Currently, the model of the research evaluation system of traditional Chinese medicine (TCM) is set up through imitating that of western medicine. The application of quantitative research to TCM does promote the advance of modernization of TCM, which explore the part of TCM that can be measured in quantitative method. However, TCM has a background of profound philosophy and culture. The priority of TCM can not be expressed through quantitative research alone. On the contrary, qualitative research is more suitable to most research area of TCM. In TCM clinical research, the priority of TCM should be fully explored. It is very significant to set up the effectiveness evaluation system of TCM, especially by applying qualitative research to the diagnosis and the evaluation of treatment results and combining quantitative research.

The preparation method, serum stability, efficiency of cellular uptake and apoptosis induction of the cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel (C-TAT-Lipo) were investigated. The best preparation procedure was performed by orthogonal test based on single factor screening method. First, the paclitaxel (PTX)-loaded liposomes were prepared by filming-rehydration method, evaluated with entrapment efficiency and polydispersity index. The morphology of C-TAT-Lipo was characterized by transmission electron microscopy. Turbidity variations were monitored in the presence of fetal bovine serum (FBS) to evaluate the serum stability of the liposomes developed here. Next, the efficiency of cellular uptake of different Rho-PE-labeled liposomes on B16F1 cells in vitro was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. The quantitative analysis of apoptosis induced by different PTX-loaded liposomes was performed by Annexin V-FITC/PI double staining. The optimal formulation was as follows: Chol : lipid: 1 : 8 (molar ratio); drug : lipid: 1 : 40 (mass ratio); lipid concentration: 3 mmol x L(-1); temperature of hydration: 25 degrees C. The mean size and polydispersity index of C-TAT-Lipo were about (97.97 +/- 3.68) nm and 0.196 +/- 0.037, the zeta potential was (-0.89 +/- 0.45) mV, the entrapment efficiency of paclitaxel was (90.16 +/- 1.53)%. The particle sizes did not exhibit significant variations in 50% FBS over 24 h at 37 degrees C. The efficiency of cellular uptake of the C-TAT-Lipo increased 1.40 fold following the cleavage of PEG. Apoptosis analysis showed 59.3% increase of the apoptosis and necrosis profile of C-TAT-Lipo after the detachment of PEG shells, which was markedly higher than that of N-TAT-LP with or without glutathione and SL, respectively. The results indicate that the C-TAT-Lipo is successfully prepared by filming-rehydration method and shows significant antitumor activities.
Animals ; Annexin A5 ; Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Liposomes ; chemistry ; Melanoma, Experimental ; Mice ; Microscopy, Confocal ; Paclitaxel ; pharmacology ; Particle Size ; Polyethylene Glycols ; chemistry

To screen and identify the mimotopes for lipopolysaccharide(LPS) epitope, a rand om phage displayed dodecapeptide library was screened with a monoclonal antibody 2B4 spe ci fically against LPS conservative epitope. The positive clones were identified by phage ELISA and competitive inhibition assay by either S.typhi T 8-61 LPS or E.coli O111:B4 LPS. After three rounds of biopanning,the clones binding with 2B4 antibody were well enriched with positive rate of 80%. The bindings between 12 of positive phage clones and screening antibody were competitively inhibited by the two kinds of LPS,indicating that the positive clones have similar epitope with LPS. The positive peptide sequences were deduced from the corresponding DNA sequences. There were identical sequences among them. The seq uences were GPPQWFFSQPQL （5/12，41.7%），LPQYFWNTATTA （3/12，25%），FPQNHWNVP WAT（2/12，16.6%），HSQSFWNAPLAM and AHPWTHGYFPPL （1/12，8.3%） respectively . The results demonstrate that the peptides screened with 2B4 antibody are mimot opes for LPS conservative epitope.

Objective To establish a RP-HPLC method to determine the content of geniposide in Xiaoyan Tuire Patch. Method The column of Agilent ZORBAX SB-C18 (4.6 mm?250 mm 5 ?m) was used. The mobile phase was composed of water-acetonitrile-phosphoric acid (86∶14∶0.01). The flow rate was 1 mL/min, with UV detection at 238 nm, and the temperature was 30 ℃. Results The linear range was 0.07～0.70 ?g (r =0.99997). The average recovery was 98.60% and the RSD was 1.96%. Conclusion The method is simple, sensitive and accurate, and can be used for the determination of Xiaoyan Tuire Patch.

Objective To establish a RP-HPLC method to determine the content of ephedrine?HCl in Xiaoyan Zhike Plaster. Method The sample was prepared according to 2005 edition of Chinese Pharmacopocia, the column of Inertsil-3 ODS (4.6 mm?250 mm, 5 ?m) was used. The mobile phase was composed of methanol-0.1% of phosphoric acid (5∶95). The flow rate was 1 mL/min, with UV detection at 207 nm. Results The linear range was 0.041～0.41 ?g (r=0.999 8). The lowest limit of detection was 0.025 6 ?g. The average recovery and the precision was 99.55% and 2.58%. Conclusion The method is simple, sensitive and accurate, and can be used for the quality control of Xiaoyan Zhike Plaster.

Objective To find out the bioactive constituents in the stem of Lonicera japonica and to carry out a systematic study on the chemical constituents of the plant. Methods Three compounds were isolated from the plant by means of solvent extraction and column chromatography, and their structures were elucidated by spectral analyses. Results All these compounds were identified as luteolin (Ⅰ), loga-nin (Ⅱ), r-1-(4-hydroxy-3-methoxy-phenyl)-trans-2, cis-3-dihydroxymethyl-7, 8-dihydroxy-6-methoxy-1, 2, 3, 4-tetrahydro-naphthalene (Ⅲ), respectively. Conclusion Compound Ⅲ is a new compound named as japenol.

Objective To optimize the alcohol extraction technique of the medical herbs such as Rhizoma Corydalis and Radix Angelicae Dahuricae. Methods To optimize the alcohol extraction technique with L9(34) orthogonal test. The content of the tetrahydropalmatine in Rhizoma Corydalis,which was used as evaluating criteria,was determined by HPLC. Results The optimized extraction technique was as follows: the medicinal material was extracted for 3 times with 3 times amount of 75% alcohol,1.5 hours per time. Conclusions The optimized technique is reasonable and the extraction rate was high.

Objective: To investigate VEGF expression in NIH3T3 cells infected by adenovirus containing hVEGF165 gene and its influence on proliferation of NIH3T3 cells, and to observe the expression of hVEGF and its angiogenic effect in vivo. Methods: Adenoviral vector containing hVEGF165 gene was constructed and was used to infect NIH3T3 cells. The infection efficiency of adenovirus vector was examined by immunofluorescence and flow cytometry. Expression of VEGF in NIH3T3 cells and its levels in the culture medium were examined by immunohistochemical (IHC) staining, RT-PCR, and ELISA. The infected NIH3T3 cells were implanted in skin defect at rat back and the acellular dermis on the wound was obtained one week later; the expression of hVEGF was detected by IHC in the dermis and the density of vessels was determined under microscope. Results: NIH3T3 cells were effectively transfected by adenovirus containing VEGF gene in vitro, the transfection efficiency was in a dose-effect manner with multiplicities of infection (MOI) of the adenovirus. When MOI was 100, the infection efficiency was more than 95%. The expression of VEGF mRNA and protein was detected by RT-PCR and IHC 24 h after transfection. ELISA result showed that the high level of VEGF on the 3rd day after transfection and the level reached its peak 7 d after infection (1 052 pg/ml); VEGF expression was detectable 13 d after transfection. MTT assay demonstrated no significant difference in cellular proliferation between the transfection and non transfection group. Expression of hVEGF was also detected in vivo in mice, and the density of vessels in the experimental group was significantly higher than that in the control group (P<0.01). Conclusion: Adenoviral vector can effectively transfect VEGF gene into NIH3T3 cells; VEGF gene can be detected in vitro and in vivo; and it can promote neovascularization in the transplanted tissues.

Objective: To prepare NIH3T3 cells harboring microencapsulated VEGF gene and investigate the proliferation, activity and metabolic function of the modified cells. Methods: Microencapsulated VEGF modified NIH3T3 cells were prepared through an alginate-BaCl2 process. Morphological appearance of the microencapsulation and the cell morphology were observed under inverted phase microscope; untreated NIH3T3 cells served as control. The concentrations of VEGF in the culture supernatant (collected every 48 hours) were measured by ELISA; the proliferation and vitality of the cells were examined by MTT assay and flow cytometry with PI staining. Results: The microcapsules were round in shape and the cells grew well. There was no significant difference in the concentrations of VEGF,MTT values and vitalities of cells between the 2 groups. Conclusion: The growth and metabolic functions of NIH3T3 cells are not influenced by microencapsulated NIH3T3 cells harboring VEGF gene. The bio-properties of modified cells are similar to those of the control cells,which lays a foundation for transplantation of microencapsulated VEGF modified NIH3T3 cells in vivo.

Objective To explore the role of cell cycle regulators in tuberculosis with lung carcinoma.Methods p53,MDM_2,p21~ ras and p21~ WAF1 proteins were detected by LsAB immunohistochemical technique in 69 cases of tuberculosis with lung carcinoma.Results As compared with primary tuberculosis with lung carcinoma,the expression rate p53 or MDM_2 protein in relapsing NPC was similar(78% to 80%,84% to 83%),and the expression rate of p21~ ras and p21~ WAF1 protein in relapsing NPC was obviously descended(73% to 93%,52% to 84%);The high-expression rate of p53 protein in relapsing NPC was similar(42% to 51%),the high-expression rate of MDM_2 protein in relapsing NPC was obviously risen(57% to 32%),and the high-expression rate of p21~ ras and p21~ WAF1 protein in relapsing NPC was obviously descended(16% to 65%,17% to 46%).Among of them,the significant rise of MDM_2 protein expression level in relapsing NPC mainly occurred in the patients of group 2 which relapsing-interval was shorter than 34 months(P