优化明胶酶谱法检测自发性高血压大鼠血浆MMP-2和MMP-9活性

目的以优化明胶酶谱法检测自发性高血压大鼠(SHR)血浆基质金属蛋白酶-2(MMP-2)与-9(MMP-9)的活性。方法以含明胶的SDS-聚丙烯酰胺凝胶电泳酶谱法为基础,改变孵育时间、工作液成份,采用不同pH值的孵育液及不同冻融次数的样品,观察对基质金属蛋白酶(MMPs)活性的影响。并以优化的酶谱法检测Wistar及自发性高血压大鼠血浆MMPs的相对活性。结果凝胶孵育时间由42 h缩短为17 h不影响酶谱法检测结果。省略漂洗步骤及洗脱液仅使用2.5%Triton X-100、孵育液中去除NaN3及NaCl且电泳后步骤将去离子水更换为蒸馏水也不影响实验结果。孵育液pH值在7.2～8.8范围内均可用于酶谱法。血浆样品反复冻融多达6次不影响酶谱法检测结果。用优化的酶谱法检测结果显示,与Wistar大鼠相比,SHR大鼠血浆MMP-2与MMP-9活性显著增高。结论优化酶谱法与常规方法相比更为简便经济,检测大鼠血浆MMP-2与MMP-9活性所得结果一致。     

血红素加氧酶-1过表达延长肝脏低温保存时间的研究

目的 研究血红素加氧酶-1(heme oxygenase-1,HO-1)过表达延长肝脏低温保存的时间及其机制.方法 利用大鼠肝脏离体再灌注模型,用钴-原卟啉(cobalt protoporphyrin,CoPP)和锌-原卟啉(zincprotoporphyrin,ZnPP)特异地诱导和抑制HO-1,观察肝脏保存0、6、24h,灌注2h后的胆汁生成量,灌流液AST、LDH、TNF-α和IL-6的活性,肝脏MDA的含量,肝组织HO-1蛋白表达的Western印迹,细胞凋亡情况等.结果 CoPP诱导了肝组织HO-1的表达,与未诱导24h保存组相比,CoPP诱导组的肝脏灌流液AST、LDH、TNF-α和IL-6的活性以及肝脏MDA含量显著降低,胆汁生成量明显增加,凋亡细胞数量减少(P<0.05),并与未诱导6h保存组接近.给予ZnPP后,这些保护作用消失.结论 HO-1过表达延长了肝脏低温保存时间,原因可能与抗氧化应激、抑制炎性因子的表达和细胞凋亡有关.     

钴-原卟啉诱导大鼠血红素加氧酶-1适度过表达的研究

目的研究钴-原卟啉(CoPP)诱导大鼠血红素加氧酶-1(HO-1)的适度过表达,并观察其抗肝缺血/再灌注损伤(IRI)的作用。方法建立大鼠肝脏IRI模型,按体重给予不同诱导剂量的CoPP(5、2.5mg/kg及2.5mg/kg体重2次),关用锌原卟啉(ZnPP)来抑制HO-1的活性。观察血浆谷草转氨酶(AST)、乳酸脱氢酶(LDH)、肝组织丙二醛(MDA)的变化,肝组织光镜和电镜下的改变,肝组织HO-1蛋白表达的Western印迹。结果CoPP诱导组中均有明显的HO-1蛋白表达;与缺血/再灌注(IR)组比较,CoPP诱导组动物血浆AST、LDH活性以及肝MDA含量明显降低,肝组织损伤减轻。3个剂量组中以2.5mg/kg体重诱导2次效果最佳。ZnPP的加入阻断了CoPP诱导组的这些保护作用。结论CoPP诱导HO-1适度过表达的诱导剂量为2.5mg/kg体重2次,在这一诱导剂量下HO-1过表达对肝IRI具有重要的保护作用。     

肾性高血压大鼠主动脉血红素加氧酶-1表达增加

目的探讨肾性高血压时血红素加氧酶(HO)/一氧化碳(CO)系统的变化,以明确血红素加氧酶在肾性高血压发病过程中的意义及作用。方法①制作两肾一夹(2K1C)肾性高血压大鼠模型,分别于2、4、6、8周记录血压,用放免法测定血浆中血管紧张素II的含量。②分光光度比色法测定主动脉微粒体中HO的活性。③用W estern b lot方法测定主动脉HO-1蛋白的表达。结果从术后2周起,2K1C组血压明显高于假手术组(P<0.01);同时血浆AngII含量显著增加(P<0.01)。2K1C组在4周和6周时主动脉HO活性升高明显(P<0.05),但8周时有所回降,但仍高于对照组;2K1C组4周时HO-1蛋白含量明显升高,比假手术组高25.38%(P<0.05)。结论在肾性高血压大鼠,血浆AngII增高诱导了HO-1的表达增加来对抗压力负荷对血管的损害。     

AcSDKP对大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的影响

目的探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)对10%血清和血小板源性生长因子(PDGF)诱导的大鼠心成纤维细胞MMP-2、MMP-9活性和MMP-1表达的调节作用。方法明胶酶谱法检测心成纤维细胞MMP-2、MMP-9的活性。Western blot法检测心成纤维细胞MMP-1的表达。结果10%血清和PDGF使心成纤维细胞MMP-2、MMP-9活性增强,也促进MMP-1的表达;AcSDKP能够进一步增加由10%血清和PDGF诱导的心成纤维细胞MMP-2、MMP-9的活性,并促进MMP-1的表达。结论AcSDKP上调了由PDGF介导的心成纤维细胞MMPs活性或表达,这可能与AcSDKP抗心肌纤维化的作用相关。     

银杏内酯B对新生大鼠缺血性损伤海马MMP-2及MMP-9表达的影响

目的:观察银杏内酯B(GB)对缺氧缺血性脑损伤(HIBD)新生大鼠海马内基质金属蛋白酶MMP-2及MMP-9的影响,探讨GB的脑保护作用机制。方法:192只新生7天SD大鼠随机分为正常组(N组)、假手术组(S组)、HIBD模型生理盐水组(H组)和银杏内酯B治疗组(G组)。选择模型成功后24 h、4 d、10 d三个时间点,应用免疫组化、RT-PCR法检测各组大鼠海马CA1区MMP-2、MMP-9蛋白及mRNA的表达变化。结果:HIBD后海马MMP-2蛋白表达明显升高,4 d达高峰,10 d后开始下降;同时MMP-9蛋白表达也明显升高,但表达高峰在HIBD后24 h,4 d后开始下降。mRNA的表达趋势与蛋白表达相一致。N组可见极少量阳性表达细胞,S组中可见少量阳性表达细胞,G组的大鼠海马CA1区MMP-2、MMP-9的表达也明显升高,但是小于H组,G组的MMP-2、MMP-9的表达与H组相比差异有显著性(P<0.05)。结论:HIBD可引起MMP-2、MMP-9表达异常,并且呈动态变化;GB对脑保护作用机制之一可能与其抑制MMP-2、MMP-9高表达有关。     

肢体缺血-再灌注大鼠应用氨基胍后脑、肝脏及肾脏HO-1表达的变化

目的探讨大鼠肢体缺血-再灌注(I-R)时,脑、肝脏及肾脏组织内高表达的iNOS-NO是否对诱导型血红素氧合酶(HO-1)表达具有诱导作用.方法对肢体I-R大鼠应用氨基胍(AG)抑制iNOS后,用RT-PCR及免疫组化染色法观测其脑、肝及肾组织HO-1mRNA及蛋白表达的变化.SD大鼠随机分为I-R6h+AG、I-R12h+AG两个实验组及I-R6h、I-R12h两个对照组.通过夹闭大鼠双侧股动脉根部4h、开放6h或12h,制备肢体I-R6h、I-R12h组模型,I-R6h+AG、I-R12h+AG组于去夹再灌注前20min经腹腔注射AG(10mg/kg).结果(1)I-R6h组脑组织HO-1mRNA的相对表达量为0.645±0.049,I-R6h+AG组较前者显著下降(P＜0.01),为0.143±0.008;I-R12h组为0.808±0.016,I-R12h+AG组为0.329±0.014,I-R+AG组较前者显著下降(P＜0.01),为0.412±0.025.I-R12h组为1.116±0.085,I-R12h+AG组为0.870±0.040,两者比较,P＜0.01.(2)I-R6h组肝组织HO-1mRNA的相对表达量为0.605±0.014,两者相比,P＜0.01.(3)I-R6h组肾组织HO-1mRNA的相对表达量为0.706±0.042,I-R6h+AG组为0.286±0.052,两者相比,P＜0.01;I-R12h组为1.668±0.065,I-R12h+AG组较前者显著升高(P＜0.01),为3.176±0.109.(4)免疫组化染色显示,脑、肝及肾组织内HO-1蛋白生成的变化与mRNA表达的变化一致.讨论与结论在肢体I-R的情况下,脑等远隔多器官的iNOS及HO-1均表达上调,抑制iNOS活性使这些器官的HO-1表达水平显著下降,提示,在肢体I-R的状态下,远隔多器官高表达的iNOS-NO对HO-1基因表达具有上调性诱导作用.肾有别于脑与肝脏,应用AG12h后,肾血管内堆积大量破坏的红细胞,而血红素是HO-1表达的强效诱导剂,I-R12h+AG组肾HO-1表达上调可能是血红素的上调性诱导作用逆转了AG对HO-1的下调作用.     

筋脉通胶囊通过增强背根神经节Nrf2和HO-1的表达改善糖尿病大鼠的周围神经痛

目的观察中药筋脉通胶囊对链尿佐菌素(STZ)诱导的糖尿病(DM)大鼠背根神经节(DRG)的核因子E2相关因子2(Nrf2)和血红素加氧酶-1(HO-1)的表达以及血浆一氧化碳(CO)含量的影响。方法腹腔内注射STZ诱导建立DM大鼠模型,随机分为模型组、筋脉通小、中和大剂量组及硫辛酸组,并设对照组。成模后每天1次灌胃给药,持续12周。电子Von Frey仪检测机械痛阈值,免疫组化法及Rt-PCR检测DRG的Nrf2和HO-1蛋白及mRNA表达,并检测血浆一氧化碳血红蛋白(COHb)含量。结果与对照组相比,糖尿病大鼠的机械痛阈值、血浆COHb含量以及DRG的Nrf2和HO-1蛋白及mRNA表达水平均明显下降(P0.01);各治疗组的上述指标均显著回升(P0.01或P0.05)。在提高DRG的HO-1蛋白表达上,筋脉通中剂量组疗效显著优于硫辛酸组(P0.05)。结论筋脉通胶囊可通过增强DRG的Nrf2、HO-1和CO表达来改善糖尿病大鼠的周围神经痛。     

HO-1/CO系统抑制AngⅡ诱导的大鼠心肌细胞凋亡

目的 探讨血红素氧合酶-1/一氧化碳(HO-1/CO)系统对血管紧张素Ⅱ(AngⅡ)诱导的大鼠心肌细胞凋亡的影响.方法 原代培养新生Wistar大鼠心肌细胞,随机分为:对照组、AngⅡ组、AngⅡ+氯化血红素(hemin)(HO-1诱导剂)组和AngⅡ+锌原卟啉-9(ZnppIX)(HO-1抑制剂)组.用real-time PCR及Western blot检测心肌细胞HO-1mRNA和蛋白的表达,比色法测定细胞培养上清液中碳氧血红蛋白(COHb)含量,流式细胞仪检测细胞凋亡.结果 AngⅡ组心肌细胞HO-1 mRNA、蛋白、COHb含量和细胞凋亡均明显高于对照组(P＜0.05),AngⅡ+hemin组HO-1mRNA、蛋白、COHb含量进一步升高(P＜0.05),而细胞凋亡回降(P＜0.05),但仍高于对照组(P＜0.05),AngⅡ+ZnPPIX组仅细胞凋亡湿著升高(P＜0.05),其他指标无显著变化.结论 HO-1/CO系统对AngⅡ诱导的心肌细胞凋亡具有抑制作用.     

硫化氢在脂多糖所致大鼠急性肺损伤中的作用

目的探讨硫化氢(H2S)在脂多糖(LPS)所致大鼠急性肺损伤(ALI)中的作用及可能的机制。方法将64只SD大鼠随机分为对照组、LPS组(经气管内滴注LPS复制ALI)、NaHS LPS组和炔丙基甘氨酸(PPG) LPS组。给药后4 h或8 h处死,测定肺系数;光镜观察肺组织形态学改变;化学法检测血浆H2S和CO含量、肺组织丙二醛(MDA)含量、胱硫醚-γ-裂解酶(CSE)和血红素加氧酶(HO)活性;用免疫组织化学法检测肺组织HO-1蛋白表达及吸光度值。结果气管内滴注LPS可引起肺组织明显的形态学改变;肺系数和肺组织MDA含量增加;血浆H2S含量和肺组织CSE活性下降;肺组织HO活性和HO-1蛋白表达增强;血浆CO含量增加。预先给予NaHS可显著减轻LPS所致上述指标的改变。而预先给予PPG可加重LPS所致肺损伤,但对CO含量、HO活性和HO-1蛋白表达无明显影响。结论H2S/CSE体系的下调在LPS所致ALI的发病学中有一定作用,而外源性给予一定量的NaHS对肺组织具有保护作用,该作用可能与其抗氧化效应和上调CO/HO-1体系有一定关系。     

Expression of MMP-10, MMP-21, MMP-26, and MMP-28 in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive cutaneous tumor with poor outcome and increasing incidence. We examined by immunohistochemistry the expression of three novel matrix metalloproteinases (MMPs)—MMP-21, MMP-26, and MMP-28—in 44 primary MCC tumors and six lymph node metastases while MMP-10 served as a positive control. Their mRNA expression was also studied in the UISO MCC cell line basally and after various stimulations using quantitative real-time PCR. MMP-28 was observed in tumor cells of 15/44 samples especially in tumors <2 cm in diameter (p = 0.015) while 21/44 specimens showed MMP-28 in the tumor stroma. Expression of MMP-21 was demonstrated in tumor cells of 13/43 samples. MMP-26, instead, was positive in stromal cells (17/44) and its expression associated with tumors ≥2 cm in diameter (p = 0.006). Stromal expression of MMP-10 was the most frequent finding of the studied samples (31/44), but MMP-10 was detected also in tumor cells (17/44). Most of the metastatic lymph nodes expressed MMP-10 and MMP-26. MMP-10, MMP-21, and MMP-28 mRNAs were basally expressed by the UISO cells, and the corresponding proteins were detectable by immunostaining of cultured cells. IFN-α and TNF-α downregulated MMP-21 and MMP-28 expression. Our results suggest that novel MMPs may have a role in MCC pathogenesis: especially that MMP-26 expression in stroma is associated with larger tumors with poor prognosis. Expression of MMP-21 and MMP-28 seems to associate with the tumors of lesser malignant potential. We also confirm the previous finding on the role of MMP-10 in MCC pathogenesis.     

子宫内膜异位症组织中MMP-2和MMP-9基因表达的意义

目的：探讨MMP-2和MMP-9在子宫内膜异位症发生中的作用。方法：采用免疫组织化学SP法研究25例子宫内膜异位症和22例正常子宫内膜组织中MMP-2及MMP-9的蛋白表达情况；采用RT-PCR法研究33例子宫内膜异位症的异位子宫内膜组织和30例正常子宫内膜组织中MMP-2和MMP-9基因的mRNA表达情况。结果：子宫内膜异位症组中的MMP-2和MMP-9的蛋白表达及mRNA表达均明显高于各自的正常子宫内膜对照组（P<0.05）。结论：MMP-2和MMP-9在子宫内膜异位症的发病过程中可能起重要的作用。     

Immunolocalization of MMP-2 and MMP-9 in human rheumatoid synovium

Matrix metalloproteinase (MMP)-2 and MMP-9, two important members of the matrix metalloproteinase family, have been shown critical contributions in intra-tumor angiogenesis and invasion of tumor progression, and they might also play important roles in the angiogenesis as well as the pannus formation of rheumatoid arthritis (RA). In the present study, we used the immunohistochemistry, the immunofluorescence staining and the con-focal scanning methods to characterize the immunolocalization of MMP-2 and MMP-9 in RA synovium tissues. Our results showed that both MMP-2 and MMP-9 immunostaining could be found in synoviocytes and vascular endothelial cells. Moreover, our con-focal scanning also showed that MMP-2 could be found in infiltrating CD14⁺ monocytes and CD68⁺ macrophages, and MMP-9 could be found in infiltrating CD68⁺ macrophages in RA synovium tissues, while weak or negative staining of these two MMPs could be found in infiltrating CD20⁺B cells and CD3⁺T cells in RA synovium. Thus, our finding suggests that both MMP-2 and MMP-9 expressed by synoviocytes as well as certain infiltrating immune cells role importantly in the angiogenesis in RA progression.     

MMP-2 activation and stepwise progression of pulmonary adenocarcinoma: analysis of MMP-2 and MMP-9 with gelatin zymography

Small pulmonary adenocarcinomas can be classified on the basis of their histological characteristics and prognosis, and when classified as such, the prognosis of replacing-type adenocarcinoma with active fibroblast proliferation is significantly worse than adenocarcinoma without fibroblast proliferation. In order to clarify the biological mechanisms of the key to the morphological changes associated with active fibroblast proliferation, we examined the activities of matrix metalloproteinase (MMP)-2 and MMP-9, which are important enzymes in the stromal invasion by cancers. The active MMP-2 and MMP-9 content of 40 pulmonary adenocarcinomas that were less than 20 mm in diameter was measured by the gelatin zymography method. The quantity of active MMP-2 in the pulmonary adenocarcinomas with active fibroblast proliferation was higher than in the pulmonary adenocarcinomas without proliferation (P < 0.001), but there were no correlations between the histological features and the activation of MMP-9. The presence of active fibroblast proliferation in small pulmonary adenocarcinomas suggests that the cancer cells have acquired the ability to invade through the action of active MMP-2, and this is thought to be one of the reasons for the worse prognosis of pulmonary adenocarcinoma with active fibroblast proliferation.     

MMP-2、MMP-9及EMMPRIN在子宫内膜异位症中的表达及临床意义

目的研究基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）、细胞外基质金属蛋白酶诱导因子（EMMPRIN）在子宫内膜异位症（EMs）的表达和意义。方法应用免疫组化二步法检测EMs患者异位内膜42例、在位内膜42例及正常内膜20例中的MMP-2、MMP-9、EMMPRIN的表达情况,并对它们的EMMPRIN、MMP-2、MMP-9蛋白表达水平进行相关性分析。结果 MMP-2、MMP-9、EMMPRIN在异位内膜组中阳性表达率分别为95.24%、92.86%和90.48%,显著高于在位内膜组、正常内膜组（P〈0.05）;而在位内膜组和正常内膜组差异无统计学意义（P〉0.05）。异位内膜组中,EMMPRIN分别和MMP-2,MMP-9呈正相关性（P〈0.01）。结论 MMP-2、MMP-9、EMMPRIN共同参与了子宫内膜异位症的发生发展;EMMPRIN可能通过促进MMP-2和MMP-9的合成与分泌发挥其作用。     

Activity of MMP-2 and MMP-9 in sera of breast cancer patients

Gelatinase A (MMP-2) and gelatinase B (MMP-9) are proteolytic enzymes involved in the process of tumor invasion, and they are considered as possible tumor markers in breast cancer patients. In this study, we examined serum activity of proMMP-2 and proMMP-9 in relation to TNM stage, tumor size, lymph node involvement, grade of differentiation of tumors, as well as steroid and Her2/neu receptor status in breast cancer patients. The activity of gelatinase in the sera of 52 patients was analyzed by SDS-PAGE zymography. The activity of proMMP-2 and proMMP-9 significantly increased with each advancing clinical stage of disease (p=0.02–0.0009) and compared to controls (p=0.015 to p<0.01). We found a positive correlation between the activity of proMMP-2 and proMMP-9 and tumor size (p=0.007; p=0.05). Patients with lymph node-positive cancer have higher proMMP-2 and proMMP-9 activity than those with node-negative cancer. ProMMP-2 and proMMP-9 activity is not associated with the expression of Her2/neu receptors, but patients with Her2/neu overexpression (3+) showed increased proMMP-2 activity. Steroid receptor score is not associated with enhanced gelatinase activity. The relationship between the increase in proMMP-2 and proMMP-9 activity in serum and tumor size and lymph node status suggests the usefulness of these enzymes as staging markers of breast cancer patients.     

Expression of matrix metalloproteinases MMP-1, MMP-11 and MMP-19 is correlated with the WHO-grading of human malignant gliomas

Glioblastomas (GBM) are the most prevalent type of malignant primary brain tumor in adults. They may manifest de novo or develop from low-grade astrocytomas (LGA) or anaplastic astrocytomas. They are characterized by an aggressive local growth pattern and a marked degree of invasiveness, resulting in poor prognosis. Tumor progression is facilitated by an increased activity of proteolytic enzymes such as matrix metalloproteinases (MMPs). Elevated levels of several MMPs were found in glioblastomas compared to LGA and normal brain (NB). However, data for some MMPs, like MMP-1, are controversially discussed and other MMPs like MMP-11 and MMP-19 have as yet not been analysed in detail. We examined the expression of MMP-1, MMP-9, MMP-11 and MMP-19 in NB, LGA and GBM by semiquantitative RT-PCR, Western blotting and immunohistochemistry and found an enhanced expression of these MMPs in GBM compared to LGA or NB in signal strength and in the percentage of tumors displaying MMP expression. The transition from LGA to GBM was characterized by a shift of pro-MMP-11 to expression of the active enzyme. Therefore, MMP-1, MMP-11 and MMP-19 might be of importance for the development of high-grade astrocytic tumors and may be promising targets for therapy.