AIM: To investigate the role of HGF/c-Met signaling pathway in crizotinib-induced apoptosis of different lung carcinoma cell lines and to analyze its potential regulatory mechanisms .METHODS: EML4-ALK positive cell line H2228, c-Met proliferation cell line H1993 and control cell line A549 were treated with crizotinib at different doses for different time periods .The viability of the cell lines was measured by MTT assay .The apoptosis was analyzed by flow cytometry with PI staining.The protein levels of MET and phosphorylated MET (p-MET) of HGF/c-Met signaling pathway as well as its down-stream key proteins AKT , ERK, p-AKT and p-ERK in the cell lines before and after crizotinib treatment were examined by Western blot .RESULTS:The growth of H1993, H2228 and A549 cell lines was inhibited after crizoti-nib treatment for 72 h in a dose-dependent manner .Apoptotic rates of H1993 cells and H2228 cells were increased with the crizotinib concentration and exposure time .Down-regulation of p-MET, p-AKT and p-ERK at protein levels in H1993 cells and H2228 cells after exposure to crizotinib for 72 h was confirmed by Western blot .No obvious change of the related-pro-teins of HGF/c-Met signaling pathway was found in A 549 cell line.CONCLUSION: HGF/c-Met signaling pathway may contribute to crizotinib-induced apoptosis of H1993 cells and H2228 cells, which provides the experimental basis for MET-targeting treatment of lung cancer .

Objective: To establish an HPLC-fluorescence detection method for the determination of thioguanosine-monophosphate (TGMP), thioguanosine-diphosphate (TGDP) and thioguanosine-triphosphate (TGTP) in red blood cells (RBC), as well as quantify the individual thioguanine nucleotides metabolites in kidney transplant recipients with azathioprine (AZA) therapy.Methods: The individual thioguanosine phosphates were extracted from RBC by dichloromethane and subsequently oxidized by potassium permanganate.The separation was achieved on a Nucleosil C18 column (150 mm×4.6 mm,5 μm) with an ion pairing reagent and detected by a fluorescence detector (excitation at 315 nm, emission at 390 nm).The mobile phase consisted of 20 mmol·L-1 potassium phosphate buffer (pH was adjusted to 6.8 by 5 mmol·L-1 tetrabutylammonium hydrogensulfate)-acetonitril (80:20) with the flow rate of 1.0 ml·min-1.Results: TGMP, TGDP and TGTP were quantified from RBC within the range of 50-500, 50-1000 and 100-5 000 pmol·ml-1, respectively.The limit of quantification (LOQ) was 50, 50 and 100 pmol·ml-1 RBC for TGMP, TGDP and TGTP, respectively.The intra-and inter-day RSDs were below 7.0% with the method recovery between 95.0% and 103.6%.The mean extraction recovery was above 90%.The assay was applied in the blood samples of 30 kidney transplant recipients with AZA therapy, and the results indicated that TGTP was the predominant phosphate metabolite in RBC.Conclusion: The method is simple, rapid, sensitive and specific, and it can quantitatively determine the individual thioguanosine phosphates in RBC of kidney transplant recipients with AZA therapy.

AIM:To detect the changes of active status of bypass signaling pathways in EML4-ALK positive lung cancer cell line H3122 treated with alectinib, hepatocyte growth factor (HGF), epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), and to explore the potential mechanisms.METHODS:EML4-ALK positive cell line H3122 was treated with increasing concentrations of alectinib or/and induced by HGF, EGF and TGF-α.The cell viability was measured by CCK-8 assay.The cell apoptosis was analyzed by flow cytometry.The protein levels and phosphorylation status of ALK, c-Met and EGFR, and the downstream molecules AKT, ERK, p-AKT and p-ERK were examined by Western blot.RESULTS:The viability of the H3122 cells was inhibited by alectinib in a dose-dependent manner after administrated for 72 h, and the IC50 value was 0.042 μmol/L.The concentration-growth curves of the H3122 cells shifted to the right after induced by HGF, EGF and TGF-α.After treatment with alectinib at 0.05 μmol/L for 48 h, the apoptotic rate of H3122 cells was (20.12±1.36)%, while the apoptotic rates of the cells in the groups of alectinib combined with HGF, EGF or TGF-α were (7.85±1.03)%, (5.60±0.79)% and (4.58±1.00)%, respectively.Those values were remarkably lower than those in alectinib single treatment group (P<0.05).Alectinib inhibited the protein levels of p-ALK and its downstream signaling pathway molecules, while HGF significantly up-regulated the protein levels of p-Met and its downstream p-AKT and p-ERK.Besides, EGF and TGF-α remarkablely up-regulated the protein levels of p-EGFR and its downstream p-AKT and p-ERK.Combined treatment with crizotinib and 17-DMAG successfully inhibited the viability of the H3122 cells even in the presence of the HGF and EGFR ligands, respectively.CONCLUSION:Bypass signaling pathways are activated by HGF, EGF and TGF-α in EML4-ALK positive lung cancer cell line H3122, which may be linked to alectinib resistance.

Type 1 diabetes is a autoimmune disease that occurs under the influence of both genetic predisposition and environmental factors,mediated mainly by T lymphocytes with various kinds of other involved immune cells.The autoimmune attacks eventually lead to the islet beta cell destruction and insulin insufficiency.Multiple fundamental studies and clinical trials have revealed that umbilical cord blood pluripotent stem cells have the potential to help restore immune balance,induce immune tolerance,stop the autoimmune attacks against beta cells,and promote beta cell regeneration in type 1 diabetes mellitus (T1DM),by means of cell to cell contact and soluble cytokine secretion.For the past few years,the National Clinical Research Center for Metabolic Diseases of Second Xiangya Hospital has been leading the research on stem cell education therapy and has performed the therapy for 35 juvenile type 1 diabetes patients from China and foreign countries,introducing a novel treatment for T1DM.

Objective:To study the association of RAC1 gene polymorphisms with protein expression levels of Rac1-GTP. Methods:A total of 182 healthy Hans population in Hubei were recruited. The 4 tag-SNPs in RAC1 gene were genotyped by Real time TaqMan-MGB genotyping assay. And the Rac1-GTP protein levels in plasma samples from all participants were determined by enzyme linked immunosorbent assays ( ELISA ) . The distribution characteristics of Rac1-GTP expression levels were also analyzed. Furthermore,the expression levels of Rac1-GTP were compared among different genotypes of the 4 tag-SNPs in RAC1 gene. Results:The distribution of Rac1-GTP expression levels was positive skewed in healthy Chinese Hans population. The expression levels were significantly higher in females than in males (P<0. 05),and appeared in decreased trend with age,but without significant differences (P>0. 05). Different expression levels of Rac1-GTP were observed in different genotypes for rs702482 and rs10951982 (P<0. 05). However,no significant difference was found for rs702483 and rs6954996 (P>0. 05). Conclusion:RAC1 genetic polymorphisms can potentially affect the expression levels of Rac1-GTP protein in healthy Chinese Hans population.

Objective:To develop a method for the detection of 10 single nucleotide polymorphisms(SNPs) of Nm23 gene,and to explore the genotypic and allelic distributions of the 10 SNPs in Chinese Hans population in Wuhan.Methods: Two hundreds healthy subjects ,115 men and 85 women included ,were enrolled as DNA sample donors.The real time TaqMan-MGB genotyping assay was used for the determination of the 10 SNPs selected ,and the results were validated by direct gene sequencing.Results:The method established could accurately and quickly screen the genotypes of the 10 SNPs of Nm23/NDPK gene.Distribution frequencies of the 10 SNPs were similar to these in other researches as well as these of HCB.All the loci follow the Hardy-Weinberg equilibrium.Highly linkage disequilibriums were found between rs 16949649 and rs7207370 , rs16949649 and rs34214448 , rs2159359 and rs2302254 , rs2159359 and rs8075231 ,rs2159359 and rs2041296 ,as well as rs2159359 and rs8071647 ,respectively.Four Tag SNPs:rs34214448 , rs2302254 ,rs11868380 and rs2318785 were initially selected by Heploview software.Conclusion:The method established for SNP gen-otyping can meet the needs for rapid analysis of Nm 23 gene polymorphisms ,and may have great values in investigating the association between gene polymorphisms and diseases as well as adverse drug reactions.

[Objective] To assess the predilection age,clinical features,treatment and pognostic factors of primary cutaneous B-cell lymphoma (PCBCL).[Methods] A total of 31 patients with PCBCL were registered in Tianjin Cancer Central Registry from January 1970 to September 2010,and 26 patients had complete medical recortds.A retrospective analysis was conducted on the 26 patients.The following indices were analyzed,including gender,age,body sites of initial involvement,extent of cutaneous involvement,pathological subtypes,metastatic sites,treatment,survival and prognosis.Data were processed with SPSS16.0 software,survival analysis was carried out by using Kaplan-Meier method,univariate analysis of prognostic factors by Log-rank test,multivariate analysis of prognostic faetors by COX proportional hazards model,and variables were selected by Forward LR method.[Results] Clinical manifestafions were atypical in these patients.The ratio of man and women was 1:1.6.Of the 26 cases,12 were primary cutaneous follicle-center lymphoma (PCFCL),8 were primary cutaneous marginal zone lymaphoma (PCMZL),and 4 were PCLBCL,leg type.Follow-up revealed lymph node involvement in 8 patients (4 in cervical nodes and 4 in inguinal nodes),distant metastasis of organs in 3 patients ( 1 in lung and pleura,1 in bone marrow and 1 in central nervous system).Most patients received surgical treatment combined with chemotherapy or chemoradiation.The 5-year overall survival rate was 80.8％(21/26).Statistical analysis showed that the prognosis of PCBCL was associated with histological subtype,lactate dehydrogenase (LDH) level and globulin level,but unrelated to gender,age,site of initial involvement,extent of cutaneous involvement,involvement of lymph nodes and organs,presence of B symptoms,treatment strategies,number of relapses,level of β2-globulin and hemoglobin,or lymphocyte absolute value.[Conclusion]s PCBCL,as a kind of rare extra-nodal lymphoma,usually occurs with atypical clinical mainfestations in persons aged from 39 to 66 years.The 5-year overall survival rate was 80.8％ in these patients.The prognosis of PCBCL seems to be related to histological subtypes,LDH and globulin levels.

AIM:To investigate the protective effect of bactericidal/permeability-increasing protein (BPI) on sepsis induced by intra-abdominal infection in rats and its mechanism.METHODS:Intra-abdominal infection induced sepsis was reproduced by cecal ligation and puncture (CLP). BPI or equal volume of physiological saline was intra-abdominally given immediately after CLP and 12 hours after CLP respectively (2.5 mg/kg of BPI each time). Plasma endotoxin levels were determined with limulus amebocyte chromogenic assay.RESULTS:(1)The survival time in BPI group was significantly higher than in physiological saline (PS) group. (2)The values of MAP, LVSP, IP, d p /d t max and -d p /d t max in BPI group, although decreasing ,were markedly higher than those in PS group. (3) Plasma glutamate-pyruvate transaminase and urea nitrogen levels in BPI group, though increasing, were significantly lower than those in PS group.(4) There was no significant change of plasma endotoxin levels in BPI group, while plasma endotoxin levels were markedly increased in PS group. There was significantly different between two groups. CONCLUSIONS:BPI has an obvious protective effect on intra-abdominal infection induced sepsis, which might be related to its antagonism against endotoxin.

Objective:To establish a method for the detection of 8 single nucleotide polymorphisms (SNPs) of RAC1 gene,and to analyze the genotypic and allelic distributions of the 8 SNPs in healthy Chinese Han population.Methods:The real-time fluorescence TaqMan-MGB probes allele classification technology was used for the determination of the 8 SNPs in 150 cases of healthy volunteers in Hubei China ,and the results were validated by direct gene sequencing.Results: The method established in this study can accurately screen the genotypes of the 8 SNPs of human RAC1 gene.Highly linkage disequilibriums were found between rs 10951982 and rs9374 , rs702482 and 836488 ,respectively.All the 8 sits were in accordance with Hardy-Weinberg equilibrium.Six Tag-SNPs were selected by Heploview software:rs10951982 ,rs6954996 ,rs6951997 ,r1s2977 ,rs702482 and rs702483.The MAFs of the 8 SNPs in our study were close to the MAFs in CHB and Asian in Hap Map database .Significant distribution differences were also observed in different races.Conclusion:No significant differences are observed in this study in healthy Chinese Han population.But differences are found when compared with the data of other races in Hap Map.

Objective To investigate the prognosis of patients with Kawasaki's disease complicated with coronary artery lesion and to provide evidence for diagnosis and treatment of these patients. Methods This study was conducted during January 2002 to June 2007. All patients diagnosed as Kawasaki's disease complicated with coronary artery lesions were from the Affiliated Hospital of Guilin Medical College, the Second People's Hospital of Guangxi Province and Guilin Women and Childrens' Hospital. All cases were echocardiogram examined in month 3, 6, 12, 24 and 36 in the purpose of observing the morphology of coronary artery. The study subjects were re-categorized to the groups of mild, moderate dilatation of coronary artery and giant coronary aneurysm, based on the severity of coronary artery lesion. The results of these results of the three groups were compared. Results Eighty-four cases in the mild group, 27 cases in the moderate group and 8 cases in the giant coronary aneurysm. The recovery cases were 23 (27%), 3 (11%) and 0 in the 3 groups respectively at month 3. The above numbers were 44 (52%), 8 (30%) and 0 respectively at month 6.The numbers were 69(82%), 13 (48%) and 1 (13%) at month 12. The numbers were 78 (93%), 19 (70%), 3 (38%) at month 24. The numbers were 82(98%), 20(74%) and 4(50%) at month 36. Thirteen patients were treated with adenosine-triphosphate (ATP) stress echocardiography examiantion, 5 patients were evaluated by coronary angiography,and 4 patients were tested by 64-slice CT coronary reconstruction. Part of the patients were found to have coronary stenosis or occlusion. Conclusion Patients of Kawasaki's disease often have concurrent coronary artery lesions. Patients with mild dilatation of the coronary artery are the most commonly seen and have the best prognosis. On the contrast, patients with giant coronary aneurysm are the lest common situation and is the worst in prognosis. Part of them will develop coronary artery stenosis or occlusion in late stage which may lead to ischemic heart disease. We should stress on close follow-up of patients with Kawasaki's disease complicated with coronary artery lesion. Appropriate and timely treatment will increase their clinical outcomes.