OBJECTIVE: To set up a prostate cancer cell line in which beta-catenin expression is stably suppressed and to investigate the role of Wnt/beta-catenin signaling pathway in prostate tumorgenesis. METHODS: We select 3 sites in the complete coden sequence region of beta-catenin gene as the RNAi targets, ligated the annealed double pre-DNA strands into the retroviral vectors pSUPER-retro and transfected them into the packaging cells PA317, and then collected supernatant with retrovirus to infect DU145. After selection by puromycin and culture expansion, the stable cell clones were attained. Expression of the 2 target genes of Wnt/beta-catenin signaling pathway cyclinD1 and c-myc, was detected in the beta-catenin RNAi cells by Western blot. The effect of suppressing beta-catenin by RNAi on cell proliferation was quantified by methylthiazoletetrazolium (MTT) assay. RESULTS: Western blotting and RT-PCR showed that the expression level of beta-catenin in the 2 stable cell clones apparently decreased. CyclinD1 and c-myc expression decreased in the beta-catenin RNAi cells. MTT showed that the cell number of beta-catenin expression suppression cell clones decreased significantly (P < 0. 05), suggesting the cell proliferation was prevented. CONCLUSION The beta-catenin gene stable suppression cell line was successfully established.
Cell Line, Tumor ; Genetic Vectors ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; Retroviridae ; genetics ; Signal Transduction ; Wnt Proteins ; biosynthesis ; genetics ; beta Catenin ; biosynthesis ; genetics

OBJECTIVETo construct a phosphatidylinositol 4-kinase beta (PI4K-beta) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant.

METHODSIn line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-beta mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector.

RESULTSA mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.

CONCLUSIONThe improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.

1-Phosphatidylinositol 4-Kinase ; genetics ; Base Sequence ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; genetics ; Polymerase Chain Reaction ; methods ; Protein Engineering ; methods ; Recombinant Proteins ; genetics ; Sequence Deletion

Objective: To establish a method for isolation and purification of fetal membrane derived adherent cells (FMDACs) , and investigate their biological characteristics. Method: FMDACs were isolated with trypsin inducing and cultured in vitro. FMDACs were induced to differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique were used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: FMDACs were successfully isolated and expanded in vitro. They had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion; FMDACs have the possibility of multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs is stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.

OBJECTIVE: To identify the gene causing diffuse palmoplantar keratoderma in a Chinese pedigree. METHODS: Four normal individuals and 3 patients in a diffuse palmoplantar keratoderma family and 10 unrelated control samples were recruited. The hotspot of the mutations of keratin 9 gene was analyzed by polymerase chain reaction and direct sequencing. RESULTS: We found a G485A transition in ke ratin 9 gene, resulting in the substitution of glutamine for arginine at codon 162 in this diffuse palmoplantar keratoderma family. The mutation was not found in the 10 unrelated control samples and 4 normal individuals. CONCLUSION The mutation G485A found in keratin 9 gene is the disease-causing mutation in the diffuse palmoplantar keratoderma family.
Base Sequence ; DNA Mutational Analysis ; Female ; Heterozygote ; Humans ; Keratins ; genetics ; Keratoderma, Palmoplantar, Diffuse ; genetics ; Male ; Molecular Sequence Data ; Mutation ; Pedigree

OBJECTIVETo observe clinical characteristics of herb-induced liver injury (HILI).

METHODSGeneral conditions, medical history, clinical manifestations, biochemical indices, prognosis, and Roussed Uclaf Causality Assessment Method (RUCAM) scores were retrospectively analyzed in 595 inpatients at 302 Military Hospital between January 2009 and January 2014.

RESULTSThere were 423 cases (accounting for 71.1%) were females with multiple onset age ranging 41 to 50 years old. The median time from starting Chinese herbs to the occurrence of liver injury (LI) was 30 days (15-75 days), and 511 cases (85.9%) were classified as hepatocellular injury. Chinese herbs inducing HILI were mainly used for skin disease (102 cases, 17.1%), osteoarticular disease (57 cases, 9.6%), and gastrointestinal disease (49 cases, 8.2%), covering 207 kinds of Chinese patent medicines. Polygonum multiflorum, Psoralea corylifolia, and Corydalis ambigua were often seen in Chinese prescriptions. In RUCAM scoring, 451 HILI patients (accounting for 74.1%) were very possibly associated with Chinese herbs. Liver failure occurred in 47 HILI patients (accounting for 7.9%), cirrhosis in 45 patients (accounting for 7.6%), chronic HILI in 80 patients (accounting for 13.4%), 27 (4.5%) died, and only 2 (0.3%) underwent liver transplantation.

CONCLUSIONSChinese herbs could cause LI or even death. Attention should be paid to herbal hepatotoxicity and improving monitoring system of HILI.

Chemical and Drug Induced Liver Injury ; diagnosis ; Female ; Humans ; Prognosis ; Retrospective Studies

OBJECTIVETo investigate changes in serum insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-binding protein-3 (IGFBP-3) and their significance in children with left-to-right shunt congenital heart disease (CHD) associated with heart failure (HF).

METHODSTwenty healthy children (control group), 20 children with HF, without basic heart disease (HF group), 20 children with left-to-right shunt CHD, without HF (CHD group), and 30 children with left-to-right shunt CHD associated with HF (CHD+HF group) were included in the study. These groups were compared in terms of serum IGF-1 and IGFBP-3 levels. According to the New York Heart Association (NYHA) Functional Classification, the CHD+HF group was further divided into NYHA-II, NYHA-III and NYHA-IV subgroups and the subgroups were compared in terms of serum IGF-1, IGFBP-3, and cardiac troponin I (cTnI) levels. The correlation of serum IGF-1 and IGFBP-3 levels with serum cTnI level in the CHD+HF group was analyzed.

RESULTSThe CHD group showed decreased serum IGF-1 and IGFBP-3 levels compared with the control group (P<0.01). The CHD+HF group showed a significantly decreased serum IGF-1 level compared with the control group (P<0.01) and CHD group (P<0.05). The HF group had significantly increased serum IGF-1 and IGFBP-3 levels compared with other groups (P<0.01). The NYHA-II subgroup had the highest serum IGF-1 level and the NYHA-IV subgroup had the lowest serum IGF-1 level (P<0.01). In the CHD+HF group, serum IGF-1 and IGFBP-3 levels were negatively correlated with serum cTnI level (r=-0.692, P<0.05; r=-0.530, P<0.05).

CONCLUSIONSSerum IGF-1 level can be used as an objective condition evaluation indicator for CHD, and low serum IGF-1 level is a risk factor for HF. This also provides a clinical basis for treatment of HF using exogenous IGF-1.

Child, Preschool ; Female ; Heart Defects, Congenital ; blood ; Heart Failure ; blood ; Humans ; Infant ; Insulin-Like Growth Factor Binding Protein 3 ; blood ; Insulin-Like Growth Factor I ; analysis ; Male ; Troponin I ; blood

Objective　To investigate the protective role of glycyrrhizin on experimental obstructive nephropathy in rats. Methods 　The model of obstructive nephropathy was induced by unilateral uretera l ligation in rats and the animals were sacrificed at 12 h and 3,14,28,56 d resp ectively after operation. Specimens were taken from the cortex of kidney. In re nal stroma, routine morphological examinations and counts of karyocyte were made , the expression of collagen type Ⅰand type Ⅲ were also detected by immunohist ochemical staining,then, semi-quantified by EIG image analysis system. Results　 A progressive fibrosis was observed in renal stroma of the mod el. 12 h after operation, counts of karyocyte increased markedly i n renal stroma in groups of treatment and pretreatment with glycyrrhizin compar ed with that in sham-operation group（P<0.05）,and decreased distinctly compared with that in saline treated group（P<0.01）, but no significant di fference was found between treatment group and pretreatment group（P>0.05） ;3 d after operation, in group treated or pretreated with glycyrrhizin. The e xpression of collagen type Ⅰ increased markedly compared with that in sham-o peration group（P<0.01） in renal stroma, but decreased notably compare d with that in saline treated group（P<0.01）,while, there was not any diffe rence between treatment group and pretreatment group（P>0.05）. The expressi on of collagen type Ⅲ in renal stroma was almost the same as that of coll agen type Ⅰ. Conclusion　Glycyrrhizin has some therapeutical bu t no preventive effect to experimental obstructive nephropathy in rats.

Calcium ; metabolism ; Calcium Signaling ; Epinephrine ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Humans

Decoction is one of the most commonly used dosage forms of traditional Chinese medicine. The stability of chemical constituents in decoction is closely related to the clinical efficacy and safety. There were few reports about the influence of metal ions in the stability of chemical constituents in traditional Chinese medicine. However, there is no evidence that metal ions in decoction water need to be controlled. In this study, 2,3,5,4'-tetrahydroxy stilbene-2-O-β-D-glucoside (THSG), one of the main constituents in Polygoni Multiflori Radix was studied. Ordinary tap water, deionized water, and water containing different metal ions were used to investigate and compare the influence on THSG. The results showed that after storage in a dark place at the room temperature for 10 days, the degradation of THSG was 7% in deionized water, while undetectable in tap water. The content of THSG could be decreased by different kinds of metal ions, and the effect was concentration-dependent. Moreover, Fe3+ and Fe2+ showed the greatest influence at the same concentration; and our study has shown that THSG decreased more than 98% in Fe and Fe2+ solutions at 500 ppm concentration. In the same time we found out p-hydroxybenzaldehyde (molecular weight: 122.036 7) and 2,3,5-trihydroxybenzaldehyde-2-O-glycoside (molecular weight: 316.079 4) were the main degradation products of THSG in tap water and water containing Cu2+, Ca2+, Zn2+, Mg2+ and Al3+. The product of THSG dimer with a water molecule was found in water containing Fe3+ and Fe2+. The above results showed that the metal ions in water could significantly influence the stability of THSG in water, indicating that the clinical efficacy and safety of decoction would be affected if the metal ions in water were not under control. It's suggested that deionized water should be used in the preparation of decoction containing Polygoni Multiflori Radix in the clinic to avoid degradation of THSG. Meanwhile, decoction prepared by tap water should be taken by patients in a short time. Our investigation provides important information and reference about the influence of metal ions on the stability of decoctions in other traditional Chinese medicine that have unstable groups such as hydroxyls and unsaturated bonds, etc.
Drugs, Chinese Herbal ; chemistry ; Glucosides ; chemistry ; Ions ; chemistry ; Metals ; chemistry ; Plant Roots ; chemistry ; Polygonaceae ; chemistry ; Stilbenes ; chemistry

OBJECTIVETo construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.

METHODSThe recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.

RESULTSRestrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.

CONCLUSIONThe minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.

Animals ; COS Cells ; Cercopithecus aethiops ; Dystrophin ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Models, Genetic ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection