5-Fluorouracil dose escalation generated desensitized colorectal cancer cells with reduced expression of protein methyltransferases and no epithelial-to-mesenchymal transition potential

Background:Colorectal cancer(CRC)is one of the most frequently diagnosed cancers.In many cases,the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil(5-FU).The epithelial-to-mesenchymal transition(EMT)and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers.This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC.Materials and Methods:HCT-116,Caco-2,and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU.The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays.This was followed by aWestern blot which analyzed the protein expressions of the epithelial marker E-cadherin,mesenchymal marker vimentin,and the EMT transcription factor(EMTTF),the snail family transcriptional repressor 1(Snail)in the parental and desensitized cells.Western blotting was also conducted to study the protein expressions of the protein methyltransferases(PMTs),Euchromatic histone lysine methyltransferase 2(EHMT2/G9A),protein arginine methyltransferase(PRMT5),and SET domain containing 7/9(SETD7/9)along with the global lysine and arginine methylation profiles.Results:The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU.The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells.This was reflected in the observed reduction in E-cadherin,vimentin,and Snail in the desensitized cell lines.Additionally,the protein expressions of EHMT2/G9A,PRMT5,and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment.Conclusion:This study showed that continuous,dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.

Unleashing the pathological role of epithelial-to-mesenchymal transition in diabetic nephropathy: The intricate connection with multifaceted mechanism

Renal epithelial-to-mesenchymal transition(EMT)is a process in which epithelial cells undergo biochemical changes and transform into mesenchymal-like cells,resulting in renal abnormalities,including fibrosis.EMT can cause diabetic neph-ropathy through triggering kidney fibrosis,inflammation,and functional impair-ment.The diverse molecular pathways that drive EMT-mediated renal fibrosis are not utterly known.Targeting key signaling pathways involved in EMT may help ameliorate diabetic nephropathy and improve renal function.In such settings,un-derstanding precisely the complicated signaling networks is critical for develo-ping customized therapies to intervene in EMT-mediated diabetic nephropathy.

Role of Insulin-like Growth Factor II Receptor in Transdifferentiation of Free Silica-induced Primary Rat Lung Fibroblasts

Objective To study the role of insulin-like growth factor II receptor in free silica-induced transdifferentiation of primary rat lung fibroblasts Methods Rat lung fibroblasts and rat alveolar macrophages were cultured. A transdifferentiation model of primary rat lung fibroblasts was induced by free silica. Levels of a-SMA protein, IGF-liR protein and mRNA were measured by immunocytochemistry, Western blot and RT-PCR, respectively. Lung fibroblasts were treated with Wortmannin. Results The expression levels of a-SMA concentration and decreased after Wortmann and IGF-IIR increased with the increasing free silica n was used. Conclusion The IGF-IIR plays an important role in free silica-induced transdifferentiation of primary rat lung fibroblasts.

In vitro transdifferentiation of corneal epithelial-like cells from human skin-derived precursor cells

The damage of human corneal cells encounter with the problem of availability of corneal cells for replacement. Limitation of the source of corneal cells has been realized. An attempt of development of corneal epithelial-like cells from the human skin-derived precursor (hSKPs) has been made in this study. Combination of three essential growth factors: epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) could demonstrate successfully induction of hSKPs to differentiation into corneal cells.The induced cells expressed the appearance of markers of corneal epithelial cells as shown by the presence of keratin 3 (K3) by antibody label and Western blot assay. The K3 gene expression of induced hSKPs cells as shown by reverse transcription-polymerase chain reaction (RT-PCR) technology was also demonstrated. The presence of these markers at both gene and protein levels could lead to our conclusion that the directional transdifferentiation of hSKPs cells into corneal epithelial cells was successfully done under this cell induction protocol. The finding shows a newly available stem cell source can be obtained from easily available skin. Cells from autologous human skin might be used for corneal disorder treatment in future clinical application.

Epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions in the colon

Epithelial-to-mesenchymal and mesenchymal-to-epi- thelial transitions are well established biological events which have an important role in not just normal tissue and organ development, but in the pathogenesis of diseases. Increasing evidence has established their presence in the human colon during colorectal carcinogenesis and cancer invasion, chronic inflammation-related fibrosis and in the course of mucosal healing. A large body of evidence supports the role for transforming growth factor-13 and its downstream Smad signaling, the phosphatidylinositol 3＇-kinase/Akt/mTOR axis, the Ras-mitogen-activated protein kinase/Snail/Slug and FOXC2 pathway, and Hedgehog signaling and microR- NAs in the development of colorectal cancers via epi- thelial-to-mesenchymal transition. C-met and Frizzled-7, among others, seem to be the principle effectors of mesenchymal-to-epithelial transition, hence have a role not just in mucosal regeneration but in the progression of colonic wall fibrosis. Here we discuss a role for these pathways in the initiation and development of the transition events. A better understanding of their induction and regulation may lead to the identification of pathways and factors that could be potent therapeu- tic targets. The inhibition of epithelial-to-mesenchymal transition using mTOR kinase inhibitors targeting theATP binding pocket and which inhibit both mTORC1 and mTORC2, RNA aptamers or peptide mimetics, such as a Wnt5A-mimetic, may all be useful in both cancer treatment and delaying fibrosis, while the induction of mesenchymal-to-epithelial transition in induced pluripotent stem cells may enhance epithelial healing in the case of severe mucosal damage. The preliminary results of the current studies are promising, but more clinical investigations are needed to develop new and safe therapeutic strategies for diseases of the colon.

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity

AIM To establish a model to enrich and characterize stemlike cells from murine normal liver and hepatocellular carcinoma(HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition(EMT).METHODS In this study,we utilized a stem cell conditioned serumfree medium to enrich stem-like cells from mouse HCC and normal liver cell lines,Hepa 1-6 and AML12,respectively.We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating theRNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR(q RTPCR).Next,we examined the relationship between stem cells and EMT using q RT-PCR.RESULTS Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor,basic fibroblast growth factor and heparin sulfate.The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell(CSC) marker Cd44 compared to parental cells grown as adherent cultures.We report that epithelial markers E-cadherin and ZO-1 were downregulated,while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres.The 3-dimensional spheres also exhibited changes in expression of Snai,Zeb and Twist family of EMT transcription factors.CONCLUSION Our novel method successfully enriched stem-like cells which possessed an EMT phenotype.The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

The Effect of Connective Tissue Growth Factor on Human Renal Tubular Epithelial Cell Transdifferentiation

To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5.0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9 %, 65.5 % vs 2.4 %, P<0.01) .α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).

Role of epithelial-to-mesenchymal transition in the pulmonary fi brosis induced by paraquat in rats

BACKGROUND:This study aims to explore the characteristics of the epithelial-to-mesenchymal transition(EMT)process and its underlying molecular mechanisms in the period of paraquat(PQ)-induced pulmonary fi brosis(PF).METHODS:Picrosirius red staining and collagen volume fraction were utilized to evaluate the pathological changes of PQ-induced PF in rats.Immunohistochemistry,Western blot,and real-time reverse transcriptase-polymerase chain reaction(RT-PCR)were used to measure the protein and gene expression of EMT markers,EMT-associated transcription factors,and regulators of EMT-related pathways,respectively.RESULTS:The collagen deposition in the alveolar septum and increased PF markers were characteristics of pathological changes in PQ-induced PF,reached a peak on day 14 after PQ poisoning,and then decreased on day 21.The protein and gene expression of the fibrosis marker,EMT markers,transcription factors,and regulators of EMT-related signaling pathways signifi cantly increased at diff erent time points after PQ poisoning compared with corresponding controls(P<0.05),and most of them reached a peak on day 14,followed by a decrease on day 21.The gene expression of EMT markers was significantly correlated with PF markers,transcription factors,and regulators of EMT-related signaling pathways(P<0.05).The mRNA expression of transcription factors was signifi cantly correlated with that of TGF-β1 and Smad2(P<0.05 or P<0.01),instead of Wnt2 andβ-catenin(P>0.05).CONCLUSIONS:EMT process plays a role in the PQ-induced PF,in which most PF and EMT markers have a peak phenomenon,and its underlying molecular mechanisms might be determined by further studies.

Transdifferentiation of pancreatic α-cells into insulinsecreting cells: From experimental models to underlying mechanisms

Pancreatic insulin-secreting β-cells are essential regulators of glucose metabolism. New strategies are cur-rently being investigated to create insulin-producing β cells to replace deficient β cells, including the differentiation of either stem or progenitor cells, and the newly uncovered transdifferentiation of mature non-β islet cell types. However, in order to correctly drive any cell to adopt a new β-cell fate, a better understanding of the in vivo mechanisms involved in the plasticity and biology of islet cells is urgently required. Here, we review the recent studies reporting the phenomenon of transdifferentiation of α cells into β cells by focusing on the major candidates and contexts revealed to be involved in adult β-cell regeneration through this process. The possible underlying mechanisms of transdifferentiation and the interactions between several key factors involved in the process are also addressed. We propose that it is of importance to further study the molecular and cellular mechanisms underlying α- to β-cell transdifferentiation, in order to make β-cell regeneration from α cells a relevant and realizable strategy for developing cell-replacement therapy.

Combination of cell signaling molecules can facilitate MYOD1-mediated myogenic transdifferentiation of pig fibroblasts

Background:Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression,which is facilitated by various signaling pathways associated with myogenesis.In this study,we attempted to transdifferentiate pig embryonic fibroblasts(PEFs)myogenically into skeletal muscle through overexpression of the pig MYOD1 gene and modulation of the FGF,TGF-β,WNT,and cAMP signaling pathways.Results:The MYOD1 overexpression vector was constructed based on comparative sequence analysis,demonstrating that pig MYOD1 has evolutionarily conserved domains across various species.Although forced MYOD1 expression through these vectors triggered the expression of endogenous muscle markers,transdifferentiated muscle cells from fibroblasts were not observed.Therefore,various signaling molecules,including FGF2,SB431542,CHIR99021,and forskolin,along with MYOD1 overexpression were applied to enhance the myogenic reprogramming.The modified conditions led to the derivation of myotubes and activation of muscle markers in PEFs,as determined by qPCR and immunostaining.Notably,a sarcomere-like structure was observed,indicating that terminally differentiated skeletal muscle could be obtained from transdifferentiated cells.Conclusions:In summary,we established a protocol for reprogramming MYOD1-overexpressing PEFs into the mature skeletal muscle using signaling molecules.Our myogenic reprogramming can be used as a cell source for muscle disease models in regenerative medicine and the production of cultured meat in cellular agriculture.

Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells

BACKGROUND Senescence is characterized by a decline in hepatocyte function,with impairment of metabolism and regenerative capacity.Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism,liver diseases,and organ regeneration.The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors.However,the impact of senescence on HepaRG cells is not yet known.AIM To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells.METHODS We compared the transdifferentiation capacity of cells over 10(passage 10[P10])vs P20.Aging was evaluated by senescence-associated(SA)beta-galactosidase activity and the comet assay.HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry(expression of cluster of differentiation 49a[CD49a],CD49f,CD184,epithelial cell adhesion molecule[EpCAM],and cytokeratin 19[CK19]),quantitative PCR analysis(expression of albumin,cytochrome P4503A4[CYP3A4],γ-glutamyl transpeptidase[γ-GT],and carcinoembryonic antigen[CEA]),and functional analyses(albumin secretion,CYP3A4,andγ-GT).Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide(NAD^(+))/NAD with hydrogen(NADH)content were also measured.RESULTS SAβ-galactosidase staining was higher in P20 than P10 HepaRG cells;in parallel,the comet assay showed consistent DNA damage in P20 HepaRG cells.With respect to P10,P20 HepaRG cells exhibited a reduction of CD49a,CD49f,CD184,EpCAM,and CK19 after the induction of transdifferentiation.Furthermore,lower gene expression of albumin,CYP3A4,andγ-GT,as well as reduced albumin secretion capacity,CYP3A4,andγ-GT activity were reported in transdifferentiated P20 compared to P10 cells.By contrast,the gene expression level of CEA was not reduced by transdifferentiation in P20 cells.Of note,both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells.Finally,both ATP and NAD^(+)/NADH were depleted in P20 cells with respect to P10 cells.CONCLUSION SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells,with consequent impairment of metabolic and regenerative properties,which may alter applications in basic studies.

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells.METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, &#x003b2;-catenin, N-cadherin, collagen type&#x02005;I&#x02005;(COL-I), vimentin, &#x003b1;-smooth muscle actin (&#x003b1;SMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids.RESULTS: The E-cadherin/&#x003b2;-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and &#x003b1;SMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/&#x003b2;-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression.CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids.

支气管肺发育不良发生机制中肺泡上皮细胞转分化关键信号通路的研究进展

支气管肺发育不良(bronchopulmonary dysplasia,BPD)是早产儿严重呼吸系统并发症,重症病例仍缺乏有效治疗手段。BPD是多因素疾病,发病机制主要包括肺泡简单化和肺微血管发育障碍。肺泡上皮细胞是肺泡的主要构成部分,包括肺泡Ⅰ型(alveolar type 1,AT1)和肺泡Ⅱ型(alveolar type 2,AT2)细胞,其中AT1细胞参与气血屏障构建,发挥气体交换作用,AT2细胞具有增殖分化的干细胞特性,维持肺内环境稳态、修复肺损伤。肺损伤修复的核心是AT2细胞向AT1细胞的转分化,而激活转分化的信号转导机制尚未明确。本文通过文献检索和分类总结,探讨肺泡上皮细胞转分化的关键信号转导通路及研究进展,为阐述BPD发病机制及探索BPD新的治疗方案提供参考。

Protein Expression in Silica Dust-induced Transdifferentiated Rats Lung Fibroblasts

Objective To analyze the expression of different proteins in free silica-induced transdifferentiated rat lung fibrob^asts. Methods Rat lung fibroblasts and alveolar macrophages were cultured. A transdifferentiation model of rat lung fibroblasts was established. Free silica was used as a stimulator for rat lung fibroblasts. Changes in a-SMA were detected by immunohistochemistry and Western blot, respectively. Protein of lung fibroblasts was extracted and analyzed by two-dimensional electrophoresis （2-DE）. Results Six protein spots were identified by mass spectrometry, including glyceraldehyde 3-phosphate-dehydrogenase, peroxiredoxin 5, heterogeneous nuclear ribonucleoprotein A2, transgelin 2, keratin K6 and vimentin. Conclusion Some proteins are changed in free silica-induced transdifferentiaed rat lung fibroblasts

尿酸盐在慢性肾病并发肾间质纤维化中的作用及机制

目的探讨尿酸盐在慢性肾病并发肾间质纤维化过程中的作用及机制。方法利用0.2%腺嘌呤的饲料喂养小鼠9周,构建慢性肾病小鼠模型。造模结束后,小鼠眼眶后静脉丛采血,检测肾功能和血尿酸含量;HE染色和PAS染色分析肾脏组织病理变化;Masson染色观察肾脏纤维化程度;尿酸盐染色观察肾组织中尿酸盐沉积;Western blot和免疫组化检测目标分子的表达变化。利用不同浓度尿酸刺激原代培养小鼠肾小管上皮细胞(mRTECs)及人肾小管上皮细胞系(HKC);划痕实验观察尿酸对细胞迁移的影响。结果动物水平上,生化分析检测结果显示,模型组小鼠血清尿素氮(P=0.0064)、肌酐(P=0.0080)、血尿酸(P=0.0007)水平较对照组明显增加;HE和PAS染色结果显示,模型组小鼠出现严重肾小管损伤及炎症细胞浸润;Masson染色结果显示,模型组胶原沉积明显增加;尿酸盐染色结果显示,与对照组比较,模型组小鼠肾组织出现大量尿酸盐结晶;Western blot和免疫组化结果显示,模型组小鼠波形蛋白、平滑肌肌动蛋白和转化生长因子β1(TGF-β1)水平较对照组升高,E钙黏蛋白水平较对照组降低。细胞水平上,划痕结果显示,尿酸刺激促进肾小管上皮细胞迁移;Western blot检测结果与免疫组化一致。结论尿酸可通过促进肾小管上皮细胞分泌TGF-β1,进而促进上皮-间质细胞转分化,加速肾间质纤维化的发生。

水蛭素对缺氧诱导心脏微血管内皮细胞间质转分化的作用及机制研究

目的探讨通络药物水蛭素对缺氧诱导的人心脏微血管内皮细胞(HCMECs)间质转分化(EndMT)的作用及可能机制。方法取常规培养的HCMECs细胞,随机分为对照组、缺氧组、水蛭素组(包括0、20、40、80、100μg/ml 5个浓度)。对照组常规培养不做任何处理,缺氧组置入低氧培养箱72 h,水蛭素组预加入Hirudin工作液,4 h后置入低氧培养箱72 h。MTS比色法检测HCMECs增殖能力;倒置显微镜观察HCMECs形态;免疫荧光鉴定HCMECs间质转分化情况,Western-blot检测内皮间质转分化相关蛋白:包括内皮细胞标记血小板—内皮细胞黏附分子(PECAM-1/CD31)、血管内皮钙黏蛋白(VE-cadherin),间质细胞标记α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异性蛋白-1(FSP-1),以及低氧诱导因子1α(HIF-1α)、转化生长因子β1(TGF-β1)、Smad同源物2/3(Smad2/3)、锌指转录因子(snail)等相关信号通路的蛋白表达。结果MTS法检测显示,缺氧显著抑制细胞活性(P<0.01),水蛭素在20~100μg/ml浓度范围内可提高细胞活性,且呈现浓度依赖性,当浓度为100μg/ml时细胞活性最强(P<0.01)。各组细胞培养72 h后,倒置显微镜下观察发现:对照组细胞呈铺路石样或鹅卵石状结构,缺氧组细胞由鹅卵石状结构变为分散的长梭形,接近成纤维细胞形态,水蛭素组长梭形细胞形态明显改善,细胞恢复鹅卵石样。Western-blot与免疫荧光结果显示:与对照组比较,缺氧组CD31、VE-cadherin蛋白水平降低(P<0.01),且vWF表达减少,α-SMA、FSP-1蛋白水平升高(P<0.01),且vimentin表达增强;与缺氧组比较,水蛭素明显增加CD31、VE-cadherin蛋白表达(P<0.01),并增强vWF表达,下调α-SMA、FSP-1蛋白表达(P<0.01),并减弱vimentin表达;与对照组相比,缺氧组信号通路HIF-1α、TGF-β1、p-smad2/3、snail蛋白表达均升高(P<0.01),与缺氧组比较,水蛭素下调HIF-1α、TGF-β1、p-smad2/3、snail蛋白表达(P<0.01)。结论水蛭素可以改善缺氧诱导的HCMECs细胞发生EndMT,其机制可能与调控HIF-α/TGF-β1/smad/snail通路有关。

清肾颗粒对5/6肾切除大鼠肾组织线粒体自噬及肾小管上皮-间质细胞转化的影响

目的:探讨清肾颗粒对5/6肾切除大鼠肾组织线粒体自噬及肾小管上皮细胞转分化(Epithelial-mesenchymal,EMT)的影响。方法:雄性SD大鼠30只,随机分为假手术组、模型组、清肾颗粒组,每组各10只。除假手术组外,其余20只大鼠均制作5/6肾切除模型。清肾颗粒各组分别给予相应剂量清肾颗粒水溶液;假手术组、模型组以生理盐水灌胃。连续给药8周后,无菌采取腹主动脉血,代谢笼留取24h尿液,摘取左侧肾脏。检测血、尿肌酐浓度,并计算内生肌酐清除率;Western blot法检测肾组织中Pink1、Parkin、LC3-Ⅱ、α-SMA;免疫荧光法检测肾组织中LC3-Ⅱ和线粒体膜蛋白VDAC1共定位表达;HE和Masson染色光镜观察大鼠肾脏病理改变;透射电镜检测肾小管上皮细胞中线粒体的超微结构。结果:与假手术组比较,模型组大鼠的线粒体自噬相关蛋白Pink1、Parkin、LC3Ⅱ表达量均显著下降,肾小管EMT标志蛋白α-SMA显著升高(P<0.05)。与模型组比较,清肾颗粒组大鼠的Pink1、LC3Ⅱ蛋白表达量显著升高,α-SMA表达量显著降低(P<0.05),Parkin亦有所升高,但差异无统计学意义(P>0.05)。结论:清肾颗粒能够上调NRK-52E细胞内miR-23b-5p表达,并通过增强PINK1/Parkin通路介导的线粒体自噬活性,抑制NRK-52E细胞转分化进程。

Neuronal conversion from glia to replenish the lost neurons

Neuronal injury,aging,and cerebrovascular and neurodegenerative diseases such as cerebral infarction,Alzheimer’s disease,Parkinson’s disease,frontotemporal dementia,amyotrophic lateral sclerosis,and Huntington’s disease are characte rized by significant neuronal loss.Unfo rtunately,the neurons of most mammals including humans do not possess the ability to self-regenerate.Replenishment of lost neurons becomes an appealing therapeutic strategy to reve rse the disease phenotype.Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain,but it carries the risk of causing gene mutation,tumorigenesis,severe inflammation,and obstructive hydrocephalus induced by brain edema.Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss,which may overcome the above-mentioned disadvantages of neural stem cell therapy.Thus far,many strategies to transfo rm astrocytes,fibroblasts,microglia,Muller glia,NG2 cells,and other glial cells to mature and functional neurons,or for the conversion between neuronal subtypes have been developed thro ugh the regulation of transcription factors,polypyrimidine tra ct binding protein 1(PTBP1),and small chemical molecules or are based on a combination of several factors and the location in the central nervous system.However,some recent papers did not obtain expected results,and discrepancies exist.Therefore,in this review,we discuss the history of neuronal transdifferentiation,summarize the strategies for neuronal replenishment and conversion from glia,especially astrocytes,and point out that biosafety,new strategies,and the accurate origin of the truly co nverted neurons in vivo should be focused upon in future studies.It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transc ription factors or downregulation of PTBP1 or drug interfe rence therapies.

In situ direct reprogramming of astrocytes to neurons via polypyrimidine tract-binding protein 1 knockdown in a mouse model of ischemic stroke

In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1(PTB) knockdown has been shown to reprogram astrocytes to functional neurons in situ. In this study, we used AAV-PHP.e B-GFAP-sh PTB to knockdown PTB in a mouse model of ischemic stroke induced by endothelin-1, and investigated the effects of GFAP-sh PTB-mediated direct reprogramming to neurons. Our results showed that in the mouse model of ischemic stroke, PTB knockdown effectively reprogrammed GFAP-positive cells to neurons in ischemic foci, restored neural tissue structure, reduced inflammatory response, and improved behavioral function. These findings validate the effectiveness of in situ transdifferentiation of astrocytes, and suggest that the approach may be a promising strategy for stroke treatment.