RNAi技术沉默Bcl-2基因对人膀胱癌细胞株T24增殖影响的研究

目的探讨RNA干扰(RNAi)技术沉默Bcl-2基因表达对人膀胱癌细胞株T24增殖的影响。方法针对Bcl-2 mRNA序列设计合成3对编码小干扰RNA(siRNA)的DNA模板,构建pGenesil-1-Bcl-2 siRNA重组质粒,转染T24细胞。采用Western blot检测重组质粒对Bcl-2蛋白表达的影响,MTT法观察重组质粒对T24细胞体外生长的抑制作用,Annexin-V-PI双染法流式细胞术(FCM)检测转染重组质粒后细胞的凋亡状况。结果成功构建了pGenesil-1-Bcl-2 siRNA重组质粒,并成功转染T24细胞。重组质粒抑制Bcl-2基因的表达接近70%;转染重组质粒后,T24细胞的活力降低为(66.9±5.6)%;重组质粒组的T24细胞凋亡率为34.55%～45.39%。结论pGenesil-1-Bcl-2 siRNA重组质粒明显下调Bcl-2在膀胱癌细胞中的表达,并抑制肿瘤细胞生长,促进其凋亡。     

抑癌基因RUNX3在膀胱癌T24细胞株中的表达以及对凋亡的影响

目的 明确RUNX3抑癌基因在正常膀胱组织和膀胱癌T24细胞株的甲基化状态,并将野生型RUNX3基因转染T24细胞,探讨RUNX3基因恢复表达后对膀胱癌细胞凋亡的影响. 方法应用RT-PCR、甲基化特异性PCR和非甲基化PCR检测正常膀胱组织、膀胱癌T24细胞中RUNX3基因的表达以及基因启动子区域CpG岛的甲基化状态.构建真核载体的RUNX3-EGFP-pDest质粒,在LipefectamineTM2000介导下转染膀胱癌T24细胞,转染实验设立3组:空白对照组、空质粒EGFP-pDest转染组以及RUNX3-EGFP-pDest转染组.Western blot检测RUNX3蛋白表达,流式细胞仪检测细胞凋亡情况. 结果正常膀胱组织RT-PCR可检测出1248bp的RUNX3基因条带,而膀胱癌T24细胞中无法检测出RUNX3基因的表达.正常膀胱组织RUNX3基因甲基化PCR检测阴性,非甲基化PCR阳性;T24细胞反之.正常膀胱粘膜组和RUNX3-EGFP-pDest质粒转染组Western blot检测,均表达RUNX3蛋白,而膀胱癌T24细胞未表达RUNX3蛋白.流式细胞仪检测,空白对照组的凋亡率为(3.1±0.46)%,而EGFP-pDest转染组和RUNX3-EGFP-pDest质粒转染组的凋亡率分别为(10.1±1.62)%、(41.20±1.53)%,应用方差分析的LSD法和SNK法进行均数的多重两两比较,RUNX3-EGFP-pDest质粒转染组与EGFP-pDest转染组、空白对照组凋亡率之间的差异均具有显著性,P<0.01.结论 正常膀胱组织RUNX3基因正常表达,未发生启动子区域CpG岛甲基化,而膀胱癌T24细胞可能因RUNX3基因启动子区域CpG岛发生甲基化,致RUNX3基因无法表达.转染野生型RUNX3抑癌基因后促进了膀胱癌T24细胞的凋亡.     

胸腺素β4基因沉默对膀胱移行细胞癌上皮间质转化的逆转作用

目的 观察胸腺素β4(Tβ4)基因沉默对膀胱癌细胞上皮间质转化(EMT)的逆转,探讨其在肿瘤侵袭转移中的作用机制.方法 使用针对Tβ4的慢病毒载体(knti-Tβ4)转染膀胱癌细胞株T24.采用定量逆转录-聚合酶链反应(RT-PCR)、Western blot法检测Tβ4、整合素连接激酶(ILK)和上皮性标记基因E-cadherin、β-catenin的表达改变;Immunofluorescence法检测ILK、E-cadherin、β-catenin在转染后124细胞中的表达改变;细胞划痕实验、Boyden小室体外侵袭实验、AO/EB荧光染色法反映细胞转移潜能和凋亡的变化.结果 转染后48 h的T24细胞,Tp4、ILK、β-catenin mRNA或蛋白表达开始下降,以转染后96 h明显;而上皮标记基因E-cadherin在转染96h后,表达显著增加(P<0.05);Immunofluorescence显示ILK和β-catenin在细胞质、细胞核中表达减弱,而E-cadherin在胞膜中表达增强,细胞形态向正常上皮细胞转化;转染后的324细胞体外迁移能力与侵袭力下降[(10.4±1.2)%比(73.5±1.4)%],细胞凋亡增多(12.3%比36.6%).结论 肿瘤细胞中的Tβ4表达下调可以逆转肿瘤细胞的间质表型,而向正常上皮表型转化,降低肿瘤转移潜能.     

siRNA沉默高迁移率蛋白A2基因对人膀胱移行细胞癌细胞T24增殖凋亡的影响

目的 观察siRNA靶向沉默HMGA2基因在人膀胱移行细胞癌T24细胞中表达的变化,探讨抑制HMGA2基因对T24细胞增殖、周期和凋亡的影响.方法 针对人HMGA2基因构建siRNA干扰片段,瞬时转染T24细胞后,采用Western-blot方法检测转染siRNA干扰片段48h后T24细胞蛋白表达量的变化,CCK-8法检测T24细胞增殖和流式细胞仪（FCM）检测T24细胞周期和凋亡情况.结果 转染后48h的T24细胞HMGA2蛋白表达水平较空白对照组（CON）和阴性对照组（NC）显著下降,差异有统计学意义（P＜0.05）,转染HMGA2-siRNA的实验组抑制T24细胞增殖,T24细胞S期细胞比例（35.47±0.23）％高于CON组和NC组（P＜0.05）,实验组T24细胞凋亡率为（17.25±0.24）％,明显高于CON组和NC组（P＜0.05）.结论 HMGA2基因的特异性siRNA可以抑制T24细胞增殖,细胞周期阻滞在S期,并促进其凋亡.     

人抗凝血酶Ⅲ基因转染血管内皮样细胞的表达

目的 观察人抗凝血酶Ⅲ(human antithrombin-Ⅲ,hAT-Ⅲ)基因转染血管内皮样细胞(VELCs)后的表达情况. 方法 体外分离、培养和扩增的人骨髓间充质干细胞(BMMSCs),定向诱导分化产生血管内皮样细胞,然后将血管内皮样细胞随机分为实验组和对照组,实验组采用脂质体转染方法,将携带hAT-Ⅲ基因的质粒DNA转染血管内皮样细胞,分别于转染后72h、96h,采用逆转录-聚合酶链反应(RT-PCR)、免疫组织化学法、蛋白免疫印迹法(Western-Blotting)和发色底物法检测细胞hAT-Ⅲ的表达;对照组细胞采用空白TE缓冲液代替质粒DNA,其余方法完全同实验组. 结果 RT-PCR显示实验组细胞扩增出hAT-Ⅲ特异片段,对照组阴性;免疫组化显示实验组细胞阳性表达hAT-Ⅲ,对照组阴性;Western-Blotting显示实验组细胞培养上清液中有hAT-Ⅲ特异条带,对照组阴性; 发色底物法检测显示实验组细胞hAT-Ⅲ活性为9.50%±1.52%,对照组为1.83%±1.17%,两组比较差异有统计学意义(t=7.910,P<0.01). 结论 hAT-Ⅲ基因可以成功转染血管内皮样细胞并进行表达.     

肾母细胞瘤过表达基因对肾癌细胞的作用

目的 探讨肾母细胞瘤过表达( nephroblastoma overexpressed,NOV)基因对肾癌细胞增殖、黏附、侵袭和迁移能力的影响. 方法 构建NOV蛋白表达真核细胞重组表达质粒pEGFP-C1-NOV,转染人肾癌细胞株786-O.通过计数细胞绘制生长曲线、水溶性四氮唑法(WST-1)检测细胞生长抑制率,通过细胞黏附实验、侵袭实验和细胞迁移实验,比较转染pEGFP-C1 -NOV组(实验组)与转染空载体组(空载组)及未转染组(空白组)的细胞增殖、黏附、侵袭和迁移能力的差异. 结果 与空白组比较,实验组48、72 h抑制率分别为29.14％、32.46％,空载组分别为9.25％和- 8.16％,实验组与空白组比较差异有统计学意义(P＜0.05),空载组与空白组相比差异无统计学意义(P＞0.05).与层粘连蛋白黏附,实验组A值为0.26±0.03,高于空白组(0.15±0.01)和空载组(0.14 ±0.02),差异有统计学意义(P＜0.05);与人纤维连接蛋白黏附,实验组A值为0.28±0.04,高于空白组(0.124±0.095)和空载组(0.128±0.082),差异有统计学意义(P＜0.05).实验组穿越Matrigel基质胶的细胞数为240.25±23.12,显著高于空白组(56.16 ±6.25)和空载组(50.28 ±7.13),差异有统计学意义(P＜0.05);实验组迁移通过聚碳酸酯微孔滤膜的细胞数为267.25±20.94,显著高于空白组(66.10 ±5.68)和空载组(56.28 ±4.11),差异有统计学意义(P＜0.05). 结论 NOV可抑制肾癌细胞的增殖,促进肾癌细胞786-O的黏附、侵袭和迁移.     

Cdx2对胃癌细胞生物学性状的影响

目的 观察Cdx2对人胃癌细胞MGC-803生物学性状的影响.方法 利用脂质体将pCMV-Cdx2-HA和pCMV-HA质粒分别转染MGC-803细胞,应用逆转录.聚合酶链反应(RT-PCR)和Western blot技术检测MGC-803细胞中Cdx2基因的表达;应用体外实验及流式细胞仪分别检测Cdx2对MGC-803的侵袭力、黏附力、增殖力、迁移力和凋亡率的影响.结果 转染pCMV-Cdx2-HA质粒的MGC-803细胞中可以检测到高Cdx2的表达,其侵袭能力[穿膜细胞数:(64.33±11.94)个/视野下降到(23.93±8.95)个/视野]、黏附能力[(1.172±0.042)]、增殖力[(26.13±1.60)%]和迁移能力[(42.87±2.19)%]较对照组明显降低(P＜0.05),而且凋亡率升高,48 h和72 h的凋亡率分别为(11.40±0.36)%、(9.72±0.50)%(P＜0.05).结论 Cdx2高表达对MGC-803具有抑制其侵袭转移的作用,并促进肿瘤细胞的凋亡.     

miRNA-101对激素非依赖型前列腺癌细胞系LNCaP EZH2表达的影响

目的:探讨microRNA-101(miRNA-101)对激素非依赖型前列腺癌细胞系LNCa P的果蝇zeste基因增强子的人类同源基因(EZH2)表达的影响。方法:实验分为空白组、阴性对照组和转染组。转染组通过基因重组技术构建miRNA-101表达载体,应用脂质体将其转染到LNCa P细胞中,荧光显微镜检测转染效率。3组细胞通过qRT-PCR检测细胞中EZH2 mRNA的表达。阴性对照组和转染组用Western印迹检测EZH2蛋白的水平。结果:转染组细胞培养24 h后,70%以上的细胞内出现绿色荧光基因信号。阴性对照组和转染组细胞培养72 h后,转染组细胞miRNA-101表达增加明显(P<0.01);转染组EZH2 mRNA的表达水平(0.01±0.10)显著低于空白组(0.95±0.40)和阴性对照组(0.86±0.30)(P均<0.01)。阴性对照组EZH2蛋白表达水平随培养时间延长而增加,但转染组EZH2蛋白表达水平随培养时间延长则下降。结论:miRNA-101可以抑制LNCa P细胞的EZH2的表达,具有成为前列腺癌生物治疗靶点的可能。     

小干扰RNA抑制雄激素受体基因表达对人膀胱癌T24细胞增殖及凋亡的影响

目的 观察小干扰RNA技术(siRNA)抑制雄激素受体(AR)基因的表达对人膀胱癌T24细胞增殖及凋亡的影响.方法 化学合成针对AR的siRNA,用脂质体转染T24细胞.采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)和Western blot检测AR mRNA和蛋白的变化.转染细胞48 h后用噻唑蓝(MTT)比色法检测细胞体外增殖活性的变化,流式细胞术检测细胞的凋亡状况.结果 成功的转染T24细胞,实时荧光定量RT-PCR筛选出一条AR mRNA抑制效率最高的siRNA,行Western blot检测可抑制AR蛋白表达至70.3%.转染细胞48 h后,T24细胞增殖活性抑制率达到(25.9±5.4)%,细胞凋亡率达到21.61%.结论 应用siRNA干扰技术能有效的抑制AR基因的表达,同时可抑制人膀胱癌T24细胞的体外增殖活性及促进其凋亡的发生.     

膜反应性溶解抑制物在细胞及小鼠体内的表达

目的 观察外源基因在猪血管内皮细胞及小鼠体内表达情况.方法 体外培养猪血管内皮细胞,脂质体转染法将pcDNA3-ICAM-2-Enhancer CD59cDNA转染猪的血管内皮细胞,以未转染的猪血管内皮细胞为阴性对照,人血细胞为阳性对照,经G418筛选(浓度为100 mg/L)获得具有抗性的克隆,通过流式细胞仪检测转染细胞的表达情况.通过显微注射将CD59基因片段导入小鼠授精卵,对出生的小鼠进行基因检测.结果 pcDNA3-ICAM-2-Enhancer-CD59cDNA重组质粒在细胞中表达强度为63.1%;转CD59基因小鼠外周血单核细胞(PBMCs)表达强度为73.38%.结论 以人细胞间黏附因子Ⅱ(ICAM-2)为启动子并带增强子的CD59重组基因在猪的血管内皮细胞及小鼠体内均高效特异表达.     

Rho激酶在烧伤大鼠血清诱导的血管内皮细胞骨架变化中的作用

目的　观察烧伤血清刺激下血管内皮细胞骨架的变化及Rho信号转导通路所起的作用。　方法　常规培养人脐静脉血管内皮细胞系ECV304,随机分为空白对照组、实验对照组、烧伤组、Y -27632组、烧伤 Y -27632组、Y- 27632 烧伤组、溶血磷脂酸(LPA)组和LPA Y- 27632组,分别用正常大鼠血清、烧伤大鼠血清、30μmol/LRho激酶抑制剂Y -27632、13μmol/LRhoA激动剂LPA单独刺激或联合刺激。采用HE染色于光镜下观察各组内皮细胞形态。于荧光倒置相差显微镜下观察内皮细胞骨架变化,除Y -27632组外,各组均在刺激后6、7、8h3个时相点进行观察。用流式细胞仪检测实验对照组、烧伤组、Y -27632组、烧伤 Y- 27632组、LPA组和LPA Y 27632组刺激6h的肌动蛋白含量。　结果　空白对照组、实验对照组细胞呈梭形或多边形,生长良好;丝状肌动蛋白主要分布在细胞周边,形成周边肌动蛋白丝带,细胞生长融合为单层后呈网状结构;球状肌动蛋白集中在细胞中央。烧伤组烧伤血清刺激6h,细胞贴壁差,丝状肌动蛋白重组,应力纤维形成,周边肌动蛋白丝带模糊;球状肌动蛋白松散,胞浆中可见散在分布的絮状球状肌动蛋白,且这些反应随着刺激时间延长而增强。烧伤 Y- 27632组或Y -27632 烧伤组细胞生长、贴壁良好,丝状肌动蛋白的分布与空白对照组、实验对     

Upregulation of cell adhesion through delta Np63 silencing in human 5637 bladder cancer cells

Some researchs have demonstrated that the loss of delta Np63 is associated with aggressive phenotypes and poor prognosis. However, other research indicates that delta Np63 is considered to have oncogenic properties. Delta Np63 overexpression is often observed in association with the oncogenic growth of squamous cell carcinomas and bladder cancer. In this study, we investigated the oncogenic role of delta Np63 in regulating cell adhesion in transitional cell carcinoma of the bladder (TCCB). The cells were stably transfected with the delta Np63 short hairpin RNA (shRNA) plasmid. Immunocytochemistry was performed to determine the knockdown efficiency. Tumour cells were studied for their ability to adhere to vascular endothelial cells. Confocal microscopy was used to analyse the changes in cytoskeletal F-actin. F-actin expression was measured by flow cytometry. Cell invasion ability was assessed using transwell chambers. The delta Np63-silenced tumour cells were shown to adhere more tightly than controls to vascular endothelial cells (P<0.05). The content of F-actin in the delta Np63-silenced cells was enhanced (P<0.05). The Matrigel invasion assays showed that human 5637 bladder cancer cells had a lower degree of motility when transfected with pdelta Np63-shRNA (P<0.05). In conclusion, silencing of the delta Np63 expression can enhance the adhesiveness of 5637 cells by inducing F-actin cytoskeleton production, and it will possibly inhibit the TCCB invasion and metastasis.     

非小细胞肺癌K-ras突变小分子对吉非替尼耐药细胞的作用

目的 观察针对K-ras突变小分子NSC-741909是否可特异性杀伤吉非替尼原发耐药细胞,并探讨其机制.方法 选取吉非替尼耐药细胞;观察NSC-741909作用后细胞增殖与凋亡的变化;共聚焦显微镜观察细胞骨架改变;Western blot检测K-ras、JNK、P-JNK的改变.结果 NSC-741909可使耐药细胞在作用24 h后,在1 μmol/L和2 μmol/L时,FITC-A+/PE-A+细胞增加至(6.9±0.6)%和(21.1±3.2)%(P＜0.01);作用30 min后,K-ras表达在2 h下降达70%;p-JNK表达增加,并持续至少15 h,而总JNK蛋白表达无改变;细胞骨架蛋白F-actin呈稀疏、不规则、发散状排列.结论 针对K-ras突变的小分子NSC-741909可特异性杀伤吉非替尼原发耐药细胞,这是通过持续激活JNK途径从而导致细胞凋亡实现的.

Abstract：

Objective To study whether a recently identified novel anticancer agent NSC-741909 can suppresses the growth of non-small cell lung cancer cell line ( NSCLC) which has primary resistance to Gefitinib and explore its molecular mechanisms. Methods Select NSCLC cell line which is resistant to Gefitinib. Observe the cell growth supression effect of NSC-741909 to the cell line, apoptosis and actin cytoskeleton changement. Observe K-ras, JNK, p-JNK protein expression by Western blotting. Results NSC-741909 can induce apoptosis of Gefitinib resistant cell lines at 24 h. At that time point, FITC-A +/ PE-A + increased to (6. 9 ±0.6)% and (21. 1 ±3.2)% (P ＜0. 01) at 1 and 2 μmol/L;K-ras protein decreased to 70% at 2 h; p-JNK expression was increased and lasted for at least 15 h and total JNK remained the same. Cell cytoskeleton F-actin presented as loose, irregular and radiation arrangement. Conclusion NSC-741909 which supress the mutant K-ras expression can induce the apoptosis of the NSCLC which is primary resistant to Gefitinib. This inhibition was mediated by sustained JNK activation.     

An ex vivo analysis of Sertoli cell actin dynamics following gonadotropic hormone withdrawal

The receptors for the steroid hormone testosterone and the peptide hormone follicle-stimulating hormone are localized to the somatic Sertoli cell in the seminiferous epithelium. In the rat, prolonged gonadotrophic hormone withdrawal has been shown to result in substantial germ cell apoptosis. Previous studies have shown that, coincident with the loss of germ cells following hypophysectomy, the actin cytoskeleton of the Sertoli cell becomes disorganized and diffuse throughout the cell's cytoplasm. The molecular mechanisms that govern Sertoli cell actin filament dynamics in response to the loss of gonadotrophic hormones remain undefined. It was therefore hypothesized that hypophysectomy brings about a decrease in the amount of polymerized actin (F-actin) within the Sertoli cell and that this decrease is associated with changes in the expression of genes known to govern Sertoli actin dynamics. To this end, Sertoli cells were isolated from adult control and hypophysectomized rats. Sertoli cells from hypophysectomized rats were found to contain significantly less (72%) F-actin relative to untreated controls, although overall, beta-actin protein and mRNA expression remained constant. The expression levels of genes known to directly influence the amount of F-actin in cells were then examined by Northern blot analysis. Cofilin and profilin I gene expression was unaffected by hypophysectomy, whereas the expression of profilin II and espin both decreased significantly (47% and 42%, respectively). Taken together, these results suggest that, following hypophysectomy, the actin cytoskeleton of the Sertoli cell shifts to a predominantly depolymerized state, perhaps in part because of decreases in profilin II and espin gene products.     

Role of actin cytoskeleton in prostaglandin-induced protection against ethanol in an intestinal epithelial cell line

Prostaglandins (PGs) protect a variety of gastrointestinal cells against injury induced by ethanol and other noxious agents. This investigation attempted to discern the mechanism of cytoprotection as it relates to the relationship between actin and PGs in IEC-6 cells (a rat intestinal epithelial cell line). IEC-6 cells were incubated in Dulbecco's modified Eagle's medium +/- 16,16-dimethyl prostaglandin E(2) (dmPG, 2.6 microM) for 15 min and subsequently incubated in medium containing 1, 2.5, 5, 7.5, and 10% ethanol (EtOH). Cells were then processed for immunocytochemistry using FITC-phalloidin in order to stain the actin cytoskeleton, and cell viability was determined by trypan blue exclusion. Quantitative Western immunoblotting of fractioned G-actin (nonpolymerized; S1) and F-actin (polymerized; S2) was also carried out. EtOH concentrations equal to and greater than 5% led to the collapse of the actin cytoskeleton as depicted by extensive disorganization and fragmentation. In addition, these same EtOH concentrations significantly decreased the S2 fraction and increased the S1 pool of actin. Preincubation with dmPG prevented collapse of the actin cytoskeleton, significantly increased the S2 polymerized fraction as determined by quantitative immunoblotting, and increased cell viability in EtOH-treated cultures. Prior incubation with cytochalasin D, an actin disruptive agent, not only reduced cell viability but also prevented the cytoprotective effects of dmPG. Phalloidin, an actin stabilizing agent, had effects similar to that of dmPG as demonstrated by stability of the actin cytoskeleton and increased cellular viability. Such findings indicate that PGs are important in the organization and stability of actin under in vitro conditions. These effects on actin may play an essential role in the mechanism of PG-induced cytoprotection.     

与侵袭性相关胶质瘤细胞骨架构筑特点

目的 探讨与侵袭性相关胶质瘤细胞骨架构筑特点.方法 流式细胞术(FCM)检测细胞内F-肌动蛋白(F-actin)荧光强度;原子力显微术(AFM)表征整体骨架及骨架成分三维形貌,胶质瘤细胞微管直径、长度为(27.69±3.42)、(439.96±43.20)am,胶质细胞微管直径、长度为(24.70±2.25)、(136.76±13.78)nm.比较胶质瘤细胞与胶质细胞骨架形态结构差异,探讨与侵袭性相关胶质瘤细胞骨架构筑特点.结果 胶质瘤细胞整体骨架呈网架结构,边界不齐,呈"树根样";微丝密集;中间丝呈"鱼网状",无极性多点联系;微管较大较长,紧密结合成束,束间联系密切.胶质细胞整体骨架边界整齐;微丝稀疏;中间丝密集交织;微管联系疏松.胶质瘤细胞内F-actin荧光强度(202.54±11.06)明显强于胶质细胞内F-actin荧光强度(62.64±10.23),差异有统计学意义(P＜0.01).结论 整体骨架边界不齐,呈"树根样",微丝密集,中间丝高度网络结构,微管又大又长,紧密结合成束,束间联系密切,以及高F-actin含量均与胶质瘤侵袭性相关.     

肿瘤坏死因子-α对血管内皮细胞通透性影响的实验研究

目的：观察人肿瘤坏死因子-α（TNF—α）对人血管内皮细胞（EA.hy926）单层通透性的影响并初步探讨其作用机制。方法：测定异硫氰酸荧光素（FITC）标记的葡聚糖透过Transwell小室的荧光强度,以表示EA．hy926细胞单细胞层的通透性大小;激光共聚焦显微镜观察细胞骨架肌动蛋白（F—actin）和血管内皮钙黏蛋白（VE—cadherin）的形态分布;蛋白免疫印迹检测钙黏蛋白的表达。结果：与对照组比较,TNF-α使EA.hy926内皮细胞的通透性明显增加（P〈0．05）,诱导肌动蛋白重新分布及应力纤维形成,并使钙黏蛋白排列紊乱、断裂、细胞问裂隙形成增多。免疫印迹检测表明TNF-α减少钙黏蛋白表达呈剂量和时间依赖性。结论：TNF-α诱导内皮细胞通透性增高,其机制可能与其破坏内皮细胞屏障功能的完整性有关。     

Microfilament disruption occurs very early in ischemic proximal tubule cell injury.

Experimental ischemic acute renal failure results in disruption of proximal tubule apical membranes. Previous work utilizing immunofluorescence with an anti-actin antibody has demonstrated that the apical cytoskeleton of proximal tubule cells is disrupted during ischemic injury. In this study, using rhodamine-phalloidin which stains only filamentous actin, we demonstrate that graded durations of ischemia resulted in progressive disruption of proximal tubule apical microfilaments. Quantification using spectrofluorometry showed that 5, 15 and 50 minutes of ischemia resulted in 32.8 +/- 4%, 48.8 +/- 2.5%, and 58.4 +/- 2.6% decreases in apical F-actin relative to controls. Ischemia did not qualitatively affect either glomerular or distal tubule F-actin structure, though there were nonprogressive increases in glomerular fluorescence. In summary, rhodamine-phalloidin staining can be used to qualitatively and quantitatively assess proximal tubule microfilaments in vivo. We conclude that ischemia results in very early loss of proximal tubule apical microfilaments, with the majority of F-actin loss occurring within five minutes.     

Alterations in aortic endothelial cell morphology and cytoskeletal protein synthesis during cyclic tensional deformation

Previous studies have shown that bovine aortic endothelial cells (ECs) in culture respond to repetitive tensional deformation with an increase in deoxyribonucleic acid synthesis and cell proliferation. This study was designed to determine whether cyclic tensional deformation of ECs in vitro induces different morphologic or protein synthetic responses. ECs from passages 6 through 9 were seeded in 35 mm2 well silicone rubber plates at 2 x 10(5) cells/well and allowed to attach for 24 hours. The experimental group was placed in a vacuum-operated stress-providing device that exerted an elongation of 24% at maximum downward deflection of the culture plate bottom and was subjected to repetitive cycles of 10 seconds of 24% maximum elongation and 10 seconds of relaxation for 5 days. The control group was subjected to similar incubation conditions but without stretch. 35S-methionine (500 muCi/well) was added to the plates 24 hours before harvesting, and two-dimensional gels of the harvested lysates (isoelectric focusing with pH 3 to 10 ampholytes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% gels) were performed and the labeled proteins visualized by autoradiography. The data indicate that there is a differential synthesis of proteins, with synthesis of some proteins decreased or ablated whereas other proteins were increased in response to cyclic mechanical tension. The actin filament organization was evaluated after staining with rhodamine phalloidin, a fluorescent F-actin probe. The ECs subjected to tension had a more polygonal shape and demonstrated pseudopods and actin stress fibers, whereas ECs cultured under static conditions were more rounded and did not express actin stress cables.(ABSTRACT TRUNCATED AT 250 WORDS)     

Podocytes respond to mechanical stress in vitro

Glomerular capillary pressure is thought to affect the structure and function of glomerular cells. However, it is unknown whether podocytes are intrinsically sensitive to mechanical forces. In the present study, differentiated mouse podocytes were cultured on flexible silicone membranes. Biaxial cyclic stress (0.5 Hz and 5% linear strain) was applied to the membranes for up to 3 d. Mechanical stress reduced the size of podocyte cell bodies, and processes became thin and elongated. Podocytes did not align in the inhomogeneous force field. Whereas the network of microtubules and that of the intermediate filament vimentin exhibited no major changes, mechanical stress induced a reversible reorganization of the actin cytoskeleton: transversal stress fibers (SF) disappeared and radial SF that were connected to an actin-rich center (ARC) formed. Epithelial and fibroblast cell lines did not exhibit a comparable stress-induced reorganization of the F-actin. Confocal and electron microscopy revealed an ellipsoidal and dense filamentous structure of the ARC. Myosin II, alpha-actinin, and the podocyte-specific protein synaptopodin were present in radial SF, but, opposite to F-actin, they were not enriched in the ARC. The formation of the ARC and of radial SF in response to mechanical stress was inhibited by nonspecific blockade of Ca(2+) influx with Ni(2+) (1 mM), by Rho kinase inhibition with Y-27632 (10 microM), but not by inhibition of stretch-activated cation channels with Gd(3+) (50 microM). In summary, mechanical stress induces a unique reorganization of the actin cytoskeleton in podocytes, featuring radial SF and an ARC, which differ in protein composition. The F-actin reorganization in response to mechanical stress depends on Ca(2+) influx and Rho kinase. The present study provides the first direct evidence that podocytes are mechanosensitive.