Traditional Chinese medicines (TCM) is one of the most important sources of new drugs.The rapid development of modern science and technology has brought new opportunities for TCM.Admittedly,new academic theory is getting into a golden period of innovation.Key technologies that enbody TCM features and adapt to modern drug-screening requirements are urgently needed.After five years' endeavor,the authors' group has made great progress in the new theories and methodologies for the discovery of bioactive compounds from TCM.In this review,a total of five key technologies:library-bioactivity-structure integration,biological and chemical fishing technology,ligand-and receptor-based virtual screening,profile-bioactivity relationship and the technology for discovering bioactive equivalent combinatorial components (BECCs),were introduced.In the text,several valuable demonstrations over the TCM-based drug discovery were provided,for uncovering the scientific basis of TCM and accelerating the process of TCM modernization.

Objective To obtain highly expressing cell lines by inserting the glutamine synthetase (GS) screening system and replacing the promoter of the vector.Methods The mutation of the point BamHⅠwas induced to build a new vector pIRES2-EGFP.The marker gene GS was inserted by AseⅠ and NheⅠ, and the promoter hCMV was replaced by PacⅠand NheⅠ.The new vector pHGS1.0 and the vector pIRES2-enhanced screen fluorescein protein( EGFP)-B were inserted by the recombinant protein TEM8 ( 1-227 )-VEGFR1 domain2-IgG2 ( TV-IgG2 ) gene to analyze the advantages of the expression.Results The glutamine synsthetase is successfully inserted, the human cytomegalovirus replaced, and recombinant protein is increased 5-fold by human immunoglobulin quantification kit.Conclusion The GS system is a highly protein expressing system.

Objective To study the value of flow cytometry in identifying metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Methods Twenty-six cell lines representing ten epithelial neoplasms, one lymphoma cell line and one human T cell lymphoblast-like cell line were purchased from American Tissue Culture Collection. From July 2009 to June 2010, five nonhematopoietic neoplasms,fifteen hematopoietic neoplasms and fifteen control patients with complete remession after hematopoietic stem cell transplantation were collected in Beijing Daopei Hospital. Cryopreserved cell lines were thawed and cultured until they entered log phase. After permeabilization, cell lines were analyzed by staining with cytoplasmic CK-FITC antibody using four-color flow cytometer. The percent CK positivity was measured by comparing with negative control. Bone marrow samples were stained with membrane and cytoplasmic antibodies according to our routine methods. Based on lineage markers and blast markers as well as CK expression, the relevant hematopoietic diseases were diagnosed or excluded according to 2008 World Health Organization diagnosis standards. Results All epithelial neoplasm cell lines expressed CK, with average positive percentage 81.1%. All the lymphoid tumor cell lines didn't express CK. Two epithelial neoplasms were CK positive, 100. 0% in thyroid carcinoma and 98. 2% in lung carcinoma, respectively. Hematopoietic tumor and control samples didn't express CK. They expressed relevant hematopoietic markers, such as CD45 as well as lineage markers, or CD138 and cytoplasmic immunoglobulin light chain. Three nonepithelial nonhematopoietic neoplasms didn't express CK. CK positive or negative nonhematopoietic neoplasms didn't express hematopoietic markers such as CD45, HLA-ABC and HLA-DR DP DQ, as well as lineage specific markers. Besides, CK positive might be helpful to suggest epithelial origin. Conclusion Flow cytometry with hematopoietic markers and CK can effectively exclude hematopoietic tumor and identify metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow.

Objective To evaluate the clinical effect of ultrasound - guided sclerosing agent lauromacrogol injection in treating lymph leakage. Methods A total of 31 patients with postoperative lymph leakage were selected for this study. Of the 31 patients, successful conservative oppression treatment was accomplished in 16, and lauromacrogol injection had to be carried out in 15 as conservative oppression treatment failed. The patients were followed up and the results were analyzed. Results In 15 patients receiving lauromacrogol injection treatment, complete cure of lymph leak was obtained in 14 with a success rate of 93.33%. Among the 14 cases, the second lauromacrogol injection was employed in 3 at one week after the first injection. Infection occurred in another case one day after the injection , which was cured after dressing change for 15 days. Conclusion For the treatment of lymph leakage, ultrasound-guided sclerosing agent lauromacrogol injection is effective and safe.

Objective To study the effect of metformin on the growth of megakaryocytic leukemia cell line Dami and to explore the molecular mechanisms of the inhibitory effect of metformin on the proliferation of Dami. Methods The Dami cells were cultured and divided into control and 1,2,4,8,16 and 32 mmol·L-1 metformin groups.Then MTT test was performed to detect the inhitory rate of proliferation of Dami cells after treated with different concentrations of metformin. Flow cytometry was used to examine the distribution of cell cycle, and Western blotting was carried out to analyze the expressions of Cdc2 and CylinB1 and the phosphorylation of Cdc2. Results The MTT results showed that compared with control group,the inhibitory rates of proliferation of the Dami cells in 32 mmol·L-1 metformin groups at 0,24,48,72 and 96 h (35.1%±2.3%,49.7%±5.1%, 78.85±0.9%,79.1%± 3.0%%,and 85.2%± 3.2%)were significantly increased(P<0.01),Furthermore, after metformin treatment for 72 h,the inhibitory rates of proliferation of the Dami cells in 1,2,4,8,16 and 32 mmol·L-1 metformin groups were (33.8 ± 0.3)%,(51.9 ± 0.2)%,(59.4 ± 1.6)%,(65.5 ± 2.0)%, (75.5±0.9)%,and (79.1±3.0)%,respectively. Metformin inhibited the growth of Dami cells in a time-and dose-dependent manner. The flow cytometry results results revealed that compared with control group, the percentages of Dami cells in G2/M phase in 1,2 and 4 mmol·L-1 metformin groups were increased from (26.0± 0.5)% to (38.5 ± 1.5 )%, (48.4 ± 1.1 )%, and (58.2 ± 2.7 )%;there was significant difference in the percentages of Dami cells in G2/M phase between control group and 4 mmol·L-1 metformin group (P<0.01). Western blotting analysis showed that compared with control group, the expressions of Cdc2 and CyclinB were evidently reduced, the phosophorylation of Cdc2 at Tyr1 5 was up-regulated, and the phosphorylation at Thr1 6 1 was down-regulated.Conclusion Metformin can inhibit the growth of Dami cells and induce G2/M arrest,and its mechanism may be related to inhibiting the activation of Cdc2/CyclinB1 complex.

Objective To investigate the protective effect of umbilical cord mesenchymal stem cells (UCMSCs) on traumatic brain injury (TBI) in rats.Methods Thirty healthy Sprague-Dawley rats (10 rats for each group) were randomly divided into normal control group (normal),model group (injection of saline after TBI) and UCMSCs transplantation group (injection of UCMSCs after TBI).The rats in experimental groups were sacrificed on the 10th day after UCMSCs transplantation.The percentage of UCMSCs in brain tissue was detected by flow cytometry.The pathological changes of brain tissue were observed by hematoxylin-eosin (HE) staining method.The expressions of vascular endothelial growth factor (VEGF),glial fibrillary acidic protein (GFAP) and brain-derived neurotrophic factor (BDNF) in brain tissue were measured by immunohistochemistry and immunofluorescence double staining.The neurological deficit was evaluated by neurological deficit degree.Results The percentage of CD90,CD73 and CD105 cells in the UCMSCs transplantation group was significandtly higher than that in the model group (0.4％ vs 0.1％,P＜0.05).The results of HE staining showed that the brain injury of the transplanted group was alleviated compared with the model group (P＜0.05).The VEGF of the brain tissue in injury area in the UCMSCs transplantation group was higher than that in the model group (P＜0.05).The number of GFAP and BDNF positive cells in the UCMSCs transplantation group was higher than that in the model group (P＜0.05),and the neurological deficit score was also higher than that in the model group (P＜0.05).Conclusions UCMSCs transplantation for the treatment of TBI rats can effectively reduce the vascular damage in the injury area and promote nerve recovery.

Polyethylene glycol (PEG) of different lengths were prepared to investigate their effects on oral absorption of nanostructured lipid carrier (NLCs).Three kinds of PEG-modified NLCs with different chain lengths, including polyethylene glycol (100) monostearate (S100), polyethylene glycol (55) monostearate (S55), polyethylene glycol (40) monostearate (S40), were prepared by film dispersion method.Coumarin 6 was chosen as a fluorescent probe to characterize the physicochemical properties of NLCs with different lengths.Meanwhile, the stability of NLCs in simulate buffer, the release behavior, cytotoxicity of NLCs, the uptake kinetics and cellular uptake mechanisms were evaluated. This work demonstrated that the thickness of the hydrated layer increased with the increase of PEG length. Of note, S100-modified NLCs (pNLC-EG100) exhibited higher cellular uptake efficiency compared with other formulations. Thus, S100 was optimized as the best molecular weight for PEG-modified NLCs on oral drug delivery system.

Objective To establish a simple but quick method to improve the high fidelity of the synthesis of DNA frag -ments.Methods High fidelity DNA unidirection synthesis method (HFUS) was presented and used that involved Phusion DNA polymerase, BsrDⅠrestriction enzymes and λexonuclease.Using the same system at different temperatures , HFUS method synthesized one positive single-strand DNA and several reverse single -stranded DNA one by one into the target DNA fragment.Results Two random sequences DNA fragments of 340 bp and 450 bp were synthesized using HFUS meth-od.Conclusion This article explores a new method for the synthesis of genes .Through the harvest of 450 bp DNA, HFUS may promise to be a new approach to the synthesis of DNA .

OBJECTIVETo analyze the sequence of STK11 gene coding region in 20 patients with Peutz-Jeghers syndrome and identify the point mutations in STK11 gene associated with the occurrence of the disease.

METHODSBlood samples were collected from 20 inpatients with Peutz-Jeghers syndrome treated in our center between January 2009 and October 2010. The sequence of STK11 gene coding region was analyzed using PCR and DNA sequencing and compared with the normal sequence of STK11 gene.

RESULTSOf the 20 patients with Peutz-Jeghers syndrome, 14 showed STK11 gene mutations in the coding region, including 1 patient having two mutations and 13 patients with a single mutation site. In one case, sequence analysis of the STK11 gene identified a novel type of STK11 germline mutation, in which the cytosine (C)460 was substituted by guanine (G) in exon 3 to result in a new amino acid at codon 154. Four patients from 2 families were found to have a common mutation. The remaining 6 patients were not found to have mutations in STK11 gene coding region.

CONCLUSIONMutations of STK11 gene is a major cause of Peutz-Jeghers syndrome. The missense mutation of 460 C→G in exon 3 of STK11 gene is a novel mutation associated with Peutz-Jeghers syndrome.

Adult ; Asian Continental Ancestry Group ; genetics ; Codon ; DNA Mutational Analysis ; Exons ; Female ; Humans ; Male ; Mutation ; Pedigree ; Peutz-Jeghers Syndrome ; genetics ; Protein-Serine-Threonine Kinases ; genetics

To construct nanostructured lipid carriers(NLCs)with different particle sizes but the same other physicochemical properties, central composite design was adopted. Coumarin-6(C-6)was selected as the model drug due to its high lipophilicity and high fluorescence intensity. Physicochemical properties of NLCs with 100 nm, 200 nm and 300 nm in particle size could remain stable during certain time in K-R solution and PBS. Release experiments in vitro showed that cumulative release of C-6 in NLCs was less than 7% after 24 h. The MTT assay indicated that both blank NLCs and C-6 loaded NLCs showed low toxicity. To confirm the integrity of NLCs in gastrointestinal tract, DiR-loaded NLCs were prepared and the distribution in vivo was monitored by fluorescence imaging. After 6 h oral administration, intact DiR-loaded NLCs could stiu be found, suggesting that NLCs could be used to characterize the uptake in gastrointestinal tract.