Analysis study of serum miRNA expression profile in patients with non-obstructive azoospermia / 重庆医学

Objective To analyze the serum miRNA expression levels in non-obstructive azoospermia (NOA) patients and healthy sperm donors.Methods Serum miRNA levels in NOA patients and healthy sperm donors were analyzed by adopting the miRNA expression profiles chip.The data were processed and an alyzed by using the GenePix proV6.0 software to find out the differentially expressed miRNA,then the difference was verified by RQ-PCR,finally the bioinformatic software was utilized to predict the miRNA target gene.Results Compared to healthy sperm donors,71 cases of NOA had miRNAs expression difference,miRNA expression were increased in 47 cases and miRNAs expression was decreased in 24 cases.Moreover,Realtime PCR analysis verified the chip accuracy.The bioinformatic software target gene prediction showed that the potential target gene of these differential miRNA were involved in spermatogenesis.Conclusion The specific miRNA exists in serum miRNA of NOA,which helps to study the molecular mechanism of spermatogenesis.

The effect of the recombinant human resistin on lipid metabolism by AMPK pathway in HepG2 cells / 实用医学杂志

Objective To investigate the effect of rh-resistin on lipid metabolism in HepG2 cells and to elucidate its relation to AMPK pathway. Methods We treated the HepG2 cells with 50 ng/ml rh-resistin and 0.5 mmol/L palmitic acid,used siRNA technique to inhibite α2 subunite expression of AMPK in HepG2 cells and quantitative RT-PCR to detect ACC1,ACC2,and HL mRNA expression levels of related lipid metabolism genes. The P-AMPK-Thr172 of AMPK and P-ACC-Ser79 of ACC were determined by Western blotting. The Lipid accumu-lation in cells was determined by images of Laser Scanning Confocal Microscope after Nile red staining. Results Rh-resistin decreased the AMPKα2,HL mRNA expressions and the phosphorylation level of AMPK and ACC in both basal and insulin-stimulated conditions (P 0.05),while it increased ACC2 mRNA expressions and cytoplasmic lipid droplets in the same conditions (P <0.05). Conclusion Rh-resistin may affect lipid metabolism via AMPK pathway with increase of fatty acid synthe-sis and inhibition of triglyceride catabolism,which leading to lipid accumulation in HepG2 cells.

A novel double heterozygote of HBB c.219T>A;220G>T: gene diagnosis and pedigree analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify a novel hemoglobinopathy applied by direct sequencing and clone sequencing.</p><p><b>METHODS</b>EDTA anticoagulated blood of proband and his parents were analyzed by hematology analyzers and Capillarys hemoglobin electrophoresis (CE). Then thalassemia genetypes were screened by gap-PCR and reverse dot blot (RDB). Proband was suspected with abnormal hemoglobin combine alpha beta compound thalassemia. The mutation of beta-globin was identified by direct sequencing and clone sequencing.</p><p><b>RESULTS</b>Hb analysis showed that probands Hb A2 variant was eluted in Z (C) zone and his father's in Z (A2) zone on CE,and proband's mother elevated HbA2 of 4.6%. Screened by RDB, the proband was CD71-72(+A) homozygote and showed the mismatch with his parents. Through direct sequencing and clone sequencing, we deduced that our proband inherited the mutations of HBB c.[219T>A;220G>T] from his father and inherited the Southeast-Asian deletion and HBB c.216-217insA from his mother.</p><p><b>CONCLUSION</b>A novel double heterozygote of HBB c.[219T>A; 220G>T] was identified in south China. This mutation enriches the beta-thalassemia gene mutation spectrum in Chinese population.</p>

Effect of recombinant human resistin on glucose metabolism by AMPK pathway in HepG2 cells / 实用医学杂志

Objective To investigate the effect of rh-resistin on glucose metabolism in HepG2 cells and to elucidate whether the underlying mechanisms are related to AMPK pathway. Methods Cells transfected with control siRNA or AMPKα2 siRNA were cultured in 6-well plates and then treated with 50 ng/mL rh-resistin for 24 hours , while untransfected cells were treated with or without 50 ng/mL rh-resistin on the same conditions , followed by serum-starving in glucose-free DMEM for 3 ~ 5 hours in the continued absence or presence of rh-re-sistin. Then the cells were treated with or without insulin for 2 hours. AMPKα2, G6Pase, PEPCK and Glut2 mRNA expression levels were determined by quantitative RT-PCR. The phosphorylation state of AMPK was deter-mined by Western blotting. Glycogen synthesis was measured by the incorporation of D-[U-14C] glucose to glycogen. Results Rh-resistin suppressed the AMPKα2, Glut2 mRNA expressions, and reduced the phosphory-lation level of AMPK and glycogen synthesis on both basal and insulin-stimulated conditions (P < 0.05), while it accelerated G6Pase and PEPCK mRNA expressions on the same conditions (P < 0.05). The mRNA expression levels of G6Pase , PEPCK , Glut2 and the phosphorylation level of AMPK and glycogen synthesis were signifi-cantly different between the rh-resistin group and the rh-resistin in conjunction with AMPKα2 siRNA-treated group. Conclusion Rh-resistin may affect glucose metabolism in HepG2 cell via AMPK pathway.

Cloning of activating adenosine monophosphate-activated protein kinase alpha 2 subunit gene and construction of its wild-type and mutant eukaryotic expression vectors / 中国组织工程研究

BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation.TIME AND SETTING: The experiment was performed in the Clinical Molecular Biology Laboratory, the Second Affiliated Hospital of Sun Yet-sen University from April 2007 to January 2008.MATERIALS: QuikChange II Site-Directed Mutagenesis Kit was produced by Stratagene. Eukaryotic expression vector pcDNA3.1(+) and E. coll DH5a were provided by the laboratory. Human skeletal muscle tissue was from patients who received amputation surgery in the Second Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from the patients, and fresh samples were collected and frozen in liquid nitrogen.METHODS: The human AMPKo2 subunit gone was amplified from human skeletal muscle by RT-PCR, cloned into T vector, and the recombinant plasmid was confirmed by sequencing. In vitro site-directed mutagenesis was carded out with Quickchange site-directed mutagenesis kit. The wild-type and mutant coding genes were subcloned into eukaryotic expression vector pcDNA3.1, and the recombinant plasmids were validated by enzyme digestion and sequencing.MAIN OUTCOME MEASURES: ①The cloning of aim gone; ②site-directed mutagenesis; ③ eukaryotic expression plasmid. RESULTS: The human AMPKα2 subunit gene (about 1700 bp) was successfully cloned, with 99％ homology to the reported AMPK α2 gene. A GenBank accession number was EF056019, The achieved mutation of the 45<'th> Lysine (AAA) was found to Arginine(AGA). The wild-type and mutant pcDNA-AMPKα2 recombinant plasmids were constructed successfully. CONCLUSIONS: The human AMPKα2 subunit gene was cloned successfully from human skeletal muscle and its wild-type and mutant eukeryotic expression vectors were constructed successfully in the experiment.

Dengue Type Ⅰ Virus Isolated in Guangzhou Detected by Using TaqMan MGB Real-time PCR / 中华医院感染学杂志

OBJECTIVE To detect serotype of dengue virus in sera from patients with fever.METHODS A pair of degenerated primers and one MGB probe were designed targeting the conserved region at the E gene of dengue type 1 virus.TaqMan MGB real-time PCR assay was developed with plasmid including E gene of dengue type 1 virus as standard sample.The sera of 10 patients with fever were used to extract RNA,and convert into cDNA.Then cDNA were detected by TaqMan MGB real-time PCR assay and the amplified products were analyzed at the same time.RESULTS The sera of 9 patients from 10 samples were observed to generate a fluorescent signal,and about 100 bp fragment was obtained simultaneously.CONCLUSIONS Dengue fever on 2006 in Guangzhou is caused by the dengue type 1 virus.TaqMan MGB real-time PCR assay is rapid and sensitive to detect dengue virus infections.

Detection of West Nile Virus Using SYBR GreenⅠ Fluorescent Quantitative PCR Assay / 中华医院感染学杂志

OBJECTIVE To establish SYBR Green Ⅰ fluorescent quantitative PCR assay for the detection of West Nile virus(WNV),which could be used for early laboratory diagnosis.METHODS A fragment of WNV gene was amplified by PCR,then cloned into pMD-18 T vector.The combinant plasmid was sequenced and analyzed by means of BLAST program,and used as the positive DNA in place of WNV.The SYBR GreenⅠfluorescent quantitative PCR assay was established based on positive plasmid.The sensitivity and specificity of the assay were performed.RESULTS The combinant plasmid was confirmed by sequencing and the fragment belonged to WNV.Ten copies of WNV RNA were detected by SYBR GreenⅠfluorescent quantitative RT-PCR assay.Results of the other members of Flaviviridae were negative,which indicated this assay was specific for WNV.CONCLUSIONS SYBR GreenⅠfluorescent quantitative PCR assay established in this study is highly sensitive and specific,and so it can be used for early diagnosis of WNV infection.

Effects of insulin on osteoblast and its post-receptor mechanism / 中国病理生理杂志

AIM: To study the effects of insulin on the proliferation and function of osteoblasts and the relationship between insulin post-receptor change in osteoblasts and osteoblastic cell growth.METHODS: The effects of different levels of insulin on osteoblasts were assessed by MTT colorimetry.Osteocalcin in medium was measured by RIM.IGF-1 mRNA expression levels were determined by RT-PCR.The concentrations of free IGF-1 protein in serum-free medium were measured by ELISA.In addition,the protein level and phosphorylated protein of P~(44/42)MAPK were determined by Western blotting analysis.RESULTS: Insulin enhanced the proliferation of osteoblasts,depending on its dose and exposure time.Insulin at concentration of 10~(-7) mol/L showed the strongest effect,and the action attained the plateau phase beyond 96 h.The best concentration that stimulated synthesis of osteocalcin by insulin was 10~(-7) mol/L.When the insulin concentration beyond 10~(-7) mol/L,the osteocalcin concentration was decreased.Exposure time had no effect on insulin-stimulated synthesis of osteocalcin of osteoblastic cells.When the concentration of insulin reaches 10~(-6) mol/L,the IGF-1 mRNA expression stimulated by insulin was also decreased.The concentrations of free IGF-1 protein in insulin-stimulated groups were all higher than that in control group(P0.05).Insulin acute stimulation rapidly induced the activity of tyrosine phosphorylation of P~(44/42)MAPK.The degree of tyrosine phosphorylation of P~(44/42)MAPK was increased step by step along with the increasing doses of insulin from 0 to 10~(-7) mol/L(P

Expression of survivin in pancreas in streptozotocin-induced diabetic mice / 中国病理生理杂志

AIM: To investigate the expression of survivin in pancreas in the streptozotocin-induced diabetic mice. METHODS: Low dose of streptozotocin was used to induce diabetes mellitus in BALB/c mice. Body weight and blood glucose concentrations were examined at 1, 2, 3 and 4 weeks after the streptozotocin injection. Expression of survivin mRNA was detected by real-time FQ-PCR. RESULTS: Survivin was expressed in the pancreas of normal BALB/c mice. Low dose of streptozotocin provoked hyperglycaemia with increased survivin expression in the pancreas, but blood glucose concentration and expression of survivin was not significantly changed in control group. CONCLUSION: Survivin is expressed in the pancreas of normal BALB/c mice. Streptozotocin increases survivin expression in the pancreas, which may be related with islets regeneration.