BACKGROUND:Previous study has found that hsa-miR-182 is probably related to the apoptosis-related genes such as cytochrome C (Cycs C) and calcineurin subunit CnB (PPP3R1) in nucleus pulposus cells.
OBJECTIVE:To determine whether miR-182 plays a regulatory role in nucleus pulposus cel apoptosis by detecting the relative gene expression levels after transfecting miR-182 with Cycs C and PPP3R1 into nucleus pulposus cel s via plasmid delivery.
METHODS:After a bioinformatics prediction about miR-182, miR-182 and target genes were transfected into the nucleus pulposus cel s, and at the same time, blank control group was established. Then the expression levels of the target genes were detected through cel lysis.
RESULTS AND CONCLUSION:miR-182 significantly inhibited the expression of Cycs C in nucleus pulposus cel s compared with the blank control group (P<0.05). Compared with the blank control group, miR-182 made no inhibitory effect on the expression of PPP3R1. These findings suggest that miR-182 may play a regulatory part in nucleus pulposus cel apoptosis by inhibiting the expression of Cycs C.

Objective To investigate whether the accelerator image beam line (IBL) full scan and extend field of view(EFOV) scan mode megavoltage cone beam CT(MV CBCT) images can be used for dose calculation in adaptive radiotherapy.Methods The large aperture CT and MV CBCT were used to scan the CIRS 062M electron density modules,the CT value was established to electron density curves in the Pinnacle treatment planning system.Also,CT and MV CBCT were used to scan the head and neck,chest,abdomen and pelvis phantom.The intensity modulated radiotherapy(IMRT) plans were made with CT images and transplanted to MV CBCT images.The dose of targets and organs with their electron density curves was calculated,and two type IMRT plans with different CT images were compared.Results The dose distribution of head and neck phantom was acceptable,compared with the reference plan,the difference was within 3 ％.The dose distribution of chest and.pelvis was significantly reduced from reference plans,and the difference was 5％ and 10％ separately.This difference was beyond the scope of clinical acceptance.Conclusions MV CBCT images of accelerator IBL full scan mode in patients with head and neck site scan could be used for dose calculation in adaptive radiotherapy,chest and pelvic sites in EFOV mode scanning MV CBCT images could only be used for image guidance.

Objective To observe the expression of matrix metalloproteinases(MMP)-1,MMP-3 and iNOS in cartilage of experimental rabbit osteoarthritis at different time intervals after anterior cruciate ligament transection(ACLT)operation.The aim of this study is to provide the theoritical evidence for using ACLT rabbit model in Osteoarthritis(OA)research.Methods Unilateral ACLT was performed on 27 randomly selected while rabbits and underwent unilateral arthrotomy was performed on the other 9 white rabbits as the control group.Nine randomly selected white rabbits in experimental group were killed and 3 white rabbits in the control group at 4th,8th and 12th week respectively.Cartilage degradation of femoral condyles was evaluated macr-oscopically,mRNA expression level and protein expression level of MMP-1,MMP-3 and iNOS was measured by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry respectively.Results Forepart OA cartilage degradation was observed at the 4th week and became more severe at the 8th week after ACLF.Afterpart cartilage degradation was evident at the 12th week after ACLT while cartilage still remained normal in the control group,mRNA expression level and protein expression level of MMP-1.MMP-3 and iNOS were increased at the 4th week and became higher gradually at the 8th,12th week after ACLT compared with the control group.Expression distribution of MMP-1,MMP-3 and iNOS bad different patterns respectively.Conclusion It is suggested that the process of OA cartilage degradation can be simulated by ACLT model and MMP-1,MMP-3 and iNOS may be good markers in therapeutical research of OA.

Animals ; Bromodeoxyuridine ; metabolism ; Cerebral Cortex ; injuries ; Immunohistochemistry ; Male ; Nerve Regeneration ; Rats ; Rats, Sprague-Dawley

Objective To determine the peripheral dose ( PD) of a Trilogy accelerator under different conditions and the feasibility of PD measurement using the semiconductor diode ionization chamber. Methods In a solid water phantom, a CC13 air?filled ionization chamber and a semiconductor diode ionization chamber were used for PD measurements with different distances (13 measurement locations within 1?31 cm) , depth ( 3, 5, 15 cm) , field sizes ( 10, 20, 30 cm) , wedge ( W15, W45, VW15, VW45) , and beam energy (6, 18 MV). The relationship of PD with PDleakage and PDscat er was determined by removing the scatter phantom. Simulating the patients with cervical cancer undergoing radiotherapy, a CIRS phantom received volumetric modulated arc therapy ( VMAT) , step?shoot intensity?modulated radiotherapy ( IMRT) , and sliding?window IMRT to measure PDs of the breast, thyroid, and lens. All the data were normalized to the isocenter. Results PD was gradually reduced with the increase in distance ( 13?41% at 1 cm from the edge to 0?25% at 31 cm from the edge) . With a fixed distance from the edge of the radiation field, there was no significant difference in PD between different depths. A radiation field with a size of 30 cm had a PD about two?fold higher than that with a size of 10 cm. PD increased with the increase in the physical wedge angle and increased by 1% compared with the open field;PD decreased with the increase in the virtual wedge angle and decreased by 2?3% compared with the open field. PD decayed from 13?35% at 1 cm to 0?23% at 31 cm under 6 MV X?ray and from 11?06% at 1 cm to 0?20% at 31 cm under 18 MV X?ray. Dscat er was dominant in the regions close to the edge of radiation field and decreased from 62?45% at 1 cm to 5?71% at 25 cm. In all measurements under 6 MV X?ray, the maximum proportion difference between CC13 ionization chamber and diode ionization chamber was less than 1%. PDs of the breast, thyroid, and lens were 6?72, 2?90, and 2?37 mGy in VMAT mode, 7?39, 4?05, and 2?48 mGy in step?shoot IMRT mode, and 9?17, 4?61, and 3?21 mGy in sliding?window IMRT mode, respectively. Conclusions For the measurement of PDs, the CC13 air?filled ionization chamber and semiconductor diode ionization chamber have good consistency and feasibility under 6 MV X?ray. In clinical practice, the understanding of the relationship of PD with different radiation conditions helps to reduce the doses to organs at risk. Shielding and protective techniques can further reduce dose deposition.

OBJECTIVETo develop a quantitative real-time polymerase chain reaction (PCR) for detecting Bartonella henselae.

METHODSAccording to the 16S-23S rRNA intervening sequences (IVS) specific for B. henselae, one pair of primers and one TaqMan-MGB probe were designed. A quantitative real-time PCR was developed with the primers, the probe, and the IVS, a standard template, in DNA sequence detection system (ABI 7900HT).

RESULTSThe standard curve was established with the standard template and the relationship between the value of threshold cycle (Ct) and the DNA copy number was linear (r = 0.997). The sensitivity of this quantitative real-time PCR was about 1000 times higher than that of a common PCR used to detect homologous DNA. By this quantitative real-time PCR, the DNA sample of B. henselae was positively detected but not from other rickettsial or bacterial DNA samples. The variation coefficients of intra- and inter-assay reproducibility were 0.2%-1.9%. Using the real-time quantitative PCR to detect samples from mice that were experimentally infected with B. henselae, the small amount of B. henselae DNA was detected in blood samples on days 2, 3, and 5 and large amount of B. henselae DNA was detected in spleen samples on days 1 and 2 after infection.

CONCLUSIONResults from our study suggested that this quantitative real-time PCR was highly specific, sensitive and with good repeatability for detection of B. henselae. It seemed quite useful for rapid detection of tiny DNA of B. henselae in various samples and laboratory diagnosis of bartonellosis caused by B. henselae.

Animals ; Bartonella Infections ; diagnosis ; Bartonella henselae ; genetics ; DNA, Bacterial ; analysis ; Mice ; Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Sensitivity and Specificity

OBJECTIVETo develop a real-time quantitative polymerase chain reaction(PCR) assay for detecting Rickettsia rickettsii.

METHODSThe primers and TaqMan-MGB probe were designed according to the ompB gene of R. rickettsii. A DNA fragment of ompB gene amplified from R. rickettsii by PCR was used as a standard template for the development of the method.

RESULTS5 copies of ompB fragments of R. rickettsii were detected. The genomic DNA of R. rickettsii was detected by the developed quantitative PCR assay. However, the genomic DNA from another rickettsial or bacterial agent was not determined. Through this developed method, the positive results were obtained from the animals and cells, artificially infected with R. rickettsii.

CONCLUSIONThe real-time quantitative PCR assay seemed to be highly sensitive and specific which might be used to rapidly detect R. rickettsia DNA in various samples and to early diagnose patients infected by R. rickettsii.

DNA Primers ; Humans ; Polymerase Chain Reaction ; methods ; Rickettsia rickettsii ; genetics ; Rocky Mountain Spotted Fever ; diagnosis ; Sensitivity and Specificity

OBJECTIVEIn order to study the feature of T cell TCR-CD3 complex-mediated gene in lead poisoning patients.

METHODSReal-time PCR with SYBR Green I technique was used for determination of the expression levels of CD3 genes in peripheral blood mononuclear cells of 46 cases lead poisoning patients (11 cases in observation group and 35 cases in mild lead poisoning group) and 31 cases in control group.

RESULTSThe median expression levels of CD3γ gene in observation group and mild lead poisoning group (6.89%, 5.87 %) were higher than the control group (P < 0.05). The median expression levels of CD3δ gene in observation group and mild lead poisoning group (0.54%, 0.70%) were lower than the control group (P < 0.05). The median expression levels of CD3ε gene in observation group and mild lead poisoning group (10.22%, 6.08%) were higher than the control group (P < 0.05). A significant Positive correlation was found between CD3γ, CD3ε and seniority in lead poisoning patients. A significant negative correlation was found between CD3ε and blood ZPP, urea δ-ALA (r = -0.358, P < 0.05; r = -0.385, P < 0.05), but there was no significant correlation between them after controlling for blood lead, urea lead. The expression levels of CD3 genes prove to be a descending order of CD3γ, CD3ε, CD3δ in control group, while it was changed for CD3ε, CD3γ, CD3δ in the observation group as well as in mild lead poisoning group.

CONCLUSIONExpression of T cell TCR-CD3 complex-mediated gene was changed in lead poisoning patients, it might be related to the body immunodeficiency. The expression level of CD3ε gene can be used as sensitive immune function screening indicator in Lead poisoning patients.

Adolescent ; Adult ; Aged ; Female ; Humans ; Lead Poisoning ; immunology ; Male ; Occupational Diseases ; immunology ; Receptor-CD3 Complex, Antigen, T-Cell ; metabolism ; Young Adult

OBJECTIVETo investigate the mechanism of plasma bilirubin level increase after hepatic ischemia-reperfusion in rats.

METHODSRats were divided into a sham operation group (A group), a 20 min ischemia-reperfusion group (B group) and a 35 min ischemia-reperfusion group (C group). Study time points were 6 hours and 1, 3, and 5 days after the reperfusion. Pathological changes in the livers were studied with histological slides stained with hematoxilin and eosin. Routine biochemistry methods were used to detect the bilirubin level of blood plasma and the bile drained from the ischemic hepatic lobes. RT-PCR was used to analyze the expression of the multidrug resistance-associated protein 2 (MRP2) and mRNA. Immunohistochemistry was used to analyze the localization of MRP2 in the canalicular membrane.

RESULTSB and C groups showed a mild inflammatory reaction without hepatocyte necrosis. At 6 h and 1 day after reperfusion, there was a significant increase of the plasma bilirubin level and a decrease of the bilirubin level of the drained bile in B group. These changes lasted to the day 3 and day 5 in C group. MRP2 mRNA down-regulation was found at 6 h only in the B and C groups. No localization of MRP2 in the canalicular membrane was found but it appeared in "esicules" under the canalicular membrane in C group.

CONCLUSIONSAbsence of MRP2 localization in the canalicular membrane could be the cause of the blood plasma bilirubin level increase after liver ischemia-reperfusion.

Animals ; Bilirubin ; blood ; Liver Diseases ; blood ; Male ; Multidrug Resistance-Associated Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; blood

OBJECTIVETo investigate the effect of demethylating agent 5-Aza-CdR (5-aza-2'- deoxycytidine) on demethylation and transcription-regulating of RASSF1A gene in gastric cancer cell SGC7901 in vitro, as well as on the growth inhibition of cells.

METHODSAfter SGC7901 cells were treated with 5-Aza-CdR, MTT assay, flow cytometry, and Annexin V-FITC staining were performed to analyze the cell proliferation, cell cycle and apoptotic rate respectively. Methylation- specific PCP (MSP), RT-PCR and Western blotting were used to detect methylation state, expression of mRNA and protein of RASSF1A gene.

RESULTSAfter SGC7901 cells were treated with different concentrations of 5-Aza-CdR, the cell growth was inhibited(P<0.05), the cell cycle was blocked at G(1) phase, and the apoptotic rate increased significantly(P<0.05). Hypermethylation was detected in the promoter region of RASSF1A gene in SGC7901 cells, and no expression of RASSF1A mRNA and protein was found. After treated with 5-Aza-CdR, demethylation occurred in RASSF1A gene,which subsequently induced re-expression of this gene at both mRNA and protein level.

CONCLUSIONDemethylating agent 5-Aza-CdR can regulate demethylation and re-expression of RASSF1A gene in gastric cancer cell SGC7901,and inhibit its growth.

Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Stomach Neoplasms ; genetics ; metabolism ; Tumor Suppressor Proteins ; metabolism