To construct a cDNA subtractive library of genes transactivated by hepatitis C virus core protein with suppression subtractive hybridization technique. mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-)-core and pcDNA3 1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNA were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, and then it was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain JM109. The cDNA were sequenced and analyzed in GenBank with Blast search after PCR. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR product showed that 213 clones contained 100~ 1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes before. The full length sequences were obtained with bioinformatics method,which had been accepted by GenBank. It suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.

To screen anti idiotypic single chain variable fragments(anti Id scFv)against hepatitis C virus NS4A(HCV NS4A)so as to lay a foundation for developing anti Id scFv vaccine againist the hepatitis C virus. The recombinant phage antibody library was panned by hepatitis C virus NS4A monoclonal antibody which was coated in a microwell plate. After five rounds of biopanning, 82 clones specific to HCV NS4A antibody were determined with the enzyme linked immunoadsorbent assay(ELISA). The specificity of anti idiotypic scFv was identified by ELISA and competitive inhibition assay. The DNA sequence of the positive clone was determined. The result showed that HCV NS4A anti Id scFv had a specific combination character with hepatitis C virus NS4A monoclonal antibody. The DNA sequence data showed that the anti Id scFv coding gene included 789 bp. The results suggested that the anti Id scFv fragments to HCV NS4A monoclonal antibody could be successfully selected by recombinant phage antibody library screening technique, which might pave a way for the study of new preventive and therapeutic strategy of hepatitis C using anti Id scFv.

To screen HCV core mimotopes,HCV core monoclonal antibody was used as immobilized molecule,and a 12 mer phage peptide library was biopanned and positive clones were selected by enzyme linked immunoadsorbent assay(ELISA), competitive inhibition assay, and DNA sequencing. 10 positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results,one epitope was confirmed as a mimotope of HCV core protein. The study might provide a new approach for HCV therapy and vaccine development.

Objective To investigate the xenotransplantation model of fetal pig skin precursor tissue and its development after transplantaion. Methods Porcine skin precursor tissue was obtained from the embryo of gestation day 56 (E56), and made into microskin or stamp skin graft. The microskin was transplanted to the dorsal wound in BALB/c nude mice, then covered with human corpse skin. The stamp skin graft was imbedded subcutaneously into the back of nude mice, and microskin was injected subcutaneously into the auricles of nude mice. Their growth and development were observed and they were examined by HE staining at 6th and 12th week after transplantation respectively. Two-sample t test was used to analyze the size of newly grown skin tissue. Results Porcine skin precursor tissue graft in three models above survived and continued growth after transplantation, and growth ability of the dorsal wound transplantation model was significantly stronger than that of the auricle model. Epidermis and hypodermis were detected in newly grown skin tissues. Hair follicles, a few of sebaceous glands, but no sweat glands were observed in auricle model, while many sebaceous glands and sweat glands were observed in the dorsal wound model. Conclusion Transplantation of microskin to dorsal wound is the optimal model of investigating the xenotransplantation of fetal pig skin precursor tissue and its development after transplantion.

Objective To investigate the clinical effectiveness of coronary artery bypass grafting through descending thoracic aorta in elderly patients with coronary heast disease and to decrease the post-operative complication.Methods Thirteen elderly patients underwent coronary bypass surgery with minimally invasive direct coronary artery bypass (MIDCAB).Age range from 70 to 82 years with a mean of(72.1?6.0)years.Patients suffered from multi vessel disease.Many minimally invasive techniques of“Y”blood vessel graft anastomosis,anastomosis of blood vessel graft to descending aorta,minimally invasive direct,thoracoscope assist were used.Results All patients were survived.The mean duration of intubation was (6.9?0.9) hours.The average ICU stay was (2.5?0.5)days.No patients received blood transfusion.During the short-term follow-up(3 to 14 months) patients had no complaint of angina,Conclusions The technique of“Y”blood vessel graft anastomosis,descending aorta blood vessel graft,minimally invasive direct and thoracoscope assist in combination with coronary artery bypass grafting is a safe and cost-effective new procedure for elderly patients with multi-coronary artery disease.

OBJECTIVETo explore the mechanism of electroacupuncture therapy on Parkinson's disease (PD).

METHODSFifty Wistar rats were randomly divided into a normal group, a sham-operation group, a model group, a Fengfu-Taichong group and a Shuanggu Yitong group. PD model was duplicated by microinjection of 6-Hydroxyl-Dopamine into right corpora striata, and by microinjection of normal saline in sham-operation group. Rats in normal group, sham-operation group and model group were not treated. In Fengfu-Taichong group, the rats were treated by electroacupuncture at "Fengfu" (GV 16) and "Taichong" (LR 3) on the basis of the PD model, and by electroacupuncture at "Fengfu" (GV 16), "Taichong" (LR 3), "Guanyuan" (CV 4) and "Zusanli" (ST 36) in Shuanggu Yitong group, once daily for 2 weeks. GDNF and Ret expression were detected by immunohistochemistry and western blotting, respectively.

RESULTSThe number of GDNF positive cells and the content of Ret receptor increased significantly in the two electroacupuncture groups compared with those in the other groups (all P < 0.01), and the expression of GDNF increased significantly in Shuanggu Yitong group compared with that in Fengfu-Taichong group (P < 0.01).

CONCLUSIONElectroacupuncture can not only increase the expression of GDNF, but also enhance its effect. "Shuanggu Yitong" method is better than simple acupuncture at "Fengfu" (GV 16) and "Taichong" (LR 3) in increasing expression of GDNF.

Animals ; DNA-Binding Proteins ; genetics ; metabolism ; Disease Models, Animal ; Electroacupuncture ; Gene Expression ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Humans ; Nuclear Proteins ; genetics ; metabolism ; Parkinson Disease ; genetics ; metabolism ; therapy ; Random Allocation ; Rats ; Rats, Wistar

BACKGROUNDMicrovascular decompression (MVD) is a well accepted surgical treatment strategy for trigeminal neuralgia (TN) with satisfying long-term outcome. However, considerable recurrent patients need more effective management. The purpose of this study was to evaluate the effectiveness of radiofrequency thermocoagulation rhizotomy (RTR) on patients with recurrent TN after MVD.

METHODSTotally 62 cases of recurrent TN after MVD undergoing RTR from January 2000 to January 2010 were retrospectively evaluated. Based on surgical procedures undertaken, these 62 cases were classified into two subgroups: group A consisted of 23 cases that underwent traditional RTR by free-hand; group B consisted of 39 cases that underwent RTR under the guidance of virtual reality imaging technique or neuronavigation system. The patients in group A were followed up for 14 to 70 months (mean, 40 ± 4), and those in group B were followed up for 13 to 65 months (mean, 46 ± 7). Kaplan-Meier analyses of the pain-free survival curves were used for the censored survival data, and the log-rank test was used to compare survival curves of the two groups.

RESULTSAll patients in both groups A and B attained immediate pain relief after RTR. Both groups attained good pain relief rate within the first two years of follow-up: 92.3%, 84.6% and 82.6%, 69.6% respectively (P > 0.05). After 2 years, the virtual reality or neuronavigation assisted RTR group (group B) demonstrated higher pain relief rates of 82.5%, 76.2% and 68.8% at 3, 4 and 5 years after operation respectively, while those in group A was 57.2%, 49.6%, and 36.4% (P < 0.05). Low levels of minor complications were recorded, while neither mortalities nor significant morbidity was documented.

CONCLUSIONSRTR was effective in alleviating the pain of TN cases suffering from unsuccessful MVD management. With the help of virtual reality imaging technique or neuronavigation system, the patients could attain better long-term pain relief.

Adult ; Aged ; Electrocoagulation ; methods ; Female ; Humans ; Male ; Microvascular Decompression Surgery ; Middle Aged ; Radiosurgery ; methods ; Retrospective Studies ; Rhizotomy ; methods ; Treatment Outcome ; Trigeminal Neuralgia ; surgery ; therapy

Objective Non-invasive detection is the focus of intense research in diagnosis of acute renal allograft rejection currently. Urine protein is considered the cue to reflect the pathological changes in kidney disease. In this study, we explored the urine markers for early acute renal allograft rejection. Methods The urine protein of two patients with acute renal allograft rejection were examined by 2D gel electrophoresis and bioinformatics. We adopted pH 4-7 ready strip IPG and stained the gel with Sypro-Ruby. The digitized 2D maps of urine protein were quantitatively analyzed using 2D-analysis software packages. By analyzing the differential expressions of proteome between different time points (1, 2, 3 days before acute rejection and 7, 14, 21 days after acute rejection), 30 protein spots were selected and analyzed by MALDI-TOF-MS/MS. Results We obtained 2D gel electrophoresis maps of urine protein of the patients with acute renal allograft rejection, which are of good reproducibility and resolution. Sixteen protein spots were identified, resulting in thirteen corresponding proteins. Out of these proteins, we screened three proteins (alpha-1-antichymotrypsin, tumor rejection antigen gp96, Zn-Alpha-2-Glycoprotein) closely related to acute rejection. Conclusion The urine protein spots on 2D gel electrophoresis maps for the patients with acute renal allograft rejection were of obvious difference when detected at different time points of acute rejection. Alpha-1-antichymotrypsin, tumor rejection antigen gp96 and Zn-Alpha-2-Glycoprotein might be the candidate protein markers to diagnose acute renal allograft rejection after renal transplantation.

Objective To investigate the pharmacokinetics of mycophe-nolate mofetil ( MMF) in Chinese adult patients with autoimmune disea-ses.Methods Fourteen patients with autoimmune disease were included in this study.Oral dose ( MMF 0.75 g, q12 h) was given for the first time and 7 days later steady -state.Serum concentrations of the myco-phenolic acid ( MPA) , an active metabolite of the pro -drug MMF, and its phenol glucuronide metabolite ( MPAG ) were determined by HPLC method.Pharmacokinetic parameters of MPA and MPAG were calculated and influences of renal function on MPA drug exposure and MPAG drug exposure in steady -state were investigated.Results The main pharma-cokinetic parameters of MPA after first -dose and steady -state were as follows: Cmax were ( 8.45 ±7.54 ) , ( 10.89 ±4.37 ) mg · L-1 , AUC0-12 h were (41.07 ±49.26 ), (55.09 ±41.74 ) mg· h· L-1 and the main pharmacokinetic parameters of MPAG after first -dose and steady-state were as follows: Cmax were (41.24 ±28.57 ), (67.63 ± 36.98) mg · L-1, AUC0-12h were (487.25 ±326.53 ), (720.79 ± 413.86 ) mg· h· L-1.A large variability existed in pharmacokinetic parameters of MPA and MPAG.The differences in pharmacokinetic parameters are larger between individual patient of the two groups , the AUC0-12h of steady -state compared with the first administration were both obviously greater ( P<0.05 ).There was an obviously inverse relation-ship between MPAG drug exposure and renal function ( P<0.05 ) , however , MPA drug exposure was less affected by renal function ( P>0.05 ).Conclusion In patients with autoimmune disease receiving a fixed MMF dose , there is a large inter-individual variability of MMF pharmacokinetics and accumulation of MPA and MPAG drug exposure .In addition, a significant accumulated phenomenon was found.

OBJECTIVETo evaluate the clinicopathological characteristics and surgical treatment of esophageal carcinosarcoma.

METHODSThe patients with esophageal carcinosarcoma were divided into two types according to barium swallow: intraluminal carcinosarcoma (n=20) and fungating carcinosarcoma (n=2). Only one esophageal carcinosarcoma case was diagnosed by esophagoscopic biopsy preoperatively. Twenty patients underwent left thoracic approach esophagectomy and esophagogastrostomy above aortic arch, and two patients underwent esophagectomy and esophagogastrostomy below aortic arch.

RESULTSAll the cases survived during operation and had no severe complication. Post-operative biopsy revealed that 21 cases had definite boundary between the carcinoma and the sarcoma. Only one case showed the invasion of carcinomatous tissues into sarcomatous tissues and mixed growth. Four cases had lymph node metastases (18.2%). The 1-, 3- and 5-year survival rates were 90.9% (20/22), 77.3% (17/22) and 68.2% (15/22) respectively.

CONCLUSIONSEsophageal carcinosarcoma is a rare malignant tumor with little invasiveness, low lymph node metastasis, early clinical symptom occurrence, low preoperative accurate diagnostic rate and good prognosis. Surgical resection is the main treatment for esophageal carcinosarcoma.

Adult ; Aged ; Carcinosarcoma ; pathology ; surgery ; Esophageal Neoplasms ; pathology ; surgery ; Female ; Humans ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Prognosis