Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Camptothecin ; administration & dosage ; analogs & derivatives ; Chemotherapy, Adjuvant ; Colorectal Neoplasms ; drug therapy ; pathology ; surgery ; Disease-Free Survival ; Fluorouracil ; administration & dosage ; Humans ; Leucovorin ; administration & dosage ; Postoperative Care ; Treatment Outcome

Objective To analyze the suspicious results of serum HBV DNA by fluorescence quantitative PCR and develop appropriate countermeasures in order to improve the quality of detection of HBV DNA.Methods Blood samples of patients from the First Affiliated Hospital of Wenzhou Medical College from 2008 to 2011 were analyzed for HBV DNA by fluorescence quantitative PCR.1969 cases of suspicious results,judged by the rule of review the results of serum HBV DNA combined with the historical results,PCR amplification curve,HBV serum markers and clinical diagnosis,were analyzed and redetected by using of two different reagents,careHBV PCR Kit and careHBV PCR Kit V2,at the same time.The consistency and inconsistency ratio of the results were evaluated.Both the reasons of inconsistent and the undetected rates of careHBV PCR Kit were analyzed.The two reasons for the inconsistent results included the reagent related factors,e.g,showing no amplification curve caused by the false negative and abnormal low efficiency of amplification curve,and the non reagent related factors such as operating pollution and other sample factors.Results There were 115 154 blood samples were detected for HBV from 2008 to 2011 and 1969 samples (1.71％) with suspicious results were redetected.The consistency and inconsistency results were 1588 (80.65％) and 381 (19.35％),respectively.Every year from 2008 to 2011,the percentage of the inconsistent results caused by the reagent related factors were 18.87％,20.23％,51.33％ and 59.57％ respectively,which showed an increasing trend,and the percentage of inconsistent results caused by the nonreagent related factors were 81.13％,79.77％,48.67％ and 40.43％ respectively,which showed a declining trend year by year.The undetected rates of careHBV PCR Kit were 2.49％,4.08％,10.09％ and 14.47％ respectively,showing an increasing trend.Conclusions The redetection for the specimens with the suspicious results by using of different reagents can avoid the blind detection of HBV DNA and reduce the experimental error.All the clinical samples for quantitative HBV DNA including the mutations of HBV gene can be measured accurately and effectively,which is helpful to hepatitis B patients for antiviral therapy.

Objective To explore the role of OAZ gene in the pathogenesis of systemic lupus erythe-matosus (SLE) by using RNA interfering technique. Methods Peripheral blood mononuclear cells (PBMC) from SLE patients were collected. Each sample was equally divided into four groups for cell culture in 96 well plates. Specific siRNA for OAZ and GAPDH were concordantly added to the experimental group and the positive control group, while nonspecific siRNA was added to the negative control group and only culture medium was added to the Mock control group. Cells and supernatants were harvested after culturing for 72 hours, then RNA was extracted and reverse transcripted to cDNA. OAZ, Id1, Id2, Id3, Id4 mRNA expression levels were analyzed by using real-time PCR. Levels of IFN-γ, IL-4, IL-10, IL-12, IL-21, CCL2, ANA in the supernatant were tested by ELISA. Relationships between the expression levels of OAZ mRNA with levels of cytokines and ANA were analyzed. Results OAZ, Id1, Id2, Id3 gene mRNA expression levels (△Ct: 12.5±1.4, 8.9±1.5, 4.3±0.8, 8.04±1.1) in the experimental group were significantly decreased comparing to those in the negative control group (△Ct: 10.2±1.1, 6.5±1.2, 2.4±1.3, 6.2±1.2 respectively, P<0.05). Levels of IFN-γ, IL-10, IL-12, IL-21 and ANA in the experimental group were significantly lower than those in the negative control group (P< 0.05), but level of CCL2 was higher than the negative control group (P<0.05). Difference of OAZ mRNA expression levels (△△Ct) between the experimental group and the negative control group was negatively correlated with changes of ANA, IL-21 levels, but positively correlated with changes of Th1/Th2, CCL2. Conclusion OAZ siRNA can effectively reduce the expression of genes involved in the OAZ signaling pathway in SLE. OAZ may lead to abnormal production of ANA via regulating Id genes and cytokines.

Objective To depermine if a double mainpenance dose of clopidogrel can improve phe clinical oupcome in papienps who have clopidogrel htpo-responsiveness ( CH) afper percupaneous coronart inpervenpion (PCI) and analtze correlapive risk facpors of CH. Methods We had enrolled 134 consecupive papienps undergoing PCI for spable coronart arpert disease in our cenper bepween Januart 2014 po June 2015. CH was depermined bt plapelep aggregapion measured bt phrombelaspographt ( TEG). Blood samples were paken 24 h and 3 monphs afper PCI procedure. All subjecps were divided inpo 2 groups (i. e phe CH group and phe clopidogral sensipive group) according po pheir responsiveness bt TEG. The CH group (n = 45) received a double mainpenance dose of clopidogrel as 150 mg/ d and phe clopidogrel sensipive group (n = 89) received a spandard mainpenance dose as 75 mg/ d. Changes in clopidogrel responsiveness and correlapive risk facpors were observed afper 3 monphs of clopidogrel preapmenp. Major adverse cardiac evenps (MACEs) and bleeding incidenps were recorded during follow-up lease 6 monphs. Results The clopidoprel htpo-responsive rape decreased from 33. 6% (45 / 134 papienps) po 11. 9% (16 / 134 papienps) afper 3 monphs of preapmenp. No spapispical difference found bepween phe 2 groups in morpalipt rape and non-fapal mtocardial infarcpion ( P >0. 05). Rapes of overall MACE (33. 3% vs. 22. 5% ), rehospipalizapion (26. 7% vs. 16. 9% ) and pargep vessel revascularizapion (11. 1% vs. 6. 7% ) were significanp higher in phe CH group ( all P < 0. 05) . Mulpivariape regression analtsis showed: smoking ( OR 4. 498, 95% CI 1. 378 - 4. 018, P = 0. 036), diabepes (OR 4. 385, 95% CI 1. 370 - 7. 552,P = 0. 026) and clopidogrel dosage ( OR 0. 597, 95% CI 1. 005 - 2. 676, P = 0. 019 ) were phe risk facpors for CH. Conclusions For papienp wiph htpo-responsiveness po clopidogrel afper PCI, a higher mainpenance dose of clopidogrel as 150 mg/ d for 3 monphs can provide equivalenp clinical benefip in serious adverse evenp (including morpalipt and non-fapal mtocardial infarcpion) compared po spandard mainpenance dose for clopidogrel responsive papienps.

Objective To establish a method of reverse hybridization to detect five subfamilies of low risk Human Papillomaviruses(HPV6,11,42,43 and 44)and eighteen subfamilies of high risk HPV (HPV16,18,31,33,35,39,45,51,52,53,56,58,59,66,68,73,83 and MM4)in one reaction.Methods Special probes for twenty-three HPV subfamilies were fixed on nylon membrane bars,biotin labeled general primers mediated polymerase chain reaction(GP-PCR)were applied in HPV DNA amplification.PCR amplified DNA fragments were reversely hybridized with special probes that were fixed on the membranes. All samples(136)detected by reverse hybridization method were paralleled with the methods of Hybridization Capture Ⅱ(HC-Ⅱ)and sequencing.Results Positive rate of the 136 samples detected by reverse hybridization was 41.9%,while HC-Ⅱ 42.6% and sequencing 40.4%.Reverse hybridization detection indicated coherence with the other two methods(Kappa 0.8644 and 0.9089,respectively).While sequencing was lab standard for DNA test,the sensitivity was 96.36%,specificity was 95.06%,accuracy was 95.59%.Conclusions Method of reverse hybridization is adaptable to 23 kinds of HPV subfamilies, which can confirm the exactly subfamilies of HPV infection.This method is adaptable in clinical detection of HPV,with high sensitivity,high specificity,simply and convenient operation and the results are easily to be read.

AIM: To investigate whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress Polo- like kinase- 1 (Plk 1 ) expression and its effects in A549 cells. METHODS: A recombinant plasmid containing siRNA targeting Plk1 ( psiRNA - hH1 - Plk1 ) was transfected into A549 cells with Lipofectamine 2000.Expressions of Plk1, cyclin B1 and p53 protein were detected by Western blotting. Cell proliferation was evaluated by direct cell counting, while cell cycle and apoptosis were examined by flow cytometry, and expression of α - tubulin was detected by immunofluorescence. RESULTS: The results demonstrated that sequence specific siRNA targeting Plk1 was capable of suppressing Plk1 expression, and reflecting in lower kinase activity in A549 cells. The level of Plk1 protein was reduced by at least 70％ after 48 h of psiRNA - hH1 - Plk1 treatment relative to controls. Expressions of cyclin B1 and p53 were increased greatly after Plk1 depletion, and cells showed absence of microtubule polymerization and spindle abnormalities in staining for α -tubulin. Growth inhibition, G2/M arrest and apoptosis were observed in psiRNA -hH1 -Plk1 transfected group. CONCLUSION: All these data suggest that siRNA targeted against human Plk1 may be a valuable tool in cancer therapy.

ObjectiveTo study changes of molecular markers of prothrombotic state:Platelet granule membrane protein ( GMP-140 ),Von Willebrand factor ( vWF:Ag),thrombomodulin (TM),Two-D dimer ( DD),antithrombin Ⅲ ( AT- Ⅲ ) in plasma and puerarin for treatment functions of acute pancreatitis (AP).MethodsIn 78 patients with AP [ severe acute pancreatitis (SAP):26 cases,mild acute pancreatitis (MAP):52 cases ],using a random number table,the patients were given puerarin treated base (n =40) and conventional treated base group (n =38 ).The two groups were given fast,continuous gastrointestinal decompression,correction of electrolyte and acid-base balance disorders,vein support,antisecretory drugs,antibiotics inhibit pancreatic secretion and inhibition of trypsin activity of drug treatment.Puerarin group:Puerarin injection 0.5 g in 5％glucose injection intravenous infusion of 500 ml,1 time a day.GMP-140 vWF:Ag,TM,DD were measured by the methods of analysis of enzyme-linked immunosorbent assay and AT-Ⅲ was measured by the methods of analysis of chromogenic substrate method preformed in all patients,plasma amylase and uric amylase were determined by the method of somogyi and after the treatment.And 22 healthy people were selected as normal controls ( NC,Group C,n =22).ResultsCompared with the Group C and MAP,the plasma GMP-140 [ ( 86.26 ± 15.28 )ng/Lvs (32.56 ± 18.17) ng/L and (58.68 ± 15.86)ng/L],vWF[(236.22 ±31.78)％vs (95.12 ±31.68)％ and (126.68 ± 17.06)％ ],TM [(65.70 ± 12.27) μg/L vs (4.26 ±0.92) μg/L and (9.80 ± 6.98) μg,/L],DD [ (0.87 ±0.04) mg/L vs (0.36 ±0.06) mg/L and (0.56 ±0.05) mg/L] were significantly elevated,however the AT-Ⅲ [ (56.13 ± 15.78) U/ml vs (98.76 ±22.68) U/ml and (80.38 ± 18.29)U/ml )was significantly decreased SAP ( P ＜ 0.01 ).There were significant differences on the levels of GMP-140 [ (31.52 ± 15.81 ) ng/L vs (59.62 ± 13.73 ) ng/L,t =- 23.283 ],vWF [ ( 93.32 ± 28.62) ％ vs ( 128.81 ±16.23)％,t=-28.205,P＜0.01 ],TM[ (4.36 ± 0.82) μg,/L vs (11.23 ± 7.62)μg/L,t =-43.419,P ＜0.001],DD[ (0.32 ±0.05) mg/L vs (0.68 ±0.04) mg/L,t =- 15.642,P ＜0.001],AT-Ⅲ ((97.68 ±21.69) U/ml vs (76.86 ± 17.92) U/m,t =14.967,P ＜ 0.01 ) between puerarin treated base group and conventional treated base group.Comparing with treated base,the group given puerarin obviously shortened the increased of plasma [ ( 81.26 ± 17.12) U/L vs ( 119.63 ± 51.87 ) U/L,t =- 7.618,P ＜ 0.001 ],uric amylase [ (416.37 ± 116.50) U/L vs (576.32 ± 126.58) U/L,t =- 36.659,P ＜ 0.001 ],the time of abdominal pain relief and therapy to spend [ ( 2.18 ± 0.76 ) d vs ( 5.26 ± 0.58 ) d,t =- 13.619,P ＜ 0.001 ].Conclusion The molecular markers of prothrombotic state:GMP-140,vWF:Ag,TM,DD,AT- Ⅲ might all play key roles in the development of AP.Puerarin can improve the pancreatic microcirculation and adjust molecular markers of prothrombotic state,and had certain treatment functions with AP.

ObjectiveTo study the changes of the plasma thrombomodulin(TM) and von Willebrand factor (vWF) levels and their clinical significance associated with the extent and severity of acute ischemie colitis.MethodsThe plasma TM and vWF levels were determined by enzyme linked immunosorbent assay in 46 patients with acute ischemic colitis (acute ischemic colitis group),42 patients with ulcerative colitis (ulcerative colitis group) and 40 healthy subjects (control group).ResultsThe plasma TM was (49.6 ±2.3) μg/L,and vWF was(198.8 ±8.9)％ in acute ischemic colitis group.The plasma TM was (38.2 ± 3.8) μ g/L,and vWF was ( 162.6 ± 7.6)％ in ulcerative colitis group.The plasma TM was (23.8 ±2.3) μg/L,and vWF was ( 116.7 ± 6.2)％ in control group.The plasma TM and vWF levels in acute ischemic colitis group were higher than those in ulcerative colitis group and control group (P ＜ 0.05 or ＜ 0.01 ).The plasma TM and vWF levels in ulcerative colitis group were higher than those in control group (P＜ 0.05).The plasma TM levels[(49.9 ± 0.3 ) μg/L] and vWF [(210.6 ± 8.2 ) ％] in all colon disease were higher than those in partial colon disease (P ＜ 0.05 ).ConclusionThe changes of plasma TM and vWF levels can be used as one of the indicators for assessment of the development and the prognosis of acute ischemic colitis.

Purpose To analyze the gait of patients following reconstruction of posterior cruciate ligament(PCL) and posterolateral corner(PLC).Methods Between March 2007 and April 2008,16 patients with combined PCL and PLC injuries-deficient knee underwent the reconstruction with allograft.Dial test were performed before surgery,and gait analysis and dial test were assessed 1 year after reconstruction.Sixteen healthy volunteers were used as controls.Results There are no differences in the time-distance factors and kinematics between patients and controls(P>0.05).The torque of knee extension revealed significant difference between patients and controls(P<0.01).Mean abtorsion angle of the patients increased 16.5°±6.2° before surgery;whereas the angle increased-4.4°±7.8° 1 year after surgery(P<0.01).The maximal mean abtorsion angnlation from gait analysis was 14.1°±15.7° and from dial test was 29.7°±1 5.2°,respectively.There was a strong linear correlation(r=0.9671,P<0.05)between the outcomes from dial test and gait analysis.Conclusion After reconstruction of the PCL and PLC,the gait of patients became almost normal 1 year after operation,except the torque of knee extension.