Objective To optimize the method for total DNA extraction and RAPD analysis of Nervilia fordii (NF),and to study the genetic diversity of different breeds of NF,the substitute of NF and the fake of NF at molecular level.Methods We used low-pH extraction medium with high salt to extract total DNA,and used randomly amplified polymorphic DNAs (RAPD) to select polymorphism primer from 49 random primers.Twenty-two kinds of NF samples were analyzed by RAPD,and classified by SPSS.Genetic diversity were estimated by Shonnon's index and Nei's index.Results A higher puritiy of DNA can be obtained from fresh HF than that from medical materials.We selected 19 polymorphism primers for the cluster analysis of fresh NF and dried medical material.For medical materials of NF,the distance of amplification band of small-leave breed is close to that of middle-size leave breed,but is far away from that of,big-leave breed and Nervilia plicata.For the fresh materials of NF,there breeds of Nervilia fordii can be classified into one kind,the distance of fresh NF band is far from that of Nervilia plicata,Pachyrhizus erosus and the cultured breed,and more far awary from that of Plantago asiatica and Centella asiatica.Shonnon's genetic diversity is 0.463,Nei's genetic diversity is 0.267.Intra-population genetic variation is obvious compared to iner-population genetic variation.The estimated gene flow from Gst (Nm) is 0.94.Conclusion The molecular difference in different breeds of Nervilia Fordii can be used to identify Nervilia Fordii.The genetic diversity of Nervilia fordii is mainly caused by the geography environment.

Objective IFN-? is a pleiotropic cytokine with potent immunomodulatory effects and antiviral activity. To study the mechanism of IFN-? clearing duck hepatitis B virus (DHBV) in ducks, it is essential to establish a method to quantify expression of DuIFN-? in immune response. In the present study,a semi-quantitative competitive RT-PCR was developed to quantify expression of duck IFN-?(DuIFN-?) mRNA by PBMCs. Methods Based on ?-actin consensus sequence, fishing the ?-actin gene as house keeping gene from duck PBMC by RT-PCR. A competitive internal control was constructed and the competitive RT-PCR system could be used to quantify the transcription of DuIFN-? mRNA. Results After duck PBMCs were stimulated in vitro with PHA, the peak of DuIFN-? expression was at 24-36h. Then RT-PCR method was applied to detect DuIFN-? mRNA transcription by PBMCs from DHBV infected ducks immunized with DuIFN-? plasmid plus DNA vaccine or DNA vaccine alone. Results showed that expression of DuIFN-? in ducks co-immunized with DuIFN-? plasmid were higher than other groups immunized without DuIFN-? plasmid as adjuvant. Conclusions The results indicated that DuIFN-? gene could be a useful adjuvant to develop vaccines. The semi-quantitation of DuIFN-? mRNA by competitive RT-PCR provides the basis for future study of the mechanism of IFN-? in duck hepatitis B virus persistent infection.

Objective To investigate the effect of nursing with solution focused approach in advanced schistosomiasis pa?tients. Methods Fifty three patients with advanced schistosomiasis in the Liujiahu Hospital for Schistosomiasis Control of Yiyang City were randomized selected and separated into an intervention group and a control group. The patients of the interven?tion group received the conventional nursing plus solution focused approach,while the patients of the control group received on?ly the conventional nursing. The effectiveness of nursing and treatment was evaluated in the two groups comparatively. Results The knowledge of self?care skills,medical compliance behaviors and services satisfaction degree in the intervention group were higher than those in the control group(c2=3.78,2.87,4.09 respectively,all P<0.05). The occurrence rate of upper gastroin?testinal bleeding in the intervention group was significantly lower than that in the control group(c2=4.894,P<0.05). The aver?age hospitalization duration of the intervention group was shorter than that of the control group(t=4.17,P<0.05). Conclu?sions The solution focused approach is a feasible and effective method in course of nursing of advanced schistosomiasis pa?tients. It enhances the confidence of the patients and the trustiness of the patients to the health care providers. The complications and the hospitalization duration of the patients are reduced.

In drowning cases, it is difficult to judge whether the deceased died from drowning or throw-ing into the water after killed or identificate the cause of death of the decom posed corps in forensic practices. The diatom test is still considered as the im portant assistant evidence in drowning cases. This paper reviews research progress on technologies in recent years of forensic diatom test, and the applica-tion value of the newdeveloping approach in the field of forensic m edicine.

OBJECTIVE:To establish the quality standards for Shenshuaikang granule. METHODS:TLC was used for the quali-tative identification of Astragali Radix and Rhei Radix et Rhizoma in the preparation. HPLC was used for the contents determina-tion of emodin and chrysophanol ,the column was Agilent HC-C18 with mobile phase of methanol-0.1% phosphoric acid (85:15 , V/V)at a flow rate of 1.0 ml/min,the detection wavelength was 254 nm,the column temperature was 30 ℃ and the injection vol-ume was 10 μl. RESULTS:TLC showed clear spots and good separation. The linear range was 1.9-60.8 μg/ml for emodin(r=0.999 9, n=6) and 1.6-51.2 μg/ml for chrysophanol (r=0.999 9),RSDs of precision,stability and reproducibility tests were lower than 3%,recoveries were 95.76%-103.66%(RSD=2.83%,n=9)and 97.24%-104.34%(RSD=2.65%,n=9),respectively. CONCLU-SIONS:The standard can be used for the quality control of Shenshuaikang granule.

Multidrug resistance (MDR) plays a major obstacle to successful gastric cancer chemotherapy. The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine (Nef) in adriamycin (ADM) resistant human SGC7901/ADM gastric cancer cells. The MDR cells were heated at 42°C and 45°C for 30 min alone or combined with 10 μg/mL Nef. The cytotoxic effect of ADM was evaluated by MTT assay. Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique. Intracellular accumulation of ADM was monitored with high performance liquid chromatography. Mdr-1 mRNA, P-glycoprotein (P-gp), γH2AX expression and γH2AX foci formation were determined by real-time PCR, Western blot and immunocytochemical staining respectively. It was found that different heating methods induced different cytotoxic effects. Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells. The water submerged hyperthermia increased the cell membrane fluidity. Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM. The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp. The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells. The higher temperature was, the worse effect was. Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

Objective To produce a comprehensive transcript dataset of Oncomelania hupensis before and after Schistosoma japonicum infection,so as to provide experimental data for perfecting genetic structural information and excavating related mo-lecular markers of O. hupensis infected by S. japonicum. Methods O. hupensis snails were divided into the following 3 groups:one week after S. japonicum miracidium infection,4 weeks after S. japonicum miracidium infection,and normal condition. Mil-lion high-quality reads were obtained from the normalized cDNA of the pooled samples,which were assembled into transcripts. Results A total of 63686 unigenes were identified and were classified into 4 main categories,including general functional pre-diction(15.36%),signal transduction mechanism(11.75%),posttranslational modification(8.89%),and functional unknown (12.20%). Conclusions The transcriptome information of O. hupensis snail after the invasion of S. japonicum shows that sever-al genes are significantly up-regulated or down regulated expression,and that the availability of transcriptome information might provide a strong foundation for further understanding the schistosome-snail interaction at the molecular level.

ObjectiveTo study the effects and molecular mechanisms of Shuanglong Pill on angiogenesis of ischemic myocardium. Methods63 Wistar rats were ligated left anterior descending branch to be made into models of acute myocardial infarction(AMI).All rats were randomly divided into large-dosage group of Shuanglong Pill(LDG),small-dosage group of Shuanglong Pill(SDG),The control of acute MI(MIC).Other 11 rats were set as normal control(NC).Rats in every group had been treated half for 2 weeks and half for 4 weeks. New vessels' quantity in ischemic myocardium of acute MI rats were detected by immunohistochemistry double steps assay;the expressions of vessel endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) on ischemic myocardium were detected by immunohistochemistry SP assay;the expressions of VEGF mRNA and bFGF mRNA on ischemic myocardium were detected by reverse transcription polymerase chain reaction(RT-PCR) assay.ResultsThere were more new capillaries on ischemic myocardium of LDG, SDG compared with that in MIC at every stage,moreover,there was significantly increase at the end of 4 weeks compared with that at 2 weeks.At the end of 2 weeks, the expression of VEGF in LDG were significantly elevated compared with that in MIC; and the expression of bFGF in LDG were significantly elevated compared with that in SDG and MIC.Whereas the expression of VEGF in LDG were significantly decreased at the end of 4 weeks compared with 2 weeks. There were significant differences of above comparison (P<0.05-0.01).At every stage the expression of VEGF mRNA in LDG, SDG were significantly elevated compared with that in MIC; but its expression was significantly decreased at the end of 4 weeks compared with that 2 weeks. The expression of bFGF mRNA was significantly elevated in LDC at the end of 2 weeks compared with that in other groups.ConclusionsShuanglong Pill could promote angiogenesis in ischemic myocardium,application of large dose of Shuanglong Pill could significantly upregulate the expression of VEGF, bFGF, VEGF mRNA and bFGF mRNA.

OBJECTIVE:To screen and identify endophytic fungi from Schisandra chinensis with antioxidant activity. METH-ODS:The tissue isolation skill was used to isolate endophytic fungi from roots,leaves,stems and fruits of S. chinensis. And anti-oxidant activity of endophytic fungi were screened by DPPH radical scavenging assay and hydroxyl radical scavenging assay. The to-tal DNA were extracted;the 18S rDNA ITS were amplified and sequenced with primer ITS1 and ITS4;the results of sequencing were analyzed comparatively based on homology to confirm the classification of active strains. RESULTS:23 strains were isolated from S. chinensis. GSR-12,isolated from roots of S. chinensis,had strong antioxidant activities. The scavenging rate on DPPH and the hydroxyl radical were 87.96% and 82.31% respectively. GSR-12 strain was identified as Clonostachys rosea by analyzed com-paratively. CONCLUSIONS:1 strain of C. rosea,isolated from roots of S. chinensis,has strong antioxidant activity.

AIM: To elucidate the relationship of inflammation, coagulation and cholesterol gallstone formation. METHODS: Hamsters were divided into four groups: control group (feeding normal diet), lithogenic diet (LD) 2 weeks group, LD 6 weeks group and LD+aspirin 6 weeks group. Gallstone incidence, antithrombin antigen (AT-Ⅲ:Ag), antithrombin activity (AT-Ⅲ:Ac), thrombin (F-Ⅱa:Ac), plasminogen activator inhibitor activity (PAI:Ac), plasmin activity (Plm:Ac), D-dimer:Ag and C-reactive protein (CRP) in gallbladder bile were observed as read-out parameters. RESULTS: The incidence of gallstones in control group, LD 2 weeks group, LD 6 weeks group and LD+aspirin group were 0%, 20%, 73% and 25%, respectively. AT-Ⅲ:Ag, F-Ⅱa:Ac, D-dimer and CRP in LD 2 weeks group and LD 6 weeks group were significantly higher than those in the control group (P