Cell Transplantation ; adverse effects ; Graft Rejection ; prevention & control ; Hepatocytes ; transplantation ; Humans ; Liver Failure ; surgery

Objective To create Mucin gene 1 (MUC1) antisense peptide nucleic acid (PNA),and to observe its effects on MKN-45 cell invasion and explore the mechanism. Methods The sequence of antisense PNA was designed according to MUC1 gene sequence and transfected into human gastric cancer cells (MKN-45) by liposome,and the empty vector group (randomized control group)and blank control group (negative control group) were involved. The expression of MUC1 was detected by real time quantitative PCR and the changes of E-cadherin expression were also observed.The effects on gastric cancer cell invasion were tested with transwell chamber assays.Results The expression of MUC1 gene was effectively suppressed by the 3 created antisense PNA,and their expression level (0.62±0.18,0.49±0.12 and 0.60±0.21) was significantly lower than that of negative control group (1.18 ± 0.03,P ＜ 0.01). There was no significant difference between radomized control group and negative control group (1.00±0.04,P=0.657).After MUC1 PNA transfected,the capability of gastric cancer cell invasion decreased significantly (P=0.005).And the expression of E-cadherin at mRNA and protein level was up-regulated.Conclusions There is negative correlation between MUC1 and E-cadherin expression in gastric cancer cell MKN-45.The capability of tumor cell invasion is significantly inhibited by suppressing MUC1 gene expression.

Objective To investigate the expression of microRNA (miRNA)-10a in the intestinal mucosa,serum and peripheral blood mononuclear cell (PBMC) of patients with inflammatory bowel disease (IBD) and explore its role and relevance in the pathogenesis of the disease.Methods The intestinal or colonic mucosal biopsy specimens of nine active ulcerative colitis (UC) patients,11 active Crohn's disease (CD) patients and eight patients with negative colonoscopy result as control were collected.The sera of 12 active UC patients,13 active CD patients and nine healthy controls were collected.The PBMC of nine active UC patients,11 active CD patients and eight healthy controls were collected.The expression of miRNA-10a in the intestinal mucosa,sera and PBMC and the expression of IL-12/IL-23 p40 in the intestinal mucosa were detected by real-time polymerase chain reaction (PCR).Each 8 cases of active UC and CD patients were collected.The intestinal mucosa before infliximab (IFX) treatment and six weeks after three times of IFX treatment were collected.And at same time,the intestinal mucosa of 11 active UC patients and 10 active CD patients were collected and cultured for 18 hours stimulated with IFX in vitro and then the expression of miRNA-10a in the intestinal mucosa was tested.One-way analysis of variance was used for comparison in three samples.Paired t-test was used for two samples comparison.Spearman test was used for correlation analysis.Results Compared with healthy controls,the expression of miRNA-10a in the intestinal mucosa,serum and PBMC of UC and CD patients significantly decreased (F=38.45,30.46 and 14.74,all P＜0.05).There was no statistic significance between UC and CD groups.The expression of IL-12/IL-23 p40 in the intestinal mucosa of UC and CD patients significantly increased (F=32.90,P＜0.05).The expression of IL-12/IL-23 p40 was negatively correlated with the expression of miRNA-10a in the intestinal mucosa of CD patients.After three times of IFX treatment,the expression of miR-10a in the intestinal mucosa of IBD patients significantly increased (t=3.341,3.382,both P＜0.05).After stimulated with IFX in vitro,the expression of miRNA-10a in the intestinal mucosa significantly increased (t=3.095,7.193,both P＜0.05).Conclusions miRNA-10a was closely correlated with the inflammation of IBD patients and with the role of targeting IL-12/IL-23 p40.miRNA-10a might be a new target for the IBD treatment.

Emerging evidence has demonstrated that genomes are organized into higher-order structures in vivo and long range interactions between genomic regions largely contribute to the regulation of gene expression. Hematopoiesis, orchestrated by the precise spatial regulation and organization of hematopoietic transcription factors, serves as a good model for exploring these issues. The chromosome conformation capture (3C) methodology and its high throughput based technology provide an innovative solution for analyzing the regulation of functional elements through inter-chromosomal and intra-chromosomal interactions, and contacts of functional components in nuclei, thus leading to a more comprehensive understanding of human genome and gene expression. This review focuses on the recent progress of 3C and its derivatives, and their applications in unraveling the mechanisms of transcriptional regulation in hematopoiesis.
Chromosomes ; Gene Expression Regulation ; Genetic Techniques ; Genomics ; Hematopoiesis ; genetics ; Humans ; Nucleic Acid Conformation

OBJECTIVETo design and synthesize ribozymes targeting 138 and 218 sites of the mRNA nucleotide of mouse caspase-12, a key intermedium of ER stress mediated apoptosis, and to identify their activities through in vitro transcription and cleavage.

METHODSThe mouse caspase-12 gene fragment was obtained by RT-PCR and cloned into the PGEM-T vector under the control of T7 RNA polymerase promoter. The transcription product of the target was labeled with a-32P UTP, while ribozymes were not labeled. Ribozyme and target RNA were incubated for 90 min at 37 degree C in a reaction buffer to perform the cleavage reaction.

RESULTSIt was found that under a condition of 37 degree C, pH 7.5 and with Mg2+ in a concentration of 10 mmol/L, Rz138 and Rz218 both cleaved targets at predicted sites, and the cleavage efficiency of Rz138 was 100%.

CONCLUSIONRz138 and Rz218 prepared in vitro possess the perfect specific catalytic cleavage activity. Rz138 has excellent cleavage efficiency. It may be a promising tool to prevent ER stress induced apoptosis through catalytic cleavage of caspase-12 mRNA in vivo. It also can be used to verify whether caspase-12 is necessary in ER stress induced apoptosis.

Animals ; Base Sequence ; Caspase 12 ; genetics ; Endoplasmic Reticulum ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Oxidative Stress ; genetics ; RNA, Catalytic ; chemistry ; genetics ; RNA, Messenger ; genetics

OBJECTIVETo design hammerhead ribozymes against mouse caspase-7 and to study their expression and cleavage activity in vitro.

METHODSThe secondary structures of ribozyme and caspase-7 genes were analyzed and simulated by computer. Ribozymes DNA sequences were synthesized by automatic synthetic apparatus. Caspase-7 DNA sequence was acquired by reverse transcription PCR. Ribozymes and caspase-7 DNA sequences were separately cloned into pBSKneo U6 and pGEM-T vectors. Ribozymes and caspase-7 mRNA were obtained by transcription in vitro, and ribozymes cleavage activity was identified by cleavage experiment in vitro.

RESULTSTwo ribozymes named Rz333 and Rz394 targeting 333 and 394 sites in caspase-7 mRNA were designed by computer software, and their DNA sequences were synthesized. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiments showed that Rz333 cleaved caspase-7 mRNA and produced 243nt and 744nt segments. The cleavage efficiency is 67.98%, while Rz394 cannot cleave caspase-7 mRNA.

CONCLUSIONSRz333 can site-specifically cleave caspase-7 mRNA.

Animals ; Base Sequence ; Caspase 7 ; Caspase Inhibitors ; Caspases ; genetics ; Cloning, Molecular ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; RNA, Catalytic ; biosynthesis ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

This study was aimed to investigate the effect of GATA-2 over-expression on function of mouse fetal liver hematopoietic stem cells. GATA-2 was introduced into mouse fetal liver cells via retrovirus mediated transduction with GFP as a detecting marker. Flow cytometry, colony-forming assay and cell cycle assay were used to detect the biologic changes of these retrovirus infected mouse fetal liver hematopoietic stem cells. The results showed that GATA-2 over-expression increased the Lin(-)Sca1(+)C-Kit(+) (LSK) population dramatically. Cell cycle of LSK cells didn't show abnormal, while colony forming ability decreased significantly. These data indicated that GATA-2 over-expression inhibited definitive differentiation of mouse fetal liver hematopoietic stem cells. It is concluded that over-expression of GATA-2 can significantly raise the LSK cell proportion in mouse fetal liver and inhibit the differentiation capability, the underlying mechanisms may be related to up-regulation of Hes-1, which may lead to the blocking of cell differentiation at the stem/progenitor cell stage.
Animals ; Cell Differentiation ; Cells, Cultured ; Female ; GATA2 Transcription Factor ; genetics ; Hematopoietic Stem Cells ; cytology ; Liver ; cytology ; Male ; Mice ; Mice, Inbred C57BL

Acute Disease ; Female ; Hepatitis A ; complications ; Humans ; Jaundice ; complications ; Middle Aged ; Myelinolysis, Central Pontine ; complications ; virology

OBJECTIVETo screen the mutations of RET proto-oncogene in sporadic patients with pheochromocytoma.

METHODSForty-two cases of sporadic pheochromocytoma were tested for mutations of RET gene. Of these 42 DNA samples, 12 were extracted from peripheral blood cells and 30 from paraffin-embedded pheochromocytoma specimens. The PCR product of exon 10 and exon 11 was used to molecular analysis of the RET proto-oncogene.

RESULTSAmong 42 patients, 2 were found to have RET gene mutations. One of mutations located at codon 634 (TGC>TAC) in exon 11 of RET proto-oncogene. Another one located at codon 632 (GAG>AAG).

CONCLUSIONSome patients with apparently sporadic pheochromacytoma were carrier of mutations, a routine genetic analysis for mutations of RET gene is indicated for these patients.

Adrenal Gland Neoplasms ; diagnosis ; genetics ; Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; DNA Mutational Analysis ; Female ; Genetic Predisposition to Disease ; genetics ; Genetic Testing ; Humans ; Male ; Middle Aged ; Mutation ; Pheochromocytoma ; diagnosis ; genetics ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-ret ; genetics

TRESK is the most recently reported two-pore domain K+ channel, and different from other two-pore domain channels in gene, molecular structure, electrophysiological and pharmacological properties. Although the current knowledge of this potassium channel is inadequate, researches have demonstrated that TRESK is remarkablely linked to acute and chronic pain by activation of calcineurin. The fact that TRESK is sensitive to volatile anesthetics and localization in central nerve system implies that TRESK may play a very important role in the mechanism mediating general anesthesia. The further research of TRESK may contribute to explore the underlying mechanism of some pathological conditions and yield novel treatments for some diseases.
Amino Acid Sequence ; physiology ; Anesthetics, General ; pharmacology ; Animals ; Calcineurin ; metabolism ; Cell Membrane ; drug effects ; metabolism ; Central Nervous System ; drug effects ; metabolism ; Humans ; Neurons ; drug effects ; metabolism ; Pain ; drug therapy ; metabolism ; physiopathology ; Peripheral Nervous System ; drug effects ; metabolism ; Potassium Channels ; chemistry ; drug effects ; physiology