prepared against rabbit fibroblast collagenase. Both enzyme preparations revealed, with either antibody, identical bands of approximate molecular masses 57,000, 52,500, and 45,000. These antibody preparations variously inhibited RP cell collagenase activity. Intracellular collagenase in RP cells in culture was demonstrated by the indirect immunofluorescence antibody technique using polyclonal anti-fibroblast collagenase antibody. RNA samples from RP cells hybridized with rabbit fibroblast collagenase cDNA (clone H9) and showed a distinct band at 2.7 kb. Both control and PMA-stimulated RP cells and PMA-stimulated reference skin fibroblasts demonstrated strong cytoplasmic hybridization between H9 and collagenase mRNA. The results indicate that RP cell collagenase is identical to rabbit fibroblast collagenase, and that the RP cell line provides a useful in vitro reference system for the study of collagenolysis in the rabbit model.     

Regulation of collagenase and collagenase mRNA production in early- and late-passage human diploid fibroblasts

The levels of collagenase and collagenase mRNA produced by early-passage (less than 40% of lifespan completed) and late-passage (greater than 80% of lifespan completed) cultures of human fibroblasts were analyzed. The constitutive levels of collagenase and collagenase mRNA produced by the late-passage cultures were 10-30 x greater than the levels observed in similarly treated early-passage cultures. Immunofluorescence analysis established that the percentage of collagenase-positive cells was also greater (77% vs. 4%) in the late-passage cultures. To determine whether the difference in collagenase production resulted from cell-derived regulatory factors, collagenase production was examined in cultures plated onto substrates coated with fibroblast extracellular matrix (ECM). Collagenase and collagenase mRNA production was enhanced in both types of cultures, although amounts produced by ECM-induced early-passage cultures was significantly less than that produced by similarly treated late-passage cultures. Collagen-coated substrates also induced collagenase synthesis.     

Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.     

Monoclonal antibodies to type IV collagenase recognize a protein with limited sequence homology to interstitial collagenase and stromelysin

Type IV collagenase is a metalloproteinase associated with metastatic tumor cells. It specifically cleaves the triple helical basement membrane (type IV) collagen molecule at a single site. Monoclonal antibodies which block the activity of the human type IV collagenase were developed and used to purify this antigen. The purified type IV collagenase was partially sequenced following cyanogen bromide and trypsin cleavage. The amino acid sequence of the human type IV collagenase fragments revealed a region homologous to the human interstitial collagenase and stromelysin. However, several sequences in type IV collagenase were identified which are distinct from the latter. Polyclonal antibodies were raised against a synthetic peptide derived from such a sequence. Following affinity purification, the antibodies recognized the denatured human type IV collagenase in Western immunoblotting. These data indicate that type IV collagenase is a distinct member of a general family of metalloproteinases.     

Calmodulin regulates the interleukin 1-induced procollagenase production in human uterine cervical fibroblasts

Interleukin 1 (IL-1) stimulates the synthesis of collagenase in human uterine cervical fibroblasts. This inductive effect of IL-1 on collagenase production was augmented by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific inhibitor of calmodulin, in a dose-dependent manner. The apparent collagenase activity observed in the culture medium of the cells treated with IL-1 and 40 microM W-7 was about three times higher than that produced by the cells treated with IL-1 alone. The immunoblotting with the specific antibody against human collagenase showed that the increased collagenase activity resulted from the accelerated biosynthesis of collagenase. Another calmodulin inhibitor, trifluoperazine, enhanced the effect of IL-1 on collagenase production similarly. However, the effect of N-(6-aminohexyl)-1-naphthalenesulfonamide, the weakest inhibitor of calmodulin, was negligible. These results suggest that W-7 enhances the collagenase production by specifically inhibiting calmodulin and that calmodulin may act as a suppressor of the IL-1-induced collagenase production in human uterine cervical fibroblasts.     

Production of latent collagenase by human umbilical vein endothelial cells in response to angiogenic preparations

Three preparations known to be angiogenic in vivo and which stimulate production of latent collagenase by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate production of latent collagenase by cultured human umbilical vein endothelial (HUVE) cells. Bovine retinal extract and murine adipocyte-conditioned medium had no effect on production of latent collagenase by HUVE cells at concentrations that were effective in stimulating production of latent collagenase by BCE cells. However, with higher concentrations of bovine retinal extract, production of latent collagenase by HUVE cells was stimulated. Human hepatoma cell sonicate stimulated production of latent collagenase by HUVE cells in a dose-dependent manner. The concentration of human hepatoma cell sonicate which stimulated production of latent collagenase by HUVE cells was lower than the concentration that was effective for the stimulation of production of latent collagenase by BCE cells. Plasminogen activator production by HUVE cells was unaffected by human hepatoma cell sonicate. Varying the concentration of serum in HUVE cultures did not affect the stimulation of latent collagenase production by human hepatoma cell sonicate, suggesting that serum components neither block nor stimulate the action of the collagenase-inducing factor. Although human hepatoma cell sonicate is reported to stimulate endothelial cell multiplication, purified and partially purified endothelial cell mitogens had no effect on production of latent collagenase. Thus, at least two preparations which contain angiogenic activity will stimulate production of latent collagenase by HUVE cells.     

Equine retained placenta: technique for and tolerance to umbilical artery injections of collagenase

Under laboratory conditions and in clinical experiments, bacterial collagenase has proven to be effective in hydrolyzing placenta and detaching cotyledon from caruncle in the bovine species. Laboratory studies in which placental samples were incubated with collagenase have also demonstrated that collagenase is 3.7 times more effective in hydrolyzing equine placenta than bovine placenta. This led to the hypothesis that collagenase may be a potential treatment for mares with retained placenta. However, that collagenase may hydrolyze the uterine wall and perforate the uterus was a concern. It was the purpose of this study thus to determine any adverse effects of collagenase on the equine uterus and to develop a method for intraplacental injection of collagenase. Three normally expelled intact placentas from Arabian mares, 10 cyclic mixed-breed mares, and 4 mares of various breeds with retained placenta were used. Fluoroscein dye and latex were used to study the placental vasculature and to determine a suitable dose of collagenase; placentas were hydrolyzed by collagenase solution in vitro. Bacterial collagenase solution (40,000 units, 200 ml) was infused into the uterine lumen of each cyclic mare. Uterine biopsies were obtained from the mares before collagenase infusion and again at 16 h and 26 d after infusion. In the mares with retained placenta, each placenta was infused via its umbilical cord vessels with 200,000 units of bacterial collagenase in 1 L of saline. Results showed that none of the uteri from cyclic mares were damaged by collagenase treatment. During a 4-wk period of monitoring (including endoscopy) mares with retained placenta did not show any abnormalities. Retained placentas were expelled in less than 6 h after collagenase treatment. It was concluded that intraplacental injections of collagenase are a safe and potentially effective treatment for retained placenta in mares.     

The gelatinolytic activity of rat uterus collagenase

The collagenase produced by rat uterine cells in culture has been examined for its ability to degrade denatured collagen. Acting as a gelatinase, rat uterus collagenase was able to successfully degrade the denatured chains of collagen types I through V. In addition, the enzyme produced multiple cleavages in these chains and displayed values for Km of 4-5 microM, compared to values of 1-2 microM when native collagen was used as substrate. Furthermore, rat uterus collagenase degraded the alpha 2 chain of denatured type I collagen at a significantly faster rate than the alpha 1 chain, as previously observed for human skin fibroblast collagenase. In contrast to the action of human skin collagenase, however, the rat uterus enzyme was found to be a markedly better gelatinase than a collagenase, degrading the alpha chains of denatured type I guinea pig skin collagen at rates some 7-15-fold greater than native collagen. Human skin collagenase degrades the same denatured chains at rates ranging from 13-44% of its rate on native collagen. Rat uterus collagenase, then, is approximately 50 times better a gelatinase than is human skin collagenase. In addition to its ability to cleave denatured collagen chains at greater rates than native collagen, the rat uterus collagenase also attacked a wider spectrum of peptide bonds in gelatin than does human skin collagenase. In addition to cleaving the Gly-Leu and Gly-Ile bonds characteristic of its action on native collagen, rat uterus collagenase readily catalyzed the cleavage of Gly-Phe bonds in gelatin. The rat enzyme was also capable of cleaving Gly-Ala and Gly-Val bonds, although these bonds were somewhat less preferred by the enzyme. The cleavage of peptide bonds other than Gly-Leu and Gly-Ile appears to be a property of the collagenase itself and not a contaminating protease. Thus, it appears that the collagenase responsible for the degradation of collagen during the massive involution of the uterus might also act as a gelatinase to further degrade the initial products of collagenolysis to small peptides suitable for further metabolism.     

Additive enhancement of neutrophil collagenase activity by HOCl and cathepsin G.

Using preparations of latent collagenase derived from neutrophil specific granule extracts, the relative effects of cathepsin G and HOCl on activation of neutrophil collagenase were determined using a quantitative collagenase assay. Enhancement of collagenase activity by cathepsin G and physiologically relevant concentrations of HOCl were comparable, but HOCl mediated collagenase activation was reversible in the presence of HOCl scavenger. Collagenase activity in preparations treated with cathepsin G and HOCL simultaneously or sequentially was significantly greater than activity in preparations treated with HOCl or cathepsin G alone. The results indicated additive, yet independent enhancing effects of HOCl and cathepsin G on activity of neutrophil collagenase.