Colonic Neoplasms ; genetics ; pathology ; CpG Islands ; genetics ; DNA Methylation ; Humans ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; methods

Eukaryotic expression plasmid pCI-neo-mdr1 which contains human multidrug resistance gene 1(mdr1),was constructed and transferred into human hepatocarcinoma HepG2 cells by use of liposome. G418 was used to screen the cells successfully with mdr1 and the selected cells was named HepG2/mdr1 Morphological and biological properties of HepG2/mdr1 cells were observed. The results show that the constructed HepG2/mdr1 cell line was high efficient and stationary in the expression of mdr1. The work was valuable and desirable for the establishment of multidrug resistant cell models,and for the study of MDR in human hepatoma. Furthermore,the work also provided a perfect model for the research of relationship between insulin resistance and MDR in hepatocarcinoma cells.

Adult ; African Continental Ancestry Group ; ethnology ; C-Reactive Protein ; metabolism ; Calcinosis ; complications ; Coronary Disease ; complications ; Female ; HIV Infections ; complications ; pathology ; physiopathology ; HIV-1 ; Heart Diseases ; pathology ; physiopathology ; Humans ; Lipid Metabolism ; Male ; Middle Aged ; Myocardium ; pathology

OBJECTIVETo determine the characteristics of GR6 gene and putative GR6 protein, and to evaluate the expression of GR6 gene in colorectal cancer and normal mucosa.

METHODSBioinformatic software and databases were applied to analyze the characteristics of GR6 gene and putative GR6 protein. Semi-quantitative RT-PCR was used to detect the expression of GR6 gene in colorectal carcinoma, adenoma and normal mucosa.

RESULTGR6 gene, encoding putative GR6 protein, consisted of 3 exons and contained 4 CpG islands by sequence analysis. It was predicted that putative GR6 protein included one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, and three N-myristoylation sites. PSORT II software analysis predicted that putative GR6 protein was located in nucleus (reliability: 76.7%). At the level of mRNA, the expression of GR6 gene was high in normal mucosa, moderate in mucosa adjacent to cancer and adenoma tissue, low in colorectal carcinoma tissue. Significant differences were demonstrated between normal mucosa and adenoma (P<0.05), normal mucosa and carcinoma (P<0.01).

CONCLUSIONThe putative GR6 protein, encoded by GR6 gene may predictably function as an important nuclear signal transduction molecule. Decreased expression of GR6 gene may play an important role in the initiation and promotion of colorectal neoplasia.

Amino Acid Sequence ; Base Sequence ; Blood Proteins ; chemistry ; genetics ; Colorectal Neoplasms ; genetics ; Computational Biology ; CpG Islands ; DNA Methylation ; Fetal Proteins ; chemistry ; genetics ; Humans ; Molecular Sequence Data ; Oncogene Proteins ; chemistry ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo screen the differentially methylated DNA sequences between mucosa adjacent to colorectal cancer (MACC) and normal colonic mucosa.

METHODSThe methylated DNA sequences were enriched by methylation CpG islands amplification (MCA), and the differentially methylated DNA sequences between MACC and normal colonic mucosa were isolated by representational difference analysis (RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with BLAST program system in GenBank. With the separated fragment 1A12 as probe, dot blot was used to study its distribution between RDA products (No. 1-4 rounds), MACC (tester) and normal colonic mucosa(driver).

RESULTSTwenty-five differentially methylated DNA sequences were obtained. Preliminary studies indicated that 1A01 fragment was concerned with two different genes (LOC256866 and CECR7), it was located in the first exon of CECR7. 1A12 fragment was located in 5 flanking region of GR6 gene. By dot blot with 1A12 probe, hybridized signals were detected in MCA product of MACC and RDA products of No. 1-4 rounds, respectively. No signal was detected in MCA product of normal colonic mucosa.

CONCLUSIONThe differentially methylated DNA sequences can be isolated effectively between two different tissues with MCA coupled with RDA. Different methylated DNA fragments exist between MACC and normal colonic mucosa and these fragments may be concerned with colorectal cancer.

Adult ; Base Sequence ; Colorectal Neoplasms ; diagnosis ; genetics ; CpG Islands ; genetics ; DNA Methylation ; DNA, Neoplasm ; chemistry ; isolation & purification ; metabolism ; Genetic Testing ; Humans ; Intestinal Mucosa ; metabolism ; Polymerase Chain Reaction ; Sequence Analysis, DNA

AIM:To study the telomere maintenance mechanism in mesenchymal stem calls(MSCs).METHODS:MSCs were isolated from healthy human bone marrow by their adherence to plastic and then were checked with CD14-FITC,CD45-FITC,CD44-FITC,HLA-DR-FITC,CD34-PE,CD29-PE and CD166-PE.Telomere length and ECTR DNA in MSCs were detected by Southern blotting.The localization of TRF1 and promyelocytic leukemia(PML)in MSCs were detected with immunofluorescence staining.TRAP protocol was performed to detect the telomerase activity in MSCs and MSCs-derived adipocytes.Western blotting and TRAP protocol were applied to measure telomerase activity of MSCs,which were synchronized by serum starvation and aphidicolin treatment.RESULTS:The telomere in length seemed shorter and relatively more homogeneous in MSCs and HeLa cells than that in WI-38-2RA cells.TRF1 did not concide with PML nuclear body in MSCs and HeLa cells while it exclusively did in WI-38-2RA cells.ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38-2RA cells.Telomerase was negative in MSCs but it was positive in MSCs-derived adipocytes detected by TRAP.Moreover,a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and aphidicolin treatment.Untreated MSCs expressed very low level of telomerase probed by Western blotting with 2C4 mAb,but the telomerase level had significantly increased when these cells were trapped in S phase.CONCLUSION:The telomere of MSCs is maintained by telomerase pathway instead of alternative lengthing of telomere(ALT)and the level of telomerase expression is associated with cell cycle stage.[

Objective To repair segmental mandibular defects with autogenous bone marrow stromal cells(BMSCs)and?-triealcium phosphate.Methods Isolated BMSCs were in vitro expand- ed.A 3 cm-long segmental mandibular defect was created at right mandible in 12 canines,of which de- fects in six canines were repaired with BMSCs and?-tricalcium phosphate(?-TCP)and that in other six cases repaired with?-TCP,which was used as control.The engineered bone was evaluated by X-ray, CT,DXA,gross and histological examination,immunohistochemistry and biomechanical test 4,12,26,32 weeks after operation respectively.Results In induced BMSCs,histochemistry showed AKP activity. Oral X-ray showed obvious callus formation 4-26 weeks after operation in experimental group but minimal bone formation in control group.At 32 weeks after operation,gross observation,X-ray and CT demonstra- ted well bony-union in experimental group but bony-nonunion in control group.DXA indicated that the bone density of experimental group was significantly higher than that of control group.Biomechanical test revealed no statistical difference upon mechanical strength of mandibula between experimental group and normal group.Conclusions Canine segmental mandibular defects can be well repaired with the tissue- engineered bone generated by autogenous osteogenic BMSCs and?-TCP scaffold.

AIMThe purpose of this study is to compare the effects of the two clutches on recording the condylar movement.

METHODOLOGYTen subjects (6 women, 4 men; mean age 25.4 years) participated in the study. The mandibular movement, sagittal condylar inclination angle, and transversal condylar inclination angle of each subject were recorded with the CADIAX using the two clutches, respectively. The characteristics of the tracings of the protrusion, opening, and mediotrusion were analyzed with the t-test statistics at a = 0.05 level. The Kappa values were calculated for an assessment of the congruence of the tracings.

RESULTSThe results showed that the contour, direction, and dimension of the tracings in the two clutches were approximately same, but the tracings determined by the functional occlusal clutch were more regular and congruent. In the group segment recorded with the tray clutch, opening/closing paths of one subject showed crossed and time curves of three subjects appeared peak-like changes of velocity, but none were statistically different (P>0.05).

CONCLUSIONThe research suggests that the functional occlusal clutch should be preferred in the evaluation of the mandibular function, as the tracings with the tray clutch are more likely to produce false positive results.

Adult ; Dental Occlusion ; Diagnosis, Computer-Assisted ; instrumentation ; Female ; Humans ; Jaw Relation Record ; instrumentation ; Male ; Mandibular Condyle ; physiology ; Movement ; Temporomandibular Joint ; physiology

OBJECTIVETo develop a rapid and effective method for genomic DNA extraction with magnetic bead-based semi-automatic system.

METHODSDNA was extracted from whole blood samples semi-automatically with nucleic acid automatic extraction system.The concentration and purity of samples was determined by UV-spectrophotometer. Orthogonal design was used to analyze the main effect of lysis time, blood volume, magnetic bead quantity and ethanol concentration on the DNA yield; also the 2-way interaction of these factors.

RESULTSLysis time, blood volume, magnetic bead quantity and ethanol concentration were associated with DNA yield (P<0.05), but no interaction existed. DNA yield was higher under the condition with 15 min of lysis time, 100 μl of blood volume, 80 μl of magnetic beads and 80 % of ethanol. A significant association was found between the magnetic bead quantity and DNA purity OD260/OD280 (P=0.008). Interaction of blood volume and lysis time also existed (P=0.013). DNA purity was better when the extracting condition was 40 μl of magnetic beads, 15 min of lysis time and 100 μl of blood volume. Magnetic beads and ethanol concentration were associated with DNA purity OD260/OD230 (P=0.017 and P<0.05), the result was better when magnetic beads was 40 μl and ethanol concentration was 80 %.

CONCLUSIONThe results indicate that the optimized conditions with 40 μl magnetic beads will generate higher quality of genomic DNA from the whole blood samples.

Analysis of Variance ; DNA ; blood ; isolation & purification ; Humans ; Immunomagnetic Separation ; methods