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cdc42 deletion yielded enamel defects by disrupting mitochondria and producing reactive oxygen species in dental epithelium

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Genes & Diseases 2024年第5期11卷 335-349页

作者： Jinxuan Zheng Rongcheng Yu Yiqi Tang Sihui Su Sainan Wang Chenxi Liao Xuecong Li Jiabin Liao Dongsheng Yu Tingting Ai Wei Zhao Vicky Yau Chufeng Liu Liping Wu Yang Cao Hospital of Stomatology Guanghua School of StomatologyGuangdong Provincial Key Laboratory of StomatologySun Yat-sen UniversityGuangzhouGuangdong 510055China Guangdong Provincial Key Laboratory of Oral Diseases GuangzhouGuangdong 510055China Department of Cariology and Endodontology Peking University School and Hospital of Stomatology&National Center for Stomatology&National Clinical Research Center for Oral Diseases&National Engineering Research Center of Oral Biomaterials and Digital Medical DevicesBeijing 100081China Department of Oral and Maxillofacial Surgery University at BuffaloBuffaloNY 14214USA Department of Orthodontics Stomatological HospitalSouthern Medical UniversityGuangzhouGuangdong 510280China

Developmental defects of enamel are common due to genetic and environmental factors before and after ***42,a Rho family small GTPase,regulates prenatal tooth development in ***,its role in postnatal tooth development,especially enamel formation,remains ***,we investigated cdc42 functions in mouse enamel development and tooth repair after ***42 showed highly dynamic temporospatial patterns in the developing incisors,with robust expression in ameloblast and odontoblast ***,epithelium-specific cdc42 deletion resulted in enamel defects in *** differentiation was inhibited,and hypomineralization of enamel was observed upon epithelial cdc42 *** analysis showed that abnormal mitochondrial components,phosphotransferase activity,and ion channel regulator activity occurred in the cdc42 mutant dental *** oxygen species accumulation was detected in the mutant mice,suggesting that abnormal oxidative stress occurred after cdc42 ***,cdc42 mutant mice showed delayed tooth repair and generated less calcified *** dysfunction and abnormal oxygen consumption were evidenced by reduced Apool and Timm8a1 expression,increased Atp5j2 levels,and reactive oxygen species overproduction in the mutant repair ***-specific cdc42 deletion attenuated ERK1/2 signaling in the labial cervical *** Sox2 expression in the mutant labial cervical loop after clipping might lead to delayed tooth *** findings suggested that mitochondrial dysfunction,up-regulated oxidative stress,and abnormal ion channel activity may be among multiple factors responsible for the observed enamel defects in cdc42 mutant ***,cdc42 exerts multidimensional and pivotal roles in enamel development and is particularly required for ameloblast differentiation and enamel matrix formation.

关键词： cdc42 Enamel defects Mitochondrial dysfunction Rho GTPase Tooth repair

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cdc42基因的激活和抑制对烟曲霉极性生长影响的初步探究

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中国真菌学杂志 2024年第1期19卷 1-7页

作者：张梦楠于婉婉马彦山西医科大学第二医院皮肤性病科太原030000

目的构建烟曲霉cdc42基因显性激活性(dominant active,DA)和显性抑制性(dominant negative,DN)点突变菌株,初步了解这两种点突变对烟曲霉极性生长以及RAC1基因表达的影响。方法运用分子生物学方法构建cdc42基因激活性(cdc42^(G14V))和... 详细信息

目的构建烟曲霉cdc42基因显性激活性(dominant active,DA)和显性抑制性(dominant negative,DN)点突变菌株,初步了解这两种点突变对烟曲霉极性生长以及RAC1基因表达的影响。方法运用分子生物学方法构建cdc42基因激活性(cdc42^(G14V))和抑制性(cdc42^(T19N))菌株;光学显微镜观察点突变菌落特征;RT-PCR方法检测Ku80、DA cdc42和DN cdc42菌株中RAC1的表达量。结果成功构建烟曲霉cdc42显性激活性(DA)和显性抑制性(DN)点突变菌株并经测序确认;烟曲霉DA cdc42和DN cdc42菌株的菌落生长直径、形成分生孢子数和各时间点孢子发芽率较对照株Ku80都明显降低,且差异均有统计学意义(Pcdc42和DN cdc42菌株中的RAC1的表达量均较对照株Ku80有所上升(Pcdc42的激活和抑制都会对烟曲霉的极性生长产生负面影响;且会影响RAC1基因的表达。

关键词：烟曲霉 cdc42 点突变极性生长

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髓系特异性敲除cdc42对小鼠肝脏缺血再灌注损伤的影响及其机制探究

髓系特异性敲除Cdc42对小鼠肝脏缺血再灌注损伤的影响及其机制探究

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作者：唐梦雨南昌大学

学位级别：硕士

实验目的:肝脏缺血再灌注损伤（Hepatic Ischemia-reperfusion Injury,HIRI）常见于肝脏部分切除手术或肝移植手术,骨髓来源的巨噬细胞介导的炎症在其中起关键作用,而细胞分裂周期蛋白42（cdc42）参与调节细胞迁移、细胞骨架重排和细胞... 详细信息

实验目的:肝脏缺血再灌注损伤（Hepatic Ischemia-reperfusion Injury,HIRI）常见于肝脏部分切除手术或肝移植手术,骨髓来源的巨噬细胞介导的炎症在其中起关键作用,而细胞分裂周期蛋白42（cdc42）参与调节细胞迁移、细胞骨架重排和细胞极性等方面。在本课题中我们旨在探究髓系巨噬细胞cdc42缺失对HIRI的影响及其潜在机制。实验方法:***42flox/flox小鼠与髓系细胞特异性表达Cre重组酶的Lyz2-Cre小鼠杂交,得到髓系巨噬细胞特异性敲除cdc42(cdc42Mye)小鼠和未敲除(cdc42Flox)小鼠。ROSA小鼠与cdc42Mye小鼠杂交得到cdc42MyeROSAm Tm G（cdc42 ROSA Lyz）小鼠,Lyz2-Cre小鼠与ROSA小鼠杂交得到ROSAm Tm G（ROSA Lyz）小鼠。2.选取8-12周龄的雄性小鼠,血管夹夹闭肝左叶和肝右叶的门静脉、肝动脉和总胆管缺血1h,复灌12h制备HIRI模型。将cdc42Mye与cdc42Flox,cdc42ROSA Lyz与ROSA Lyz小鼠分别设置对照组和实验组。3.收集小鼠血清,检测肝功能相关指标ALT和AST的含量;制备肝脏组织匀浆,检测肝脏内氧化应激相关指标LDH、MDA和SOD的含量;通过小鼠细胞因子抗体微阵列检测血清中细胞因子分泌情况。4.采用4%多聚甲醛将小鼠肝脏组织固定后,对其脱水和石蜡包埋,最后进行切片。通过HE染色评估小鼠肝脏坏死情况;通过TUNEL荧光染色观察肝细胞凋亡情况;通过荧光、免疫组化和免疫荧光观察髓系巨噬细胞浸润及极化情况。5.收集小鼠血液,通过血常规检测小鼠血液内各成分含量;通过流式细胞术标记血液内中性粒细胞和单核细胞,标记M1型和M2型巨噬细胞,观察髓系细胞的浸润及巨噬细胞极化情况。6.收取cdc42 ROSA Lyz小鼠和ROSA Lyz小鼠的肝脏组织,4%多聚甲醛固定24h后通过30%蔗糖对组织脱水,OCT包埋后放入-80℃冰箱,后续冰冻切片7μm,荧光显微镜观察肝脏组织内髓系细胞的浸润情况。7.提取肝组织蛋白,Western Blot检测肝组织内i NOS、IL-6、IL-10和ARG1的表达水平;提取肝组织的总RNA,RT-q PCR检测IL-6、TNF-α、CD206和IL-1β的表达水平。8.在肝脏缺血再灌注术前给予小鼠腹腔注射ML141（10mg/kg）2次,观察抑制cdc42活性对小鼠HIRI的影响。通过HE染色观察肝组织受损情况,通过多重染色免疫荧光观察髓系巨噬细胞对肝脏的浸润及其极化情况。9.体外采用集落刺激因子刺激促使C57BL/6野生型小鼠的骨髓细胞分化为巨噬细胞,然后,采用ML141抑制cdc42活性,分别检测经典促炎诱导剂LPS和抗炎因子IL-4刺激后巨噬细胞中极化相关蛋白STAT家族及SOCS3蛋白的表达情况。实验结果:1.成功构建髓系特异性敲除cdc42小鼠和cdc42 ROSA Lyz小鼠。2.髓系特异性敲除cdc42显著减轻小鼠HIRI后肝损伤情况;减少肝细胞凋亡;保留了肝功能。3.髓系特异性敲除cdc42减少HIRI小鼠中髓系巨噬细胞的浸润;抑制巨噬细胞M1极化并且促进巨噬细胞M2极化。4.髓系特异性敲除cdc42减轻了HIRI小鼠诱导的炎症反应。5.抑制cdc42活性可减轻小鼠HIRI。6.体外实验中髓系cdc42缺失或失活明显抑制巨噬细胞M1极化,减少炎性因子的分泌。7.髓系特异性敲除cdc42通过抑制STAT1的磷酸化,促进STAT3和STAT6的磷酸化增强巨噬细胞M2极化。结论:抑制cdc42的活性或髓系cdc42的缺失可以通过抑制髓系巨噬细胞的浸润、抑制促炎反应和促进巨噬细胞的M2极化来保护肝脏免受HIRI。

关键词：肝脏缺血再灌注损伤 cdc42 巨噬细胞极化

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SKA1通过cdc42介导高尔基体堆叠调控胰腺癌细胞侵袭与转移

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现代生物医学进展 2024年第7期24卷 1201-1208页

作者：李彤郭婧李东中山静子余湘南徐馨复旦大学附属中山医院肝肿瘤内科上海200032 复旦大学附属中山医院徐汇医院消化内科上海200031 上海嘉会国际医院普通外科上海200233 复旦大学附属中山医院消化内科上海200032

目的:探讨SKA1调控胰腺导管腺癌(PDAC)侵袭与转移的分子机制。方法:利用免疫组化染色法检测胰腺导管腺癌组织样本及癌旁组织中SKA1与下游分子cdc42的表达水平、用免疫印迹方法在不同胰腺癌细胞系及正常胰腺导管上皮细胞中进行验证;利用... 详细信息

目的:探讨SKA1调控胰腺导管腺癌(PDAC)侵袭与转移的分子机制。方法:利用免疫组化染色法检测胰腺导管腺癌组织样本及癌旁组织中SKA1与下游分子cdc42的表达水平、用免疫印迹方法在不同胰腺癌细胞系及正常胰腺导管上皮细胞中进行验证;利用慢病毒转染技术构建SKA1稳定敲减和过表达的胰腺癌细胞株;利用免疫荧光方法检测PDAC细胞内SKA1敲低或过表达对高尔基体结构的影响,及cdc42抑制剂ZCL278对高尔基体结构变化的调控;并利用Transwell实验及细胞划痕实验检测ZCL278对SKA1促癌作用的影响;应用免疫印迹方法检测SKA1及cdc42表达对自噬标志物的影响。结果:在胰腺导管腺癌组织与细胞中SKA1与cdc42表达均显著高于正常组织或细胞,且二者表达呈显著正相关;并且发现SKA1低表达的患者具有更长的总体生存期,而cdc42表达高低与总体生存期无关。接着我们发现SKA1可促进PDAC细胞内高尔基体堆叠,并且在SKA1过表达的细胞中cdc42抑制剂ZCL278可以抑制这种堆叠现象。进一步我们发现抑制cdc42可逆转SKA1过表达对胰腺癌侵袭与转移的促进效应,具有统计学差异,而在SKA1非高表达的细胞中无显著差异,最后我们发现cdc42可受SKA1的表达调控诱导PDAC细胞发生自噬。结论:SKA1通过调控cdc42介导的高尔基体致密堆叠进而影响胰腺癌发生自噬,最终促使胰腺癌细胞迁移和侵袭。

关键词：胰腺癌 SKA1 高尔基体 cdc42

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Ischemic accumulation of succinate induces cdc42 succinylation and inhibits neural stem cell proliferation after cerebral ischemia/reperfusion

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Neural Regeneration Research 2023年第5期18卷 1040-1045页

作者： Lin-Yan Huang Ju-Yun Ma Jin-Xiu Song Jing-Jing Xu Rui Hong Hai-Di Fan Heng Cai Wan Wang Yan-Ling Wang Zhao-Li Hu Jian-Gang Shen Su-Hua Qi School of Medical Technology Xuzhou Key Laboratory of Laboratory DiagnosticsXuzhou Medical UniversityXuzhouJiangsu ProvinceChina College of Pharmacology Xuzhou Medical UniversityXuzhouJiangsu ProvinceChina Research Center for Biochemistry and Molecular Biology and Jiangsu Key Laboratory of Brain Disease Bioinformation Xuzhou Medical UniversityXuzhouJiangsu ProvinceChina School of Chinese Medicine The University of Hong KongHong Kong Special Administrative RegionChina

Ischemic accumulation of succinate causes cerebral damage by excess production of reactive oxygen species. However, it is unknown whether ischemic accumulation of succinate affects neural stem cell proliferation. In this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery. We found that succinate levels increased in serum and brain tissue(cortex and hippocampus) after ischemia/reperfusion injury. Oxygen-glucose deprivation and reoxygenation stimulated primary neural stem cells to produce abundant succinate. Succinate can be converted into diethyl succinate in cells. Exogenous diethyl succinate inhibited the proliferation of mouse-derived C17.2 neural stem cells and increased the infarct volume in the rat model of cerebral ischemia/reperfusion injury. Exogenous diethyl succinate also increased the succinylation of the Rho family GTPase cdc42 but repressed cdc42 GTPase activity in C17.2 cells. Increasing cdc42 succinylation by knockdown of the desuccinylase Sirt5 also inhibited cdc42 GTPase activity in C17.2 cells. Our findings suggest that ischemic accumulation of succinate decreases cdc42 GTPase activity by induction of cdc42 succinylation, which inhibits the proliferation of neural stem cells and aggravates cerebral ischemia/reperfusion injury.

关键词： cdc42 cerebral ischemia/reperfusion injury GPR91 neural stem cells neurogenesis proliferation SIRT5 succinate succinylation

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Maternal miR-202-5p is required for zebrafish primordial germ cell migration by protecting small GTPase cdc42

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Journal of Molecular Cell Biology 2020年第7期12卷 530-542页

作者： Yilin Jin Wei Liu Yangxi Xiang Wanwan Zhang Hong Zhang Kuntong Jia Meisheng Yi School of Marine Sciences Sun Yat-sen University Guangzhou 519082China Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering Sun Yat-sen UniversityGuangzhou 519082China Southern Marine Science and Engineering Guangdong Laboratory(Zhuhai) Sun Yat-sen UniversityGuangzhou 519082China

In many lower animals,germ cell formation,migration,and maintenance depend on maternally provided determinants in germ *** zebrafish,these processes have been extensively studied in terms of RNA-binding proteins and other coding *** role of small non-coding RNAs in the regulation of primordial germ cell(PGC)development remains largely unknown and poorly investigated,even though growing interests for the importance of miRNAs involved in a wide variety of biological ***,we reported the role and mechanism of the germ plasm-specific miRNA miR-202-5p in PGC migration:(i)both maternal loss and knockdown of miR-202-5p impaired PGC migration indicated by the mislocalization and reduced number of PGCs;(ii)cdc42se1 was a direct target gene of miR-202-5p,and overexpression of cdc42se1 in PGCs caused PGC migration defects similar to those observed in loss of miR-202-5p mutants;(iii)cdc42se1 not only interacted with cdc42 but also inhibited cdc42 transcription,and overexpression of cdc42 could rescue PGC migration defects in cdc42se1 overexpressed ***,miR-202-5p regulates PGC migration by directly targeting and repressing cdc42se1 to protect the expression of cdc42,which interacts with actin to direct PGC migration.

关键词： primordial germ cell miR-202-5p cdc42se1 cdc42 migration

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Inactivation of cdc42 in embryonic brain results in hydrocephalus with ependymal cell defects in mice

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Protein & Cell 2013年第3期4卷 231-242页

作者： Xu Peng Qiong Lin Yang Liu Yixin Jin Joseph E.Druso Marc A.Antonyak Jun-Lin Guan Richard A.Cerione Department of Systems Biology and Translational Medicine College of MedicineTexas A&M Health Science CenterTempleTX 76504USA Department of Molecular Medicine Cornell UniversityIthacaNY 14853USA Department of Internal Medicine-Division of Molecular Medicine and Genetics and Department of Cell and Developmental Biology University of Michigan Medical SchoolAnn ArborMI 48109USA

The establishment of a polarized cellular morphology is essential for a variety of processes including neural tube morphogenesis and the development of the ***42 is a Ras-related GTPase that plays an essential role in controlling cell polarity through the regulation of the actin and microtubule cytoskeleton *** studies have shown that cdc42 plays an indispensable role in telencephalon development in earlier embryo developmental stage(before E12.5).However,the functions of cdc42 in other parts of brain in later embryo developmental stage or in adult brain remain ***,in order to address the role of cdc42 in the whole brain in later embryo developmental stage or in adulthood,we used Cre/loxP technology to generate two lines of tissue-specific cdc42-knock-out *** of cdc42 was achieved in neuroepithelial cells by crossing cdc42/flox mice with Nestin-Cre mice and resulted in hydrocephalus,causing death to occur within the postnatal *** analyses of the brains from these mice showed that ependymal cell differentiation was disrupted,resulting in aqueductal *** of cdc42 in the cerebral cortex also induced obvious defects in interki-netic nuclear migration and *** further explore the role of cdc42 in adult mice brain,we examined the effects of knocking-out cdc42 in radial glial cells by crossing cdc42/flox mice with human glial fi brillary acidic protein(GFAP)-Cre *** of cdc42 in radial glial cells resulted in hydrocephalus and ependymal cell *** together,these results highlight the importance of cdc42 for ependymal cell differentiation and maintaining,and suggest that these functions likely contribute to the essential roles played by cdc42 in the development of the brain.

关键词： cdc42 small GTPase neuron glial cell polarity development

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miRNA-145/miRNA-205 inhibits proliferation and invasion of uveal melanoma cells by targeting NPR1/cdc42

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International Journal of Ophthalmology(English edition) 2020年第5期13卷 718-724页

作者： Yang Li Jing-Ting Luo Yue-Ming Liu Wen-Bin Wei Beijing Tongren Eye Center Beijing Key Laboratory of Intraocular Tumor Diagnosis and TreatmentBeijing Key Laboratory of Ophthalmology and Visual SciencesBeijing Tongren HospitalCapital Medical UniversityBeijing 100730China

AIM:To investigate the role of microRNA-145(miRNA-145)and microRNA-205(miRNA-205)in proliferation and invasion of uveal melanoma(UM)***:The expression level of miRNA-145 and miR NA-205 from samples of UM patients were determined by real-time polymerase chain reaction(RT-PCR).The growth and invasion inhibitory effects were observed by the transfection of UM cells with miRNA-145 and *** epithelial-to-mesenchymal transition(EMT)-related proteins were screened by Western *** clinical samples from The Cancer Genome Atlas(TCGA)were applied to search for potential protein ***’s correlation analysis was applied to estimate co-expression between ***-luciferase reporter assay was used to verify the binding sites on target protein for miRNA-145 and ***:The expression levels of miRNA-145 and miRNA-205 in the samples from patients with UM were significantly lower than those in the normal tissue *** growth and invasion inhibitory effects were observed in human UM cells with miRNA-145 and miR NA-205 *** miRNA-145 and miRNA-205 could decrease the expression level of cell division control protein 42(cdc42).After database searching and sequence alignment,we identified that Neuropilin 1(NRP1)had binding sites for both miRNA-145 and ***:The miRNA-145 and miRNA-205 can reduce the proliferation,migration and invasion of UM cells by targeting the mRNA of its upstream protein NRP1 to down-regulate the expression level of cdc42.

关键词： uveal melanoma microRNA-145 microRNA-205 cdc42 NRP1

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cdc42介导乳腺癌紫杉醇耐药的初步研究

CDC42介导乳腺癌紫杉醇耐药的初步研究

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作者：应佳霏中南大学

学位级别：硕士

研究背景:乳腺癌是女性最常见的肿瘤,近几十年来,乳腺癌的发病率逐渐增加。即使乳腺癌早期诊断和治疗的方法很多,但依旧有许多患者存在极大的复发风险。cdc42是Rho家族的一个成员,研究证实cdc42与细胞增殖、谷氨酰胺代谢、细胞侵袭和迁... 详细信息

研究背景:乳腺癌是女性最常见的肿瘤,近几十年来,乳腺癌的发病率逐渐增加。即使乳腺癌早期诊断和治疗的方法很多,但依旧有许多患者存在极大的复发风险。cdc42是Rho家族的一个成员,研究证实cdc42与细胞增殖、谷氨酰胺代谢、细胞侵袭和迁移、调节细胞极性等过程有关。cdc42与乳腺癌细胞的运动及化疗耐药密切相关,探究乳腺癌细胞中cdc42的表达及cdc42对乳腺癌化疗耐药的具体机制,有望为乳腺癌治疗提供新的抗癌靶点。研究目的:本文旨在通过生物信息学方法探究cdc42与乳腺癌化疗耐药之间的关系,并通过细胞实验进行验证。研究方法:通过生物信息学方法探究cdc42基本性质、在乳腺组织中的表达水平、与乳腺癌治疗敏感性和预后之间的关系,通过细胞实验验证cdc42与乳腺癌化疗耐药的关系。以MDA-MB-231细胞构建PTX耐药细胞MDA-MB-231/PTX,构建cdc42过表达细胞MDA-MB-231/cdc42-OE,加入cdc42选择性抑制剂ML141制备MDA-MB-231/PTX/ML141细胞。PCR及Western Blot分别检测耐药基因、cdc42、GLS1的表达水平,PTX处理各组细胞,CCK-8检测各组细胞活性。研究结果:cdc42过表达是乳腺癌预后不良的危险因素之一,并且可导致乳腺癌细胞对紫杉类和蒽环类药物敏感性降低。细胞实验验证cdc42介导了乳腺癌的紫杉醇耐药,并且耐药机制可能与谷氨酰胺代谢有关。cdc42、GLS1在MDA-MB-231/PTX细胞中表达水平显著升高,cdc42可降低乳腺癌细胞对PTX的敏感性。研究结论:cdc42的表达上调与乳腺癌发生发展及不良预后相关,cdc42介导了乳腺癌的化疗耐药,并且耐药机制可能与谷氨酰胺代谢有关。cdc42具有成为乳腺癌治疗新的抗癌靶点的潜力。全文图34幅,表格4个。参考文献50篇

关键词： cdc42 乳腺癌化疗耐药生物信息分析

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cdc42在谷氨酰胺介导胰岛素合成与分泌中的作用及其分子机制

Cdc42在谷氨酰胺介导胰岛素合成与分泌中的作用及其分子机制

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作者：刘丽欣南昌大学

学位级别：硕士

目的:糖尿病（DM）的发生与胰岛素绝对或相对不足有关,它是一种以高血糖为特征的进行性代谢紊乱疾病。研究表明,细胞分裂周期蛋白cdc42在胰岛素的合成与分泌中起关键作用。此外,非必需氨基酸-谷氨酰胺可能参与胰岛素的合成与分泌,其具... 详细信息

目的:糖尿病（DM）的发生与胰岛素绝对或相对不足有关,它是一种以高血糖为特征的进行性代谢紊乱疾病。研究表明,细胞分裂周期蛋白cdc42在胰岛素的合成与分泌中起关键作用。此外,非必需氨基酸-谷氨酰胺可能参与胰岛素的合成与分泌,其具体机制尚不明确。我们旨在探讨cdc42在谷氨酰胺介导胰岛素合成与分泌中的作用及其分子机制,为糖尿病的防治提供新的策略。方法:1.特异性敲除胰岛β细胞cdc42小鼠的制备和基因型鉴定:利用cdc42-Flox小鼠与RIP-CRE小鼠（携带大鼠胰岛β细胞特异性胰岛素启动子的CRE重组酶）交配繁殖获得胰岛β细胞特异性敲除cdc42的小鼠。通过PCR对小鼠尾尖DNA进行基因型鉴定。利用胶原酶P（CLG）法手工挑选金黄色胰岛细胞团,进行Western blot和RT-q PCR实验,检测胰岛细胞中cdc42蛋白和m RNA敲除情况。2.小鼠葡萄糖耐量实验小鼠饥饿处理16h（禁食不禁水）,首先检测空腹（0min）血糖浓度,然后腹腔注射20%葡萄糖溶液（2g/kg）5min后,注射7.5%谷氨酰胺（Ala-GLN）溶液（0.75g/kg）,检测15min、30min的血糖浓度。3.小鼠胰岛素合成与分泌的检测RT-q PCR检测小鼠胰岛细胞系β-TC6细胞胰岛素m RNA表达水平;体外分离小鼠胰岛细胞团,进行Western blot和ELISA试剂盒检测胰岛素的合成与分泌。4.检测胰岛β细胞中谷氨酰胺酶表达情况使用cdc42特异性抑制剂ML141,配合不同浓度葡萄糖或谷氨酰胺刺激小鼠胰岛细胞系β-TC6细胞6h,利用免疫荧光检测细胞内谷氨酰胺酶（GLS1）和胰岛素的表达;体外分离小鼠（F/F+＆F/F-）胰腺组织,经固定、脱水、包埋、切片、染色,进行免疫组化,检测胰岛细胞内GLS1和胰岛素的分布情况。5.检测调控谷氨酰胺酶表达的相关蛋白1)使用cdc42特异性抑制剂ML141处理或PCMV6-cdc42（T17N）-Flag质粒（显性失活型）转染小鼠胰岛细胞系β-TC6细胞,或体外分离小鼠（F/F+＆F/F-）胰岛,不同浓度葡萄糖或谷氨酰胺刺激相应时间,利用Western blot检测和比较PAK1、P38和STAT3蛋白磷酸化表达情况。2)采用谷氨酰胺酶抑制剂968处理小鼠胰岛细胞系β-TC6细胞,利用Western blot检测P38和STAT3蛋白磷酸化表达情况。结果:1.成功构建胰岛β细胞特异性敲除cdc42小鼠cdc42-Flox小鼠与RIP-CRE小鼠交配成功,获得胰岛β细胞特异性敲除cdc42的小鼠。同时带有Lox P和CRE序列为特异性敲除鼠（F/F+＆KO）;仅带有Lox P序列为对照鼠（F/F-＆WT）。Western blot和RT-q PCR实验结果显示,敲除组小鼠的胰岛细胞中cdc42的蛋白和m RNA水平明显低于对照组,表明特异性敲除胰岛β细胞cdc42的小鼠构建成功。2.胰岛β细胞缺失cdc42明显降低谷氨酰胺在小鼠葡萄糖耐量反应中的敏感性和协同作用,减少胰岛素的合成与分泌小鼠葡萄糖耐量和ELISA实验结果显示:谷氨酰胺明显改善腹腔注射葡萄糖介导的小鼠糖耐量反应,明显增加胰岛素分泌;但胰岛β细胞缺失cdc42（KO）小鼠的血糖值未得到改善,血液中胰岛素含量显著下调,且谷氨酰胺与葡萄糖共同介导的胰岛组织中胰岛素含量明显减少。3.抑制或敲除cdc42显著抑制胰岛β细胞中谷氨酰胺酶和胰岛素表达抑制cdc42活性后,无论低糖、高糖还是谷氨酰胺刺激,胰岛β细胞中谷氨酰胺酶和胰岛素的表达均显著下降;胰岛β细胞特异性敲除cdc42小鼠胰腺组织中谷氨酰胺酶和胰岛素表达明显减少。4.抑制或敲除cdc42显著降低胰岛β细胞中P38和STAT3的磷酸化抑制cdc42活性显著降低胰岛β细胞PAK1、P38和STAT3磷酸化;胰岛β细胞缺失cdc42基因后,胰岛P38磷酸化也呈现相同趋势。5.抑制谷氨酰胺酶活性对胰岛β细胞中STAT3的磷酸化水平无明显影响Western blot结果显示:968抑制GLS1活性后,P38磷酸化显著降低,但STAT3磷酸化未见明显变化。结论:我们的研究表明,胰岛β细胞缺失cdc42显著减弱谷氨酰胺改善机体葡萄糖耐量以及谷氨酰胺与葡萄糖协同促进胰岛素合成与分泌的作用。胰岛β细胞缺失cdc42或活性减弱显著抑制胰岛β细胞表达谷氨酰胺酶和胰岛素。cdc42可能通过信号转导与转录激活因子3（STAT3）参与调控胰岛β细胞中谷氨酰胺酶的表达,进而通过谷氨酰胺代谢影响胰岛素的合成与分泌。

关键词： cdc42 谷氨酰胺酶谷氨酰胺代谢胰岛素

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