| Part 1Influences of p55PIK ,a regulatory subunit of Phosphoinositide 3-kinases (PI3Ks)and N-terminal 24 amino acids within it on imflammation cytokines releasing of human keratinocyte HacaT cell line on LPS stimulationObjectives1.Observe the influence of N24,the 24 amino acids on the N-terminal of p55PIK which is a regulatory subunit of Phosphoinositide 3-kinases (PI3Ks),on the proliferation of human keratinocyte HacaT cell line2.Draw the releasing curve of TNF-αof human keratinocyteHacaT cell line on the stimulation of different concentrations of LPS3.Observe the influence of N24,the 24 amino acids on the N-terminal of p55PIK which is a regulatory subunit of PI3Ks ,on the releasing of TNF-α,IL–6 or IL–8 in human keratinocyteHacaT cell lineMethodsTo effectively express N24 and p55PIK in cells , TAT-N24 fusion peptide;AD-N24- GFP adnovirus and AD-p55PIK-GFP adnovirus were constructed. Human keratinocy- teHacaT cell line was used as the main study objective on the stumilation of LPS.To find out the most effective stimulation time point or the most effective stimulation concentration of LPS on HaCaT cell line, Cell cycle analysis ,BrdU incorporation assay or CFDA labeling method was used to examine the cell cycle distribution ,the DNA synthesis and the proliferation respectively.ELISA and Real-time PCR were used to examine the expression of TNF-α,IL–6 or IL–8 on the treatment of TAT-N24 fusion peptide,AD-N24-GFP adnovirus or AD-p55PIK-GFP.Conclusions1 . TAT-N24 fusion peptide,AD-N24-GFP adnovirus or AD-p55PIK-GFP was successfully constructed.Cell cycle was arrested in G0/G1 phase by TAT-N24.The cell cycle distributions were G0/G1 phase (58.54±2.06)%, S phase (26.34±1.69)%, G2/M phase (15.12±1.91)% in TAT-N24 group, G0/G1 phase (60.14±2.06)%, S phase (25.32±1.78)%, G2/M phase (14.34±1.39)% in control group ,difference between two groups was significantly(p<0.05);Proportion of BrdU positively stained cell was (8.37±2.06)% in TAT-N24 group, (28.14±4.69)% in control peptide group or (26.73±3.58)% in Blank group,which suggested DNA synthesis of HaCaT cell line was inhibited by TAT-N24 .The proliferation Index(PI) of CFDA labeling was 30.75±3.66 in TAT-N24 group,47.81±5.66 in control peptide group or 50.16±5.33 in Blank group, difference between two groups was significantly(p<0.05),which suggested proliferation of HaCaT cell line was inhibited by TAT-N24.2.The LPS stimulated imflammation cytokine releasing model of HaCaT cell line was successfully constructed. TNF-α,IL–6 or IL–8 was auto-secreted on physiological condition and was increased on the stimulation of LPS,which was similar with the pathology process of psoriasis. ELISA was utilized to find out the most effective dose and the best examine time point of LPS stimulation..3.The releasing of TNF-α,IL–6 and IL-8 stimulated by LPS were inhibited by TAT-N24. ELISA results show that TNF-α,IL–6 and IL-8 concentration were 208.06±30.18pg/ml,86.4±9.78pg/ml and 260.59±54.05pg/ml respectively in LPS stimulation group and were 121.78±22.26pg/ml,53.18±7.36pg/ml and 125.08±35.17pg/ml respectively in TAT-N24 treated LPS stimulation group.4.Real-time PCR results show that the expression of TNF-α,IL–6 and IL-8 were up-regulated in LPS stimulation group than none LPS stimulated cell and was inhibited by TAT-N24 or by AD-N24-GFPon LPS stimulation. In contrary, the expression of TNF-α,IL–6 and IL-8 was up-regulated on LPS stimulation and was further enhanced by treated with AD-p55PIK-GFP on LPS stimulation.Conclusions1.Been over-expressed with N24 in HaCaT cell line,cell cycle distribution was arrested at G0/G1 phase ,DNA synthesis and proliferation were inhibited.2.Releasing of TNF-α,IL–6 or IL-8 on LPS stimulation was inhibited by TAT-N24 or AD-N24-GFP3 . Releasing of TNF-α,IL–6 or IL-8 on LPS stimulation was increased by AD-p55PIK-GFP Part 2The influences and mechanisms involved of p55PIK,a regulatory subunit of Phosphoinositide 3-kinases (PI3Ks), and the N-terminal 24 amino acids within it in human keratinocyte HacaT cell line on LPS stimulationObservethe influences of p55PIK and N24 on the expression of TLR2/TLR4 and find out the mechanisms on it in human keratinocyte HacaT cell line on LPS stimulationMethodsImmunocytochemistry was used to examine the expression of NF-κB P65in HacaT cell line; Immunofluorescence was performed to observe the translocation of NF-KB P65in HacaT cell line; Western Bloting was utilized to detects the expression of TLRs signal transductional pathway related proteins treated with AD-N24-GFP or AD- p55PIK-GFP.Results1,Immunocytochemistry results show that protein NF-κB P65 in nuclear on LPS stimulation was higher than no-LPS-stimulated cell ,which may be inhibited by AD-N24- GFP.2,Immunofluorescence results show that protein NF-κB P65 mainly located in cytoplasma and trans-located at nuclear on LPS stimulation,but located back to cytoplasma again in treatment of AD-N24-GFP on LPS stimulation.3,Western Bloting results show that the expression of protein TLR2 or protein TLR4 was up-regulated, which reached peak at 4th hour and was lasted for at least 24 hours,on LPS stimulation.. AD-p55PIK-GFP treatment but not AD-N24-GFP treatment increased the expression of TLR2 or TLR4.4,The expression of protein MyD88 was up-regulated, which was reach the peak level at 8th hour and decreased for at least 24 hours ,on LPS stimulation.. AD-p55PIK-GFP treatment increased the expression of MyD88, but AD-N24-GFP treatment in opposite.5,The expression of Phosphorylated-Akt protein was up-regulated, which was reach the peak level at 2th hour and decreased to nomal level at 24 hours on LPS stimulation. AD-N24-GFP did not influence the expression of Phosphorylated-Akt,but AD- p55PIK -GFP treatment do add the expression of p-Akt6,ERKs was Phosphorylated and activated at 30mins and reached peak at 4h and then decreased but higher than nomal at 24th hours on LPS stimulation group. Both AD- p55PIK-GFP treatment and AD-N24-GFP treatment did not influence the activity of ERKs.7,The expression of IκB-αwas down-regulated at 30mins and reached hollow at 4h and lasted to 16h and then up-regulated but higher than nomal at 24th hours on LPS stimulation group. AD-N24-GFP treatment but not AD-p55PIK-GFP treatment increased the expression of IκB-α.ConclusionAD-N24-GFP and AD-p55PIK-GFP influences the releasing of imflammation cytokines through regulating the TLRs/MyD88 signal transductional pathway in HaCaT cell.
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