Objective To construct a eukaryotic expression plasmid carrying the PKCI-1/HINT1 gene,to investigate its expression in A375 melanoma cells after transfection,and to evaluate its effects on apoptosis and autophagy of A375 cells.Methods The PKCI-1/HINT1 gene sequence was amplified by reverse transcription PCR (RT-PCR) with total RNA extracted from A375 cells as the template,then inserted into the eukaryotic expression plasmid PCDNA3.1 (+) to construct a recombinant plasmid,PCDNA3.1 (+)-PKCI-1/HINT1.Some A375 cells were classified into two groups to be transiently transfected with the recombinant plasmid (PCDNA3.1 (+)-PKCI-1/HINT1 group) or the empty plasmid PCDNA3.1 (+) (control group).After additional 48-hour culture,RT-PCR and Western blot analysis were performed to quantify the mRNA and protein expressions of PKCI-1/HINT1 respectively,Hoechst 33342 staining was conducted to detect apoptosis of A375 cells,Western blot analysis to detect the expressions of intracellular caspase-3 and autophagy-associated protein beclin1,and cell autophagy was observed by using the green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) labelling method combined with confocal laser scanning microscopy.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of A375 cells at 24,48,72 and 96 hours after transfection.Results Enzyme digestion and sequencing analysis confirmed that the eukaryotic expression plasmid PCDNA3.1 (+)-PKCI-1/HINT1 was successfully constructed and effectively expressed in the transfected A375 cells.MTT assay showed that PKCI-1/HINT1 could obviously inhibit the proliferation of A375 cells,and the number of live cells was decreased by 17.0％,25.6％ and 29.4％ in the PCDNA3.1 (+)-PKCI-1/HINT1 group at 48,72 and 96 hours,respectively,compared with the control group (all P ＜ 0.05).Hoechest 33258 staining revealed that PKCI-1/HINT1 could promote the formation of apoptotic bodies in A375 cells.Confocal laser scanning microscopy demonstrated that the overexpression of PKCI-1/HINT1 increased GFP-LC3 puncta formation in A375 cells.In addition,Western blot analysis indicated that PKCI-1/HINT1 up-regulated the protein expressions of caspase-3 and beelin1 in A375 cells.Conclusions The eukaryotic expression plasmid PCDNA3.1 (+)-PKCI-1/HINT1 was successfully constructed,and PKCI-1/HINT1 could be effectively expressed in A375 cells.High-level expression of PKCI-1/HINT1 could suppress cellular proliferation,promote apoptosis,and induce autophagy,of A375 cells.

Objective The expression of TGF-β1 and IL-10 in the grafts of inbred mice with rejection of heart transplantation were studied and the interaction of them in the rejection of heart transplantation in inbred mice investigated.Methods Allografts were divided into 2 groups:control group (n =70),cyclosporin A-treated group (CsA group,n =70).Hearts from inbred BABL/c mice were transplanted into a cervical location in the other recipients and the survival time of the allografts was observed.The local expression of TGF-β1 and IL-10 was detected at day 1,3,7,11,14,21 by reverse transcription polymerase chain reaction(RT-PCT) respectively.Results The survival time of the allografts was (20.3 ± 1.7) days in control group,and(32.2 t 3.4) days in CsA group (P ＜ 0.01).The levels of the two cytokines expression were up-regulated in CsA group.The up-regulation of TGF-β1 was closely correlated with the survival of the grafts.Conclusion The expression and production of the two cytokines is up-regulated probably cause of administ ration of cyclosporin A,and favorite to the heart graft survival,and action of these two cytokines are probably interrelated.

Objective To explore the relationship between the types of female breast density and age and breast cancer .Methods By accepting the digital mammography X -ray examination for 5 006 women cases and according to the ACR BI -RADS standard in the fourth edition ,the breast density assessment was quantified . We analysed the relationship between the breast density and age and breast cancer .Results In 5 006 cases,the average female age was between 44.22 ±8.09 years old,median age was 43 years old.The components of the breast density were fat type , small amount type , large amount type and compact type each count were 256 (5.11%),726(14.51%),3 719(74.29%),305(6.09%)respectively.By dividing into different age -group to analyze the breast density,there was significant statistical differences of the breast density among age -groups(P<0.001).Among them the breast cancer were 184 cases.Age was between 51.26 ±10.15 years old.Breast cancer in each breast density were fat type 10.16%(26/256),small amount type 9.09%(66/726),large a-mount type 2.45%(91/3719)and compact type 0.33%(1/305).There were statistical differences among age -groups and breast densities and breast (P<0.001).Conclusion Age plays a very important effects on the fe-male breast density .The lower breast density is a high risk factor to breast cancer occurrence .

Naringin is mainly present in Rutaceae Citrus Pomelo,grapefruit,lime,and its variants of the peel and fruit,which belongs to dihydrogen flavonoids.Studies have shown that naringin has anti-type 1 and type 2 diabetes pharmacological effects,the mechanism of action by inhibiting diabetes-related oxidative stress injury,inflammation,abnormal metabolism of glucose,and other aspects,while a certain degree of delay in diabetes complications (including diabetic cardiomyopathy,diabetic nephropathy,diabetic retinopathy,and diabetic neuropathy).The mechanism of naringin on the mechanism of diabetes mellitus and its complications was studied in order to provide the basis for the development of antidiabetic drug.

Objective To explore the miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malignant melanoma.Methods Bioinformatics analysis was conducted by using online software miRanda and TargetScan,so as to predict the miRNA targeting CCL18 gene.Three kinds of C CL18 3'UTR dual-luciferase reporter vectors,including mutant 3'UTR vector (mutant 3'UTR group),wildtype 3'UTR vector (wild-type 3'UTR group) and empty vector (blank control group),as well as miRNA vectors carring selected miRNAs were constructed according to human gene sequence analysis,and then were used to co-transfect 293T cells.After 48-hour treatment,the cells were lysed for detection of luciferase activity.Real-time fluorescence-based quantitative PCR was performed to measure the expression of CCL 18 and selected miRNA in 14 fresh malignant melanoma tissue specimens and 14 paracancerous normal skin tissue specimens (control tissues),and their correlations were analyzed.Results Online software analysis showed that some miRNAs were identified to target the 3'UTR of CCL18 gene,including miR-183,miR-128 and miR-33a.Luciferase reporter vectors and miRNA vectors were constructed successfully.As luciferase activity assay showed,when miR-183 and miR-128 were bound to the CCL18 3'UTR,the luciferase activities were significantly higher in their mutant 3'UTR groups (11.63 ± 0.42;8.80 ± 0.49) than in their wild-type 3'UTR groups (4.86 ± 0.39;5.01 ± 0.54;both P ＜ 0.05) and blank control groups (2.41 ± 0.13;2.39 ± 0.05;both P ＜ 0.01),while there were no significant differences between miR-33a-hinding mutant 3'UTR group (6.41 ± 0.47) and miR-33a-binding wild-type 3'UTR group (6.16 ± 0.22,P ＞ 0.05).Real-time fluorescence-based quantitative PCR revealed higher mRNA expression of the CCL18 gene (3.52 ± 1.68),but lower expression of miR-183 (0.49 ± 0.32),miR-128 (0.30 ± 0.20) and miR-33a (0.46 ± 0.40) in the malignant melanoma tissues compared with the control tissues.The mRNA expression of the CCL18 gene was negatively correlated with the expression of miR-128 (rs =-0548,P ＜ 0.05),but showed no significant correlations with the expression of miR-183 and miR-33a (both P ＞ 0.05).Conclusion miR-128 may play a role in regulating the potential malignant melanoma-promoting gene CCL18.

Objective: To compare the efifcacy and safety of sirolimus-eluting stent (SES) and everolimus-eluting stent (EES) for treating the patients with non-ST segment elevation acute coronary syndrome (NSTE-ACS). Methods: A total of 400 NSTE-ACS patients treated in Jining Medical College Hospital from 2013-09 to 2014-09 were studied. According to different stents, the patients were divided into 2 groups: SES group,n=220 and EES group,n=180. A prospective follow-up study was conducted for 1.5 years to compare the incidence rate of major adverse cardiovascular events (MACE). The patients were further stratiifed by GRACE scores as Low risk group (score<109), Medium risk group (score 109-140) and High risk group (score>140). MACE free survival was studied by Kaplan-Meier curve and analyzed by Long-rank test, predictive value of GRACE for 1.5 year MACE incidence rate was examined. Results: There were 355/400 (89%) patients completed (16.7 ± 5.7) months of follow-up study including 205 in SES group and 150 in EES group. MACE occurrence rates were similar between SES group and EES group (16.10% vs 18.0%), P>0.05. By GRACE score stratiifcation, MACE rates in High risk SES group were higher than High risk EES group (48.00%vs 16.00%),P<0.05; while they were similar between Medium risk groups (14.49% vs 28.00%) and Low risk groups (9.11% vs 12.86%), allP>0.05. ROC curve indicated that the predictive value of GRACE score for 1.5 year MACE incidence was for AUC=0.762, 95% CI (1.026-1.050),P<0.001. Conclusion: Implanting of EES would be more beneifcial for NSTE-ACS patients with high GRACE risk; GRACE score has the better predictive value for their long-term prognosis.

Objective To screen microRNAs(miRNAs)related to early mycosis fungoides(MF). Methods A high?throughput miRNA PCR array was used to determine miRNA expression profiles in skin lesions of 6 patients with early MF (early MF group) and 6 patients with lichen planus (control group), followed by screening of differentially expressed miRNAs between the two groups. Then, real?time fluorescence?based quantitative PCR(RT?qPCR)was performed to verify the differentially expressed miRNAs in lesional specimens from 13 patients with early MF and 13 patients with eczema or lichen planus, as well as in Myla cells and normal human T?lymphocytes. Results The high?throughput miRNA PCR array showed that the expressions of hsa?miR?378a?5p, hsa?miR?107 and hsa?miR?302c?3p were significantly higher in the early MF group than in the control group(all P<0.05). For skin lesions, the results from RT?qPCR were similar to those from the miRNA array assay. Compared with normal human peripheral blood T?lymphocytes, Myla cells showed significantly increased expressions of hsa?miR?378a?5p and hsa?miR?107, which was consistent with the results from the miRNA array assay. However, no significant difference was observed in the expression of hsa?miR?302c?3p between the two kinds of cells. Conclusion MiRNA expression profiles in early MF are different from those in inflammatory skin diseases.

Objective:To explore and discuss the differences of pregnancy contents and inform formats in in-formed consent form ( ICF) for the drug clinical trial between China and foreign countries. Methods:We collected Chinese and foreign ICFs for drug clinical trial that had been audited by the Ethics Committee of the third Xiangya Hospital for the past five years. Based on the relevant domestic and foreign law, we concluded the element stand-ards and inform formats about pregnancy inform. By analyzing the integrity of the whole elements, the inform rate of every element and the using rate of every inform format, we compared the differences of pregnancy contents and in-form formats between Chinese ICFs and foreign ICFs. Results:The total number of ICFs was 177 in this study, in-cluding 107 Chinese ICFs and 70 foreign ICFs. The integrity rate of pregnancy in Chinese ICFs was statistically lower than them in foreign ICFs (19% vs. 56%, P=0. 000). Compared with foreign ICFs, the low informed ele-ments were the study of the pregnancy risk (32% vs. 73%, P=0. 000), the pregnancy test during the following-up period (33% vs. 56%, P=0. 002) and the measurements for contraception (22% vs. 53%, P=0. 000). Conclusion:The integrity level of pregnancy content in Chinese ICFs was lower than that of the foreign ICFs. And the three elements including pregnancy risk study, pregnancy test during the following-up period and measure-ments for contraception was obviously defected. Pregnancy informing forms of informed consent in China was inferi-or to abroad.

Objective To measure histidine triad nucleotide?binding protein 1(HINT1)protein expression and gene promoter methylation, and to analyze the relationship between HINT1 gene promoter methylation and clinical pathological features of melanoma. Methods Fifty?six patients with melanoma and 51 patients with nevus were enrolled as subjects and controls, respectively. Methylation?specific PCR (MSP) was performed to measure the methylation of HINT1 gene promoter in lesional and paratumoral tissue specimens from the patients with melanoma, as well as in lesional specimens from the patients with nevus. Immunohistochemistry was carried out to quantify the expression of HINT1 protein in these tissue specimens. Results MSP showed that the methylation rate of HINT1 gene promoter was significantly higher in melanoma tissues than in paratumoral and nevus tissues(76.8%[43/56]vs. 33.9%[19/56]and 35.3%[18/51], χ2 = 20.810 and 18.749, respectively, both P < 0.05), but was insignificantly different between paratumoral and nevus tissues(χ2=0.022, P>0.05). Immunohistochemistry revealed that the expression rate of HINT1 was 21.4%(12/56)in melanoma tissues, compared to 82.4%(42/51)in nevus tissues(χ2 = 39.633, P <0.01). There was a significant difference in the methylation rate of HINT1 promoter between HINT1?positive and ?negative melanoma tissues(6/12 vs. 37/44[84.1%], P<0.05), and between Clark levelⅠ-ⅡandⅢ-Ⅴmelanoma tissues(59.1%[13/22]vs. 88.2%[30/34],χ2=6.365,P=0.012). Conclusions HINT1 protein is lowly expressed in melanoma, which may be associated with high methylation of its gene promoter. Moreover, the high methylation ofHINT1 gene promoter may be involved in the initiation and progression of melanoma.

To report a case of neonatal succinate-CoA ligase deficiency with mild methylmalonic aciduria caused by SUCLG1 compound heterozygous mutation. Our proband developed the disease at the 2nd day of life, manifested by vomiting and milk rejection. Blood gas analysis indicated metabolic acidosis and hyperlactacidemia. Laboratory tests showed mild hyperammonemia, mild elevation of serum aminotransferase and normal homocysteine. Imageology examination showed brain dysplasia and multiple cardiac malformations. Mild methylmalonic aciduria was suggested by blood acylcarnitines and urine organic acids analysis. Two novel mutations, c.40A>G (p.M14V) and a heterozygous deletion of exon 6 to 9, were identified by whole-exome sequencing in SUCLG1, which came from the mother and father, respectively. Metabolic acidosis turned better by volume expansion and acidosis correction, but lactic acidosis was persistent. Edema and respiratory failure occurred, after which the parent gave up treatment and the patient died after discharged from hospital. The mutations of SUCLG1 gene lead to succinate-CoA ligase deficiency and mitochondrial DNA depletion syndrome. There is no specific treatment for this disease. For infants with feeding difficulties, lactic acidosis and multiple organ damage, metabolic abnormality screening should be performed. Genetic analysis is the diagnostic method.