Objective To recombine the tissue plasminogen activator (TPA) gene and establish the expressing model in vitro to provide both theoretical basic and technical guidance for gene therapy of ischemic heart disease and prevention of postoperative vessel re-stenosis. Methods Expression vector pcDNA3.1 TPA was constructed, and transfected into Chinese hamster ovary(CHO) cells. Then the exogenous TPA expression was observed. Results The expression quantity of TPA in transfected CHO cells was higher than that in non-transfected cells. The exogenous TPA activity was 12 296 IU/10 6cell/24hr in the transfected cells, when measured by chromogenic substrate assay, while that of non-transfected cells was 3 176IU/10 6cell/24hr. The TPA quantity of non-transfected cells was 9 608 ng/10 6cell/24hr, when measured by enzyme-linked immunosorbent assay (ELISA), while that of transfected cells was 586 172 ng/10 6 cell/24hr, and the latter was the 60 times of the former. Conclusions When pcDNA3 1(+)TPA was transfected into CHO cells, exogenous TPA can efficiently be expressed, which provides theoretical basic for TPA clinical gene therapy.

To clone and express 27 kDa cysteine protease (CP) gene of Spirometra erinacei plerocercoid,and analyze the biology characteristics,a total of RNA was extracted from the plerocercoids and reversely transcribed into cDNA.The 27kDaCP gene was amplified by PCR and cloned into pM-19T vector for sequencing.The accurate sequence was subcloned into the expression vector pET-28a (+).The recombinant plasmid was transformed into Transetta (DE3) and the expression protein induced by IPTG.The recombinant protein was purified by Ni2 + affinity chromatography,and analyzed by SDS-PAGE and Western blotting.The 27 kDa CP gene and its expression protein were predicted and analyzed by bioinformatics analysis tools such as NCBI and ExPASy.Results showed that the ORF length of 27 kDa CP gene sequence was 1 011 bp,and the removed signal peptide sequence was 954 bp with the submission number of ANA52569 in GenBank.The whole sequence of 27 kDa CP (Mr 35 669.9,pI 5.92) was 317 amino acids conferred from cDNA,which belongs to the Peptidase_C39_like superfamily.The pET-28a (+)-27kDa-CP was expressed under the induction of IPTG.Western blotting analysis showed that the purified protein reach expectancy,and had better response with positive serum of Spirometra erinacei plerocercoid infection.In conclusion,the 27 kDa CP gene of Spirometra erinacei plerocercoid is successfully cloned and expressed and knowing coded sequences and bioinformatic.

Objective To establish the fingerprint chromatogram of Buzhong Yiqi Decoction(BYD) by multiple wavelength method,and to analyze the substance of formulas as a whole.Methods HPLC-PDA method was adopted.The chromatographic conditions were as follows:Hypersil ODS2 analytical column,the mobile phase consisting of 0.05 %phosphate acid and acetonitrile with gradient elution,detecting wavelength at 254 and 280 nm,the flow rate being 1.0 mL/min and the temperature of the column at 30 ℃.Results The methodological results were good.The results of dual-wavelength fingerprint chromatogram showed the all-around information of the fingerprints at 254 and 280 nm.The similarity of 10 batches of BuzhongYiqi Decoction was over 0.98.Conclusion Dual-wavelength fingerprint chromatogram can realize the analysis of a formulas as a whole,which supplies references for improving the quality control of BuzhongYiqi Decoction.

Objective To observe the immunoglobulin A secreting cells (IgASCs) and secretory IgA (sIgA) level in jejunum of mice infected with plerocercoids,and to understand the roles of resistance to invasion processes of the plerocercoids.Methods A total of 100 Kunming mice (half males and half females) were chosen,the weight was 20-25 g,they were randomly divided into control and experiment groups according to their body weight via the random number table method,50 per group.Mice of experiment group were fed each with 5 plerocercoids in snakes,and mice of control group were not infected,testing time and methods were the same in the two groups.Ten mice were randomly sacrificed from one group on days 1,7,14,28 and 56 after infection,to collect empty intestinal juice and jejunal segment.The immunohistochemical method was used to examine the quantity of IgASCs in jejunal mucosa,while enzyme-linked immunosorbent assay (ELISA) was applied to test the level of slgA of jejunal fluid.Results The IgASCs were distributed in lamina propria of the jejunal mucosa,and the percentage of positive IgASCs reached the peak value [(64.24 ± 0.60)％] at d 1 after infection in experiment group,then decreased,and it was lower than control group at d 14 [(41.98 ± 0.42)％ vs (43.52 ± 0.94)％,t =-4.727,P ＜ 0.01].The sIgA level reached the peak value [(22.05 ± 1.43) mg/L] at d 7 after infection in experiment group,then decreased,and there was no statistical significant difference between control and experiment groups at d 56 [(20.00 ± 0.42) mg/L vs (21.26 ± 2.59) mg/L,t =1.516,P ＞ 0.05].There was a positive correlation between the percentage of positive IgASCs in the jejunal mucosa and sIgA level in the jejunal fluid at d 7 after infection (r =0.663,P ＜ 0.01),and there was a negative correlation between them at d 14 after infection (r =-0.542,P ＜ 0.05).Conclusion The plerocercoids infection might induce high level expressions of IgASCs and sIgA,they show positive correlation at d 7 after infection.

Objective:To test the validity and reliability of the General Alienation Scale (GAS)in college students sample.Methods:Totally 1200 college students were surveyed,using classical test theory (CTT)and i-tem response theory (IRT)to test the psychometric properties of GAS.Under the framework of CTT,item selec-tion was performed with all data.Then,the sample was divided into two groups based on the input order to exam-ine the construct validity.Exploratory factor analysis (EFA)and parallel analysis (PA)were performed with odd group,while confirmatory factor analysis (CFA)was performed with even group.Criterion validity was examined with the short-form of the University of California Los Angeles Loneliness Scale (ULS-8),General Self-Efficacy Scale (GSES),Rosenberg Self-Esteem Scale (RSES).Under the framework of IRT,all data was fitted the rat-ing scale model (RSM),including the principal component analysis of residuals (PCAR),the item fit indexes (Infit and Outfit),measurement reliability (item separation index,item reliability,person separation index and person reliability)and the Wright map.Results:Under the framework of CTT,the GAS was retained 13 items except item 2 and 5.The results of both EFA and PA showed that 13 items of GAS was one-dimensional,and there was a good fit of CFA (χ2 /df=2.689,P <0.001,CFI =0.897,TLI =0.877,RMSEA =0.055,SRMR =0.046).The correlation coefficients were 0.57, -0.60 and -0.40 with the GAS,ULS-8,GSES,RSES
scores respectively (Ps <0.001 ).The Cronbach 's αcoefficient and confidence interval were 0.81 (0.80, 0.83).Under the framework of IRT,the ratio of first-paired eigenvalue was 1.6 in PCAR.The infit and outfit of retaining 13 items in CTT were from 0.6 to 1.4 excluding item 4.The item separation index,item reliability,per-son separation index and person reliability of the 12-item GAS were10.91,0.99,2.14 and 0.82 respectively.The item difficulty can matched with person ability,and they were closed to the normal distribution.Conclusion:It suggests that the General Alienation Scale (GAS)is of good validity and reliability,and it could be used as an ef-fective tool for measuring the alienation of college students.

AIM: To establish an effective and convenient method for applying HPLC fingerprints to quality control in the production of Xiasangju Granules(Spica prunellae,Folium mori,Flos chrysanthemi). METHODS: Komasil Sunfrie C_(18)(150 mm?4.6 mm,5 ?m) analytical column was used and eluted with a gradient program consisted of phase A(1% acetic acid) and phase B(methanol) and detected at 290 nm.The fingerprints of aqueous extract,alcohol-precipitated extract,concentrate and finished product were compared with. RESULTS: The fingerprint method for Xiasangju Granules was established.The similarity among 10 batches of Xiasangju Granules was no less than 0.970.The difference between extracts and finished product of Xiasangju Granules was obvious. CONCLUSION: This validated method is available for quality evaluation and quality control in Xiasangju Granule's production.

ObjectiveTo explore the effects of hemofiltration on serum enzyme (SE),endotoxin (ET) and malonaldehyde (MDA) of dogs with heat stroke caused shock.MethodsSixteen healthy male hybrid dogs were randomly divided into 2 groups,8 for each:as heat stroke group (HS group ) and hemofiltration group (HF group).Severe heat stroke model was induced with high temperature.The dogs were taken out of the heating cabin when it reached heat stroke level,and then observed under normal temperature without treatment.The dogs in HF group was immediately treated with hemofiltration.The changes of SE,ET,MDA of two groups of dogs were observed and the survival time between two groups was compared,ResultsThe time from heat exposure to shock was ( 107.00 ± 28.52 ) and ( 111.38 ± 22.24 )minutes in HS group and HF group respectively ( t =- 0.354,P =0.729 ).The SE ( CK,LDH,ALT,AST) of the dogs were all higher after heat stroke,and the dogs of two groups showed no siginificant difference (P ＞ 0.05).At three hours after heat stroke,the SE increased apparently in HS group and HF group,but the level was significantly lower in HF group. Before heat stroke,the serum ET showed no siginificant difference between two groups ( P ＞ 0.05 ).After heat stroke,the serm ET was much higher than before ( P ＜0.01 ),but there was still no siginificant difference between two groups ( P ＞0.05 ).At three hours after heat stroke,the ET increased both in HF group and HS group,but the level was lower in HF group.Before heat stroke,the serm MDA had no siginificant difference between two groups ( P ＞ 0.05 ).After heat stroke,the serm MDA was much higher than before ( P ＜ 0.0l ),but there was still no siginificant difference between two groups (P ＞ 0.05 ).After heat stroke in three hours,the MDA of HS group rose apparently while HF group slowly declined.The median survival time of HF group was 180 min while HS group was 75 min,the survival rate showed siginificant difference (P ＜ 0.01 ).Conc4usions HF can improve the prognosis of dogs with heat stroke caused shock,prolong its survival time,reduce mortality.The mechanism is probably that HF clear serum MDA,partially clear serum ET and then eventually reduce cell and tissue injury and reduce SE.

Objective To study the change of Hesperidin content in various combinations of medicine from Buzhong Yiqi Decoction(BYD) by RP-HPLC.Methods The hesperidin content was determined by HPLC and analyzed by L8(27) orthogonal design and statistic analysis(SPSS).Results Principal herbs and ministerial herbs of BYD had a significant effect on the hesperidin content of adjunctive herbs(P

Objective To study the pathogenesis of Entamoeba gingivalis ( E\^g .) and its relation to periodontal diseases.\ Methods Rats were treated with immuno\|inhibitor for one week and the neck of incisor teeth of the rats was bound with steel wire. They were randomly divided into three groups: the first group was infected by E\^g . in the periodontal tissue, the second group was infected by symbiotic bacteria (s.b.),and the third group was given physiological saline as control.Observation on the periodontal inflammation was made for each group of rats, and the purulent secretion from periodontal abscess was examined for living pathogens.\ Results The incidence of periodontal diseases in rats infected by E\^g . was higher than that of symbiotic bacteria group and that of control ( P

Objective: To establish the optimum preparation process for Xuesaitong Dropping Pills(total saponin of Radix Notoginseng). Methods: On the basis of orthogonal design, preparation conditions were selected among sort of polyglycol (as substance), consumption of polyglycol and processing temperature of usoline (as cooling agent). Results: Under the condition of using polyglycol 6000, and processing temperature at 10～15 ?C , the quality of Xuesaitong Dropping Pills is good.