Correlation Of Gene NALP1to Tumorigenesis And Development Of Osteosarcoma

Osteosarcoma is a sort of highly malignant tumors origin of mesenchymal tissue，highincidence of adolescence，high rate of early metastasis and poor prognosis.However，thecauses of osteosarcoma is not clear and the main treatment are surgery andchemotherapy.Gene NALP1is an important immune molecules recently discovered，whichcontaining death domain.The function of gene NALP1is involved in the inflammatoryresponse and apoptosis.In this research，the main purpose is to study the expression of gene NALP1inosteosarcoma cells，and affection in osteosarcoma cells in which the expression of geneNALP1was raised.To explain concretely，we divided the experiment into three parts.Part1，we identify the different mRNA and protein expression of gene NALP1among thehuman osteosarcoma cell lines MG-63、 U-2OS and human osteoblast cell linehFOB1.19.Part2，we construct the model of osteosarcoma cells which overexpressing geneNALP1.Part3，we analyse the difference between the overexpressing NALP1and thenomal human osteosarcoma cell in experiment including vitro migration and vitroapoptosis.Part One The different expression of gene NALP1among the human osteosarcomacell lines MG-63、U-2OS and human osteoblast cell line hFOB1.19Objective：To identify the different expression of gene NALP1among the humanosteosarcoma cell lines MG-63、U-2OS and human osteoblast cell line hFOB1.19.Methods：Take the human osteosarcoma cell lines MG-63、U-2OS and the humanosteoblast cell line hFOB1.19to the object of study.Real time-polymerase chain reaction（RT-PCR）and Western blot were used for detecting the mRNAand protein expression ofof gene NALP1.Results：The expression of mRNA and protein of gene NALP1in thehuman osteosarcoma cell lines MG-63、U-2OS is lower obviously than that in the humanosteoblast cell line hFOB1.19. Conclusion：NALP1expression is down-regulated in thehuman osteosarcoma cell.Part Two Construct the model of osteosarcoma cells which overexpressing gene NALP1Objective：Construct NALP1cDNA eukaryotic expression plasmid，then transfect therecombinant plasmid into the human osteosarcoma cell lines MG-63、U-2OS.Methods：Aeukaryotic expression recombinant plasmid for pcDNA3.1/NALP1was constructed byusing of recombinant DNA technique and was transfected into the human osteosarcomacell lines MG-63、U-2OS by using liposome-mediated method.The mRNA and protein expression of NALP1were detected by RT-PCR and Western-blot.Results： Therecombinant eukaryotic expression plasmid was demonstrated by sequencing.Theexpression of NALP1in osteosarcoma cell up-regulation of NALP1was detected byRT-PCR and Western-blot methods.Conclusion：The recombinant plasmid was constructedsuccessfully and transfected the human osteosarcoma cell lines correctly.We got theosteosarcoma cells up-regulation of NALP1.PartThree Vitro migration and vitro apoptosis experiment in osteosarcoma cellsup-regulation of NALP1Objective：To investigate the affection in osteosarcoma cell up-regulation of NALP1we used the experiment of vitro migration and vitro apoptosis.Methods：Application ofTranswell experiment to test the ability of migration in MG-63transfected by recombinantplasmid. Application of flow cytometry to investigate the apoptosis in MG-63and U-2OStransfected by recombinant plasmid，compared with two groups，one is MG-63and U-2OStransfected by empty plasmid，another is MG-63and U-2OS.Results：The migrative abilityof the MG-63cells transfected by recombinant plasmid decreased obviously. The earlyapoptosis rate in MG-63and U-2OS transfected by recombinant plasmid was higherobviously than the compared groups.Conclusion：Overexpressed NALP1in osteosarcomacell would decrease the ability of migration and induce apoptosis of osteosarcoma cell，which also could improve the sensitivity of osteosarcoma cell to the chemotherapy drugmethotrexate.