| PART ONECONSTRUCTION AND IDENTIFICATION OF THEEUKARYOTIC EXPRESSION VECTOR OF CILP2GENEObjective: To construct and identify the human recombinant plasmidpIRES2-EGFP-CILP2-Flag.Methods: RNA was extracted from HEK293cells and cDNA wasobtained by reverse transcript-polymerase chain reaction (RT-PCR).CILP2-Flag fragment DNA was obtained by PCR with high fidelityenzyme and digested with BglII and EcoRI enzyme. After purification,CILP2-Flag was ligased with pIRES2-EGFP vector. The recombinantplasmid was identified by double enzymes digestion and DNA sequenceanalysis.Results: After BglII and EcoRI digestion, the products weredetermined by agarose gel electrophoresis, and there showed two bands at3.5kb and5.3kb, respectively, which was accordance with the molecularweight of CILP2and plasmid fragments. Sequencing results were analyzed by DNA Blast and the nucleotide sequence was matched with the CDScode sequence of human CILP2gene completely in Genbank. We hadsuccessfully constructed the recombinant plasmid.Conclusion: The recombinant plasmid of human pIRES2-EGFP-CILP2-Flag has been constructed successfully. It will help to explore thephysiological functions of CILP2gene. PART TWODISTRIBUTION OF CILP2GENE IN DIFFERENTTISSUES OF MICE AND DIFFERENTIAL EXPRESSIONOF CILP2IN VARIOUS PATHOLOGICAL MODELSObjective: To observe mRNA distribution of cilp2gene in differenttissues of healthy C57BL/6J mice and the differential mRNA and proteinexpression of cilp2in ApoE-/-mice and patients with CAD.Methods: The12-week-old C57BL/6J mice were killed by cervicaldislocation and RNA were extracted from heart, liver, kidney, brain, spleen,lung, stomach, intestine, and muscle, and reversed transcripted into cDNA.Real-time fluorescent quantitative PCR was performed to measure themRNA levels of cilp2in different tissues and repeated three times. Muscle、 aorta and coronary arteries tissues were obtained from male12-week-oldApoE-/-mice and C57BL/6J mice, cilp2mRNA and protein expressionwere measured by RT-PCR and western blot; Muscle tissues were obtainedfrom patients with CAD and controls, CILP2mRNA and protein weremeasured by RT-PCR and western blot.Results: The cilp2gene highly expressed in muscle, followed by theheart, brain, kidney, lung, stomach, spleen, liver and intestine; the cilp2mRNA and protein expression of muscle and coronary ateries in ApoE-/-mice were higher than that in C57BL/6J; the CILP2mRNA and proteinexpression of muscle tissue in patients with CAD were higher than that incontrols.Conclusion: The cilp2gene highly expresses in atherosclerosis modeland may involve in the development of atherosclerosis. PART THREEIDENTIFICATION OF CILP2AS A SECRETED PROTEINObjective: To verify CILP2as a secreted protein.Methods: HEK293T cells were transfected with pIRES2-CILP2-Flagplasmid and empty pIRES2-EGFP vector for48hours, culture medium andcell lysate were immunoprecipitated with M2-Flag antibody beads, precipitates were separated by polyacrylamide gel electrophoresis,immunoblotted with CILP2antibody and then detected by western blotting;tree hundred microliter of plasma from3individuals was diluted30-foldwith PBS, the protein level of CILP2in normal and coronary heart diseasepatients were detected by10%SDS-PAGE western blotting.Results: Culture medium of HEK293T cells transfected CILP2-Flagplasmid contained CILP2protein; CILP2protein can be detected in humanplasma; CILP2protein had a higher expression level in CAD patients thanin normal.Conclusion: CILP2is a secreted protein and have a higher expressionin CAD patients than normal. PART FOREEFFECT OF CILP2ON THP1DERIVED FOAM CELLFORMATIONObjective: To investigate the possible pathogenic roles of CILP2inTHP1foam cell formation.Methods: THP1macrophages were stimulated by oxLDL in differentconcentration and time, and CILP2mRNA were detected by RT-PCR.CILP2adenovirus infected THP1macrophages48hours and SR-AI, CD36, CD68, LOX-1, TF and PAI-1mRNA were detected by RT-PCR. THP1macrophages were costimulated with oxLDL and CILP2, the accumulationof neutral lipids was determined by red O staining.Results: CILP2mRNA expression in THP1macrophages wereincreased in a dose-and time-dependent manner accompanied with theoxLDL, and reached the peak at20ug/ml and24h, respectively; scavengerreceptors SR-AI and CD68mRNA elevated significantly in THP1macrophages infected CILP2adenovirus for48hours compared to controls(p<0.05); Oil red O staining showed that in the presence of oxLDL, lipidaccumulation in the THP1macrophages infected CILP2increasedsignificantly (p<0.05); However, there were no significant differences inTF and PAI-1mRNA expression.Conclusion: CILP2may contribute to foam cell formation bystimulating the expression of SR-AI and CD68and the accumulation oflipids. |
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