Objective To develop a DNA microarray for HLA-DR1,DR51 group genotyping. Methods According to the specific allelic sequences coding HLA-DR1,DR51 loci,HLA- DR1,DR51 group typing probes which were immobilized on a glass support were synthesized.A pair of group-specific primers labeled by the Cy5-dCTP were designed,then the primers were used in the PCR,thus the PCR products were labeled with Cy5.The labeled PCR products were hybridized with array.The signals were scanned by scanner and analyzed by image software.The typing results were confirmed by standard DNA and PCR-SSO. Results A total of 130 samples were typed by this DNA array.There were 34 HLA-DR1,DR51 group loci typed by DNA array.Among them,18 loci were DR15,8 were DR16,6 were DR10 and 2 were DR1.No false positive or false negative typing results occurred.The accuracy and reproducibility were 100% and the overall time of genotyping was about 3.5 hours. Conclusions DNA array technique is a precise,rapid molecular method of high resolution power and high specificity for HLA-DR1,DR51 genotyping,which is applicable to clinical transplant practice.

Objective To develop an oligoneucleotide array for HLA-DR52 group rapid genotyping.Methods According to the special allele sequences of HLA-DRB loci in Chinese Han's population, HLA-DR52 group typing probes which were immobilized on a glass supports were synthesized. A pair of group-special primers labeled by the Cy5-dCTP were designed and were used in the PCR. The labeled PCR products with Cy5 were hybridized with array. The signals were scanned by a scanner and analyzed by Image software. 83 samples were typed by this array and the results were compared with PCR-SSP typing.Results Among 57 HLA-DR52 group loci typed by PCR-SSP,2 samples had no HLA-DR52 loci typed by array,3 DR52 group homozygotes typed by PCR-SSP were actually heterozygotes by array. The other 1 non-DR52 group homozygote identified by PCR-SSP was a heterozygote with one DR52 group locus. Conclusion The oligoneucleotide array technique is a precise, rapid molecular method for HLA-DR52 genotyping. Compared with PCR-SSP method, the genotyping chip is more sensitive and intuitionistic and suitable for clinic practice.

Objective:To investigate the expression of CXCL1 2 and CXCR4 in adenoid cystic carcinoma(ACC)and to explore its re-lationship with clinicopathologic characteristics and prognosis of the patients.Methods:The expression of CXCL1 2 and CXCR4 in af-fected tissue was detected immunohistochemically in 62 cases of ACC.Both of the two factors and clinicalpathology factors were pro-cessed in accordance with the Kaplan-Meier method and the COX regression model.Results:The positive rates of CXCL1 2 and CX-CR4 expression were 54.8%(34/62)and 77.42%(48/62)respectively.Patients with the 2 factor expression had a shorter survival time than those without them(P<0.05).Multivariate analysis revealed that CXCR4 expression,clinical stage,histological differentia-tion and metastasis/recurrence were independent risk factors for the prognosis of ACC patients.Conclusion:The expression of CXCR4 may be correlated with the malignancy of ACC.CXCR4 expression,clinical stage,metastasis/recurrence and histological differentia-tion can indicate the prognosis of ACC patients.

Objective To determine the effect of intravenous administration of vascular endothelial growth factor (VEGF) on myocyte apoptosis and the expression of P 53 , Fas, Bax and Bcl 2 in an experimental model rat with acute myocardial infarction Methods Thirty three male Sprague Dawley rats were subjected to left coronary artery ligation The rats were randomized to receive VEGF 165 heparin(treated group) or heparin saline(control group), respectively, 24 hours after surgery VEGF 165 (2 ?g/1 ml)with heparin(50 U)or heparin saline(50 U/1 ml)was administered daily via tail vein for 7 days(treated group, n=8;control group, n =10) and 14 days(treated group, n=7;control group,n =8)separately Apoptotic index and the expression of the P 53 , Fas, Bax and Bcl 2 in myocardium were measured at 9 or 16 days after coronary artery ligation Results The number of apoptotic myocytes in VEGF treated group was less than that in control group\[(10?2)% vs (28?2)%, P

Objective To investigate the anti-fibrogenesis property and mechanism of short hairpin RNA (shRNA ) targeting heat shock protein 47 (HSP47) in schistosomiasis liver fibrosis mice model . Methods Sixty female BALB/c mice (SPF level) were infected percutaneously with (16 ± 1) Schistosoma japonicum cercariae . Twelve mice which were not infected with Schistosoma japonicum were set as uninfected control .The 60 mice were randomly divided into five groups including 1 mg/kg HSP47-shRNA intervention group ,2 mg/kg HSP47-shRNA intervention group ,4 mg/kg HSP47-shRNA intervention group ,unrelated shRNA intervention group and infected control group ,with 12 mice each group .From 6 to 14 weeks post infection ,mice in HSP47-shRNA intervention groups were intravenously injected with HSP47-shRNA weekly and those in unrelated shRNA intervention group were intravenously injected with 2 mg/kg of unrelated shRNA vector weekly .Survival rate ,liver and spleen in general and liver histology of mice in each group were observed at week 14 which was considered as end of intervention . The expressions of HSP47 mRNA and protein were determined by real-time polymerase chain reaction (PCR) and Western blot .Collagen type Ⅰ ,collagen type Ⅲ ,tissue inhibitor of metalloproteinase-1 (TIMP-1) , matrix metalloproteinase-9 (MMP-9) ,plasminogen activator inhibitor-1 (PAI-1) ,transforming growth factor-β1 (TGF-β1 ) , connective tissue growth factor (CTGF ) , interleukin (IL )-13 and IL-17 at transcriptional level were measured by real-time PCR .Means among groups were compared using student-t test .Results In vivo intervention of HSP47-shRNA mainly localized in hepatic stellate cells of mice . The survival rate of 1 mg/kg ,2 mg/kg and 4 mg/kg HSP47-shRNA intervention groups were 25 .00% , 25 .00% and 33 .33% , respectively .Mice received 4 mg/kg dose of HSP47-shRNA had significantly higher survival rate than the infected controls (χ2 = 4 .168 ,P= 0 .043) .In both 4 mg/kg HSP47-shRNA intervention group and the infected control group , hematoxylin and eosin staining showed chronic granuloma change ,of which ova were wrapped by the spindle-shaped collagen and inflammatory cell infiltrated .The percentage of positive staining for Masson trichrome and sirius red showed the quantity of the collagen deposition was significantly reduced by the HSP 47-shRNA intervention ,compared to the infected control group (t = 3 .191 ,P = 0 .039) .HSP47-shRNA significantly reduced HSP47 expression , and reduced the transcriptional levels of collagen type Ⅰ ,collagen type Ⅲ ,TIMP-1 ,PAI-1 ,TGF-β1 , CTGF ,IL-13 and IL-17 (all P < 0 .01) ,and increased the expression of MMP-9 mRNA ( P < 0 .01) . Conclusion Delivery of shRNA targeting HSP47 to the hepatic schistosomiasis mice model can silence the expression of HSP47 and improve the liver fibrosis .Collagen degradation and related cytokines release through upregulation of MMP-9 and downregulation of PAI-1 may be one of the anti-fibrotic mechanisms in HSP47-shRNA intervention .

ObjectiveTo explore the effect of preoperative chemotherapy on DNA repair in hepatocellular carcinoma(HCC) patients. MethodsThe expression of hOGG1 portein in HCC and the surrounding liver tissue was detected by immunohistochemistry assay. ResultsThe expression of hOGG1 protein in HCC tissue was significantly higher in patients undergoing preoperative chemotherapy than that in control cirrhotic tissues,that of paracancerous tissues,and in patients without preoperative chemotherapy( ?~2=4.8297,?~2=4.0292,all P

Objective To detect the existence of vasculogenic mimicry in hepatocellular carcinoma (HCC). Methods In this study 42 patients with a total of 47 HCC nodules underwent radical resection.Histological and immunohistochemical double staining of CD31 and PAS were applied to observe the existence of vasculogenic mimicry ( VM ). Reverse tanscription PCR (RT-PCR) were applied to study the expression of VE-cadherin, EPHA2 and MMP-2 genes. Results VM was found in 16 of the 42 (38. 1% )HCC cases. The typical forms of VM in the microscope are vessel-like structure formed by tumor cells,without endothelial cells and the PAS-positive looping pattern. The existence of VM in HCC correlates to a higher Edmondson grade, higher capacity of intrahepatic disseminating and poorer tumor-free survival time (P＜ 0. 05). Comparing the difference of VE-cadherin gene, EPHA2 gene and MMP-2 gene expression between VM positive nodes and in VM negative nodes by RT-PCR method demonstrated that VE-cadherin gene, EPHA2 gene and MMP-2 gene have a more intense expression in VM positive nodes than in VM negative nodes ( P ＜ 0. 05 ). Conclusion VM exists in human hepatocellular carcinoma. VM occurred more frequently in higher malignant HCC and predicts a higher rate of tumor recurrence and poorer prognosis.

To establish an HPLC method to determine osimertinib mesylate,Agilent ZORBAX Eclipse Plus C18 column (4.6 mm × 250 mm,5 μm) was used with a mobile phase consisting of methanol-buffer solution (20 mmoL/L NaH2PO4,pH 3.0 adjusted with 85％ H3PO4) (50 ∶ 50) at the flow rate of 1.0 mL/min.The detection wavelength was 210 nm,and the column temperature was kept at 35 ℃.The calibration curve was liner over the range from 50％ to 150％ of determination concentration (0.201 1-0.603 2 mg/mL,r =0.999 9).The limit of quantitation (LOQ) and limit of detection (LOD) were 0.32 μg/mL and 0.08 μg/mL,respectively.The contents of osimertinib mesylate in samples were 100.1％,99.5％ and 99.7％.Good chromatographic separation of osimertinib mesylate and its related substances,including synthetic impurities and degradation products,were obtained.The established HPLC method is specific,accurate,simple and durable,and could be used for the determination of osimertinib mesylate.

Objective To detect the levels of IL-1, IL-6,TNF-αand IFN-γin serum and colon tissue of rat mod-els of ulcerative colitis with spleen and kidney Yang deficiency, and to explore their roles in the pathogenesis of ulcerative colitis ( UC) .Methods The rat model of ulcerative colitis with Yang deficiency of spleen and kidney was induced by perfusion of rhubarb decoction plus intramuscular injection of hydrocortisone and combined with TNBS (2,4,6-trinitro-benzenesulfonic acid) and ethanol enema.Sixty SPF wistar rats ( body weight 180 ±10 g, male:female=1:1) were ran-domly divided into blank control group, UC model with spleen kidney Yang deficiency for 7 days, 14 days and 21 days groups, respectively.The levels of IL-1, IL-6, TNF-αand IFN-γin serum and colon tissue were detected by ELISA.Re-sults Compared with the blank group, the levels of IL-1, IL-6, TNF-αand IFN-γin serum and colon tissue of rat UC model group with spleen kidney Yang deficiency were greatly increased (P<0.05), especially evidently increased in the model group at 21 days.Conclusions The pro-inflammatory cytokines IL-1, IL-6, TNF-αand IFN-γplay an important role in the pathogenesis of ulcerative colitis with syndrome of spleen and kidney Yang deficiency.

Objective To study the expression and significance of Th17 and Tc17 cells in the peripheral blood,skin and lung in a murine model of bleomycin (BLM)-induced systemic sclerosis (SSc).Methods Thirty female BALB/c mouse were randomly divided into 3 groups,including a control group ( A group),a injected with BLM 4 week without pulmonary fibrosis(PF) group( B group) and with obviously PF group(C group).Pathological changes of skin and lung were detected.The proportion of CD4+,CD8+,CD4+IL-17+(Th17),CD8+IL-17+(Tc17) cells in the peripheral blood,skin and lung of mouse was determined by flow cytometry.The mRNA expressions of RORγt,IL-17A in skin and lung of mouse were evaluated by real-time PCR.Enzyme linked immunosorbent assay(ELISA) was used to measure the levels of IL-17 in serum.Results Dermal hydroxyproline(HYP) contents and the score of PF were significantly increased in C group [ (3.07±1.26) μg/mg,4.0±1.41 ]and B group [ (2.43±0.61) μμg/mg,1.50±0.76]as compared with A group [ (1.45±0.40) μg/mg,0.60±0.70 ],and pulmonary HYP contents was obviously increased in C group than in A and B groups,all P＜0.05.Compared with the A group,the percentage of CD4+ and Th17 cells in the peripheral blood,skin and lung of B and C groups,Tc17 cells of C group was significantly increased,and CD8+ cells was significantly decreased(all P＜0.05).The ratio of Th17/CD4+CD8+ in the peripheral blood,skin and lung of B and C groups [ ( 1.41 ±0.36)％,( 1.79±0.77)％ ],[ (2.58±1.07)％,(5.23±2.34)％ ]and [ (3.50±1.20)％,(4.02±1.32) ％ ]was significantly increased compared with A group (0.71±0.25)％,(1.15±0.59)％,(0.99±0.46)％.The ratio of Tc17/CD4+CD8+ in the lung of C groups( 1.62±0.53) ％ and in the skin of B and C groups [ (1.70±0.70) ％,( 1.63±0.63 ) ％ ]was significantly increased compared with A group [ ( 1.00±0.47 ) ％,( 1.1 1 ±0.34 ) ％ ],all P＜0.05.Compared with the A group,the mRNA levels of IL-17A,RORγt in skin of B and C groups,and in lung of C group were higher and the levels of IL-17 in serum was significantly increased,all P＜0.05.Th17 cells and the levels of IL-17 in blood were positive correlation with dermal and pulmonary inflammation,fibrosis and H YP contents,all P＜0.01.The frequency of Th17 and Tc17 cells in skin and lung respectively had a positive correlation with dermal and pulmonary inflammation,the score of fibrosis,and HYP contents of skin and lung,all P＜0.01.Conclusion Th17 and Tc17 cells were significantly increased in the peripheral blood,skin and lung of a murine model of SSc,and Th17 cells is dominated.They correlated with the inflammation and fibrosis of skin and lung,and may participate in the pathogenesis of SSc through secrete IL-17.