Identification of serum differential proteins in colorectal adenoma and early malignantly transformed adenoma by proteomics

AIM: To investigate the associated proteins and sensitive biomarkers for early diagnosis of colorectal adenocarcinoma by comparing the results of differential proteomic analysis between colorectal adenoma and early malignantly transformed adenoma. METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expressions of colorectal adenoma and early malignantly transformed adenoma. Proteins expressed differentially among groups were detected, cut out and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of colorectal adenoma and early malignantly transformed adenoma were analyzed with gel-analysis software, an average of 1 672 spots in adenoma, 1 732 in early malignantly transformed adenoma were observed. 28 spots of a 1.5-fold change were found, including 15 proteins down-regulated and 13 up-regulated in early malignantly transformed adenoma, in which 23 proteins were identified by mass spectrometry, the rate of identification was 82.14%. 13 differential proteins were attained, 8 were up-regulated and 5 were down-regulated, which was classified to 6 categories, including protease inhibitor, complement, immunoglobulin, keratoproteins, signal transduction protein and function-unknown proteins. CONCLUSION: The changes of serum proteins in early malignantly transformed adenoma from adenoma can be identified by proteomic technology. Proteins detected in the study may provide new biomarkers correlated with biological behavior of colorectal adenocarcinoma.     

Proteomic analysis of colon cancer

AIM: To investigate the cancer associated proteins and sensitive biomarkers in colon cancer. METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expression in cancer and matched morphologically normal colonic mucosa from 8 patients. Proteins that showed differential expression of a 2-fold change were cut and analyzed by MALDI-TOF mass spectrometry. RESULTS: Two-dimensional protein maps of cancer and matched normal tissue were gained successfully. Gel-analysis software identified an average of 3289 spots in cancer while 3066 in matched normal tissue and statistical filtering yielded 31 spots of a 2-fold change, 18 of which were identified by using mass spectrometry, including keratin 8, S100A6, protein disulfide isomerase, etc. Functional analysis revealed that these proteins were associated with cancer cellular oncogenesis, proliferation, differentiation and metastasis. CONCLUSION: Proteomic analysis can identify the proteins with variance of colon cancer versus normal colonic tissue as well as providing probable new biomarkers correlated with biological behavior of colon cancer.     

Analysis of serum biomarkers in human knee osteoarthritis

AIM: To analyze the proteomic components of the sera from knee osteoarthritis patients and normal people, and to search proteins that might serve as serum biomarkers for osteoarthritis diagnosis, treatment or pathogenesis. METHODS: Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was applied to analyze the sera obtained from the patients with knee osteoarthritis (n=4) and normal controls (n=4). The differentially expressed proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western blotting analysis was applied to confirm the results. RESULTS: Comparative proteomic data of serum from the patients with osteoarthritis was successfully obtained. Eight differentially expressed protein spots were observed. Five up-regulated and 3 down-regulated proteins were identified. Western blot analysis confirmed that α₂-macroglobulin was increased. CONCLUSION: There are significant differences between serum proteins obtained from the patients with knee osteoarthritis and normal controls. α₂-Macroglobulin might be utilized as potential biomarkers for the diagnosis and treatment of osteoarthritis.     

Expression of alpha-smooth muscle actin in scar tissue and its relationship with apoptosis

AIM: To examine the expression of alpha-smooth muscle actin in scar tissue, and observe the phenomenon of apoptosis and its involvement in the process of pathological scarring and the presence of myofibroblasts or absence of cell in the dermis. To investigate the potential role of reparative cell apoptosis in hyperplastic scar formation. METHODS: The samples of scar were obtained from post-burn patients undergoing plastic operation in our burn unit recently, and the samples of control came from skin donor site of the same patient correspondingly. TUNEL assays were performed to evaluate the number of apoptotic cells in scar versus normal skin. In situ hybridization and immunohistochemistry staining technique were employed to determine the expression of different dermis cells markers in scar tissue and normal skin. RESULTS: There existed evident difference in apoptotic cells in the dermis between scars tissue and normal human skin. The expression positive cells were much more in hyperplastic scars than that in normal human skin; the apoptotic cells of proliferative stage were slight more than that of mature stage. However, in proliferative stage, the number of apoptotic cells was higher for the combination of hyperplastic scar than normally healed flat scars. But in mature stage, no obviously difference was detected between hyperplastic scar and normally healed flat scar. The monoclonal anti-α smooth muscle actin (ASMA) expression was significantly stronger in proliferative stage than that of mature stage. CONCLUSIONS: With reconstitution of dermal tissue, myofibroblasts containing alpha-SM actin disappear under normal wound healing, probably as a result of apoptosis. The myofibroblast play a critical role in wound closure and in the pathologic sequelae of healing.     

橡胶树死皮病黄色体差异表达蛋白的初步分析

橡胶树"死皮病"给橡胶种植业带来严重的危害.为了更好地了解和阐明死皮病发生、发展的分子机制,研究应用双向凝胶电泳技术(two-dimensional gel electrophshiya/oresis,2-DE)比较橡胶树死皮株与健康株胶乳黄色体蛋白质组表达的差异.采用TCA/丙酮沉淀法提取橡胶树死皮株与健康株黄色体蛋白质并采用固相pH梯度(immobjlized pH graalient,IPG)双向凝胶电泳分离两种材料蛋白质,凝胶经银染显色后,用PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质.成功获得橡胶树死皮株与健康株胶乳黄色体的双向凝胶电泳图谱.鉴定出13个蛋白差异点,其中10个上调表达,3个下调表达.并应用质谱技术鉴定了其中部分表达差异的蛋白质点,对渗调蛋白进行功能分析,认为渗调蛋白在死皮株中表现下调的情形可能与死皮病的发生有一定的关系.     

Downregulation of beta tropomyosin protein expression in colon adenocarcinoma

AIM: To investigate the cancer associated proteins and sensitive biomakers for early diagnosis in colon adenocarcinoma by using proteomic technique.METHODS: Two-dimensional gel electrophoresis was used to define patterns of protein expression in adenocarcinoma tissue from 8 patients with matched normal colonic mucosa. Proteins expressed differently of a 2-fold change were cut and analyzed by MALDI-TOF/TOF mass spectrometry. RESULTS: Two-dimensional protein maps of adenocarcinoma and normal colonic mucosa were gained successfully. Gel-analysis software identified an average of 3 289 spots in adenocarcinoma while 3 066 in normal mucosa and statistical filtering yielded 31 spots of a 2-fold change, 18 of which were identified by using mass spectrometry, including cytokeratin 8, cytokeratin 10, S100A6, beta tropomyosin (TMβ), protein disulfide isomerase, etc. Functional analysis revealed that these proteins were associated with adenocarcinoma cellular oncogenesia, proliferation, differentiation, metastasis and so on. The results of Western blotting validated that the expression level of TM β in colon adenocarcinoma was much lower than that in matched normal colonic mucosa.CONCLUSION: Proteomic analysis can identify the proteins with variance of colon adenocarcinoma versus normal colonic mucosa. Downregulation of TM β might serve as a new biomarker of colon adenocarcinoma.     

Functional analysis of differential microRNA expression profile in mouse fibrotic liver tissues induced by CCl4

AIM:To discover the expression profile of microRNAs (miRNAs) in mouse fibrotic liver tissues induced by carbon tetrachloride (CCl4), and to investigate the functions of these differential miRNAs based on the gene ontology (GO) analysis and KEGG Pathway analysis. METHODS:The mice were randomly divided into normal group and model group. Liver fibrosis was induced by subcutaneous injection of CCl4. miRNA expression profile of the liver tissues was assayed by a mouse miRNA microarray (Agilent 12.0). The differential expression of miRNAs between the normal and model mice was screened, and GO analysis and KEGG Pathway analysis were performed to determine the functions of these differential miRNAs. RESULTS:Thirty-nine miRNAs with differential expression were discovered in the model mice compared with the normal mice, among which 23 were up-regulated and 16 were down-regulated. GO analysis and KEGG Pathway analysis indicated that most pathological processes of liver fibrosis regulated by miRNAs included cell proliferation and activation, cell apoptosis, cell cycle, cell adhesion, inflammatory reaction, cell migration, transforming growth factor β (TGF-β) signaling pathway, Wnt signaling pathway and proteometabolism process. GO analysis revealed that the key up-regulated miRNAs were mmu-miR-322, mmu-miR-15b, mmu-miR-195, mmu-miR-200b and mmu-miR-214, and the key down-regulated miRNAs were mmu-miR-16, mmu-miR-130a, mmu-miR-101b, mmu-miR-30a and mmu-miR-30e. Analyzing the target genes screened out by GO analysis and Pathway analysis simultaneously, we found that the key up-regulated miRNAs included mmu-miR-200b, mmu-miR-322, mmu-miR-106b, mmu-miR-23a and mmu-miR-15b, and the key down-regulated miRNAs included mmu-miR-16, mmu-miR-30e, mmu-miR-30c, mmu-miR-30a and mmu-miR-130a. CONCLUSION: Differential expression of miRNAs is discovered in mouse fibrotic liver tissues induced by CCl4 compared with the normal liver tissues. Most of the pathological processes involved in liver fibrosis may be regulated by miRNA, such as cell proliferation and activation, cell adhesion and apoptosis, cell migration and differentiation, metabolism, TGF-β receptor signaling pathway and so on.     

Gene and protein alterations in the hippocampus of rats with temporal lobe epilepsy

AIM:To examine the expression profiles of both genes and proteins in hippocampus of rats with temporal lobe epilepsy (TLE) for revealing the molecular mechanisms of TLE and looking for the candidate targets and new therapeutic approaches in clinical practice.METHODS:Rat temporal lobe epilepsy was induced by administration of lithium chloride and pilocarpine (LiCl-PILO).The expression spectra of genes and proteins were constructed through the techniques of cDNA microarray,two-dimensional (2D) electrophoresis and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Subsequently,the differentially expressed genes and proteins were identified and analyzed.RESULTS:There were 192 genes of differential expression observed in hippocampal tissues of LiCl-PILO-induced temporal lobe epilepsy,and 159 genes have been registered in Genbank database,in which 84 genes were up-regulated while 75 genes were down-regulated.78 protein spots of differential display were screened out,in which 31 proteins were detected to be down-regulated and 47 were up-regulated.Finally,5 proteins were identified.CONCLUSION:These genes and proteins found in our study may play pivotal roles in the pathogenic mechanisms of epilepsy and may promise new therapeutic targets for refractory epilepsy in the future.     

Proteomic analysis of human endometrium during the implantation window

AIM: To establish a method for determining the differential expression of proteins in human endometrium during the implantation window and to analyze the correlation between altered expression of the proteins and endometrial receptivity. METHODS: A comparative proteomic strategy in a combination of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was adopted to search the proteomic alternations in the endometria of pre-receptive pre-receptive [day 2 after luteinizing hormone surge (LH+2 d)] state versus the endometria of receptive (LH+7 d) state. Validation of annexin IV was performed by Western blotting. RESULTS: Approximate 2 555±98 polypeptide spots were revealed by densitometry analysis of the 2D protein maps in LH+2 d and LH+7 d endometrial tissues resolved in the linear range of pH 3~10 on the 2D gel, in which 31 proteins were found to be significantly changed, including 17 proteins up-regulated and 14 proteins down-regulated in LH+7 d samples. These 31 identified proteins were classified into 6 functional categories of the correlation with implantation process: cell migration or assimilation, enzymic activity, signal transduction and gene regulation, immunoregulation, vascularization, and blood clotting or fibrinolysis system. The same expression trend of annexin IV was confirmed by Western blotting. CONCLUSION: Human endometrium has a differential proteomic repertoire during the window of implantation. The 6 functional categories of differentially expressed proteins in the receptive endometrium indicate that they play an important role in transforming of the endometrium during the receptive state.     

Analysis of expressive proteome in human hepatocarcinoma cell HepG2 transfected with miR-26a mimics

AIM: To determine whether microRNA-26a(miR-26a) is involved in development of liver cancer by analysis of proteomic expression profile of human hepatocarcinoma cell HepG2 transfected with miR-26a mimics.METHODS: HepG2 cells were cultured by a routine method and transfected with miR-26a mimics for 48 h for cell cycle analysis. The expressive proteome profiles of HepG2 cells with or without miR-26a mimics treatment were established by the methods of two-dimensional electrophoresis separation following lysis of the cells and extraction of the proteins. The proteomic expression profiles were analyzed by comparative proteomics technique to discover the important protein spots with differential expression. The identification of the proteins was conducted by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS). RESULTS: miR-26a brought down the proliferation of HepG2 cells. Total 11 protein spots with alteration of expressive amounts more than 2 times were successfully identified in the proteomic expression profile of HepG2 cells treated with miR-26a mimics, including annexin A1, peroxiredoxin 4, proliferating cell nuclear antigen, apolipoprotein A1, cytochrome C oxidase subunit 5A, cyclin E2, ribose-phosphate pyrophosphokinase 3, cyclin-dependent kinase 1 and phosphatidylethanolamine-binding protein 1. Among these, the expression of 3 protein spots was up-regulated and 8 of them was down-regulated.CONCLUSION: miR-26a contributes to the anti-cancer effect by expressive regulation of the proteins mentioned above, or directly or indirectly controls the proliferation, differentiation and death of hepatocarcinoma cells.     

HSP27 and αB-crystallin are highly up-regulated in corpus uteri myometrium at labor

［ABSTRACT］AIM: To investigate the expression of heat-shock protein 27 (HSP27) and α-cystallin B chain (αB-crystallin) proteins in corpus uteri myometrium in not-in-labor and in-labor situations.METHODS: Comparative proteomics technique was used to identify HSP27 and αB-crystallin in corpus uteri myometrium in not-in-labor and in-labor situations. The methods of half quantitative RT-PCR, Western blotting analysis and immunohistochemistry were performed to determine the differential expression levels of HSP27. RESULTS: The protein levels of HSP27 and αB-crystallin were highly up-regulated in corpus uteri myometrium at term spontaneous labor (P<0.05). Four HSP27 spots were identified with identical molecular weight and different isoelectric points from 2 two-dimensional gel electrophoresis profiles of corpus uteri myometrium. ImageMaster 2D Platinum software analysis showed that only one HSP27 spot had differential significance (P<0.05),and the rest spots had no significant difference between the 2 profiles (P>0.05). CONCLUSION: The protein levels of HSP27 and αB-crystallin are highly up-regulated in corpus uteri myometrium at term spontaneous labor, suggesting that the two small heat-shock proteins participate in human myometrial contraction at labor and will be potential targets for future tocolytic design.     

笃斯越橘响应低温和短光周期的蛋白质组分析

以3年生笃斯越橘苗为材料,将苗木分别置于对照(23℃,日照长度14 h),低温短日照(4℃,日照长度10 h)和低温长日照(4℃,日照长度14 h)的人工气候室中。处理21 d后,采用同位素标记相对和绝对定量(iTRAQ)蛋白质组学技术测定枝条蛋白质表达变化情况。结果显示:通过质谱鉴定出5 972个蛋白质,600个差异表达蛋白,其中在低温短日照与对照比对组中,丰度显著上调蛋白有140个,丰度显著下调蛋白有114个;在低温长日照与对照比对组中,丰度显著上调蛋白有255个,丰度显著下调蛋白有122个;在低温短日照与低温长日照比对组中,丰度显著上调蛋白有39个,丰度显著下调蛋白有187个。这些蛋白主要参与(1)RNA代谢,(2)蛋白质翻译后修饰,(3)碳水化合物转运和代谢,(4)能量代谢和转换,(5)脂质代谢,(6)次生代谢产物合成,(7)抗氧化与胁迫防御,(8)光合作用,(9)无机离子转运和代谢。结果表明与抗冻性有关的蛋白差异表达主要受低温诱导,少数蛋白表达受低温和光周期共同影响,低温短光周期更有利于抗冻性形成。低温锻炼可显著提高RNA代谢、蛋白质翻译后修饰、抗氧化和防御反应、次生代谢产物合成以及脂质代谢过程中许多蛋白质的丰度,提示这些代谢途径和相关蛋白可能在笃斯越橘抗冻机制中起重要作用。     

Proteomic study on mice hepatic cell membrane proteins after exposed to hypoxia

AIM: To identify the differential expression of mice hepatic cell membrane proteins after hypoxic exposure.METHODS: The hepatic cells of C57BL/6 mice were cultured for 8 h under hypoxic or normoxic conditions and the membrane proteins were extracted. The differentially expressed membrane proteins were separated by two-dimensional electrophoresis. The technique of matrix-assisted laser desorption/ionization mass time-of-flight spectrometry(MALDI-TOF-MS) was used to analyze the proteins.RESULTS: Compared with the proteins extracted from the cells under normoxic condition with a threshold of 1.5-fold, 28 differentially expressed proteins were identified in the proteins extracted from the cells under hypoxic condition according to the database NCBInr 20071130, in which 9 were down-regulated and 7 were up-regulated, including prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP.CONCLUSION: The results suggest that prohibitin, cytochrome b-c1 complex subunit 1 and p22HBP might play important roles in sensing of cellular oxygen and the related signal transduction pathways.     

Differential expression profiling of proteome for systemic lupus erythematosus with quantitative proteomic technique

AIM: To identify and quantify the total proteins in peripheral blood mononuclear cells (PBMC) of systemic lupus erythematosus (SLE) patients with quantitative proteomic technique, and to establish a differential expression profile of proteome for SLE. METHODS: Four-plex isobaric tags for relative and absolute quantification coupled with multiple chromatographic fractionation and tandem mass spectrometry were used to analyze the total proteins in PBMC from healthy controls and the patients of stable SLE, active SLE and rheumatoid arthritis. The proteins were identified by database searching with peptide mass fingerprinting. The differential expression of the proteins was compared. RESULTS: More than 400 proteins were identified. Compared with healthy controls, 44 proteins were discovered to be significantly expressed by more than 2 folds in stable SLE and active SLE, among which 9 proteins were up-regulated and 35 proteins were down-regulated. Compared with rheumatoid arthritis group, 52 proteins displayed 2 or more folds of changes in stable SLE and active SLE, including 19 up-regulated proteins and 33 down-regulated ones. The up-and down-regulated proteins between active SLE and stable SLE were 17 and 13, respectively. CONCLUSION: Quantitative proteomic technique is efficiently applicable for protein identification and relative quantitation in human peripheral blood mononuclear cells. Determination of the differentially expressed proteomic profile of SLE is helpful for better understanding the pathogenesis of SLE and developing new strategies for diagnosis and treatment of SLE.     

Gene microarray analysis of ovarian serous cystadenocarcinoma-related genes based on TCGA

AIM: To detect the differentially expressed genes associated with ovarian serous cystadenocarcinoma (OV) by microarray and to analyze the participated signaling pathway. METHODS: We analyzed 16 datasets of Affymetrix GeneChip Human Exon 1.0 ST Arrays from The Cancer Genome Atlas (TCGA), including 8 OV and 8 normal ovary samples. The function of differential genes was determined by pathway and gene ontology (GO) analysis. The probable functions of the key genes were predicted according to intergenic signal transduction network. RESULTS: The 1 144 genes were identified as distinctively expressed in OV (P<0.05), 747 of which were up-regulated and 397 were down-regulated. The GO analysis results showed that the altered genes were involved in 362 up-regulated and 160 down-regulated significant functions (P<0.05) related to cell cycle, DNA replication, cell proliferation, cell apoptosis, cell adhesion, etc. The pathways of the different genes were involved in the 59 enrichment-related pathways (P<0.05), 45 of which were up-regulated and 14 were down-regulated. Among the 59 pathways, cell cycle, P53 signaling pathway, DNA replication, pathways in cancer, PI3K-Akt signaling pathway, ECM-receptor signaling pathway, cell adhesion molecules and cell apoptosis were related to tumor genesis, development and metastasis. As a result, 229 genes with significant functions and pathways in GO and pathway analysis were selected to construct signal transduction network (Signal-Net), 4 of which, CDK1, PLK1, MCM3 and PGK1, were found to play key roles in OV signal regulation network. CONCLUSION: The OV shows abundant differentially expressed genes that play key roles in cancer-related signal pathways.     

Serum comparative proteomic analysis of radiation-induced hepatic injury in cirrhotic rats

AIM: To investigate the differential expression of serum proteins in cirrhotic SD rats for exploring the pathogenesis and identifying the potential biomarkers of radiation-induced hepatic injury. METHODS: Liver cirrhosis was induced in 8 healthy SD rats with subcutaneous injection of carbon tetrachloride (CCl₄) for 6 weeks and then the animals were randomly divided into 2 groups (4 rats in each group): control group (CCl₄ alone) and experimental group (CCl₄ plus radiation). The latter received hemi-liver radiation with single dose of 15 Gy while the former did not receive radiation. Total serum proteins of the 2 groups were extracted 6 h after radiation. Two-dimensional gel electrophoresis (2-DE) were performed to look for differentially expressed proteins. These proteins were then analyzed and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western bloltting was used to validate the expression of heparanase and hepatocyte growth factor receptor in all independent series of serum samples. RESULTS: Two-dimensional gel images were acquired with good resolution and repetition. Thirty-three differentially expressed proteins were selected and 12 proteins were successfully identified by MS, in which 5 were up-regulated while 7 were down-regulated. The increased level of heparanase and decreased level of hepatocyte growth factor receptor were further confirmed by Western blotting. CONCLUSION: Twelve proteins associated with radiation-induced hepatic injury are successfully characterized by serum comparative proteomics. Heparanase and hepatocyte growth factor receptor might be useful for detecting and monitoring radiation-induced hepatic injury.     

Comparative proteomic analysis of lens in congenital inherited cataract mice

AIM: To separate total lens proteins of congenital inherited cataract in mice and to observe the alteration of proteins after gene mutation.METHODS: We studied the mice with a spontaneous mutation of crystallin gamma S (Crygs) transmitted as a recessive trait. Total proteins were extracted and separated using immobilized pH gradient (IPG), two-dimensional electrophoresis (2-DE) and colloidal Coomassie brilliant blue (CBB) staining. The image analysis was carried out using PDQuest 7.30 software package. Several significantly differential proteins in gel were identified by matrix assisted laser adsorption/ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS). RESULTS: As the 882 μg sample was added, we detected (417±53) spots and (370±41)spots in cataract and normal lens, respectively. As the 190 μg sample was loaded, we detected (60±7) spots and (57±5) spots in cataract and normal lens, respectively. Seven kinds of differential proteins were identified, including BFSP/filensin, γS, γF, βA1, βB1, βB2 and αB. In crystalline lens of mutant mice, γS and beaded-filament structure protein (BFSP/filensin) were not detected. γF was down-expressed (＜4 fold) while βA1, βB1, βB2 and αB were over-expressed (＞4 fold) in mutant cataract. The latter proteins were less MW than normal, suggesting that they were possibly truncated.CONCLUSION: 2-DE and mass spectrometry can help to assess and analyze the function of proteins as a novel approach. The mutant Crygs gene can lead to the abnormity of γS crystallin, which can induce the changes of skeletonal protein (BFSP/filensin) and other crystallins (γF, βA1, βB1, βB2 and αB), and then evoke cataract secondarily.     

Starvation induces autophagy of hypertrophic scar fibroblasts

［ABSTRACT］AIM: To investigate the starvation-induced autophagy of hypertrophic scar fibroblasts. METHODS: Primary human fibroblasts from hypertrophic scars were isolated and the fibroblasts in logarithmic growth phase were cultured with amino acid-free Earle's balanced salt solution (EBSS) instead of the DMEM medium. The cells were collected at the time points of 0 h, 1 h, 2 h and 3 h after EBSS culture. The expression of microtubule-associated protein 1 light chain 3 (LC-3) and autophagy-related protein Beclin-1 was detected by Western blotting and qRT-PCR. Autopagosomes in fibroblasts were observed by electron microscopy and monodansylcadaverine (MDC) staining. RESULTS: The expression of LC3 and Beclin-1 increased at 1 h after starvation, reached to the highest level at 2 h after starvation, and then began to decline, which were still higher than that in control group. Compared with control group, the autopagosomes were observed in the fibroblasts under fluorescence microscope and election microscope at 2 h after starvation. CONCLUSION: Autophagy in fibroblasts of hypertrophic scars can be induced by starvation, which may be related to the formation of hypertrophic scars.     

Screen of novel candidate regulators involved in oxidative stress-reprogrammed LPS signaling pathway by comparative phosphoprotein-affinity profiling

AIM: To determine the differences in phosphoproteome between LPS stimulated THP-1 cells with and without previous oxidative stress for screening of more potential regulators.METHODS: Differentiation of THP-1 cells into macrophages was induced by treatment with 100 μg/L PMA for 36 h. Differentiated cells were rested for additional 36 h without PMA treatment, then treated with 100 μmol/L H₂O₂ or medium for 1 h followed by LPS or medium treatment for 30 min. After desalted, phosphoproteins were enriched by phosphoprotein metal affinity column, and were run on 2-D electrophoresis, then the spots were analyzed to show the difference between LPS group (cells treated with LPS alone) and H₂O₂+LPS group (LPS stimulated cells also pretreated with H₂O₂). Finally, some of these spots were identified by MS and subsequent bioinformatic analysis was also conducted. RESULTS: Compared to LPS group, 29 reproducibly changed spots on the 2-D map in H₂O₂+LPS group were visualized and selected for MS analysis. Among these, 12 down-regulated spots (include those disappeared), 17 up-regulated spots (include those newly emerged) were selected. Up to now, 5 of these were identified, which were shown to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, proteasome beta-4 subunit, which was dramatically down-regulated in H₂O₂+LPS group, was a major component of the proteasome complex and might participate in LPS signalling through various ways.CONCLUSION: With comparative phosphoprotein-affinity profiling, the interference brought by highly abundant house-keeping proteins is minimized, rendering us to detect less abundant signalling molecules. Aforementioned 5 proteins, especially proteasome beta-4 subunit, might be involved in LPS pathway reprogrammed by oxidative stress.     

Effect of lentivirus-mediated DKK3 overexpression on apoptosis of hypertrophic scar fibroblasts

AIM:To investigate the effect of lentivirus-mediated DKK3 overexpression on the apoptosis of hypertrophic scar fibroblasts. METHODS:Human hypertrophic scar fibroblasts were isolated and cultured in vitro. The cells were divided into control group, vector (negative control lentivirus infection) group and DKK3 (pcDNA3.1-DKK3 lentivirus infection) group. The overexpression effect was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels of cleaved caspase-9, collagen type Ⅱ (COL Ⅱ), COL I and cleaved caspase-3 in the cells, and cytochrome C in the cytoplasm and mitochondrion were detected by Western blot. RESULTS:After transfection with pcDNA3.1-DKK3, the expression of DKK3 at mRNA and protein levels was increased in the hypertrophic scar fibroblasts (P<0.05). The viability of hypertrophic scar fibroblasts in DKK3 group was decreased, and the apoptotic rate was increased. The protein levels of cleaved caspase-9 and cleaved caspase-3 were increased in the cells, and the protein levels of COL Ⅱ and COL I were decreased. The protein level of cytochrome C was increased in the cytoplasm, while the protein level of cytochrome C in the mitochondrion decreased. Compared with vector group, these differences were statistically significant (P<0.05). CONCLUSION:Lentivirus-mediated DKK3 overexpression induces apoptosis and reduces collagen synthesis in the fibroblasts from hypertrophic scars.