ObjectiveTo evaluate the sensitivity and specificity of reverse transcriptase polymerase chain reaction (RT PCR), immunohistochemistry (IHC) and micropartical enzyme immunoassay (MEIA) in detecting metastasis of human colonic carcinoma (HCC) in a nude rat xenograft model. Methods A metastatic model of HCC in nude rats was established by injecting HT 29 cells, a HCC cell line, into the mesenteric vein of the rats. Hepatic and pulmonary metastasis were detected by RT PCR for cytokeratin 20 (CK20) expression, APAAP immunostaining with monoclonal antibody (McAb) KL 1 (anti cytokeratin) for human CK expression, routine pathology (RP) and MEIA for tissue carcinoembryonic antigen (CEA) determination, respectively. Results The detection rate of HCC metastasis by RT PCR, IHC, RP or MEIA was 73 8%, 64 3%, 50% and 33%, respectively. The detection rate of RT PCR was higher than that of RP (? 2 =5 048, P 0 05). The detection rate of IHC was higher than that of MEIA (? 2 =8 052, P 0 05). RT PCR, IHC and MEIA were all highly specific in detecting HCC cells in nude rats. ConclusionsRT PCR for CK20 expression and APAAP immunostaining with the McAb KL 1 for human CK expression were highly sensitive and specific diagnostic techniques in detecing HCC metastasis. MEIA for tissue CEA determination was highly specific, but with a low sensitivity.

Objective:To study the inhibited function of CTLA4 Ig on T cell activation and its mechanism Methods:After separating fresh PBMC,the effects of CTLA4 Ig on T lymphocyte transformation and mixed lymphocyte reaction were analyzed,and its inhibitory effect on the CD25 expression on T cell surface was observed At last,the changes of the nuclear protein in active T cell were analyzed by the EMSA method.Results:CTLA4 Ig has an inhibitory effect on the lymphocyte transformation,MLR and CD25 expression on T cell surface,and it can inhibite the activation of RE/AP binding factor related to IL 2 expression. Conclusion:CTLA4 Ig inhibit the activation of T cell by multipathway,one of the mechanism might be its inhibitory effect on the activation of RE/AP binding factor.

Objective To construct a cDNA library of three-year old Polygonum multiflorum leaf tissues so as to further research the gene regulation of secondary metabolite biosynthesis of medicinal plants. Methods Total RNA from leaf tissues of P.multiflorum was extracted and mRNA was purified,which were synthesized to double strand cDNA through reverse transcription.After the cDNA termini was blunted,the 5' end of EcoR Ⅰ adapters phosphorylated was conjoined,and then digested by Xho Ⅰ,cDNA fragments were fractionated by Sepharose CL-2B spin column.The fragments longer than 400 bp were linked to Uni-ZAP XR vector.The primary cDNA library was established after the recombinants had been packaged.Uni-ZAP XR Vector might fleetly release pBluescript SK-phasmids at the presence of ExAssist helper phage of coinfection and inverted E.coli SOLR.Finally,PCR and double enzymes digestion were used to analyze the range of inserts,respectively. Results The titer of cDNA primary library was 1.07?106pfu/mL and the length of exogenous insert was at about 0.5-2.0 kb with 5.4?105 recombinants,the recombinants of amplified library were 4.25?1011 and the rate of recombination was 98.5%. Conclusion The results indicate that the cDNA library of P.multiflorum leaf tissues has enough volume for screening the desired genes and sets up a basis for studying on gene regulation of secondary metabolite biosynthesis of medicinal plants besides.

Objective To investigate the expression and correlation of hypoxia-inducible factor-1?(HIF-1?),vascular endothelial growth factor(VEGF)and sFlt-1 in the preeclampsia placenta,and discuss their significance in the pathogenesis of preeclampsia.Methods Placentas were collected from 20 pregnant women with preeclampsia as study group and 15 normal pregnant women as control group.The expressions of HIF-1?,VEGF and sFlt-1 protein were semi-quantitatively analyzed with immunohistochemical assay and mRNA level was determined using reverse transcription polymerasc chain reaction(RT-PCR)technique. Results(1)the expression of HIF-1?and sFlt-1 protein in preeclampsia group obviously increased.Strong (+++)positive expression was observed in 9 and 11 cases respectively,significantly higher than in control group(2 and 3 cases)(P＜0.05),however,VEGF expression obviously reduced in preeclampsia group(P＜0.01).(2)the level of HIF-1?and sFlt-1 mRNA in preeclamptic placenta was 0.604?0.013, 0.898?0.041,significantly higher than 0.208?0.007 and 0.559?0.244 in normal placenta(P＜0.05). Although the level of VEGF mRNA increased in preeclampsia placenta,it was not significantly different from that in normal placenta(P＞0.05).The ratio of VEGF mRNA/sFlt-1 mRNA obviously reduced in preeclampsia group and was significantly lower than in control group(P＜0.05).(3)in preeclampsia group,HIF-1?mRNA expression was positively correlated with the expression of sFlt-1 mRNA(r=0.577, P＜0.05),and negatively correlated with the ratio of VEGF mRNA/sFlt-1 mRNA(r=-0.376,P＜0.05).Conclusion Abnormal high HIF-1?expression in preeclampsia placenta indicates that HIF-1?might play an important role in the pathogenesis of preeclampsia,possibly through affecting the cytotrophoblastic invasion and placental vascular reconstruction via the modulation of VEGF and sFlt-1 gene transcription.

Objective To investigate the kidney function changes and the risk factors in elderly patients aged 80 years and over.Methods A total of 1054 hospitalized patients aged ≥80 years in our hospital were selected.Serum levels of creatinine,urea,uric acid and urinate were tested.Cockcroft-Gault equation was used to estimate glomerular filtrate rate (GFR).Results The renal function in 87.1 ％ of the patients was reduced.The percentage of patients with 30≤eGFR ＜60 ml · min-1 · 1.73 m-2 was 66.2％.The percentage of patients with 15≤eGFR ＜30 ml · min-1 · 1.73 · m-2had a gender difference (P＜0.05).Serum creatinine level was not an ideal indicator for early renal dysfunction in the elderly,especially in emaciated elderly people.Multivariate logistic regression analysis showed that gender,hypertension,coronary heart disease and hyperuricemia were independent risk factors for the decline of renal function (OR=1.937,1.602,1.842,7.020,respectively,all P＜0.05).Conclusions The rate of renal dysfunction is high in very old people.Many risk factors affect renal dysfunction.

Objective To determine the chlorpyrifos in human blood by liquid chromatography-tandemmass spectrometry and to validate its application in poisoning cases. Methods The samples were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Capcell Pack C18 mG II column (250 mm×2.0 mm, 5μm) using an isocratic elution of solvent A (0.1% formic acid-water with 2 mmol/L ammoniumacetate) and solvent B (methanol with 2 mmol/L ammoniumacetate) at 5∶95 (V∶V).Results The linearranged from5 to 500ng/mL (r=0.9987).Thelimitofdetection (LOD) and the lower limit of quantification (LLOQ ) were 2 ng/mL and 4 ng/mL , respectively. For this method, the precision and accuracy of intra-day and inter-day were <10% and 97.44%-101.10%, respectively. The re-sults in stability test of long-termfrozen were satisfied. The matrix effect, recovery and process efficien-cy were 64.97%-86.81%, 76.70%-85.52%, and 55.57%-66.58%, respectively. Conclusion This method can provide a rapid approach to chlorpyrifos extraction and determination in toxicological analysis of forensic and clinical treatment.

OBJECTIVE: To determine the chlorpyrifos in human blood by liquid chromatography-tandem mass spectrometry and to validate its application in poisoning cases. METHODS: The samples were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Capcell Pack C18 MGII column (250 mm x 2.0 mm, 5 μm) using an isocratic elution of solvent A (0.1% formic acid-water with 2 mmol/L ammonium acetate) and solvent B (methanol with 2 mmol/L ammonium acetate) at 5:95 V:V). RESULTS: The linear ranged from 5 to 500 ng/mL (r = 0.998 7). The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 2 ng/mL and 4 ng/mL, respectively. For this method, the precision and accuracy of intra-day and inter-day were < 10% and 97.44%-101.10%, respectively. The results in stability test of long-term frozen were satisfied. The matrix effect, recovery and process efficiency were 64.97%-86.81%, 76.70%-85.52%, and 55.57%-66.58%, respectively. CONCLUSION This method can provide a rapid approach to chlorpyrifos extraction and determination in toxicological analysis of forensic and clinical treatment.
Chlorpyrifos/blood* ; Chromatography, High Pressure Liquid/methods* ; Chromatography, Liquid/methods* ; Humans ; Limit of Detection ; Poisoning ; Reproducibility of Results ; Tandem Mass Spectrometry/methods*

Objective To explore the clinical usage of allograft vessels for portal venous reconstruction during radical resection for pancreatic cancer.Methods We retrospectively analyzed clinical data of 14 patients who underwent pancreaticoduodenectomy with vascular resection (PDVR) and reconstruction by allograft vascular form June 2009 to May 2013.Results Patient's mean age was (59.1 ±10.4) years old.The mean diameter of tumors was (3.9 ± 1.3) cm,mean lcngth of resected vessels was (4.4 ±0.5)cm,and the R0 resection rate was 71.4％.The implanted vessels in 4 cases were allograft portal veins,and in the other 10 cases were allograft iliac arteries.In morphological classification,9 cases used straight vessels and 5 cases used Y shaped vessels.The overall median survival time of the group was (14.0 ± 3.0) months.The 1,2 year' s survival rate was 52.0％ and 40.0％,respectively.Conclusions Allograft vessels for portal venous reconstruction during PDVR have biological and structural advantages.

Objective To investigate the prevalence of glomerular microthrombosis in lupus nephritis (LN) and the significance of antibodies to anti-coagulation related factors and anti-phospholipid antibodies in glomerular microthrombosis (GMT).Methods Kidney biopsy specimens and plasma samples were obtained consecutively from 124 patients with LN. Kidney biopsy specimens were examined for the presence of glomerular microthrombi.Plasma samples from 25 LN patients with GMT (LN-GMT group) and 99 LN patients without GMT (LN-non-GMT group) were tested for lupus anticoagnlant (LA) and antibodies to cardiolipin (ACL),β2 glycoprotein I (β2GP I ),plasmin,thrombin,tissue plasminogen activator (t-PA) and Annexin A II.Results The prevalence of GMT in LN patients was about 20.2%.Compared to LN-non-GMT group,LN-GMT group had elevated SLE disease activity indices (SLEDAI),elevated activity and chronicity indices of kidney tissue injury,and elevated serum creatinine,blood urea nitrogen and proteinuria levels,and also had a higher frequency of hypertension (P＜0.01).The positive rates of LA,IgG class anti-β2GP I and anti-thrombin antibodies were higher in LN-GMT group than in LN-non-GMT group (P＜0.05).The positive rates of IgG class antibodies to ACL,plasmin,t-PA and Annexin A II in LN-GMT group were not statistically different from those in LN-non-GMT group (P＞0.05).No difference was found in the positive rate of any IgM class antibody between the two groups (P＞0.05).Conclusion This study has shown that GMT occurs approximately in 20.2% of the LN patients.Patients with GMT have more severe kidney tissue injury and more poor renal outcomes than patients without GMT.LA and antibodies to β2GP I and thrombin play a role in glomerular microthrombosis in lupus nephritis.

Objective To evaluate hepatic artery resection and microsurgical reconstruction in radical resection of Klatskin's tumor.Methods We retrospectively reviewed clinical data of 7 patients with advanced hilar cholangiocarcinoma (Klatskin's tumor) who underwent left hemihepatectomy combined with right hepatic artery resection and microsurgical reconstruction with or without portal vein reconstruction from August 2008 to March 2012.Results Right hepatic artery was reconstructed with end-to-end anastomosis,using the reserved left hepatic artery (n =1),the remanent right hepatic artery (n =1),the hepatic artery proper (n =4) and the gastroduodenal artery (n =1),among those 2 patients underwent concomitant portal vein reconstruction.Post-operative pathology showed middle to low differentiated adenocarcinoma in 2 patients,low differentiated adenocarcinoma in 3 and papillary adenocarcinoma in 2.R0 resection was achieved in 6 patients.There was no post-operative liver failure,biliary-enteric anastomotic leakage or perioperative deaths.Conclusions Hepatic artery resection and microsurgical reconstruction increases the radical resection rate of advanced hilar cholangiocarcinoma and decreases postoperative complications.