miR-24对心脏成纤维细胞生长和迁移的影响及机制研究

目的:探讨miR-24对心脏成纤维细胞的生长和迁移的影响及机制。方法:采用RT-PCR检测心肌细胞和心脏成纤维细胞中mi R-24的表达水平。在心脏成纤维细胞中转染mi R-24 mimics、mimics control、mi R-24 inhibitors、inhibitors control后,通过Western blot检测细胞中Col-1、α-SMA的表达,MTT检测细胞增殖情况,流式细胞仪检测细胞凋亡情况,Transwell小室检测细胞迁移能力。通过靶基因预测库预测mi R-24的靶基因,荧光素酶鉴定靶基因的正确性,并通过RT-PCR和Western blot检测转染后细胞中Furin的表达。结果:心脏成纤维细胞HEH2中mi R-24的表达水平与心肌细胞H9C2相比差异显著(P0.01),心脏成纤维细胞中mi R-24表达上调。mi R-24 mimics组中Col-1、α-SMA的表达水平明显低于mimics control组(P0.01)。mi R-24 mimics组中细胞OD值较mimics control组显著降低(P0.01),mi R-24 inhibitors组中细胞OD值较inhibitors control组显著升高(P0.01)。mi R-24 mimics组、mimics control组、mi R-24 inhibitors组、inhibitors control组细胞凋亡无显著性差异(P0.05)。mi R-24 mimics组细胞迁移数目明显低于mimics control组,差异显著(P0.01),mi R-24 inhibitors组细胞迁移数目高于inhibitors control组,差异显著(P0.05)。mi R-24 mimics组细胞中Furin蛋白和m RNA水平明显降低,野生型Furin和mi R-24 mimics共转染的细胞中荧光素酶活性最低。结论:mi R-24在心脏成纤维细胞中高表达,通过靶基因Furin抑制心脏成纤维细胞合成Col-1、α-SMA,抑制心脏成纤维细胞增殖和迁移。     

RhoA-Rho激酶信号转导通路参与TGF-β1诱导的血管外膜成纤维细胞表型转化为肌成纤维细胞(英文)

血管外膜成纤维细胞表型转化为肌成纤维细胞是血管重塑的重要病理特征。本研究旨在探讨小分子G蛋白RhoA及其下游Rho激酶信号通路在转化生长因子β1(transforming growth factor β1,TGF-β1)诱导的血管外膜成纤维细胞/肌成纤维细胞表型转化中的作用。用10ng/mLTGF-β1诱导体外培养的大鼠胸主动脉外膜成纤维细胞表型转化为肌成纤维细胞,使用亲和沉淀法检测RhoA活性、使用免疫印迹检测RhoA、Rho激酶蛋白表达和Rho激酶活性;使用免疫印迹和免疫细胞化学检测肌成纤维细胞标记蛋白的表达。结果显示,TGF-β1上调体外培养的血管外膜成纤维细胞RhoA蛋白表达和RhoA活性。TGF-β1增加Rho激酶下游底物肌球蛋白磷酸酶目标亚单位的磷酸化,但不改变Rho激酶的蛋白表达,提示TGF-β1增加Rho激酶活性。腺病毒Ad-N19RhoA-hrGFP感染和Rho激酶特异性抑制剂Y27632都呈剂量依赖性地抑制TGF-β1诱导的肌成纤维细胞标记分子α平滑肌肌动蛋白和钙结合蛋白Calponin的蛋白表达。本研究证明RhoA-Rho激酶信号通路参与了TGF-β1诱导的血管外膜成纤维细胞/肌成纤维细胞表型转化。     

bFGF不能调节人结膜下Tenon's囊成纤维细胞表型改变

探讨了碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）能否对人结膜下Tenon’S囊成纤维细胞（human subconjunctival Tenon＇s capsule fibroblast,HTCF）表型改变起诱导作用。在5例白内障手术中取人结膜下Tenon＇s囊组织块培养成纤维细胞。用不同浓度bFGF（0、5、10、20ng／ml）诱导HTCF 48 h。免疫细胞化学技术、蛋白质印记免疫技术检测其平滑肌肌动蛋白（α-smoothmuscleactin,α-SMA）表达与正常HTCF有否差异。实验结果表明用不同浓度（0-20ng／ml）bFGF诱导HTCF48h,虽能明显促进HTCF细胞的生长,但均不能上调细胞内α-SMA的表达。在0-20ng／ml范围内,体外用bFGF诱导HTCF48h,不能诱导其改变为肌成纤维细胞。     

碱性成纤维细胞生长因子对培养的瘢痕成纤维细胞FN合成的调控作用

目的探讨碱性成纤维细胞生长因子（bFGF）对成纤维细胞纤维连结蛋白（FN）合成的调控作用。方法采用细胞培养、ELISA法、RT-PCR方法观察bFGF在不同剂量下对瘢痕来源的成纤维细胞FN合成的影响。结果FN的表达在低bFGF浓度组与对照组无明显差异,随着浓度的升高表现为增高趋势,以50、100、500ng／ml最显著,与对照组之间有显著性差异（P〈0．05）。FN mRNA表达在50-100ng／ml组明显升高,与对照组间有显著性差异（P〈0．05）。mRNA表达趋势与上清中蛋白的表达具有一致性。结论高浓度bFGF刺激FN合成可能是bFGF促进创面愈合的重要原因。     

骨桥蛋白增强自发性高血压大鼠血管外膜成纤维细胞的迁移活性

血管外膜成纤维细胞迁移参与形成新生内膜是一些血管疾病的共同发病过程。研究高血压动物模型的外膜成纤维细胞是否与对照组不同将有利于阐述高血压血管重塑的机制。本实验比较自发性高血压大鼠(spontaneously hy-pertensive rats,SHR)与正常对照大鼠(Wistar-Kyoto rats,WKY)的血管外膜成纤维细胞在体外培养条件下迁移能力的差别,并对其机制进行了探讨。采用大鼠胸主动脉的培养血管外膜成纤维细胞,用Transwell技术测定培养细胞的迁移能力。用实时定量PCR技术检测mRNA表达。结果表明,在血清和bFGF趋化作用下,SHR培养血管外膜成纤维细胞的迁移活性显著强于WKY(每个视野平均迁移细胞数目,血清:35.20±5.26 vs 22.2±3.27,P<0.05;bFGF:30.23±4.54vs 19.20±4.47,P<0.05)。进一步研究发现,SHR培养血管外膜成纤维细胞中的骨桥蛋白(osteopontin,OPN)mRNA水平显著高于WKY(1863.23±43.91 vs 326.24±68.29,P<0.01)。反义OPN(100 μmol/L)对血清诱导的SHR血管外膜成纤维细胞迁移有抑制作用(每个视野平均迁移细胞数目 38.60±5.98 vs 26.61±3.84,P<0.05)。而正义及错配义OPN组均无此效应。反义OPN对SHR细胞迁移的抑制作用呈浓度依赖性。上述结果证实SHR培养血管外膜成纤维细胞的迁移能力强于WKY,OPN在细胞迁移中     

神经生长因子对成纤维细胞生长的负调节作用

从小鼠颌下腺分离所得的神经生长因子(nervegrowth factor,NGF)是分子量为130000的多肽,其沉降系数为7S,由三种亚基α,β,γ组成,化学式为α_2βγ_2,其中β亚基可以完全表现NGF的生理效应.NGF的生物学效应比较广泛,它对神经系统的发育、分化有重要促进作用,在神经系统损伤后起修复、营养作用.另外,NGF对免疫系统、生殖系统也有一定作用.近年来,NGF与肿瘤关系的研究尤其     

改构型酸性成纤维细胞生长因子对细胞增殖活性的影响

目的:研究aFGF和MaFGF对正常的肾小管上皮细胞及胃癌细胞增殖的影响。方法:用不同浓度的aFGF和MaFGF分别作用于肾小管上皮细胞及胃癌细胞,48h后采用WST-8法测定aFGF和MaFGF对两种细胞的促增殖活性。结果:在各浓度下,MaFGF组对肾小管上皮细胞和胃癌细胞的促增殖作用都显著低于aFGF组。结论:MaFGF对肾小管上皮细胞及胃癌细胞的促分裂活性较aFGF明显下降。     

SIRT1对TGF-β1活化肾脏成纤维细胞的抑制机制

[目的]探讨沉默信息调节因子1(SIRT1)对转化生长因子β1(TGF-β1)活化肾脏成纤维细胞的抑制作用及机制。[方法]设立成纤维细胞NRK-49F组、TGF-β1刺激组(10 ngl/mL)、SIRT1蛋白低、中、高剂量组(10.0μg/mL、100.0μg/mL、1 000.0μg/mL),以上各组每孔设6个平行样,培养72 h。采用MTT法测定各组细胞活力,流式细胞术测定细胞凋亡水平,ELISA法测定细胞纤维化程度α-SMA、FN、Vimentin蛋白水平,RT-PCR法及蛋白印迹法测定Smad3、SOSC1 mRNA和蛋白水平。[结果] TGF-β1刺激组OD值、存活率、α-SMA、FN、Vimentin蛋白、Smad3、SOSC1 mRNA和蛋白表达水平高于成纤维细胞NRK-49F组,凋亡率低于成纤维细胞NRK-49F组(P<0.05)。SIRT1蛋白低、中、高剂量组OD值、存活率、α-SMA、FN、Vimentin蛋白、Smad3、SOSC1 mRNA、Smad3、SOSC1蛋白表达水平低于TGF-β1刺激组,凋亡率高于TGF-β1刺激组,随着SIRT1蛋白剂量逐渐...     

三磷酸腺苷对小鼠骨骼肌成纤维细胞NOR-10迁移作用及分子机制初步研究

目的:探讨三磷酸腺苷(ATP)对小鼠骨骼肌成纤维细胞的迁移作用及其可能机制。方法:细胞划痕实验检测1μM、10μM、100μMATP对NOR-10细胞愈合率的影响;细胞迁移小室实验检测空白对照组、100μM ATP组、30μM PPADS+100μM ATP组、100μM RB2+100μM ATP组细胞的迁移率。结果:细胞划痕实验及迁移实验表明高浓度ATP能够促进NOR-10细胞的迁移能力,100μM ATP促细胞迁移能力最强(P0.05),并且其促迁移作用能被30μM PPADS,100μM RB2所抑制(P0.05),但100μM RB2的抑制作用更强(P0.05)。结论:高浓度ATP(10μM)能够促进NOR-10细胞的迁移能力,并且其促迁移可能通过激活P2Y受体作用大于P2X受体。     

蛋白聚糖和溶血磷脂酸对不同细胞周期心脏成纤维细胞生长的影响

本文研究了蛋白聚糖（pG和）和溶血磷酸（LGA）对培养的处于不同细胞周期的SD乳鼠心脏成纤维细胞生长的影响及PG对LPA生物学活性的调节食用。采用流式细胞术测定细胞所处的周期;3H-TdR参人法测定细胞的DNA合成。研究结果表明（1）培养至次融汇状态的乳鼠心脏成纤维细胞经低血清（0.4%FBS）饥饿培养48小时,G0/G1期细胞占88.5%;G0/G1期细胞经2%FBS刺激24小时,G2期细胞占91。7%。（2）PGs对G0/G1期和G2期的心脏成纤维细胞的DNA合成均有摄制作用。2.94-47.04μg/ml PGs对G0/G1期细胞DNA合成的摄制率为80%-93%对G2期的抑制率为13%-94%.(3)1-80μmol/L范围内,LPA以浓度依赖方式促进不同细胞周期的心脏成纤维细胞DNA合成增加,50μmol/L LPA诱导G0/G1期和G2期细胞DNA合成的增加分别为78%±和122%±21%。（4）10μmol/L LPA存在下,2.94%-47.04μg/ml PGs使G0/G1期细胞和G2期细胞的DNA合成分别下降为对照的36%±11%-15%±10%和91%±13%-3%±1%,说明PGs可以抑制LPA诱导的心脏成纤维细胞DNA合成.上述研究结果提示PGs和LPA对乳鼠心脏成纤维细胞G1期至S期的转换有重要的调节作用,并可能通过调节心脏成纤维细胞的生长影响心肌肥厚的形成和发展.     

A model for signaling specificity of Wnt/Frizzled combinations through co-receptor recruitment

Wnts control mammalian developmental morphogenesis and are critical for adult stem cell maintenance. Wnts initiate several intracellular signaling cascades, such as Wnt/β-catenin-, Wnt/Ca²⁺- and Wnt/ROR2-signaling. Signaling preference of Wnts for these various pathways is thought to depend on the repertoire of receptors present on recipient cells. Here, we propose a further refinement of this receptor model and hypothesize that Wnt signaling specificity depends on co-receptor recruitment upon binding of Wnt to Frizzled receptor molecules. In this model, recruitment of LRP5/6 leads to activation of Wnt/β-catenin signaling, whereas signaling through other pathways is mediated by recruiting ROR2.     

Increased connexin 43 expression improves the migratory and proliferative ability of H9c2 cells by Wnt-3a overexpression

The change of connexin 43 (Cx43) expression and the biological behaviors of Cx43 in rat heart cell line H9c2, expressing Wnt-3a (wingless-type MMTV integration site family, member 3A) were evaluated in the present study. Plasmid pcDNA3.1/Wnt-3a was constructed and transferred into H9c2 cells. The cell model Wnt-3a~ -H9c2 steadily expressing Wnt-3a was obtained. Compared with H9c2 and pcDNA3.1-H9c2 cells, the expression of Cx43 in Wnt-3a~ -H9c2 cells was clearly increased, the proliferation of Wnt-3a~ -H9c2 cells was significantly changed, and cell migration abilities were also improved (P<0.05). In comparison with H9c2 and pcDNA3.1-H9c2 cells, the G_2 phase of the cell cycle increased by 11% in Wnt-3a~ -H9c2 cells. Thus, Wnt-3a overexpression is associated with an increase in Cx43 expression and altered migratory and proliferative activity in H9c2 cells. Cx43 might be one of the downstream target genes regulated by Wnt-3a.     

Secreted Wnt antagonist Dickkopf-1 controls kidney papilla development coordinated by Wnt-7b signalling

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.     

Estradiol regulates the insulin-like growth factor-I (IGF-I) signalling pathway: a crucial role of phosphatidylinositol 3-kinase (PI 3-kinase) in estrogens requirement for growth of MCF-7 human breast carcinoma cells

Estrogens can stimulate the proliferation of estrogen-responsive breast cancer cells by increasing their proliferative response to insulin-like growth factors. With a view to investigating the molecular mechanisms implicated, we studied the effect of estradiol on the expression of proteins implicated in the insulin-like growth factor signalling pathway. Estradiol dose- and time-dependently increased the expression of insulin receptor substrate-1 and the p85/p110 subunits of phosphatidylinositol 3-kinase but did not change those of ERK2 and Akt/PKB. ICI 182,780 did not inhibit estradiol-induced IRS-1 and p85 expression. Moreover, two distinct estradiol-BSA conjugate compounds were as effective as estradiol in inducing IRS-1 and p85/p110 expression indicating the possible implication of an estradiol membrane receptor. Comparative analysis of steroids-depleted and steroids-treated cells showed that IGF-I only stimulates cell growth in the latter condition. Nevertheless, expression of a constitutively active form of PI 3-kinase in steroid-depleted cells triggers proliferation. These results demonstrate that estradiol positively regulates essential proteins of the IGF signalling pathway and put in evidence that phosphatidylinositol 3-kinase plays a central role in the synergistic pro-proliferative action of estradiol and IGF-I.     

PKD1 inhibits cancer cells migration and invasion via Wnt signaling pathway in vitro

The approximately 14 kb mRNA of the polycystic kidney disease gene PKD1 encodes a large ( approximately 460 kDa) protein, termed polycystin-1 (PC-1), that is responsible for autosomal dominant polycystic kidney disease (ADPKD). The unique organization of its multiple adhesive domains (16 Ig-like domains/PKD domains) suggests that it may play an important role in cell-cell/cell-matrix interactions. Here we demonstrated that PKD1 promoted cell-cell and cell-matrix interactions in cancer cells, indicating that PC-1 is involved in the cell adhesion process. Furthermore in this study, we showed that PKD1 inhibited cancer cells migration and invasion. And we also showed that PC-1 regulated these processes in a process that may be at least partially through the Wnt pathway. Collectively, our data suggest that PKD1 may act as a novel member of the tumor suppressor family of genes.     

Enolase1 overexpression regulates the growth of gastric cancer cells and predicts poor survival

Gastric cancer has become the third most common cancer around the world. In patients with gastric cancer, the 5-year survival rate is still low. However, the mechanism underlying gastric cancer remains largely unknown. As a glycolytic enzyme, enolase 1 (ENO1) is widely expressed in most tissues. The functions of ENO1 have been reported in various types of cancer. Here in this study, we identified that ENO1 promoted the growth of gastric cancer cells through diverse mechanisms. Our immunohistochemical, bioinformatic and Western blot data showed that ENO1 was significantly overexpressed in human gastric cancer cell lines and tissues. The survival analysis revealed that ENO1 overexpression predicted poor survival in the patients suffering gastric cancer. Knockdown of ENO1 expression repressed the rate of proliferation and capacity of colony formation in two human gastric cancer cell lines (MGC-803 and MKN-45). In addition, knockdown of the expression of ENO1 led to the arrest of the cell cycle at the G1 phase and promoted the apoptosis of MKN-45 and MGC-803 cells. The further microarray and bioinformatic analysis revealed that ENO1 regulated the expression of diverse genes, many of which are involved in the progress of cancer. Taken together, our data demonstrated that ENO1 was an oncogene-like factor and might serve as a promising target for the treatment of human gastric cancer.     

Wnt5a regulates distinct signalling pathways by binding to Frizzled2

Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the β‐catenin‐independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin‐mediated route in response to Wnt5a, and inhibition of clathrin‐dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a‐dependent lipoprotein receptor‐related protein 6 (LRP6) phosphorylation and β‐catenin accumulation. Wnt3a‐dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a‐dependent accumulation of β‐catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the β‐catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin‐dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization‐dependent and ‐independent mechanisms.