ABSTRACT
OBJECTIVE@#To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).@*METHODS@#Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay.@*RESULTS@#Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.@*CONCLUSION@#IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.
Subject(s)
Animals , Mice , Autophagy , Calcium/metabolism , Chondrocytes , Endoplasmic Reticulum/metabolism , Endoribonucleases/pharmacology , Homeostasis , Inositol , Mice, Knockout , Protein Serine-Threonine Kinases , RNA, Messenger/metabolism , Sirolimus/pharmacology , Tunicamycin/pharmacologyABSTRACT
The clinical results of the application of pedicled vascularized bone graft (VBG) from Lister's tubercle vs. traditional bone graft (TBG) were evaluated and compared. Thirteen cases of symptomatic scaphoid nonunion were treated between January 2011 and December 2012, including 7 cases subject to VBG and the rest 6 cases to TBG, respectively. Outcomes were assessed by modified Mayo wrist score system. All cases were followed up for an average period of 3.5 months after operation. The results showed that total scores in VBG group were 86.4±9.4 after operation with excellent result in 4 cases, good in 2 and acceptable in one, and those in TBG group were 71.7±9.3 after operation with good result in 2 cases, acceptable in 3 and disappointing in one. Total score of wrist function was significantly improved in VBG group as compared with TBG group (P<0.05). Our study suggests that VBG method is more effective for treating scaphoid nonunion than TBG method.
ABSTRACT
As a result of the complex anatomy in upper cervical spine, the operative treatment of axis neoplasms is always complicated. Although the procedure for the second cervical vertebra (C2) surgery had been described previously in diverse approaches and reconstruction forms, each has its own limitations and restrictions that usually result in less satisfactory conclusions. The purpose of this study was to evaluate the operation efficacy for axis tumors by using a combined anterior (retropharyngeal) cervical and posterior approach in achieving total resection of C2 and circumferential reconstruction. Eight consecutive C2 tumor patients with mean age of 47.6 years in our institute sequentially underwent vertebra resection and fixation through aforementioned approach from Jan. 2006 to Dec. 2010. No surgical mortality or severe morbidity occurred in our group. In terms of complications, 2 cases developed transient difficulty in swallowing liquids (one of them experienced dysphonia) and 1 developed cerebrospinal fluid leakage (CSFL) that was resolved later. During a mean follow-up period of 31.9 months, the visual analogue scale (VAS) and Japanese orthopedic association (JOA) score revealed that the pain level and neurological function in all patients were improved postoperatively, and there was no evidence of fixation failure and local recurrence. It is concluded that the anterior cervical retropharyngeal approach permits a visible exposure to facilitate the C2 vertebra resection and perform an effective anterior reconstruction at the same time. The custom-made mesh cage applied in our cases can be acted as a firm and convenient implant in circumferential fixation.
ABSTRACT
As a result of the complex anatomy in upper cervical spine, the operative treatment of axis neoplasms is always complicated. Although the procedure for the second cervical vertebra (C2) surgery had been described previously in diverse approaches and reconstruction forms, each has its own limitations and restrictions that usually result in less satisfactory conclusions. The purpose of this study was to evaluate the operation efficacy for axis tumors by using a combined anterior (retropharyngeal) cervical and posterior approach in achieving total resection of C2 and circumferential reconstruction. Eight consecutive C2 tumor patients with mean age of 47.6 years in our institute sequentially underwent vertebra resection and fixation through aforementioned approach from Jan. 2006 to Dec. 2010. No surgical mortality or severe morbidity occurred in our group. In terms of complications, 2 cases developed transient difficulty in swallowing liquids (one of them experienced dysphonia) and 1 developed cerebrospinal fluid leakage (CSFL) that was resolved later. During a mean follow-up period of 31.9 months, the visual analogue scale (VAS) and Japanese orthopedic association (JOA) score revealed that the pain level and neurological function in all patients were improved postoperatively, and there was no evidence of fixation failure and local recurrence. It is concluded that the anterior cervical retropharyngeal approach permits a visible exposure to facilitate the C2 vertebra resection and perform an effective anterior reconstruction at the same time. The custom-made mesh cage applied in our cases can be acted as a firm and convenient implant in circumferential fixation.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Axis, Cervical Vertebra , Diagnostic Imaging , General Surgery , Combined Modality Therapy , Laminectomy , Methods , Radiography , Plastic Surgery Procedures , Methods , Spinal Neoplasms , Diagnostic Imaging , General Surgery , Treatment OutcomeABSTRACT
The clinical results of the application of pedicled vascularized bone graft (VBG) from Lister's tubercle vs. traditional bone graft (TBG) were evaluated and compared. Thirteen cases of symptomatic scaphoid nonunion were treated between January 2011 and December 2012, including 7 cases subject to VBG and the rest 6 cases to TBG, respectively. Outcomes were assessed by modified Mayo wrist score system. All cases were followed up for an average period of 3.5 months after operation. The results showed that total scores in VBG group were 86.4±9.4 after operation with excellent result in 4 cases, good in 2 and acceptable in one, and those in TBG group were 71.7±9.3 after operation with good result in 2 cases, acceptable in 3 and disappointing in one. Total score of wrist function was significantly improved in VBG group as compared with TBG group (P<0.05). Our study suggests that VBG method is more effective for treating scaphoid nonunion than TBG method.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Bone Transplantation , Methods , Fractures, Ununited , General Surgery , Hand Strength , Physiology , Pain , Range of Motion, Articular , Physiology , Retrospective Studies , Scaphoid Bone , Wounds and Injuries , General Surgery , Surgical Flaps , Treatment Outcome , WristABSTRACT
<p><b>OBJECTIVE</b>To investigate the surgical results of one stage total en bloc spondylectomy (TES) by anterior and posterior approaches for lumbar vertebral tumors and evaluate its benefit for these tumors.</p><p><b>METHODS</b>A total of 21 patients with the lumbar vertebral tumor treated with on stage TES by posterior and anterior approaches from April 2003 to August 2007 were reviewed, which included 14 males and 7 females with an average age of 47.6 years. Thirteen patients were suffered with the primary lumbar vertebral tumors and 8 patients were diagnosed for the lumbar vertebral metastasis tumors. There were 8 of S3, 3 of I A and 2 of II according to Ennekinng stage system. And there were 1 of Grade B, 4 of Grade C, 8 of Grade D, and 6 of Grade E according to Frankel grade system. The spinal reconstruction was obtained by titanium mesh filled with autograft for benign and low-grade malignant tumors and methylmethacrylate-filled titanium mesh for malignant tumors. The spinal stability was enhanced by posterior internal fixation with rod-screw system.</p><p><b>RESULTS</b>The operation time was 250 min and bleed loss was 2100 ml on average. The follow-up period lasted from 1.0 to 5.5 years. All cases had pain before operation, among which 14 cases obtained complete relief and 7 cases obtained partly relief after operation. In all cases with neurological deficit, they improved neurologically by more than one grade using the Frankel grading system. Up to now, 1 patient had be local recurrence after operation and 4 patients were dead on the following time. The others still are alive and no local recurrence.</p><p><b>CONCLUSION</b>One-stage TES by anterior and posterior approaches for lumbar vertebral tumor is feasible, safe and effective to lumbar vertebral tumor resection and stability reconstruction, which has many advantages such as controlling local recurrence, spinal cord decompression thoroughly, relieving the pain, improving the life quality and prolonging the lifetime.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Feasibility Studies , Follow-Up Studies , Lumbar Vertebrae , Spinal Neoplasms , General Surgery , Spine , General Surgery , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To analyze the transcription activation and possible regulation mechanism of human X-box binding protein 1(XBP1)gene 5'upstream DNA sequence in different cell lines.</p><p><b>METHODS</b>Six kinds of XBP1 promoter deletion mutants were cloned into pGEM-Teasy vector, which included XBP1 gene 5' upstream -1039 to 66 bp,-859 to 66 bp,-623 to 66 bp,-351 to 66 bp,-227 to 66 bp,-227 to -45 bp respectively. Every deletion mutant sequence was cut from Teasy-XBP1p by KpnI and Xho I, and subcloned into pCAT3-Basic to produce a set of constructs termed as p1-XBP1p, p2-XBP1p, p3-XBP1p, p4-XBP1p, p5-XBP1p, p6-XBP1p, respectively. The transcription activity of each construct was detected after transiently transfecting K562, HepG2,NIH-3T3 and L0(2)cell with FuGENE 6 transfection reagent. Cells transfected by pCAT3-Basic or pCAT3-Promoter were used as negative and positive controls. The activity of chloramphenicol acetyltransferase(CAT), which reflects the transcription activation of the XBP1 gene promoter, was detected by ELISA after 48 hours of transfection.</p><p><b>RESULTS</b>The reporter vectors of six kinds of XBP1 promoter deletion mutants were successfully constructed, as confirmed by restriction enzyme digestion and sequencing. The activities of p4-XBP1p and p5-XBP1p were higher than the other deletion mutants in K562 and HepG2. And the activity of p5-XBP1p was the highest in HepG2. There was no activity detected from any transfected NIH-3T3.</p><p><b>CONCLUSION</b>The XBP1 gene promoter can transactivate its downstream gene to transcription. The core sequence of XBP1 promoter was implied between -227 bp and 66 bp. This sequence was connected with the transcriptional activity of XBP1 promoter closely. Its transcription activity varies with different cell lines. XBP1 promoter might drive gene expression with cell-type specificity.</p>
Subject(s)
Animals , Humans , Mice , 3T3 Cells , 5' Flanking Region , Genetics , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase , Metabolism , DNA , DNA-Binding Proteins , Genetics , Gene Deletion , Gene Expression Regulation , Physiology , Genes, Reporter , K562 Cells , Molecular Sequence Data , Nuclear Proteins , Genetics , Promoter Regions, Genetic , Genetics , Regulatory Factor X Transcription Factors , Transcription Factors , Transcription, Genetic , Physiology , Transcriptional Activation , Transfection , Tumor Cells, Cultured , X-Box Binding Protein 1ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of tacrolimus on expression of heat shock protein 70 (HSP 70) after spinal cord injuries (SCI) in rats and the relationship between expression of HSP 70 and apoptosis of neural cells.</p><p><b>METHODS</b>Seventy-two male rats were divided randomly into three groups: the sham-operation group, the injury group and the group treated with tacrolimus, and the latter two groups were SCI with a weight-drop impactor at the T(10) vertebrae level (10 g weight was dropped from a 4.0 cm height). The tacrolimus group was injected with tacrolimus 5 minutes after SCI, while the other groups received 0.9% saline likewise. The inclined plate and BBB (Basso, Beattie and Bresnahan) scales were used to evaluate hindlimb neurological function. The expression of HSP 70 mRNA after SCI was detected by using reverse transcription polymerase chain reactions (RT-PCR) and immunohistochemistry staining was performed to determine the protein expression of HSP 70 and Caspase-3. The apoptosis of neural cells was assessed with the terminal deoxynucleotidyl transferase-mediated deoxyuredine triphosphate-digoxin nick end labeling (TUNEL) method.</p><p><b>RESULTS</b>Compared with the injury group, the expression of HSP 70 was significantly higher in the tacrolimus group, and the peak expression of HSP 70 mRNA and protein was respectively observed at 6, 24 h after SCI. Caspase-3-positive or TUNEL-positive cells were significantly less in the tacrolimus group than in the injury group. Neurological function score of the tacrolimus group was significantly better than that of the injury group.</p><p><b>CONCLUSIONS</b>Tacrolimus may inhibit activity of Caspase-3, attenuate apoptosis of neural cells and ameliorate neurological function recovery after SCI by inducing high expression of HSP 70.</p>
Subject(s)
Animals , Male , Rats , Acute Disease , Apoptosis , Caspase 3 , Genetics , Metabolism , Disease Models, Animal , Gene Expression , HSP70 Heat-Shock Proteins , Genetics , Metabolism , Immunohistochemistry , Immunosuppressive Agents , Pharmacology , In Situ Nick-End Labeling , Neurons , Metabolism , Pathology , RNA, Messenger , Genetics , Metabolism , Random Allocation , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord Injuries , Tacrolimus , PharmacologyABSTRACT
<p><b>OBJECTIVES</b>To investigate the regulating effect of HBV pre-S2 protein on iNOS gene promoter and the molecular biological mechanisms of pre-S2 protein in HBV pathogenicity.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) technique was employed to amplify the sequence of iNOS promoter and 3 deletion mutants using HepG2 genomic DNA as the template, and the products were cloned into the pGEM-T vector. The iNOS gene and 3 deletion mutants were cut from T- iNOS by Kpn I and Xho I, and then cloned into pCAT3-Basic. The resulting vectors were named p1-iNOSp, p2-iNOSp, p3-iNOSp, and p4-iNOSp. Each of the reporter vectors was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-pre-S2 by FuGENE 6 transfection reagents. The HepG2 cells transfected with pCAT3-Basic were used as a negative control. The activity of CAT in HepG2 cells transfected was detected by an ELISA kit 48 hours after the transfection, which reflected the regulating effect of HBV pre-S2 protein on iNOS gene promoter activity.</p><p><b>RESULTS</b>The expressive vector pcDNA3.1(-)-pre-S2 and report vector pCAT3-iNOSp were constructed and confirmed by restriction enzyme digestion and sequencing. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells could down-regulate the activity of p1-iNOSp, p3-iNOSp, and the inhibition rate was 54.7% and 79.5%, respectively. The expression of pcDNA3.1(-)-pre-S2 in HepG2 cells had no regulatory effects on p2-iNOSp and p4-iNOSp.</p><p><b>CONCLUSION</b>It is suggested that HBV pre-S2 protein can down-regulate iNOS gene promoter.</p>
Subject(s)
Humans , Down-Regulation , Hepatitis B Surface Antigens , Genetics , Nitric Oxide Synthase Type II , Genetics , Promoter Regions, Genetic , Genetics , Protein Precursors , Genetics , Transcriptional Activation , TransfectionABSTRACT
Objective To investigate the regulating effect of hepatitis B virus(HBV)SPⅠbinding protein 1(SBP1)on inducible nitric-oxide synthase(iNOS)gene promoter activity.Methods Polymerase chain reaction(PCR)technique was employed to amplify the coding sequence of iNOS promoter DNA by using HepG2 genomic DNA as template,and 3 deletion mutants were amplified. The products were cloned into pGEM-T vector,respectively.The iNOS gene and 3 deletion mutants were cut from T-iNOS by KpnⅠand XhoⅠ,and then were cloned into pCAT3-Basic.The construc- ted vectors were named as p1-iNOSp,p2-iNOSp,p3-iNOSp and p4-iNOSp,respectively.Each of the vectors containing different iNOSp DNA fragments was transfected into the HepG2 cell line and cotransfected into HepG2 cells with pcDNA3.1(-)-SBP1 by FuGENE 6 transfection reagents.The HepG2 cells transfected with pCAT3-Basic were used as negative control.The activity of chloram- phenicol acetyltransferase(CAT)in transfected HepG2 cells was detected by an enzyme-linked immu- nosorbent assay(ELISA)kit after 48 hours,which would reflect the regulating effect of SBP1 on iNOS gene promoter activity.Results The expression vector pcDNA3.1(-)-SBP1 and report vector pCAT3-iNOS were constructed and confirmed by restriction enzyme digestion and sequencing.The expression of pcDNA3.1(-)-SBP1 in HepG2 cells up-regulated the activity of p1-iNOSp and down- regulated the activity of p3-iNOSp.The inhibiting rate was 31.3%.Conclusions It is suggested that SBP1 can regulate iNOS gene promoter bidirectionally by influencing the binding sites of nuclear factor (NF)-IL6,A activator domain binding site and NF-?B in iNOS gene promoter.
ABSTRACT
Objective To investigate the transactivating effect of hepatitis C virus(HCV)core protein on inducible nitric oxide synthase(iNOS)gene promoter and the molecular biological mecha- nisms of HCV pathogenesis.Methods Polymerase chain reaction(PCR)technique was employed to amplify the sequence of iNOS promoter by using HepG2 genomic DNA as template,and the product was cloned into pGEM-T vector.The iNOSp gene was cut from T-iNOSp by KpnⅠand XhoⅠ,and then was cloned into pCAT3-Basic,the constructed vector was named as pCAT3-iNOSp,pCAT3-iN- OSp was transfected into the LO_2 cell line.LO_2 cell was also cotransfected with pcDNA3.1(-)-core and pCAT3-iNOSp by FuGENE 6 transfection reagents.The LO_2 cells transfected with pCAT3-Basic was used as negative control.The activity of CAT in LO_2 cells was detected by an ELISA kit after 48 hours,which reflected the transactivating function of HCV core protein to iNOS gene promoter.Re- sults The expressive vector pcDNA3.1(-)-core and report vector pCAT3-iNOSp had been construc- ted and confirmed by restriction enzyme digestion and sequencing.The expression of CAT in LO_2 cells transfected with pCAT3-iNOSp and peDNA3,1(-)-core was 11 times as higher as that of pCAT3-bas- ic,and 6 times as higher as that of pCAT3-iNOSp.Conclusion It is suggested that HCV core protein can transactivate iNOS gene promoter.