Validation of a fast and reliable liquid chromatography tandem mass spectrometry for simultaneous quantitation of Voriconazole, Posaconazole and Linezolid in human serum / 中华检验医学杂志

Objective:To establish a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of voliconazole (VRC), posaconazole (PCZ), and linazolam (LNZ) in human serum.Methods:This study is a methodological validation by LC-MS/MS. The blood concentration results of VRC, PCZ, and LNZ in our hospital′s anti-infection patients were collected. Voriconazole, Posaconazole, and Linezolid were accurately weighed and prepared. Linezolid-[2H3] was used as the internal standard. After gradient elution on the ACE PFP column, the residuals were analyzed by LC-MS/MS in the positive electrospray ionization mode and multiple reaction monitor (MRM) mode. The method′s linearity, precision, lower limit of detection, and recovery rate were validated according to standard guidelines.Results:The linear correlation coefficient ( r) of the standard curve was above 0.99 ( r>0.99). The linear range of VRC and PCZ were 0.10 mg/L~10.00 mg/L, and the lower limit of detection were 0.01 mg/L. The linear range of LNZ was 0.50 mg/L~50.00 mg/L, and the lower limit of detection was 0.05 mg/L. The recoveries of VRC, PCZ and LNZ were 90.96%-103.18%, 91.84%-99.17%, and 97.04%-100.41%, respectively. Intra-and inter-batch precision (% CV) for VRC were less than 8.30%. Intra-and inter-batch precision (% CV) for PCZ was less than 9.78%. Intra-and inter-batch precision (% CV) for LNZ was less than 7.14%. Drug concentrations in 155 cases of VRC, 44 cases of PCZ, and 59 cases of LNZ were detected. Conclusion:We have established an LC-MS/MS method for the rapid, accurate, highly specific determination of VRC, PCZ, and LNZ concentrations in human serum. This method is suitable for analyzing large clinical sample sets.

Value of urinary C-terminal agrin fragment in monitoring the progression of early kidney injury in type 2 diabetic patients / 中华检验医学杂志

Objective:This study aimed to explore the feasibility and clinical value of monitoring the progression of early kidney injury in type 2 diabetic patients by assessment of the urinary C-terminal agrin fragment (uCAF) with enzymatic chemiluminescence immunoassay.Methods:A total of 251 patients with type 2 diabetes, who attended the Second Affiliated Hospital of Wenzhou Medical University from October 2018 to March 2020, were included in this retrospective analysis. One hundred and fifty-six participants undergoing health check-up at the Second Affiliated Hospital of Zhejiang University School of Medicine in February 2021 served as controls. Basic clinical information, glycosylated hemoglobin type A 1c and serum creatinine values were recorded, and urine specimens were collected for urinary creatinine, urinary α 1 microglobulin(uα 1M), urinary immunoglobulin G (uIgG), urinary albumin, urinary N-Acetyl-B-D-glycosaminidase (uNAG) and uCAF measurements. Based on the estimated glomerular filtration rate (eGFR), 251 patients were classified into G1~G5 stage groups with 116, 22, 28, 55 and 30 patients in each group. One hundred and sixty-six patients with early diabetic kidney disease (stage G1-G3) were divided into subgroups A1 (79), A2 (48) and A3 (39) according to the urinary albumin/creatinine ratio (UACR), the uα1M levels were divided into uα1M subgroup 1 (83 cases), uα1M subgroup 2 (42 cases), and uα1M subgroup 3 (41 cases), and uIgG subgroup 1 (83 cases), uIgG subgroup 2 (42 cases), and uIgG subgroup 3 (41 cases) according to uIgG levels. The Spearman method was used to analyze the correlation between uCAF levels and eGFR, UACR, uα1M and uIgG levels. Results:(1) The linear range of the uCAF detected by enzymatic chemiluminescence immunoassay was 3.97-2 000.00 ng/ml, with a detection limit of 2.28 ng/ml, intra-batch coefficients of variation of 1.15% and 1.57%, inter-batch coefficients of variation of 1.63% and 5.78%, and a biological reference interval of <95.35 μg/g Cr. (2) The uCAF level and positive rate (UACR≥30 mg/g) increased with the decrease of eGFR from G1-G3, uCAF level was negatively correlated with eGFR value ( r=-0.543, P<0.000 1), and the positive rate increased from 24.14% (28/116) to 85.71% (24/28) from G1-G3. The uCAF level and positivity rate decreased with the decrease of eGFR from G4 to G5. uCAF level was positively correlated with eGFR value ( r=0.495, P<0.001), and the positivity rate decreased from 30.91% (17/55) to 23.33% (7/30) from G4 to G5. (3) In patients with early diabetic kidney disease, uCAF levels and positivity rates increased gradually with the increase of UACR. uCAF levels were positively correlated with UACR values ( r=0.602, P<0.001), and the uCAF positivity rate reached 21.52% (17/79) in the A1 subgroup. (4) uCAF level was positively correlated with uα1M and uIgG levels in patients with early diabetic kidney disease ( r=0.757, 0.596, both P<0.001). Conclusion:Analytical performance of enzyme chemiluminescence immunoassay for the detection of CAF is satisfactory and could be used a biomarker for monitoring damage and progression of early diabetic kidney disease in patients with type 2 diabetes.

Analysis of application value on acute kidney injury biomarkers / 中华检验医学杂志

Acute kidney injury is a common and severe clinical event, the morbidity and mortality of acute kidney injury are increasing steadily, which is related with heavy burden to the country and social economy. Early diagnosis and intervention could significantly reduce the occurrence and progression of acute kidney injury. Therefore, it is of particular importance to find sensitive, specific, and economic biomarkers for acute kidney injury. In recent years, efforts have been made on exploring proteins in blood and urine as biomarkers of acute kidney injury. Present review summarizes progress in this field based on results of recent basic and clinical research.

Importance of the standardized clinical application and evaluation of renal injury markers / 中华检验医学杂志

Chronic Kidney Disease (CKD) has become one of the main diseases severally threatening human health, which has aroused global attention. Acute Kidney Injury (AKI) is the main cause of CKD. Early diagnosis of AKI is the most effective way to prevent CKD. More and more new biomarkers of renal injury will be found through genomics, proteomics, and metabolomics methodologies. The clinical application of a single marker in the real world will be subject to certain restrictions because of the etiological diversity and personal differences. Kidney damage monitoring models will be established by artificial intelligence method. However, standardizing the evaluation of its clinical application value is essential to screen out truly valuable kidney injury biomarkers or monitoring models, to promote the clinical application of kidney injury markers, and to effectively reduce the incidence of CKD.

Effect of virus inactivation on weak positive results of nucleic acid test for 2019 novel coronavirus / 中华检验医学杂志

Objective:To investigate the effect of virus inactivation on weak positive result of 2019 novel coronavirus(2019-nCoV) nucleic acid test.Methods:A retrospective study was conducted on the nasopharyngeal swabs of three patients with positive PCR nucleic acid test for 2019-nCoV at different concentrations in the Second affiliated Hospital of Zhejiang University Medical College from January to February 2020.The virus in nasopharyngeal swab specimens were inactivated by water bath at 56 ℃ for 30 min, dry bath at 56 ℃ for 60 min and dry bath at 60 ℃ for 30 min respectively. After treatment, these samples RNA were extracted and then detected by three new commercial quantitative real-time polymerase chain reaction reagent kits for 2019-nCoV.Cycle threshold (Ct) value was used to evaluate the effect of virus inactivation on nucleic acid detection of 2019-nCoV.Results:There was no significant difference between the groups before and after inactivation. Ct values of ORF1ab gene before inactivation were 23.28±0.28, 25.25±0.25, 28.93±0.44, 32.06±0.47, 35.20±0.38, 32.89±0.38, 36.24±0.23, 33.30±0.46, and those after inactivation were, group 1:23.60±0.20, 27.29±0.30, 31.83±0.51, 37.41±0.46, group 2: 24.25±0.34, 27.18±0.42, 31.84±0.61, 34.99±1.01, 34.89±0.45,group 3: 23.37±0.17, 26.89±0.52, 32.05±0.50.Ct value of N gene before inactivation were 24.38±0.09, 26.64±0.11, 30.35±0.12, 33.29±0.33, 36.93±0.11, 34.50±0.12, 35.63±0.12, those after inactivation were, group 1: 24.66±0.11, 28.52±0.14, 32.71±0.14, 37.00±0.13;group 2: 25.41±0.10, 28.79±0.15, 33.29±0.28; group 3: 23.37±0.11, 28.68±0.11, 33.54±0.13, 37.18±0.23(ORF1ab gene: t=-1.416; N gene: t=-1.379, P>0.05). There was no significant difference among the three inactivation groups, the specific Ct values are shown above(ORF1ab gene: t=-0.460; N gene: t=-0.132, P>0.05). However, the Ct values of the inactivated groups (1,2,3) and the non-inactivated group at different dilution times were different (10 ×:Ct value of ORF1ab was 25.25±0.25 in the non-inactivated group, and 27.29±0.30, 27.18±0.42 and 26.89±0.52 in the inactivated group1,2 and 3, t(ORF1ab)=-7.327, P<0.01.Ct value of N gene in the non-inactivated group was26.64±0.11, those in inactivated group 1, 2 and 3 were 28.52±0.14, 28.79±0.15 and 28.68±0.11, respectively, t (N)=-19.340, P<0.01. 100 ×:Ct value of ORF1ab was 28.93±0.44 in the non-inactivated group, and 31.83±0.51,31.84±0.61 and 32.05±0.50 in the inactivated group1,2 and 3, t (ORF1ab)=-9.462, P<0.01. Ct value of N gene in the non-inactivated group was 30.35±0.12, those in the inactivated group 1, 2 and 3 were 32.71±0.14, 33.29±0.28 and 33.54±0.13, respectively, t (N)=-18.583, P<0.01. The positive detection rate of the non-inactivated group (7/11, 8/11, 5/11) was significantly different from that of the inactivated group (inactivated group 1:4/11, 4/11, 3/11, inactivated group 2:3/11, 3/11, 3/11, and inactivated group 3:3/11, 3/11, 2/11) ( Z=-2.670, P<0.01). There were no significant difference among the inactivated groups(inactivated group 1:4/11, 4/11, 3/11, inactivated group 2:3/11, 3/11, 3/11, inactivated group 3:3/11, 3/11, 2/11) ( Z=4.413, P>0.05) and among the three reagents(reagent 1:7/11, 4/11, 3/11, 3/11, reagent 2:8/11, 4/11, 3/11, 3/11, reagent 3:5/11, 3/11, 3/11, 2/11)(χ 2=1.199, P>0.05). Conclusion:The virus inactivation can degrade the nucleic acid of the 2019-nCoV, resulting in the decrease of the Ct value and the false negative results of the low-concentration specimens.

A weak D type 59 case identified in the Chinese Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study a case with weak D59 phenotype identified among ethnic Han Chinese population.</p><p><b>METHODS</b>Routine serological tests were used to analyze the reaction patterns, and the RhD epitopes were verified with 12 monoclonal antibodies. Sequence-specific primer PCR was applied for typing the weak RhD and RhD zygosity in the proband and his family members.</p><p><b>RESULTS</b>A c.1148T>C variant was identified in the proband, for which serological test indicated a weak D phenotype. RHD zygosity testing confirmed that the proband had a RHD+ /RHD- genotype.</p><p><b>CONCLUSION</b>A weak D59 phenotype was firstly identified in a Chinese individual.</p>

Imperatorin increased sensitivity of lung cancer CD133 +cell subsets to gefitinib by down-regulating c-met expression / 中国病理生理杂志

AIM:To investigate the role of imperatorin in reversing the resistance of the PC 9 CD133+cell sub-sets to gefitinib.METHODS:MTT assay was performed to evaluate the viability of PC 9 cells treated with imperatorin and gefitinib.The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot .The percentage of CD133 +cell subsets population and the apoptotic rate of the PC 9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry .RESULTS:The sensitivity of the PC 9 CD133 +cell subsets to gefitinib was significantly lower than that of the PC9 CD133 -cell subsets.Treatment with gefitinib alone significantly inhibited the protein levels of EGFR /PI3K/AKT in the PC9 CD133 -cell subsets but not the PC 9 CD133 +cell subsets .Treatment with gefitinib alone increased the percentage of CD133 +cell subsets population in the PC9 cells.However, combination of gefitinib with imperatorin signifi-cantly inhibited the enrichment of CD 133 +cell subsets population .Imperatorin down-regulated c-met expression , sugges-ting the c-met was the target of imperatorin in the PC9 CD133 +cell subsets.The results of MTT assay, Western blot analy-sis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI 3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC 9 CD133 +cell subsets.CONCLUSION:Imperatorin increases the sensitivity of lung cancer CD 133 +cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib .

Development and application of prostate cancer gene 3 in prostate cancer / 中华检验医学杂志

The incidence of prostate cancer ( PCa ) is rising steadily among males in many countries.Serum prostate-specific antigen ( PSA ) is widely applied in clinical diagnosis and screening of PCa.However, the grey area of PSA levels has a low specificity in PCa screening and may lead to a high rate of negative biopsy and overtreatment.The PCA3 gene is strongly and specifically overexpressed in PCa cells and malignant prostate tissue.The gene has been identified as a molecular biomarker for PCa detecting.The diagnostic significance of PCA3, however, is awaiting further researches.In this review, the progress of molecular biological characteristics of PCA3,and its applications in diagnosis and treatment of prostate cancer were discussed.

The progress of biomarkers in prostate cancer / 中华检验医学杂志

Affected by many factors such as environment and lifestyle change , prostate cancer has become common malignancy in older men . The introduction and widespread adoption of PSA has revolutionized the way prostate cancer is diagnosed and treated .However , the use of PSA has also led to over-diagnosis and overtreatment of prostate cancer resulting in controversy about its use for screening .PSA also has limited predictive accuracy for predicting outcomes after treatment and for making clinical decisions about adjuvant and salvage therapies .Hence, there is an urgent need for novel biomarkers to supplement PSA for detection and management of prostate cancer .In this review, we discuss the traditional and new relevant molecular markers of early diagnosis and prognosis of prostate cancer for clinical diagnosis and prognosis of prostate cancer providing a reference .

Microfluidic chip digital PCR technology and its clinical application / 中华检验医学杂志

Microfluidics-based digital PCR depends on microfluidic chip to split PCR reaction mixture into many tiny equal-volume units.Quantitative assessment of target DNA template can be obtained by counting the number of fluorescence-positive units after thermocycling.Microfluidics-based digital PCR exhibits many advantages including absolute quantification, high sensitivity and accuracy, and shows great promise in a variety of applications, such as infectious diseases diagnose, early cancer detection and prenatal diagnose. There are already several microfludics-based digital PCR products produced from sereval companies.It is believed that as the technology improves, microfluidics-based digital PCR will find broader applications and become the next-generation tool for genetic tests.

Association of polymorphism in the promoter region of PCA3 gene with risk of prosate cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism in the promoter region of PCA3 gene and its relationship with risk of prostate cancer (PCa).</p><p><b>METHODS</b>The promoter region of PCA3 gene of the DNA of peripheral blood mononuclear cells was detected by sequence analysis in the 186 PCa and 141 BPH patients and 135 healthy control individuals. If the samples were detected with polymorphism of insection/deletion, clone sequence analysis was used with pBS-T carrier to verify it.</p><p><b>RESULTS</b>There were 5 polymorphisms. TAAA repeat times: 4, 5, 6, 7, 8, and 8 genotypes (TAAA 4/5, TAAA 4/6, TAAA 5/5, TAAA 5/6, TAAA 5/7, TAAA 5/8, TAAA 6/6, and TAAA 6/7) were detected in the promoter region of PCA3 gene. The eight genotypes were divided into three groups: ≤10TAAA, 11TAAA, ≥12TAAA. Unconditional logistic regression analysis models were used to analyze the relationship between different genotypes and cancer risks adjusted by sex and age. The type 11TAAA and ≥12TAAA was associated with higher relative risk for prostate cancer than the group ≤10TAAA [OR=1.74, 95% CI=1.06-2.87 (for type 11TAAA); OR=5.63, 95% CI=1.85-17.19 (for type ≥12TAAA)]. In the 186 PCa patients, there was 62.4% allele of PCA3 gene with AG/CA mutation found in the promoter 18-19 bp region of PCA3 gene and it had a close relation with the development of prostate cancer.</p><p><b>CONCLUSIONS</b>Short tandem repeats are found in the promoter region of the PCA3 gene in PCa patients, and the increase of TAAA repeat sequences highly enhance the relative risk of prostate cancer development. The occurrence of such STR might be related to the mutations in their upstream loci.</p>

The current status and future prospects of laboratory biomarkers in the diagnosis of liver cancer / 中华检验医学杂志

Liver cancer has a high morbidity and mortality in China.With new technologies and diagnostic instruments being developed,it is a common goal for researchers to discover some new diagnostic biomarkers of high sensitivity and specificity on liver cancer.The clinical laboratory is responsible to choose and understand the diagnosis value of these laboratory indicators and variation in the liver cancer progress.Then,these new biomarkers in screening for early malignancy,aiding cancer diagnosis,determining prognosis can be used effectively in the clinical laboratory to improve laboratory diagnostic capacity in order to serve the clinic more effectively and better.

The effects of 5-Aza-2-deoxycytidine, a methylation inhibitor, on the proliferation of androgen-sensitive prostate cancer cell line, and on the regulation of methylation of glutathione s-transferase P1 and retinoic acid receptorβ2 gene / 中华老年医学杂志

Objective To investigate the effects of 5-Aza-2'-deoxycytidine (5-Aza-dc),a methylation inhibitor,on the proliferation of androgen-sensitive prostate cancer line (LNCaP),and on its regulation of methylation on glutathione s transferaseP1 (GSTP1) and retinoic acid receptorβ2 (RARβ2) gene.Methods LNCaP cells were treated with 5-Aza-dc in different concentration,CCK8 method was used to detect the growth of LNCaP cells.The methylation of GSTP1 and RARβ2 gene in LNCaP cell was detected by nested methylation specific polymerase chain reaction (nMSP).Results The proliferation of LNCaP cells was inhibited after exposed to 5-Aza-dc.The methylation of GSTP1 and RARβ2 gene was changed from hypermethylation to demethylation by the 5-Aza-dc.These effects were dose-and time-dependent within certain concentration of 5-Aza-dc,but LNCaP cells grew better after 72 h than within 48 h when exposed to 5 Aza dc below 1.0 μmol/L.Also the methylation of GSTP1 and RARβ2 gene changed from hypermethylation to demethylation by the 5-Aza-dc was not different when exposed to 5-Aza-dc below 1.0 μmol/L within 72 h and 48 h.Conclusions 5-Aza-dc may effectively inhibit the growth of LNCaP cells and reverse the DNA methylation damage in some tumor suppressor genes,but the continuity and stability of low dose 5-Aza-dc is changeable.The study will provide a research basis for clinical treatment.

The applications of chromatin immunoprecipitation in the study on the downstream target genes of androgen receptor in prostate cancer cells / 中华检验医学杂志

Prostate cancer is one of the most common malignant tumor of male population in the West.Via binding to androgen response element in the genome and interacting with co-regulators,androgen receptor can participate in the development of prostate cancer and the convertion from androgen dependent prostate cancer to androgen independent prostate cancer.Chromatin immunoprecipitation ,coupled with PCR , chip,and high-throughput sequencing ,makes a huge progress in the study on the downstream target genes of androgen receptor and provide a new way to understand the molecular mechanisms of castration -resistant prostate cancer.

Analysis of suspicious results of serum HBV DNA detected by fluorescence quantitative PCR / 中华检验医学杂志

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.

The quality of POCT ensured by the hospital's internal management / 中华检验医学杂志

Point of care testing (POCT) is a laboratory-medicine discipline that is evolving rapidly in analytical scope and clinical application.Medical-laboratory tests can be performed at the point of care,shortening the time caused by sample transportation and preparation.At present,POCT contains many items,ranging from blood-glucose measurement to coagulation assays.But,errors in results in POCT practical applications are usually caused by personnel and methodology.Management of POCT detection within the hospital should be strengthened to ensure the quality of POCT in order to provide better services for patients.

An experimental research on effect of LTβR and P65 in progression of bladder cancer / 中华检验医学杂志

ObjectiveTo investigate the relationship of lymphotoxin β receptor (LTβR) and classical nuclear factor-κB (NF-κB) activation pathway in the pathogenesis and progress of cystitis and bladder cancer.MethodsThe LTβR and P65 mRNA expression were detected by Real-time quantitative PCR in 108 cases of fresh bladder tissue specimens (75 cases of bladder cancer,10 cases of inflammation and 23 normal bladder mucosa cases grouped by the tissue classification ),and protein expression were analyzed by immunohistochemistry assay in 118 cases of paraffin-embedded biopsy specimens (73 cases of bladder cancer,30 cases of cysitis and 15 normal bladder mucosa cases).The correlation analysis between the expressions of LTβR and P65 with clinical pathological data was then performed.Differences between LTβR and P65 mRNA and protein expression level were compared in different groups of bladder tissues using Kruskal-Wallis H test and the Chi-square test.Results( 1 )The mRNA expressions of LTβR and NF-κB/P65were higher in bladder cancer than those in normal group ( LTβR:29.4 ( 14.2 - 46.7 ) × 10 - 3/1.2 ( 0.3 -7.0) ×10-3,Z=-5.508; P65:9.7 (2.7 -21.1) ×10-3/1.0(0.8 ～1.8) ×10-3,Z=-5.030,P＜0.05 ).There were significantly differences between bladder cancer with different histological grades ( LTβR:18.2(2.1-31.3) × 10-3/ 28.4(16.6-36.2) × 10-3/47.9(34.3 -70.5) ×10-3,x2K-W=20.378;P65:4.9(1.3 - 12.0) × 10-3/7.4(3.0-21.9) × 10-3/17.0(10.0 ～28.3)× 10-3 ,x2K-W2 =15.219,P all ＜0.05) and lymph node metastasis (LTβR:27.2(9.7-40.1) ×10-3/39.4(26.7 -52.6) ×10-3,Z=-2.552; P65:7.4(2.3-15.6) ×10-3/13.4(6.7-23.3) ×10-3,Z=-2.026,P＜0.05).(2)The positive rates of LTβR and phosphorylated P65 ( p-P65 ) protein in cancer were higber than those of normal group (LTβR:69.8％/13.3％,x2 =16.600 ; p-P65:56.2％/6.7％,x2 =12.220,P ＜ 0.05 ).Upregulation of LTβR and p-P65 were associated with the histological grade (LTβR:56.3％/70.0％/90.4％,x2 =7.055; p-P65:40.6％ /60.0％/76.2％,x2 =6.679,P ＜0.05) and with lymph node metastasis (LTβR:58.3％/92.0％,x2 =8.849; p-P65:52.1％/64.0％,x2 =5.088,P ＜0.05).(3)There was a positive correlation between LTβR and P65 expression ( mRNA:r =0.654,P ＜ 0.05,protein:r =0.399,P ＜ 0.05 )in the bladder cancer and cystitis (r =0.521,P＜0.05).ConclusionsThe activation of LTβR and P65 was associated with progression and metastasis of bladder cancer.The activation of classical NF-κB pathway by LTβR may be achieved by P65.

Significance of tyrosine kinase receptor gene and its variant expressions in bladder tumor tissues / 中华检验医学杂志

ObjectivesTo explore the clinical significance of tyrosine kinase receptor RON mRNA expression and it's splicing variant in bladder tumors. Methods Sixty-three cases of transitional cell carcinoma of the bladder (TCCB), including 30 cases of pathological grade I , 15 cases of pathological grade Ⅱ and 18 cases of pathological grade Ⅲ (44 cases of clinical stage Tis + T1, 15 cases of T2 + T3 +T4), 7 inverted papilloma of the bladder ( IPB), 9 cases of papillary urothelial neoplasm of low malignant potential (PUNLMP) and 12 cases of normalbladder mucosa RT-PCR was employed with the internal standard (GAPDHmRNA) to detect the expression of RON mRNA. PCR and direct sequencing was then utilized to identify the potential RON mRNA splicing variants. Finally, the variants' positive rates of expression were analyzed among the different tissues, diverse TCCB pathological grades and clinical stages. ResultsThe expression levels of RON mRNA/GAPDH mRNA among TCCB, IPB, PUNLMP and normal control were 4. 9 × 10-3 ( 1. 8 × 10-3-1.0 × 10-2 ), 3. 8 × 10-3 (2. 4 × 10-3-1.7 × 10-2 ), 4. 9 ×10 -3 ( 1.7 × 10 -3-1.1 × 10 -2 ) and 1.0 × 10-3 (4. 5 × 10-4-2. 8 × 10-3 ) respectively. The difference had a statistical significance (x2K-W = 17. 278 ,P ＜0. 05 ). The expression levels among pathological grade I, Ⅱ andⅢ were 3.7 × 10-3( 1.3 × 10-3-7.5 × 10-3) , 4. 9 × 10-3(1.9 × 10-3-1.1 × 10-2) and 8.9 × 10-3(2. 7 ×10 -3-8.0 × 10 -2 ) respectively. The erpression levels among the clinical stage Tis + T1 and T2 + T3 + T4were 3.5 × 10-3 ( 1.2 × 10 -3-7. 7 × 10-3 ) and 9. 7 × 10 -3 ( 2. 9 × 10-3-5. 3 × 10-2 ). The differences between expression levels were of statistical significance among the different pathological grades ( x2k-W =7. 341, P ＜0. 05 ) and clinical stages ( Z = - 2. 306, P ＜ 0. 05 ). The positive rates of exon 11deletion(E11△) in TCCB, IPB and PUNLMP were 71％ (45/63), 57％ (4/7) and 67％ (6/9) respectively, andthe total positive rate in bladder tumor tissues was 70％. Meanwhile, expression of the novel RON variant wesnot detected in the normalbladder mucosa. The positive expression rate of E1 1△ has no significant correlationamong the different clinical pathological tissues (x2 = 0. 620, P ＞ 0. 05 ). There was no statistical significancein expression positive rate between different pathological grades ( Z =0. 221, P ＞0. 05 ) and clinical stages( Z = 0. 538, P ＞ 0. 05) as well. A novel RON splice variant, deletion of RON exon 11 3 476 - 3 539 ( E3476 -3539△) was fond in the pathological tissue. The positive expression rates of E3 476 -3 539 in TCCB,IPB and PUNLMP were 57％ (36/63), 43％ (3/7) and 56％ (5/9) respectively, and the total positive expression rate was 56％ (44/79). The positive rates of E3 476 -3 539△ in pathological grade I , Ⅱ and Ⅲ were 40％ ( 12/30), 67％ (10/15) and 78％ (14/18), and it's positive rates in clinical stage Tis +T1and T2 +T3 + T4 were 48％ (21/44) and 80％ (12/15). The differences in each group had significantly statistical significance ( Z = 7. 285, 5. 041, P ＜ 0. 05 ) . However, the positive rates amongdifferent pathological tissues had no significance (x2 = 0. 517, P ＞ 0. 05 ). Conclusions The expression level of RON mRNA is significantly associated with histological grading and clinical stage. RON may play an important role in the progression ofTCCB. Compared with the normal control, the increased RON variant expression may contribute to the carcinogenesis of the bladder tumor.

Prevalence of human papillomavirus infection in women with uterine cervical cancer in Wenzhou / 中华临床感染病杂志

Objective To investigate the prevalence of human papillomavirus(HPV)infections in women with uterine cervical cancer in Wenzhou.Methods Exfoliated cells samples of cervix uteri were collected from 198 patients with cervical cancer. Flow-through hybridization technique was used to detect HPV and its genotypes.The relationship of HPV infection with cervical cancer stage,histological type and differentiation degree were analyzed.The prevalence of HPV infections in patients with different cervical diseases was observed.SPSS 13.0 was used for statistical analysis.Results In 198 patients with cervical cancer,HPV infection was occunrred in 147 (74.24%), of whom 101patients were superinfected (51.01%),and 129 patients(65.15%)were infected with the high-risk HPVs,which were significantly higher than those in cervicitis and cervical dysplasia(x2 = 28.28,65.34 and 95.22,P ＜ 0.01).HPV positive rate was not correlated with clinical stages,differentiation degree of cervical cancer(x2 = 0.475 and 0.969,P＞0.05).HPV positive rates in squamous cancer and adenocarcinoma had no statistical difference (x2 =0.582,P＞0.05).The logistic regression analysis showed that HPV 16/58 infection and age over 40might increase the risk of carcinogenesis of the cervix.Conclusion sHPV infection and superinfection are popular in women with cervix cancer in Wenzhou.HPV16/58 infection and age over 40 years are the risk factors of cervical cancer.

Detection of aberrant methylation of RARβ2, GSTP1 and DAPK gene in prostate cancer and its clinical significance / 中华老年医学杂志

Objective To explore hypermethylation of RARβ2, GSTP1 and DAPK gene in prostate cancer tissues, and to explore its correlation with clinicopathological features of prostate cancer and its diagnostic value. Methods Hypermethylation of RARe2, GSTP1 and DAPK gene was detected by nested methylation-specific PCR (NMSP) in 57 prostate cancer (PCa) tissues and 35 benign prostate hyperplasia (BPH) tissues. The correlation between hypermethylation and clinicopathological features of prostate cancer and its diagnostic value were analyzed. Results The hypermethylation frequencies of RARβ2, GSTP1 and DAPK gene in PCa were significantly higher than those in BPH (RARβ2: 52.6% vs. 0% GSTP1: 61.4% vs. 2.9%;DAPK: 43.9% vs. 8.6%;all P<0.01). The methylation rate of RARβ2 gene was directly correlated with distinct Gleason scores and clinical stage (4～7 score vs. 8～10 score: 34.8% vs. 64.7%;stage B, C vs. stage D: 37.0% vs. 66.7%;x2=4.927 and 5.004, P=0.026 and 0.025). The methylation rate of GSTP1 gene was significantly different in patients with different Gleason scores (4～7 score vs. 8～10 score: 43.5% vs. 73.5%;x2 =11.530, P=0.001), but had no difference in patients with distinct clinical stage (stage B, C vs. stage D: 51.9% vs. 70.0%;x2=1.975, P=0.16) . There was no difference in DAPK gene methylation rate among patients with different Gleason scores and distinct clinical stage (4 ～7 score vs. 8～10 score: 39.1% vs. 50.0%;stage B, C vs. stage D: 33.3% vs. 53.3%;x2= 1.290 and 2.309, both P～0.05). GSTP1 gene showed the highest sensitivity of 61.4% (35/57)with specificity of 97.1%(34/35), while RARβ2 gene had the highest specificity of 100% (35/35) with the sensitivity of 52.6% (30/57). The sensitivity and specificity of DAPK gene were 43.9% and 91.4% (25/57 and 32/35), respeetively. When the hypermethylation of RARβ2, GSTP1 and DAPK gene were detected together, the diagnostic sensitivity was increased, but the specificity was decreased. Conclusions The aberrant methylation of RARβ2, GSTP1 and DAPK gene is correlated with tumorigenesis and progression of prostate cancer, which may be used as an effective diagnostic marker for prostate cancer.