Establishment and differential protein identification of two-dimensional gel electrophoresis for proteomics in the spinal cord of morphine-tolerant rats / 中南大学学报(医学版)

To establish a two-dimensional gel electrophoresis (2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance, and to detect differentially expression proteins that are associated with chronic morphine tolerance.
 Methods: Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group (MT group, n=8) and a saline group (NS group, n=8). The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software. Identification of differential protein spots was conducted by MALDI-TOF-MS, and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis. Western blotting was used to verify the expression of some differentially expressed proteins.
 Results: A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group, and 36 proteins were significantly differentially expressed in the MT group compared with the NS group. Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software, and a total of 14 proteins were obtained. Among them, 2 protein spots were down-regulated in the MT group compared with that in the NS group, and 12 protein spots were up-regulated in the MT group compared with that in the NS group. Two kinds of proteins (NUDAA, ENOG) were verified by Western blotting and the results were consistent with proteomics data.
 Conclusion: The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily, which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.

Clinical value assessment of chemiluminescence for urine asymmetric dimethylarginine / 中华检验医学杂志

Objective To explore the clinical application value of chemiluminescence detection of urine asymmetric dimethylarginine ( ADMA ) in pregnancy-induced hypertension ( PIH ) . Methods Collected the 24 h urine from 60 normal pregnancy women and 72 PIH pregnancy women who were admitted to Huzhou Maternal and Child Health Hospital from May 2014 to April 2015 by the case-control study , Determination of urine ADMA content by chemiluminescence ( CLIA ) and high performance liquid chromatography ( HPLC ) , the results between two assays were analyzed by the Rank sum test , receiver operating characteristic ( ROC) curve and pearson correlation analysis .Results Compared with the normal control group , the urine ADMA concentration in the PIH group was significantly increased by HPLC and CLIA, and the concentration of ADMA by CLIA in the PIH group was 68.18(57.25,81.55)μmol/L higher than that of the normal control group 30.11(22.69,42.97)μmol/L(Z=-8.139,P<0.001),and the concentration of ADMA by HPLC in the PIH group by HPLC was 71.11(57.65,82.89)μmol/L higher than that of the normal control group 28.11(21.06,42.99)μmol/L(Z=-8.356,P<0.001).The difference was statistically significant .The two methods of urine ADMA concentration were highly positively correlated with PIH blood pressure.The correlation coefficient r values were 0.746 and 0.763, respectively, the P values were 0.007 and 0.008 respectively.Conclusions CLIA can better detect the ADMA concentration in urine of pregnant women with PIH , and has a good clinical diagnosis ability .The ADMA concentration in urine is related to the blood pressure level of PIH .

Clinical value of prenatal MRI in the diagnosis of fetal simple expansion of lateral ventricle and follow-up after birth / 中华妇产科杂志

Objective To explore the value of prenatal MRI in the diagnosis of fetal simple expansion of lateral ventricle(ventriculomegaly), and follow up the nervous system development status after birth. Methods Simple expansion of the lateral ventricle fetus by prenatal MRI examination were collected in Huzhou Maternal and Child Care Hospital from May 2013 to June 2015, 126 cases of live births in expansion group, 50 normal cases were recruited in the same period as the control group. In expansion group, fetal subgroup analysis was done:(1) unilateral or bilateral lateral ventricle expasion:one group was 98 cases was lateral ventricle expansion (77.8%, 98/126), expansion of bilateral ventricle group was 28 cases (22.2%, 28/126). (2) Prenatal MRI in the diagnosis of the lateral ventricle of expansion: expansion of the lateral ventricle width was greater than 10.0 mm, if both sides were expanding, the expand width was the heavier one side, divided into 3 subgroups: ①Expansion in group A (lateral ventricle width 10.0-12.0 mm) were 88 cases (69.8%, 88/126).②Expansion in group B (lateral ventricle width 12.1-15.0 mm) were 29 cases (23.0%, 29/126). ③Expansion of group C (lateral ventricle width> 15.0 mm) were 9 cases (7.12%, 9/126). All 176 cases were followed up after birth at the 3rd, 6th, 12th, 18th month (corrected age was used for premature babies), and Gesell developmental schedules (GDS) were used to evaluate the neurobehavioral development. Results (1) The MRI results after birth:21 cases were followed up by MRI after birth. In group A, 11 cases had MRI and 9 were normal (the ventricular width0.05). (3) The GDS results among the subgroups:in each evaluation after birth, there were no statistically significant differences between group A and the control group (all P>0.05). The GDS results of group B at the 3rd and 6th month were lower than those of the control group (P0.05). And for group C, statistically significant differences were found compared to the control group at each follow-up time (all P0.05). But when the result at the 3rd month was compared to the results of the 12th or 18th month, the differences were statistically significant (P0.05). There was no statistically significant difference between the results at the 12th and 18th month (P>0.05). (5) The GDS results in unilateral and bilateral ventricle expansion:at the 18th month, among the 98 unilateral cases, 86 (87.8%, 86/98) had normal GDS results(>85 scores);8 (8.2%, 8/98) had borderline results (75-85 scores);4 (4.1%, 4/98) had delayed results (0.05). Conclusions Among the simple expansion of lateral ventricle, those whose ventricular width are≤12.0 mm may not need clinical treatment. If the width is between 12.1 to 15.0 mm, closely follow-up and targeted rehabilitation training after birth are recommended. When the width is more than 15.0 mm, the risk of the central nervous system function delay is significantly increased, and early intervention might improve the prognosis.

Construction of eukaryotic expression plasmid of human CKB and establishment of NCI-H520 cell line stably transfected with the constructed plasmid / 中国医师杂志

Objective To construct an eukaryotic expression vector pEGFP-N1-CKB and establish a stably transfected NCI-H520 cell line.Methods Human CKB gene was amplified by PCR with human CKB cDNA library as the template and the fragment was combined with plasmid pEGFP-N1.The recombinant expression vector,pEGFP-N1-CKB,was transfected to NCI-H520 using Lipofectamin.The stably transfected cell line was established after G418 selection and the expression level of CKB gene before and after transfection was detected by Western blot.Results After identification by restriction enzyme digestion and sequencing,the eukaryotic expression vector,pEGFP-N1-CKB,was successfully constructed.The expression level of CKB in NCI-H520 transfected by pEGFP-N1-CKB was significantly higher than that in control.CKB gene had a stable transfection in NCI-H520 cells.Conclusions An eukaryotic plasmid encoding CKB (pEGFP-N1-CKB) has been constructed and a cell line expressed CKB stably has been successfully prepared.

Screening for methylation-silenced genes in acute myeloid leukemia HL-60 cell line by a quantitative proteomic approach / 中南大学学报(医学版)

Objective To screen for new methylation association genes in HL-60 to reveal the pathogenesis of leukemia, and provide important theoretical and scientific basis for the prevention and cure of leukemia. Methods Two-dimensional fluorescence difference gel electrophoresis (F-2D-DIGE) was performed to separate the total proteins from acute myelogenous leukemia (AML) cell line HL-60 cells with or without 5-aza-2-deoxycytidine (5-aza-2-dC) treatment. Imaging software Decyder 6.5 and PDQuest were used to detect the differential expression protein spots, and matrix-assisted laser desorption/ionizaion time-of-flight mas spectrometer (MALDI-TOF MS) was adopted to identify the differential expression proteins. Results F-2D-DIGE maps of 5-aza-2-dC-untreated HL-60 and-treated HL-60 cells were established. A total of 53 differential protein spots were detected, and 35 differential proteins were successfully identified. Of the identified proteins, 32 proteins were up-regulated, and 3 proteins were down-regulated in HL-60 cells after 5-aza-2-dC treatment.Conclusion Thirty-five differential proteins may be associated with methylation in HL-60 cell line, which provides the important clues for epigenetic study of leukemia.

Establishment of protein expression profile of laser capture microdissection-purified nasopharyngeal carcinoma tissue / 中南大学学报(医学版)

OBJECTIVE@#To establish the protein expression map of nasopharyngeal carcinoma (NPC), and provide a basis for proteomic study of NPC.@*METHODS@#Laser capture microdissection (LCM) was used to isolate cancer cells from NPC tissues. Two-dimensional gel electrophoresis(2-DE) was used to separate the total proteins of LCM purified NPC cells. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins, and bioinformatics was used to construct the 2-DE database of NPC proteome.@*RESULTS@#A 2-DE reference map of NPC was established. On the 2-DE map, a total of (1 312+/-30) protein spots were detected, and 427 protein spots representing 241 non-redundant proteins were identified. The 2-DE database of NPC proteome was constructed. These data could be accessed at our website (http://www.xyproteomics.org).@*CONCLUSION@#A protein expression profile of LCM purified NPC tissues has been established for the first time, which provides useful information and source for proteomic study of NPC.

Comparative proteome analysis of laser capture microdissection for purified primary tumor and lymph node metastatic tumor in human lung squamous carcinoma / 中南大学学报(医学版)

Objective To search for lymph node metastasis-associated proteins in human lung squamous carcinoma (hLSC).Methods Laser capture microdissection (LCM) was used to purify the target cells from lung primary tumor and matched lymph node metastatic tumor in hLSC. Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected tumor cells from lung primary tumor and matched lymph node metastatic tumor. PDQuest software was applied to analyze 2-DE images. Differential protein spots between the two types of tissues were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The expression of Rho-GDIα, one of the differential proteins, in the microdissected lung primary tumor cells (LPTC) and matched lymph node metastatic tumor cells (LNMTC) was detected by Western blot. Results In the present study, 2-DE patterns of microdissected LPTC and LNMTC were established, and 22 differential proteins in the above two tissues were identified, of which 14 were down-regulated in LNMTC and 8 were up-regulated in LNMTC.Conclusion The 22 differential proteins may play some roles in the process of lymph node metastasis in hLSC, and the data provide new clues for metastasis-associated biomarker screen and mechanism of hLSC.

Screening for nasopharyngeal carcinoma antigens that induce humoral immune response by serological proteomics analysis / 中华检验医学杂志

Objective To identify proteins eliciting a humoral immune response in patients with nasopharyngeal carcinoma (NPC) by a serological proteome analysis, and provide candidate biomarkers for diagnosis and treatment of NPC.Methods Two-dimensional (2-DE) electropboresis was used to separate total cellular proteins from 19 NPC tissues.Separated proteins were transferred onto PVDF membranes and sera from 19 NPC patients and 19 healthy subjects were individually screened by western blotting for antibodies that react against separated proteins.Each tissue samples was subjected to three 2-DE gels and coomassie staining was performed in one of them.The protein spots which selectively reacted with the patient sera were excised from the preparative gels and subjected to further analysis of MOLDI-TOF MS and ESI-QTOF MS/MS.The proteins were identified based on peptide mass fingerprints and peptide sequence tags followed by searching database.Results In this study, 13 NPC associated antigens (HSP 70, HAS,HSP 60, CK 15, LAP 3, α-enolase, EBP 1, CK 19, ribosomal protein P 0, pyrovate dehydrogenase E1,guanine nucleotide-binding protein, prohibitin, Rho-GDI 2) that elicited an antibody response in most of NTC patients were identified.The positivities of these proteins were more than 21% all in NPC patients, but were lower or even absent in normal subjects.Conclusion These 13 NPC associated antigens and their autoantibodies may be useful for NPC diagnosis and treatment.

Expression of Raf kinase inhibitor protein in human gastric cancer and its clinical significance / 中华消化杂志

Objective To investigate differential expression of Raf kinase inhibitor protein (RKIP)in gastric cancer and its clinical significance.Methods Pure gastric adenocarcinoma cells (GAC)and paracancerous gastric epithelial cells(NGEC)in 12 patient with gastric cancer were obtained using laser capture microdissection(LCM).After trypsin digestion all specimens were labeled with18O/16O.Quantitatively identification of differentially expressed proteins between GCC and NGEC was conducted using Nano-HPLC-MS/MS.The expression of RKIP in GAC was determined by Western blotting.Immunohistochemistry(IHC)was used to detect the expression of RKIP in gastric cancer(n=118),non-cancerous mucosa tissues(n=70)and matched positive lymph node tissues(n=35).Results A total of 78 differentially expressed proteins were identified and RKIP was downexpressed in GCC compared with NGEC(1:4.37).IHC examination revealed that positive expression of RKIP in gastric cancer had negative relation with deeper invasion,TNM stage and lymphoid node metastasis,while it had positive relation with histological differentiation(P＜0.05).Conclusion Down-regulation of RKIP in gastric cancer tissues might affect the biological behavior of gastric cancer.

Screening of EGFR-regulated Secreted Proteins in Human NPC Cell Line CNE2 / 生物化学与生物物理进展

In order to screen EGFR-regulated secreted proteins in human nasopharyngeal carcinoma(NPC), and to reveal the role and mechanism of epidermal growth factor receptor(EGFR) in the pathogenesis of NPC. NPC cell line CNE2 cells were cultured in serum-free medium and stimulated by transforming growth factor-? (TGF-?) for 24 h in experimental group. Control CNE2 cells were cultured at the same condition but without TGF-? stimulation. The culture medium of control and experimental cells was desalted and concentrated through ultrafiltration to prepared the total secreted proteins. Two-dimensional gel electrophoresis (2-DE) was used to separate the secreted proteins of control and experimental cells, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the control and experimental cells were identified by desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The 2-DE patterns of the secreted proteins of TGF-? stimulated and un-stimulated CNE2 cells were established, 22 differential proteins spots between the two groups of cells were found, and 8 non-redundant proteins were identified with MALDI-TOF-MS, the functions of which were involved in invasion, metastasis, apoptosis and proliferation of cancer cells. The data will be valuable for further to study the role and mechanism of EGFR in the pathogenesis of NPC.

Quantitative Proteomics Analysis of LCM Purified Stroma of Nasopharyngeal Carcinoma and Normal Nasopharyngeal Mucosa / 生物化学与生物物理进展

The mechanism of how stroma plays an important role in tumor carcinogenesis is now a hotspot. To delineate the features of stromal protein between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal mucosa(NNM), laser capture microdissection (LCM) was performed to purify stromal cells from the NPC and NNM, respectively. The protein expressed profiles of the stroma of NPC and NNM were compared using fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) and 34 differential protein spots between tumor stroma and normal stroma were chosen to be identified by mass spectrometry (MS). A total of 20 differential proteins were identified, and three differential proteins (CapG, L-plastin and S100A9) were selectively further validated by Western blotting and immunohistochemical analysis to confirm the results of 2D-DIGE. 2D-DIGE patterns of the stroma of NPC and NNM were established for the first time, the results suggested that differentially expressed proteins in the stroma of NPC and NNM may be useful for understanding the relationship between NPC cells and their surrounding microenvironment. Further studying of these proteins will be helpful to elucidate the mechanisms of NPC carcinogenesis and provide new thoughts on therapy of NPC through stroma.

Comparative Proteomics Analysis of LCM Purified Nasopharyngeal Carcinoma and Normal Nasopharyngeal Epithelial Tissue / 生物化学与生物物理进展

To search for nasopharyngeal carcinoma (NPC) biomarkers,laser capture microdissection (LCM) was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues (NNET). Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected NPC and NNEC,PDQuest software was applied to analyze 2-DE images,and the differential protein spots between the two types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. The expression of cytokeratin 8(CK8),one of the differential proteins,in the microdissected NPC and NNEC as well as 4 NPC cell lines with different differentiated degrees and/or metastatic potentials was detected by Western blot. Immunohistochemistry was also used to detect the expression of CK8 in paraffin-embedded tissues including 63 cases of primary NPC,28 cases of NNET and 20 cases of cervical lymphonode metastasis. In the present study,2-DE patterns of microdissected NPC and NNEC were established,and 29 differential proteins in the above two tissues were identified,of which 15 only expressed or up-regulated in NPC and 14 only expressed or up-regulated in NNET. The expression level of differential protein CK8 between the NPC and NNET was selectively confirmed,and was found to be related to the differentiation and/or metastasis of NPC cell lines. Significant down-regulation of CK8 was observed in NPC compared with NNET,and significant up-regulation of CK8 was also observed in lymphonode metastasis compared with primary NPC. The data suggest that CK8 may be related to the differentiation and lymphonode metastasis of NPC,and may serve as molecular biomarkers for metastasis and differentiation of NPC.

Differential Proteomic Analysis of Sera From Lung Squamous Carcinoma Patients and Healthy Individuals / 生物化学与生物物理进展

To screen for serum biomarkers for lung squamous carcinoma, two-dimensional gel electrophoresis (2-DE) was performed to separate serum proteins from healthy individuals and stage 1 lung squamous carcinoma(LSC) patients, respectively. PDquest software was used to analyze 2-DE images, and the differential serum protein spots between the healthy individuals and LSC patients were identified by ESI-Q-TOF MS/MS. Then Western blot and immunohistochemistry were used to detect the expression levels of haptoglobin-2(HP-2), one of the differential proteins, in the sera and tumor tissues in the patients with LSC, respectively. 2-DE maps of serum proteins from healthy individuals and stage 1 LSC patients were established. Ten differential serum protein spots were detected, four proteins of which were identified by MS/MS. Western blot showed that the serum level of HP-2 in the LSC patients was significantly higher than that in healthy individuals, but was not associated with LSC staging. Immunohistochemistry showed that the expression level of HP-2 in the LSC tissues was significantly higher than that in the normal bronchial epithelial tissues adjacent to tumors. The results indicated that serum HP-2 protein is a candidate biomarker for LSC, and might be useful for diagnosis of LSC. Up-regulation of HP-2 in the LSC tissues may contribute to the high serum level of HP-2 in the patients.

Identification of Proteins Associated With p53 in Nasopharyngeal Carcinoma Cell Line CNE2 by Proteomics and RNAi Techniques / 生物化学与生物物理进展

In order to elucidate the mechanisms of p53 overexpression in nasopharyngeal carcinoma (NPC) and detect proteins associated with the function of p53 in high throughout screening, p53 which knockdown human NPC CNE2 cell line (CNE2sip53) were successfully established by using stable RNA interference (RNAi). Two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of CNE2sip53 and its control cell line CNE2/pSUPER, and PDQuest software was applied to analyze 2-DE images. Twenty-two differential protein spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14-3-3? etc.) , and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, the differential expression levels of the partial proteins (HSP27, 14-3-3?, GRP75) were confirmed by Western blot analysis and compared with CNE2 and CNE2 cells transfected with pcDNA3.1-FLAG, CNE2 cells transfected with pcDNA3.1-FLAG-p53 had obvious down-regulations of HSP27 and 14-3-3?, and an up-regulation of GRP75. The 22 differentially expressed proteins could be divided into five groups based on their functions: signal transduction, chaperone, transcription and translation, metabolism and cytoskeleton, which were involved in cell cycle, the transcription regulation, cell adherence,cellular metabolism and so on. The data suggest that these differential proteins may be associated with the function of p53 in NPC, and will be valuable for further to study the mechanisms of p53 overexpression and inactivation in NPC.

Proteomic comparison of two-dimensional gel electrophoresis profiles from human lung squamous carcinoma and normal bronchial epithelial tissues / 基因组蛋白质组与生物信息学报·英文版

Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733+/-0.101 mm in IEF direction, and 0.925+/-0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241+/-88 spots were detected, 987+/-65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+/-72 spots were detected, and 875+/-48 spots were matched with an average matching rate of 73.5%. A total of 864+/-34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.