質(zhì)子感知卵巢癌G蛋白偶聯(lián)受體1（OGR1）屬于G蛋白偶聯(lián)受體超家族OGR1亞家族4個成員（TDAG8,OGR1,G2A和GPR4）之一,在腫瘤及炎性等病理酸性條件下被激活而調(diào)節(jié)細(xì)胞的生理病理過程。病理酸性微環(huán)境的形成與細(xì)胞的糖酵解途徑增強(qiáng)有關(guān),而這個過程是由低氧誘導(dǎo)因子（HIF）的過度激活及其誘導(dǎo)多種靶基因的表達(dá)來決定的。我們對腫瘤大數(shù)據(jù)進(jìn)行分析研究后發(fā)現(xiàn),HIF誘導(dǎo)的基因中也包含OGR1,所以我們推測OGR1可能與腫瘤細(xì)胞適應(yīng)低氧或酸性環(huán)境的生存能力有關(guān)。在該研究中我們將探索OGR1與腫瘤在酸性環(huán)境中存活、轉(zhuǎn)移和凋亡等復(fù)雜生物學(xué)特性之間的關(guān)系,闡明OGR1的生理功能和病理機(jī)制,為腫瘤細(xì)胞抵抗酸性的本質(zhì)方面以及在腫瘤治療戰(zhàn)略方面提出新的觀點。在研究中,首先克隆了人類OGR1基因并構(gòu)建OGR1原核表達(dá)載體。繼而又構(gòu)建OGR1真核表達(dá)質(zhì)粒pHBLV-CMV-EF1中。通過酶消化和測序鑒定確認(rèn)正確后。用lipofectamine2000將質(zhì)粒轉(zhuǎn)染到A549肺癌細(xì)胞株中。用實時熒光定量PCR基因檢測技術(shù)來檢測A549細(xì)胞中外源ORG1基因的表達(dá)。用Western Blot法檢測蛋白表達(dá);在研究... 

【文章來源】：內(nèi)蒙古大學(xué)內(nèi)蒙古自治區(qū) 211工程院校

【文章頁數(shù)】：72 頁

【學(xué)位級別】：碩士

【文章目錄】：
摘要
abstract
ABBREVIATIONS
CHAPTERⅠ: （LITERATURE REVIEW）G-PROTEIN COUPLED RECEPTORS（GPCRS）RECEPTORS AND OVARIAN CANCER G PROTEIN-COUPLED RECEPTOR
    1.1 G-PROTEIN COUPLED RECEPTORS（GPCRS）
    1.2 OVARIAN CANCER G PROTEIN-COUPLED RECEPTOR（OGR1）
    1.3 THE EXTRACELLULAR PH AND CANCER
    1.4 THE INTRACELLULAR PH AND CANCER
CHAPTERⅡ:MATERIALS AND METHODS
    2.1 EXPERIMENTAL MATERIALS AND MAIN REAGENTS
        2.1.1 MAIN REAGENTS AND CELLS
        2.1.2 MAJOR INSTRUMENTS AND EQUIPMENTS
    2.2 EXPERIMENTAL METHODS
        2.2.1 PREPARATION OF LB CULTURE MEDIUM
        2.2.2 PREPARATION OF SOLID MEDIUM（AMP RESISTANCE）
        2.2.3 PREPARATION OF50×TAE ELECTROPHOPHORESIS BUFFER
        2.2.4 PREPARATION OF1%AGAROSE GEL
        2.2.5 EXTRACTION OF TOTAL RNA
        2.2.6 CLONING OF OGR1 GENE
        2.2.7 ENZYMATIC DIGESTION OF OGR1 GENES AND VECTORS
        2.2.8 LIGATION OF OGR1 GENES AND VECTORS
        2.2.9 TRANSFORMING OGR1-CMV RECOMBINANT VECTOR INTO DH5ΑE.COLI
        2.2.10 EXTRACTION OF RECOMBINANT PLASMID
        2.2.11 ENZYME DIGESTION VERIFICATION
        2.2.12 RECOVERY OF A549 CELLS
        2.2.13 PROPAGATION OF A549 CELLS
        2.2.14 LENTIVIRUS TRANSFECTION INTO A549 CELLS
        2.2.15 DETECTION OF OGR1 GENE EXPRESSION BY REAL-TIME PCR
        2.2.16 WESTERN BLOT DETECTION OF OGR1 PROTEIN EXPRESSION
        2.2.17 DETECTION OF SECOND MESSENGER CA2+
        2.2.18 PROLIFERATION
        2.2.19 WOUND-HEALING TEST
        2.2.20 APOPTOSIS
        2.2.21 ANIMAL EXPERIMENT METHOD
CHAPTERⅢ:RESULTS AND ANALYSIS
    3.1 CLONING OF OGR1 GENE AND CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR
        3.1.1 AMPLIFICATION OF OGR1 GENE
        3.1.2 DOUBLE DIGESTION AND IDENTIFICATION OF OGR1 RECOMBINANT VECTOR
        3.1.3 SEQUENCING RESULTS AND BIOINFORMATICS ANALYSIS OF OGR1 GENE
    3.2 ESTABLISHMENT OF A549-OGR1 CELL LINE OVEREXPRESSING OGR1 GENE
        3.2.1 A549 CELL RESUSCITATION
        3.2.2 A549 CELL TRANSFECTED WITH VECTOR CMV AND CMV-OGR
        3.2.3 DETECTION OF OGR1 GENE EXPRESSION BY REAL-TIME PCR
        3.2.4 COMPARISON OF FOUR EXPRESSION SENSORY RECEPTORS IN LUNG CANCER CELL
        3.2.5 WESTERN BLOT DETECTION OF PROTEIN ACTIVITY
        3.2.6 CALCIUM MEASUREMENT
    3.3 STUDY ON THE EXPRESSION OF OGR1 ENHANCES CELL SURVIVAL AND PROLIFERATION UNDER ACIDIC CONDITIONS AS WELL IN VIVO AND IN VITRO
        3.3.1 EFFECT OF OGR1ON CELL PROLIFERATION
        3.3.2 EFFECT OF OGR1ON CELL APOPTOSIS
        3.3.3 WOUND HEALING TEST RESULTS（MIGRATION）
        3.3.4 ANIMAL EXPERIMENT RESULTS
Conclusion
DISCUSSION
REFERENCES


【參考文獻(xiàn)】：
期刊論文
[1]Underlying mechanism of ASIC1a involved in acidosis-induced cytotoxicity in rat C6 glioma cells[J]. Xie-chuan WENG,Jian-quan ZHENG~2,Jin LI,Wen-bin XIAOBeijing Institute of Pharmacology and Toxicology,Beijing 100850,China.  Acta Pharmacologica Sinica. 2007(11)



本文編號：3142007