Effects of leukemia inhibitory factor on endogenous neural stem cell proliferation and glycoprotein-130 expression in a mouse model of cerebral infarction

Leukemia inhibitory factor （LIF） has been shown to promote proliferation of endogenous neural stem cells. In this study, we treated mice with cerebral infarction using LIF to investigate whether the LIF receptor subunit glycoprotein （gp）130 is involved in neuroprotection. After LIF treatment, the motor function of model mice was significantly improved. Immunofluorescence histochemistry showed increased numbers of endogenous neural stem cells surrounding the infarct foci. Western blot analysis revealed that gp130 expression was significantly decreased surrounding the infarcted foci. Results demonstrated that LIF promoted the proliferation of endogenous neural stem cells by inhibiting gp130 protein expression.

Effects of leukemia inhibitory factor and basic fibroblast growth factor on free radicals and endogenous stem cell proliferation in a mouse model of cerebral infarction

The present study established a mouse model of cerebral infarction by middle cerebral artery occlusion, and monitored the effect of 25 tJg/kg leukemia inhibitory factor and （or） basic fibroblast growth factor administration 2 hours after model establishment. Results showed that following administration, the number of endogenous neural stem cells in the infarct area significantly increased, malondialdehyde content in brain tissue homogenates significantly decreased, nitric oxide content, glutathione peroxidase and superoxide dismutase activity significantly elevated, and mouse motor function significantly improved as confirmed by the rotarod and bar grab tests. In particular, the effect of leukemia inhibitory factor in combination with basic fibroblast growth factor was the most significant. Results indicate that leukemia inhibitory factor and basic fibroblast growth factor can improve the microenvironment after cerebral infarction by altering free radical levels, improving the quantity of endogenous neural stem cells, and promoting neurological function of mice with cerebral infarction.

Spatiotemporal expression of leukemia inhibitory factor receptor protein during neural tube development in embryos with neural tube defects

Leukemia inhibitory factor receptor(LIFR),as a neuroregulatory cytokine receptor,generally shows a neuroprotective effect in central nervous system injuries.In this study,to understand the effect of LIFR on pathogenesis of neural tube defects,we explored spatiotemporal expression of LIFR at different stages of fetal development in normal and neural tube defect embryos.Spina bifida aperta was induced with all-trans retinoic acid on embryonic day 10 in rats,and the spatiotemporal expression of LIFR was investigated in spina bifida aperta rats and healthy rats from embryonic day 11 to 17.Real time-polymerase chain reaction and western blot assay were used to examine mRNA and protein expression of LIFR in healthy control and neural tube defect embryos.Results of the animal experiment demonstrated that expression of LIFR protein and mRNA in the spinal cords of normal rat embryos increased with embryonic development.LIFR was significantly downregulated in the spinal cords of spina bifida aperta rats compared with healthy rats from embryonic days 11 to 17.Immunohistochemical staining showed that the expression of LIFR in placenta and spinal cord in spina bifida aperta rat embryos was decreased compared with that in control embryos at embryonic day 15.Results from human embryo specimens showed that LIFR mRNA expression was significantly down-regulated in spinal cords of human fetuses with neural tube defects compared with normal controls at a gestational age of 24 to 33 weeks.The results were consistent with the down-regulation of LIFR in the animal experiments.Our study revealed spatiotemporal changes in expression of LIFR during embryonic neurulation.Thus,LIFR might play a specific role in neural tube development.All animal and human experimental procedures were approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University,China(approval No.2016PS106K)on February 25,2016.

Leukemia Inhibitory Factor Decreases Neurogenesis and Angiogenesis in a Rat Model of Intracerebral Hemorrhage

Neurogenesis and angiogenesis can improve the neurologic function after intracerebral hemorrhage(ICH).Leukemia inhibitory factor(LIF)plays an important role in neurogenesis and angiogenesis.In this study,a rat model of autologous blood-induced ICH was used to evaluate the effect of LIF on the neurogenesis and angiogenesis following ICH.After ICH,LIF-positive neurons and dilated vessels were detected in the peri-hematomal region.It was found that LIF levels increased significantly and peaked 14 days after ICH induction.Double immunofluorescence confirmed that LIF was expressed in neurons and endothelial cells.ICH also led to increases of doublecortin(DCX)-and von Willebrand factor(vWF)-positive cells as well as proliferation of cell nuclear antigen(PCNA)+/DCX+and PCNA+/vWF+nuclei.All these ICH-induced increases were significantly attenuated by exogenous LIF in fusion.These data suggested that LIF was a negative regulator of neurogenesis and angiogenesis after ICH.

Expression of Leukemia Inhibitory Factor in Airway Epithelial Tissue of Asthmatic Rats

In order to investigate the expression of leukemia inhibitory factor （LIF） in airway epithelial tissues of normal and asthmatic rats, the influence of dexamethasone and the role of LIF in pathogenesis of asthma, 30 Sprague-Dawley （SD） rats were randomly divided into 3 groups （10 for each group）： normal group, asthma model group, and dexamethasone-interfered group. In asthma model group and dexamethasone-interfered group, asthma rat models were established by intraperitoneal （i.p.） injection of 10% ovalbumin （OVA） and challenge with 1% OVA via inhalation. Rats in dexamethasone-interfered group were pretreated with dexamethasone （2 mg/kg, i.p） 30 min before each challenge. The expression of LIF protein in lung was detected by immunohistochemistry. The results showed that LIF protein was mainly expressed in cytoplasm of bronchial epithelial cells. The expression of LIF protein in the airway epithelial tissue of asthma model group was significantly higher than that in normal group and dexamethasone-interfered group （P〈0.01）, but there was no significant difference between normal group and dexamethasone-interfered group （P〉0.05）. It was concluded that the expression of LIF was increased significantly in the airway epithelial tissue of the asthma rats, and dexamethasone could down-regulate the expression of LIF. It was suggested that LIF might play an important role in the pathogenesis of asthma as an inflammation regulator.

藏西北绒山羊子宫内膜容受性相关基因和可变剪接事件的综合分析

背景:藏西北绒山羊子宫内膜容受性是胚胎植入的关键因素,目前对于藏西北绒山羊子宫内膜容受性相关基因表达及可变剪接的认识还未明确。目的:分析并挖掘藏西北绒山羊子宫内膜容受性相关的基因和可变剪接事件。方法:在妊娠第5天和第15天(分别代表容受前子宫内膜组和容受性子宫内膜组),分别随机选取3只藏西北绒山羊,采集子宫内膜组织,苏木精-伊红染色观察组织形态,免疫组织化学检测子宫内膜容受性标志蛋白白血病抑制因子、血管内皮生长因子的表达水平;提取子宫内膜组织总RNA,质量检测合格后进行转录组测序,寻找差异表达的mRNA、长链非编码RNA、环状RNA和miRNA,进行功能预测,并分析与子宫内膜容受性相关的可变剪接mRNA和长链非编码RNA。结果与结论:(1)与容受前子宫内膜组比较,容受性子宫内膜组子宫内膜组织中白血病抑制因子和血管内皮生长因子蛋白的表达水平明显升高;(2)测序结果显示,差异表达基因多为mRNA和长链非编码RNA,包括250个上调的mRNA、193个上调的长链非编码RNA、135个下调的mRNA和123个下调的长链非编码RNA,显著富集于Wnt、Hedgehog和Hippo信号通路;(3)可变剪接事件分析显示有8个差异表达的可变剪接转录本,均为mRNA分子,其中2个下调、6个上调,与血管内皮生长因子受体信号、细胞运动和胚胎发育有关。

Synchronous gastric cancer complicated with chronic myeloid leukemia (multiple primary cancers):A case report

BACKGROUND With the advancement of medical technology and improvement in living standards,the incidence of multiple primary cancers has gradually increased.In particular,tumors of the digestive system account for a large proportion of multiple primary cancers.The diagnosis and treatment of chronic myeloid leukemia,particularly with synchronous gastric cancer,at the first consultation is relatively rare.CASE SUMMARY Herein,we present the case of a middle-aged man who was referred to the Department of Hematology owing to an elevated white blood cell count.After the examination,he was diagnosed with chronic myeloid leukemia and was administered imatinib.Three months after the initial diagnosis,he visited our hospital again for abdominal pain,and further examination revealed gastric malignancy.After discussion with a multidisciplinary team,S-1(Tegafur,Gimeracil,and Oteracil Potassium Capsules) combined with oxaliplatin—SOX regimen—was initiated.Later,the patient’s condition rapidly progressed.He developed colonic obstruction and underwent an ostomy;however,he died less than 6 months after the initial diagnosis.CONCLUSION Multiple primary cancers are influenced by environmental and genetic factors;a standardized multidisciplinary discussion plays a key role in treatment.

GATA3通过调控LIFR抑制人乳腺癌细胞的迁移能力

目的探究GATA结合蛋白3(GATA binding protein 3,GATA3)对乳腺癌细胞迁移能力的影响。方法在MCF7细胞中利用慢病毒载体介导的基因干涉技术敲低GATA3基因,使用实时定量荧光PCR(qRT-PCR)和蛋白质印迹检测GATA3和LIFR的mRNA和蛋白表达水平,Transwell实验检测MCF7细胞的迁移能力。在MCF7和T47D细胞中用染色质免疫沉淀(ChIP-qPCR)实验检测GATA3在LIFR的启动子区的结合位点。在敲低GATA3基因的MCF7细胞中回补LIFR,通过细胞划痕实验和Transwell实验检测MCF7细胞的迁移能力。结果与对照组相比,敲低GATA3基因的MCF7细胞的迁移能力增强(P_(均)<0.05)。与对照组相比,敲低GATA3基因的MCF7细胞的LIFR表达水平降低(P_(均)<0.05)。乳腺癌细胞MCF7与T47D中GATA3在LIFR的启动子区有结合(P_(均)<0.05)。在敲低GATA3基因的MCF7细胞中稳定过表达LIFR可以部分挽救GATA3基因敲低引起的细胞迁移能力的增强(P_(均)<0.05)。结论GATA3通过转录激活LIFR抑制乳腺癌细胞MCF7的迁移。

参芪地黄汤化裁联合胰岛素治疗二甲双胍控制血糖不佳气阴两虚证糖尿病的效果

目的探讨参芪地黄汤化裁联合胰岛素治疗在二甲双胍控制血糖不佳气阴两虚证糖尿病患者中的应用价值。方法选取2019年3月—2022年6月就诊的100例二甲双胍控制血糖不佳气阴两虚证糖尿病开展回顾性研究,均为2型糖尿病。根据治疗方法不同分为研究组和常规组,每组50例。研究组采用参芪地黄汤化裁联合胰岛素治疗,常规组采用胰岛素治疗。比较2组疗效、中医证候积分、血糖相关指标[糖化血红蛋白(HbA1c)、稳态模型胰岛素抵抗指数(HOMA-IR)、餐后2 h血糖(2 h PBG)、空腹血糖(FBG)、胰岛β细胞功能指数(HOMA-β)]、炎性指标[C反应蛋白(CRP)、单核细胞趋化蛋白-1(MCP-1)、白血病抑制因子(LIF)、高迁移率族蛋白B1(HMGB1)]、预后指标[脂蛋白相关磷脂酶A2(Lp-PLA2)、血尿酸(SUA)、甲状腺球蛋白(TG)]及安全性。结果研究组临床总有效率94.00%(47/50)高于常规组80.00%(40/50)(P<0.05)。治疗后研究组各中医证候积分及总积分低于常规组(P<0.05)。治疗后研究组HbA1c、2 h PBG、FBG、HOMA-IR低于常规组,HOMA-β高于常规组(P<0.05)。治疗后研究组CRP、MCP-1、LIF、HMGB1、Lp-PLA2、SUA、TG低于常规组(P<0.05)。2组不良反应发生率比较差异无统计学意义(P>0.05)。结论参芪地黄汤化裁联合胰岛素治疗二甲双胍控制血糖不佳气阴两虚证糖尿病患者效果显著,可有效降低血糖水平,改善临床症状,减轻炎症反应,且安全性高。

BMPRⅡ^(+)neural precursor cells isolated and characterized from organotypic neurospheres:an in vitro model of human fetal spinal cord development

Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not well understood.Type-ⅡBMP receptor(BMPRⅡ),the cognate receptor,is expressed by neural precursor cells during embryogenesis;however,an in vitro method of enriching BMPRⅡ^(+)human neural precursor cells(hNPCs)from the fetal spinal cord is absent.Immunofluorescence was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRⅡand leukemia inhibitory factor(LIF).Regions of highest BMPRⅡ^(+)immunofluorescence localized to sensory columns.Parenchymal and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/CD39.LIF immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons,mirroring the expression of LIF receptor/CD118.A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages,while synergistically increasing the proportion of neurospheres with a stratified,cytoarchitecture.These neurospheres were characterized by BMPRⅡ^(+)/MAP2ab^(+/–)/βⅢ-tubulin^(+)/nestin^(–)/vimentin^(–)/GFAP^(–)/NeuN^(–)surface hNPCs surrounding a heterogeneous core ofβⅢ-tubulin^(+)/nestin^(+)/vimentin^(+)/GFAP^(+)/MAP2ab^(–)/NeuN^(–)multipotent precursors.Dissociated cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage cells.Fluorescence-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in culture.This is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal cords.Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterized by surface BMPRⅡ^(+)hNPCs.Our study has provided a new methodology for an in vitro model capable of amplifying human fetal spinal cord cell numbers for>10 passages.Investigations of the role BMPRⅡplays in spinal cord development have primarily relied upon mouse and rat models,with interpolations to human development being derived through inference.Because of significant species differences between murine biology and human,including anatomical dissimilarities in central nervous system(CNS)structure,the findings made in murine models cannot be presumed to apply to human spinal cord development.For these reasons,our human in vitro model offers a novel tool to better understand neurodevelopmental pathways,including BMP signaling,as well as spinal cord injury research and testing drug therapies.

白血病抑制因子对牙本质发育的影响

目的:探究白血病抑制因子(leukemia inhibitory factor, LIF)对牙本质发育的影响。方法:免疫组织化学染色观察LIF在出生后2天(post-natal 2,PN2)小鼠成牙本质细胞中的表达。建立小鼠切牙机械损伤模型,统计切牙生长速率;通过微计算机断层扫描技术(micro computed tomography, Micro-CT)、显微硬度计、电子探针测量Lif-/-小鼠及其对照切牙的牙本质密度、硬度及钙磷比(Ca/P);碱性磷酸酶(ALP)染色和茜素红染色观察LIF对成牙本质细胞矿化的影响;免疫荧光染色观察成牙本质细胞Ⅰ型胶原α1(Col1α1)、牙本质基质蛋白1(DMP1)表达情况。结果:LIF随着成牙本质细胞分化程度增加表达含量升高。Lif-/-小鼠切牙新生速率减慢。Lif-/-小鼠切牙近釉牙本质界侧牙本质密度、硬度及Ca/P均降低。ALP及茜素红染色结果显示Lif敲低的成牙本质细胞ALP水平降低、矿化结节数量减少,而加入LIF外源性刺激后可以逆转这一现象。免疫荧光结果显示Lif-/-小鼠成牙本质细胞Col1α1、DMP1表达含量降低。结论:LIF缺失抑制小鼠牙齿牙本质形成与矿化。

白血病抑制因子在肿瘤发生发展中的作用与靶向治疗策略

白血病抑制因子(LIF)属IL-6家族,是一种多效性细胞因子,因最早被发现能够抑制小鼠髓系M1白血病细胞增殖并诱导其终末分化而得名。LIF广泛参与器官、神经发育与再生和免疫调节等反应,对于肿瘤的发展同样具有重要的作用。与抑制白血病细胞生长的作用相反,LIF通常促进多种类型实体瘤的发展,高表达的LIF能够促进肿瘤的发生发展、转移、耐药和肿瘤免疫逃逸等,与患者的不良预后相关。聚焦LIF生理和病理的功能作用及其所调控信号通路的整体性,寻找新的靶向药物,对于LIF通路靶向治疗策略的开发具有重要意义。

白血病抑制因子对牛卵母细胞体外发育能力的影响

[目的]探究白血病抑制因子(leukemia inhibitory factor, LIF)对牛卵母细胞和早期胚胎发育能力及多潜能基因表达的影响。[方法]将牛卵丘-卵母细胞复合体(cumulus oocyte complexes, COCs)分为4组,对照组:在卵母细胞体外成熟(in vitro maturation, IVM)和胚胎体外培养(in vitro culture, IVC)过程中均不添加LIF溶液;处理组1(T1组):IVM过程添加25 ng/mL LIF,IVC过程不添加LIF;处理组2(T2组):IVM过程不添加LIF,IVC过程添加25 ng/mL LIF;处理组3(T3组):IVM和IVC全过程均添加25 ng/mL LIF。用Fluo-3荧光指示剂检测卵母细胞胞内游离钙离子([Ca2+]i)浓度;挑选有第一极体排出的卵母细胞进行孤雌激活,统计各组卵母细胞卵裂率和囊胚率;利用实时荧光定量PCR检测囊胚中多潜能基因的相对表达量。[结果]与对照组相比,培养8 h后,T1组牛卵母细胞胞内[Ca2+]i浓度极显著下降(P<0.01);培养24 h后,T1组牛卵母细胞胞内[Ca2+]i浓度极显著升高(P<0.01)。T1和T3组孤雌激活后卵裂率和囊胚率均显著高于对照组,且T3组囊胚率显著高于T1和T2组(P<0.05)。实时荧光定量PCR结果显示,T3组囊胚中POU结构域5类转录因子1(POU5F1)和同源框蛋白(NANOG)基因表达量均显著高于对照组,尾型同源盒转录因子2(CDX2)基因表达量显著低于对照组(P<0.05)。[结论]在IVM过程中添加LIF可提高卵母细胞的发育能力,在IVM+IVC全过程添加LIF对早期胚胎发育和多潜能基因的表达有积极作用。

Neural Stem Cells Transfected with Leukemia Inhibitory Factor Promote Neuroprotection in a Rat Model of Cerebral Ischemia

Leukemia inhibitory factor(LIF) contributes to the neuroprotection by neural stem cells(NSCs) after ischemic stroke. Our aim was to explore whether LIFtransfected NSCs(LIF-NSCs) can ameliorate brain injury and promote neuroprotection in a rat model of cerebral ischemia. To accomplish this goal, we transfected NSCs with a lentivirus carrying the LIF gene to stably overexpress LIF. The LIF-NSCs reduced caspase 3 activation under conditions of oxygen-glucose deprivation in vitro.Transient cerebral ischemia was induced in rats by 2 h of middle cerebral artery occlusion(MCAo), and LIF-NSCs were intravenously injected at 6 h post-ischemia. LIF-NSC treatment reduced the infarction volume and improved neurological recovery. Moreover, LIF-NSCs improved glial cell regeneration and ameliorated white matter injuryin the MCAo rats. The NSCs acted as carriers and increased the expression of LIF in the lesions to protect against cerebral infarction, suggesting that LIF-NSCs could be a potential treatment for cerebral infarction.

Buyang Huanwu Decoction(补阳还五汤)Attenuates Glial Scar by Downregulating Expression of Leukemia Inhibitory Factor in Intracerebral Hemorrhagic Rats

Objective: To evaluate the effect of Buyang Huanwu Decoction(补阳还五汤,BYHWD) on glial scar after intracerebral hemorrhage(ICH) and investigate the underlying mechanism.Methods: Collagenase type Ⅶ(0.5 U) was injected stereotaxically into right globus pallidus to induce ICH model.One hundred and twenty SpragueDawley rats were randomly divided into 3 groups according to a random number table,including normal group(n=40),ICH model group(n=40) and BYHWD group(n=40),respectively.After ICH,the rats in the BYHWD group were intragastrically administered with BYHWD(4.36 g/kg) once a day for 21 days,while the rats in ICH group were administered with equal volume of distilled water for 21 days,respectively.Double immunolabeling was performed for proliferating cell nuclear antigen(PCNA)+/glial ?brillary acidic protein(GFAP)+ nuclei.The expression of GFAP and leukemia inhibitory factor(LIF) was evaluated by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR).Results: The astrocytes with hypertrophied morphology around the hematoma was observed on day 3 after ICH.The number of GFAP positive cells and GFAP m RNA levels increased notably on day 3 and reached the peak on day 14 post-ICH(P<0.01).PCNA+/GFAP+ nuclei were observed around the hematoma and reached the peak on day 14 post-ICH(P<0.01).In addition,LIF-positive astrocytes and LIF m RNA level in the hemorrhagic region increased signi?cantly till day 14 post-ICH(P<0.01).However,BYHWD not only reduced the number of PCNA+/GFAP+ nuclei,but also decreased GFAP and LIF levels(P<0.05).Conclusion:BYHWD could attenuate ICH-induced glial scar by downregulating the expression of LIF in the rats.

miR-92a-1-5p通过调控SOCS3和P53促进急性白血病细胞增殖的机制研究

目的探讨miR-92a-1-5p通过调控细胞因子信号转导抑制因子3(SOCS3)、P53促进急性白血病(AL)细胞增殖的机制。方法收集20例初诊AL患者和15例正常人的骨髓标本,检测miR-92a-1-5p、SOCS3、P53 mRNA、蛋白质的表达水平,剖析其与临床特征和预后的关联性;利用基因工程手段,在急性淋巴细胞白血病细胞系HL-60和急性髓系白血病细胞系K562中分别提高和降低miR-92a-1-5p的表达水平,以评估其对SOCS3和P53蛋白表达水平的调控作用;研究miR-92a-1-5p对细胞周期、增殖和凋亡的影响。结果miR-92a-1-5p的表达水平与白细胞计数、骨髓增生程度、分化程度、髓外浸润和预后均呈负相关,与SOCS3和P53 mRNA表达水平均呈正相关。在HL-60和K562细胞中,上调miR-92a-1-5p表达水平能抑制SOCS3和P53 mRNA的表达,促进细胞周期进入S期,增加细胞增殖能力,抑制细胞凋亡;下调miR-92a-1-5p表达则产生相反的效果。结论miR-92a-1-5p通过调控SOCS3和P53促进AL细胞增殖,可能作为AL的潜在诊断标志物和治疗靶点。

Extracts from Paeonia lactiflora Pallas, Rehmannia Glutinosa var. Purpurea Makino, Perilla Frutescens var. Acuta Kudo may increase the endometrial receptivity through expression of leukemia inhibitory factor and adhesion molecules

OBJECTIVE: To find out the combination of the extracts from Paeonia lactiflora Pallas(PL), Rehmannia Glutinosa var. Purpurea Makino(RG), Perilla Frutescens var. Acuta Kudo(PF) to increase endometrial receptivity.METHODS: Herbal medicines were extracted with boiling water and polysaccharides were removed.We examined the effect of PL, RG, and PF(PRP), a most effective herbal formula deduced from constitutive ingredient herbs of Antai Yin which is composed of PRP, on the leukemia inhibitory factor(LIF) expression and endometrial receptivity.RESULTS: The combination of the extracts from PRP induced the LIF expression in Ishikawa cells and increased the adhesion between Ishikawa and JAr cells. In addition, PRP-induced attachment of JAr cells onto Ishikawa cells and expression of adhesion molecules, ITGAV, ITGB5, CD44 s, and Lselectin, are significantly reduced by knock-down of LIF expression.CONCLUSION: Induced by the combination of the PRP extracts, the adhesion between trophoblast and endometrial cells are mediated by expression of LIF and adhesion molecules. Thus, we suggest the combination of the PRP extracts may be a novel therapy for enhancing embryo implantation rate.

白血病抑制因子通过调控CD44的表达增强结直肠癌细胞干性特征

目的:探讨白血病抑制因子(leukemia inhibitory factor,LIF)通过调控肿瘤干细胞(cancer stem cells,CSCs)分子标志物CD44的表达增强结直肠癌(colorectal cancer,CRC)细胞干性特征的分子机制。方法:利用TCGA公共数据库和RNAscope原位杂交方法分析LIF基因在CRC组织中的表达情况;应用慢病毒感染系统构建稳定敲减LIF的CRC细胞系(HCT116和Caco2细胞);实验分CRC细胞对照组、CRC细胞添加LIF组、CRC细胞敲减LIF对照组和CRC细胞敲减LIF组;应用干细胞成球实验、MTT、RTCA、平板集落及迁移实验检测LIF对CRC细胞的影响;应用RT-qPCR和Western blot检测LIF对CRC肿瘤球细胞干性相关标志物CD44和转录因子ELF3表达的影响;应用RT-qPCR检测敲减LIF后CD44剪接变异体的表达变化。结果:CRC组织中LIF的表达水平高于癌旁组织(P<0.01),且LIF高表达的CRC患者无病生存期缩短(P<0.05),外源性LIF可增强CRC细胞的成球、增殖和迁移能力(P<0.05),敲减LIF可抑制CRC细胞的增殖和迁移(P<0.05)。外源性LIF可上调CRC肿瘤球细胞CD44的表达(P<0.05),而敲减LIF可抑制CD44的表达(P<0.01),同时CD44剪接变异体的转录水平降低。外源性添加LIF和敲减LIF可分别增强和降低转录因子ELF3的表达水平(P<0.05)。结论:LIF通过上调转录因子ELF3,增强CRC细胞CD44的表达,进而增强CRC细胞的干性特征,促进CRC细胞的恶性进展。

Protective Effects of Leukemia Inhibitory Factor on Retinal Vasculature and Cells in Streptozotocin-induced Diabetic Mice

Background： Leukemia inhibitory factor （LIF） has been reported to possess various pharmacological effects, including displaying vascular and neuroprotective properties, during retinal disease. The aim of this study was to investigate the vascular and structural changes in the retina of diabetic mice and to explore whether LIF prevents experimental diabetes-induced retinal injury in the early stages. Methods： Diabetes was induced in C57BI/6J mice with streptozotocin （STZ） injections. Successful diabetic animal models were randomly separated into two groups： the diabetic group （n = 15） and the LIF-treated group （n = 15）. Normal C57BL/6 mice served as the normal control group （n = 14）. Recombinant human LIF was intravitreally injected 8 weeks after the diabetic model was successfully established. Retinas were collected and evaluated using histological and immunohistochemical techniques, and flat-mounted retinas and Western blotting were performed at 18 weeks after the induction of diabetes and 2 days after the intravitreal injection of LIF. The analysis of variance test were used. Results： Histological analysis showed that there were fewer retinal ganglion cells （RGCs） and the inner nuclear layer （INL） became thinner in the diabetic model group （RGC 21.8 ± 4.0 and INL 120.2 ± 4.6 μm） compared with the normal control group （RGC 29.0 ± 6.7, t = -3.02, P = 0.007; INL 150.7 ±10.6 lain, t = -8.88, P 〈 0.001, respectively）. After LIF treatment, the number of RGCs （26.9 ± 5.3） was significantly increased （t = 3.39, P = 0.030） and the INL （ 134.5± 14.2 lain） was thicker compared to the diabetic group （t - 2.75, P = 0.013）. In the anti-Brn-3a-labeled retinas, the number of RGCs in the LIF-treated group （3926.0 ± 143.9） was obviously increased compared to the diabetic group （3507.7 ± 286.1, t = 2.38, P = 0.030）, while no significance was found between the LIF-treated group and the control group （4188.3 ± 114.7, t= -2.47, P- 0.069）. Flat-mounted retinas demonstrated that a disorganized, dense distribution of the vessel was prominent in the diabetic model group. Vessel distribution in the LIF-treated mouse group was typical and the thickness was uniform. The levels of phosphosignal transducer and activator of transcription 3 activation were obviously higher in the LIF-injected retinas than those in the diabetic control group （t = 3.85, P = 0.019） and the normal control （t = -3.20, P - 0.019）. Conclusion： The present study provides evidence that LIF treatment protects the integrity of the vasculature and prevents retinal injury in the early stages of diabetic retinopathy in STZ-induced diabetic models.

Leukemia Inhibitory Factor Enhanced the Developmental and Implantation Compatibility of Mouse Embryos in Co-culture with Human Endometrial Epithelial Cells

Objective:Among the variousin vitro embryo culture systems,co-culture has demonstrated remarkable effects in pre-implantation embryo development owing to the production of embryo-nourishing factors.Nevertheless,little is known about the secretion of these factors.Therefore,in this study,the effect of leukemia inhibitory factor(LIF),one of the most important nourishing factors in the early development of mouse embryos,in human endometrial epithelial cells(hEECs)was evaluated.Methods:Two-cell stage embryos were collected from the oviducts of hyper-stimulated and mated mice and cultivated in a co-culture with an hEEC monolayer with or without LIF.The quality and developmental and attachment potential rates of cultured embryos were evaluated by determining the levels of octamer-binding transcription factor 4(Oct4)and caudal type homeobox 2(Cdx2)transcripts.Results:LIF significantly increased the developmental rate(82.67%vs.61.04%,respectively)and attachment rate(64%vs.45.45%,respectively)of mouse embryos co-cultured with hEECs compared to those in untreated embryos.The expression levels ofOct4 andCdx2 in blastocysts cultured in the presence of LIF were higher than those in blastocysts cultured without LIF.Conclusions:Despite the secretion of LIF by hEECs during co-culture with embryos,the amount of this factor was insufficient,and its addition to the culture media could increase the developmental potential of embryos.