Objective:For developing the study of MUC1/Y vaccine,constructed th eukaryotic expression vectors expressing MUC1/Y.Used it to modify the DC inducint CTL in order to treat the gastrointestinal cancers.Methods:Obtained the MCU1/Y cDNA full length cloned it into pIRES2 EGFP that expressing green fluorescence protein and pcDAN3 1+ respectively.Selected 8 cases of gastrointestinal cancer whose HLA phenotype was A2,inducing DC using rhIL 4 combined with GM CSF in vitro.Transfected DC with pcDAN3.1 MUC1/Y then co cultured DC with T cell to induce CTL (named as T pcDAN3 1 MUC1/Y).At the same time,used pIRES2 EGFP MUC1/Y to detect transfection efficiency.SW620 (HLA A2+,MUC1/Y+),the gastric cancer cell line was used as specific target cell;Lovo(HLA A2 ,MUC1/Y+)and Raji(HLA A2 ,MUC1/Y )were used as unspecific target cells.The cytolytic of specific CTL was measured by LDH releasing assay.The apoptosis of target cells were detected by ANNEXIN V FITC kit.The ability of IFN ? releasing of T cells was measured by ELISA.Results:The transfection efficiency of plasmid was about 8%.There was significant difference between the cytolytic activity of T pcDNA3 1 MUC1/Y to SW620,Lovo and Raji.The cytolytic activity was about (42 8?6 15),(27 26?1 96)% and (22 73?2 15)% respectively.Compared with T IL 2)CTL induced by PBMC stimulated of IL 2(100 U/ml)) and T pcDNA3 1(CTL induced by DC transfected by pcDNA3 1+),the cytolytic activity of T pcDNA3 1 MUC1/Y against SW620 cell line showed a significant increase.The results of ANNEXIN V-FITC experimences showed that T pcDNA3 1 MUC1/Y could induce apoptosis of specific target cell.Conclusion:The expressing of MUC1/Y mRNA has important sense in gastrointestinal cancer.Constructed eukaryotic vector pIRES2 EGFP MUC1/Y that contains full length MUC1/Y cDNA could be used to study the transfection efficiency and select the positive clone;pcDAN3 1 MYC1/Y could induce powerful CTL immunoresponse.

Objective:To observe the effect of combining Chinese medicine and tuina along the meridians on motor function and activities of daily living (ADL) in patient with post-stroke upper limb spasticity. Methods:A total of 220 patients with post-stroke upper limb spasticity were randomly allocated into a treatment group (n=110) and a control group (n=110). Patients in the treatment group received tuina along the meridians combined with spasticity-alleviating and collateral-unblocking Chinese medicine, whereas patients in the control group received routine rehabilitation therapy. Patients in both groups were treated for 3 weeks. Then the patients’ motor function, ADL and muscle tone were evaluated before and after treatment using the Fugl-Meyer assessment scale (FMA), modified Barthel index (MBI) and modified Ashworth scale (MAS). Results:After treatment, the FMA scores, MBI scores, and muscle (shoulder intortor, elbow flexors and wrist flexors) tones were significantly improved (P<0.05), but the improvement was more significant in the treatment group than that in the control group (P<0.05). Conclusion:Tuina along the meridians combined with spasticity-alleviating and collateral-unblocking Chinese medicine can substantially alleviate muscle tone on the affected side and remarkably improve the patients’ motor function and ADL.

Objective To study microsatellite instability(MSI) in multiple primary colorectal carcinoma(MPCC) and solitary colorectal tumor(SCT), and to explore the relationship between the expression of mismatch repair(MMR)、p53、Bax、PCNA and MSI. Methods The expression of MMR、p53、Bax、 PCNAwere detected by immunohistochemical staining, and MSI at five microsatellite loci were examined by PCR-SSLP in 51 tumors from 38 MPCC patients and 35 SCT cases. Results The replication errors positive phenotype was observed in 27 of 51(53%) tumor foci from MPCC cases, and in 6 of 35(17%) SCT cases. There was an inverse correlation between replication errors (RER) positive and expression of p53; the PCNA labeling index of RER positive tumors were significantly lower than of RER negative tumors; RER positive related strongly with poor differentiation, the proclivity for proximal colon. Conclusions MSI may play an important role in the development of MPCC and may be used as a tumor marker of MPCC.

Objective:To study the use of left renal vein as a graft vessel in reconstruction after portal vein/superior mesenteric vein (PV-SMV) resection in pancreaticoduodenectomy.Methods:A retrospective study was conducted on 5 of these patients who underwent surgery from July 2008 to December 2017 at Chinese PLA General Hospital. The operative, complication and follow-up data were analysed.Results:There were 4 males and 1 female, with an average age of 57 (33-72) years. The mean operative time was 6.8 (5.4-9.1) h and the mean tumor size was 3.8 (2.8-4.8) cm. The average length of the PV-SMV defect left after resection was 3.8 (3.2-4.6) cm. The average length of the left renal vein used was 3.4 (3.0-4.1) cm. The operations were carried out in 3 patients with pancreatic cancer and in 2 patients with colon cancer pancreatic metastasis. The average postoperative hospital stay was 12 (10-25) days. Perioperative complications included 1 patient each with ascites, diarrhea and delayed gastric emptying. The creatinine levels ranged from 70-98 μmol/L preoperatively, with a transient creatinine rise to 80-156 μmol/L after operation and became 62-107 μmol/L upon discharge from hospital. The follow-up time was 4.3-17.8 months. Two patients died of recurrence/metastasis at 14.2 and 17.8 months after surgery.Conclusions:The left renal vein has the appropriate diameter and rich collateral branches. It has a sufficient length and it is conveniently located in the surgical field. This study showed that there was a minimal effect on renal function after its excision, and it can be used as a graft vessel for reconstruction in pancreaticoduodenectomy after PV-SMV resection.

Site-directed mutagenesis method was used to introduce two desired mutations, which were confirmed by DNA sequencing, into mouse complement receptor Type II gene(MCR2). Then the constructed eukaryotic expression vectors containing wild type mouse CR2/1(wtMCR2/1), mutant type mouse CR2/1 (mtMCR2/1) and human CR2 (hCR2) cDNA were transferred into mouse SP2/0 cells by electroporation. After two-week screening by G418, the stably transfected clones were obtained. Several ways including PCR, RT-PCR, and immunohistochemistry were utilized to screen those clones with interesting genes integrated and expressed. Then Epstein-Barr virus(EBV) was used to infect these transfected cells and EBER-1 (EBV encoded RNAs) hybridization results showed that only hCR2 and mtMCR2 transfected SP2/0 cells could be infected by EBV, but positive rate of the former was much higher than the latter. This study sets groundwork for elucidating the mechanism by which EBV enters the cells and for establishing the animal model of EBV-related nasopharyngeal carcinoma (NPC).

AIM: To establish nasopharyngeal carcinoma(NPC) cell lines stable expressing NPC-derived latent membrane protein 1(LMP1) gene. METHODS: General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed by using recombinant techniques, then transfected these vectors into a poor differentiated NPC cell line named CNE-2 ,integration and expression of N-LMP1 in CNE-2 cells were detected by PCR,RT-PCR and Western blot. RESULTS: (1) General expression vector and epithelium-specific expression vector of NPC-derived LMP1 gene were constructed successfully.(2) It showed that N-LMP1 gene expressed in CNE-2 cells correctly. CONCLUSION: The first NPC cell lines which stable express NPC-LMP1 were established. The cell lines obtained will provide important basis for exploring the role of NPC-LMP1 in nasopharynx carcinogenesis.

Objective To investigate the effect of acute irradiation by high power millimeter wave on the pathological changes of mouse lung tissue. Methods The BALB/c mice were vertically placed under the high power millimeter wave equipment with working frequency of 34. 1 GHz, and the mean output power were 5,10 and 12 W while the distance between the animal and the bottom of the irradiation horn were 10 mm and 20 mm, respectively. The mice were tied on the platform and continuously received irradiation until death. After immediate dissection, the mouse lung was quickly rinsed with 0.9% NaCl solution, fixed in 10% formaldehyde solution and mounted for paraffin section. After HE staining and image taken with a CCD camera, the Image Pro Plus software and quantitative image analysis by combining the mean optical density and area was used to determine the pathological injuries of the lung. Result Using the HA23. 16 and HA9. 92 pyramid horns with different physical parameter, the mice exposed to irradiation with high mean power of 12 W were dead most quickly, the death time was only about 110 s. Meanwhile, the death time was about 30 min after irradiation with the mean power of 5 W. There was significant hemorrhage in the mouse lung with high power millimeter irradiation, although the hemorrhage degree was different under different irradiation parameters. When the mean power were 10 and 12 W.the hemorrhage degree of lung was extremely high, where the bronchia and blood vessel of lung was markedly broken. A lot of cells of bronchia had been released. However, when the mean power was 5 W, the hemorrhage degree of lung was less observed, while the bronchia and blood vessels had not severe fracture. Conclusions High power millimeter wave wave irradiation has remarkable effect on mice lung. The damage degree of lung tissue is highly correlated with the mean power of millimeter wave irradiation. As the high power millimeter used in this study could result in significant thermal effect, the acute heat-induced response might lead to animal death by causing serious lung injury.

Objective To investigate the clinical effect of modified storage type of autologous blood transfu-sion combined with shed blooding retransformation technique after OrthoPAT for artificial total knee arthroplasty. Methods 70 patients with total knee replacement were randomly divided into observation group and control group, 35 cases in each group.The observation group was treated with the modified storage autotransfusion combined shed blooding retransformation technique after OrthoPAT,while the control group was given conventional allogeneic blood transfusion.The hemoglobin values and blood coagulation function of the two groups at immediately before anesthesia and surgery,10min before autologous blood transfusion and after reinfusion of 15min,after 6h and 24h of surgery were recorded,and the drainage blood total value,allogeneic blood transfusion measurement issues and transfusion rate after 24h were recorded.Results The hemodynamics of the two groups were stable at each time,there were no difference at urine volume (all P >0.05).The coagulation conditions were normal of the two groups at each time,there were no statistically significant differences between the two groups (all P >0.05).The average volume and homologous blood transfusion rate in the observation group were (126.3 ±6.5)mL,1 /35,which were significantly lower than those in the control group [(476.4 ±10.6)mL,2 /35],the differences were statistically significant (t =10.73,χ2 =6.31,all P <0.05).The incidence rate of postoperative complication of the observation group was 5.7%,which was signifi-cantly lower than 22.9% of the control group,the difference between the two groups was statistically significant (χ2 =4.93,P <0.05).Conclusion The improved storage type of autologous blood transfusion combined with shed bloo-ding retransformation technique after OrthoPAT has exact effect for artificial total knee arthroplasty,the incidence of adverse reactions is low,as well as the low blood transfusion rate.

Objective To study the effect of total knee arthroplasty after limb position on postoperative hemorrhage,to provide basis for clinical diagnosis and treatment.Methods 270 cases of total knee arthroplasty were selected.The patients were divided into groupⅠ,group Ⅱand group Ⅲ according to the random number table method, 90 cases in each group.Patients of group Ⅰ with limb hip and knee were straight,group Ⅱ hip joint elevation of 45 degrees,70 degrees of knee flexion,group Ⅲ hip joint elevation of 45 degrees,the knee extension.All the patients were intervened for 12h after operation,were placed drainage bag 24 hours.The lead flow,preoperative,postoperative hemoglobin and 5 days after the knee joint activity were compared in the three groups.Results Induced flow after surgery in group Ⅰ was (433.4 ±25.3)mL,which was significantly higher than (402.6 ±19.6)mL and (403.5 ± 21.5)mL in group Ⅱand group Ⅲ,and the differences were statistically significant (t =5.253,5.301,all P <0.05),there was no significant difference of induced flow between groupⅡ and group Ⅲ(P >0.05).The hemoglobin levels of the three groups were (92.3 ±4.2)g/L,(114.9 ±6.4)g/L and (113.2 ±7.5)g/L,which were significantly decreased after operation,the differences were statistically significant compared with before operation (t =5.083, 6.034,7.893,all P <0.05),the hemoglobin after surgery of group Ⅰ was significantly lower than group Ⅱ and groupⅢ,the differences were statistically significant (t =6.423,7.043,all P <0.05),there was no significant difference between group Ⅱ and group Ⅲ (P >0.05).There was no significant difference of range of motion in the three groups after 5 days of operation (P >0.05).Conclusion Hip flexion can effectively reduce bleeding after total knee arthro-plasty,the flexion and extension of knee joint had no significant effect on postoperative hemorrhage.

BACKGROUND: As the preliminary experiment for gene therapy in intervertebral disc degeneration, this study aims to construct a recombinant plasmid containing fluorescent pAAV-hSOX9-IRES-tdTomato for adeno-associated virus packaging, in a broader attempt to lay the foundation for late experiments in vitro and in vivo.OBJECTIVE: To construct human SOX9 gene overexpressing adeno-associated virus, pAAV-hSOX9-IRES-tdTomato, packaging. METHODS: The plasmid pAAV-IRES-tdTomato and plasmid pUC57-hSOX9 were connected into pAAV-hSOX9-IRES-tdTomato by enzyme digestion method. The adeno-associated virus was packaged with plasmid co-transfections method. The recombinant pAAV-hSOX9-IRES-tdTomato was transfected into 293AAV cell by calcium phosphate transfection. The purification and drop of adeno-associated virus was tested by determination of biological titer. RESULTS AND CONCLUSION: The results of BLAST sequence comparison analysis showed that, pAAV-hSOX9-IRES-tdTomato exactly matched the synthetic gene sequence hSOX9. The titer is 1×107 TU/mL. Human gene SOX9 recombinant adenoviruses, pAAV-hSOX9-IRES-tdTomato, have been constructed successfully.