Proteomics Study On Human Skin Keratinocytes

The effects of time, pathologic status (psoriasis) and keratinocyte-specific factor (fibroblast growth factor 7 and fibroblast growth factor 10, hFGF-7 and hFGF-10) stimulations on the proteome of human skin kerotinocytes were preliminarily investigated on the basis of establishment of the pivotal technological platforms for human skin kerotinocytes and epidermis. The results may provide the theoretical evidences for further exploring the possible mechanisms of the effects of long-term culture on human skin kerotinocytes, studying the pathogenesis of skin diseases (psoriasis), and investigating the possible mechanisms of the effects of keratinocyte-specific factors on human skin kerotinocytes.Chapter 1 The effects of long-term culture on human skin keratinocytesAIM. To establish a method for isolation, culture and passage of human skin keratinocytes, to set up a pivotal technological platform for studying the proteomics of human skin keratinocytes, and investigate the effects of long-term culture on the proteome of keratinocytes, providing a basis for further exploring the possible mechanisms of the effects of long-term culture on human skin kerotinocytes.METHODS: DispaseⅡwas used to isolate the epidermis from the foreskin of children. The keratinocytes were cultured in Defined K-SFM medium. Trypsin combined with EDTA was used to digest the keratinocytes for passage. The cells were observed under the Phase Contrast Microscope. Immunocytochemistry was applied to identify the cultured cells. The Population Doubling Time (PDT) of the cells from the primary to the seventh-passage was assessed with haemocytometer.The whole-cell proteins were prepared from the primary and the seventh-passage human skin keratinocytes respectively, and applied to Two Dimension Gel Electrophoresis (2-DE). The nonlinear IPG dry strip (pH3-10, 17cm) was used for isoelectric focusing of the first dimension, and 12% SDS-PAGE was performed for the second dimension. The conditions for 2-DE were optimized. The 2-DE patterns of the whole-cell proteins from the primary and the seventh-passage human skin keratinocytes were analyzed by using PDQuest software. Two differentially expressed protein spots were selected for trypsin digestion, and the trypsin-digested peptides were subjected to tandem time-of-flight mass spectrometry (ABI4700 TOF-TOF) analysis. Proteins were identified by comparison of the experimental data to the NCBInr database with MASCOT online search tool, and the mRNA levels of two differentially expressed proteins identified were detected by semi-quantitative RT-PCR.RESULTS: The primary keratinocytes were round or elliptic. The shape of the cells of higher passage number became irregular and many vacuolus appeared around the nucleus. The cytoplasm of the cells presented Kelly fluorescence under the Fluorescent Microscope, indicating that the cells were keratin positive. The primary keratinocytes proliferated slowly and about 10 days were needed to reach confluence. The PDT was 116h. The cells grew faster after passage. The PDT of the cells of passage 2, 3 and 4 reduced markedly. The proliferation rate of the cells became slower and the PDT elongated after passage 5. The cells of passage 8 could not reach confluence even cultured for long time.The method of 2-DE of the whole-cell proteins from human skin keratinocytes was established and optimized to get the 2-DE patterns with high resolution. The matched spots were 751 and 806 in the gels of the primary and the seventh-passage keratinocytes respectively. The corresponding matched rates were 75.3 % and 80.8 %. The Peptide Mass Fingerprints corresponding to the selected two proteins with high resolution and down-regulated expression levels in the seventh passage keratinocytes were obtained by tandem time-of-flight mass spectrometry analysis. By comparison of the experimental data to the NCBInr database with MASCOT online search tool, the differentially expressed protein 1 was identified as Psoriasis Associated Fatty Acid Binding Protein (PA-FABP) related to the fatty acid transportation and metabolism, and the differentially expressed protein 2 was identified as Galectin-7 related to apoptosis regulation. Finally, the transcription levels of both differentially expressed proteins were much lower in the seventh-passage keratinocytes than that in the primary keratinocytes. The biological functions of both PA-FABP and Galectin-7 and their roles in the effects of long-term culture on human skin keratinocytes were also discussed.CONCLUSIONS: The method for isolation, culture and passage of human skin keratinocytes, and the pivotal technological platform for studying the proteomics of human skin keratinocytes were successfully established. Compared with the primary keratinocytes, the expression levels of both PA-FABP and Galectin-7 in the seventh-passage keratinocytes were down-regulated, indicating that the transportation and/or metabolism may change and the capacity of apoptosis regulation possibly reduced in keratinocytes after long-term culture.Chapter 2 Proteomics study on the normal and the psoriatic lesion epidermisAIM: To establish a pivotal technological platform for studying the proteomics of human skin epidermis, to investigate the difference of the proteome between the normal and the psoriatic lesion epidermis, and further to speculate the possible pathogenesis of psoriasis via differentially expressed proteins identified.METHODS. DispaseⅡwas used to isolate the epidermis from the foreskin and the psoriatic lesion skin. The whole-cell proteins were prepared from the normal and the psoriatic lesion epidermis respectively, and applied to Two Dimension Gel Electrophoresis (2-DE). The nonlinear IPG dry strip (pH3-10, 17cm) was used for isoelectric focusing of the first dimension, and 12% SDS-PAGE was performed for the second dimension. The 2-DE patterns of the whole-cell proteins from the normal and the psoriatic lesion epidermis were analyzed by using PDQuest software. Six differentially expressed protein spots were selected for trypsin digestion, and the trypsin-digested peptides were subjected to tandem time-of-flight mass spectrometry (ABI 4700 TOF-TOF) analysis. Proteins were identified by comparison of the experimental data to the NCBInr database with MASCOT online search tool, and the mRNA level of one of the differentially expressed proteins identified,αB-Crystallin, was detected by semi-quantitative RT-PCR.RESULTS: The method of 2-DE of the whole-cell proteins from human skin epidermis was established to get the 2-DE patterns with high resolution. PDQuest software analysis of the 2-DE patterns of the normal and the psoriatic lesion epidermis showed that the matched spots were 876 and 794 in the gels of the normal and the psoriatic lesion epidermis respectively, and the corresponding matched rates were 79.6 % and 72.2 %. The Peptide Mass Fingerprints corresponding to the selected six proteins with high resolution, among which five were up-regulated and one was down-regulated in the psoriatic lesion epidermis, were obtained by tandem time-of-flight mass spectrometry analysis. By comparison of the experimental data to the NCBInr database with MASCOT online search tool, five up-regulated proteins were identified as S100 calcium binding protein A7-like 1, Psoriasin,αB-Crystallin, Peroxiredoxin 2 isoform b, and Transglutaminase 3 precursor, and the other down-regulated protein was Cystatin B. These differentially expressed proteins were involved in cell proliferation, apoptosis, differentiation, and inflammatory infiltration. Finally, the transcription level of one of the differentially expressed proteins,αB-Crystallin, was much higher in the psoriatic lesion epidermis than that in the normal epidermis. The possible roles of the differentially-expressed proteins in the pathogenesis of the psoriasis were also discussed.CONCLUSIONS: The pivotal technological platform for studying the proteomics of human skin epidermis was successfully established. The differentially expressed proteins identified are possibly closely related to the hyperproliferation, reduced spontaneous apoptosis, and abnormal differentiation of the psoriatic lesion keratinocytes.Chapter 3 Proteomies study on the effects of hFGF-7 and hFGF-10 on HaCat cellsAim: To construct the recombinant adenovirus containing the genes of keratinocyte-specific factors, human fibroblast growth factor 7 (hFGF-7) and human fibroblast growth factor 10 (hFGF-10), to secreted express hFGF-7 and hFGF-10 in HaCat cells infected with the recombinant adenovirus, to investigate the effects of expressed hFGF-7 and hFGF-10 on the proteome of HaCat cells, and further to speculate the possible mechanisms of the effects of hFGF-7 and hFGF-10 on HaCat cells via differentially expressed proteins identified on the basis of establishing the pivotal technological platform for studying the proteomics of human skin keratinocytes.METHODS. The gene fragments of hFGF-7 and hFGF-10 were synthesized by PCR and cloned into the vector pET-3c to construct the recombinant plasmids pET-3c-hFGF-7 and pET-3c-hFGF-10. The gene fragments of hFGF-7 and hFGF-10 with BglⅡsite at the 5' terminus and HindⅢsite at the 3' terminus were amplified by PCR using the above recombinant plasmids as the templates, and ligated with the shuttle vector pAdTrack-CMV to get the recombinant plasmids pAdTrack-CMV-hFGF-7 and pAdTrack-CMV-hFGF-10, which were linearized with PmeⅠand transferred into Escherichia coli B J5183 containing the adenoviral bone plasmid pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral plasmids pAdEasy-hFGF-7 and pAdEasy-hFGF-10. The recombinant adenoviral plasmids were then transfected into HEK-293 cells to package and amplify the recombinant adenovirus rAd-hFGF-7 and rAd-hFGF-10. The expressions of hFGF-7 and hFGF-10 in HaCat cells infected with the recombinant adenovirus were detected by Western blotting. The influences of the recombinant adenovirus on the proliferation and cell cycle of HaCat cells were checked by MTT and FACS respectively.The whole-cell proteins were prepared from four group cells including HaCat, Ad infected HaCat, rAd-hFGF-7 infected HaCat and rAd-hFGF-10 infected HaCat respectively, and applied to Two Dimension Gel Electrophoresis (2-DE). The nonlinear IPG dry strip (pH3-10, 17cm) was used for isoelectric focusing of the first dimension, and 12% SDS-PAGE was performed for the second dimension. The 2-DE patterns of the whole-cell proteins from four group cells were analyzed by using PDQuest software. Four differentially expressed protein spots were selected for trypsin digestion, and the trypsin-digested peptides were subjected to tandem time-of-flight mass spectrometry (ABI 4700 TOF-TOF) analysis. Proteins were identified by comparison of the experimental data to the NCBInr database with MASCOT online search tool, and the mRNA and protein levels of one of the differentially expressed proteins identified, VDAC2, were detected by semi-quantitative RT-PCR and Western blotting.RESULTS: The recombinant adenovirus rAd-hFGF-7 and rAd-hFGF-10 containing hFGF-7 and hFGF-10 gene were successfully constructed, which could effectively infect HaCat cells. The result of Western blotting showed that the culture media of the infected HaCat cells could react with hFGF-7 and hFGF-10 antibody. The recombinant adenovirus could stimulate the proliferation of HaCat cells and change the cell cycle with increased cell ratio in S phase and G2 phase.The 2-DE patterns with high resolution were obtained. PDQuest soft-ware analysis of the 2-DE patterns of the whole-cell proteins from four group cells showed that the matched spots were 516 and 535 in the gels of the control group and Ad infected group respectively, and the corresponding matched rates were 67.5 % and 70.0%, while the matched spots were 602 and 587 in the gels ofrAd-hFGF-7 infected group and rAd-hFGF-10 infected group respectively, and the corresponding matched rates were 78.8 % and 76.8 %. The Peptide Mass Fingerprints corresponding to four selected proteins with high resolution and up-regulated expression levels in the groups infected with the recombinant adenovirus were obtained by tandem time-of-flight mass spectrometry analysis. By comparison of the experimental data to the NCBInr database with MASCOT online search tool, the differentially expressed proteins were identified as VDAC2, Proteasome alpha 1 subunit, isoform 2, Gelsolin-like capping protein, and Protein disulfide isomerase-associated 3 precursor. These differentially expressed proteins were involved in cell apoptosis, cytoskeleton regulation and protein degradation. Finally, the mRNA and protein levels of one of the differentially expressed proteins, VDAC2, were higher in the groups infected with the recombinant adenovirus than those in the control and Ad infected groups. The possible roles of VDAC2 in the biological functions of hFGF-7 and hFGF-10 were also discussed.CONCLUSIONS: The recombinant adenovirus rAd-hFGF-7 and rAd-hFGF-10 were successfully constructed by homologous recombination in bacteria. HaCat cells infected with the recombinant adenovirus expressed and secreted hFGF-7 and hFGF-10 respetively, which promoted the proliferation and changed the cell cycle of HaCat cells.The differentially expressed protein, VDAC2, identified by the methods of proteomics may be related to the biological activities of anti-apopotosis of hFGF-7 and hFGF-10.