Fluorogenic Quantitative Polymerase Chain Reaction Research Articles

Effects of FAM96B on the proliferation, apoptosis, invasion and metastasis of hepatocellular carcinoma HepG2 cells

Objective To investigate the effects of family with sequence similarity 96 member B (FAM96B) on the proliferation, apoptosis, invasion and metastasis of hepatocellular carcinoma (HCC) HepG2 cells. Methods FAM96B mRNA and protein were examined respectively by real-time fluorogenic quantitative polymerase chain reaction (Real-time PCR) and Western blotting in L02 and HepG2 cells. Moreover, the eukaryotic expression vector of FAM96B was constructed and transfected into HepG2 cells. The cell proliferation of each group at 0, 24, 48, 72 and 96 h was tested by methyl thiazol tetrazolium (MTT). Then the apoptosis, invasion and metastasis were tested respectively by Flow cytometry, wounding healing assay and Transwell. Meanwhile, the subcutaneous tumorigenic model of nude mice were established by 10 male and aged 4-5 weeks BALB/c-nu/nu mice. The model mice were randomized into control group and FAM96B group, 5 mice in each group. And which were injected with 0.3 ml 1×1010 pcDNA3.1-HepG2 cells and pcDNA3.1-FAM96B HepG2 cells. The weight and volume of tumors were measured every three days. All the mice were executed on the 30th day. Statistical analysis was performed using SPSS 21.0. The measurement data were expressed as mean±standard deviation (Mean±SD). The mean comparison between the two groups was performed by t test. Results The FAM96B mRNA expression was decreased in HepG2 cells than in L02 cells (t=25.343, P<0.01). Meanwhile, The FAM96B protein expression was decreased in HepG2 cells than in L02 cells. Compared with control group, the proliferative ability of FAM96B group was significantly reduced (P<0.01). The apoptosis rate in FAM96B group was increased in FAM96B group than in control group [(8.61±1.05)% vs. (47.34±4.82)%, t=23.544, P<0.01]. Compared with control group, the migration ability of FAM96B group was obviously reduced [(222.33±32.78) μm vs. (284.13±46.18) μm, t=4.227, P<0.01], and the invasion ability was obviously reduced [(45.47±4.05) vs. (101.13±5.54), t=31.413, P<0.01]. Moreover, in the subcutaneous tumorigenic model of nude mice, the tumour volume and weight were all decreased in FAM96B group than in control group (tvolume=0.610, tweight=7.186, P all<0.05). Conclusion FAM96B in hepatocellular carcinoma HepG2 cells is low expression, up-regulated its expression levels can inhibit the proliferation, invasion, metastasis and induce apoptosis of HepG2 cells. And up-regulated FAM96B expression levels can inhibit growth of tumors in the subcutaneous tumorigenic model of nude mice. Key words: Family with sequence similarity 96 member B; Hepatocellular carcinoma; HepG2 cells; Proliferation; Apoptosis; Invasion and metastasis

Sa1220 QUANTITATIVE ANALYSIS OF GASTRIC CANCER SUSCEPTIBILITY GENE METHYLATION IN EARLY DIAGNOSIS OF GASTRIC CANCER

To investigate the relationship between methylation of relevant genes and the development of gastric cancer based on quantitative analysis of methylation of gastric cancer-related genes. Real-time fluorogenic quantitative polymerase chain reaction (PCR) was applied to detect the methylation value of MGMT, GSTP1 and gastric cancer -specific gene MINT25. A total of 181 cases were included and divided into three groups, 74 gastric cancer, 58 precancerosis containing polyps, intestinal metaplasia and atypical hyperplasia, and 49 normal gastric mucosa. The methylation rates of the MGMT, GSTP1 and MINT25 in gastric cancer group were 59.46%, 75.67% and 68.92%, respectively, which were significantly higher than that of precancerosis and control groups (P < 0.05). Meanwhile, quantitative analysis of methylation showed there was statistically significant in three groups and between any two groups (P < 0.05).The quantitative levels of each gene methylation were gently increasing from normal mucosa, precancerosis to gastric cancer. Moreover, similar results were also observed in the malignancy, pericarcinomatous (5 cm to lesions) and normal mucosa (P < 0.05). The methylation of MGMT, GSTP1 and gastric cancer -specific gene MINT25 is involved in the carcinogenesis of gastric cancer. Quantitative analysis of methylation of gastric cancer-related genes by using fluorogenic quantitative PCR may be used for diagnosis of early gastric cancer.Tabled 1GenegrouppositivenegativeMethylation positive rate (%)PMethylation (X±S)PNon-Methylation (X±S)PMGMTGastric cancer group443059.46<0.057.32±1.07<0.053.25±1.26<0.05Precancerous lesion group203834.48<0.055.29±0.98<0.055.84±1.41<0.05Normal group84116.33<0.052.83±1.27<0.057.65±1.17<0.05GSTP1Gastric cancer group561875.67<0.057.12±1.32<0.055.27±1.39<0.05Precancerous lesion group283048.28<0.054.78±1.53<0.056.62±1.08<0.05Normal group4458.16<0.052.51±0.56<0.057.93±1.22<0.05MINT25Gastric cancer group512368.92<0.056.83±1.26<0.055.16±1.24<0.05Precancerous lesion group302851.72<0.055.04±1.45<0.056.5±0.84<0.05Normal group3466.12<0.053.14±0.51<0.057.4±1.16<0.05GenetissueMethylation level (%)PMGMTGastric carcinoma67.21<0.05Paracancerous tissue43.12<0.05Normal mucosa28.48<0.05GSTP1Gastric carcinoma72.58<0.05Paracancerous tissue51.72<0.05Normal mucosa18.46<0.05MINT25Gastric carcinoma67.46<0.05Paracancerous tissue48.53<0.05Normal mucosa12.35<0.05 Open table in a new tab

Quantitative assessment of gene promoter methylation in non-small cell lung cancer using methylation-sensitive high-resolution melting.

DNA methylation is closely associated with aberrant epigenetic changes. Previous studies have identified various genes associated with non-small cell lung cancer (NSCLC), but the precise combination responsible for its etiology is still debated. The aim of the present study was to select a new set of NSCLC-related genes using methylation-sensitive high-resolution melting. The promoter methylation status of six selected genes, consisting of protocadherin γ subfamily B, 6 (PCDHGB6), homeobox A9 (HOXA9), O6-methylguanine-DNA methyltransferase (MGMT), microRNA (miR)-126, suppressor of cytokine signaling 3 (SOCS3) and Ras association domain family member 5, also termed NORE1A, was evaluated in 54 NSCLC patients. From these samples, genome-wide DNA was extracted and bisulfite conversion was performed along with fluorogenic quantitative polymerase chain reaction to detect methylation values of the six selected promoters. The present results revealed frequent methylation on PCDHGB6, HOXA9 and miR-126, which contrasted with infrequent methylation on MGMT. The results indicated no methylation on either SOCS3 or NORE1A. The sensitivity and specificity of the methylation assessment were 85.2 and 81.5%, respectively, and the analysis results were validated by pyrosequencing. Furthermore, minute comparison of the association between DNA methylation and clinical features was performed. Overall, these results may provide potential information for the development of better clinical diagnostics and more targeted and effective therapies for NSCLC.

The correlation between SNPs within the gene of adrenergic receptor and neuropeptide Y and risk of cervical vertigo.

The current investigation was aimed to explore the potential associations of SNPs within ADRB2, ADRB1, NPY, and ADRA1A with risk and prognosis of cervical vertigo. Altogether 216 patients with cervical vertigo and 204 healthy controls were gathered, and their DNAs were extracted utilizing the whole-blood DNA extraction kit. Besides, the PCR reactions were conducted using the TaqManR single nucleotide polymorphism (SNP) genotyping assays, and the SNPs were detected on the 7900HT real-time fluorogenic quantitative polymerase chain reaction (PCR) instrument. Finally, the severity of cervical vertigo was classified according to the JOA scoring, and the recovery rate (RR) of cervical vertigo was calculated in light of the formula as: [Formula: see text] RESULTS: The SNPs within ADRA1A [rs1048101 (T>C) and rs3802241 (C>T)], NPY [rs16476 (A>C), rs16148 (T>C), and rs5574 (C>T)], ADRB1 [rs28365031 (A>G)] and ADRB2 [rs2053044 (A>G)] were all significantly associated with regulated risk of cervical vertigo (all P<.05). Haplotypes of ADRA1A [CT and TC] and NPY [CCT and ATT] were also suggested as the susceptible factors of cervical vertigo in comparison with other haplotypes. Furthermore, the SNPs within ADRA1A [rs1048101 (T>C)], NPY [rs16476 (A>C), rs16148 (T>C)], as well as ADRB1 [rs28365031 (A>G)] all appeared to predict the prognosis of cervical vertigo in a relatively accurate way (all P<.05). Ultimately, the haplotypes of ADRA1A (CC) and NPY (CCT) tended to decrease the RR. The SNPs within ADRB2, ADRB1, NPY, and ADRA1A might act as the diagnostic biomarkers and treatment targets for cervical vertigo.

Construction of prokaryotic expression system for IL-8 of Carassius auratus var. Qihe and preparation of polyclonal antibody

PDF HTML阅读 XML下载 导出引用 引用提醒 淇河鲫IL-8原核表达系统的构建及多克隆抗体的制备与鉴定 DOI: 作者: 作者单位: 1. 华南农业大学 兽医学院, 广东 广州 510642;2. 河南师范大学 生命科学学院, 河南 新乡 453007;3. 河南师范大学 水产学院, 河南 新乡 453007;4. 河南省水产动物养殖工程技术研究中心, 河南 新乡 453007 作者简介: 王俊丽(1971-),女,副教授,研究方向为动物营养与免疫.E-mail:wangjunli1971@126.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31372545）；河南省高校科技创新团队支持计划项目（14IRTSTHN013）；河南省科技计划基金项目（142300410159）；河南省教育厅科学技术重点研究项目（14A180007）；河南省自然科学基金项目（162300410165）. Construction of prokaryotic expression system for IL-8 of Carassius auratus var. Qihe and preparation of polyclonal antibody Author: Affiliation: 1. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;2. College of Life Sciences, Henan Normal University, Xinxiang 453007, China;3. College of Fisheries, Henan Normal University, Xinxiang 453007, China;4. Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Xinxiang 453007, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为揭示淇河鲫（）白细胞介素-8（interleukin-8，IL-8）的功能，本研究构建了原核表达系统，并制备了兔抗鲫IL-8多克隆抗体。首先采用RT-PCR法扩增淇河鲫基因编码序列中不含信号肽的基因片段，克隆到pET-32a（+）载体后转化入Rosetta菌株构建原核表达系统。经IPTG诱导表达出相对分子质量约27.8 kD的目标融合蛋白。将纯化的融合蛋白免疫家兔制备多克隆抗体，效价达1：105。经免疫亲和层析纯化的抗体能特异性识别重组和天然的淇河鲫IL-8蛋白。比较不同组织的免疫组化和荧光定量PCR结果，IL-8蛋白量和mRNA的表达量在不同组织间变化趋势一致，在肌肉、脾和头肾均检测到较高的表达量，肠道的表达量较低。本研究为进一步探讨淇河鲫IL-8的生物学功能奠定了基础。 Abstract:Interleukin-8 (IL-8), a chemokine that participates in the early inflammatory process, plays a key role in mediating resistance to infection in animals. However, there has been no information about IL-8 at the protein level for characterizing the regulatory role of this molecule during the immune response in fish. In this work, we constructed a prokaryotic expression system and prepared a polyclonal antibody against IL-8 of ). The coding region without a signal peptide sequence was first amplified and cloned into pET-32a(+), a prokaryotic expression plasmid. The recombinant plasmid was then transformed into Escherichia coli Rosetta. A soluble fusion protein was expressed after induction by isopropyl β-D-1-thiogalactopyranoside. The purified protein was detected as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a predicted molecular mass of 27.8 kD, suggesting that the prokaryotic expression system for IL-8 of crucian carp was successfully constructed. The purified recombinant protein was used as an immunogen to raise polyclonal antibodies in New Zealand rabbits. The serum antibody titer reached 1:105, as detected by indirect enzyme-linked immunosorbent assay. The antibody was purified by affinity chromatography and had a high binding activity and specificity for the recombinant protein antigen, as measured by Western Blot. Immunohistochemistry results showed that the polyclonal antibody can also specifically bind to endogenous IL-8 in crucian carp tissues. Comparing the results of immunohistochemistry with that of fluorogenic quantitative polymerase chain reaction, the expression of IL-8 was consistent between the protein and mRNA levels, and its expression ranked among tissues as follows:muscle > spleen > head kidney > intestine. This study provides a material basis for further research on the role of IL-8 in the immune response of . 参考文献 相似文献 引证文献

Effects of hippocampal stimulus on α₅ subunit of extrasynaptic GABA(A) receptor in kainic acid-induced epileptic rats

To observe the effects of electrical hippocampal stimulation of α₅ subunit of extrasynaptic GABA(A) receptor in kainic acid-induced epileptic rats, explore the optimal therapeutic parameters and elucidate the possible mechanism of electrical stimulation for hippocampal epilepsy. A total of 40 healthy male Sprague Dawley (SD) rats were randomly divided into 5 groups of epilepsy, sham operation, low-frequency stimulation (LFS), high-frequency stimulation (HFS) and normal control (n = 8 each). The expression of α₅ subunit of extrasynaptic GABA(A) receptor as well as the association with the curative effects of LFS were evaluated by behavioristics, real-time fluorogenic quantitative polymerase chain reaction (PCR) and Western blot. The kainic acid kindling rats had class V attack with a modeling success rate of 67%. After treatment, as compared with sham operation and epilepsy groups, the number of seizures decreased significantly in LFS and HFS treatment groups (P < 0.01). But LFS and HFS treatment groups were compared, the number of seizures has no statistical significance (P > 0.05). When sham operation and epilepsy groups were compared, the number of seizures was not statistically significant (P > 0.05). The paired inter-group comparisons showed that LFS and HFS treatment groups and sham operation and epilepsy groups had significant differences in α₅ subunit mRNA and protein (P < 0.01). No statistical significance existed between LFS and HFS treatment groups (P > 0.05) or sham operation and HFS treatment groups (P > 0.05). As compared with normal control group, LFS treatment and HFS groups had significant difference (P < 0.01). Electrical hippocampal stimulation can effectively inhibit seizures. Both LFS and HFS are equally effective, but the former offers greater benefits. And an inhibition of extrasynaptic α₅ subunit plays an important role in seizure during hippocampal stimulation.

Formononetin exhibits anti-hyperglycemic activity in alloxan-induced type 1 diabetic mice.

The aim of this study was to investigate the anti-hyperglycemic activity and mechanism of formononetin in alloxan-induced type 1 diabetic mice by determining its effect on some diabetes-related indices as described below. Body weight, fasting blood glucose, hepatic glycogen, serum insulin, and serum glucagon were determined by electronic scales, glucometer, and ELISA kits. Fas, Caspase-3, pancreatic and duodenal homeobox-1 , insulin receptor substrate 2, glucokinase and glucose transporter 2, mRNA and proteins levels in pancreas tissue, and glucokinase and glucose-6-phosphatase mRNA, and proteins levels in liver tissue were detected by fluorogenic quantitative-polymerase chain reaction and Western blot assays. The results indicated that formononetin (5, 10, and 20 mg/kg; oral administration) reversed the alloxan-induced increase of some indices (fasting blood glucose level and Fas and Caspase-3 mRNA and proteins levels in pancreas tissue) and reduction of some indices (body weight gain, oral glucose tolerance, insulin activity, hepatic glycogen level, pancreatic and duodenal homeobox-1, insulin receptor substrate 2, glucokinase and glucose transporter 2, mRNA and proteins levels in pancreas tissue, and glucokinase mRNA and protein levels in liver tissue). The glucagon level and glucose-6-phosphatase mRNA and protein levels in liver tissue were not affected by the drugs administration. In conclusion, formononetin exhibited anti-hyperglycemic activity in alloxan-induced type 1 diabetic mice by inhibiting islet B cell apoptosis and promoting islet B cell regeneration, insulin secretion, hepatic glycogen synthesis, and hepatic glycolysis.

MicroRNA-126 Overexpression Inhibits Proliferation and Invasion in Osteosarcoma Cells

This study investigated the biological effects of microRNA-126 overexpression in human MG63 osteosarcoma cells. A recombinant plasmid expressing microRNA-126, pcDNA6.2-microRNA-126, was constructed and transfected into MG63 cells. Using real-time fluorogenic quantitative polymerase chain reaction, the microRNA-126 expression was measured in microRNA-126-MG63 group, Ctrl-MG63 group, and blank group. Cell proliferation, cell cycle distribution, cell migration, and invasion were analyzed using methyl thiazolyl tetrazolium assay, flow cytometer, wound-healing assay, and transwell assay, respectively. As expected, microRNA-126 expression was higher in microRNA-126-MG63 group than in Ctrl-MG63 group and blank group (both P < .05). After 48/72 hours of transfection, cell proliferation in microRNA-126-MG63 group was significantly reduced compared to blank group (both P < .05). Compared to blank group, cell population in G0/G1 stage was significantly higher in microRNA-126-MG63 group, accompanied by lower cell numbers in the S and G2/M phases and decreased proliferation index (all P < .05). Wound-healing assay showed a wider scratch width in microRNA-126-MG63 group and reduced cell migration than blank group (both P < .05). Cells overexpressing microRNA-126 exhibited reduced ADAM9 expression levels compared to other 2 groups (all P < .05), suggesting ADAM9 is a target of microRNA-126. Cell proliferation, migration, and invasion rates were reduced in microRNA-126 group after 48/72 hours of transfection, compared with blank group (all P < .05). Cotransfection of pcDNA6.2-microRNA-126 and pMIR-ADAM9 into MG63 cells led to higher cell proliferation, invasion, and migration rates, compared with transfection of pcDNA6.2-microRNA-126 alone (all P < .05). In summary, our data show that microRNA-126 inhibits cell proliferation, migration, and invasion in human osteosarcoma cells by targeting ADAM9.

Relationship between the expressions of indoleamine 2, 3-dioxygenase in hepatocellular carcinoma and clinicopathological parameters

To explore the expressions of indoleamine 2, 3-dioxygenase (IDO) in hepatocellular carcinoma and analyze its relationship with clinicopathological parameters. Quantitative real-time polymerase chain reaction (PCR), fluorogenic quantitative PCR, immunohistochemical and immunofluorescence were used to detect the expression of indoleamine 2, 3-dioxygenase in hepatocellular carcinoma. The IDO mRNA expression in cancerous tissues increased markedly than that in the corresponding non-cancerous tissues (2(-ΔΔCT) = 1.71, P = 0.001) . The immunohistochemical and immunofluorescence results showed that IDO protein was expressed in cytoplasm of hepatocellular carcinoma and tumor-surrounding tissues. But there was no expression in normal liver tissues from benign hepatic lesions and corresponding non-cancerous tissues. An over-expression of IDO protein was detected in 43 patients (48.3%) , a low expression in 25 (28.1%) and no expression in 21 (23.6%). Relationship between IDO expression and clinicopathological parameters: an over-expression of IDO in HCC was associated with recurrence, survival time, metastasis and TNM stage (P < 0.05), but not associated with patient's cirrhosis, AFP level, histological differentiation type, Barcelona clinic liver cancer stage, gender, age, HbsAg positivity, number of tumors and tumor size (P > 0.05). An over-expression of IDO in HCC patients may affect patient prognosis.

Effect of serum containing Jinmaitong Capsule (筋脉通胶囊) on Rats’ Schwann cell apoptosis induced by high glucose concentration

To evaluate the effect of serum containing Jinmaitong Capsule (JMT) on apoptosis of Schwann cells (SCs) that are cultured in high glucose at the cellular and molecular levels. SCs were cultured in Dulbecco's modified Eagle's medium (control group), high glucose (50 mmol/L) medium supplemented with 20% rat serum (HG group), and 50 mmol/L glucose medium supplemented with serum containing JMT (JMT group). SC apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling kit. The expression of Bcl-2 and the caspase-3 p20 subunit in SCs were detected by realtime fluorogenic quantitative polymerase chain reaction and confocal laser scanning microscopy, respectively. No apoptosis was detected in SCs that were cultured in the control group. The percentage of apoptosis of SCs cultured in the HG group was much higher than that in the control group. The apoptosis of SCs in the JMT group was lower than that in the HG group. Fluorescence intensity of Bcl-2 and the expression of Bcl-2 mRNA in SCs that were cultured in the HG group were much lower than those in the control group and much higher than those in the JMT group (P<0.01). The fluorescence intensity of caspase-3 p20 and the expression of caspase-3 p20 mRNA in SCs that were cultured in the HG group were much higher than those in the control group (P<0.01), and they were remarkably lower in the JMT group (P<0.01). JMT effectively prevents SC apoptosis that is induced by high glucose. This effect may be because of increased expression of Bcl-2 mRNA and protein and decreased expression of caspase-3 p20 mRNA and protein.

The effects of low-frequency electric stimulus on hippocampal of Effects of low-frequency electric stimulus on hippocampal of α5 subunit of extra synapse GABAA receptor in kainic acid-induced epilepsy rats

To explore the effects of low-frequency electric stimulus (LFS) on hippocampal of α5 subunit of extrasynaptic GABAA receptor in kainic acid-induced epilepsy rats and explore the possible mechanism of LFS on hippocampus to treat epilepsy. A total of 32 male Sprague-Dawley rats were divided randomly into 4 groups:epileptic, pseudo-surgery, LFS treatment and control. The expression of α5 subunit of extrasynaptic GABAA receptor and the association with the curative effects of LFS were evaluated by behavioristics, real-time fluorogenic quantitative polymerase chain reaction (PCR) and Western blot. After treatment, the number of seizures in LFS group were significantly lower than that in the epileptic and pseudo-surgery groups (2.88 ± 0.83 vs 8.50 ± 0.93 and 8.88 ± 0.83) (P < 0.01). The results of real-time PCR demonstrated that the expression of mRNA of α5 subunits in LFS treatment group was obviously higher than that in epileptic and pseudo-surgery groups (0.74 ± 0.20 vs 0.30 ± 0.16 and 0.31 ± 0.16) (P < 0.01), but was lower than 1.10 ± 0.23 in control group. Western blot demonstrated that the protein expression of α5 subunits in LFS (0.75 ± 0.09) and control (0.88 ± 0.09) groups were dramatically higher than that in epileptic (0.22 ± 0.08) and pseudo-surgery (0.26 ± 0.08) groups (P < 0.01). Low-frequency electric stimulus on hippocampus is effective in controlling epileptic seizures. And the mechanism may be related with the expression level of α5 subunits of extrasynaptic GABAA receptor.

Association of the plasma riboflavin levels and riboflavin transporter (C20orf54) gene statuses in Kazak esophageal squamous cell carcinoma patients

To evaluate the association of the plasma riboflavin level in Kazak esophageal cancer patients and their riboflavin transporter (C20orf54) gene statuses. Plasma riboflavin levels were detected by high performance liquid chromatography in Kazak patients with esophageal squamous cell carcinoma (ESCC) and healthy controls. C20orf54 mRNA and protein expression were analyzed by real-time fluorogenic quantitative polymerase chain reaction and immunohistochemistry in samples from 61 ESCC patients consisting of both tumor and normal tissue, respectively. C20orf54 mRNA expression was decreased in ESCC (0.279 ± 0.102) than in normal counterpart tissue (0.479 ± 0.287; P = 0.049) significantly. Tumors exhibited low C20orf54 protein expression (42.6, 26.2, 18.0 and 13.1% for no C20orf54 staining, weak staining, medium staining and strong staining, respectively), which was significantly lower than that in the normal mucous membrane (13.1, 26.2, 41.0 and 19.7% for no C20orf54 staining, weak staining, medium staining and strong staining, respectively). Defective expression of C20orf54 in tumor cells was significantly associated with poor differentiation. However, other parameters such as depth of invasion and lymph node metastasis had no significant relationship with C20orf54 expression. The average blood concentration of riboflavin was 2.6468 ± 1.3474 ng/ml in ESCC patients lower than control group (4.2960 ± 3.2293 ng/ml, P = 0.015). A positive correlation of plasma riboflavin levels with defective expression of C20orf54 protein was found in ESCC patients (F = 8.626; P = 0.038). Defective expression of C20orf54 is associated with the development of Kazak esophageal squamous cell carcinoma and this may represent a mechanism underlying the decreased plasma riboflavin levels in ESCC.

Decreased blood riboflavin levels are correlated with defective expression ofRFT2gene in gastric cancer

To investigate the relationship between blood riboflavin levels and riboflavin transporter 2 (RFT2) gene expression in gastric carcinoma (GC) development. High-performance liquid chromatography was used to detect blood riboflavin levels in patients with GC. Real-time fluorogenic quantitative polymerase chain reaction and immunohistochemistry were used to analyze the expression of RFT2 mRNA and protein in samples from 60 GC patients consisting of both tumor and normal tissue. A significant decrease in the RFT2 mRNA levels was detected in GC samples compared with those in the normal mucous membrane (0.398 ± 0.149 vs 1.479 ± 0.587; P = 0.040). Tumors exhibited low RFT2 protein expression (75%, 16.7%, 8.3% and 0% for no RFT2 staining, weak staining, medium staining and strong staining, respectively), which was significantly lower than that in the normal mucous membrane (10%, 16.7%, 26.7% and 46.7% for no RFT2 staining, weak staining, medium staining and strong staining, respectively; P < 0.05). Tumors with low RFT2 expression were significantly associated with tumor stage and histological grade. Moreover, a significantly decrease in Uyghur patients was observed compared with Han patients. However, other parameters-gender, tumor location and lymph node metastasis-showed no significant relationship with RFT2 expression. Blood riboflavin levels were reverse correlated with development of GC (1.2000 ± 0.97569 ng/mL in high tumor stage patients vs 2.5980 ± 1.31129 ng/mL in low tumor stage patients; P < 0.05). A positive correlation of plasma riboflavin levels with defective expression of RFT2 protein was found in GC patients (χ² = 2.619; P = 0.019). Defective expression of RFT2 is associated with the development of GC and this may represent a mechanism underlying the decreased plasma riboflavin levels in GC.

Chinese herbal medicine Jianpi Jiedu Formula down-regulates the expression of vascular endothelial growth factor in human gastric cell line MKN45 induced by Helicobacter pylori by inhibiting cyclooxygenase-2

To investigate the effects of Jianpi Jiedu Formula (JPJDF), a compound Chinese herbal medicine, on vascular endothelial growth factor (VEGF) expression induced by Helicobacter pylori (Hp) in human gastric cancer cells, and to explore the possible mechanism. Human gastric MKN45 cancer cells were infected with standard Hp NCTC11637 by coculture, and the cells were divided into 7 groups: normal control group, Hp group, NS398 inhibitor group, blank serum group, and 5%, 10% and 20% JPJDF groups. The expressions of VEGF and cyclooxygenase-2 (COX-2) mRNAs in human gastric cancer cells infected by Hp were evaluated by real-time fluorogenic quantitative polymerase chain reaction (PCR). After inhibiting the expression of COX-2 with COX-2 specific inhibitor NS398 (50 μmol/L), VEGF mRNA expression induced by Hp in human gastric cancer MKN45 cells was evaluated by real-time fluorogenic quantitative PCR. Real-time fluorogenic quantitative PCR results showed that COX-2 mRNA expression increased significantly after MKN45 cells were infected with Hp for 6 h (P<0.01), and VEGF mRNA expression also increased significantly after MKN45 cells were infected with Hp for 12 h as compared with the normal control group (P<0.01). After inhibiting COX-2 expression with COX-2 specific inhibitor NS398, Hp-induced VEGF mRNA expression significantly reduced (P<0.01). 5%, 10% and 20% JPJDF-containing sera all down-regulated VEGF and COX-2 mRNA expressions induced by Hp in a dose-dependent manner as compared with the Hp infection group. Hp could induce the expressions of COX-2 and VEGF in human gastric cancer cells, and JPJDF down-regulates Hp-induced expression of VEGF by inhibiting COX-2, which might be one of the important mechanisms of its prevention and treatment of gastric cancer.

Hepatitis B virus inhibition in mice by lentiviral vector mediated short hairpin RNA

BackgroundChronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication.MethodsHepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively.ResultsCo-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group.ConclusionLentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.

Effects of Yisui Jiedu Recipe on JAK2-STAT5 signal transduction pathway in bone marrow hematopoietic cells from patients with myelodysplastic syndrome-refractory anemia

To investigate the effect of Yisui Jiedu Recipe (YSJDR), a compound traditional Chinese herbal medicine, on cytokines and their corresponding just another kinase 2-signal transducers and activators of transcription 5 (JAK2-STAT5) signal transduction pathway in bone marrow hematopoietic cells from patients with myelodysplastic syndrome-refractory anemia (MDS-RA). Fluorogenic quantitative polymerase chain reaction (FQ-PCR) method was established to detect the levels of JAK2, STAT5 and Bcl-xL mRNA expressions, and JAK2-STAT5 signal transduction pathway was activated by granulocyte-macrophage-colony stimulating factor (GM-CSF) in cultured bone marrow hematopoietic cells from 10 patients with MDS-RA. The levels of interleukin-2 (IL-2), interleukin-3 (IL-3), gamma-interferon (gamma-INF) and tumor necrosis factor-alpha (TNF-alpha) in the cultural supernatant of untreated control, AG490-treated and YSJDF-treated cells were measured by enzyme-linked immunosorbent assay. The levels of IL-2 and TNF-alpha in YSJDR-treated group were significantly lower than those in untreated control group and AG490-treated group (P<0.01, P<0.05), and IL-3 level in YSJDP-treated group was remarkably higher than that in the other two groups (P<0.01). There were no significant differences in the levels of IL-2 and IL-3 between AG490-treated group and untreated control group (P>0.05), while the TNF-alpha level in AG490-treated group was decreased obviously as compared with the untreated control group (P<0.01). There was no significant difference in gamma-INF level between YSJDR-treated group and AG490-treated group (P>0.05), while TNF-alpha level in the two groups were significantly lower than that in the untreated control group (P<0.01). The expressions of JAK2, STAT5 and Bcl-xL mRNAs were significantly down-regulated in the YSJDR-treated and the AG490-treated groups as compared with those in the untreated control group (P<0.05, P<0.01), while there were no differences in the expressions of JAK2, STAT5 and Bcl-xL mRNAs between YSJDR-treated group and AG490-treated group. YSJDR can modulate cytokine level in bone marrow hematopoietic cells of MDS-RA, suppress JAK2-STAT5 signal transduction, and inhibit the Bcl-xL mRNA expression.

Hepatitis B virus is inhibited by RNA interference in cell culture and in mice

Background and aimsFor chronic hepatitis B virus (HBV) infection the effects of current therapies are limited. Recently, RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we studied the effects of HBV-specific 21-bp short hairpin RNAs (shRNAs) targeted to the surface antigen (HBsAg) region and the core antigen (HBcAg) region both in a cell culture system and in a mouse model for HBV replication. MethodsHBsAg and hepatitis B e antigen (HBeAg) in the media of the cells and in the sera of the mice were analyzed by time-resolved immunofluorometric assay, intracellular HBcAg by immunofluorescence assay, HBsAg and HBcAg in the livers of the mice by immunohistochemical assay, HBV DNA by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and HBV mRNA by semi-quantitative reverse transcriptase PCR (RT-PCR). ResultsTransfection with the shRNAs induced an RNAi response. Secreted HBsAg was reduced by >80% in cell culture and >90% in mouse serum, and HBeAg was also significantly inhibited. Immunofluorescence detection of intracellular HBcAg revealed 76% reduction. In the liver tissues by immunohistochemical detection, there were no HBsAg-positive cells and >70% reduction of HBcAg-positive cells for shRNA-1. And for shRNA-2 the detection of HBsAg and HBcAg also revealed substantial reduction. The shRNAs caused a significant inhibition in the levels of viral mRNA relative to the controls. HBV DNA was reduced by >40% for shRNA-1 and >60% for shRNA-2. ConclusionsRNAi is capable of inhibiting HBV replication and expression in vitro and in vivo and thus may constitute a new therapeutic strategy for HBV infection.

Clinical analysis of the infection with human papillomavirus in women

To determine the relationship between human papillomavirus infection, cervical carcinoma, pre-cancerous lesion and condyloma acuminatum. From January 2004 to August 2005, 1086 inpatients in department of dermatology and department of gynaecology and obstetrics in Southwest Hospital and No. 302 Hospital with cervical lesions and condyloma were reviewed. All specimens were detected for HPV-DNA using techniques of Gene Array and fluorogenic quantitative polymerase chain reaction (FQ-PCR). All detections of HPV-DNA were performed in the first admission before the patients underwent any examination or treatment. The positive rates of HPV-DNA detection were 100% in cervical intraepithelial neoplasia (CIN) I, and II and cervical carcinoma. Among these, the main subtype was HPV 16. But some of the patients were found to be positive for more than 2 subtypes of HPV. While the commonest HPV subtype was HPV 18 in endometrial cancer. Some of the patients were detected to be positive for more than 2 subtypes of HPV. In 636 female patient with condyloma acuminatum, the infection rates of HPV6, HPV11 accounted for 44.97% and 29.40%, respectively, HPV 16 and/or HPV 18 infection constituted a small percentage. In a few cases, infection with more than 2 subtypes was detected. Cervical carcinoma including pre-cancerous lesion differs from condyloma acuminatum in dominate infectious subtype of HPV. The former is mainly associated with HPV 16 and HPV 18 infections, and the latter mainly associated with HPV 6 and HPV 11 infections. But in both of the above lesions, a mixed infection with more than 2 types may occur and make the pathological changes and clinical treatment more complicated. The early diagnosis and supervision of HPV infection may be of great value for improvement of prognosis and quality of life.

Association of interleukin-12 p40 gene 3'-untranslated region polymorphism and outcome of HCV infection.

To investigate the effect of interleukin-12 p40 gene (IL12B) 3'-untranslated region polymorphism on the outcome of HCV infection. A total of 133 patients who had been infected with HCV for 12-25 (18.2+/-3.8) years, were enrolled in this study. Liver biochemical tests were performed with an automated analyzer and HCV RNA was detected by fluorogenic quantitative polymerase chain reaction. B-mode ultrasound was used for liver examination. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the detection of IL12B (1188A/C) polymorphism. Self-limited infection was associated with AC genotype (OR = 3.48; P = 0.001) and persistent infection was associated with AA genotype (OR = 0.34; P = 0.014) at site 1188 of IL12B. In patients with persistent HCV infection, no significant differences were found regarding the age, gender, duration of infection and biochemical characteristics (P>0.05). According to B-mode ultrasound imaging and clinical diagnosis, patients with persistent infection were divided into groups based on the severity of infection. No significant differences were found in the frequency of IL-12 genotype (1188A/C) between different groups (P>0.05). The polymorphism of IL12B (1188A/C) appears to have some influence on the outcome of HCV infection.