Role of Proton Sensing receptor OGR1 in Lung cancer cells

声明

ABBREVIATIONS

ChapterⅠ:G-protein coupled receptors(GPCRs)receptors and ovarian cancer G protein-coupled receptor (Literature review)

1.1G-protein coupled receptors (GPCRs)

1.2Ovarian cancer G protein-coupled receptor (OGR1)

1.3Theextracellular pHand cancer

1.4The intracellular pH and cancer

Chapter Ⅱ: 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

2.2.2Preparation of solid medium(Amp resistance)

2.2.3Preparation of 50×TAE electrophophoresis buffer

2.2.4Preparation of 1% agarose gel

2.2.5Extraction of total RNA

2.2.6Cloning of OGR1 gene

2.2.7Enzymatic digestion of OGR1 genes and vectors

2.2.8Ligation of OGR1 genes and vectors

2.2.9 Transforming OGR1-CMV recombinant vector into DH5α E.coli

2.2.10Extraction of recombinant plasmid

2.2.11 Enzyme digestion verification

2.2.12 Recovery of A549 cells

2.2.13 Propagation of A549 cells

2.2.14Lentivirus transfection into A549 cells

2.2.15Detection of OGR1 gene expression by Real-Time PCR

2.2.16Western blot detection of OGR1 protein expression

2.2.17Detection of second messenger Ca2+

2.2.18 Proliferation

2.2.19 Wound-healing test

2.2.20 Apoptosis

2.2.21Animal experiment method

Chapter Ⅲ: Results and Analysis

3.1 Cloning of OGR1 gene and construction of eukaryotic expression vector

3.1.1Amplification of OGR1 gene

3.1.2Double digestion and identification of OGR1 recombinant vector

3.1.3Sequencing results and bioinformatics analysis of OGR1 gene

3.2.1 A549 cell resuscitation

3.2.2A549 cell transfected with vector CMV and CMV-OGR1

3.2.3Detection of OGR1 gene expression by Real-Time PCR

3.2.4Comparison of four expression sensory receptors in lung cancer cell

3.2.5Western blot detection of protein activity

3.2.6Calcium measurement

3.3 Study on the expression of OGR1 enhances cell survival and proliferation under acidic conditions as well in vivo and in vitro

3.3.1Effect of OGR1 on cell proliferation

3.3.2 Effect of OGR1on cell apoptosis

3.3.3Wound healing test results(migration)

3.3.4 Animal experiment results

Discussion

参考文献