Comparison of BCR::ABL (P210) mRNA levels detected by dPCR and qPCR methods in patients with chronic myeloid leukemia / 中华血液学杂志

Objective: To compare digital polymerase chain reaction (dPCR) and real-time quantitative PCR (qPCR) measurements of BCR::ABL (P210) mRNA expression in patients with chronic myeloid leukemia (CML) . Methods: In this non-interventional, cross-sectional study, BCR::ABL (P210) mRNA was simultaneously measured by dPCR and qPCR in peripheral blood samples collected from patients with CML who underwent tyrosine kinase inhibitor therapy and who achieved at least a complete cytogenetic response from September 2021 to February 2023 at Peking University People's Hospital. The difference, correlation, and agreement between the two methods were evaluated using the Wilcoxon signed-rank test, Spearman's correlation, and Bland-Altman analysis, respectively. Results: In total, 459 data pairs for BCR::ABL mRNA expression measured by dPCR and qPCR from 356 patients with CML were analyzed. There was a significant difference in BCR::ABL mRNA expression between the two methods (P<0.001). When analyzed by the depth of the molecular response (MR), a significant difference only existed for patients with ≥MR4.5 (P<0.001). No significant difference was observed for those who did not achieve a major MR (no MMR; P=0.922) or for those who achieved a major MR (MMR; P=0.723) or MR4 (P=0.099). There was a moderate correlation between the BCR::ABL mRNA expression between the two methods (r=0.761, P<0.001). However, the correlation gradually weakened or disappeared as the depth of the MR increased (no MMR: r=0.929, P<0.001; MMR: r=0.815, P<0.001; MR4: r=0.408, P<0.001; MR4.5: r=0.176, P=0.176). In addition, the agreement in BCR::ABL mRNA expression between the two methods in those with MR4.5 was weaker than other groups (no MMR: ▉= 0.042, P=0.846; MMR:▉=0.054, P=0.229; MR4:▉=-0.020, P=0.399; MR4.5:▉=-0.219, P<0.001) . Conclusions: dPCR is more accurate than qPCR for measuring BCR::ABL (P210) mRNA expression in patients with CML who achieve a stable deep MR.

Correlation of quantitative polymerase chain reaction with clinical characteristics of patients with viral retinitis

Purpose: To evaluate the correlation of quantitative real?time polymerase chain reaction (qRT?PCR) to the clinical characteristics of patients with viral retinitis.Methods: Retrospective case series. Results: Aqueous or vitreous samples of 20 out of 35 eyes showed qRT?PCR positivity for virus etiology (57.14%). Cytomegalovirus (CMV) was most commonly identified in nine eyes (45%). The mean DNA copy number was 2,68,339.65 copies/mL (range: 90–3205397). DNA copy number significantly correlated with the extent of clinical involvement (P = 0.013); however, there was no correlation between DNA copy number and presenting visual acuity (P = 0.31), macular involvement (P = 0.675), optic nerve involvement (P = 0.14), and development of retinal detachment (P = 0.73). There was a significant correlation between the number of DNA copies and the timing of sampling (P = 0.0005). Samples taken earlier in the course of the disease had higher viral copies than later ones. Conclusion: qRT?PCR is useful in confirming a viral etiology in over 50% of cases of suspected viral retinitis. It correlates well with the extent of clinical involvement and timing of sampling

Application and Prospect of RNA Profiling Analysis in Forensic Body Fluid Identification / 法医学杂志

In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.

Research Progress of DNA-Based Technologies for Postmortem Interval Estimation / 法医学杂志

In criminal investigations, postmortem interval (PMI) is important information to be inferred in homicide investigations, as well as the focus and the difficulty in forensic pathology research. Because the DNA content in different tissues is relatively constant and shows changes regularly with the extension of PMI, it has become a research hotspot of PMI estimation. This paper reviews the recent progress of PMI estimation technologies including DNA-based single cell gel electrophoresis, image analysis, flow cytometry, real-time fluorescence quantitative PCR and high-throughput sequencing, hoping to provide references for forensic medicine practice and scientific research.

Correlation between DNA Methylation of ZNF772 Promoter Region and Cervical Cancer / 中国医学科学院学报

To investigate the DNA methylation in ZNF772 promoter region and its mRNA and protein expressions and analyze the clinical significance of DNA methylation of ZNF772 gene in cervical cancer. Cervical squamous cell carcinoma (SCC) tissues were harvested from three patients (SCC group),and normal cervical tissues from healthy individuals of the same age were used as the control group. Hyper-methylation and lower transcripts were screened by whole-genome bisulfite sequencing (WGBS) and RNA sequencing. Furthermore,in 40 cervical tissue samples in SCC group and 45 normal cervical tissues in the control group,DNA methylation status and mRNA expression of ZNF772 were measured by using real-time quantitative polymerase chain reaction (RT-qPCR) and bisulfite sequencing polymerase chain reaction (BSP). The protein expression was detected by immunohistochemistry. In the SCC group,the potential relationships of DNA methylation status in ZNF772 promoter and mRNA expression with the clinicopathological parameters of cervical cancer were analyzed. As shown by WGBS and RNA sequencing,the abnormal DNA methylated gene ZNF772 was associated with mRNA expression. RT-qPCR verified that the mRNA expression of ZNF772 was significantly lower in SCC group than in control group (=8.351,=0.016). Immunohistochemistry further confirmed that the positive expression of ZNF772 protein was down-regulated in SCC group (=3.802,=0.005). BSP showed that the DNA methylation rate of ZNF772 promoter region (-420,-422 locus) in SCC group was significantly higher than that in control group (=8.566,=0.038;=6.332,=0.043). Spearman correlation analysis showed that,in SCC group,DNA hypermethylation in ZNF772 promoter was negatively correlated with the mRNA expression (=-0.351,=0.045;=-0.349,=0.032) and was significantly correlated with HPV16/18 infection,tumor size,World Health Organization pathological grade,and International Federation of Gynecology and Obstetrics clinical stage (=0.018,=0.012,=0.009,and =0.035,respectively). The DNA hypermethylation in the promoter region of ZNF772 gene is involved in the occurrence and development of cervical cancer.

Diagnostic ability of real-time quantitative polymerase chain reaction versus immunohistochemistry for Ki-67 assessment in breast cancer: An Indian perspective

Background & objectives: Breast cancer is the most common cancer of women. Inferior prognosis in some patients has been attributed to the higher proliferative capability of the tumour. Immunohistochemistry (IHC) for Ki-67, despite being a simple and cost-effective method, has not become a valid tool to evaluate this biomarker. This is ascribed to variation in pre-analytical and analytical techniques, variable expression, hotspot distribution and inter-and intra-observer inconsistency. This study was aimed at defining the analytical and clinical validity of real-time quantitative polymerase chain reaction (RT-qPCR) as an alternative to IHC evaluation. Methods: This study included a total of 109 patients with invasive breast cancers. Ki-67 IHC visual assessment was compared with the mRNA value determined by RT-qPCR. Concordance between both the methods was assessed. Receiver operating characteristic (ROC) curve analysis and Cohen's kappa value with intraclass correlation were performed. Results: The threshold value for Ki-67 by RT-qPCR obtained by ROC curve was 22.23 per cent, which was used to divide breast cancer cases into high proliferative and low proliferative groups. A significant correlation was observed between both the breast cancer groups formed using RT-qPCR threshold as well as median laboratory value of Ki-67 labelling index by IHC. Interpretation & conclusions: The study results showed a significant correlation between the two methods. While IHC is subject to technical and interpretative variability, RT-qPCR may offer a more objective alternative.

Expression and Clinical Significance of Pin1 and CyclinD1 in Gastrointestinal Stromal Tumor / 中国医科大学学报

Objective To investigate the relationship between Pin1 and CyclinD1 expression and the development of gastrointestinal stromal tu?mor(GIST). Methods The protein and mRNA expression of Pin1 and CyclinD1 in 85 samples of GIST and adjacent non?cancerous tissues were detected by immunohistochemistry and real?time quantitative polymerase chain reaction. Results The expression rate of Pin1 protein in GIST tis?sues(64.7%;55/85)was higher than that in adjacent non?cancerous tissues(26.7%;4/15). Similarly,the expression rate of CyclinD1 protein in GIST tissues(42.3%;36/85)was higher than that in adjacent non?cancerous tissues(6.7%;1/15). The expression of Pin1 and CyclinD1 mRNA in GIST tissues was 7.03 and 5.53 times that in adjacent non?cancerous tissues ,respectively. There was no obvious correlation between the expres?sion of Pin1 and clinicopathological parameters. The expression of CyclinD1 was positively correlated with the grade of NIH and tumor diameter (P<0.05). There was a significant correlation between the expression of Pin1 and CyclinD1 in GIST tissues. Conclusion The expression of both Pin1 and CyclinD1 was up?regulated in GIST tissues. The significant correlation between the expression of Pin1 and CyclinD1 in GIST tissues sug?gests that their synergistic effect promotes carcinogenesis and the development of GIST.

Study on Expression Interferon Stimulating Gene and Related Signal Pathway Gene in Peripheral Blood Mononuclear Cells of Patients with Chronic Hepatitis C / 现代检验医学杂志

Objective To detect mRNA expressions of STING and type Ⅰ interferons(IFN-α and IFN-β) in peripheral blood mononuclear ceils (PBMCs) of patients with chronic hepatitis C(CHC).Methods 113 patients with CHC and 94 healthy controls were collected from Renmin Hospital of Wuhan University during January 2015 and January 2016.Expressions of mRNA of STING,RIG-1,IFN-α and IFN-β were detected by real-time quantitative PCR,and their relative expression values were obtained by 2-△△Ct method and the results were statistically analyzed.Results The expression of mRNA of STING,RIG-1,IFN-α and IFN-β in CHC were 0.018,0.361,0.578 and 0.573 times than its in healthy controls respectively and the differences were statistically significant (t-8.28,4.26,2.18 and 2.07,all P ＜ 0.05).Pearson correlation analysis showed that mRNA expressions of STING in healthy controls were positively correlated with that of IFN-α mRNA and IFN-β mRNA (r=0.487 and 0.207,all P＜0.05),which had no correlation in CHC (r=0.174 and 0.091,all P＞0.05).The expressions of STING mRNA was positively correlated with that of RIG-1 mRNA in healthycontrols (r=0.222,P＜0.05),but there was no correlation of expression in CHC between STING mRNA and RIG-1 mRNA (r=-0.029,P＞0.05).Conclusion Compared with healthy controls,the expression of STING,RIG-1,IFN-α and IFN-β mRNA were all reduced.There was positive correlation between STING and RIG-1,IFN-α and IFN-β in healthy controls,but no correlation in CHC.

Expression of miR-139-5p in small cell lung cancer tissue and its clinical significance / 吉林大学学报(医学版)

Objective:To explore the expression of miR-139-5p in small cell lung cancer (SCLC)tissue and its clinical significance, and to clarify the role of miR-139-5p in the occurrence and development of SCLC. Methods:The biological function of miR-139-5p was examined by cell growth,apoptosis and cell cycle analysis. The expressions of miR-139-5p in 50 cases of cancer tissue and paracarcinoma normal tissue were examined by QRT-PCR.Combined with the clinical data,the role of miR-139-5p in clinic was anzlyzed.Results:The expression level of miR-139-5p in SCLC tumor tissue was lower than that in normal lung tissue (P 0.05).The expression level of miR-139-5p in the patient at LD stage was lower than that of the patients at ED stage (P <0.01).The expression level of miR-139-5p in the resistant patients was higher than that of the patients sensitive to chemotherappy (P <0.01).The expression level of miR-139-5p in the survival patients was lower than that in the death patients (P <0.01).Cox regression analysis indicated that miR-139-5p expression and disease stage were the independent prognostic factors for SCLC.Conclusion:miR-139-5p in participates in the occurrence and development of SCLC by inhibiting the cell proliferation,promoting apoptosis and inducing the cell cycle arrest;it may be used as a target gene to evaluate the prognosis of SCLC patients.

Identiifcation and validation of a novel gene expression signature for diagnosing tumor tissue origin / 中国癌症杂志

Background and purpose:Cancer of unknown primary (CUP) represents approximately 5%~10%of malignant neoplasms. For CUP patients, identiifcation of tumor origin allows for more speciifc therapeutic regimens and improves outcomes.Methods:By retrieving the gene expression data from ArrayExpress and Gene Expression Omnibus data repositories, we established a comprehensive gene expression database of 5 800 tumor samples encom-passing 22 main tumor types. The support vector machine-recursive feature elimination algorithm was used for feature selection and classiifcation modelling. We further optimized the RNA isolation and real-time quantitative polymerase chain reaction (RTQ-PCR) methods for candidate gene expression proifling and applied the RTQ-PCR assays to a set of formalin-fixed, paraffin-embedded tumor samples.Results:Based on the pan-cancer transcriptome database, we identiifed a list of 96-tumor speciifc genes, including common tumor markers, such as cadherin 1 (CDH1), kallikrein-re-lated peptidase 3 (KLK3), and epidermal growth factor receptor (EGFR). Furthermore, we successfully translated the microarray-based gene expression signature to the RTQ-PCR assays, which allowed an overall success rate of 88.4% (95%CI: 83.2%-92.4%) in classifying 22 different tumor types of 206 formalin-fixed, paraffin-embedded samples. Conclusion:The 96-gene RTQ-PCR assay represents a useful tool for accurately identifying tumor origins. The assay uses RTQ-PCR and routine formalin-ifxed, paraffn-embedded samples, making it suitable for rapid clinical adoption.

Quantitative analysis of intestinal bacteria in Caenorhabditis elegans using qPCR / 军事医学

Objective To establish a method for detecting intestinal bacteria in Caenorhabditis elegans.Methods The gut flora number of 1, 20 or, 50 C.elegans was quantified and compared using real-time quantitative PCR (qPCR). Results The gut flora of a single C.elegans could be detected using qPCR method , which could also reflect the difference in the number of gut bacteria between different samples .Conclusion The qPCR method can be uased to accurately quantify intestinal bacteria even in only one C.elegans and has the advantages of low-cost, high-sensitivity and good-specificity.

Utility of routine real time quantitative PCR monitoring of HCV infection in haemodialysis patients.

Background: Hepatitis C virus (HCV) infection causes signifi cant morbidity and mortality in patients of end stage renal disease (ESRD) on haemodialysis (HD). Stringent screening methods can help in its early diagnosis. Objective: The study addresses the utility of real-time quantitative polymerase chain reaction (RQ-PCR) in the diagnosis and monitoring of HCV infection especially on seronegative and normal serum alanine aminotransferase (ALT) HD patients. Material and Methods: This retrospective study was carried out from January 2010 to December 2012. Patients of ESRD on maintenance HD and on whom all the three assays HCV antibody serology, PCR and ALT were done were included in the study (n = 123). Group 1 (n = 57), comprised of patients with negative serology and normal ALT, and Group 2 (n = 66), had either raised ALT and or a positive or equivocal serology. Results: Out of the 123 cases studied, HCV serology was positive in 36.5% (45), ALT raised in 18.6% (23) and PCR positive in 67.4% (83) cases. PCR positivity was signifi cantly higher than serology and raised ALT. Group 2 had a signifi cantly higher PCR positivity than Group 1 (P = 0.0004), but 50.9% patients of Group 1, were also PCR positive and 69% of them had a high viral count of >8 × 105 IU/ml at the time of detection. Conclusion: Regular routine screening of HCV by RQ-PCR in ESRD patients can help in early diagnosis of HCV infection in patients with low index of suspicion.

Establishment and preliminary application of real time PCR assay for quantitative detection of CRLF2 / 国际检验医学杂志

Objective To establish a real‐time quantitative PCR method for the detection of cytokine receptor‐like factor 2 (CRLF2) expression .Methods Specific primers amplification target gene CRLF2 and housekeeping genes ABL were designed ,the purified PCR products were performed the TA cloning .After bacterial colony PCR screening and sequencing ,then the recombinant plasmids DNA was extracted and measured by using UV spectrophotometer and converted to copies/mL concentration .Finally it was diluted for preparing the plasmid standard substance ,then the standard curve was drawn for observing the sensitivity and linear rang ,meanwhile the stability of the plasmid DNA was evaluated .This method was initially applied to detect the CRLF2 level of bone marrow mononuclear cells in 10 cases of healthy children and 10 cases of newly diagnosed acute lymphoblastic leukemia (ALL) .Results CRLF2 PCR product had a single specific melting curve;the linear detection range of the standard substance was 103 - 108 copies /ml;the plasmid standard substance by freeze‐thawing for 3 times remained stable;the CRLF2 level of clinical sample was within the linear detection range of standard substance .Conclusion The real‐time quantitative PCR method for CRLF2 established by our laboratory has good specificity ,linearity range and stability ,which can be applied to the quantitative detection of CRLF2 gene in clinical ALL children .

mRNA expressions of Toll-like receptor 3, 7 and type Ⅰ interferons in peripheral blood of patients with chronic hepatitis C / 中华临床感染病杂志

Objective To investigate mRNA expressions of Toll-like receptors (TLR3 and TLR7)and type Ⅰ interferons (IFN-α and IFN-β) in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis C (CHC).Methods Thirty patients with CHC and 30 healthy controls were collected from Renmin Hospital of Wuhan University during September 2013 and May 2014.Liver function was detected using enzyme-linked immunosorbent assay (ELISA).mRNA expressions of TLR3,TLR7,IFN-α and IFN-β were detected by real-time quantitative PCR,and their relative expression values were obtained by 2-△△Ctmethod.The t test was performed for the comparison of mean differences between CHC patients and healthy controls,and Pearson analysis was used to analyze the correlations between TLR mRNA and IFN mRNA,liver function,HCV RNA load.Results Taking mRNA expressions in healthy control group as 1,the relative mRNA expressions of TLR3,TLR7,IFN-α and IFN-β in CHC group were 0.086,0.633,0.145 and 0.423 respectively (t =4.25,2.39,2.37 and 2.80,all P ＜ 0.01).Pearson correlation analysis showed that mRNA expressions of TLR3 and TLR7 were positively correlated with that of IFN-β (r =0.381 and 0.487,all P ＜ 0.05),but not correlated with IFN-α mRNA expression,HCV RNA load,ALT and AST (all P ＞ 0.05).Conclusion The mRNA expressions of TLR3,TLR7 and IFN-α,IFN-β decrease in CHC patients,and the mRNA expressions of TLR3 and TLR7 are positively correlated with that of IFN-β,indicating that increasing the expression of TLRs might be a new strategy in CHC treatment.

Prognostic Implications of Expression of the Wilms Tumor 1 (WT1) Gene in Acute Leukemia (Experience from South Egypt).

Background: Wilms’ tumor (WT1) gene expression has been reported in the majority of acute leukemia patients at diagnosis and has been evaluated as a prognostic and minimal residual disease (MRD) marker but its role is still controversial. Methods: Real-time quantitative polymerase chain reaction was used on bone marrow samples from 100 newly diagnosed adults and pediatrics acute leukemia patients (50 AML and 50 ALL patients). WT1 expression were examined at diagnosis and at the end of induction. Results: WT1 was expressed in (14%) ALL and in (36%) AML patients. We found no statistically significant impact of WT1 expression at diagnosis on response p= 0.054, 0.057, DFS (P = 0.591, 0.858), or OS (p= 0.339, p= 0.331) in ALL and AML patients respectively. Persistence of WT1 expressions at the end of induction didn't show any effect on relapse rate in AML however, it showed significant results in ALL p=0.045. Conclusion: Our results suggest that WT1 expression in patients with acute leukemia doesn't have any implication on response or survival however, significant association was found in predicting ALL relapse but the small sample size should be considered.

Multiplex real-time quantitative polymerase chain reaction for simultaneous detection of Escherichia coli and Candida albicans genes in human whole blood / 中华临床营养杂志

Objective To establish a multiplex real-time quantitative polymerase chain reaction (MRQPCR) assay for fast and simultaneous detection of Escherichia coli (E.coli) and Candida albicans (C.albicans) genes in human whole blood,in order to facilitate differentiation of the types of microorganism and evaluation of the severity of bacterial or fungi translocation due to impaired gut barrier,hence providing help to select specific antimicrobial agents.Methods The β-D-galactosidase gene of E.coli and ITS2 gene of C.albicans were selected as the target genes for designing primers and probes.E.coli and C.albicans genomes were extracted with QIAamp(R) DNA Blood Mini Kit,and the 25 μl TaqMan MRQ-PCR amplification reaction system was established.18 simulated human whole blood samples and 10 whole blood samples from febrile surgical patients were detected for E.coli and C.albicans genes using MRQ-PCR.Results The specificity of the primers and probes were excellent.The correlation coefficients of the standard curves of E.coli and C.albicans were 0.994-0.999 and 0.994-0.998,respectively;and the efficiency of amplification were 0.894-1.022 and 0.905-1.028,respectively.In the standard samples,the lowest detection limits of E.coli and C.albicans were 13.9 copies/μl and 0.8 cfu/μl,respectively;the sensitivity was 100％ and 99.69％,the specificity was 100％ and 94.73％,respectively;the average recovery rates were (101.89 ± 5.69)％ and (103.74 ± 4.64)％ respectively;the intra-batch coefficients of variance (CV) in detecting the genes were (13.14 ± 10.27)％ and (19.18 ± 8.54)％,respectively,and the inter-batch CV were (14.35 ± 9.34)％ and (18.31 ± 10.25) ％,respectively.In human whole blood,the lowest detection limits of E.coli and C.albicans were 12 455.2 copies/ml and 800.3 cfu/ml,respectively;the average recovery rates were (111.60 ± 11.06) ％ and (99.96 ± 6.16) ％,respectively;the intra-batch CV in detecting the genes were (11.02 ± 5.65) ％ and (8.14 ± 7.29)％,respectively,and the average inter-batch CV were (12.88 ± 7.59)％ and (18.62 ± 9.14)％.Conclusions MRQ-PCR is a rapid,sensitive,specific,accurate,and reproducible method for simultaneous detection of E.coli and C.albicans genes in human whole blood,with sample-,cost-,and time-saving advantages.It is a promising technique for rapid differentiation between fungi and bacteria,which could help targeted administration and evaluation of antimicrobial agents,and help to assess the consequence of gut barrier damage and the efficacy of treatment.

Correlations of enterobacteria DNA load in venous blood determined by real-time quantitative polymerase chain reaction with vital signs and blood cell count in febrile surgical patients / 中华临床营养杂志

Objective To determine enterobacteria DNA load in venous blood of febrile surgical patients using real-time quantitative polymerase chain reaction (RQ-PCR), to study the correlations between DNA load and vital signs/blood cell count, and to compare the difference between different detection methods in terms of positive rates.Methods A total of 72 blood samples were obtained for bacterial cuhure and RQ-PCR.The correlations of enterobacteria DNA load with body temperature, heart rate, while blood cell count,and percentages of leukocyte and lymphocyte were then analyzed.Results The enterobacteria positive rate determined by RQ-PCR (63.89％) was significantly higher than that by bacterial culture (9.72％) (F =4.383, P =0.036).The DNA load was significantly correlated with both body temperature and heart rate (P =0.006, r =0.323;P =0.000, r =0.411), but not with white blood cell count, percentages of leukocyte and lymphocyte, and age (P=0.438, r=0.093;P=0.825, r=0.027;P=0.451, r=-0.090;P =0.096, r =0.198).Conclusions RQ-PCR can quickly determine the enterobacteria DNA load in peripheral blood with high sensitivity.Routine blood cell count may not accurately reflect the enterobacteria DNA load in blood.Body temperature and heart rate may be influenced by various factors.

The relationship between microRNA-146a and TNF-α in lipopolysaccharide-stimulated alveolar macrophages of rats / 中华急诊医学杂志

Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a)expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate,and stimulated with 1 μg/ml of LPS for 0 h,3 h,6 h and 12 h separately after 90 min.Cells were harvested and supernatant were collected 0 h,3 h,6 h and 12 h after incubation.The expressions of miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant of cells were assayed by enzyme-linked immunosorbent assay ( ELISA ).Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA.Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge ( 359.80 ±57.54) pg/ml (P ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.

Comparison of two real-time quantitative polymerase chain reaction for detecting HCV RNA virus load in plasma / 中华传染病杂志

Objective To compare the plasma hepatitis C virus(HCV)RNA levels detected by the fully automated viral load detection system(COBAS TaqMan)and the national real-time quantitative polymerase chain reaction(PCR)kit,and to investigate the clinical application value of these two methods in clinical practice.Methods A total of 168 serial plasma samples collected from 26 patients with chronic hepatitis C(CHC)before and at week 2,4,12,24,36 and 48 of antiviral treatment were detected by both COBAS Taqman 48 analyzing system and the national real-time quantitative PCR kit.The results of two methods were compared by chi square test and t test.Resnlts Both COBAS and national kit showed great positive detecting results when HCV RNA≥1×104IU/mL(at week O),and the virus load value detected by national kit was significantly higher than that detected by COBAS(t=2.05,P0.05).Conclusions The national TaqMan real-time quantitative PCR kits could be used to screen the suspected cases of HCV infecrion and to diagnose CHC cases with high HCV virus load.COBAS detection is more sensitive in cases with low HCV virus load and in on-treatment monitor during anti-HCV therapy.

Effects of simvastatin on renal expression of Fas/Fas-L in aged diabetic rats after uninephrectomy / 第二军医大学学报

Objective: To explore the effects of hydroxy-methyl-glutaryl coenzymeA reductase inhibitor (HMG-CoA RI) simvastatin on renal expression of apoptosis related gene Fas/Fas-L in aged diabetic rats after uninephrectomy. Methods: Elderly uninephrectomized SD rats were randomly divided into uninephrectomized control group (group C), streptozocin(STZ)-induced diabetic model group(group D), and diabetes + simvastatin group (group S). The apoptosis indices in renal tisses of three groups were detected by terminal deoxynucleotide mediated nick end labeling 12 weeks later. Immunofluorescence and real-time quantitative PCR were employed to observe the expression changes of Fas/Fas-L genes at the transcription and translation levels. Results: Compared with group C, group D had significantly higher apoptosis index and expression level of Fas/Fas-L; the above parameters were lower in group S compared with those in group D (P<0.05). The expression of Fas/Fas-L protein was mainly in the renal tubules. Conclusion: Both Fas and Fas-L genes are associated with the development and progression of diabetic nephropathy in aged rats. Simvastatin can down-regulate the translation and transcription of the genes, inhibit cell apoptosis and protect the kidney.