炎症介质对人腹膜间皮细胞透明质酸合成酶mRNA表达和透明质酸合成的影响

目的 了解炎症介质对人腹膜间皮细胞(HPMCs)透明质酸合成酶mRNA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随机分为4组:正常对照组、脂多糖组(LPs,10 ng/ml)、肿瘤坏死因子-α(TNF-α,100 U/ml)组,白介素-1β(IL-1β,100 U/ml)组,给予上述刺激后继续培养24 h。人腹膜间皮细胞HAS-2及HAS-3 mRNA表达以逆转录多聚酶链反应(RT-PCR)法检测;细胞衣样结构合成以微粒排除法检测;细胞培养上清液透明质酸(HA)的浓度以放射免疫分折法检测。结果 LPS组、TNF-α组HAS-2 mRNA表达分别较对照组增强1.2倍和1.3倍(P均<0.05);IL-1β组HAS-2 mRNA表达虽较对照组增强,但差异无显著性意义(P>0.05)。LPS组、1L～1β组、TNF-α组HAS-3 mRNA表达分别较对照组增强1.7倍、19倍、8.5倍(P均<0.05)。对照组、LPS组、IL-1β组、TNF-α组细胞胞衣样结构面积与细胞体面积的比值各组间差异无显著性意义(P>0.05);对照组、LPS组、IL-1β组、TNF-α组细胞培养上清液透明质酸(HA)的浓度均显著高于对照组(P均<0.05)。结论炎症因子可上调HPMCs透明质酸合成酶HAS-2 mRNA、HAS-3 mRNA的表达和促进透明质酸的合成。由于其主要增强HAS-3 mRNA的表达,提示炎症状态时有大量小相对分子质量透明质酸合成。     

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。     

高糖对大鼠腹膜间皮细胞分泌FOXP3mRNA的影响及苦参碱的干预作用

目的:研究高糖刺激下大鼠腹膜间皮细胞分泌叉头状螺旋转录因子(FOXP3mRNA)及苦参碱的干预作用,探讨腹膜纤维化发生机制。方法:通过体外培养大鼠腹膜间皮细胞(PMCs),依据加入药物浓度分成5组:2.5%葡萄糖组(n=10,A);2.5%葡萄糖+苦参碱组(n=10,B);4.25%葡萄糖组(n=10,C);4.25%葡萄糖+苦参碱组(n=10,D);空白对照组(仅加不含血清的DMEM/F12培养基),分别于第24 h、48 h、72 h收集培养上清液,采用RT-PCR检测FOXP3mRNA的表达,同时观察各组大鼠腹膜间皮细胞光镜下的表现。结果:光镜下,可见对照组和B组腹膜间皮细胞小,呈圆形、梭形或不规则形,生长密集。A、C、D组腹膜间皮细胞大、呈梭形、分布较稀疏;在含2.5%高糖A组,FOXP3mRNA的表达相对水平较对照组增加,差异有统计学意义(P<0.05);而在4.25%高糖C组,FOXP3mRNA的表达较对照组增加明显,差异有统计学意义(P<0.01)。在含苦参碱溶液的B组和D组中,PMCs表达FOXP3mRNA的相对水平B组较A组减少,但差异无统计学意义(P>0.05);D组较C组下降明显,差异有统计学意义(P<0.05)。结论:苦参碱能拮抗高糖致大鼠腹膜间皮细胞分泌FOXP3mRNA,从而保护腹膜间皮细胞。     

透明质酸合成酶2在肾癌中的表达及临床意义

目的：研究透明质酸合成酶2在肾癌中的表达,并探讨其潜在的临床意义。方法：运用实时定量聚合酶链反应（qPCR）方法检测五种肾癌细胞（ACHN、Caki-1、OS-RC-2、786-O、SN12PM6）透明质酸合成酶三种亚型（HAs1、HAS2、HAs3）mRNA的表达,运用Westernblot方法进一步检测mRNA表达含量高的HAS亚型在五种肾癌细胞系及肾透明细胞癌（ccRCC）组织中的蛋白表达。结果：在五种肾癌细胞系中HAS2mRNA表达水平均明显高于正常肾小管上皮细胞（HK-2）,其中在。肾癌SN12PM6细胞系中表达水平最高（均P〈0．05）,HAS1mRNA的表达水平均明显低于正常肾小管上皮细胞（均P〉0．05）,而HAS3mRNA表达水平除肾癌Caki-1细胞系外均低于正常肾小管上皮细胞（P〉0．05）。在五种肾癌细胞系中HAS2蛋白均明显表达,而正常‘肾小管上皮细胞无明显表达。在肾透明细胞癌组织中HAs2蛋白表达也明显高于相应癌旁组织。结论：透明质酸合成酶2在多种肾癌细胞系和肾透明细胞癌组织中均明显表达,提示透明质酸合成酶2可能在肾癌尤其在肾透明细胞癌发生发展过程中起着至关重要的某种作用,并可能成为新的肾透明细胞癌分子标志物。     

人膀胱移行细胞癌组织中成纤维细胞内透明质酸合成酶优势亚型的确定及意义

目的探讨膀胱移行细胞癌组织中成纤维细胞内透明质酸合成酶（HAS）的优势亚型，为膀胱移行细胞癌组织中透明质酸的功能研究奠定基础。方法于GeneBank中检索得到三种透明质酸合成酶亚型（HASl、HAS2、HAS3）的mRNA序列，应用Primer5．O软件，根据这些mRNA序列分别设计、合成三对引物；常规分别提取膀胱移行细胞癌组织中成纤维细胞和正常膀胱黏膜组织中成纤维细胞的总RNA，逆转录半定量聚合酶链反应（PCR，25循环）后进行常规琼脂糖凝胶电泳、图像扫描和分析、凝胶DNA回收和序列测定。结果正常膀胱组织中成纤维细胞mRNA中扩增出较明显的HAS1条带，HAS2、HAS3条带不明显，而膀胱癌组织中成纤维细胞mRNA中扩增出非常明显的HAS3条带和较明显的HASl条带，无HAS2条带。扫描图像分析后比较两者HASl条带凝胶灰度值，差异无统计学意义（P〉0．05），膀胱癌组织中成纤维细胞的HAS3条带和HASl条带的凝胶灰度值差异有统计学意义（P〈0．01）。结论膀胱癌组织中成纤维细胞出现HAS3的大量合成，且为其透明质酸合成酶的优势亚型。     

RNAi阻断人膀胱癌组织中成纤维细胞内透明质酸合成酶3的表达研究

目的探讨RNA干扰技术阻断人膀胱移行细胞癌组织中成纤维细胞内透明质酸合成酶3（hyaluronic acid synthase 3,HAS3）的表达与膀胱移行细胞癌生物学行为之间的关系。方法设计、体外化学合成HAS3 mRNA序列特异性、非特异性小干涉双链RNA（small interferencing RNA,siRNA）,分别与脂质体LipofectamineTM 2000结合后转染体外培养的人膀胱移行细胞癌组织中的成纤维细胞。实验分为A、B、C、D四组：孵育48h后,取各组细胞培养液进行放免法透明质酸浓度测定、免疫细胞化学染色了解透明质酸的合成、RT—PCR法检测HAS3 mRNA的表达。结果与A组相比,D组细胞HAS3 mRNA表达减少了78．2％,HA染色明显变淡,细胞培养液中HA浓度下降了60．3％（P〈0．01）;B、C组mRNA表达分别减少了4．7％、5．4％,HA染色几乎无变化,HA浓度分别下降了5．2％、5．8％（P〉0．05）。结论RNA干扰技术可以显著地减少膀胱移行细胞癌组织中成纤维细胞的HAS3的表达,从而大幅度地降低该细胞内透明质酸的合成。     

TGF-β1对大鼠腹膜间皮细胞ROS和NADPH氧化酶亚基p67phox表达的影响及黄芪的干预作用

目的:探讨TGF-β1对大鼠腹膜间皮细胞(RPMCs)活性氧(ROS)和NADPH氧化酶亚基p67phox表达的影响及黄芪注射液(AGI)对其的干预作用。方法:体外培养SD大鼠原代腹膜间皮细胞至第二代,静止24h后,随机分为:正常对照组(A组),AGI(2g/ml)组(B组),TGF-β1(10ng/ml)组(C组),TGF-β1+AGI(2g/ml)组(D组,AGI预处理1h)。用荧光染料(DCF)及激光共聚焦显微镜检测细胞内活性氧(ROS)。RT-PCR检测NADPH氧化酶亚基p67phox mRNA的表达;Western印迹检测p67phox的蛋白表达。结果:TGF-β1可显著增加大鼠腹膜间皮细胞ROS产生,刺激20min后,ROS的表达较对照组显著上升(P<0.05)。AGI可显著抑制TGF-β1刺激后ROS的产生(P<0.05);大鼠腹膜间皮细胞经TGF-β1刺激后,NADPH氧化酶亚基p67phox mRNA和蛋白的表达均上升,AGI可抑制TGF-β1诱导的p67phox mRNA和蛋白的表达上调,差异有统计学意义(P<0.05)。结论:TGF-β1可诱导大鼠腹膜间皮细胞产生的ROS增加、NADPH氧化酶亚基p67phox表达上调;AGI可抑制NADPH氧化酶的表达和活性ROS的产生,从而为AGI防治腹膜纤维化提供了理论依据。     

透明质酸在膀胱移行细胞癌、乳头状瘤组织中的表达及其意义

目的　探讨人膀胱移行细胞癌 (BTCC)、乳头状瘤组织中透明质酸的表达情况及其意义。方法　应用免疫组织化学染色方法检测 60例BTCC、2 5例膀胱乳头状瘤和 15例正常膀胱黏膜上皮组织标本中透明质酸表达情况并进行统计学分析。结果　正常对照组HA染色 :(-) 11例 ,( ) 4例 ,染色位于上皮细胞间质。膀胱乳头状瘤组染色 :(-) 3例 ,( ) 10例 ,( ) 12例 ;G1级膀胱移行细胞癌组染色 :(-) 2例 ,( ) 7例 ,( ) 11例 ;G2 级 :( ) 2例 ,( ) 13例 ,( ) 5例 ;G3级 :( ) 1例 ,( ) 8例 ,( ) 11例 ;染色均位于癌细胞胞浆和间质中。正常对照组与BTCC、膀胱乳头状瘤组 ,膀胱乳头状瘤组与BTCC组比较 ,差异均有极显著性 (P <0 .0 0 1) ;G1级组与G2 级组、G1级组与G3 级组之间差异有显著性 (P =0 .0 13 2 ,0 .0 0 3 5 ) ,G2 级组与G3 级组比较 ,差异无显著性 (P =0 .0 5 2 8) ;膀胱乳头状瘤组与G1级组之间的差异无显著性 (P =0 .64 0 7)。结论　透明质酸与BTCC的生物学特性有直接的关联 ,膀胱移行细胞癌细胞本身具有合成透明质酸的功能 ;膀胱乳头状瘤的生物学行为有恶性倾向     

外源性透明质酸对创面纤维黏连蛋白表达的影响

目的　探讨外源性透明质酸延迟创面愈合的作用机理。方法　成年日本大耳白兔 18只 ,建立兔耳皮肤创伤愈合模型 ,随机分 2 %透明质酸治疗组 (A组 )、1%透明质酸治疗组 (B组 )和磷酸盐缓冲液对照组 (C组 )。观察大体形态、组织学变化及平均愈合时间 ,未愈合创面面积及纤维黏连蛋白的表达情况。结果　①三组平均愈合时间为 (11.7± 0 .6 )天 ,(11.3± 0 .6 )天 ,(10 .8± 1.0 )天 ,三组之间有显著差异 (P <0 .0 5 )。A、B组与C组比较各时间点未愈合面积也有显著差异 (P <0 .0 5 )。②组织学观察 ,A、B组胶原纤维较细、排列整齐。C组胶原纤维较粗大、排列紊乱。③纤维黏连蛋白的表达 ,A、B组纤维黏连蛋白的表达少于C组 (P <0 .0 1)。结论　①外源性透明质酸抑制创面纤维黏连蛋白的表达是延迟创面愈合的原因之一。②透明质酸的这一作用与其浓度有依赖关系。     

奥曲肽联合透明质酸钠预防兔术后腹膜粘连的疗效评价

目的: 观察奥曲肽联合透明质酸钠预防兔术后腹膜粘连的效果。方法:建立兔术后腹膜粘连模型,然后分为4组：（1）术中不用药物处理设为模型对照组;（2）关腹前局部涂抹透明质酸钠设为透明质酸钠组;（3）关腹前腹腔内注射奥曲肽设为奥曲肽组;（4）关腹前局部涂抹透明质酸钠同时腹腔内注射奥曲肽设为联合组。术后14d剖腹观察,判定腹膜粘连程度等级。结果:4组粘连发生率比较无统计学意义(χ2＝3.51, P>0.05);联合组重度粘连发生率(8.3%)显著低于其它3组（分别为66.7%,33.3%,25.0%）(均P<0.01)。奥曲肽组和透明质酸钠组两组的粘连级别近似（P> 0.05）。结论:奥曲肽和透明质酸钠均可减轻实验性腹膜粘连的程度和重度腹膜粘连发生率,两者合用其作用更明显,表明两药合用具有降低粘连的协同作用。     

Hyaluronan and proximal tubular cell migration

BACKGROUND: The ubiquitous polysaccharide hyaluronan has been associated with both acute renal injury and progressive renal disease. The aim of this study was to examine the effect of hyaluronan on proximal tubular cell migration. METHODS: The proximal tubular cell line, HK-2 cells, were grown in monolayer culture, and cell migration following addition of hyaluronan characterized in an in vitro model of injury that we have previously developed and characterized. RESULTS: Addition of well-defined preparations of exogenous hyaluronan increased cell migration; however, optimum enhancement of migration was seen with hyaluronan of high molecular weight. Activation of the mitogen-activated protein kinase (MAPK) signaling cascade, as assessed by increased expression of the dually phosphorylated active form of MAPK, could be demonstrated following addition of hyaluronan. This was blocked by the addition of a specific antibody to the hyaluronan receptor, CD44. Hyaluronan-dependent enhanced migration was also abrogated by addition the CD44 blocking antibody, and by inhibition of MAPK kinase (MEK) activity. Generation of a denuded area also led to increased synthesis of endogenous hyaluronan and activation of MAPK, and blockage of either CD44 or MAPK activation inhibited proximal tubule cell (PTC) migration and re-epithelialization under nonstimulated conditions. CONCLUSION: We have demonstrated that hyaluronan activation of the MAPK pathway through binding to its receptor CD44, enhances proximal tubule cell (PTC) migration. In addition, the results suggest that mechanical injury of PTC stimulated hyaluronan generation. These observations may have implications for both recovery from acute tubular injury and progressive renal fibrosis.     

The TGF-beta-induced gene product, betaig-h3: its biological implications in peritoneal dialysis.

BACKGROUND: TGF-beta is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-beta induces betaig-h3 in several cell lines, and betaig-h3 may be a marker for biologically active TGF-beta. However, no study has reported induction of betaig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes. METHODS: We used cultured HPMCs to investigate the biological roles of betaig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in betaig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model. RESULTS: In vitro, TGF-beta1 induced betaig-h3 in cultured HPMCs, and betaig-h3-mediated mesothelial cell adhesion occurred via alphavbeta3 integrin. betaig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, betaig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and betaig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that betaig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. betaig-h3 protein expression was higher in the dialysis group. CONCLUSION: In vitro, betaig-h3 induced by TGF-beta1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, betaig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.     

Leptin augments myofibroblastic conversion and fibrogenic activity of human peritoneal mesothelial cells: a functional implication for peritoneal fibrosis.

BACKGROUND: Myofibroblastic conversion of mesothelial cells is proposed to play an important role in pathological changes following serosal membrane injury. METHODS: Human peritoneal mesothelial cells (HPMCs) were isolated and maintained in culture. The gene expression was assessed by RT-PCR. Activation of signal transduction was determined by western blot and densitometry. Morphological changes were observed by phase-contrast and electron microscopy. RESULTS: In vitro study showed that TGF-beta1-induced myofibroblastic growth of HPMCs was significantly enhanced in the presence of leptin. Augmented expression of alpha-smooth muscle actin, fibronectin and type I collagen mRNA in HPMCs induced by leptin were TGF-beta1-dependent, suggesting that leptin promoted peritoneal fibrogenesis through synergistic activation of the TGF-beta1 signaling system. Leptin and TGF-beta1 synergistically augmented activation of signalling components of mitogen-activated protein kinase (MAPK), STAT3 and Smad but did not modulate the expression of LEPR-B. CONCLUSION: Leptin may act as a profibrogenic TGF-beta1 activated cytokine in peritoneal bioenvironment associated with TGF-beta1 activated pathogenic processes.     

Expression of Na＋-dependent glucose transporter 1 and 2 in peritoneum of peritoneal dialysis patients

Objective To investigate the expression of Na＋-dependent glucose transporter (SGLT) in human peritoneal mesothelial cells (HPMCs) and vascular endothelial cells in peritoneal tissues of peritoneal dialysis (PD) patients at different dialysis vintages, and to study the influence of high glucose treatment on the expression of SGLT1 and SGLT2 in primary HPMCs. Methods According to the dialysis vintage, PD patients were divided into four groups: 0 year group, ＞0-2 years group, ＞2-4 years group and＞4 years group. HE and Masson staining were used to observe the morphologic changes of peritoneal tissues in PD patients. Immunohistochemical staining was used to detect the expression of SGLT1 and SGLT2 in peritoneal HPMCs and vascular endothelial cells. The primary HPMCs were extracted from the peritoneal dialysis fluid, and treated with high-glucose or high-mannitol for 0 h, 12 h, 24 h, 48 h, 72 h and 96 h. Western blotting was used to investigate the SGLT1 and SGLT 2 expression in HPMCs. The cell viability was detected by using cell counting kit (CCK-8). Results HE and Masson staining showed that the peritoneum of PD patients in 0 year group was smooth and continuous, with a flat layer of HPMCs. The number of HPMCs in＞0-2 years group decreased compared with that in 0 year group. The HPMCs size increased in＞2-4 years group, but the number decreased. The peritoneum of PD patients in＞4 years group was significantly thickened and fibrotic, and HPMCs almost disappeared. Immunohistochemical staining showed that the expression of SGLT1 and SGLT2 in HPMCs gradually decreased with the increase of dialysis vintage (P＜0.05). The wall of peritoneal blood vessel became thicken, but the expression of SGLT1 and SGLT2 was not statistically different among four groups (P＞0.05). SGLT1 in primary HPMCs could be up-regulated (0 h, 12 h and 24 h), and then down-regulated (24 h, 48 h, 72 h, 96 h) with the treatment of 60 mmol/L glucose (P=0.029); but there was no significant difference of SGLT2. Conclusion High glucose and the increase of dialysis vintage can reduce the number and the viability of HPMCs, and decrease the expression of SGLT1 and SGLT2, but there was no significant influence on SGLT1 and SGLT2 in peritoneal vascular endothelial cells.