Objective To study the expression of Gli1 protein and vascular endothelial growth factor (VEGF)-C in papillary thyroid carcinoma and their relationship with cervical lymph node metastasis.Methods The expression of Gli1 protein and VEGF-C were investigated by immunohistochemical EnVision method in 67 cases of papillary thyroid carcinoma and 32 cases of nodular goiter pathological specimen tissues.Nascent microvascular and micro-lymphatic of all pathological speimen tissues were examined by immunohistochemical monoclonal antibody CD34 and D2-40 staining assay respectively,and the microvascular density (MVD) and micro-lymphatic vessel density (MLVD) were calculated.The relationship between the positive expression of Gli1 protein and MVD,between the positive expression of VEGF-C and MLVD,and their relationship with cervical lymph node metastasis were analyzed.Results The positive rates of Gli1 protein,VEGF-C and MVD,MLVD were significantly higher in papillary thyroid carcinoma than those in nodular goiter [67.16％(45/67) vs.43.75％(14/32),70.15％(47/67) vs.31.25％(10/32),23.14 ± 2.06 vs.2.14 ± 0.31,13.36 ± 1.32 vs.3.53 ± 0.65,P ＜ 0.05].The positive expression of Gli1 protein was related to the patient' s age and the tumor size (P ＜ 0.05),while the positive expression of VEGF-C was not related to the patient' s age and the tumor size (P ＞ 0.05).The positive expression of Gli1 protein and VEGFC were significantly higher in TNM stage Ⅲ-Ⅳ of papillary thyroid carcinoma than those in TNM stage Ⅰ-Ⅱ (P ＜ 0.01) and also was significantly higher with cervical lymph node metastasis than without cervical lymph node metastasis (P ＜ 0.01).MVD and MLVD were significantly higher with cervical lymph node metastasis than without cervical lymph node metastasis (P ＜ 0.05).The positive expression of Gli1 protein was positively correlated with MVD (r =0.784,P＜ 0.01),the positive expression of VEGF-C was positively correlated with MLVD (r =0.529,P ＜ 0.01),the positive expression of Gli 1 protein was positively correlated with the positive expression of VEGF-C (r =0.586,P ＜0.01).Conclusions Gli1 protein which may participate in the formation of nascent microvascular is abnormally activated by the Hedgehog signaling pathway to express,VEGF-C which may be participate in the formation of nascent micro-lymphatic is mediated and started by the MAPK signaling pathways to express.Expressions of Gli1 protein is positively correlated with expressions of VEGF-C,therefore the Hedgehog signaling pathway may be associated with the MAPK signal pathway.Suppressing nascent microvascular and micro-lymphatic may become new target to blockingup papillary thyroid carcinoma cervical lymph node metastasis.

OBJECTIVE To evaluate the role ofpreoperative ultrasonograpy in detecting cervical lymph node metastasis. METHODS Medical records between February 1998 and February 2002,consisting of 51 cases (58 sides) of well-differentiated thyroid carcinoma with cervical lymph nodes metastasis, were reviewed. Patients were divided into 2 groups: group 1,34 cervical sides with palpable cervical lymph nodes preoperatively and group 2,24 cervical sides with impalpable nodes but positive for nodal metastasis ultrasonically. All patients underwent modified neck dissection. The preoperative ultrsonographic results and preoperative pathologic findings were compared. RESULTS Of the 58 sides with positive preoperative ultrsonographic results, 53 sides had been demonstrated to have cervical lymph nodes metastasis pathologically. The sensitivity of ultraonography was 91.4 %(53/58). Four patients had developed lateral cervical recurrence during the course of the follow-up, yielding a recurrence rate of 7.5 %. Ultrasonography detected cervical lymph node believed to be uninvolved by physical examination in 39.6 % of patients. The most frequent involvement site was middle neck according to ultrasonography [71.7 %(38/53)] and level Ⅲ according to pathological findings [67.9 %(36/53)]. CONCLUSION Preoperative ultrasonograpy is a basis for detecting cervical lymph nodes metastasis in thyroid cancer patients. It can detect metastatic cervical lymph nodes and their localizations. All thyroid cancer patients should undergo preoperative ultrasonography.

Objective To characterize the profile and clinical significance of hepatitis B virus (HBV) quasispecies in patients infected with hepatitis B virus based on the sequence of reverse transcriptase (RT) region.Methods Fifty HBV infected treatment-naive patients were enrolled and divided into three groups,asymptomatic carriers (ASC) group (10 cases),chronic hepatitis B (CHB) group (30 cases) and liver cirrhosis (LC) group (10 cases).HBV genomes were extracted from serum samples.The sequence of RT region was amplified by polymerase chain reaction (PCR) and cloned into vectors.Fifteen to thirty clones per sample were selected,sequenced and analyzed by bioinformatics software.The mean values among groups were compared by analysis of variance.The median values among groups were compared by nonparametric statistics.The enumeration data were analyzed by x2 test.Results Totally 1221 HBV RT region nucleotide sequences were obtained (152from ASC patients,780 from CHB patients and 289 from LC patients).Genotype distribution showed no difference among three groups.However,the quasispecies complexity showed significant differences among the three groups,LC group ＞CHB group＞ ASC group (F=33.400,P＜0.05).The quasispecies diversity was LC group ＞CHB group＞ ASC group,and that of LC group was significantly different from the other two groups (F=18.070,P＜0.05),while there was no significant difference between CHB and ASC patients.Conclusions The HBV isolated from patients in immune clearance phase have higher variability than those isolated from patients in immune tolerance phase.The longer the infection persists and the more severe the disease is,the more variable HBV quasispecies are.

Objective To characterize serum hepatitis B virus(HBV)full-length genome quasispecies and to investigate its ralationship with severe exacerbation of chronic hepatitis B(CHB).Methods HBV full-length genome was amplified and cloned from four treatment naive CHB patients and four treatment naive CSHB patients.Fourteen to sixteen clones per sample were selected,sequenced and analyzed by bioinformatics software.The measurement data was compared by independent-samples t test and count data was analyzed by x2 test. Results Totally 120 HBV fulllength genome sequences were obtained.All the patients had either genotype B or C virus monoinfection.One hundred percent clones(60/60)from CSHB patients showed mutations including G1896A,A1762T/G1764A(one patient even carried A1762T/G1764A/C1766T mutations),T1753C/G and start codon mutations in preS2,preS1,which were more common than those from CHB patients(46/60,76.7%;x2=15.85,P<0.01).The quasispecies complexity and diversity were higher in CSHB patients than CHB patients within full-length genome,S,X,P genes and reverse transcriptase region,but lower within C gene at both nucleotide and amino acid levels.But the difference were not statistically significant in all regions.Conclusion The mutation frequency and quasispeeies heterogeneity in HBV genome are higher in CSHB patients than in CHB patients,which may play a role in the severe exacerbation of CHB and needs further investigation in large scale studies.

Objective To investigate the value of the reverse shock index multiplied by GlasgowComa scale score (rSIG) and serum translocator protein 18000 in the prognosis of patients with severe traumatic brain injury. Methods One hundred and fifteen patients with severe traumatic brain injury were divided into the survival group and death group. SPSS 20.0 software was used to compare the vital signs, rSIG and TSPO between the two groups, and the relationship between rSIG and TSPO was analyzed. Receiver operating characteristic (ROC) curve was used to predict the value of rSIG and TSPO and their combination in the prognosis of patients with severe traumatic brain injury. According to the best cut-off value of rSIG and TSPO of ROC curve, patients were divided into the rSIG ≤ 14.8 group and rSIG>14.8 group, and the TSPO ≤ 1.84 ng/mL group and TSPO>1.84 ng/mL group, and the mortality between the groups was compared. Results In 115 patients, rSIG of the survival group was significantly higher than that of the death group, and TSPO was significantly lower than that of the death group [(10.5±4.4) vs. (6.4±4.1), 1.0(0.3,1.9) ng/mL vs.3.4 (2.0, 4.6) ng/mL, P<0.01]. The ability of rSIG combined with TSPO to forecast the mortality of patients with severe traumatic brain injury is not superior to the predictive power of these two indicators alone. The serum TSPO value and 28-day mortality in the rSIG > 4.15 group were significantly higher than those in the rSIG ≤ 4.15 group. The rSIG value of the TSPO ≤ 1.84 ng/mL group was significantly higher than that of the TSPO>1.84 ng/mL group; the 28-day mortality was significantly lower than that in the TSPO>1.84 ng/mL group. The rSIG value was negatively correlated with serum TSPO value (r=-0.611, P<0.01). Conclusions rSIG value and serum TSPO value have good predictive value for the prognosis of patients with severe traumatic brain injury, and can provide certain guiding significance in clinical practice.

Objective·To analyze the expression changes of adhesion G protein-coupled receptor F1(ADGRF1)in the occurrence and development of pancreatic ductal adenocarcinoma(PDAC),and explore the impact of ADGRF1 on the proliferation of PDAC cells and the potential molecular mechanisms that promote PDAC progression.Methods·The expression of ADGRF1 at mRNA level was analyzed based on the Gene Expression Omnibus(GEO)database and The Cancer Genome Atlas(TCGA)database,respectively.The expression of ADGRF1 in normal pancreatic ductal epithelial cells(hTERT-HPNE)and various PDAC tumor cells was detected by using real-time fluorescence quantitative PCR(qPCR)and Western blotting.Immunohistochemical staining(IHC)was used to detect the differential expression of ADGRF1 in cancer tissues and adjacent tissues of PDAC patients.After knocking down ADGRF1 with small interfering RNA(siRNA)transfection,the changes in the proliferation ability of PDAC AsPC-1 and SW1990 cells were detected through CCK8 assay and plate cloning experiment.Stable overexpression of ADGRF1 was constructed in PDAC Patu8988 cell line,and the proliferation changes induced by overexpression of ADGRF1 were evaluated through CCK8 assay.RNA sequencing(RNA-seq),gene set enrichment analysis(GSEA),and immune infiltration analysis were utilized to predict signaling pathways associated with ADGRF1-mediated promotion of PDAC cancer progression.Results·Analysis of the TCGA database and GEO database revealed higher expression of ADGRF1 mRNA in PDAC tissues compared to normal pancreatic tissues(all P=0.000).qPCR and Western blotting results demonstrated up-regulation of ADGRF1 mRNA and protein levels in various PDAC cells compared to hTERT-HPNE cells(all P<0.05).IHC results confirmed higher ADGRF1 expression in PDAC cancer tissues compared to adjacent tissues.Furthermore,downregulation of ADGRF1 inhibited the proliferation of PDAC AsPC-1 and SW1990 cell lines,while overexpression of ADGRF1 promoted the proliferation of Patu8988 cells(all P<0.05).RNA-seq,GSEA enrichment analysis,and immune infiltration analysis revealed that ADGRF1 expression was related to signaling pathways such as interferon-α(IFN-α),tumor necrosis factor-α(TNF-α),and nuclear factor κB(NF-κB).Conclusion·ADGRF1 is highly expressed in PDAC cells and tissues,and promotes the proliferation of PDAC cells via immune-related signaling pathways.

Objective·To establish a mouse model with dormant cancer and no obvious metastasis by combining the preimmune strategy with the mVenus-p27K-cell G0 phase indicator system,the DTR-HSV/TK suicide gene system,and the Luc2-tdTomato tracer system.Methods·The KPC1199 mouse pancreatic cancer cell line was transfected with the mVenus-p27K-cell G0 phase indicator system,the DTR-HSV/TK suicide gene system,and the Luc2-tdTomato tracer system to construct a stable expression cell line,KPC1199-PDL.After being cultured in the serum-free condition,KPC1199-PDL cells were sorted into mVenus(+)cells and mVenus(-)cells by flow cytometry,and the expression of G0 phase-related genes was verified by real-time fluorescence quantitative PCR(qPCR).Sensitivity of KPC1199-PDL cells to diphtheria toxin(DTX)and ganciclovir(GCV)was evaluated by CCK-8 assay.A transsplenic portal vein-hepatic metastasis model was constructed in wild-type C57BL/6 mice to validate the function of KPC1199-PDL cells in vivo by immunofluorescence technology.The KPC1199-PDL cells were injected subcutaneously into C57BL/6 mice,followed by in situ injection of DTX and GCV to ablate subcutaneous tumors 5 d later,to obtain preimmunized mice.The transsplenic portal vein-hepatic metastasis models were constructed in these mice.Bioluminescence imaging was used to evaluate subcutaneous tumor ablation and hepatic metastasis in the mice,and immunofluorescence assay was used to detect the distribution and dormant state of tumor cells in the livers of preimmunize mice.Results·The three tool systems were stably expressed in KPC1199-PDL cells,and their proliferative ability was not affected.In the serum starving condition,some KPC1199-PDL cells expressed the mVenus protein,indicating entry into the G0 phase;the mVenus(+)cells sorted out by flow cytometry exhibited significantly higher expression of G0 phase-related genes(all P<0.05)and significantly lower expression of the proliferation-related gene compared with mVenus(-)cells(P<0.05).The CCK-8 assay demonstrated high sensitivity of KPC1199-PDL cells to DTX and GCV.In vivo experiments confirmed that KPC1199-PDL cells could be effectively traced through tdTomato protein expression,and could indicate entry into the G0 phase through mVenus protein expression.Following subcutaneous tumor implantation and drug ablation,preimmunized mice were successfully obtained.In the subsequent transsplenic portal vein-hepatic metastasis model,no metastatic signals were observed in the liver by bioluminescence imaging,but single or small clusters of G0 phase tumor cells expressing both mVenus and tdTomato,not expressing the proliferation marker Ki67,were detected in liver tissue sections by immunofluorescence analysis.Conclusions·A recognizable and traceable dormant cancer model is constructed with the combination of the preimmune mouse model of pancreatic cancer,the mVeneus-p27K-indicator system,the DTR-HSV/TK suicide gene system,and the Luc2-tdTomato tracer system.