HCV RNA positive serum was first selected by RT-PCR test kit from several anti-HCV positive sera obtained from Xi'an.HCV RNA extrac ted from the elected sera was converted to cDNA by reverse transcription with ra ndom primer.Half-nested PCR was performed.The amplified product was 852 bp.The purified PCR product was digested by restriction endonucleases and then ligated to epressio vector pET-22b\++.Its nucleotide sequence was determined by dideoxy chain termination method.A comparison of the sequence with several isolates rep orted previously showed that the sequence belonged to HCV type Ⅱ.

Objective: To study the recombinant epitope antigens of hepatitis C virus (HCV), in order to fulfil the requirements of recombinant immunoblot assay kit. Methods: An expressing vector pBVIL1 for expression of recombinant antigens in a fusion manner with IL-1β was constructed. A series of selected genes from the HCV antigens including the C, NS3, NS4 and NS5 were amplified from HCV gene-containing plasmids using PCR and the expression plasmids for these genes were constructed in pBVIL1, respectively. The activity of the purified recombinant antigens were tested against an identified HCV antibody positive and negative panel with ELISA. Results and Conclusions: All the cloned genes of chosen antigen epitopes were highly expressed in pBVIL1 in E.coli. The activity of the C and NS4 antigens were slightly higher than the RIBA3.0 antigens, while the activity of NS3 was slightly lower than the RIBA3.0 antigen. But the total evaluation for the panel was same as RIBA3.0. That means the cloned antigens were suitable for the use in RIBA test kit.

OBJECTIVETo detect gene mutations of children with glucose-6-phosphorate dehydrogenase (G-6-PD) deficiency and of carriers of G-6-PD deficiency gene with the technique of polymerase chain reaction and denatured gradient gel electrophoresis (PCR-DGGE), and to explore the value of the technique in the diagnosis of G-6-PD deficiency and G-6-PD deficiency gene carrying.

METHODScDNAs were harvested by reverse transcription method after RNAs had been extracted from peripheral blood of 43 children with G-6-PD deficiency and of their family members (36 lineages). Electrophoresis behaviors of the fragment from exons 11-12 of G-6-PD cDNA were detected with the technique of PCR-DGGE. Gene sequencing was then performed for the abnormal electrophoresis bands.

RESULTSAbnormal electrophoresis bands were found in the 1304-1520 fragment of G-6-PD cDNA in 33 out of 36 family lineages. The G-6-PD/6-PGD ratio was below 1.00 in 9 mothers of patients. Three of them had the G-6-PD/6-PGD ratio lower than 0.50. The PCR-DGGE bands were the same in the 3 mothers. Gene sequencing showed double heterozygote in the 3 mothers, but the maternal carriers of G-6-PD deficiency gene who had normal G-6-PD/6-PGD ratio showed mono-heterozygote in gene sequencing. Three mutational sites were found in the 1304-1520 fragment, i.e., C1311TG1376T and G1388A. The electrophoresis behaviors were different among the 3 gene mutational sites.

CONCLUSIONSPCR-DGGE is a sensitive and reliable technique in the screening of gene mutations. It is useful in the diagnosis of G-6-PD deficiency, especially in the diagnosis of female G-6-PD deficiency gene carrying.

Base Sequence ; Electrophoresis, Polyacrylamide Gel ; Female ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

Objective:To observe whether the effect of electroacupuncture (EA) on improving sex hormone disorders and follicle development is by decreasing the expression of anti-Mullerian hormone (AMH) in rats with experimental polycystic ovarian syndrome (PCOS).Methods:Forty rats were randomly divided into four groups,a normal group (NG),a model group (MG),an EA at acupoints group (EAAG),and an EA at non-acupoints group (EANAG),with 10 rats in each group.The rats in the EAAG and EANAG were intervened by EA treatment for consecutive 14 d.Zhongji (CV 3) and Guanyuan (CV 4) were selected as the acupoints in the EAAG,and the tip of the tail and 1 cm up from the tail tip were selected as the non-acupoints in the EANAG.After treatment,the histomorphological changes of the ovary,the levels of aromatase P450 (P450arom),testosterone and estradiol in the ovarian tissues,and the expressions of follicle stimulating hormone (FSH) and AMH were observed.Results:After treatment,compared with the MG and EANAG,the expression of AMH decreased (P＜0.05),the levels of P450arom and estradiol increased significantly,and the level of testosterone decreased significantly (all P＜0.01) in the EAAG.Additionally,several normal follicles were present and the number of cystically dilated follicles decreased in the EAAG.Compared with the MG and EANAG,the EAAG obviously had more follicular granulosa cells.Conclusion:EA can down-regulate the abnormally increased expression of AMH to improve sex hormone disorders and follicle development in PCOS rats.

Objective: To explore the effect of herb-partitioned moxibustion (HPM) on tight junctions (TJs) of intestinal epithelial cells in Crohn disease (CD) mediated by tumor necrosis factor-α (TNF-α)-nuclear factor kappa B (NF-κB)-myosin-light- chain kinase (MLCK) pathway. Methods: Forty-eight male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an HPM group and a mesalazine (MESA) group, with 12 rats in each group. Trinitrobenzene sulfonic acid (TNBS) was administered to establish CD models. When the model was confirmed a success, the HPM group rats were treated with HPM at Tianshu (ST 25) and Qihai (CV 6), while the MESA group rats were given MESA solution by lavage. When the intervention finished, the colonic epithelial tissues were separated, purified and cultured in each group to establish the intestinal epithelial barrier model in vitro, and TNF-α was added (100 ng/mL) in the culture medium and maintained for 24 h to establish an increased epithelial permeability model. Transepithelial electrical resistance (TEER) was used to examine the permeability of the barrier; Western blot was used to observe the expressions of the proteins related to TJs of intestinal epithelial cells mediated by TNF-α-NF-κB-MLCK pathway; immunofluorescence staining was used to observe the expressions and distributions of tight junction proteins in the intestinal epithelium. Results: After TNF-α induction, compared with the MC+TNF-α group, the TEER value increased significantly in the HPM+TNF-α and MESA+TNF-α groups (both P<0.001); the expressions of nuclear factor kappa B (NF-κB) p65, MLCK, myosin light chain (MLC), tumor necrosis factor receptor-associated factor 6 (TRAF6) and receptor interaction protein-1 (RIP1) decreased significantly (P<0.01 or P<0.05), and the expression of zinc finger protein A20 (A20) increased significantly (P<0.01); the expressions of occludin, claudin-1, zonula occludens protein 1 (ZO-1) and F-actin also increased significantly (all P<0.01). Compared with the MESA+TNF-α group, the expressions of MLC, occludin, claudin-1, ZO-1 and F-actin increased significantly in the HPM+TNF-α group (P<0.01 or P<0.05). Conclusion: HPM can protect or repair the damage of intestinal epithelial barrier in CD rats, which may be achieved through modulating the abnormal TJs in intestinal epithelium mediated by TNF-α-NF-κB-MLCK pathway.

OBJECTIVETo detect the differential display code 3 mRNA (DD3 mRNA) in the urine sample of patients with prostate cancer and to evaluate its clinical significance.

METHODSDD3 mRNA in the urine collected from 48 patients with prostate cancer, 23 patients with benign prostate hyperplasia (BPH) and 9 healthy male volunteers was measured by reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSDD3 products could not be detected in the urine samples of the BPH patients and healthy male volunteers, but could in 39/48 urine samples of the patients with prostate cancer. Significant difference was found between them (P < 0.01).

CONCLUSIONThe detection of DD3 mRNA in the urine promises to be a non-invasive, simple and sensitive method for the early diagnosis and post-treatment monitoring of prostate cancer.

Aged ; Aged, 80 and over ; Antigens, Neoplasm ; genetics ; urine ; Humans ; Male ; Middle Aged ; Prostatic Neoplasms ; diagnosis ; RNA, Messenger ; urine ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVESTo introduce the method to monitor intrapelvic perfusion pressure during minimally invasive percutaneous nephrolithotomy (MPCNL), and to observe the effect of high pressure intrapelvic perfusion on recovery.

METHODSThe end of F5 ureteral catheter and aseptic transducer were connected by self-made connecter. During the operation, 46 renal calculi cases were monitored, and the early complication, such as fever, pain index, drop of hemoglobin, the stone-free rate and hospital stay were investigated. And the relationship between the variation of pressure and recovery was studied.

RESULTSIntrapelvic perfusion pressure ranged from 3 mm Hg (1 mm Hg = 0.133 kPa) to 50 mm Hg during the course of MPCNL. The definition of high pressure was the time of the pressure more than 30 mm Hg not less than 10 min. Postoperative fever rate, pain index, drop of hemoglobin and hospital stay in the high pressure were significantly higher than low pressure group (P< or =0.05). There was no difference of the stone-free rate in two groups.

CONCLUSIONSDuring the course of MPCNL intrapelvic perfusion pressure should be monitor immediately. It should be careful to maintain the time of pressure more than 30 mm Hg less than 10 min for stable postoperative recovery.

Adolescent ; Adult ; Female ; Humans ; Kidney Calculi ; physiopathology ; surgery ; Kidney Pelvis ; physiopathology ; Male ; Middle Aged ; Monitoring, Intraoperative ; Nephrostomy, Percutaneous ; methods ; Postoperative Complications ; physiopathology ; prevention & control ; Pressure ; Recovery of Function ; Retrospective Studies ; Young Adult

OBJECTIVETo detect humoral immune response against different function regions of hepatitis C virus (HCV) in chronic patients, and further to investigate the correlativity between anti-HCV antibody titers and HCV RNA concentration.

METHODSUsing recombinant dominate epitope antigens, e.g. HCV Core, NS3, NS4, NS5 and chimeric HVR1, a set of ELISA test reagents was formulated. Then, titers of antibodies against HCV different regions and the RNA concentration of HCV in chronic patient sera were detected by ELISA and quantitative RT-PCR technique, respectively.

RESULTSGreat differences have been noted in antibody titers and positive rate of different HCV function regions in chronic patients. Antibodies against HCV Core and HVR1 have the highest positive rate, then NS3, NS4, and NS5 in sequence.

CONCLUSIONThe titer of antibodies against different regions of HCV in chronic patients has good correlation with HCV RNA concentration.

Hepatitis C Antibodies ; blood ; Hepatitis C, Chronic ; immunology ; virology ; Humans ; RNA, Viral ; blood

OBJECTIVETo preliminarily study the effect of prepubertal exposure of male SD (Sprague-Dawley) rats to diethylstilbestrol (DES) on the apoptosis of spermatogenic cells after sexual maturation and its mechanism.

METHODSThirty 21-day-old male SD rats were randomly divided into 4 experimental groups, DES 0.01, 0.1, 1.0 and 10.0 microg/(kg x d) and 1 control group. The experimental groups were injected (s.c.) with different doses of DES (dissolved in corn oil) during prepuberty [from postnatal day (PND) 22 to PND 35] and the control group with medium only. The apoptosis and related proteins Bcl-2 and Bax expressions of testicular spermatogenic cells were studied with TUNEL and immunohistochemistry after the rats sexual maturation (at PND 64).

RESULTSCompared with the control group, the apoptosis of testicular spermatogenic cells in the DES 0.01 microg/kg group had no difference, but significantly increased in the DES 0.1, 1.0 and 10.0 microg/kg groups and the apoptosis increased with the increase of DES dose. In the control and DES 0.01 microg/kg groups, Bax protein expressed weakly but Bcl-2 protein strongly in spermatogenic cells. With the increase of DES exposure, Bax protein expression in spermatogenic cells increased but Bcl-2 protein expression decreased.

CONCLUSIONPrepubertal exposure of SD rats to inappropriate dose of DES can make the apoptosis of spermatogenic cells increase after sexual maturation. Bax and Bcl-2 proteins participate in the apoptotic course caused by prepubertal DES exposure.

Animals ; Apoptosis ; drug effects ; Diethylstilbestrol ; toxicity ; Dose-Response Relationship, Drug ; Male ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sexual Maturation ; Spermatids ; drug effects ; metabolism ; bcl-2-Associated X Protein ; biosynthesis

OBJECTIVETo investigate the effects of prepubertal exposure to diethylstilbestrol (DES) on the testicular development and function of Sprague-Dawley (SD) rats.

METHODSNinety 21-day-old male SD rats were randomly and equally divided into 4 experimental groups (Da, Db, Dc and Dd), which were injected with DES dissolved in corn oil at the dose of 0.01, 0.1, 1.0 and 10.0 microg/(kg x d) from postnatal day (PND) 22 to 35, and a control group (C), which received vehicle only. The testicular development of all the rats was observed, and their testes were harvested in the stages of late puberty (PND 50), sexual maturity (PND 64) and adulthood (PND 130) respectively to determine the weight and histological features of the testis and examine the quality of the sperm in the epididymal cauda of the PND 130 rats.

RESULTSThe testis descent in the C, Da, Db, Dc and Dd groups occurred on PND 26.17 +/- 1.94, 26.83 +/- 1.47, 28.68 +/- 1.03, 33.50 +/- 1.87 and 41.50 +/- 2.74 respectively, significantly delayed in the Db, Dc and Dd groups compared with the C group (P < 0.05 or P < 0.01). On PND 50, the unilateral testis weights in the C, Da, Db, Dc and Dd groups were (1.38 +/- 0.01) g, (1.38 +/- 0.12) g, (1.30 +/- 0.14) g, (0.86 +/- 0.18) g and (0.73 +/- 0.27) g respectively, significantly less in the Dc and Dd groups than in the C group (P < 0.01). Compared with the C group, there was a slight decrease in the number of the cells in the epithelia of a few seminiferous tubules in the Db group on PND 50, maldevelopment of seminiferous tubules, reduced cell number in seminiferous epithelia, blocked spermatogenesis and aplasia of Leydig cells in the Dc and Dd groups in a dose-dependent manner. On PND 64, the unilateral testis weights in the C, Da, Db, Dc and Dd groups were (1.60 +/- 0. 06) g, (1.62 +/- 0.11) g, (1.58 +/- 0.08) g, (1.47 +/- 0.10) g and (0.99 +/- 0.37) g respectively, significantly less in the Dc and Dd groups than in the C group (P < 0.05 or P < 0.01), and the histological alteration of the testis in the Dc and Dd groups was similar to or less than that on PND 50. On PND 130, no statistic difference was observed either in unilateral testis weight or in the histological features of the testis between any experimental group and the control (P > 0.05). The sperm concentration in the epididymal cauda in the C, Da, Db, Dc and Dd groups were (73.00 +/- 16.90) x 10(6)/ml, (68.00 +/- 19.67) x 10(6)/ml, (68.67 +/- 12.15) x 10(6)/ml, (35.17 +/- 15.64) x 10(6)/ml and (19.13 +/- 5.17) x 10(6)/ml, significantly lower in the Dc and Dd groups than in the C group (P < 0.01). There was a significant decrease in sperm motility in the Dd group (P < 0.01), the percentage of grade a sperm in the Db, Dc and Dd groups (P < 0.05) and the percentage of grade b sperm in the Dd group (P < 0.01).

CONCLUSIONPrepubertal exposure to low dose of DES (0.01 microg/[kg x d] x 14 d) does not significantly affect the testicular development and function of SD rats, while high dose (1.0-10.0 microg/[kg x d] x 14 d) has significant short- (PND 50 and 64) or long-term (PND 130) toxic effect, which increases with dose and decreases with age. The mechanism of the toxic effect involves the insults to the development and function of Leydig and Sertoli cells.

Animals ; Carcinogens ; toxicity ; Diethylstilbestrol ; toxicity ; Dose-Response Relationship, Drug ; Male ; Organ Size ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sexual Maturation ; drug effects ; Testis ; drug effects ; growth & development ; physiology ; Time Factors