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The Selection and Analysis of Diverse Protein of Keratinocyte Before and after Nitric Oxide Stimulation

Author: ZhangYuHeng
Tutor: LiuXiaoMing
School: Dalian Medical University
Course: Dermatology and Venereology
Keywords: NO HaCaT cell Keratinocyte 2- DE Mass spectrography
CLC: R758.63
Type: Master's thesis
Year: 2011
Downloads: 8
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Abstract


Objective: To compare the proteomic changes of HaCaT cells and keratinocytes (KC) induced by Nitric Oxide (NO). Try to seek for the target proteins of NO on mRNA level. And try to investigate the function of NO in the pathogenesis of psoriasis.Method: Culture HaCaT cells and KC in vitro. Add different concentration of GSNO to HaCaT cells and KC and measure the absorbance at different time. Curve between the absorbance and the concentration of GSNO to select the best concentration of GSNO. Add the selected amount of GSNO to HaCaT cells and KC while control groups with no treatment. Lysate cells both in the test group and in the control group to get the whole protein. Purify the protein with 2-D Clean-up Kit. Define the quantity of the protein, and then add the loading buffer in it. The total proteins of KC and HaCaT cells strain before and after GSNO were separated by immobilized pH gradient based on two dimensional gel electrophoresis (2- DE). After equilibrium and silver staining, the differential expression proteins were analyzed by using Image-Master 2D analysis software. Identify the species of proteins using mass spectrography.Results:(1) Using CCK-8 method, we draw the curve between concentrations of GSNO and increasing rates of HaCaT cells. From the curve we can see that, concentrations of GSNO and increasing rates of HaCaT cells has reverse linear relationship, and with no obvious relationship with time. (2)Average protein spots were 482±31,495±28 in HaCaT cell before and after GSNO irritation respectively and the average matching rate was 82.1%和81.9% respectively. Average protein spots were 543±27,529±29 in human KC before and after GSNO irritation respectively and the average matching rate was 80.8%,81.4%respectively. (3) The 29 differential protein spots were identified between the two types which are before and after GSNO irritation. 2 protein spots were expressed only in the cells after GSNO irritation .The expression levels of 14 protein spots in the cells after GSNO irritation were higher than those before GSNO irritation, and the expression levels of 13 protein spots in the cells after GSNO irritation were lower than those before GSNO irritation. After mass spectrography, we detect 20 different sort of proteins between the test group and the control group, of which 17 proteins are known and 3 proteins are unknown.Conclusions: (1)In this study, the well- resolved, reproducible 2- DE patterns of keratinocyte and human KC before and after NO irritation were established and some differential proteins between the two types of sample were found. These data will be helpful to the further study of NO effect on skin. (2)Via the well- resolved, reproducible 2- DE patterns of keratinocyte and human KC before and after NO irritation, we detected several distinguishing patterns of protein expression., including prohibintin, keratin, actin binding protein, et al; (3) NO regulate the cell cycle of KC via adjust the target protein expression, which make sense in the early pathogenesis of psoriasis.

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CLC: > Medicine, health > Dermatology and Venereology > Dermatology > The reason is unknown skin disease > Psoriasis (psoriasis)
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