Transcriptomic Analysis of Wuzi Yanzongwan on Testicular Spermatogenic Function in Semi-castrated Male Mice / 中国实验方剂学杂志

ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.

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.

Screening of Reference Genes of Andrographis paniculata Under MeJA and Abiotic Stresses by Real-time Fluorescence-based Quantitative PCR / 中国实验方剂学杂志

ObjectiveTo screen the appropriate reference genes for real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）analysis of the Andrographis paniculata under methyl jasmonate（MeJA）and various abiotic stresses. MethodThe actin 1（ACT1），actin 2（ACT2），elongation factor（EF-1α），glyceraldehyde-3-phosphate dehydrogenase（GAPDH），tubulin（TUB），polyubiquitin（UBQ）， and 18S rRNA（18S）gene were selected as candidate reference genes based on the RNA-seq data of high temperature，drought， UV， and MeJA. The expression of seven candidate reference genes in the A. paniculata leaves was assessed by Real-time PCR，and the stability was analyzed by geNorm，NormFinder，BestKeeper， and Refinder. ResultThe results of stability evaluated by geNorm，NormFinder， and BestKeeper were not the same due to different indicators. As analyzed by Refinder， for the stability of the expression， the genes were ranked as UBQ>18S>EF-1α>ACT2>ACT1>GAPDH>TUB under high temperature stress， ACT1>UBQ>EF-1α>18S>ACT2>GAPDH>TUB under drought stress， EF-1α>TUB>ACT2>UBQ>18S>GAPDH>ACT1 under UV stress， and ACT1>EF-1α>UBQ>ACT2>18S>TUB>GAPDH under MeJA stress. Among them，18S gene was not suitable as an internal reference gene duo to its high expressive abundance. This study also verified the relative expression level of andrographolide synthesis-related gene hydroxy-methylglutaryl-CoA synthase （HMGS） in the four stresses on the basis of transcriptome data，and found that the Real-time PCR results of appropriate internal reference genes were accurate and reliable. ConclusionUBQ-ACT1-UBQ，EF-1α-TUB，and ACT1-EF-1α were the suitable combinations under stresses of high temperature，drought，UV， and MeJA. This study is expected to provide references for the research on function regulation and expression of genes in A. paniculata under high temperature，drought，UV， and MeJA stresses.

Cloning and Prokaryotic Expression of Glycosyltransferase Genes from Paris polyphylla var. yunnanensis / 中国实验方剂学杂志

Objective:To clone the full-length glycosyltransferase genes （<italic>PpUGT</italic>1，<italic>PpUGT</italic>7） related to saponins biosynthesis in <italic>Paris polyphylla</italic> var. <italic>yunnanensis</italic>，and perform bioinformatics analysis，relative expression analysis and prokaryotic expression analysis. Method:Total RNA was isolated from <italic>P. polyphylla </italic>var. <italic>yunnanensis </italic>with use of the Eastep^® Super Total RNA Extraction Kit and converted to cDNA. Specific primers were designed according to the transcriptome data to clone the full-length gene. Relevant software was then used for bioinformatic analysis of the protein sequences. The relative gene expression levels were detected by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and the prokaryotic expression vectors were built to heterologously express recombinant protein in <italic>Escherichia coli.</italic> Result:The open reading frame （ORF） of <italic>PpUGT</italic>1 was 1 827 bp，encoding 608 amino acids，and was predicted as a steroid glycosyltransferase；the ORF of <italic>PpUGT</italic>7 was 1 380 bp，encoding 459 amino acids，and was predicted as a triterpenoid glycosyltransferase. The calculated relative molecular mass of two proteins were 67.6 kDa and 51.3 kDa respectively，and both of them were hydrophilic proteins，no transmembrane domain，no signal peptides，both showing high similarity and conservativeness with homologous sequences. The results of Real-time PCR showed that the expression level of <italic>PpUGT</italic>1 was root>leaf>flower>stem；the expression level of <italic>PpUGT</italic>7 was stem>leaf>flower>root. In addition，PpUGTs proteins were expressed in <italic>E. coli</italic>. in a soluble form. Conclusion:The genes of <italic>PpUGT</italic>1 and <italic>PpUGT</italic>7 were cloned successfully. Real-time PCR showed the genes were expressed differently in different plant organs， and their recombinant proteins were successfully expressed in <italic>Escherichia coli</italic>. This study lays a foundation for functional characterization of PpUGTs and analysis of the biosynthesis pathway of saponins in <italic>Paris polyphylla </italic>var. <italic>yunnanensis</italic>.

A new method for simultaneous quantitative determination of hepatitis B virus pregenomic RNA and DNA / 第二军医大学学报

Objective To design and establish a new method for simultaneous determination of hepatitis B virus (HBV) pregenomic RNA (pgRNA) and DNA from nucleic acid extracts. Methods We established a duplex fluorescence quantitative PCR system to determine HBV pgRNA and DNA. DNA gel electrophoresis and quantitative PCR were used to test the specificity and sensitivity. We tested the feasibility and accuracy by determining the HBV pgRNA and DNA in HepG2.2.15 cells and the culture supernatants. Results The established duplex fluorescence quantitative PCR system has a good specificity and sensitivity. When it was used to determine cell culture supernatants with different dilution ratios, the dilution ratios and results were well correlated. However, this method was more suitable for the determination of HBV pgRNA and DNA in cell culture supernatants, rather than cell samples. Conclusion Our method can avoid inaccuracy of HBV RNA determination caused by HBV DNA contaminant in nucleic acid extracts, and realize simultaneous detection of HBV pgRNA and DNA in one PCR reaction, which greatly improves the determination efficiency and has potential clinical application value..

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.

Development and Comparison of Determination of Staphylococcus aureus in Different Processed Products of Angelicae Sinensis Radix Based on Real-time PCR Technique / 中国实验方剂学杂志

Objective:To quantitatively analyze the changes of Staphylococcus aureus in different processed products of Angelicae Sinensis Radix. Method:The real-time fluorescence quantitative polymerase chain reaction method （Real-time PCR） was established to quantitatively analyze S. aureus in Angelicae Sinensis Radix decoction pieces which bought from different producing areas， different enterprises and different storage time. The fluorescence quantitative reaction system was SYBR Premix Ex Taq Ⅱ of 10 μL， each of forward primer and reverse primer （10 μmol·L-1） of 0.8 μL， template/genome DNA of 1 μL， double distilled water of 7.4 μL. The reaction conditions of the fluorescence quantitative amplification curve were pre-denaturing for 30 s at 94 ℃， denaturing for 10 s at 94 ℃， annealing for 12 s at 60 ℃， extensing for 30 s at 72 ℃， cycling 45 times， single-point detection signal at 72 ℃. The melting curve was made from 72 ℃， and the step temperature of 0.5 ℃ was kept for 15 s to collect fluorescence. According to the results of Real-time PCR， representative samples were selected from Angelicae Sinensis Radix decoction pieces for comparison between plate counting method and Real-time PCR. Result:The content of S. aureus in different processed products was sorted by rank of raw Angelicae Sinensis Radix>soil-fried Angelicae Sinensis Radix>wine-processed Angelicae Sinensis Radix. The content of S. aureus was the lowest in the samples from Weiyuan area of Gansu province by comparing with other producing areas. Compared with the retail enterprises， the content of S. aureus in raw products and wine-processed products from production and sale enterprises was lower. Different storage time had certain effect on the content of S. aureus in raw products and wine-processed products， and the content of S. aureus increased with the increase of storage time. The detection results of plate counting method were 3-4 orders of magnitude lower than that of Real-time PCR. Conclusion:The established Real-time PCR is superior to plate counting method in specificity， sensitivity， reliability and reporting period， which can provide an effective method for rapid and accurate quantitative detection of S. aureus in different processed products of Angelicae Sinensis Radix.

Expression and significance of microRNA-125b in tongue squamous cell carcinoma / 华西口腔医学杂志

OBJECTIVE@#The expression of microRNA-125b in tongue squamous cell carcinoma (TSCC) was detected and analyzed for its relationship with the clinicopathological features of TSCC.@*METHODS@#Real time fluorescence-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of microRNA-125b in 35 TSCC tissues and adjacent normal tissues from 35 TSCC cases. The relationship between the expression of microRNA-125b in TSCC tissues and the clinicopathological features of patients with TSCC was analyzed. In situ hybridization (ISH) was used to detect the expression level of microRNA-125b gene in the TSCC tissues and adjacent normal tissues.@*RESULTS@#RT-qPCR results showed that the relative expression levels of microRNA-125b in the TSCC issues was 2.32±0.69, and that of normal tissues was 0.87±0.32. The statistical results showed that the expression level of microRNA-125b was significantly higher in the TSCC tissues than in the normal tissues (P<0.001). The expression level of microRNA-125b in the TSCC tissues was not significantly correlated with age, gender, pathological grade, and lymph node metastasis but was positively correlated with TNM stage. Patients with high TNM stage had high microRNA-125b expression levels (P<0.05). The ISH results showed that the expression levels of microRNA-125b in the TSCC tissues were 0.010±0.003, and that of normal tissues was 0.004±0.001. The expression levels of microRNA-125b in the 35 TSCC tissues were significantly higher than those in the normal tissues (P<0.05).@*CONCLUSIONS@#MicroRNA-125b is highly expressed in TSCC and associated with TNM stage, suggesting that high microRNA-125b expression may be involved in the development of TSCC.

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.

Transcriptome profiling of four candidate milk genes in milk and tissue samples of temperate and tropical cattle

The expression of four genes involved in milk regulation and production in bovine milk and tissue samples profiled using quantitative PCR to identify differential gene expression. Our goal focussed on the differential mRNA expression of milk genes (KCN, PRL, BLG and PIT-1) in milk samples and different tissues from four different breeds of ecologically adapted and geographically separated cattle species. The mRNA expression identified the four milk genes understudiedmost upregulated in mammary gland and milk samples as compared with other tissues. The expression of PIT-1 gene in the brain identified to have influenced the expression of PRL and K-CN in the mammary and milk samples. Among the four genes, PRL had the highest mRNA expression (144.19-fold change) in Holstein followed by K-CN with 100.89-fold change, while the smallest relative expression for most genes in this study are in the range from 0.79 to 7.35-fold difference. White Fulani cattle was identified to have a higher expression for K-CN, PRL and BLG compared with Angus and Ndama cattle, while Holstein cattle is on top of the list on the basis of the gene expression and gene regulation for all the four genes in this study. Also, White Fulani and Holstein are in the same cluster based on their mRNA expression for milk genes. Our data showed the first evidence of the molecular identification of indigenous White Fulani cattle ofhaving potential for higher milk production.

miR-146a is deregulated in gastric cancer

Background: Gastric cancer is one of the most significant reasons for cancer-related death. miR-146a is one of the dysregulated factors associated with gastric tumorigenesis. However, deregulation of this microRNA (miRNA) has become controversial. Moreover, the inflammation-mediating role of this miRNA implies that miR-146a might be dysregulated by gastric cancer-related pathogens, such as Helicobacter pylori. However, the dysregulation of miR-146a in H. pylori-infected gastric tumors has not been widely studied. Objectives: We aimed to analyze the expression level of miR-146a in gastric cancer tissues and then to assess any potential association between miR-146a and H. pylori infection and other clinical characteristics. Materials and Methods: miR-146a expression level was quantitatively studied by reverse transcription quantitative polymerase chain reaction, in 144 fresh tissues including 44 normal and 100 gastric cancer samples. Results: A dramatic overexpression of miR-146a was observed in primary gastric tumors. miR-146a showed lower expression in progressed tumors with greater stages and lymph node metastasis. Conclusion: miR-146a is highly expressed in primary gastric tumor independent of H. pylori infection. It is highly expressed in the lower stages and lymph node-negative tumors. It might suggest the importance of upregulation and downregulation of this miRNA in the initiating/promoting and progressive steps of gastric tumorigenesis, respectively

Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata / 中国实验方剂学杂志

Objective:To detect the colony number of bacteria, yeasts and molds in fermentation process of Pinelliae Rhizoma Fermentata (PRF), microbial flora species, and quantitatively analyze the dynamic changes of four dominant microorganisms at different fermentation time points of PRF, so as to provide experimental basis for exploring the processing mechanism of PRF. Method:According to Pharmaceutical Standard Preparation of Traditional Chinese Medicine Prescription of Ministry of Health of the People's Republic of China (the 10th volume), PRF was processed. The samples at five different fermentation time points (0, 30, 60, 90, 120 h) of PRF were taken, the culturing, isolation and purification of bacteria, yeasts and molds were carried out with selective media, and the colonies were counted. Fluorescence quantitative polymerase chain reaction (PCR) technique was employed to conduct absolute quantification of Bacillus subtilis, Paecilomyces variotii, Byssochlamys spectabilis and Aspergillus niger. The recombinant plasmids of these 4 microorganisms were used as the standard substances, and the standard curves were prepared after dilution of multiple ratios, quantitative analysis was performed on these 4 microorganisms in five samples at different processing time points (0, 30, 60, 90, 120 h) of PRF. Result:During the fermentation process of PRF, the number of bacteria was low with smooth change, while molds and yeasts grew dramatically at the late stage of fermentation and reached 1×106 CFU·mL-1 at the end of fermentation. At 5 different fermentation time points, the copy numbers of Bacillus subtilis were 3.53×105, 7.56×104, 1.58×105, 1.90×106, 1.85×106 copies·g-1, the copy numbers of Paecilomyces variotii were 0, 0, 0, 3.45×107, 4.15×108 copies·g-1, the copy numbers of Byssochlamys spectabilis were 0, 0, 0, 1.04×108, 2.28×108 copies·g-1, the copy numbers of Aspergillus niger were 0, 0, 9.48×105, 1.47×106, 7.56×106 copies·g-1, respectively. Conclusion:The change trend of microflora in the fermentation process of PRF can be reflected by the dynamic change of four dominant microorganisms, and molds may play an important role in the processing of PRF. Fluorescence quantitative PCR technique has the advantages of rapidity, sensitivity, good repeatability and high specificity, it is suitable for exploring processing mechanism of PRF.

Levels of miRNA-106a in non-small cell lung cancer tissues and its clinical significance / 中国基层医药

Objective@#To investigate the level and clinical significance of miRNA-106a in non-small cell lung cancer tissues.@*Methods@#Paraffin-embedded specimens of non-small cell lung cancer tissues and adjacent tissues from 80 patients with non-small cell lung cancer from January 2012 to December 2013 in Yiwu Central Hospital were selected in this study.The real-time fluorescence quantitative polymerase chain reaction (QRT-PCR) was used to determine the level of miRNA-106a in lung cancer tissues and adjacent tissues.@*Results@#The level of miRNA-106a in non-small cell lung cancer tissues (2.42±0.23) was higher than that in adjacent tissues (1.00±0.06) (t=53.433, P=0.000). The miRNA-106a levels in lung cancer tissues of stage Ⅲ-Ⅳ, lymph node metastasis and recurrence time<6 months were high than those of the stage Ⅰ-Ⅱ, no lymph node metastasis and recurrence time ≥ 6 months(t=7.641, 11.115, 2.183, P=0.000, 0.000, 0.032). The level of miRNA-106a was not associated with age, gender, pathological type, degree of differentiation and vascular invasion (all P>0.05). The level of miRNA-106a in non-small cell lung cancer tissues was cut by 2.12.The area under the ROC curve for the diagnosis of non-small cell lung cancer was 0.823(95% CI=0.820-0.825, P=0.000), and the sensitivity was 54.37%, the specificity was 89.21%.The cumulative survival rate and progression-free survival rate of patients with low-expression of miRNA-106a in non-small cell lung cancer were higher than those with high expression of miRNA-106a (P=0.027, 0.012).@*Conclusion@#The miRNA-106a level is elevated in non-small cell lung cancer tissues.The miRNA-106a may be involved in the development of non-small cell lung cancer and is of great value in the diagnosis and prognosis evaluation of non-small cell lung cancer.

Expression of RIZ1 gene in myelodysplastic syndromes and its significance / 肿瘤研究与临床

Objective To explore RIZ1 mRNA expression level in different subtypes and risk classification groups of the patients with myelodysplastic syndromes (MDS) and to analyze the correlation between the clinical features and RIZ1 mRNA expression. Methods A total of 46 newly diagnosed as MDS patients and 10 healthy controls who were the donors of hematopoietic stem cell transplantation from Chuiyangliu Hospital Affiliated to Tsinghua University and the Second Hospital of Shanxi Medical University between January 2014 and December 2017 were collected. The real time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the expression level of RIZ1 mRNA in bone marrow cells from MDS patients and the healthy controls. Results Compared with the healthy control group, the relative expression level of RIZ1 mRNA in MDS patients was decreased [the median (P25, P75):1.003 (0.895, 1.812) vs. 0.557 (0.333, 0.815)], and the difference was statistically significant (Z= -2.991, P= 0.0003). According tothe World Health Organization (WHO) classification criteria, compared with the healthy control group, the relative expression of RIZ1 mRNA in refractory anemia/refractory anemia with ring sideroblasts/refractory cytopenia with multiple dysplasia (RA/RAS/RCMD), refractory anemia with excess blasts Ⅰ (RAEB-Ⅰ), RAEB-Ⅱand MDS transformed into acute myeloid leukemia (MDS/AML) groups had statistically significant differences (χ2= 19.500, P< 0.01). Further pairwise comparison showed that the relative expression level of RIZ1 mRNA in RAEB-Ⅰ, RAEB-Ⅱand MDS/AML groups was lower than that in the healthy control group, and the differences were statistically significant (all P < 0.05). According to the international prognostic scoring system (IPSS), compared with the healthy control group, the expression of RIZ1 mRNA in low-risk, inter-risk-1, inter-risk-2 and high-risk group had statistical differences (χ2= 19.214, P= 0.001). Further pairwise comparison showed that the relative expression of RIZ1 mRNA in inter-risk-1, inter-risk-2 and high risk group was lower than that in the healthy control group, and the differences were statistically significant (all P<0.05). And RIZ1 mRNA expression showed a decreasing trend with the increase of disease risk grade. RIZ1 mRNA expression level had no relationship with age, gender, peripheral blood white cell count, the hemoglobin, platelet count and karyotype (all P> 0.05). Conclusion RIZ1 mRNA expression level is decreased in MDS patients, and it is different in various subtypes and risk classification. RIZ1 may involve in the pathogenesis of MDS and play an important role in the progression of MDS.

Levels of miRNA－106a in non－small cell lung cancer tissues and its clinical significance / 中国基层医药

Objective To investigate the level and clinical significance of miRNA －106a in non－small cell lung cancer tissues.Methods Paraffin－embedded specimens of non －small cell lung cancer tissues and adjacent tissues from 80 patients with non －small cell lung cancer from January 2012 to December 2013 in Yiwu Central Hospital were selected in this study.The real－time fluorescence quantitative polymerase chain reaction (QRT－PCR) was used to determine the level of miRNA －106a in lung cancer tissues and adjacent tissues.Results The level of miRNA－106a in non－small cell lung cancer tissues (2.42 ±0.23) was higher than that in adjacent tissues (1.00 ± 0.06) (t＝53.433,P＝0.000).The miRNA －106a levels in lung cancer tissues of stage Ⅲ －Ⅳ,lymph node metastasis and recurrence time ＜6 months were high than those of the stage Ⅰ－Ⅱ,no lymph node metastasis and recurrence time≥6 months(t＝7.641,11.115,2.183,P＝0.000,0.000,0.032).The level of miRNA－106a was not associated with age ,gender,pathological type,degree of differentiation and vascular invasion (all P＞0.05).The level of miRNA－106a in non－small cell lung cancer tissues was cut by 2.12.The area under the ROC curve for the diagnosis of non －small cell lung cancer was 0.823(95%CI＝0.820－0.825,P＝0.000),and the sensitivity was 54.37%,the specificity was 89.21%.The cumulative survival rate and progression －free survival rate of patients with low－expression of miRNA －106a in non －small cell lung cancer were higher than those with high expression of miRNA－106a (P＝0.027,0.012).Conclusion The miRNA －106a level is elevated in non －small cell lung cancer tissues.The miRNA－106a may be involved in the development of non －small cell lung cancer and is of great value in the diagnosis and prognosis evaluation of non －small cell lung cancer.

Establishment and evaluation of different serotypes group B streptococcus method by TaqMan fluorescent probe technique / 国际检验医学杂志

Objective To establish a molecular method for the identification of different serotypes of group B streptococcus(GBS)based on TaqMan fluorescence probe technology,and to lay the foundation for the sub-sequent study of multiple fluorescent probe technology to detect different serotypes of GBS.Methods Primers and probes were designed according to the different serotypes of capsular polysaccharide(CPS).CPS se-quences were amplified by real-time fluorescence quantitative polymerase chain reaction.GBS classification methods of different serotypes were established.The results were compared with latex agglutination test and the method was evaluated from the aspects of sensitivity,specificity and detection of clinical isolates.Results The logarithmic concentration of DNA in the same serotype GBS was linearly correlated with the value of Ct. The detection limit of this method is 1 pg/μL,a probe could only detect the corresponding serotype GBS.The results of TaqMan fluorescence probe test of 10 strains were consistent with the results of latex agglutination test.Conclusion TaqMan fluorescence probe technique is a simple,rapid,highly sensitive and specific method for the detection of different GBS serotypes,and it is better than latex agglutination test for the classification of clinical isolates.

Study on CMV-DNA detection in blood and urine / 国际检验医学杂志

Objective To study the detection of cytomegalovirus (CMV)-DNA in different kinds of blood and urine sample .Methods The CMV-DNA loads in 3 different kinds of blood sample from 52 patients and 3 different kinds of urine sample from 85 patients were detected by real-time fluorescence quantitative polymer-ase chain reaction(FQ-PCR) .The differences in CMV-DNA detection rate and virus loads were compared a-mong different kinds of blood and urine samples .Results The CMV-DNA detection rates in serum ,whole blood ,plasma and peripheral blood mononuclear cell (PBMC) from 52 patients were 48 .08％ ,71 .15％ ,57 .69％ and 69 .23％ respectively .The CMV-DNA detection rates of whole blood and PBMC were higher ,the difference was statistically significant (P<0 .05) .The quantitative results of PBMC was higher .The CMV-DNA detec-tion rates of mixed urine ,urine supernatant and urine sediment from 85 patients were 72 .94％ ,62 .35％ and 84 .71％ respectively ,the CMV-DNA detection rate of urine sediment was higher ,while the quantitative re-sults of mixed urine was higher .Conclusion The CMV-DNA detection results of blood and urine have large difference ,therefore using the same kind of sample for conducting detection has an important clinical signifi-cance in the clinical diagnosis ,treatment and monitoring of CM V infection .

Construction of human YPEL5 gene eukaryotic expression vector and its expression in esophageal carcinoma cells / 国际检验医学杂志

Objective To construct human yippee-like 5(YPEL5) gene eukaryotic expression recombinant plasmid and to express in esophageal carcinoma EC9706 cells .Methods The cDNA from human normal tissue was taken as a template and amplified to YPEL5 gene coding sequence with 366 bp in length .Then this se-quence was inserted into the multiple cloning site areas of eukaryotic expression vector pCDH-CD513B for ob-taining the eukaryotic expression vector pCDH-CD513B-Flag-YPEL5 .After the bacterial colony PCR identifi-cation ,it was sent to the corporation for testing the sequence .The successfully constructed recombinant plas-mid was transfected into human esophageal carcinoma EC9706 cells .The expression of PEL5 gene in EC9706 cells was detected by QRT-PCR and Western Blot .Results The YPEL5 gene segment with 366 bp in length was successfully amplified .pCDH-CD513B-Flag-YPEL5 recombinant plasmid was obtained by double enzyme digestion ,connection ,conversion and screening .The gene sequencing identification showed that the inserted gene sequence in recombinant plasmid was consistent with that in the GenBank .After 2 d of transfecting into EC9706 cells ,the QRT-PCR and Western Blot revealed that YPEL5 gene expression was significantly up-reg-ulated .Conclusion The pCDH-CD513B-Flag-YPEL5 eukaryotic expression vector is successfully constructed and is expressed in esophageal squamous cancer cell line EC9706 ,thus which lays a foundation for studying its function in the progression of esophageal cancer .