小分子RNA干扰沉默缺氧诱导因子1α加重缺氧状态肾小管上皮细胞的生长抑制和坏死

目的 探讨沉默缺氧诱导因子1α （HIF-1α）对缺氧状态肾小管上皮细胞增殖、凋亡和坏死的影响。 方法 利用氯化钴模拟缺氧状态,并应用小分子RNA干扰（siRNA）技术沉默HIF-1α基因表达。将细胞分成5组,分别是正常培养组、缺氧培养组、转染试剂组、阴性对照组和HIF-1α siRNA组。用MTT试验检测细胞的生长抑制率;用TUNEL法、半胱氨酸天冬氨酸蛋白酶（aspase）-3蛋白定量法检测细胞的凋亡率;检测细胞上清液中乳酸脱氢酶（LDH）活力来测定细胞的坏死情况;用实时定量PCR法和Western印迹法检测细胞HIF-1α、HIF-2α、葡萄糖转运蛋白1（Glut-1）、血管内皮生长因子（VEGF） mRNA和蛋白表达水平。 结果 （1）siRNA的细胞转染效率为95%～100%。在常氧条件下,终浓度100 nmol/L 的HIF-1α siRNA沉默HIF-1α基因的效率为70%;在缺氧条件下,HIF-1α siRNA沉默HIF-1α基因的效率达到97%。（2）HIF-1α siRNA组细胞的生长抑制率显著高于其它组（P < 0.05）;细胞凋亡率在缺氧培养组、转染试剂组、阴性对照组和HIF-1α siRNA组4组间的差异无统计学意义（P > 0.05）;HIF-1α siRNA组细胞培养液中LDH水平显著高于缺氧培养组、转染试剂组和阴性对照组（P < 0.05）。（3）HIF-1α siRNA组细胞的HIF-1α、Glut-1、VEGF mRNA和蛋白表达量显著低于缺氧培养组、转染试剂组、阴性对照组（P < 0.05）;而HIF-2α mRNA和蛋白表达在缺氧培养组、转染试剂组、阴性对照组和HIF-1α siRNA组4组间的差异无统计学意义（P > 0.05）。 结论 siRNA沉默HIF-1α能显著抑制缺氧状态下肾小管上皮细胞Glut-1和VEGF表达,加重缺氧状态下肾小管上皮细胞的生长抑制和坏死。     

人白蛋白刺激人肾小管上皮细胞表达单核细胞趋化蛋白-1

目的：研究人白蛋白是否能刺激人肾小管上皮细胞产生单核细胞趋化蛋白-1(MCP－10。方法：病理标本研究中,以5例正常肾组织为对照,以13例微小病变（MCD）为病变组,应用免疫组织化学和原位杂交的方法,检测肾组织中单核巨噬细胞（Mφ）及MCP－1的分布。在细胞培养中,以不同浓度的人白蛋白（0．1－30g/L)刺激人类肾小管上皮细胞系HK－2细胞24h后,用Western Blot法检测提取物中的MCP－1。结果：（1）在MCD肾组织中,MCP－1主要呈灶状在肾小管壁分布;Mφ在各区域有少量浸润,在间质与肾小管内的浸润数量呈正相关,可见Mφ嵌入在肾小管上细胞间细胞产生MCP－1,白蛋白浓度大于2g/L时明显。结论：MCD患者大量尿蛋白时肾小管有MCP－1表达,肾组织中有少量Mφ浸润,Mφ可能在肾间质和小管间移动。人白蛋白可以刺激人类肾小管上皮细胞产生MCP－1。     

单核细胞趋化蛋白-1小分子干扰RNA真核表达质粒的构建及意义

目的构建单核细胞趋化蛋白-1（MCP-1）小分子干扰RNA（siRNA）表达载体，为进-步研究其对动脉粥样硬化（AS）的防治作用提供手段。方法设计并合成两端含有酶切位点两条DNA序列，经退火成互补双链，再克隆至载体pSilencer2．0-U6中构建重组表达载体，转化DH-5α菌株，提取质粒行双酶切鉴定，并进行序列测定。结果双酶切证实MCP-1siRNA表达载体克隆构建成功，插入片段测序结果与合成的siRNA结果-致。结论成功构建MCP—siRNA表达载体，为动脉粥样硬化的防治奠定实验基础。     

脯氨酸羟化酶2 siRNA对抗霉素诱导肾小管上皮细胞凋亡的影响

目的 探讨是否脯氨酸羟化酶2 (PHD2) siRNA 通过减少缺氧诱导因子(HIF)1的降解而减轻缺氧引起的肾小管上皮细胞凋亡。方法 构建针对人PHD2的表达质粒,将其转染到培养的人肾小管上皮细胞系(HKC)中。用RT-PCR方法观察其对PHD2表达的抑制作用。Western印迹观察HIF-1α的表达。培养的HKC细胞分为对照组、模型组和siRNA组、siRNA组经PHD2 siRNA转染后与模型组同时用抗霉素诱导,用Western印迹方法观察3组细胞HIF-1α的表达,用流式细胞仪观察3组细胞的凋亡情况。 结果 经PHD2 siRNA转染的HKC细胞PHD2 mRNA表达明显下调(P < 0.01),HIF-1α蛋白表达明显上调(P < 0.05)。经抗霉素处理后,模型组和siRNA组HIF-1α蛋白表达均明显高于对照组(P < 0.01),但siRNA组HIF-1α蛋白表达高于模型组(P < 0.05。与模型组相比,siRNA组细胞凋亡明显减轻(P < 0.05)。 结论 通过RNA干扰技术抑制PHD2的表达可以增强HKC细胞HIF-1的蛋白表达,从而增强其在缺氧条件下的抗凋亡能力。     

银杏叶提取物在TGF-β1诱导的人肾小管上皮细胞表型转化中的作用研究

目的：揭示银杏叶提取物（EGb）在转化生长因子-β1（TGF-β1）诱导的人肾小管上皮细胞（HKC）表型转化中的作用,并探讨其对单核细胞趋化蛋白-1（MCP-1）表达的影响。方法：体外培养HKC,经TGF-β1、EGb干预后,应用细胞免疫组化ABC法检测细胞角化蛋白-18与α-平滑肌肌动蛋白（α-SMA）表达,应用实时定量RT-PCR及Western blot。检测Ⅰ型胶原蛋白,应用Western blot方法检测MCP-1表达。结果：TGF-β1（8ng/ml）刺激HKC细胞72h后,细胞角化蛋白-18表达明显下调,α-SMA、Ⅰ型胶原、MCP-1表达明显上调;加入EGb25、50、100、150mg/L干预72h后,α-SMA、Ⅰ型胶原和MCP-1表达呈剂量依赖性减少,细胞角化蛋白-18表达呈剂量依赖性增加。结论：EGb以浓度依赖性方式抑制TGF-β1诱导的HKC表型转化,该作用机制可能与EGb下调MCP-1基因表达有关。     

人肾小管上皮细胞蛋白质组的双向电泳及计算机图像分析

目的 双向电泳(two—dimensional gel electrophoresis，2DGE)技术是蛋白质组研究中最为重要的蛋白质分离方法。建立并优化人肾小管上皮细胞蛋白质组分析所需的固相pH梯度等电聚焦双向电泳及相关技术。方法 由于不同的细胞系特殊性，样品处理亦不同，因此以人肾小管上皮细胞：HK-2为研究对象，对样品液组成、样品处理、上样方式、上样量、IPG胶条和SDS-聚丙烯酰胺凝胶电泳染色方法等相关技术进行了研究和条件优化后，以固相pH梯度等电聚焦为第一向和SDS均一胶(T=12．5％)的水平电泳为第二向，研究人肾小管上皮细胞蛋白质组。结果成功地得到了人肾小管上皮细胞双向电泳图谱，并通过ImageMaster 2D Elite 3．01软件进行图像分析，输出初步的检测结果。结论 建立蛋白质组研究的技术平台，为从更高的水平来寻找肾脏疾病的功能蛋白和特异性蛋白，阐明肾脏疾病发生发展的网络机制等后续研究工作奠定基础。     

核心岩藻糖基转移酶siRNA抑制肾小管上皮细胞转化生长因子β-Smad2/3通路活化

目的 探讨α-1,6核心岩藻糖基转移酶（FUT8）小分子干扰RNA（siRNA）对肾小管上皮细胞转化生长因子（TGF）β-Smad2/3信号通路的影响。 方法 HK-2细胞共分为6组：正常组、阴性对照组、TGF-β1组、TGF-β1加FUT8干扰组、TGF-β1加阴性对照组、FUT8干扰组。利用外源性TGF-β1激活HK-2细胞TGF-β-Smad2/3信号通路。应用siRNA技术沉默FUT8。用免疫荧光方法测定细胞表面核心岩藻糖链表达;用免疫沉淀、凝集素免疫印迹以及免疫双染的方法测定FUT8基因沉默后TGF-βⅡ型受体（TGF-βRⅡ）、TGF-βⅠ型受体（ALK-5）的核心岩藻糖基化变化,并检测Smad2/3蛋白表达和磷酸化（p）-Smad2/3蛋白表达及其核转位的变化。 结果 与正常组及阴性对照组相比,加入5 μg/L的TGF-β1孵育HK-2细胞48 h,能显著上调 TGF-βRⅡ和ALK-5蛋白表达水平（P < 0.05）,导致p-Smad2/3表达水平明显升高（P < 0.05）,并促使其发生核转位。HK-2细胞表面存在核心岩藻糖链表达。与正常组及阴性对照组相比,TGF-β1孵育后,TGF-βRⅡ与ALK-5两种受体的核心岩藻糖链均显著升高（P < 0.05）,FUT8 siRNA能显著抑制TGF-βRⅡ与ALK-5核心岩藻糖链表达（P < 0.05）,从而抑制p-Smad2/3表达升高（P < 0.05）及其核转位,但不影响TGF-βRⅡ和 ALK-5的蛋白表达（P > 0.05）。 结论 在肾小管上皮细胞中,TGF-βRⅡ和ALK-5蛋白翻译后的核心岩藻糖基化修饰是它们发挥生物学功能所需要的,阻止TGF-βRⅡ和ALK-5的核心岩藻糖基化,能抑制TGF-β-Smad2/3信号转导通路的活化。     

RNA干扰基因沉默单核细胞趋化蛋白-1干预动脉粥样硬化早期形成的研究

目的 观察单核细胞趋化蛋白-1(MCP-1)、小干扰RNA(siRNA)、转染兔颈动脉粥样硬化(AS)模型对局部MCP-1表达及AS早期形成的影响.方法 建立28只雄性兔颈动脉AS模型,将MCP-1 siRNA真核表达质粒通过局部定位转染的方法至病变血管,病理形态学观察血管壁中泡沫细胞浸润及内膜增厚,免疫组织化学、逆转录.聚合酶链反应(RT-PCR)、Western blot检测局部MCP-1表达.结果 模型对照组、空质粒组、RNA干扰(RNAi)组镜下内膜/中膜厚度(I:M)分别为5.55、5.27、1.46.免疫组织化学染色显示RNAi组着色较浅.RT-PeR及Western blot结果显示:RNAi组与模型对照组、空质粒组比较,局部MCP-1表达降低.结论 MCP-1 siRNA转染抑制了局部MCP-1表达,延缓了AS的发展,减轻了病变程度.     

重组人单核细胞趋化因子-1对荷骨肉瘤裸鼠的治疗作用

目的观察重组人单核细胞趋化因子－ 1 (monocyte chemoattractant protein－ 1,MCP－ 1)对骨肉瘤细胞种植及骨肉瘤生长的抑制作用。方法以融合蛋白形式在大肠杆菌中表达人 MCP－ 1后予以提取、纯化。将 50只裸鼠分为 10组 :其中 A1～ 4组在接种 4.4× 106个骨肉瘤细胞的同时注射 MCP－ 1,剂量分别为 1μ g、 10μ g、 100μ g、 1 mg; B0～ 4组在骨肉瘤细胞接种 2周形成瘤体后局部隔日注射 MCP－ 1,剂量分别为 0μ g(0.2 ml生理盐水 )、 1μ g、 10μ g、 100μ g、 1 mg,共 20 d; C组为正常裸鼠注射生理盐水作空白对照。结果 A2～ 4组中 MCP－ 1明显阻止了骨肉瘤细胞的种植, A1组中极小剂量的 MCP－ 1也明显延迟其种植生长,抑瘤指数为 69.69％; A1～ 4组裸鼠血清碱性磷酸酶 (AKP)值明显低于 B0组,差异有非常显著性意义 (P< 0.01)。此外, MCP－ 1对已形成的骨肉瘤也具有一定的抑制作用, B3组抑瘤指数达 84.32％。病理检查示各组裸鼠的重要脏器未见明显损害。结论 MCP－ 1具有显著的阻抑骨肉瘤细胞种植、抑制肿瘤生长的作用,局部应用辅助其他治疗手段用于预防手术野内肿瘤细胞的种植具有重要的临床价值。     

RNA干扰沉默酸性鞘磷脂酶1基因对人成纤维细胞凋亡的影响

目的为研发保护女性生殖细胞的小分子干扰RNA（siRNA）类药物,以人包皮成纤维细胞（HFF）为模型,检测靶向凋亡关键基因酸性鞘磷脂酶1（SMPD1）基因的siRNA对HFF凋亡的抑制作用。方法设计合成三对靶向SMPDI基因的siRNA并体外转染HFF细胞,转染后48h用丝裂霉素（MMC）诱导细胞凋亡。荧光定量逆转录-聚合酶链反应（O-RT-PCR）法及免疫印迹（Western blot）检测siRNA转染后SMPD1在mRNA水平和蛋白水平的变化,Annexin V—PI双染检测细胞凋亡率。用脂质体（lipofectamine 2000）和无干扰siRNA（NT siRNA）作为对照。结果siRNA1、2、3对SMPD1基因mRNA的抑制效率分别为（67．0±9．0）％、0％、（45．0±2．1）％,以siRNA1最高,蛋白水平的抑制也呈现相同变化。siR-NA1组细胞凋亡率为15．2％,明显低于MMC组（26．3％）。结论靶向SMPD1的siRNA可以抑制人包皮成纤维细胞凋亡。     

单核细胞趋化因子-1在间质性膀胱炎中表达的研究

目的 探讨单核细胞趋化因子-1(MCP-1)在间质性膀胱炎(IC)患者膀胱组织和尿液中的表达水平及其意义. 方法根据美国国立糖尿病、消化、肾病协会IC诊断标准确诊女性IC患者35例,感染性膀胱炎(UI)患者20例,肾囊肿患者25例作为正常对照组.IC患者平均年龄47(31～65)岁.主诉下腹酸胀/疼痛和夜尿次数多,伴有尿频尿急;已生育30例.均行24 h排尿卡记录、IC症状及问题评分表(O'Leary-Sant评分表)、钾离子敏感试验(PST)和麻醉状态下水扩张后膀胱镜检查.膀胱镜检查获取3组患者膀胱组织和尿液样本,免疫组化染色方法观察MCP-1在IC组织中的表达分布.RT-PCR技术测定3组膀胱组织中MCP-1 mRNA表达水平,酶联免疫吸附试验测定3组尿液标本中MCP-1水平. 结果 IC、UI、对照组尿液标本中MCP-1浓度分别为(74.1±36.9)、(280.6±68.9)、(10.8±6.9)pg/ml;膀胱组织中MCP-1相对定量值分别为76.2±24.0、99.5±30.1、36.1±14.1.lC组和UI组中组织/尿液中MCP-1表达水平均高于正常对照组,差异有统计学意义(P＜0.01).IC组临床症状评分为(14.9±1.8)分,与MCP-1升高水平呈正相关(r=0.686). 结论 IC患者膀胱组织和尿液中MCP-1表达升高,可能成为IC诊断的非特异性免疫指标之一.     

The role of monocyte chemoattractant protein-1 in biliary atresia

Background

The aim of this study was to explain the role of monocyte chemoattractant protein-1 (MCP-1) in biliary atresia (BA).

Methods

Concentrations of serum MCP-1 and collagen type IV were measured in 38 patients with BA by using commercially available kits. MCP-1 was also assessed in liver biopsy specimens by using immunohistochemistry. Subjects were classified into groups. Group 1 comprised BA patients with normal liver function (n = 13), group II comprised BA patients with moderate liver dysfunction (n = 18), group III comprised BA patients older than 20 years awaiting liver transplantation (n = 7), and the control group comprised age-matched patients without evidence of liver disease (n = 23).

Results

Serum MCP-1 levels were significantly increased in group II compared with group I (P < .0001) and the control group (P < .0001). Serum MCP-1 levels in group III were lower than in the control group (P < .0001). There was a significant linear correlation between serum MCP-1 levels and type IV collagen levels in group II. Group II subjects with portal hypertension (PH) had higher MCP-1 levels than those without PH (P = .0009). Biopsy specimens showed MCP-1 was expressed mainly on biliary epithelial cells, vascular endothelial cells, and hepatocytes in group II.

Conclusions

These findings suggest that MCP-1 probably plays a significant role in the development of progressive liver fibrosis in BA.     

Interleukin-1α and tumour necrosis factor-α modulate the production of monocyte chemoattractant protein-1 by cultured human glomerular visceral epithelial cells

Summary: Mediators released by glomerular macrophages may stimulate glomerular visceral epithelial cells (GVEC) to produce cytokines, growth factors or extracellular matrix components. This study describes that human GVEC produce monocyte chemoattractant protein-1 (MCP-1), a monocyte-specific chemotactic factor, and the effects of interleukin-lα (IL-1α) and tumour necrosis factor-α (TNF-a) on the production of MCP-1 by GVEC.
We observed that the intensity of MCP-1 staining in GVEC is stronger in membranous nephropathy and glomerulosclerosis than in normal kidneys. Various cell lines of GVEC produced significant amounts of MCP-1, as assessed by inhibition radio-immunoassay. the presence of IL-1α and TNF-α during culture of GVEC enhanced the production of MCP-1 in a dose- and time-dependent manner. Glomerular visceral epithelial cells in culture express mRNA for MCP-1 and the expression is upregulated 2.0- and 1.4-fold in the presence of optimal concentration of IL-1α and TNF-α, respectively. De novo synthesis of MCP-1 is supported by the observation that MCP-1 production is fully inhibited by cydoheximide. Monocyte chemoattractant protein-1 isolated from GVEC supernatants exhibits a molecular size of 12 and 10 kDa as determined by gel filtration chromatography. Both sizes of MCP-1 is chemotactically active for monocytes.
This study shows increased MCP-1 production by cultured human GVEC after stimulation with the inflammatory cytokines IL-1α and TNF-α. the expression of MCP-1 in GVEC was found to be upregulated in membranous nephropathy and glomerulosclerosis. These findings suggest that MCP-1 may be involved in glomerular injury in these diseases. the possible role of MCP-1 in the pathogenesis of human glomerulonephritis is discussed.     

Role of serotonin in the regulation of renal proximal tubular epithelial cells

In various renal injuries, tissue damage occurs and platelet activation is observed. Recent studies suggest that some factors, such as serotonin, are released into microenvironment upon platelet activation following renal injury. In the present study, we aimed to investigate whether platelets and platelet-released serotonin are involved in the functional regulation of renal proximal tubular epithelial cells (PTECs). PTECs were obtained by primary cell culture and treated with platelet lysate (PL) (2?×?10⁶/mL, 4?×?10⁶/mL, 8?×?10⁶/mL) or serotonin (1?μM or 5?μM) for 12 or 24?h. Phenotypic transdifferentiation of epithelial cells into myofibroblasts were demonstrated under light microscope and confirmed by the determination of α-smooth muscle actin gene expression. Serotonin and PL were shown to induce epithelial–mesenchymal transdifferentiation of PTECs. After stimulation of PTECs with serotonin or PL, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, and collagen-α1 gene expressions, which were reported to be elevated in renal injury, were determined by real-time PCR and found to be upregulated. Expressions of some inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and transforming growth factor-β1 were found to be increased in both protein and gene levels. Recently there is no published report on the effect of serotonin on renal PTECs. Results obtained in this study have lightened the role of serotonin and platelet-mediated effects of serotonin on fibrotic and inflammatory processes in PTECs.     

钙化性纳米微粒与人肾小管上皮细胞相互作用的实验研究

目的 探讨钙化性纳米微粒( calcifying nanoparticle,CNP)对人肾小管上皮细胞(HK-2)的损伤机制及CNP在肾结石形成中的可能作用. 方法 体外培养的HK-2加入CNP,使用NADPH氧化酶抑制剂apocynin进行干预.将HK-2分为4组:正常对照组、纳米微粒组、药物组、纳米微粒+药物组.光镜及透射电镜下观察HK-2与CNP相互作用后的形态变化.采用比色法检测各组24h后细胞培养液中乳酸脱氢酶、丙二醛和透明质酸的水平. 结果 CNP可引起HK-2形态变化,通过透射电镜观察到HK-2吞噬CNP现象.24 h后各组细胞培养液中乳酸脱氢酶含量分别为(231 ±19)、(1042±25)、(433±24)、(652±39) U/L,丙二醛分别为(1.61±0.04)、(5.41±0.10)、(1.98±0.05)、(2.69±0.10)μmol/L,透明质酸分别为(83.44±4.23)、(130.01±3.76)、(95.39±2.06)、(104.73±1.67) ng/L,组间差异均有统计学意义(P＜0.05). 结论 HK-2具有黏附、吞噬CNP的能力,CNP可引起HK-2氧化应激损伤.     

Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of extracellular matrix molecules in cultured human renal proximal tubular cells

Objective: In this study, we investigated the effect of small interfering RNA (siRNA) of connective tissue growth factor (CTGF) by pRetro-Super (PRS) retrovirus vector on the expression of CTGF and related extracellular matrix molecules in human renal proximal tubular cells (HKCs) induced by high glucose, to provide help for renal tubulointerstitial fibrosis therapy. Methods: HKCs were exposed to d-glucose to observe their dose and time effect, while the mannitol as osmotic control. Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect HKC. The expression of CTGF, fibronectin (FN) and collagen-type I (col1) were measured by semi-quantitative RT-PCR and Western blot. Results: In response to high glucose, CTGF expression in HKCs was increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Introduction of PRS-CTGF-siRNA resulted in the significant reduction of CTGF, FN, col1 mRNA (p?0.01, respectively) and CTGF, col1 protein (p?0.05, respectively) expression, while PRS void vector group did not have these effects (p?>?0.05). Conclusions: CTGF siRNA therapy can effectively reduce the levels of CTGF, FN and col1 induced by high glucose in cultured HKCs, which suggested that it may be a potential therapeutic strategy to prevent the renal interstitial fibrosis in the future.     

ERK1/2信号通路在甲状旁腺激素致人近曲小管上皮细胞分泌纤溶酶原激活物抑制剂1中的作用

目的 从体外观察ERK信号通路在甲状旁腺激素（PTH）致人近曲小管上皮细胞（HK-2）合成纤溶酶原激活物抑制物1（PAI-1）中的作用。 方法 以HK-2细胞株为研究对象,用不同浓度PTH（10-8、10-9、10-10、10-11、10-12 mol/L）作用细胞48 h,以及10-10 mol/L PTH作用细胞不同时间（12、24、36、48、72 h）,分别用RT-PCR法检测PAI-1 mRNA表达,Western印迹法检测PAI-1蛋白表达。以10-10 mol/L PTH作用细胞48 h,分别观察细胞ERK1/2抑制剂预处理前后磷酸化（p）ERK1/2、PAI-1mRNA及蛋白的变化情况。 结果 10-12 mol/L PTH可促进细胞在基因及蛋白水平合成PAI-1,随着PTH浓度逐渐上升,PAI-1mRNA及蛋白浓度均相应增加,以10-10 mol/L PTH组刺激作用最显著,分别为对照组的4.01倍和3.81倍（均P < 0.01）。但随着PTH浓度进一步增加,PAI-1mRNA及蛋白浓度却随之下降。10-10 mol/L PTH作用细胞,12 h时有少量PAI-1表达,72 h时达峰值,并呈时间依赖性,分别为0 h组的4.06倍和4.03倍（均P < 0.01）。10-10 mol/L PTH作用细胞48 h,有大量的p-ERK1/2合成（P < 0.01）,经ERK1/2抑制剂预处理后,PAI-1及ERK均显著下降（均P < 0.01）,但仍高于对照组（均P < 0.05）。 结论 ERK信号通路部分参与PTH致HK-2细胞合成PAI-1的作用。