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.
Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Morphine ; Proteome ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Spinal Cord

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 .

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 width<10.0 mm after birth) , the other 2 cases were stable (the ventricular width measured first time after birth was ≥10.0 mm, but the difference was within 2.0 mm from the MRI before birth). In group B, 4 cases had MRI, 1 was normal, 1 was stable, and 2 cases were getting better (the ventricular width measured first time after birth was ≥10.0 mm, but the width decreased more than 2.0 mm from the MRI before birth). In group C, 6 cases had MRI. 3 cases were getting better and 3 cases were stable. (2) Overall GDS results:expansion group after the birth of the 3rd, 6th, 12th, 18th month GDS evaluation results compared with control group, respectively, the differences were not statistically significant (all P>0.05). (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 (P<0.05); while there were no statistically significant differences between the 2 goups at the 12th and 18th month (P>0.05). And for group C, statistically significant differences were found compared to the control group at each follow-up time (all P<0.05). (4) GDS results at different times after birth in the expansion group:there was no statistically significant difference between the results at the 3rd and 6th month (P>0.05). But when the result at the 3rd month was compared to the results of the 12th or 18th month, the differences were statistically significant (P<0.05). GDS result of 6th months after birth compared with 12th and 18th months, respectively, there were no statistically significant differences (P>0.05). 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 (<75 scores). Among the 28 bilateral cases, 23 (82.1%, 23/28) had normal GDS results;3 (10.7%, 3/28) had borderline results; 2 (7.1%, 2/28) had delayed results. There was no statistically significant difference (P>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.

Objective To explore the related factors and nursing interventional methods of depression in patients with motor aphasia after stroke. Methods The Stroke Aphasic Depression Questionnaire-Hospital version (SADQ-H) was used to diagnose depression of 271 patients with motor aphasia after stroke. The association of post-stroke depression with social factors, vascular risk factors and stroke features were analyzed. The relationship between depression severity and aphasia severity were analyzed. A total of 166 patients with depression were divided into the control group and the observation group to carry out different nursing interventional methods. Results The incidence of post-stroke depression in patients with motor aphasia was 61.3%(166/271). The occurrence of post-stroke depression was correlated with female (χ2=5.580, P=0.02) , age less than 60 years old (χ2=4.390, P=0.04) , living alone (χ2=5.400, P=0.02) , recent negative events (χ2=4.420, P=0.04). The multi-factor Logistic regression analysis showed that the occurrence of post-stroke depression was independently associated with female (OR=2.58, 95%CI 2.12-3.86, P<0.01), age less than 60 years old(OR=2.62, 95%CI 2.16-3.94, P<0.01), living alone(OR=2.59, 95%CI 2.13-3.92, P<0.01). After 4 weeks of nursing intervention, there were significant differences in SADQ-H scores (t=2.94, P=0.002) and distribution of Boston Diagnostic Aphasia Examination (BDAE) (χ2=14.045,P=0.000) between the control group and the observation group. Conclusions Female, ages less than 60 years old, living alone are independent risk factors of post-stroke depression in motor aphasia patients. The degree of depression is associated with aphasia severity. The early nursing intervention can improve the depression status and promote recovery of the language function of patients with motor aphasia after stroke.

OBJECTIVE:To observe clinical efficacy and safety of tegafur gimeracil oteracil potassium combined with 3D-CRT and microwave hyperthermia in the treatment of elderly patients with local advance esophageal cancer. METHODS:48 cases of local advance esophageal cancer were divided into group A,B and C according to random number table method,with 16 cases in each group according to random number table method. Group A was given 3D-CRT with total dose of 60-66 Gy totnlly for 6-7 weeks;group B was additionally given tegafur gimeracil oteracil potassium 60 mg/(m2·d),bid,on the basis of group A 3 weeks for a course of treatment,until the end of radvotherapy;group C was additionally given thermotherapy on the basis of group B 3 weeks for a course of treatment,until the end of radvotherapy. Clinical efficacy,1-year survival rate,dysphagia,weight and KPS score were compared among 3 groups,and the occurrence of toxic reactions(radioactive esophagitis,radioactive pneumonia, bone marrow suppression and gastrointestinal reaction) were observed in 3 groups. RESULTS:Total effective rate,1-year survival rate and the remission rate of dysphagia of group C were 93.75%,87.50% and 93.75%,which were significantly higher than those of group A(50.00%,50.00% and 56.25%)and B(68.75%,68.75% and 68.75%),with statistical significance(P<0.05). There was no statistical significance in the rate of weight gain and KPS score gain among 3 groups(P>0.05). The incidence of radioactive esophagitis and radioactive pneumonia in group C were significantly lower than in group A and B,with statistical significance(P<0.05);there was no statistical significance in the incidence of gastrointestinal reaction and bone marrow suppression among 3 groups (P>0.05). CONCLUSIONS:Tegafur gimeracil oteracil potassium combined with 3D-CRT and microwave hyperthermia in the treat-ment of elderly patients with local advance esophageal cancer further improves clinical efficacy and survival rate,but shows low inci-dence of toxic reaction.

Objective To investigate the surgical effort of transurethral bipolar plasma kinetic resection (PKR-BT) on who cannot tolerate radical cystectomy in elderly patients with high-risk infiltration bladder urothelial cancer.Methods From 2010 January to 2013 September,data from 27 cases of elderly had been reviewed.The risk of invasive bladder urothelial cancer were treated with PKR-BT treatment.All patients met WHO elderly standards in this group of patients with severe diabetes was 7 cases,and other comlications.Results All 27 patients had been successful in operation and postoperative recovery.Patients were followed up for 4-45 months,8 cases occured recurrence,6 cases of PKR-BT again,2 cases missed,complete follow up 25 patients still survived,the median follow-up time was 23.6 months,the longest survival was 45 months.Conclusion PKR-BT in the treatment of invasive bladder urothelial cancer patients is safe and effective,it can be used as one of the alternate treatment for such patients.

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.

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.

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.

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.