In order to study the expression and the feasibility of scaled production of neuropeptide in the routine expression system such as E.coli with the pituitary adenylate cyclase activating polypep tide(PACAP) as an example, the following experiments were carried out. First, on the basis of the reported amino acid sequence of PACAP, DNA sequence of PACAP w as deduced and six partially complementary oligonucleotide fragments were design ed. The coding region of PACAP was obtained by renaturing the DNA fragments and ligation and identified by DNA sequencing. The coding region of PACAP was cloned into plasmid pGEX-4T-3 and transformed into E.coli BL21(DE3 ). An expression strain BLPACAP was selected. SDS-PAGE analysis revealed that t he GST-PACAP fusion protein was highly expressed and accumulated to about 30% o f the total bacterial proteins. By affinity chromatography, up to 90% GST-PACAP was purified by one step from bacterial lysate. The purified protein could prom ote neurite outgrowth of PC12 cells and the survival of spinal cord neurons.

Objective To investigate the effect of the genotypic analysis of donor from cardiac death donation on the initial dose of Tac for liver transplant recipients and provide individualized administration for the early use of Tac in liver transplantation patients.Method Thirty recipients with a different genotype of CYP3A5 from cardiac death donors were collected from March 2010 to February 2013.The matched recipients were randomly divided into experiment group and control group.There was an adjustment of initial doses of Tac according to the donors' different CYP3A5 genotypes in experiment group but not in control group.Result In experiment and control groups,the average Tac blood concentrations at the 7th day after operation were (7.47 ± 1.83) and (8.68 ± 5.14) ng/mL,and the percent of recipeints reaching the optimal Tac concentrations was 72.2％ and 38.9％,respectively (P＜0.05).In experiment and control groups,22.2％ and 55.6％ recipients needed adjustments of Tac concentrations respectively (P＜0.05).Conclusion Individualized adjustment of Tac initial doses of recipients according to cardiac death donors' different CYP3A5 genotypes was benefit for reaching optimal concentrations as soon as possible and could decrease the rate of rejection,and reduce the side effects of Tac.

Objective The aim of this study is to investigate CD+8 natural killer T cell receptors NKG2D and NKG2A expression in peripheral blood of patients with lung cancer and discuss the relation between imbalance expression of NKG2A and NKG2D and tumor immune escape. Methods Flow cytometry was used to determine the percentage of NKG2D and NKG2A-expressing of CD+8 NKT cells in peripheral blood of 95 untreated lung cancer patients and 50 healthy controls. Results NKG2D was lower expressed on CD+8 NKT in lung cancer, the level of NKG2D in patients (77.07±5.77) % was significantly lower than that in the controls (84.13±4.49) % (t =8.14, P <0.05). In the TNM stage, the level of NKG2D in patients of Ⅰ-ⅢA, ⅢB, Ⅳ stage were (81.07±5.02) %, (76.95 ±4.70)%, (72.80±5.16) %, respectively, the level of NKG2D was significantly decreased in order (F =18.74, P <0.05). NKG2A was expressed higher on CD+8 NKT in lung cancer, the level of NKG2A in patients (33.58±8.82) % was significantly higher than that in the controls (25.31 ±8.38) % (t =-5.46, P<0.05). In the TNM stage, the level of NKG2A in patients of Ⅰ - Ⅲ A, ⅢB, Ⅳ stage were (25.10±6.93) %, (33.24±3.76) %, (43.64±6.10) %, respectively, the level of NKG2A was significantly increased in order (F =75.73,P <0.05). Conclusion Imbalance expression of NKG2A and NKG2D may restrain the function of CD+8 NKT cell of lung cancer patients in peripheral blood and that may be one of important factors in tumor immunological escape.

Objective To investigate the significance and expression of PTEN, MLL in T lymphoblastic lymphoma/leukaemia(T-LBL/ALL). Methods Seventy-six cases of T-LBL/ALL were studied by using immunohistochemical EnVision method for PTEN. Fluorescence in-situ hybridization (FISH) for MLL gene (located on chromosome 11q23) was performed to detect its breakage and amplification. Results Among the 76 cases ofT- LBL/ALL, the positive rate of PTEN was 64.47 % (49/76), lower than that in reactivated lymphoid tissue (100 %, 20/20) (λ2= 19.220, P ＜0.05). PTEN expression was reversely correlated to theclinical stage, Ki-67 index and LDH level (P ＜0.05). Among the 76 cases, MLL gene with breakage of 11q23 was detected in 13 cases (17.11%), and amplification in 18 cases (23.68 %). Survival rate ot MLL gene breakage group was lower than that of non-breakage group (25.0 %, 43.6 %). Survival rate of MLL gene amplification group was lower than that of non-amplification group too (17.1%, 42.7 %). Both of breakage and amplification were related to prognosis ( λ 2 = 11.357, λ 2 = 4.533; P ＜0.05). Conclusion Anti-oncogene PTEN down-regulation may play an important role on the development and proceeding of T-LBL/ALL. MLL gene with breakage and amplification of 11q23 are helpful to predict prognosis of T-LBL/ALL. The case with MLL gene breakage and amplification of T-LBL/ALL may have a poor prognosis. It hints this group maybe a subtype of T-LBL/ALL.

Objective To study the expression of pl6, FHIT genes in cervical carcinoma and its clinical significance. Methods By immunohistochemistry SP method, the expression of pl6, FHIT in different 118 cases of cervical lesions were detected and the results were analyzed in combination with clinical pathological features. Results Of 118 patients, 15 cases suffered cervicitis;38 cases took place cervical tumor-like changes;65 cases caught cervical cancer. p16 expression rates were 0, 33.3 %, 70.0 %, 87.5 %,and 92.3 % respectively;while FHIT expression rates were 73.3 %, 75.5 %, 60.0 %, 37.5%, and 30.8 % respectively. Compared with cervicitis, pl6 and FHIT expression rates in the cervix tumor-like changes,cervical carcinoma had significant difference (P <0.05). There was positive correlation in protein expression between p16 and FHIT (x2 =33.33, P <0.001). Conclusion Combination of p16, FHIT detection can be used as early diagnostic tool of cervical lesions and cervical cancer markers;meanwhile, the method can serve as a clinical evaluation of tumor biological behavior and prognosis of auxiliary indexes.

Auto liver transplantation (ALT) has been tremendously popular in hepatic surgery for the liver masses due to lack of enough donor for allogeneic transplantation of liver.But ALT remains stagnant because it is technically more difficult than liver transplantation.Much difficulties in this field lying ahead.Related surgical technical requirements for surgeons operating ALT,complications,difficult liver resection,hypothermic liver perfusion,veno-venous bypass,ex vivo ECMO perfusion and liver trim,assess the quality and volume of autoplast,autoplast implant and vascular anastomosis.On the other hand,the therapeutic effect largely depends on the intraoperative vascular separating range,the location and size of the tumor,the scope of lymphoid infiltrates by neoplast,the intubation site for perfusion and the sequence of opening the occlusion vessel.Thus,it's necessary to set up a scientific,normative ALT procedure to improve the therapeutic effect and prognosis.

[Objective]This study was designed to construct a recombinant adenovirus vector contains LMP-1 gene,and investigate the osteoinductive activity of MSC which were transfected recombinated adenoviral vector carrying LMP-1 gene.[Methods]Total RNA was extracted from mt osteoblast and the LMP-1 gene was acquired by RT-PCR,the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology,then the entry clone and the expression vector were used to create the expression clone throush the LR recombination reaction.The adenovirus expression clone was linearized by PacI and transfected to the 293A cell line to harvest a high titer.Ad-LMP-1 was infected into the 3rd passage MSC,the expression of LMP-1 was detected by Western blot.The osteogenic activity of MSC was evaluated by the expression of collagen Ⅰ,ALP,osteocalcin and the formation of bone nodule.[Result]The LMP-1 gene was successfully acquired and confirmed,the entry clone and the expression clone were both verified by enzymes digestion,and the expression clone was further confirmed by sequenced.The expression of LMP-1 was detected successfully in MSC.The increasing expression of collagen Ⅰ,osteocalcin.ALP and bone nodule were observed by comparing to the control group.[Conclusion]Gateway technology not only make construction of the pAd-LMP-1 recombination adenovirus vector simple and fast,but also get a high transfection efficacy in MSC.LMP-1 gene can induce the osteoblast differention of MSCs,and improve its osteogenic activity.The adenovirus vector is reliable to be used in further gene therapy research.

Objective To evaluate the roles of Toll-like receptor 2 (TLR2),TLR4 and myeloid differentiation factor 88 (MyD88) in immune recognition and immune mediation in sporotrichosis by detecting their expressions in skin lesions of sporotrichosis.Methods Biopsy specimens were obtained from the skin lesions of 19 patients with sporotrichosis and normal skin of 12 healthy human controls.Immunohistochemistry was performed to observe the expressions of TLR4 and MyD88,and real-time fluorescence-based quantitative PCR to quantify the expressions of TLR2 and MyD88 mRNAs.Data were expressed as mean ± standard error.Statistical analysis was done with the SPSS17.0 software.Independent samples t-test was conducted to compare the parameters between the lesional and control specimens.A p-value of less than 0.05 was considered to be statistically significant.Results TLR4 and MyD88 were mainly observed in the whole epidermis except the stratum corneum as well as plasma cells and lymphocytes in the dermis and subcutaneous tissue in lesional skin.However,TLR4 was nearly absent and MyD88 was weakly expressed in the dermis and subcutaneous tissue in normal skin.The expression levels of TLR4 and MyD88 were significantly higher in the lesional skin than in the control skin (TLR4,63.767 ± 3.829 vs.5.167 ± 3.246,t =4.82,P ＜ 0.05; MyD88,57.236 ± 4.744 vs.10.588 ± 1.640,t =3.30,P ＜ 0.05).Similarly,the lesional skin showed significantly stronger expressions of TLR2 and MyD88 mRNAs compared with the normal skin (TLR2,1.974 ± 1.452 vs.1.430 ± 1.073,P ＜ 0.05; MyD88,2.028 ± 2.061 vs.0.688 ± 0.422,P ＜ 0.05).Conclusion Sporothrix may induce the development of sporotrichosis by interacting with host immune system via TLR signaling pathways.

Objective To study the intraoperative and postoperative complications of autologous liver transplantation (ALT),and their prevention and treatment.Methods From October 2005 to December 2011,our center carried out 36 cases of ALT for malignant (n=23) and benign diseases (n=13).Intraoperative and postoperative complications and treatment methods were analysed.Results Of the 36 patients,2 patients developed small liver syndrome in the perioperative period.Allogeneic liver transplantation was carried out for 1 of these two patients for acute liver failure.Another patient died of lung infection 16 days after the surgery.Among 36 ALT recipients and 23 patients suffering from malignant tumor,1,2,3-year survival rates were.75％,71％,68％ and 65％,59％,54％ respectively.Conclusions With adequate preoperative assessment,the incidence of serious complications after ALT should be low.Prompt prevention and treatment of intraoperative and postoperative serious complications could cut down perioperative mortality,and provide long-term survival after ALT.

Objective To explore the dilution regression method of coagulation detection(PT ,APTT) in fat blood samples . Methods We collected 40 normal blood coagulation specimens (no fat blood ,no jaundice ,no hemolysis) in Yan′an hospital of Kun-ming ,then we detected the PT and APTT of the original plasma and 3-fold diluted plasma and 5-fold diluted plasma ,the we used the data both of before dilution and diluted to do the linear regression analysis ,and finally we got the regression equations of each index .we also collected 33 fat blood samples in Yanan hospital of Kunming ,which be divide into three groups through the severity of triglycerides :mild fat blood group(1 .7 mmol/L≤TG<11 .0 mmol/L) and moderate fat blood group(11 .0 mmol/L≤TG<20 .0 mmol/L)and severe fat blood group(TG≥20 .0 mmol/L) ,then we detected the PT and APTT after 3-fold diluted plasma and high-speed centrifugation plasma ,and then we brought diluted results into the normal regression equations and the results were compared with the high-speed centrifugation results .Results Because most of the 5-fold diluted plasma can not get the effective results ,so we use 3-fold diluted plasma to get the regression equations .The results of 3-fold diluted plasma was calculated by the regression equa-tions ,which then compared with high-speed centrifugation results ,after the analysis of statistical software ,two results had not sta-tistically significant (P>0 .05) .Conclusion Dilution regression method can be used to detect the fat blood samples in the clinical coagulation detection .