探索质子感知受体OGR1在癌细胞中的作用
发布时间：2021-04-16 19:10
质子感知卵巢癌G蛋白偶联受体1（OGR1）属于G蛋白偶联受体超家族OGR1亚家族4个成员（TDAG8,OGR1,G2A和GPR4）之一,在肿瘤及炎性等病理酸性条件下被激活而调节细胞的生理病理过程。病理酸性微环境的形成与细胞的糖酵解途径增强有关,而这个过程是由低氧诱导因子（HIF）的过度激活及其诱导多种靶基因的表达来决定的。我们对肿瘤大数据进行分析研究后发现,HIF诱导的基因中也包含OGR1,所以我们推测OGR1可能与肿瘤细胞适应低氧或酸性环境的生存能力有关。在该研究中我们将探索OGR1与肿瘤在酸性环境中存活、转移和凋亡等复杂生物学特性之间的关系,阐明OGR1的生理功能和病理机制,为肿瘤细胞抵抗酸性的本质方面以及在肿瘤治疗战略方面提出新的观点。在研究中,首先克隆了人类OGR1基因并构建OGR1原核表达载体。继而又构建OGR1真核表达质粒pHBLV-CMV-EF1中。通过酶消化和测序鉴定确认正确后。用lipofectamine2000将质粒转染到A549肺癌细胞株中。用实时荧光定量PCR基因检测技术来检测A549细胞中外源ORG1基因的表达。用Western Blot法检测蛋白表达;在研究...
【文章来源】：内蒙古大学内蒙古自治区 211工程院校
【文章页数】：72 页
【学位级别】：硕士
【文章目录】：
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
【参考文献】：
期刊论文
[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)
本文编号：3142007
【文章来源】：内蒙古大学内蒙古自治区 211工程院校
【文章页数】：72 页
【学位级别】：硕士
【文章目录】：
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
【参考文献】：
期刊论文
[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)
本文编号：3142007
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