Objective To analyze the effect of eukaryotic plasmids containing wild (sense) or anti- sense methylenetetrahydrofolate reductase (MTHFR) gene on cell viability and transcription level of tumor related genes in human gastric cancer cell line.Methods Human gastric cancer cell line MKN-45 was cultured.Recombinant plasmids containing wild MTHFR (W) or antisense MTHFR (A) gene, pCMV-W and pCMV-A,were constructed.Then pCMV-W,pCMV-A and pCMV blank plasmid were transfected into MKN45 cells respectively by using lipofect.Cell viability was analyzed by 3-(4,5-bime- thylthiazolyl-2)-2,5-diphenyhetrazolium dromide(MTT).The transcription levels of Dnmt 1,c-myc, p21~(WAF1) and hMLH1 genes were detected by real-time polymerase chain reaction(PCR).Results Cell vi- ability remarkably increased in those transfected with wild MTHFR (P＜0.01),which was contrary to those transfected with antisense MTHFR(P＜0.01).The expression of those tumor related genes mRNAs were all remarkably decreased in the MKN45-W cells in comparison with those in the MKN45-pCMV cells.No significant difference in the expressions of those tumor related genes mRNAs were found between the MKN45 cells transfected with pCMV-A and blank pCMV.Conclusion MTHFR influences cell viability and the expres- sion level of tumor related genes in human gastric cancer cell line MKN45.

Objective To investigate the effects of different concentrations of glucose on growth and collagen synthesis of rat skin fibroblast cells cultured in vitro. Methods Rat skin fibroblast cells cultured in vitro were treated with glucose of different concentrations (30,35 and 40mmol/L) for 48 h (high glucose group 1,2 and 3). Cell proliferation was detected by MTT method,the hydroxyproline contents in culture media were determined by the commercial kit,and the mRNA expression of typeⅠand Ⅲ procollagen,matrix metalloproteinase-2(MMP-2)and tissue inhibitor of metalloproteinase-2(TIMP-2)were detected by RT-PCR. Cells cultured with 25mmol/L glucose were served as controls. Results With the increase of glucose concentration in culture media,the growth of skin fibroblast cells was significantly inhibited,and the hydroxyproline contents were significantly decreased (P

OBJECTIVETo evaluate the effect of PKC-delta inhibitor Rottlerin on human colon cancer cells and its mechanism.

METHODSHuman colon cancer cell line SW1116 cells were treated with Rottlerin. The transcriptional level of DNA methyltransferase (Dnmt)1, Dnmt3a and Dnmt3b was detected by real-time RT-PCR. Cell cycle distribution was evaluated by flow cytometry (FCM). In addition, cellular morphological changes were examined by light microscopy.

RESULTSPKC-delta inhibitor decreased the expression of Dnmt1, Dnmt3a mRNA, up-regulated APC, p21(WAF1) and p16(INK4A) mRNA. Demonstarted by flow cytometry, Rottlerin increased the percentage of cell cycle G0/G1 phase cell numbers (P = 0.02) and decreased the percentage of cell cycle G2/M phase cell numbers (P = 0.01). Remarkable changes of cellular morphology were observed under light microscope: The volume and cytoplasm of cells treated with Rottlerin were increased. The cell contour was not very clear, and mitotic figures were less frequently seen.

CONCLUSIONPKC-delta inhibitor Rottlerin inhibites cell division and proliferation of the colon cancer SW1116 cells through regulating DNA methylation and blocking the signaling pathway of mitogen-activated protein kinase (MAPK).

Acetophenones ; pharmacology ; Adenomatous Polyposis Coli Protein ; genetics ; Benzopyrans ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Protein Kinase C-delta ; antagonists & inhibitors ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects

OBJECTIVETo explore the effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells and underlying mechanism.

METHODSThe thoracic aorta of newborn calf was used for primary endothelial cells culture. Cells were divided into 3 groups and cultured for 48 h: control group (C, 5.5 mmol/L), constant high glucose group (HG, 30 mmol/L) and glucose fluctuation (GF, three circles of 2 h 30 mmol/L followed by 3 h 5.5 mmol/L, 30 mmol/L overnight, repeat the whole procedure on the following day) groups. The membranes fluidity of endothelial cells was detected by fluorescence polarization method. The contents of sorbierite, aldose reductase (AR), sorbitol dehydrogenase (SDH) and advanced glycation end products (AGEs) were measured. RAGE, eNOS and ET-1 mRNA expressions were detected by semi-quantitative RT-PCR.

RESULTSThe membranes fluidity of endothelial cells in HG or GF group were significantly decreased compared with the control group (all P < 0.01) and significantly lower in GF group than those in HG group (all P < 0.01). Sorbierite, AR and AGEs concentrations were significantly higher in HG and GF groups than those in control group (all P < 0.01) and AR and AGEs concentrations were significantly higher in GF group than that in HG group (all P < 0.01). SDH of endothelial cells in HG or GF group were decreased compared with the control group and lower in GF group than in HG group (all P < 0.05). In addition, the mRNA levels of RAGE, eNOS and ET-1 were significantly upregulated compared with the control group (all P < 0.01).

CONCLUSIONSGlucose concentration fluctuation can result in more severe bovine arterial endothelial cells dysfunction than high glucose via activating polyols metabolic pathways, upregulating the expression of AGEs, eNOS and ET-1. Therefore, glucose concentration fluctuation might play a crucial role on macrovascular complications of diabetes.

Aldehyde Reductase ; analysis ; Animals ; Aorta, Thoracic ; cytology ; Cattle ; Cells, Cultured ; Endothelial Cells ; metabolism ; pathology ; Endothelin-1 ; analysis ; Endothelium, Vascular ; cytology ; metabolism ; Glucose ; metabolism ; Glycation End Products, Advanced ; analysis ; L-Iditol 2-Dehydrogenase ; analysis ; Membrane Fluidity ; Nitric Oxide Synthase Type III ; analysis

Adult ; Aged ; Bone Screws ; Female ; Femoral Neck Fractures ; surgery ; Fracture Fixation, Intramedullary ; Humans ; Internal Fixators ; Male ; Middle Aged

OBJECTIVETo analyze the specificity, sensitivity and receiver operating characteristic (ROC) curve of plasma elafin for diagnosis of skin acute graft-versus-host disease (aGVHD), and to explore its clinical diagnostic value.

METHODSIncidence of skin aGVHD from fifty-three patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) were observed prospectively in Guangdong General Hospital from Apr 2010 to Aug 2011. The plasma concentrations of elafin were detected by enzyme-linked immunosorbent assay (ELISA). Skin biopsies were taken from 28 patients with skin rash, and elafin expression in the skin was detected by immunohistochemistry. Positive expression was defined as significant staining of at 50% of the depth of the epidermis, excluding the granular cell layer and the acrosyringium.

RESULTSAmong 28 patients with skin rash, twenty-five were considered as skin aGVHD by clinical diagnosis, seventeen were confirmed as skin aGVHD by pathological biopsy. 11 cases were elafin positive by immunohistochemical staining. Elafin protein was overexpressed in aGVHD skin tissue (P = 0.001). Plasma concentrations of elafin were significantly higher in patients with skin aGVHD (positive) group than in those without skin aGVHD (negative) group (P = 0.005), among which there being no statistically significant difference in plasma elafin level between patients with grade I skin aGVHD group and negative group(P = 0.971), but being statistically significant difference compared patients with grade II-IV skin aGVHD group with those with grade I skin aGVHD group (P = 0.02) and with negative group (P = 0.008). Using the pathological diagnosis as the gold standard, the estimated specificity and the sensitivity of clinical diagnosis criteria were 27.3% and 100%, respectively, and those of tissue elafin protein level were 100% and 64.7%, respectively. The area under the ROC curve was 0.909 (0.797 - 1.021) when plasma concentrations of elafin was used in diagnosis of skin aGVHD. The sensitivity was 82.4% and the specificity was 81.8 % when the critical value was set at 1456.043 µg/L.

CONCLUSIONPlasma concentration of elafin is significantly higher at the onset of skin aGVHD. It can be used as biochemical marker of skin aGVHD and has higher value in diagnosis of skin aGVHD.

Adolescent ; Adult ; Elafin ; blood ; Female ; Graft vs Host Disease ; blood ; diagnosis ; etiology ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; ROC Curve ; Sensitivity and Specificity ; Skin Diseases ; blood ; diagnosis ; etiology ; Young Adult

This study was purposed to investigate the efficacy and safety of intravenous injecting itraconazole (ITCZ) as empirical antifungal therapy in the patients with hematological malignancies. According to recommendation in IDSA guidebook, the patients suffered from fever during neutropenia and inefficacy of treatment using broad-spectrum antibiotics for 4 days should receive intravenous injection of ITCZ as empirical antifungal therapy. The results showed that the overall clinical response rate to ITCZ injection was 62.9% (22/35), and the success rate of achieving composite endpoints was 54.3% (19/35). Mild adverse reactions were observed in 6 patients (17.1%). The injection of ITCZ was stopped in 2 patents (5.7%) due to adverse reaction. Further analysis revealed that the response rate was higher in patients with fever prior to the start of ITCZ within five days than beyond five days (P = 0.031). The response rate was higher in patients with possible invasive fungus infection (IFI) than that in patients with probable and confirmed IFI (P = 0.002). The prophylactic antifungal treatment during neutropenia displayed no significant influence on efficacy of empirical antifungal therapy with itraconazole (P = 0.054). It is concluded that the good efficacy and safety of empirical ITCZ injection for hematological malignancies patients is efficient and safe.
Adolescent ; Adult ; Aged ; Antifungal Agents ; administration & dosage ; therapeutic use ; Female ; Hematologic Neoplasms ; drug therapy ; Humans ; Injections, Intravenous ; Itraconazole ; administration & dosage ; therapeutic use ; Male ; Middle Aged ; Treatment Outcome ; Young Adult

The aim of this study was to investigate the renal function in 149 patients receiving myeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT) from June 2005 to June 2010 in our hospital, and analyze the risk factors resulting in kidney insufficiency and experience in diagnose and therapy. The creatinine clearance (CrCL) and serial creatinine level were evaluated before and after allo-HSCT within 100 days and 1 year. Non-radiation conditioning regimens were used for any patients. The acute kidney insufficiency (AKI) was defined as at least a 1.5-fold rise in serum creatinine level after allo-HSCT within the first 100 days. The chronic kidney insufficiency (CKI) was defined as the creatinine clearance < basal level within 3 months to 1 year after allo-HSCT. The results showed that the kidney insufficiency was found in 41 patients, in which the incidence of AKI was 32/149 (21.5%). CsA, amphotericin B (P = 0.025) and ES (P = 0.022) were defined as risk factors for AKI. The incidence of CKI was 18/138 (13%). cGVHD (P = 0.013) and TA-TMA (P = 0.012) were associated with the development of CKI. The 2-year survival was lower in patients with kidney dysfunction than that in patients without kidney dysfunction (39% vs 74.1%, P < 0.001). The main factors resulting in kidney insufficiency were defined as infection (52%), GVHD (20%), TA-TMA (12%) and tumor relapse (12%). It is concluded that kidney insufficiency is an important complication of allo-HSCT. Careful monitoring kidney function, minimizing the use of amphotericin B, prophylaxis and effective treatment of fungal infection, GVHD and TA-TMA may be effective preventive measures to decrease the incidence of kidney insufficiency.
Acute Kidney Injury ; etiology ; Adolescent ; Adult ; Female ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; Renal Insufficiency ; etiology ; Retrospective Studies ; Risk Factors ; Transplantation, Homologous ; Young Adult

With the development of molecular pathology,many types of epidermal growth factor receptor (EGFR) mutations have been identified.The efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) patients with different types of EGFR mutations,especially in patients with single rare mutations or complex mutations (co-occurrence of two or more different mutations),has not been fully understood.This study aimed to examine the efficacy of EGFR-TKIs in NSCLC patients with different types of EGFR mutations.Clinical data of 809 NSCLC patients who harbored different types of EGFR mutations and treated from January 2012 to October 2016 at Renmin Hospital and Zhongnan Hospital,Wuhan,were retrospectively reviewed.The clinical characteristics of these patients and the efficacy of EGFR-TKIs were analyzed.Among these patients,377 patients had only the EGFR del-19 mutation,362 patients the EGFR L858R mutation in exon 21,33 patients single rare mutations and 37 patients complex mutations.Among these 809 patients,239 patients were treated with EGFR-TKIs.In all the 239 patients,the disease control rate (DCR) was 93.7％ with two patients (0.2％) achieving complete response (CR),the median progression free survival (PFS) was 13.0 months (95％ confidence interval [CI],11.6-14.4 months),and the median overall survival (OS) was 55.0 months (95％ CI,26.3-83.7 months).Subgroup analysis revealed that the DCR in patients harboring single rare or complex mutations of EGFR was significantly lower than in those with del-19 or L858R mutation (P＜0.001).Patients with classic mutations (del-19 and/or L858R mutations) demonstrated longer PFS (P＜0.001) and OS (P=0.017) than those with uncommon mutations (single rare and/or complex mutations).Furthermore,the patients with single rare mutations had shorter median OS than in those with other mutations.Multivariate Cox regression analysis identified that the type of EGFR mutations was an independent risk factor for PFS (hazard ratio [HR]=0.308,95％ CI,0.191-0.494,P＜0.001) and OS (HR=0.221,95％ CI,0.101-0.480,P＜0.001).The results suggest that the single rare or complex EGFR mutations confer inferior efficacy of EGFR-TKIs treatment to the classic mutations.The prognosis of the single rare EGFR mutations is depressing.EGFR-TKIs may be not a good choice for NSCLC patients with single rare mutations of EGFR.Further studies in these patients with uncommon mutations (especially for the patients with single rare mutations) are needed to determine a better precision treatment.