透明质酸合成酶-2在人腹膜间皮细胞透明质酸合成及胞外基质形成中的作用

目的 明确哪一种透明质酸合成酶是人腹膜间皮细胞合成透明质酸及形成细胞外基质/胞衣样结构的主要酶。方法 分离培养人腹膜间皮细胞,以半定量RT-PCR法检测其3种透明质酸合成酶HAS-1、HAS-2、HAS-3 mRNA水平表达情况。明确正常培养人腹膜间皮细胞主要表达HAS-2mRNA和微量HAS-3 mRNA后,设计HAS-2的反义寡核苷酸序列,以脂质体介导法将其转入正常培养的人腹膜间皮细胞。转染前(0 h)、转染后8、24、48 h以RT-PCR法观察HAS-2 mRNA表达情况,并以微粒排除法观察细胞衣样结构面积变化。结果 转染后8和24 h,HAS-2 mRNA表达分别下降58％和89％(P均<0.05);转染后48 h HAS-2 mRNA表达已部分恢复至正常表达的25％水平(P<0.05)。相应地,转染后24 h以微粒排除法观察人腹膜间皮细胞细胞外基质/胞衣样结构面积与细胞体面积比值,亦明显减少至几乎完全消失。作为对照的正义序列和逆转序列则无该作用。结论 HAS-2是正常培养人腹膜间皮细胞合成透明质酸以及细胞外基质/胞衣样结构形成的关键酶。     

转化生长因子β1对脂多糖刺激大鼠腹膜间皮细胞上调表达促炎症因子的影响

目的 观察转化生长因子β1（TGF-β1）对脂多糖(LPS)刺激大鼠腹膜间皮细胞上调表达促炎症因子的影响，并探讨其可能的机制。 方法 把原代培养的第2代大鼠腹膜间皮细胞（RPMCs）分成对照组、LPS刺激组（1 mg/L）、TGF-β1刺激组（5 μg/L）及LPS+TGF-β1刺激组。RT-PCR和ELISA方法检测肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6） mRNA及蛋白的表达。蛋白印迹方法检测磷酸化核因子（p-NF）-κB/NF-κB值的变化。 结果 (1)LPS可刺激RPMCs上调TNF-α和IL-6表达。LPS刺激24 h后，p-NF-κB/NF-κB值升高。(2)TGF-β1可拮抗LPS刺激大鼠腹膜间皮细胞上调TNF-α和IL-6表达，同时，也降低p-NF-κB/NF-κB值。 结论 在体外培养的大鼠腹膜间皮细胞，TGF-β1可拮抗LPS的致炎作用，其作用机制可能是通过抑制NF-κB的活化而介导。     

不同分子量透明质酸对人腹膜间皮细胞透明质酸合成酶mRNA表达的影响

目的 :了解不同分子量透明质酸对人腹膜间皮细胞透明质酸合成酶 (HAS)的调节作用及对透明质酸合成的影响。方法 :分离培养的人腹膜间皮细胞 (HPMCs)随机分为 3组 :正常对照组 (对照组 ) ;高分子量透明质酸组(分子量为 1,6 0 0 ,0 0 0道尔顿 ,浓度 0 .0 1%,HHA组 ) ;低分子量透明质酸组 (分子量为 2 80 ,0 0 0道尔顿 ,浓度 0 .0 1%,LHA组 )。给予上述刺激后继续培养 2 4h后 ,以RT -PCR法检测HAS - 2mRNA和HAS - 3mRNA的表达 ;微粒排除法观察细胞基质的面积。结果 :LHA组HAS - 2mRNA及HAS - 3mRNA表达均较对照组增强 (P值均 <0 .0 5 ) ;HHA组HAS - 2mRNA表达较对照组增强 ,而HAS - 3mRNA表达与对照组无显著差异。各组细胞胞衣样结构面积与细胞体面积的比值的比较 ,HHA组较对照组有增加趋势 ,但未达统计学差异 (P >0 .0 5 ) ,LHA组与对照组间无显著性差异。结论 :LHA和HHA均促进HPMCsHAS - 2mRNA表达 ,LHA对与炎症反应相关的HAS - 3mRNA表达也有增强作用 ,而HHA则没有这一作用。因此 ,从选择腹膜保护剂的角度分析 ,我们倾向于选择更具生物相容性的HMW -HA。     

右美托咪定通过c-Fos/NLRP 3/caspase-1级联抑制LP S诱发的小胶质细胞炎症反应

目的探讨右美托咪定通过c-Fos/NLRP3/caspase-1级联抑制脂多糖(LPS)诱发的小胶质细胞炎症反应。方法选取新生的SD大鼠,取其小胶质细胞进行原代培养及分离纯化。采用LPS诱导建立小胶质细胞炎症模型。采用MTT法选取右美托咪定抑制LPS诱导小胶质细胞炎症反应的最适宜浓度。将细胞分为三组:对照组、LPS组和右美托咪定治疗组(D组)。采用实时定量PCR法测定细胞中IL-1β和TNF-α的mRNA表达量;采用ELISA法检测细胞上清液中IL-1β和TNF-α的含量;采用Western blot法检测原癌基因c-Fos、NLRP3和caspase-1的蛋白含量。结果与对照组比较,LPS组小胶质细胞中炎性因子IL-1β和TNF-α的mRNA表达量均明显升高(P0.05);D组IL-1β和TNF-α的mRNA表达量明显低于LPS组(P0.05)。与对照组比较,LPS组中小胶质细胞中炎性因子IL-1β和TNF-α的含量均明显增加(P0.05);而D组IL-1β和TNF-α的含量明显低于LPS组(P0.05)。与对照组比较,LPS组小胶质细胞中c-Fos、NLRP3和caspase-1的蛋白含量均明显增加(P0.01);而D组c-Fos、NLRP3和caspase-1的蛋白含量明显低于LPS组(P0.05)。结论右美托咪定可抑制LPS诱发的小胶质细胞中炎性因子IL-1β和TNF-α的mRNA表达量及蛋白含量,推测其作用机制可能与抑制c-Fos/NLRP3/caspase-1级联反应有关。     

1，25（OH）2D3对脂多糖作用下大鼠腹膜间皮细胞维生素D受体及肿瘤坏死因子α、转化生长因子β1表达的影响

目的 研究脂多糖（LPS）对大鼠腹膜间皮细胞（RPMC）维生素D受体(VDR)及肿瘤坏死因子α（TNF-α）、转化生长因子β1（TGF-β1）表达的影响,从而为1,25（OH）2D3在腹膜透析相关腹膜炎中的应用提供理论依据。 方法 胰蛋白酶消化法原代培养腹膜间皮细胞、传代、经鉴定后分组：(1)正常对照组;(2)脂多糖组：不同浓度的脂多糖（1、10、100 mg/L）分别作用6 h;10 mg/L脂多糖分别作用2、6、12 h;(3)1,25（OH）2D3作用组：10 mg/L脂多糖预孵育2 h后,加1,25（OH）2D3(10－8 mol/L、10－7 mol/L、10－6 mol/L)再作用6 h。RT-PCR法检测VDR mRNA的表达;Western印迹法检测VDR蛋白表达;ELISA法检测上清液TNF-α、TGF-β1的表达。 结果 与对照组相比,LPS组RPMC VDR mRNA和蛋白表达均显著下调（均P < 0.05）。与LPS组相比,1,25（OH）2D3组VDR mRNA和蛋白表达均显著上调（均P < 0.01）。LPS组上清液中TNF-α、TGF-β1浓度均显著高于对照组（均P < 0.01）;1,25（OH）2D3组上清液中TNF-α、TGF-β1浓度均显著低于LPS组（均P < 0.01）。 结论 LPS能下调RPMC VDR mRNA和蛋白的表达,上调TNF-α、TGF-β1表达。1,25（OH）2D3可逆转LPS的作用,上调RPMC VDR mRNA和蛋白的表达,并下调TNF-α、TGF-β1表达。VDR对腹膜透析相关腹膜炎具有一定的保护作用,并具有抑制腹膜纤维化的作用。     

高糖与炎症因子及黄芪对人腹膜间皮细胞毒性的影响

目的:研究不同浓度的含糖腹透液、多种炎症因子以及二者的共同作用对人腹膜间皮细胞损伤的影响,并探索中药黄芪对此的干预作用.方法:用乳酸脱氢酶(LDH)释放法测定细胞毒性,观察各种因素(不同浓度的含糖腹透液与炎症因子的组合、多种炎症因子及其组合和中药黄芪)对体外培养人腹膜间皮细胞株损伤的影响.结果:4.25%腹透液使细胞破坏明显增加,P＜0.05;4.25%腹透液+TNF-α或(LPS+TNF-α+IL-1β)进一步加剧细胞损伤,P＜0.05和P＜0.01.黄芪明显减少细胞破坏,在1.5%腹透液情况下,P＜0.001;在TNF-α和LPS+TNF-α+IL-1β情况下,P＜0.05.结论:黄芪减少腹膜间皮细胞损伤,在炎症因子或含糖腹透液条件下,黄芪仍有保护间皮细胞的作用.     

异丙酚对LPS诱导BV-2小胶质细胞IL-1β和TNF-α释放的影响及TLR4在其中的作用

目的 评价异丙酚对LPS诱导BV-2小胶质细胞IL-1β和TNF-α释放的影响及Toll样受体4(TLR4)在其中的作用.方法 将体外培养的BV-2小胶质细胞接种于96孔培养板中,采用随机数字表法,将其随机分为4(n=12):对照组、LPS组、异丙酚组和LPS+异丙酚组.LPS组加入LPS1μg/ml孵育24h;异丙酚组加入异丙酚30 μmol/L孵育24 h;LPS+异丙酚组同时加入LPS 1 μg/ml和异丙酚30 μmol/L孵育24h.于孵育6h时,采用ELISA法检测细胞上清液TNF-α浓度,以此反映TNF-α的释放量,采用RT-PCR法测定TLR4 mRNA表达;于孵育24h时,采用ELISA法检测细胞上清液IL-1β浓度,以此反映IL-1β的释放量,采用Western Blot法检测TLR4蛋白表达.结果 与C组比较,LPS组和LPS+异丙酚组IL-1β和TNF-α的释放量升高,TLR4 mRNA及其蛋白表达上调(P＜0.05);与LPS组比较,LPS+异丙酚组IL-1β和TNF-α的释放量降低,TLR4 mRNA及其蛋白表达下调(P＜0.05).结论 异丙酚可抑制LPS诱导BV-2小胶质细胞IL-1β和TNF-α的释放,其机制与抑制TLR4的表达有关.     

电刺激对LPS诱导M1型小胶质细胞活化的影响

目的评价电刺激对LPS诱导M1型小胶质细胞活化的影响。方法将生长良好的BV2小胶质细胞采用随机数字表法分为3组(n=18):对照组(C组)、LPS组和LPS+电刺激组(LE组)。C组常规培养24 h, LPS组和LE组加入浓度为100 ng/ml的LPS培养基孵育24 h, LE组于LPS孵育前给予100 mV/mm的直流电刺激4 h。采用ELISA法检测上清液TNF-α和IL-1β浓度;免疫荧光染色法检测M1型小胶质细胞表面标志物CD32和诱导型一氧化氮合酶(iNOS)的表达水平;qRT-PCR法检测细胞CD32和iNOS的mRNA表达水平。结果与C组相比, LPS组和LE组上清液TNF-α和IL-1β浓度升高, 细胞CD32和iNOS及其mRNA表达上调(P<0.05);与LPS组相比, LE组上清液TNF-α和IL-1β浓度降低, 细胞CD32和iNOS及其mRNA表达下调(P<0.05)。结论电刺激可抑制LPS诱导M1型小胶质细胞活化, 从而减轻炎症反应。     

生物活性透明质酸对脂多糖诱导的人树突状细胞和巨噬细胞炎症应答作用

目的探讨经人透明质酸酶PH20特异处理的重组HA,即生物活性透明质酸(B-HA)对脂多糖(LPS)诱导的人树突状细胞(DC)和巨噬细胞炎症应答的影响。方法 DC细胞和人急性单核细胞白血病单核细胞株(THP-1)经不同浓度的B-HA预先处理,加入LPS刺激不同时间,收集细胞和培养上清,采用实时荧光定量PCR和ELISA方法检测细胞因子mRNA和蛋白表达水平的变化。结果 THP-1细胞实验中,与LPS组相比,B-HA 20 mg/ml不仅能够显著降低促炎因子肿瘤坏死因子-α(TNF-α)和白细胞介素-8(IL-8)的表达量,同时能够显著增加抗炎因子白细胞介素-10(IL-10)的表达量。其细胞中TNF-α、IL-8和IL-10 mRNA表达水平分别为24.41±1.94、35.76±4.19和14.99±0.07,而LPS组分别为38.48±1.47、62.7±1.93和2.27±0.16,差异均具有统计学意义(t=5.781、P=0.0290,t=5.695,P=0.0300,t=15.20、P=0.0040)。细胞上清中三者蛋白表达水平分别为(1 609.00±75.66)pg/ml、(213.10±9.14)pg/ml和(496.3±41.92)pg/ml,LPS组分别为(3 018±102.8)pg/ml、(587.4±3.140)pg/ml和(243.3±20.23)pg/ml,差异均具有统计学意义(t=11.04、P=0.0080,t=38.74、P=0.0007,t=5.434、P=0.0320)。在人外周血分化的DC细胞实验中,B-HA对细胞因子TNF-α、IL-8和IL-10的表达均无显著影响(P均0.05)。结论 B-HA能有效调节人巨噬细胞炎性因子的表达,抑制其炎症反应;对人DC细胞的炎症反应无显著影响,提示B-HA对人炎性免疫细胞的炎症反应作用具有选择性。     

Native hyaluronan produces less hypersensitivity than cross-linked hyaluronan

Hyaluronan has been used in patients with osteoarthritis to relieve the painful symptoms associated with this condition. The native form of hyaluronan and artificially cross-linked forms of hyaluronan (such as Hylan G-F 20) are widely used brands that are approved by the Food and Drug Administration for use in patients with osteoarthritis. Clinical evidence suggests that some of these hyaluronan products may induce an antigenic reaction in some patients. Therefore, it was critical to do controlled studies on the potential antigenic reaction induced by these substances. The purpose of this study was to assess the immunologic reactions resulting from the native or cross-linked forms of hyaluronan products in guinea pigs after subcutaneous injection. Guinea pigs were sensitized to hyaluronan via three subcutaneous injections. Active cutaneous or delayed-type hypersensitivity to hyaluronan was studied. The elicitation of antihyaluronan antibodies also was studied by indirect competitive ELISAs. Our results showed that Synvisc induced delayed-type hypersensitivity in guinea pigs, however, no hypersensitivity to the native hyaluronan was observed. This delayed-type hypersensitivity reaction to the cross-linked form of hyaluronan was confirmed by our finding that sera from guinea pigs sensitized to the cross-linked form showed increased antihyaluronan-specific antibodies in competitive ELISAs. In a direct comparison, the native form of hyaluronan produced significantly less hypersensitivity than an artificially cross-linked form of high molecular weight hyaluronan. The hypersensitivity to the cross-linked form of hyaluronan can be explained in part by its elicitation of ant-hyaluronan immunoglobulins in sensitized animals.     

Intra-articular hyaluronan therapy

Viscosupplementation, in which hyaluronan derivatives are injected into the intra-articular space of osteoarthritic joints, is now widely used to treat knee osteoarthritis (OA). No viscosupplements have been approved for osteoarthritic joints other than the knee. To date, no clinical trials using viscosupplements to treat ankle or foot OA have been published. However, the mechanisms thought to be responsible for viscosupplementation's therapeutic effects would likely apply in any synovial joint. A goal of this article is to stimulate interest in research to assess the potential role of viscosupplementation in treating foot and ankle OA.     

Overexpression of hyaluronan synthase 2 alters hyaluronan distribution and function in proximal tubular epithelial cells

The functional consequences of increased renal cortical hyaluronan that is associated with both acute injury and progressive scarring are unclear. The aim of this study was to characterize hyaluronan synthase-2 (HAS2)-driven HA synthesis and determine its effect on renal proximal tubular epithelial cell (PTC) function, because this is known to be the inducible form of HA synthase in this cell type. Overexpression of HAS2 mRNA increased HA generation, which in the supernatant predominantly was HA of large molecular weight, whereas there was an increase in low molecular weight HA in cell-associated fractions. This was associated with increased expression of hyaluronidases, inhibition of HA cable formation concurrent with reduction in HA-dependent monocyte binding, and increased pericellular HA matrix. Overexpression of HAS2 led to enhanced cell migration. HA can be modified by the covalent attachment of heavy chains that are derived from the serum protein inter-alpha-inhibitor (IalphaI), a process that is known to be catalyzed by TNF-alpha-stimulated gene 6 (TSG-6; an inflammation-associated protein). Enhanced migration was abrogated by blocking antibodies to either IalphaI or TSG-6. Addition of recombinant full-length TSG-6 (TSG-6Q) or TSG-6Q_Y94F, a mutant variant with impaired HA binding, increased cell migration. Both of these proteins were able to mediate the covalent transfer of heavy chains, from IalphaI and pre-alpha-inhibitor, onto HA. Addition of the isolated TSG-6-Link module (Link_TSG-6), which binds HA but is unable to form covalent complexes with IalphaI/pre-alpha-inhibitor, had no effect on migration, suggesting that TSG-6-mediated formation of heavy chain-HA complexes is critical in the formation of a pericellular HA matrix.     

Functions of hyaluronan in wound repair

Hyaluronan is a major carbohydrate component of the extracellular matrix and can be found in skin, joints, eyes and most other organs and tissues. It has a simple, repeated disaccharide linear copolymer structure that is completely conserved throughout a large span of the evolutionary tree, indicating a fundamental biological importance. Amongst extracellular matrix molecules, it has unique hygroscopic, rheological and viscoelastic properties. Hyaluronan binds to many other extracellular matrix molecules, binds specifically to cell bodies through cell surface receptors, and has a unique mode of synthesis in which the molecule is extruded immediately into the extracellular space upon formation. Through its complex interactions with matrix components and cells, hyaluronan has multifaceted roles in biology utilizing both its physicochemical and biological properties. These biological roles range from a purely structural function in the extracellular matrix to developmental regulation through effects of cellular behavior via control of the tissue macro- and microenvironments, as well as through direct receptor mediated effects on gene expression. Hyaluronan is also thought to have important biological roles in skin wound healing, by virtue of its presence in high amounts in skin. Hyaluronan content in skin is further elevated transiently in granulation tissue during the wound healing process. In this review, the general physicochemical and biological properties of hyaluronan, and how these properties may be utilized in the various processes of wound healing: inflammation, granulation and reepithelization, are presented.     

Renal hyaluronan accumulation and hyaluronan synthase expression after ischaemia-reperfusion injury in the rat.

BACKGROUND: Hyaluronan (HA) is a connective tissue component with unique water binding and pro-inflammatory properties. It has been suggested that HA is involved in normal renal water handling but also in several pathological conditions such as organ rejection and ischaemia-reperfusion (IR) injury. METHODS: In anaesthetized normal rats we investigated if renal cortical HA accumulation and the intrarenal distribution and expression of HA synthases (Has 1, 2 and 3) correlate with renal dysfunction after renal IR injury. After 20, 30 or 45 min of unilateral renal ischaemia and 72 h of reperfusion, renal function and cortical HA content were measured. Has 1, 2 and 3 mRNA were determined in control and IR kidneys subjected to 45 min ischaemia and 72 h reperfusion. RESULTS: IR kidneys had reduced urine concentrating ability, potassium excretion, glomerular filtration rate (GFR) and renal blood flow. On average, IR kidneys had more than 10 times higher amounts of cortical HA than the contralateral control kidney and their water content was elevated while medullary HA was largely unaffected. Has 2 expression in the cortex was heavily up-regulated in IR kidneys while Has 3 remained at control levels. Has 1 could never be detected. There was a direct correlation between the amount of cortical HA and the time period of ischaemia and also between the cortical amount of HA and depression of functional parameters. CONCLUSIONS: IR injury depresses parameters of renal function, which coincides with an elevated cortical HA content and Has 2 expression. The enhanced Has 2 expression indicates that the cortical HA accumulation is primarily dependent on increased HA synthesis and not impaired degradation/elimination. The water binding and pro-inflammatory properties of HA may contribute to renal dysfunction after IR.     

Role of hyaluronan in acute pancreatitis

BACKGROUND: The connective tissue component hyaluronan is accumulated locally in the damaged tissue during various inflammatory conditions. Owing to the strong water-binding capacity of this glycosaminoglycan, increased tissue content of hyaluronan is paralleled by the development of interstitial edema. The aim with the current experiment was to investigate whether hyaluronan is accumulated in acute pancreatitis and if increased levels of hyaluronan can be correlated to the inflammation of the pancreatic tissue. METHODS: Acute pancreatitis was induced in Sprague-Dawley rats by the administration of supramaximal doses of the cholecystokinin analogue caerulein. The animals were followed for 5 hours (n = 4), 24 hours (n = 6), or 48 hours (n = 5), and the pancreata were then investigated for hyaluronan and water content, hyaluronan distribution, general morphology and the presence of CD44-positive cells, macrophages, and T lymphocytes. RESULTS: Hyaluronan accumulated in the edematous interstitium during acute pancreatitis. Twenty-four hours after the induction of pancreatitis, the hyaluronan content of the pancreata had increased by more than 100%. Simultaneously, CD44-positive cells infiltrated the tissue. However, no correlation between hyaluronan and water was seen at any time point. CONCLUSIONS: This study shows that acute pancreatitis is associated with a strong but transient increase in interstitial hyaluronan and an infiltration of CD44-positive cells located mainly in the same region as the accumulated hyaluronan.     

Roles of hyaluronan in bone resorption

Background  

Hyaluronan, an unsulfated glycosaminoglycan, while being closely linked to osteoclast function several years ago, has received little attention lately. Given recent new knowledge of hyaluronan's possible cell binding abilities, it is important to re-examine the role of this polysaccharide in bone homeostasis.     

Expression of hyaluronan synthases in articular cartilage

OBJECTIVE: To investigate the mRNA expression profiles of three mammalian hyaluronan synthases (HAS1, HAS2 and HAS3) in chondrocytes from normal (undiseased) animal cartilage and osteoarthritic human cartilage maintained in experimental culture systems and exposed to catabolic or anabolic stimuli provided by cytokines, growth factors and retinoic acid. DESIGN: Chondrocytes isolated from normal bovine, porcine or from osteoarthritic human cartilage were cultured as monolayers or embedded in agarose. Cultures were maintained for 3-5 days in the presence or absence of catabolic stimuli (IL-1, TNF-alpha or retinoic acid) or anabolic stimuli (TGF-beta or IGF-1) followed by extraction of RNA and analysis of HAS mRNA expression by RT-PCR. RESULTS: Whereas mRNA for HAS1 was not detected in any sample, the mRNAs for HAS2 and HAS3 were expressed in human, bovine and porcine chondrocytes. HAS2 mRNA was present in chondrocytes from all cartilages and under all culture conditions, whereas HAS3 did not show such constitutive expression. In agarose cultures of bovine and porcine chondrocytes HAS2 mRNA was present in control, IL-1 and retinoic acid treated cultures, whereas HAS3 mRNA was only detected in IL-1 stimulated cultures. Mature bovine chondrocytes cultured in monolayers expressed mRNAs for both HAS2 and HAS3 in the presence of IL-1, TNF-alpha, TGF-beta and IGF-1, however immature bovine chondrocytes in monolayer cultures displayed virtually no HAS3 mRNA expression in the presence of these cytokines and growth factors. HAS2 and HAS3 mRNAs were also expressed by bovine chondrocytes isolated from either the superficial or deep zone of articular cartilage, and by human chondrocytes cultured either in the absence or presence of IL-1 and retinoic acid. CONCLUSIONS: Our data indicate that HAS2 and HAS3 (but not HAS1) mRNAs are expressed in several mammalian cartilages. Chondrocyte HAS2 mRNA appears to be constitutively expressed while chondrocyte HAS3 mRNA expression can be differentially regulated in an age-dependent fashion, and may be affected by local and/or systemic catabolic or anabolic stimuli provided by cytokines or growth factors.