Objective To analyze and compare the efficacy of DNA barcode,i.e.,Cytochrome b(Cytb)and 12S ribosomal RNA(12S rRNA)gene sequences,in the species identification of wildlife.Methods DNA extraction,quantification,PCR amplification of Cytb and 12S rRNA gene fragments,Sanger sequencing,and sequence alignment analysis were performed on ten wildlife samples.Results Both gene fragments were successfully amplified in six samples,while Cytb alone was successfully amplified in 1 sample,and 12S rRNA alone in 3 samples.Sequence analysis indicated that Cytb enabled species-level identification for 6 samples(Gallinula chloropus,Streptopelia orientalis,Phasianus colchicus,Falco naumanni,Myiopsitta monachus and Lynx lynx)and genus-level identification for 1 sample(Lepus).In contrast,12S rRNA achieved species-level identificaggion for 8 samples(Gallinula chloropus,Lepus sinensis,Phasianus colchicus,Myiopsitta monachus,Muntiacus reevesi,Macaca mulatta and Lynx lynx),representing seven species,and genus-level identification for 1 sample(Falco).However,by combining Cytb and 12S rRNA,all samples could be identified to the species level.Conclusion When applying DNA barcodes to wildlife identification,the Cytb and 12S rRNA gene regions analyzed here can effectively identify common species such as Gallinula chloropus and Streptopelia orientalis,but face difficulties in distinguishing closely related species within the same genus.Therefore,when conducting wildlife species identification,it is recommended to use two or more DNA barcode markers.

Objective To analyze and compare the efficacy of DNA barcode,i.e.,Cytochrome b(Cytb)and 12S ribosomal RNA(12S rRNA)gene sequences,in the species identification of wildlife.Methods DNA extraction,quantification,PCR amplification of Cytb and 12S rRNA gene fragments,Sanger sequencing,and sequence alignment analysis were performed on ten wildlife samples.Results Both gene fragments were successfully amplified in six samples,while Cytb alone was successfully amplified in 1 sample,and 12S rRNA alone in 3 samples.Sequence analysis indicated that Cytb enabled species-level identification for 6 samples(Gallinula chloropus,Streptopelia orientalis,Phasianus colchicus,Falco naumanni,Myiopsitta monachus and Lynx lynx)and genus-level identification for 1 sample(Lepus).In contrast,12S rRNA achieved species-level identificaggion for 8 samples(Gallinula chloropus,Lepus sinensis,Phasianus colchicus,Myiopsitta monachus,Muntiacus reevesi,Macaca mulatta and Lynx lynx),representing seven species,and genus-level identification for 1 sample(Falco).However,by combining Cytb and 12S rRNA,all samples could be identified to the species level.Conclusion When applying DNA barcodes to wildlife identification,the Cytb and 12S rRNA gene regions analyzed here can effectively identify common species such as Gallinula chloropus and Streptopelia orientalis,but face difficulties in distinguishing closely related species within the same genus.Therefore,when conducting wildlife species identification,it is recommended to use two or more DNA barcode markers.

To determine the effect of a recombinant lentivirus containing human stem cell leukemia (SCL) gene on the expression of c-kit protein in damaged interstitial cells of Cajal (ICC) under high glucose condition.
 Methods: After isolation of ICC, the cells were cultured for 24 hours until the cells were adherent. After identification by inverted microscope and immunofluorescence, ICC cells were divided into two groups: A control group and a high glucose group. The control group was added with a medium containing 5 mmol/L of glucose. The high glucose group was added with a medium containing 20 mmol/L of glucose. After 48 h of continuous cultivation, the high glucose group was divided into 3 subgroups: A blank group, an empty lentivirus group, and an experimental group. The blank group, the empty lentivirus group, and the experimental group were added a medium containing PBS solution, empty lentivirus, and a recombinant lentivirus containing the SCL gene with a glucose concentration of 5 mmol/L, respectively. The cultures were incubated for 24 and 48 h. The expression of c-kit protein in ICC in each group was detected by Western blot.
 Results: After 24 or 48 h, the expression of c-kit protein in ICC was significantly lower in the blank group and the lentivirus group than that in the control group, and the expression of c-kit protein in ICC was significantly higher in the experimental group than that in the blank group and the empty lentivirus group, but it was still lower than that in the control group (all P<0.05).
 Conclusion: The recombinant lentivirus of SCL gene can up-regulate the expression of c-kit protein in functionally impaired ICC under high glucose condition.
Glucose ; Humans ; Interstitial Cells of Cajal ; Lentivirus ; Leukemia, Myeloid, Acute ; Proto-Oncogene Proteins c-kit

Aim To investigate the antitumor and antiangiogenic effects of combined low-dose cyclophosphamide(CTX)and recombined VEGF protein vaccine.Methods In this experiment,H22 hepatocellular carcinoma model was established in BALB/c mice.Mice were randomly divided into four groups: control group,CTX group(CTX),VEGF protein vaccine group(V2)and CTX plus V2 group(CTX+V2).The anti-tumor efficacy and antiangiogenic effect were investigated using a subcutaneous tumor model and an intradermal tumor model.Western blot and ELISAwere further adopted to detect the specific anti-VEGF antibody.Results CTX+V2 group displayed a lower tumor volume and tumor weight than either the single therapy group in the subcutaneous tumor model(P<005 vs V2,P<001 vs CTX).Meanwhile,CTX+V2 was more effective for antagonizing tumor-associated angiogenesis compared with either the single therapy(P<005 vs V2,P<001 vs CTX).After CTX+V2 immunization,high titer of anti-VEGF antibody was detected by ELISA and verified by Western blot.Conclusion The therapy of CTX combined with V2 has significant synergistic effect against H22 hepatocellular carcinoma.

The application status and disadvantages of lecture-based learning ( LBL ) and problem-based learning (PBL) have been compared and discussed, and the imagination of combina-tion of LBL with PBL in the application of biotechnological pharmaceutics teaching proposed. LBL teaching will be mainly applied in the fundamental principles of development and production of biotechnological pharmaceutics, while PBL teaching will be mainly applied in the specific production processes of typical biopharmaceutics. The three key points in the combination teaching process includ-ing the time allocation of LBL and PBL, the design and implementation of PBL cases and the founda-tion of reasonable teaching evaluation system have also been introduced. The combination of LBL with PBL will cultivate students' self-study, comprehensive analysis and teamwork capabilities to satisfy the demands of the development of current market for biopharmaceutical talents.

Aim To investigate the anti-EAC breast cancer effects of viable human umbilical vein endothe-lial cells ( HUVECs) vaccine. Methods Subconflu-ent HUVECs were mixed with OK432 to prepare HU-VECs-OK432 vaccine, and the anti-tumor efficacy of HUVECs-OK432 was investigated using a subcutaneous tumor model of EAC breast cancer. ELISA, splenic lymphocyte proliferation assay and cytotoxic T lympho-cytes ( CTL ) killing assay were adopted to detect the humoral and cellular immune responses after HUVECs-OK432 immunization. Results HUVECs-OK432 im-munization significantly inhibited the growth of EAC tumor in mice in the prophylactic procedures. High ti-ter of anti-HUVEC antibody was elicited by HUVECs-OK432 immunization. The proliferation activity of splenocytes from mice immunized with HUVECs-OK432 was significantly increased. T lymphocytes iso-lated from HUVECs-OK432-immunized mice were dose dependently killing HUVECs in vitro. Conclusion Strong humoral and cellular immune responses targeting HUVEC are elicited by HUVECs-OK432 immuniza-tion, which results in a significant inhibition on the growth of EAC tumor in mice.

Objective To observe the clinical efficacy of midazolam intravenous therapy for children with convulsive status epilepticus (CSE).Methods 133 admitted CSE children were randomly divided into treatment group (n =6 8) and control group (n =6 5).Based on symptomatic treatment,the control group was given diazepam plus phenobarbital intravenous injection,the treatment group was given midazolam injection plus intravenous infusion scheme.The clinical efficacy and adverse drug reactions were observed and compared between the two groups.Results After treatment for 3h,the total effective rate of the treatment group was 91.2％,which was significantly higher than 76.9％ of control group(x2 =5.078,P =0.024).Among children with markedly effective and effective effect,the mean onset time (49.3 ± 10.4)min and seizure control time (112.1 + 24.7)min of the treatment group were significantly lower than those of control group (73.8 + 15.4) min,(157.2 ± 38.4) min,the differences were statistically significant(u =9.619,7.191,P =0.000).15 ineffective cases of control group were transferred into midazolam intravenous therapy,the total effective rate after 3h was 73.3％ (11/15).1 case died in both two groups.In control group,the proportion of complications such as muscle tension descending,heart rate and blood pressure variation,respiratory depression,et al.was 49.2％,which was significantly higher than 30.9％ of the treatment group (x2 =4.668,P =0.31).Conclusion Compared with diazepam plus phenobarbital scheme,midazolam intravenous administration in treatment of children with CSE takes effect faster,and with higher safety.With the increasing of midazolam dosage,alert should be taken to drug influence on respiration and heart rate.