Effect Of Transfection MA20 And Its Mutant On Endotoxemia And Experimental Study Of HA20 Mutant

Endotoxemia is the presence of endotoxins in the blood which derived from gram-negative rod-shaped bacteria in the blood or in focal infection or from lipopolysaccharide(LPS)-contaminant fluid infusion. The endotoxins in blood may lead to inflammatory factors actvation and cytokines release, inducing inflammation and anti-inflammation unbalance, resulting in systemic inflammatory response syndrome(SIRS), multi-organ disfunction syndrome ( MODS) and even multi-organ failure(MOF). Endotoxemia is the one of the major cause resulting in the death of pediatrics trauma and infectious diseases. At present, the therapeutic measures directed to endotoxemia include: antibiotics administration, immunotherapy of antiendotoxin antibodies and anticytokin antibodies, intestinal tract barria protection and so on so forth in domestic and abroard. Even though therapeutic measures continue to be up-to-dated and much improvement of support and symptomatic treatment of endotoxemia have been made, endotoxemia is still a stronghold to overcome for medical workers. The fundamental measures of endotoxemia treatment is to effectively control the waterfall-like effect of endotoxemia induced cytokins cascade amplification.Zinc finger protein A20 is a ubiquitin-editing protein that present in the endochylema. It has been already a hot topic of anti-inflammatory and anti-apoptotic research. A study showed that the inhibition effect on TNF-induced nuclear factor-κB(NF-κB) activation of A20 protein with only four zinc fingers was compatible to that of wild-type A20 protein with 7 zinc fingers. Studies showed no strict requirement for a particular zinc finger structure, since the first four zinc fingers mutant A20 protein is as potent as the last four zinc fingers mutant protein. Another study showed that A20 inhibited NF-κB activation by dual ubiquitin-editing (de-ubiquitination and ubiquitination) of its N-terminal end and C-terminal end. Zinc finger protein A20 inhibits NF-κB activation through specific signal pathways, then suppress the inflammatory mediators and cytokines releasing , and participates in the regulation of inflammatory reaction and cell apoptosis.The aim of this study is to provide an animal experiment data for clinical application through inhibiting excess endotoxemia-induced inflammatory mediators, terminating"waterfall-like"effect of cascade amplification of inflammation, correcting inflammatory reaction imbalance, and preventing endotoxemia from aggravated development by eukaryotic expression vector pCAGGS-flagmA20 and pCAGGS-flagmA20-ZnF4-7 coding mA20 and its mutant mA20-ZnF4-7 in gene level. To explore the feasibility of artificial synthesizing this neotype anti-inflammatory protein, we are planning to apply the idea of molecular designing to make a further design for A20. Successful construction of hA20 mutant plasmid pcDNA3Flag-hA20N-ZnF4-7 may achieved by protecting large portion of N-terminal end and whole C-terminal end, and deleting the gene between number 332 and 1778, and may identified through cells expression of lipsome-mediated pcDNA3hA20 and pcDNA3-FlaghA20N-ZnF4-7. It is prospected to pave a nicely path for producing anti-endotoxemia medicine.PARTⅠEFFECT OF HYDRODYNAMICS-BASED DELIVERY OF MA20 AND ITS MUTANTS PLASMID ON EXPERIMENTAL ENDOTOXEMIA IN MICEObjective To study the therapeutic effect of mA20 and its mutant mA20-ZnF4-7 plasmid in endotoxemia mice of lipopolysaccharide (LPS)- assaulting.Methods The endotoxemia animal model was established by injection of 2.5mg/kg LPS via the tail vein. The animals were randomly divided into 5 groups: group A: normal saline(NS) control, group B: LPS-assaulting, group C: naked plasmid treatment, group D: mA20 plasmid-treatment, group E: mA20-ZnF4-7 plasmid-treatment. Hydro- dynamics-based gene transfection method was achieved by intravenous injection of 10μg plasmid DNA in 1.6ml saline 30 minutes after LPS injection. The contents of plasma TNF-αand IL-1βwere detected by ELISA. The contents of WBC mA20 and its mutant mA20-ZnF4-7 mRNA were detected by Reverse transcription and polymerase chain reaction (RT-PCR). The tissue inflammatory histopathologic changes were observed under microscope.Results (1) The TNF-αexpressions of mA20 and mA20-ZnF4-7 in plasmid-treatment group at the time point of 6h, 12h and 24h were(pg/ml): (201.797±4.789), (236.580±7.497), (154.551±16.460) and(235.710±6.108), (255.130±7.827), (223.246±11.316) respectively, which were significantly lower than those in LPS- assaulting group and naked plasmid group (p<0.05). The IL-1βexpressions of mA20 and mA20-ZnF4-7 in plasmid-treatment group at the time point of 6h, 12h, 24h were(pg/ml): (87.103±3.84), (83.655±4.973), (77.678±6.555 and (101.586±2.486), (94.460±3.110), (87.103±4.826) respectively, which were significantly lower than those in LPS-assaulting group and naked plasmid group (p<0.05). (2) The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in mA20 plasmid-treatment group and mA20-ZnF4-7 plasmid-treatment group were significantly higher than those in LPS-assaulting group, naked plasmid group and normal saline group (p<0.05). The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in LPS-assaulting group and naked plasmid group were significantly higher than those values in normal saline group (p<0.05). No significant difference was found between every time point.(3) Pathological changes under microscope showed that the inflammatory damages in mA20 plasmid-treatment group and in mA20-ZnF4-7 plasmid-treatment group were lighter than those in LPS-assaulting group and naked plasmid group. No significant differences were found between mA20 plasmid-treatment group, mA20-ZnF4-7 plasmid- treatment group and NS group.Conclusions Hydrodynamics-based gene transfection can efficiently deliver plasmid DNA into livers and lungs. Transfection of mA20-ZnF4-7 plasmid showed similar therapeutic effect to mA20 plasmid on experimental endotoxemia in mice, which may provide valuable experimental base for clinical application. PARTⅡPREVENTIIVE EFFECT OF EXPERIMENTAL ENDOTOXEMIA BY HYDROD-YNAMICS-BASED DELIVERY OF MA20 AND ITS MUTANTS PLASMIDObjective To study the preventive effect of zinc finger protein mA20 and its mutant mA20-ZnF4-7 on endotoxemia mice of lipopolysaccharide (LPS)-assaulting.Methods The endotoxemia animal model was established by injection of 2.5 mg/kg LPS via the tail vein. The animals were randomly divided into 5 groups: group A: normal saline(NS) control, group B: LPS-assaulting, group C: naked plasmid treatment, group D: mA20 plasmid-prevention, group E: mA20-ZnF4-7 plasmid-prevention. Hydro- dynamics-based gene transfection method was achieved by intravenous injection of 10μg plasmid DNA in 1.6ml saline. The contents of plasma TNF-αand IL-1βwere detected by ELISA. The contents of WBC mA20 and its mutant mA20-ZnF4-7 mRNA were detected by RT-PCR. The tissue inflammatory histopathologic changes were observed under microscope.Results (1) The TNF-αexpressions in group D and group E at the time point of 6h, 12h, and 24h were(pg/ml): (201.797±4.789), (236.580±7.497), (154.551±16.460),and(235.710±6.108), (255.130±7.827), 223.246±11.316, respectively, which were significantly lower than those in group B and group C. The IL-1βexpressions in group D and group E at the time point of 6h, 12h, and 24h were(pg/ml): (87.103±3.84), (83.655±4.973), (77.678±6.555), and (101.586±2.486), (94.460±3.110), (87.103±4.826)respectively, which were significantly lower than those in group B and group C. (2) The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in group D and group E were significantly higher than those in group A, group B and group C (p<0.05). The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in group B and group C were significantly higher than those in normal saline group(p<0.05). No significant difference was found between each time point. (3) Microscope observation of pathological section The inflammatory damage under microscope showed milder changes in group D and group E than those in group B and group C. No significant differences were found between group D, group E and group A.Conclusions (1) Hydrodynamics-based gene transfection is a simple, convenient, economic and efficient method in delivering plasmid DNA into liver and lung. (2) Transfection of mA20-ZnF4-7 plasmid in vitro shows similar preventive effect to mA20 plasmid on experimental endotoxemia in mice, which may provide valuable experimental base for clinical application. PARTⅢTHEAPEUTIC EFFICACY OF LIPOSOME ENCAPSULATING MA20 AND ITSMUTANTS PLASMID TRANSFECTION IN EXPERIMENTAL ENDOTOXEMIA IN MICEObjective To study the therapeutic effect of liposome encapsulating mA20 and its mutant mA20-ZnF4-7 plasmid in mice with lipopolysaccharides (LPS) induced endotoxemia.Methods Forty-five healthy mice weighing 18-20g were randomized into five groups: normal saline control group (NS group), LPS-assaulting group, naked plasmid treatment group, liposome encap-sulating mA20 plasmid treatment group (L-mA20 group) and liposome encapsulating mA20-ZnF4-7 plasmid treatment group (L-mA20? group). The contents of plasma TNF-αand IL-1βwere detected by ELISA. The contents of WBC mA20 and its mutant mA20-ZnF4-7mRNA were detected by RT-PCR. The tissue inflammatory histopathologic changes were observed under microscope.Results (1)The TNF-αexpressions of L-mA20 and L-mA20? group at the time point of 6h, 12h and 24h were(pg/ml): (304.696±6.087), (321.217±10.579), (307.594±11.181) and (304.985±6.291), (256.580± 33.524), (280.348±11.698)respectively, which were significantly lower than those in LPS-assulting group and in naked plasmid treatment group (p<0.05). The IL-1βexpressions in L-mA20 treatment group and in L-mA20? treatment group at the time point of 6h, 12h, 24h were(pg/ml): (256.989±3.921), (205.724±4.827), (165.724±7.772) and (263.655±5.645), (154.690±1.380), (101.816±3.259) respectively, which were significantly lower than those in LPS-assulting group and in naked plasmid treatment group (p<0.05). (2) The expressions of mA20 and its mutant mA20-ZnF4-7 mRNA in mA20 plasmid-treatment group and mA20- ZnF4-7 plasmid-treatment group were significantly higher than those in LPS-assulting group, naked plasmid group and normal saline group (p<0.05). The mA20 and its mutant mA20-ZnF4-7 mRNA expressions in LPS-assulting group and in naked plasmid group were significantly higher than those in normal saline group (p<0.05). No significant differences were found between each time point. (3) Severe inflammatory damages were found in LPS-assulting group and naked plasmid treatment group, but only mild inflammatory changes were found in L-mA20 treatment group and in L-mA20? treatment group. There were no significant histopathological differences among groups of L-mA20 treatment group, L-mA20? treatment group and NS group.Conclusions mA20-ZnF4-7 plasmid transfection with liposome in vitro can achieve similar therapeutic efficacy to mA20 plasmid on experimental endotoxemia, which may provide valuable experimental base for clinical application of more simple structure A20 protein.PARTⅣMUTANT CONSTRUCTION OF HUMAN ZINC FINGER PROTEIN A20 AND PRIMARY STUDY OF EUKARYOTIC EXPRESSIONObjectives: to design and construct human zinc finger protein A20 gene mutants plasmid by molecular designing software, to transfect human zinc finger protein A20 gene mutants plasmid into mouse RAW264.7 macrophages and to detect their anti-inflammatory activities in mouse RAW264.7 macrophages stimulated by LPS.Methods: Human zinc finger protein A20 gene was identified by sequencing. The hA20 gene were analyzed by molecular designing software and the gene fragment between no. 332 to no. 1778 were deleted. The eukaryotic expression plasmid pcDNA3.0-Flag hA20 was digested linearitely by Acc III nucleate endonuclease. Then, a coupled reaction for this linearity fragment was made by DNA ligase. The recon was transfected into JM109 overnight. The real positive clone was identified from false clone by PCR and enzyme cutting. mouse RAW264.7 macrophages were randomized into three groups: LPS-treatment group, hA20 plasmid group and hA20N-ZnF4-7 plasmid group.The LPS-stimulated RAW264.7 macrophages were transfected through Lipofectamine 2000 eukaryotic transfection system. The contents of TNF-αand IL-1βin the cell culture supernatant were detected by ELISA.Results: The sequence of human zinc finger protein A20 gene was identified to be correct by sequencing. The electropherogram of PCR confirmed that fragment length deleted was 915bp. The expressions of TNF-α, IL-1βof mouse RAW264.7 cell culture supernatant in LPS- assaulting group, hA20 plasmid-treatment group and hA20N-ZnF4-7 plasmid-treatment group respectively were: 250.10±60.18, 221.33±30.02; 120.87±33.75, 69.21±13.18 and 116.51±22.82, 63.45±12.73.Conclusions: We have successfully constructed hA20 mutant plasmid, pcDNA3.0-Fflag-hA20N-ZnF4-7 which posses significant anti-inflam- matory effects on LPS-assulting mouse RAW264.7 macrophages. The Results provide valuable base in future eukaryotic expression research.