Objective To construct the recombinant plasmid of fusion protein containing respiratory syncytial virus (RSV) cytotoxicity T lymphocyte (CTL) epitope M2:81-95 and heat shock protein (HSP) 70L1, and to investigate its immunogenicity after prokaryotic expression. Methods HSP70L1 gone was cloned from SMMC7721 cells. The M2:81-95 gene fragment was amplified by polymerase chain reaction (PCR) method. Plasmid pET-HSP70L1-M2 : 81-95 (pET-HSP70L1-M2) was constructed, identified and transferred into E. coli BL21 (DE3). Expression of HSP70L1-M2 : 81-95(HSP70L1-M2) was induced by isopropy-β-D-thiogalaetosidc (IPTG). The expressed protein was purified by affinity chromatography and renatured by gradient dialysis. The BALB/c mice were immunized with this fusion protein. IgG antibodies and the subtypes were detected by enzyme-linked immunosorbent assay (ELISA), and CTL responses were determined by methyl thiazolyl tetrazolium (MTT). Results The recombinant plasmid pET-HSP70L1-M2 was successfully constructed. The fusion protein HSP70L1-M2 was expressed in E. coll. The purified protein induced strong RSV-and CTL epitope-specific CTL responses and high titer of protein specific lgG antibody 4.87±0.35. The subtypes were IgG1 (5.53±0.28) and lgG2a (4.40±0.21) and IgG1/ IgG2a ratio was balanced. The titers of lgG, IgG1 and IgG2a in PBS control group were 0.33±0.17, 0.51±0.21 and 0, respectively, which werc significantly lower than those in immunized group (t = 3.512, 3.681, 5.856; P<0.05). Conclusions The recombinant plasmid pET-HSP70L1-M2 is successfully constructed and the fusion protein is expressed and purified. HSP70L1-M2 induced strong RSV-and CTL epitope-specific CTL responses and mixed T helper cell (Th)1/Th2 response in BALB/c mice.

Objective: To investigate the regulation of respiratory syncytial virus CTL epitope fused with G protein antigen fragment G1 to the specific immunoresponses. Methods: The recombinant plasmid pET-DsbA-G1 or pET-DsbA-G1F/M2 was transferred into E.coli BL21(DE3) and the fusion protein DsbA-G1F/M2 or DsbA-G1 was expressed.The expressing product was induced and purified by affinity chromatography. The two proteins were used to immunized BALB/c mice i.p, respectively. Serum and spleen cells were collected regularly. RSV-specific CTL responses were measured by MTT, IgG and IgG1 and IgG2a antibodies by ELISA, neutralizing antibodies by plaque reduction assay. Results: The recombinant proteins were expressed successfully and the purity was over 90% after purified by affinity chromatography. The protein DsbA-G1F/M2 induced significant RSV-specific CTL responses, while DsbA-G1 without CTL epitope did not induce detctable CTL responses. Strong IgG antibody responses were elicited,indicated by both. IgG1 and IgG2a titers induced by DsbA-G1F/M2, while only IgG1 was induced by DsbA-G1.The ratio of IgG1/IgG2a was downregulated significantly. Both antigens induced high level of neutralizing antibodies, but the latter was a little lower. Conclusion: DsbA-G1F/M2, the fusion protein of a CTL epitope and G protein fragment G1 can induce both cellular immunity and humoral immunity. The activation of CTLs downregulates the ratio of IgG1/IgG2a.The more balanced immunoresponse is advantageous for improving safety of the candidate vaccine.

Objective To evaluate the value of diagnostic negative MDCT cholangiopancreatography in patients with suspected obstructive biliary diseases by receiver operating characteristic(ROC)curve. Methods Dual-phases contrast-enhanced MDCT of the abdomen was performed in 30 patients.Multiplanar reformation,minimum intensity projection and volume rendering were generated using original data in vein phase.By double-blind method,the images were readed by two radiologists.ROC curves were analysed with software SPSS11.5,the different results of reading images between two radiologists was evaluated with Kappa test.Results The Az of the area under the ROC curve of MDCT cholangiopancreatography for the two observers was 0.968 and 0.962 respectively,it was more than 0.9,which showed that the value of diagnosing the suspected obstructive biliary diseases by negative MDCT cholangiopancreatography was good.Kappa index test was satisfactory.Conclusion Negative MDCT cholangiopancreatography may be a routine choice in diagnosing the suspected obstructive biliary diseases.

Objective:To investigate whether His-tag to change the immunogenicity of recombinant protein G1F/M2 of respiratory syncytial virus.Methods:The G1 and F/M2 gene fragments were amplified by PCR method and then ligated into the expressing vector pET-His or pET-DsbA-His.Each recombinant plasmid was transferred into E.coli BL21(DE3) and the expression was induced by IPTG.The expressed His-G1F/M2 or DsbA-His-G1F/M2 was purified by affinity chromatography.The latter was digested with thrombase and G1F/M2 was purified by affinity chromatography.His-G1F/M2 or G1F/M2 was used to immunize BALB/c mice.Anti-RSV antibody was measured by ELISA and RSV-specific CTL responses by MTT.Results:No significant difference was observed between the level of anti-RSV antibody or RSV-specific CTL response induced by G1F/M2 and that by His-G1F/M2.Conclusion:His-tag does not change the immunogenicity of recombinant protein G1F/M2 of respiratory syncytial virus.

Background Rapamycin(RAPA)is a specific inhibitor of the mammalian target of rapamycin (mTOR).Researches showed that RAPA inhibits the proliferation of lens epithelium cells(LECs)and tumor cells and induces apoptosis of tumor cells.To investigate whether rapamycin has the inhibitory effect on retinal pigment epithelium(RPE)cells is very important for the prevention and management of proliferative vitreoretinopathy (PVR).Objective This study was to investigate the effects of RAPA on the proliferation and apoptosis of human RPE cells in vitro.Methods Human RPE cells(D407 strain)were cultured and passaged and then divided into regular culture group(blank control group),DMSO control group(0.1‰ DMSO +regular culture),and different concentrations RAPA-treatment groups(5,10,20,40,80,160,320 nmol/L).The proliferation(A490)of human RPE cells was detected using MTT,and the inhibitory rates of RAPA on the proliferation of RPE cells were calculated and compared among different groups at 12,24 and 48 hours.The apoptosis rates of the cells were analyzed among various groups by Hoechst staining after 12,24,48 hours.Results The inhibitory rates of RAPA on RPE cells were significantly different among various groups(F=484.451,P＜0.01)and evidently elevated in 20-320 nmol/L RAPA groups compared with DMSO control group(P ＜ 0.01).The inhibition of RAPA on the cells was considerably enhanced as the lapse of time(F=232.262,P＜0.01)with more dominant effects in 24 and 48 hours compared to 12 hours after addition of RAPA(P＜0.05-0.01).Compared with blank control group and DMSO control group,the apoptotic rates of the cells were evidently increased in 12,24,48 hours in 10 nmol/L RAPA group(all P＜0.05),and higher cellular apoptotic rates were found in 20-320 nmol/L RAPA groups(all P＜0.01).The alteration of cellular apoptotic rate showed a gradually incremental trend as the acting time of RAPA(F =625.584,P＜0.01).Karyorrhexis and mass-like density staining and chromatin substance were seen in RPE cells under the fluorescence microscope in ≥ 10 nmoL/L RAPA groups.Conclusions RAPA suppresses the proliferation and induces the apoptosis of human RPE cells in concentration-and time-dependent manner in vitro.

Objective To summarize the main nursing points of toxic epidermal necrolysis. Methods On the foundation of conventional therapy, an overall assessment was carried out among 10 patients with toxic epidermal necrolysis. On loose skin with erythema, a combination of zinc oxide and talcum powder was externally applied to skin lesions where blisters were not broken in order to promote dry-style exfoliation of the skin lesion. After infrared irradiation, gauze containing MEBO was applied externally to skin lesions with eroded secretions to moisturize them, thus facilitating healing of the skin lesion. Meanwhile, mucosa of special part of patient's body was well nursed. Protective isolation was enhanced in order to reduce secondary infection. The patient's conditions were observed closely. Diet guidance was also done. Results All the patients were dry-style exfoliated with treatment ranges reaching up to 30%to 60%of the affected area. Dry-style exfoliation time was between 5 to 10 days, with an average of 7.20 ±1.69 days. The area of skin lesion erosion ranged from 10% to 60%. Following the external application of MEBO gauze to moisturize and heal, skin lesion healing time ranged from 7 to 18 days with an average of 13.70 ±3.40 days. Conclusion According to the specific situation of toxic epidermal necrolysis, targeted nursing and treatment can promote the dry-style exfoliation of skin lesions, reduce the area of skin erosions, alleviate the suffering of patients and promote healing of the skin lesion.

Objective To observe the effects of ketamine on the patients with depression re-ceiving modified electric conulsive therapy (MECT).Methods Sixty patients with depression were randomly divided into ketamine group and propofol group (n =30 each group).Atropine 0.5-1.0 mg, propofol 1.0 mg/kg or ketamine 0.8 mg/kg i.v.were given before MECT,Scoline 0.7-1.0 mg/kg i. v.was given after the eyelash reflex disappeared.Hamilton Depression Rating Scale (HAMD)was completed after the 2 nd ,4 th and 6 th MECT,the time of convulsion,twitch index,energy percentage, respiratory recovery time and adverse reactions were recorded.Results The total score of HAMD was significantly decreased with the increasing times of MECT in both groups,compared with propo-fol group,ketamine group's HAMD total score decreased faster,especially after the 4th MECT,the score decreased significantly in ketamine group (P <0.05).The time of convulsion,twitch index,en-ergy percentage, respiratory recovery time, adverse reactions all had no statistical significance between the two groups.Conclusion Compered with propofol,ketamine,as an anesthetic of MECT, can effectively lower the score of HAMD.

Objective To investigate the causes and managements of dys-seal syndrome (DSS) developed after esophageal stent placement. Methods From June 2001 to June 2008, esophageal stenting was performed in 98 consecutive patients with malignant esophageal obstruction. A total of 99 metallic stents were used. Of 98 patients. gastroesophageal anastomosis stricture was seen in 19, preoperative radiotherapy history in 26 and tracheoesophageal fistula in 34. Results DSS occurred in 7 patients, with an occurrence rate of 7.14% ,which was significant higher than that in patients with preoperative radiotherapy history and in patients showing marked dilated esophagus proximal to the obstructed site (X~2=0.017, 0.005, P=0.036, 0.013, respectively). After treatment, such as fasting, IPN or nasogastric feeding,only 1 case retumed to semi-liquid diet. Among the rest 6 cases of DSS, an additional stent was employed in one (but in vain), nasogastric feeding tube was used in 2, and removal of the stent under endoscopic guidance was carried out in 3. Conclusion DSS is one of the complications developed after esophageal stent placement, its prognosis is rather poor. Removal of the inserted stent may be the optimal treatment. The prevention of DSS includes proper pre-operation evaluation, selection of suitable stent, enhancement of perioperative nutritional support, etc.

Objective To evaluate the efficacy of Fluency stent-graft (Bard Corp) in transjugular intrahcpatic portosystemic shunt (TIPS).Methods The clinical data of 21 consecutive patients treated by TIPS using Fluency stent-grafts were retrospectively reviewed.All of them were recurrent variceal bleeding secondary to portal vein hypertension,1 was bleeding secondary to primary hepatic carcinoma with port vein thrombns,and 1 was Budd-Chiari syndrome.They were followed-up after (10.1 ± 4.6) months (2.0 to 24.0 months).Stent-grafts patancy,portal vein pressure and liver function were recorded and compared.Results Twenty-five stent-grafts were successfully implanted in 21 patients,23 stent grafts were 8 mm 2 were 10 mm in diameter.The covered length of the stents varied from 6 to 8 cm.The bleeding was stopped and the portal vein pressure decreased significantly from (25.4 ± 3.5) mm Hg to (15.4 ± 2.8) mm Hg (t = 12.495,P < 0.01).During the follow-up period,The patient with primary HCC and portal vein thrombosis died 4 months after the procedure. One case had a new primary HCC during the follow-up and died 24 months after the procedure.One ease with variceal bleeding secondary to portal vein hypertension died of muhisystem organ failure.One case occluded in the hepatic vein and had another stcnt graft implanation.The other 17 cases had no stenosis after 7 to 17 months follow-up.Ultrasound showed that the stents were patent 1 week before the patients died.Three cases had transient symptoms of hepatic encephalopathy and recovered after treatment.The Child scores of the 19 patients survived more than 6 months were 6.3 ±1.4 before and 6.4 ± 1.9 after the procedure without significant difference (t = 0.645,P > 0.05).Conclusion The Fluency stent-grafts could increase the patency of the TIPS,but its efficacy on the long-term effect and hepatic encephalopathy need further investigation.

Objective To investigate the effect of dexmedetomidine on excitatory aminoacid (EAA)and inhibition of amino acid(IAA)in cerebro-spinal fluid(CSF)of patients undergoing in-tracranial tumor surgery,and to explore the cerebral protective mechanism of dexmedetomidine in neurosurgery.Methods Sixty patients aged 18-64 years old,ASA Ⅰ or Ⅱ,weighing 50-90 kg un-dergoing elective intracranial tumor surgery were randomly divided into dexmedetomidine group (group D)and control group(group C).Dexmedetomidine 1 μg/kg was infused before anesthesia in-duction for more than 10 minutes and pumped continously with 0.2-0.7 μg·kg-1·h-1 in group D, while in group C midazolam 0.03-0.05 mg/kg was injected followed by intermittent administration of 0.03-0.05 mg/kg.BIS value was maintained between 40-50.MAP and HR was recorded at the time points before induction(T0 ),dura mater incision(T1 ),tumor resection(T2 ),at the end of the surgery (T3 ).And we collected CSF at T0 ,T3 ,6 hours after the surgery(T4 ),12 hours after the surgery (T5 ),24 hours after the surgery(T6 ),then the concentrations of EAA and IAA were determined with high-performance liquid chromatography (HPLC)at T0 ,T3 ,T4 ,T5 and T6 .Results The MAP and HR in group D at T1-T3 were much lower than that in T0 and in group C(P <0.05).Compared with T0 ,the Glu and Asp in CSF significantly increased in group C at T3-T6 and were much higher than those in group D (P < 0.05 ),GABA was significantly decreased and much lower than group D(P <0.05).Compared with T0 ,the Glu and Asp in group D at T3-T5 were increased and GABA was decreased, but without statistic significance. At T6 , the values recovered to the level at T0 . Conclusion Dexmedetomidine can be used to maintain hemodynamic stability in intracranial tumor surgery,and may play a role in cerebral protection through inhibiting expression of Glu and Asp (EAA).