上皮-间质转化的信号转导机制

上皮-间质转化(epithelial-mesenchymal transition,EMT)出现在各种生理和病理情况下,在胚胎发生与器官发育、肿瘤形成和转移以及纤维化过程中均有表现。TGF-β是EMT的一个重要诱导因素,其通过Smad和非Smad依赖通路对EMT的发生进行调节,对肿瘤的形成、转移和扩散起到了重要的作用。本文综述了近年来EMT信号转导机制的研究进展。     

转录因子Sp1促进肾小管上皮间质转化的体外实验研究

目的:研究转录因子Sp1促进肾小管上皮间质转化(EMT)以及肾脏纤维化的作用和机制.方法:用30 mL/L浓度的低氧培养肾小管上皮细胞HK-2,采用Real-time PCR及Western blot法分别检测细胞中转录因子Spl,E-cadherin和α-SMA的mRNA和蛋白表达水平.构建SP1基因的小干扰RNA载体,并将其转染低氧处理的HK-2肾小管上皮细胞.Real time PCR及Western blot法检测转录因子SP1对HK-2细胞EMT标志物的影响.结果:低氧处理后的HK-2细胞上皮标志物E-cadherin表达显著下降,而间皮标志物α-SMA的表达显著增加,提示HK-2细胞发生EMT.同时发现低氧处理后HK-2中转录因子Sp1的mRNA水平和蛋白水平表达逐渐增加.将Sp1的小干扰RNA载体转染HK-2细胞,得到下调Sp1表达的肾小管上皮细胞系.结果显示,与对照组相比,敲减Sp1的低氧处理的HK-2细胞中上皮细胞标志物E-cadherin 表达未见显著下降,而间质细胞标志物α-SMA的表达未见显著增加,提示此时的HK-2细胞未发生EMT.结论:转录因子Sp1可促进HK-2肾小管上皮细胞的EMT.     

转录因子在上皮间质转化中分子机制研究进展

上皮细胞-间充质转化(epithelia-mesenchymal transition,EMT),是指上皮细胞通过特定程序转化为具有间质表型细胞的生物学过程,目前研究证实这种表型的转化与转录因子密不可分.与其他转录因子相比,转录因子SNAIL、TWIST、ZEB为促进EMT过程的主要转录因子,本文总结国内外与EMT相关研究并对转录因子及其在EMT过程中的分子机制加以综述.     

EAF2表达与前列腺癌上皮-间质转化的关系及临床意义

目的探讨EAF2表达与前列腺癌上皮-间质转化(epithelial-mesenchymal transition, EMT)的关系及临床意义。方法采用免疫组化EnVision法检测82例前列腺癌及46例良性前列腺组织中EAF2和EMT标志蛋白的表达,分析蛋白表达与前列腺癌临床病理特征的相关性。结果 EAF2在前列腺癌组织中的阳性率(40.24%)明显低于良性前列腺组织(69.57%),差异有统计学意义(P0.05)。EAF2表达与前列腺癌临床分期、WHO/ISUP分组以及患者生存相关(P0.05)。EAF2与E-cadherin表达呈正相关(r=0.363,P=0.001),与vimentin(r=-0.255,P=0.021)和N-cadherin(r=-0.271,P=0.014)表达呈负相关。结论 EAF2表达与前列腺癌的EMT表型、患者预后及生存具有相关性。     

上皮-间质转化与肿瘤的浸润和转移

上皮-间质转化(epithelial-mesenchymal transitions,EMT)是指上皮细胞失去极性,经过细胞骨架重塑,转变成具有迁移能力的间充质表型的过程,是多细胞生物胚胎发生发育过程中的基础。细胞因子TGF-β、转录因子Snail、基质金属蛋白酶MMPs、病毒癌基因可以诱导EMT。肿瘤细胞EMT与肿瘤细胞的浸润、转移、信号转导有关,研究肿瘤细胞EMT将为寻找抗肿瘤治疗靶点奠定基础。     

上皮-间质转化与乳腺癌干细胞

乳腺癌干细胞是导致乳腺癌复发、转移和耐药的根源之一。上皮间质转化(epithelial-mesenchymal transition,EMT)不仅赋予乳腺癌细胞迁移和侵袭特征,还可使癌细胞获得自我更新能力而具有干细胞的特性,从而促进乳腺癌干细胞的产生。EMT可以作为乳腺癌干细胞研究的一个新的切入点。本文就近年来该方面的研究进展做简要综述。     

上皮细胞间质转型与肿瘤转移

皮细胞间质转型 (epithelial-mesenchymal transitions,EMT)是具有极性的上皮细胞转换成为具有活动能力的间质细胞并获得侵袭和迁移能力的过程,它存在于人体多个生理和病理过程中。EMT的发生涉及到多个信号转导通路和复杂的分子机制,与钙连接素、生长因子、转录因子、微环境等有关。EMT与肿瘤细胞的侵袭和转移关系密切。     

Twist基因逆转录病毒载体的构建及其诱导正常乳腺上皮细胞发生上皮间质转化

目的 构建Twist基因逆转录病毒载体,研究其对人正常乳腺上皮细胞MCF10A的影响.方法 酶切pcDNA3/myc-Twist获得myc-Twist,克隆到逆转录病毒载体pBABE-puro中,构建重组质粒pB AB E-myc-Twist.通过酶切、测序鉴定Twist基因正确后,在体外将质粒pBABE-myc-Twist和其对照分别与包装质粒pAmpho共同转染人胚肾上皮细胞系293T细胞,进行逆转录病毒的包装,并感染人正常乳腺MCF10A上皮细胞,用嘌呤霉素(puromycin)筛选,获得成功转入Twist的MCF10A-Twist细胞和MCF10A-Vector对照细胞.通过RT-PCR和Western blot法验证Twist细胞在MCF10A-Twist和MCF10A-Vector细胞内的表达.免疫荧光技术和Western blot法检测细胞内上皮间质转化(EMT)标志蛋白的表达.Transwell(R)法检测细胞的迁移和侵袭能力.结果 重组逆转录病毒载体质粒pBABE-myc-Twist经酶切和测序鉴定构建正确;Twist基因被逆转录病毒成功导入MCF10A细胞,并在靶细胞内稳定表达,RT-PCR检测到Twist mRNA在MCF10A-Twist细胞中表达,Western blot法检测发现myc-Twist蛋白在MCF10A-Twist细胞中表达;免疫荧光和Western blot法检测显示在MCF10A-Twist细胞中E-cadherin表达显著下调、vimentin表达明显上调;与MCF10A-Vector细胞相比,Transwell(R)法检测发现MCF10A-Twist细胞迁移和侵袭能力明显增加(P＜0.01).结论 Twist诱导MCF10A细胞发生EMT转化,并促进其迁移和侵袭.     

上皮间质转化机制及其与肿瘤浸润转移关系的进展

上皮间质转化(epithelial-mesenchymal transitions,EMT)是指在胚胎发育过程中,上皮细胞通过一系列改变,转分化成间叶表型的细胞.有证据显示,EMT由宿主微环境诱导,经多条信号转导途径,受控于细胞内不同的转录因子,在多细胞生物体胚胎发育过程中促进细胞运动和新的组织类型生成,是一种进化上高度保守的重要机制;     

颗粒蛋白前体(PGRN)促进小鼠乳腺癌4T1细胞上皮间质转化与侵袭和迁移

目的 探讨颗粒蛋白前体(PGRN)对小鼠乳腺癌4T1细胞侵袭和迁移的影响及其机制.方法 采用1 μg/mL PGRN处理乳腺癌4T1细胞24 h,通过TranswellTM侵袭实验检测4T1细胞的侵袭能力、划痕实验检测细胞的迁移能力,实时荧光定量PCR检测4T1细胞上皮钙黏蛋白(E-cadherin)、波形蛋白(vim...     

上调RI对小鼠黑色素瘤B16-F10细胞EMT及转移的影响

目的研究核糖核酸酶抑制因子基因表达对小鼠黑色素瘤B16-F10细胞EMT及转移的影响。方法构建RI真核表达质粒p IRES2-EGFP-RI,稳定转染B16-F10细胞。RT-PCR、Western blot和免疫荧光检测RI的表达;HE染色及相差显微镜观察细胞形态;FITC标记的鬼笔环肽染色,激光共聚焦观察细胞骨架;黏附实验、划痕实验和Transwell法检测细胞黏附、迁移和侵袭能力的变化;Western blot检测EMT及转移相关蛋白的表达;分别将各组B16-F10细胞眼眶静脉注射到c57/BL小鼠,建立肺转移动物模型,注射3周后处死小鼠。取肺称重,在解剖镜下计数肺转移结节数;肺组织切片HE染色观察肿瘤细胞转移;免疫组化检测肺转移瘤组织中转移及EMT相关蛋白的表达。结果 RI表达上调后,细胞由间质型向上皮型转换,细胞骨架重排;B16-F10-RI细胞组黏附、迁移和侵袭能力下降;与对照组相比,B16-F10-RI细胞中MMP2、MMP9、snail、slug、vimentin、twist和N-cadherin的表达明显降低,而E-cadherin,nm23-H1蛋白的表达明显增加(P0.01或P0.01)。实验组与对照组比小鼠肺的转移结节明显减少,同时EMT及转移相关蛋白在瘤组织中表达与体外细胞一致。结论上调核糖核酸酶抑制因子能够显著抑制B16-F10细胞EMT及侵袭、转移,RI可望作为治疗黑色素瘤的靶蛋白。     

Calcium-sensing receptor in the T lymphocyte enhanced the apoptosis and cytokine secretion in sepsis

Calcium-sensing receptor (CaSR) is a member of the G protein-coupled receptor superfamily that existed in lymphocytes and promoted cytokine secretion. Lymphocytes are also involved in sepsis. However, the role of CaSR in lymphocytes in sepsis is unclear. In this study, we want to examine whether the CaSR in lymphocytes in sepsis is involved in the cytokine secretions and apoptosis and make clear the relationship between NF-κB and MAPK signal transduction pathways. We investigated the issues mentioned earlier using Western blotting, ELISA, and Flow Cytometry. The sepsis was remodeled by cecal ligation and puncture (CLP). We found that CaSR protein expression increased in the peripheral blood T lymphocytes in CLP rats. The calcimimetic R568 (NPS R568) promoted, whereas the calcilytic NPS 2143 attenuated, signaling pathways proteins P65 (subunit of NF-κB), ERK1/2, and JNK (one subgroup of MAPKs) phosphorylation. However, P-P38 and P-JAKs exhibit no significant changes. Furthermore, the production TNF-α and IL-4 was greater in CLP rats than in normal rats, and NPS R568 promoted secretion of these cytokines. Simultaneously, the apoptotic ratio of T cells in CLP increased, and NPS R 568 exacerbated the apoptosis degree. However, these effects could also be inhibited by U0126 or SP600125 (MAPKs pathway inhibitor) or Bay-11-7082 or (NF-κB pathway inhibitor). From these results, we can conclude that, in the sepsis, CaSR activation promoted T-cell apoptosis and the secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokines IL-4 probably through NF-κB and partial MAPK signal transduction pathways.     

特异性ILK siRNA对膀胱癌EJ细胞EMT及移植瘤生长的影响

目的研究整合素连接激酶(ILK)特异siRNA沉默ILK基因对膀胱癌EJ细胞上皮间质转化(EMT)及移植瘤生长的影响。方法用特异性ILK siRNA表达载体p Gensil-1siRNA1质粒和对照质粒p Gensil-1siRNA3稳定转染EJ细胞,实验分为EJ siRNA组、EJ vector组和EJ细胞组;激光共聚焦显微镜下观察细胞骨架;Western blot检测p-AKT、p-GSK-3β蛋白的表达;细胞免疫荧光检测p-AKT、p-GSK-3β的表达;用免疫荧光检测整合素连接激酶和核糖核酸酶抑制因子在EJ细胞中的共定位;MTT法检测细胞增殖;流式细胞术检测细胞周期;建立裸鼠移植瘤模型,肿瘤和肺组织切片HE染色,免疫组化检测瘤组织中ILK、RI、NM23-H1和E-cadherin蛋白的表达;免疫荧光检测p-AKT、p-GSK-3β和CD31蛋白的表达。结果 EJ siRNA组细胞运动能力减弱;EJ siRNA组p-AKT、p-GSK-3β蛋白表达明显降低(P0.05);MTT法结果提示实验组细胞增殖明显受到抑制;下调ILK通过阻滞G0/G1期抑制细胞增殖;下调ILK抑制膀胱癌EJ细胞移植瘤生长转移。结论特异性ILK siRNA可以改变EJ细胞特性,抑制EMT的发生,引起增殖侵袭能力下降。     

Chloride-related current fluctuation in amphibian skin

Fluctuation in transepithelial current was analysed in skins of Rana esculenta and Bufo viridis. Perturbation of the tissues to serosa + 40 mV or + 80 mV activated a pathway for conductive Cl transport and led to the appearance of a Lorentzian component in the power density spectrum. The presence of Cl on the mucosal side was mandatory for the observation of Lorentzians. Corner frequencies, f _c, ranged between 48 Hz and 102 Hz. The mean value was slightly larger at 40 mV than at 80 mV (88.3±4.4 versus 72.5±6.4 Hz). Plateau values, S _o, were proportional to the magnitude of the activated Cl current. Single-channel currents of 48.3 ±5.2 fA and 93.0±8.7 fA were calculated at 40 mV and 80 mV, respectively, with the assumption of equal open and closed probabilities of the channels. From these data, a voltage-independent open-channel conductance of about 1.2 pS is obtained. Activation of Cl conductance was associated with an increase in channel density. Stimulation of Cl conductance by procaine did not affect S _o and f _c despite considerable stimulation of transepithelial Cl current. This could indicate that non-gated channels participate in the voltage-sensitive Cl conductance. Mucosal application of inhibitors of Cl conductance (3,5-dichlorodiphenylaminocarboxy acid, MK-196) decreased S _o in a concentration-dependent manner, but had no effect on f _c. The data could support the hypothesis that the voltage-sensitive transepithelial Cl transport is localized to a cellular compartment, which is most likely the mitochondria-rich cells. The possibility cannot be excluded, on the other hand, that the fluctuation in current originates from highly organized, specific sites in the junctional complexes.     

miR-451 inhibits invasion and proliferation of bladder cancer by regulating EMT

MicroRNAs (miRNAs) have recently been reported play a crucial role in some tumors. In order to investigate the association of miR-451 with bladder cancer, we investigate the expression of miR-451 in bladder cancer tissues and its role in biological behavior of T24, 5637 and J28 bladder cancer cell lines. Quantitative RT-PCR results showed miR-451 was significantly down-regulated in bladder cancer tissues and paracancerous tissues compared with normal bladder tissues. miR-451 expression was significantly associated with histological differentiation degree and TNM stage. Over-expression of miR-451 was established by transfecting miR-451 mimics into T24, 5637 and J28 cells, and its effects on the biological behavior of bladder cancer were studied using transwell assay, migration assay, adhesion assay, MTT and flow cytometry. Results indicated over-expression of miR-451 significantly inhibited cell proliferation, migration, invasion and induced apoptosis of the bladder cancer cells. Furthermore, we investigated the expression level of EMT related proteins in transfected 5637 cells by western blot. Results shown E-cadherin was up-regulated more significantly than N-cadherin, vimentin and Snail. N-cadherin and vimentin were up-regulated significantly when miR-451 was inhibited in miR-451 inhibitor group, however, no significant changes in mimics group. In conclusion, miR451 should be a tumor-suppressing gene in bladder cancer. miR-451 could maintain the bladder tumor cells in epithelial phenotype, inhibit EMT process, thereby reducing the invasion and migration of tumor cells.     

MicroRNA-21 regulates biological behavior by inducing EMT in human cholangiocarcinoma

MicroRNAs (miRNAs) have recently been demonstrated to play a crucial role in malignant progression including differentiation, proliferation, metastasis and invasion, MicroRNA-21 (mir-21) also has been reported to have association with tumor invasion and metastasis in some tumors including cholangiocarcinoma (CCA). In this study, we further investigated the association of mir-21 with CCA biological behavior by transfecting miR-21 mimics or mir-21 inhibitor into QBC939 and RBE cells accompanied with the tumor xenografts experiment. Results indicated that over-expression of miR-21 significantly promoted cell migration, invasion and xenografts growth, whereas contrary phenomenon was observed in mir-21 inhibitor group. Furthermore, we explored the expression of EMT related proteins in CCA cells and tumor xenografts. Results showed that E-cadherin was decreased and N-cadherin, Vimentin were up-regulated significantly when miR-21 was over-expressed. In conclusion, microRNA-21 is crucial for CCA carcinogenesis and metastasis, which could induce EMT process, thereby promote the invasion and migration of CCA cells. These findings may provide new strategy for prevention and treatment of CCA in the future.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

目的 探讨结缔组织生长因子(CTGF)对肾小管上皮细胞向间充质细胞转化的影响.方法 体外培养的人肾小管上皮细胞(HK-2),按是否用CTGF处理将其分为实验组和对照组.直接免疫荧光法、间接免疫化学检测培养72 h后肾小管上皮细胞E-cadherin、波形蛋白、α-平滑肌肌动蛋白((α-SMA)、细胞外信号调节激酶2(ERK2)的表达;Western blot方法 在72 h检测E-cadherin、波形蛋白和ERK2的表达;Boyden小室在1、3、5 d检测细胞迁移能力的变化.结果 (1)实验组上皮细胞表型标志蛋白E-cadherin随着时间的延长而表达递减,波形蛋白表达渐增,ERK2表达上调;(2)在第1天,实验组与对照组细胞迁移能力差异无统计学意义,第3天实验组迁移至滤膜下面细胞多于对照组,第5天实验组细胞迁移数明显高于对照组[(45.0±1.1):(14.0±1.2),P<0.05].结论 在体外,人肾小管上皮细胞由CTGF刺激表现出向间充质细胞转化的特性,并且ERK2信号转导通路可能参与了对这一过程的调控效应.     

SNAIL诱导MCF-7细胞上皮间质转化致阿霉素耐药性增加

 目的 观察SNAIL诱导后,MCF-7细胞上皮间质转化（EMT）的发生和P-糖蛋白(P-GP)的表达,探讨EMT致乳腺癌细胞耐药性增强的作用。方法 构建SNAIL真核表达载体,转染MCF-7细胞,用免疫荧光检测上皮标记物E-cadherin,间质标志物Vimentin的表达;MTT法评价细胞对阿霉素(ADM)的耐药性;流式细胞术检测细胞中P-GP和SNAIL的表达;Real-Time-PCR检测SNAIL、E-cadherin、Vimentin、MDR1 mRNA的表达。结果 MCF-7细胞转染SNAIL后E-cadherin表达较亲本细胞显著降低,Vimentin表达显著升高(P＜0.01),对ADM的耐药性及SNAIL、P-GP的表达也显著升高(P＜0.01),与Real-Time-PCR结果一致。结论 SNAIL诱导MCF-7细胞EMT后,增强了乳腺癌细胞中P-GP介导的对ADM的耐药。     

神经营养因子信号转导与神经细胞凋亡的分子机制

细胞凋亡(Apoptosis)是一种程序化的细胞死亡过程,其中神经细胞的凋亡对胚胎期神经系统的生长、发育、分化、成熟及生后各种原因所致的脑损伤,神经退行性疾病的发生都起着至关重要的作用。神经营养因子(Neurotrophins,NTS)通过其与相应靶细胞膜受体的活化而引发相关信使分子的级联激活而减缓神经细胞凋亡、促进神经细胞的存活,发挥其生物学功能。对NTS家族信号转导与神经细胞凋亡的分子机制的认识有助于早期干预和治疗各种原因所致神经发育残障,脑损伤及神经退性行疾病。