Experimental chemonucleolysis with recombinant human matrix metalloproteinase 7 in human herniated discs and dogs

Background contextChemonucleolysis has been proposed as a less invasive technique than surgery for patients with lumbar disc herniation. Once chymopapain had been approved as a chemonucleolysis drug, it was withdrawn because of serious complications. A novel agent with fewer complications would be desirable.PurposeThe purpose of this study was to investigate the effects of recombinant human matrix metalloproteinase 7 (rhMMP-7) in experimental chemonucleolysis in vitro and in vivo and examine its effects on tissue damage.Study designThe study design is the experimental study using human herniated discs and enzyme substrates in vitro and dogs in vivo.MethodsThe effects of rhMMP-7 on the degradation of human herniated discs were examined by measuring the wet weight in vitro. The correlations between the decrease in wet weight by rhMMP-7 and the conditions associated with herniated discs were also analyzed. The effects of rhMMP-7 on the proteoglycan and water contents were respectively examined with alcian blue staining and T₂-weighted magnetic resonance imaging at 7 days after intradiscal injection in dogs. The distribution of [¹²⁵I]-labeled rhMMP-7 was investigated by autoradioluminography at 7 days after intradiscal injection in dogs. An epidural injection study with rhMMP-7 was performed to evaluate the effects on the tissue damage around the discs at 1 and 13 weeks after the treatment in dogs. The Type 1 and 2 collagen cleavage rates were measured and compared with those of aggrecan in vitro.ResultsRecombinant human matrix metalloproteinase 7 concentration dependently decreased the wet weight of herniated discs in vitro. The decrease in wet weight of the discs by rhMMP-7 did not significantly correlate with the conditions associated with herniated discs. Intradiscal injection of rhMMP-7 reduced the proteoglycan and water contents, with an increase in the serum keratan sulfate levels. Radioactivity of [¹²⁵I]-labeled rhMMP-7 was detected in the nucleus pulposus and annulus fibrosus but not in the muscle. Epidural injection of rhMMP-7 had no effect on the injection site or the nerve tissues. The Type 1 and 2 collagen cleavage rates of rhMMP-7 were 1,000-fold weaker than those of aggrecan.ConclusionsThis study demonstrated experimental chemonucleolysis with rhMMP-7 in vitro and in vivo. The effects of rhMMP-7 were not affected by the conditions associated with herniated discs. The epidural injection study together with the autoradioluminography and in vitro enzyme assay suggests that intradiscal injection of rhMMP-7 may not induce tissue damage around the discs because of its distribution and substrate selectivity. Recombinant human matrix metalloproteinase 7 may be a novel and promising chemonucleolysis agent.     

Vascular endothelial growth factor (VEGF)-induced angiogenesis in herniated disc resorption.

Intervertebral disc herniation is a major cause of low back pain and sciatica. Spontaneous resorption of herniated disc (HD) is frrequently detected by magnetic resonance imaging (MRI). Marked infiltration by macrophages and neo-vascularization are observed upon histogical examination of HD. In addition, enhanced MRI studies suggest that HD resorption occurs more frequently in those completely exposed to the epidural space and that this correlates with their degree of vascularization. We have postulated that the angiogenic factor, vascular endothelial growth factor (VEGF), may be implicated in the neo-vascularization of HD tissues. Here we demonstrate that VEGF and its receptors VEGFR-1 and VEGFR-2 are expressed in human surgical samples of HD. Using a co-culture system comprised of murine peritoneal macrophages and intervertebral disc tissue as a model of the acute phase of HD developed previously, an increase in macrophage VEGF protein and mRNA expression was observed upon exposure to disc tissue. Tumor necrosis factor alpha (TNF-alpha) was required for this induction of VEGF. Use of a novel angiogenesis assay revealed that addition of the conditioned media from the co-culture system resulted in an increase of vascular tubule formation. This effect was strongly inhibited by anti-VEGF antibody, but augmented by recombinant VEGF. We conclude that VEGF induction, under the co-culture conditions tested can result in neo-vascularization of intervertebral disc tissue and may thus play a role in the resorption of HD.     

Sequential dynamics of inflammatory cytokine, angiogenesis inducing factor and matrix degrading enzymes during spontaneous resorption of the herniated disc.

Intervertebral disc herniation (HD) is one of the most common orthopaedic conditions. MRI analysis of HD has revealed a spontaneous resorption mechanism related with neo-vascularization. It appears that the interaction of activated macrophages with disc tissues leads to the generation of inflammatory cytokines. Moreover, inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) is required for the induction of angiogenesis inducing factors such as vascular endothelial growth factor (VEGF) or matrix degrading enzymes such as MMP-3, MMP-7 and plasmin. We hypothesized that these molecules play a crucial role during spontaneous HD resorption. In this study, we have examined the sequential expression of these molecules using a co-culture system which is composed of the interaction of activated macrophages and disc tissues as a model of the acute response of inflammation occurred in HD. We have also considered the mechanism of activating latent MMPs during HD resorption process. Current our results indicate that upregulation of both TNF-alpha mRNA and protein expressions occur first in the inflammation induced by HD. VEGF upregulation follows the increased level of TNF-alpha expression. Both plasmin and MMP-3 are upregulated at later time points. We also demonstrate that both TNF-alpha and VEGF induce upregulated expression of urokinase-type plasminogen activator (u-PA). Our previous work has demonstrated that TNF-alpha could upregulate the expression of VEGF, MMP-3 and MMP-7 in the co-culture system. It has been reported that plasmin could affect to activate latent MMPs. Based on these findings, we suggest that TNF-alpha acts as the initiator of inflammation following contact between macrophages and disc chondrocytes and that plasmin and u-PA play a crucial role in activation of MMPs. We propose a spontaneous HD resorption cascade. Further understanding of the resorption process may provide future novel therapies for HD.     

Influence of macrophage infiltration of herniated disc tissue on the production of matrix metalloproteinases leading to disc resorption.

STUDY DESIGN: Herniated lumbar disc specimens were cocultured with peripheral blood mononuclear cells, and cells isolated from extruded disc were cultured to study the production of matrix metalloproteinases. OBJECTIVE: To investigate the role of peripheral blood mononuclear cells infiltrating extruded discs and disc-derived cells in the production of matrix metalloproteinases. SUMMARY OF BACKGROUND DATA: Magnetic resonance imaging analysis of herniated disc patients revealed a progressive decrease in the size of herniated discs. Spontaneous regression of herniated disc is associated with infiltrating macrophages, and matrix metalloproteinases have been implicated in this phenomenon. However, the correlation between infiltrating macrophages and the production of matrix metalloproteinases has received little research attention. METHODS: Each disc specimen was incubated with homologous peripheral blood mononuclear cells. The numbers of peripheral blood mononuclear cells attached to the surfaces of herniated discs were counted and the culture media was assayed for MMP-3. The cells isolated from herniated discs were incubated with cytokines and the production of matrix metalloproteinases was measured. Total RNA was extracted from herniated discs and RT-PCR was carried out. RESULTS: Significantly larger numbers of peripheral blood mononuclear cells were attached to the surfaces of extruded discs, and higher amounts of MMP-3 were detected than those of control discs. The culture medium of extruded discs showed higher MMP-1 and MMP-3 production than those from controls. Significant enhancement of MMP-1 and MMP-3 mRNA expression was observed in the disc-derived cells stimulated with cytokines. CONCLUSION: These results suggest that peripheral blood mononuclear cells infiltrating extruded discs may secrete a variety of biologic materials capable of further recruiting monocytes into herniated discs in an autocrine fashion. Disc cells stimulated with cytokines showed enhanced production of matrix metalloproteinases, which might play an important role in spontaneous regression of disc materials.     

Transthoracic approach for the treatment of calcified giant herniated thoracic discs

Objective

This study aimed at reporting the results of a transthoracic approach in the treatment of patients with calcified giant herniated thoracic discs (HTDs).

Methods

Fifteen consecutive patients, 11 males and 4 females with a mean age of 46 years (range 33–61), with calcified giant HTDs underwent transthoracic decompression and segmental instrumentation with interbody fusion from November 2004 to September 2010. Clinical data retrospectively examined and compared were levels and types of disc herniation, operative time, blood loss, pre- and postoperative Frankel grades and Japanese Orthopedic Association (JOA) score, and complications.

Results

Of the 15 patients, 2 had HTDs at two levels and affected discs were primarily at the T11/12 level (60 %). Presenting symptoms included myelopathy, axial back pain, urinary symptoms, and radiculopathy. Disc herniations were classified as central (40 %) or paracentral (60 %). All discs were successfully removed without dural tears or cerebral spinal fluid leakage. The mean operation time was 179 ± 27 min (range 140–210 min), and the mean estimated blood loss was 840 ± 470 ml (range 300–2,000 ml). Frankel grades improved in 9 patients postoperatively and 12 patients at the last follow-up. The mean JOA score improved from 4.9 to 7.7. All patients reported improvement in symptoms. The average duration of follow-up was 45 ± 24 months (range 7–77 months).

Conclusions

Transthoracic decompression combined with reconstruction, fusion, and fixation is an effective method for the treatment of these lesions and is associated with a low rate of complications, morbidity, and neurological impairment.     

2002 SSE Award Competition in Basic Science: Expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption

During the process of degeneration, the intervertebral disc (IVD) shows a progressive and significant reduction in height due to tissue resorption. Intradiscal clefts and tears are major hallmarks of disc degeneration. Matrix-degrading enzymes such as matrix metalloproteinases (MMPs) are assumed to play a pivotal role in disc tissue degradation and resorption. The objective of this study was therefore to investigate the potential role of MMPs in extracellular matrix degradation leading to disc degeneration. This study was conducted on 30 formalin-fixed and EDTA-decalcified complete cross-sections of lumbar IVDs from cadavers of individuals aged between 0 and 86 years. Tissue sections were used for the immunolocalization of MMPs-1, -2, -3 and -9. The number of labeled cells was assessed by morphometric analyses, and was statistically correlated with the formation of clefts and tears, cellular proliferation, granular matrix changes and mucous degeneration. Furthermore, 30 disc specimens obtained during spinal surgery were used for in situ hybridization of MMP-2 and -3-mRNA. In addition, the enzymatic gelatinolytic activity was determined by in situ zymography in autopsy material. Immunohistochemistry showed the intradiscal expression of all four MMPs, which was confirmed by in situ hybridization, providing clear evidence for the synthesis of the enzymes within nucleus pulposus and annulus fibrosus cells. Gelatinolytic enzymatic activity was verified by in situ zymography. IVDs from infants and young adolescents remained almost completely unlabeled for all MMPs tested, while more MMPs-1 and -3 were seen in disc cells of younger adults than in those of a more advanced age; MMP-2 remained unchanged over the adult age periods, and MMP-9 was expressed in only relatively few cells. This pattern significantly correlated with the occurrence of clefts and tears. This correlation was strongest for MMP-1 ( P<0.0001), MMP-2 ( P<0.0017) and MMP-3 ( P<0.0005) in the nucleus, and MMP-1 ( P<0.0001) and MMP-2 ( P<0.038) in the annulus. In parallel, the proliferation of disc cells and matrix degeneration (granular changes and mucous degeneration) were related to MMP expression. Likewise, enzymatic activity was seen in association with cleft formation. Our data suggest that major MMPs play an important role in the degradation of the IVD. This is evidenced by the high correlation of MMP expression with the formation of clefts and tears. These findings implicate a leading function for MMPs in IVD degeneration resulting in the loss of normal disc function, eventually leading to low-back pain.     

The presence of extracellular matrix degrading metalloproteinases during fetal development of the intervertebral disc

Matrix metalloproteinases (MMPs) regulate connective tissue architecture and cell migration through extracellular matrix (ECM) degradation and are associated with both physiological and pathological processes. Although they are known to play a role in skeletal development, little is known about the role of MMPs in intervertebral disc (IVD) development. Sixteen fetal human lumbar spine segments, obtained at autopsy, were compared with five normal, non-fetal L4–L5 IVDs. Intensity and/or localization of immunohistochemical staining for MMP-1, -2, -3 and -14 were evaluated by three independent observers. MMP-2 production and activation was quantified by gelatin zymography. MMP-1 and -14 were abundantly present in the nucleus pulposus (NP) and notochordal (NC) cells of the fetal IVDs. In non-fetal IVDs, MMP-1 and -14 staining was significantly less intense (p = 0.001 and p < 0.001, respectively). MMP-3 was found in almost the entire IVD with no significant difference from non-fetal IVDs. MMP-2 staining in the NC and NP cells of the fetal IVD was moderate, but weak in the non-fetal IVD. Gelatin zymography showed a negative correlation of age with MMP-2 activity (p < 0.001). MMP-14 immunostaining correlated positively with MMP-2 activity (p = 0.001). For the first time, the presence of MMP-1, -2, -3 and -14 in the fetal human IVD is shown and the high levels of MMP-1, -2 and -14 suggest a role in the development of the IVD. In particular, the gradual decrease in MMP-2 activation during gestation pinpoints this enzyme as key player in fetal development, possibly through activation by MMP-1 and -14.     

The outcome of decompression surgery for lumbar herniated disc is influenced by the level of concomitant preoperative low back pain

Decompression surgery is a common and generally successful treatment for lumbar disc herniation (LDH). However, clinical practice raises some concern that the presence of concomitant low back pain (LBP) may have a negative influence on the overall outcome of treatment. This prospective study sought to examine on how the relative severity of LBP influences the outcome of decompression surgery for LDH. The SSE Spine Tango System was used to acquire the data from 308 patients. Inclusion criteria were LDH, first-time surgery, maximum 1 affected level, and decompression as the only procedure. Before and 12 months after surgery, patients completed the multidimensional Core Outcome Measures Index (COMI; includes 0–10 leg/buttock pain (LP) and LBP scales); at 12 months, global outcome was rated on a Likert scale and dichotomised into “good” and “poor” groups. In the “good” outcome group, mean baseline LP was 2.8 (SD 3.1) points higher than LBP; in the “poor” group, the corresponding value was 1.1 (SD 2.9) (p < 0.001 between groups). Significantly fewer patients with back pain as their “main problem” had a good outcome (69% good) when compared with those who reported leg/buttock pain (84% good) as the main problem (p = 0.04). In multivariate regression analyses (controlling for age, gender, co-morbidity), baseline LBP intensity was a significant predictor of the 12-month COMI score, and of the global outcome (each p < 0.05) (higher LBP, worse outcome). In conclusion, patients with more back pain showed significantly worse outcomes after decompression surgery for LDH. This finding fits with general clinical experience, but has rarely been quantified in the many predictor studies conducted to date. Consideration of the severity of concomitant LBP in LDH may assist in establishing realistic patient expectations before the surgery.     

Ultrastructural study of the short-term effects of chymopapain on the intervertebral disc

The initial effects of chymopapain, a chemonucleolytic agent, on the intervertebral disc of dogs were studied by light and electron microscopic techniques. Fragments of nucleus pulposus and annulus fibrosis were incubated with chymopapain up to 24 h in vitro. Proteoglycans and matrix proteins were rapidly removed, while collagen fibers remained intact up to 24 h. For several hours, most cells remained normal in appearance with only slight swelling and an increased number of vacuoles. After exposure to the protease for 24 h cells in both the annulus and nucleus showed extensive membrane damage and some were necrotic, but many survived relatively intact. These results suggest that, similar to the results of the digestion of cartilage with other proteases, the cells of the disc can survive brief chymopapain exposure during chemonucleolysis procedures and could serve as a source for regenerating tissue. The nature of the regeneration may depend on the extracellular scaffold that remains and the nutrition available to tissue as well as the age and biomechanical state of the disc. As for clinical significance, chemonucleolysis is an important nonsurgical alternative for treating prolapsed disc. The cells of nucleus and annulus can survive short-term exposure to treatment, and thus be responsible for partial regeneration of the tissue. This regeneration may be important in preventing long-term degenerative disease in the facet joints caused by increased pressure due to decreased disc height.     

Characterization of matrix metalloproteinases in human urine: alterations during adolescence

 Matrix metalloproteinases (MMPs) are a family of at least 14 zinc-dependent proteinases that have been implicated in matrix turnover under both normal and pathological conditions. Previous studies have shown that several MMPs are produced in various cell types in the kidney, suggesting that MMPs may be involved in renal morphogenesis and remodelling. Using a variety of techniques, including gelatin and casein zymography, gelatin affinity chromatography, immunoblotting, and immunoprecipitation, we have identified the major gelatinases in human urine as MMP-2 and MMP-9. Latent forms of both enzymes were detected in urine, as well as lower molecular mass species of each, consistent with activated forms of MMP-2 and MMP-9. MMP-2 and MMP-9 were also measured in individual human urine samples (n=40). No significant gender differences in MMP concentrations were detected. However, renal MMP expression appeared to be age dependent; the highest average amounts of urine MMP-2 were detected during adolescence, while the converse was true of urine MMP-9. Together, these findings indicate that under normal conditions, human urine contains MMP-2 and/or MMP-9, suggesting that these two MMPs are normally produced within the kidney, where they may regulate normal renal remodelling and matrix homeostasis in an age-specific manner. Received: 9 February 1998 / Revised: 27 July 1998 / Accepted: 28 July 1998     

经皮椎间孔镜下TESSYS技术治疗腰椎间盘突出症

[目的]评估经皮椎间孔镜下TESSYS技术治疗腰椎间盘突出症的临床疗效。[方法]2009年1月～2010年12月,新桥医院骨科采用椎间孔镜系统治疗腰椎间盘突出症245例,并与同期采用椎间盘镜系统(MED)治疗腰椎间盘突出症216例进行对比研究。观查手术时间、术中出血量、手术切口长度,采用视觉模拟评分法(VAS)评估手术前后患者腰腿疼痛缓解的情况,改良Macnab评分评估患者术后腰部功能恢复的情况,阅卷方式评估患者术后的满意度。[结果]两组病例术前VAS评分分别为(7.6±2.2)、(7.3±1.9),两者无显著性差异(P>0.05),术后VAS评分分别为(2.8±1.3)、(2.4±1.7),两者病例术后VAS评分分别与术前比较均有明显改善(P<0.05),两组之间术后VAS评分比较无显著性差异(P>0.05)。两组病例手术时间分别为(54.8±10.4)min、(60.5±15.8)min,两组比较无显著性差异(P>0.05);两组病例术中出血量分别为(81.5±20.7)ml、(9.5±4.7)ml,两组比较有显著性差异(P<0.05);两组病例手术切口长度分别为1.8 cm、0.8 cm,两组比较有显著性差异(P<0.05);两组病例Macnab评分分别为97%、95%,两组比较无显著性差异(P>0.05)。[结论]经皮椎间孔镜下TESSYS技术经椎间孔镜入路行椎间盘髓核摘除术是治疗腰椎间盘突出症的一种有效方法,具有手术切口更小、术后疼痛轻,可以早期下地活动、术后恢复更快等优点。     

Identification of human intervertebral disc stromelysin and its involvement in matrix degradation.

Human intervertebral disc when maintained in organ culture released a latent casein-degrading metalloproteinase into the medium in a manner analogous to cultures of human cartilage. This enzyme was demonstrated to be immunologically identical to prostromelysin. It was also found that the amount of procollagenase secreted by both cartilage and disc cells was considerably less than that of prostromelysin. Tissue extraction confirmed that the low level of procollagenase observed was not due to retention of the enzyme within the tissue. Human intervertebral disc link proteins were found to possess the same N-termini as those of their counterparts in human articular cartilage, where it appears that stromelysin is responsible for generating molecular heterogeneity. These results suggest that intervertebral disc cells are capable of secreting prostromelysin, which can become activated within the extracellular matrix and hence contribute to the age-related and degenerative changes in the disc.     

芹菜素在静水压下对人体腰椎间盘蛋白多糖合成的影响

[目的]探讨中药提取物芹菜素在静水压下对体外培养的人体腰椎间盘蛋白多糖合成的影响.[方法]32例后路腰椎间盘切除术的志愿患者中获取新鲜的32个椎间盘样品,每1例样品被切成1～2 mm3大小的碎块,与1ml培养基DMEM一同装入2.5ml的注射器中,放入培养椎间盘组织的压力装置.培养基中分别加入芹菜素(Apigenin)、一氧化氮合成酶的竞争性抑制剂NG-Monomethyl-L-arginine(简称L-NMMA)及一氧化氮的供体Sodium Nitroprusside (SNAP),在可调静水压装置中培养2h后提取上清液,采用考马斯亮蓝蛋白定量检测法测定上清液中蛋白多糖的含量.[结果]芹菜素3atm组和L- NMMA3atm组蛋白多糖合成最高,两组间相比无显著性差异(P＞0.05),与空白组相比有显著性差异(P＜0.05),SNAP 30 atm组蛋白多糖合成最低,与各组间相比均有显著性差异(P＜0.01).空白组、L-NMMA组、SNAP组和芹菜素组中3 atm下蛋白多糖合成较30 atm下蛋白多糖合成高,与之相比有显著性差异(P＜0.05).[结论]3 atm的静水压下,培养基中添加芹菜素使蛋白多糖合成明显增加.添加芹菜素也部分降低了 30atm的静水压下对蛋白多糖合成的抑制.     

Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model.

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.     

白蛋白刺激肾小管上皮细胞表达基质金属蛋白酶2和9

目的探讨白蛋白对近端肾小管上皮细胞表达基质金属蛋白酶2（MMP-2）和MMP-9的影响。方法大鼠近端肾小管细胞株NRK52E培养至70％或100％融合时，分别给予不同浓度（0．1～1．0g／L）去脂和非去脂牛血清白蛋白（dBSA和BSA）刺激，于24、48、72h收集培养液，用明胶酶谱和Western印迹方法检测培养液MMP-2和MMP-9活性和蛋白水平。结果与空白对照组比较，1．0g／LBSA刺激未完全融合NRK52E72h后，MMP-2和MMP-9活性分别上调276％、176％（P〈0．05）。与刺激24h比较，1．0g／LBSA刺激72h，在未完全融合NRK52E的MMP-2和MMP-9活性分别上调536％、148％；在完全融合NRK52E分别上调212％、184％（P〈0．05）。与完全融合NRK52E比较，1．0g／LBSA刺激未完全融合NRK52E 72h，MMP-2活性上调增加了274％，MMP-9活性上调减少了45．1％（P〈0．05）。dBSA刺激结果与BSA类似。结论白蛋白刺激呈剂量和时间依赖性上调近端肾小管细胞表达MMP-2和MMP-9。细胞完全融合可抑制MMP-2表达，促进MMP-9表达。     

高糖对基质金属蛋白酶2及其抑制剂在人腹膜间皮细胞中表达的影响

目的 研究高糖对人腹膜间皮细胞(HPMC)基质金属蛋白酶2(MMP2)及其组织抑制剂(TIMP)1、TIMP2基因及蛋白表达的影响,以及对人腹膜间皮下细胞外基质(ECM)降解的可能作用。方法 利用腹膜透析(腹透)患者腹透流出液标本原代培养HPMC并进行传代,采用半定量RT-PCR方法研究高糖高渗对HPMC的MMP2、TIMP1、TIMP2基因表达的影响。利用免疫组织化学(组化)及Zymography方法检测HPMC的MMP2、TIMP1、TIMP2蛋白表达。结果 HPMC存在MMP2、TIMP1、TIMP2基因及蛋白表达,4.25%葡萄糖显著上调HPMC的TIMP1基因表达(P<0.01),高渗对HPMC的TIMP1基因表达无明显影响(P>0.05),高糖高渗对HPMC的MMP2、TIMP2基因表达无明显影响(P>0.05)。结论 HPMC能够通过MMP/TIMP系统影响到腹膜ECM的降解,高糖可能通过诱导HPMC的TIMP1与MMP间表达失衡,在促进腹膜纤维化的进展中发挥一定作用。     

The relative contribution of cysteine proteinases and matrix metalloproteinases to the resorption process in osteoclasts derived from long bone and scapula

It has been suggested that functional heterogeneity exists between osteoclasts from different bone sites. This could be exploited to design therapeutics that would selectively inhibit bone resorption only at compromised sites. To further investigate the existence of functional differences between osteoclasts from different bone sites we assessed whether osteoclasts isolated from intramembranous bone differ from osteoclasts isolated from endochondral bone in the extent that they utilize cysteine proteinases and matrix metalloproteinases to degrade the organic matrix of bone. The differential involvement of the two classes of proteases was assessed by analyzing dose-dependent effects of the matrix metalloproteinase inhibitor, CT-1746, and of the cathepsin inhibitor, E64, on bone resorption. Osteoclasts isolated from the scapula (intramembranous) and long bones (endochondral) of newborn New Zealand white rabbits were seeded on cortical bovine bone slices in the presence or absence of inhibitors. Resorptive activity was evaluated by measuring the number and area of resorption pits and by measuring the release of collagen degradation products in the culture medium. In the absence of inhibitors, scapular osteoclasts and long bone osteoclasts had similar activity based on these criteria. The resorptive activity of scapular osteoclasts was inhibited to a greater extent by the MMP inhibitor CT-1746 than by the cysteine proteinase inhibitor E64. Conversely, resorption by osteoclasts derived from long bones was inhibited to a greater degree by the cysteine proteinase inhibitor. These results strongly suggest that there are functional differences between dispersed osteoclasts derived from the scapula and long bones, with scapular osteoclasts utilizing matrix metalloproteinases to a greater extent than cysteine proteinases and long bone osteoclasts using cysteine proteinases to a greater extent than matrix metalloproteinases.     

The interaction between epidermal growth factor and matrix metalloproteinases induces the development of sweat glands in human fetal skin

BACKGROUND: The development of sweat glands is a very complicated biological process involving many factors. In this study, we explore the interrelationship among epidermal growth factor (EGF), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 7 (MMP-7), and the development of sweat glands in human embryos. Furthermore, we hope to elucidate the mechanism(s) underlying the induction of epidermal stem cells into sweat gland cells. MATERIALS AND METHODS: Skin biopsies of human embryos obtained from spontaneous abortions at different gestational ages from 11 to 31 weeks were used in this study. The dynamic expression of EGF, MMP-2, MMP-7, and keratin-7 (K7) in developing sweat gland cells or extracellular stroma surrounding the sweat gland cells was examined with SP immunohistochemical methods. The localization of the cellular sources of MMP-2 and MMP-7 was examined with in situ hybridization. RESULTS: At 14-20 weeks of gestation, a gradual increase in EGF immunoreactivity was observed not only in developing sweat gland buds but also in extracellular stroma surrounding the buds, and the expression intensity of EGF peaked at 20-22 weeks of gestational age. All mRNA-positive buds or cells in developing sweat glands contained corresponding immunoreactive proteins. Positive immunostaining for K7 appeared in early sweat gland buds at 14-16 weeks of gestation, and from then on, the positive signal of K7 was concentrated in developing sweat gland cords or cells. CONCLUSIONS: The morphogenesis of sweat glands in human fetal skin begins at 14-16 weeks of gestational age, and is essentially complete by 24 weeks. There is a close relationship among EGF, extracellular matrix remodeling, and morphogenesis of the sweat glands. EGF is one of the inducers in the development and maturity of sweat gland buds or cells.     

肿瘤坏死因子α对人腹膜间皮细胞基质金属蛋白酶及其组织抑制物表达的影响

目的 观察肿瘤坏死因子α（TNF-α）对人腹膜间皮细胞(HPMC) 基质金属蛋白酶（MMP）2、MMP-9及其组织抑制物（TIMP）2、TIMP-1 mRNA和Ⅰ型胶原表达的影响，同时观察TNF-α、TGF-β、IL-1单独或协同作用对HPMC的MMP-9活性的影响。 方法 采用半定量RT-PCR法测定细胞MMP-2、MMP-9、TIMP-2及TIMP-1 mRNA 的表达。采用Biotrak MMP-9 活性检测系统来精确定量测定MMP-9活性及MMP-9原的含量。ELISA法检测Ⅰ型胶原蛋白的表达。 结果 TNF-α(1~10 μg/L)分别刺激HPMC 4、16、24及48 h后， HPMC的MMP-9 mRNA表达显著上调，为基础的2.3～4.9倍（P ＜ 0.05），呈时间依赖性。TNF-α（1 μg/L）作用48 h后显著下调TIMP-1、TIMP-2 mRNA 表达，为基础的77.2％、61.3％（P ＜ 0.05），而MMP-2 mRNA表达没有显著变化。TNF-α+TGF-β１ （1~10 μg/L）、 TNF-α+TGF-β1+IL-1(1~10 μg/L)和TNF-α(5~10 μg/L)刺激HPMC 24 h后，促其分泌MMP-9 的作用最为明显。同时，TNF-α明显上调Ⅰ型胶原蛋白表达（P ＜ 0.05）。 结论 TNF-α 明显上调HPMC的MMP-9 mRNA的表达和MMP-9 的活性；联合其他细胞因子后促MMP-9分泌的作用更明显。TNF-α单独或协同其他因子在腹膜纤维化的过程中可能发挥了重要作用。     

基质金属蛋白酶-2和基质金属蛋白酶-9在前列腺癌组织中表达的免疫组织化学研究

目的：探讨基质金属蛋白酶－2（MMP－2）和基质金属蛋白酶－9（MMP－9）在胶列腺癌组织中的表达及其与前列腺癌侵袭的关系。方法：采用免疫组织化学方法检测前列腺癌组织及良性前列腺增生组织中MMP－2和MMP－9的表达。结果：前列腺癌组织中MMP－2和MMP－9的表达显著高于良性前列腺增生组织（P＜0．01）,前列腺癌组织中MMP－2,MMP－9的表达与侵袭程度呈正相关,结论：MMP－2和MMP－9是检测前列腺癌较好的分子标志,可用于前列腺癌的辅助诊断和预后判断。