Objective To isolate and culture human adipose-derived stem cells (hADSCs),investigate the influence of hADSCs on the cellular morphology,survival rate,and function of human islet cells under the in vitro non-contact co-culture conditions,and explore its mechanism.Method hADSCs were isolated by collagenase digestion method,then cultured,and identified by morphology,immunofluorescence and multi-directional differentiation.Adult islet cells were separated and purified by Liberase enzyme and Ficoll 400,then divided into co-culture group and individual group.The cellular growth morphology of islet cells was observed by inverted phase contrast microscope.The survival rate of islet cells,insulin secretory volume,insulin stimulation index and concentration of growth factor in the supernatant were compared between the two groups.Result hADSCs of the third generation showed uniform long spindle fibrocyte-like morphology,and had multi-directional differentiational potentials of osteogenesis and adipogenesis.Immunofluorescence test of surface antigens on hADSCs revealed CD44 + and CD49d +,CD31-,CD34-and CD106-.After 14-day culture,the islet cellular morphology in co-culture group was more intact than that in individual group.The survival rate of islet cells in co-culture group was (82.83 + 2.32) ％,and that in individual group was (53.00 + 2.82) ％ (P＜0.01).Insulin secretory volumes were (23.66 + 2.11) and (7.82 +1.09) mU/L respectively in co-culture group and individual group under high glucose concentration,and 13.22 + 0.77 and 6.40 + 0.44 mU/L respectively under low glucose concentration (P＜0.01 for all).Insulin stimulation index was decreased from 1.67 + 0.10 (at 3rd day) to 1.77 + 0.13 (at 14th day) in co-culture group,and from (1.67 + 0.10) (at 3rd day) to (1.77 + 0.13) (at 14th day) in individual group (P＜0.01).After 14-day culture,the concentrations of HGF,TGF-β,VEGF and bFGF in the supernatant were higher in co-culture group than in individual group (P＜0.01).Conclusion hADSCs were isolated and cultured successfully from adult adipose tissue.They could increase the survival rate and improve the function of islet cells when co-culture with the adult islet cells in vitro through secreting HGF,TGF-β,VEGF and b-FGF.

ABSTRACT:Objective For preparation of vascularized islets , to isolate and culture human adipose derived stem cells , investigate the role of adipose derived stem cells (ADSCs ) in promoting the proliferation and vascularization of human umbilical vein endothelial cells (HUVECs ) co‐cultured in vitro , and explore its mechanism .Methods ADSCs and HUVECs were isolated by collagenase digestion method ,then cultured ,and identified by morphology ,immunofluorescence or multi‐directional differentiation .The co‐culture system of ADSCs and HUVECs was established , HUVECs cultured alone were set up for control group . The proliferation , vascularization and concentration of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b‐FGF)in the supernatant were compared between the two groups .Results The third generational ADSCs had uniform long spindle fiberous morphology and multi‐directional differentiational function . Immunofluorescence test of surface antigens on ADSCs revealed CD44/CD49d (+ ) ,CD31/CD34 (-) ,on HUVECs CD31/vWF (+ ) . High vascular density was found when co‐cultured in Matrigel of ADSCs and HUVECs than alone of HUVECs .Growth curve shown at days 3 , 4 and 5 of the logarithmic phase , HUVECs count in co‐culture group of ADSCs and HUVECs was (4 .52 ± 0 .31) × 104 ,(7 .18 ± 0 .45) × 104 ,and (8 .23 ± 0 .36) × 104 under indirect co‐culture condition , while that in individual HUVECs group was (2 .71 ± 0 .25) × 104 ,(4 .87 ± 0 .26) × 104 ,and (6 .86 ± 0 .33) × 104 ( P<0 .01) .Population doubling time of HUVECs was shorter in co‐culture group than in individual group .Also ,the OD value of HUVECs was higher in co‐culture group than in individual group when cultured at days 1 ,3 ,5 and 7 ( P<0 .01) .When cultured at days 3 ,7 and 13 ,the concentration of VEGF and b‐FGF in the supernatant was higher in co‐culture group than in individual group ( P< 0 .01 ) . Conclusion ADSCs can promote the proliferation and vascularization of HUVECs in vitro co‐culture conditions by secreting or increasing the HUVECs secretion of VEGF and b‐FGF .

Objective In this experiment,we explored the effects of rAAV\|hGDNF on protecting spinal neurons From death. Methods rAAV\|hGDNF particals were produced by recombinant virus technolgy,and infected the culture spinal neurons which were exposed to glutamate.We counted the mortality rate and detected the expression of NOS mRNA by RT\|PCR. Results In the group transfering rAAV\|hGDNF the death rate was inhibited(50%?0\^02,control 59\^25%?0\^023, P

Objective To investigate the prevalence of herpes simplex virus(HSV) infection of central nervous system(CNS) in newborn infant,and analyze its clinical characteristics.Methods Cerebrospinal fluid(CSF) was collected from 40 acute viral infection of central nervous system who were hospitalized during June 2001 to June 2002.Polymerse chain reacton techniques(nested-PCR)was used to detect HSVspecific DNA in CSF,enzyme-linked immunosorbert assays(ELISA)was applied to detect HSV-specific IgM and IgG antibody in CSF and serum specimens.Results Two cases of neonatal patients were HSV-1 DNA PCR positive in CSF,both(mo)-ther were normal during pregnancy without a history of genital herpes.Clinical presentations of one case belonged to disseminated HSV infections and the other was limited to CNS infections.HSV-2 DNA PCR of 40 cases of neonatal patients were negative in CSF.Conclusions The rate of HSV neonatal CNS infection was 5% among viral neonatal CNS infections.HSV type 1 in the period,which showed that HSV type 1 may be the common type of HSV neonatal CNS infections.The result seems to be related to low prevalence for HSV-2(among) pregnancy women in China.

The secondary metabolites of plants are important sources of natural drugs. Betula plants have abundant pharmacological value, complex mechanism and wide applications, which are closely related to the triterpenoids of theirs. Triterpenoids in Betula species are mainly divided into dammarane-type, ocotillol-type, oleanane-type, lupane-type and cycloaltunane-type. The extracts of Betula species have varieties of activities such as anti-tumor, anti-inflammatory, anti-oxidant, anti-bacterial, etc. And the biosynthetic pathways of triterpenoids after 2,3-oxidosqualene are split into four branches of dammarenediol-II, lupeol, cycloartenol and amyrin according to the different oxidosqualene cyclases. This review summarizes the chemical constituents, pharmacological activities and biosynthetic pathways of triterpenoids in Betula plants. It provides a reference for the research and development of new drugs and the production of these triterpenoids in microbial cell factories by synthetic biology methods.

Natural products are important sources of drug discovery. However, the traditional methods of extraction and isolation, as well as chemical synthesis for obtaining natural products are associated with issues such as operational complexity, high costs, low efficiency, and environmental pollution. Constructing microbial cell factories through synthetic biology methods to produce medicinal natural products has the advantages of high efficiency, low cost, and environmental protection. Nevertheless, the scope and yield improvement of the products are limited by the limitations of enzymes in microbial cell factories. Protein engineering is considered one of the most effective approaches to overcome these limitations. This article introduces commonly used methods of protein engineering technology and summarizes its specific applications in improving enzyme performance, modifying the enzymatic environment, and promoting the development of synthetic biology tools in the field of pharmaceutical natural product synthesis. Furthermore, it analyzes the current bottlenecks and challenges in protein engineering and looks forward to its future application prospects, offering insights for the development and practical use of protein engineering technology.

[Objective] To discuss and analyze the development history of TCM education in Zhejiang in modern times and provide valuable experience for modern TCM education theory and practice. [Methods] The article goes through the historical development of TCM education in modern Zhejiang, the general situation of TCM education, and the characteristics of TCM education. It deepens historical materials to search, collate and excavate the precious literature resources of historical materials and summarizes them. [Results] The modern TCM, Beiyang government, Nanjing National Government and other periods of traditional Chinese medicine business were in a difficult stage, but TCM education in these periods was actively going forward in the reform. The modern Chinese medicine school runs a comprehensive reform from the aspects of the school-running concept, enrollment system, school system and curriculum setting to traditional TCM education, creating a unique school-running model and promoting the development of TCM education. [Conclusion] Modern Chinese medicine education in Zhejiang has developed in twists and turns. Its experience has provided valuable experience for the theory and practice of Chinese medicine education the present age. promoting the development of TCM education. [Conclusion] Modern Chinese medicine education in Zhejiang has developed in twists and turns. Its experience has provided valuable experience for the theory and practice of Chinese medicine education the present age.

Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ～ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ～ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ～ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ～-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ～-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.

Objective To investigate the distribution and constituent of drug‐resistant bacteria of lower respiratory tract in‐fection among different regions (outpatient department ,wards ,RICU) to provide the basis for the clinical reasonable application of antimicrobial agents .Methods The K‐B disc diffusion method and the instrument method (VITEK‐TWO) were adopted and the detection results were interpreted according to the standards of CLSI 2010 .The detection data of 480 drug‐resistant strains isolated from the sputum ,branchoalveolar lavage fluid samples submitted in 3 regions of respiratory outpatients department by bacterial cul‐ture identification and drug susceptibility test were analyzed by using the WHONET5 .6 statistical software .Results The distribu‐tion and constituent of drug‐resistant bacteria of lower respiratory tract infection had obvious difference among 3 different regions . The top 4 of drug resistant bacteria were dominated by Gram‐negative bacteria .The drug resistance rate of Klebsiella pneumoniae in RICU was higher than that in the respiratory outpatients department and wards(P<0 .05) ,the resistance rate in the respiratory outpatients department ,wards and RICU to commonly used antibacterial drugs was similar;the multiple drug resistance of ESBLs‐producing strains was obviously higher than that of non‐ESBLs‐producing strains (P<0 .05) .Pseudomonas aeruginosa maintained the higher antibacterial activity to quinolone ,aminoglucosides ,cefepime ,imipenem ,cefoperazone/sulbactam ,and piperacillin/tazobactam ,but the resistance rate in RICU was significantly higher that in the respiratory outpatient department and wards (P<0 .05);the drug resistance of Acinetobacter baumanii in the respiratory wards and RICU was higher than that in the respiratory out‐patient department ,the resistances to imipenem were 64 .6% and 70 .4% respectively .The resistance of MRSA to rifampin in RICU was higher than that in the respiratory outpatient department and wards(P<0 .05) .Conclusion The distribution constituent and drug‐resistance rates have obvious differences among the respiratory outpatient department ,wards and RICU .Except being familiar with the drug resitant bacterial distribution and drug resistance rate monitoring situation ,clinical doctors should grasp the drug re‐sistance situation of drug resistant bacteria among different areas in various departments of own unit in order to rationally and effec‐tively use antibacterial drugs .

Objective To evaluate the clinical efficacy of the local drug resistance spectrum antibiotics and foreign guideline in the treatment of patients with community acquired pneumonia(CAP). Methods A prospective,randomized,single blind,and positive drug parallel controlled design was used in the treatment. CAP patients with no underlying disease outpatients and inpatients<48 hours were selected as the research object. The patients in the trial group were given sensitive local drug resistance spectrum antibiotics: moxifloxacin,400 mg and 1 times a day. The patients in the control group were given azithromycin tablets(each 500 mg,once daily) promulgated by the 2007 version of the IDSA / ATS adult CAP guideline. Results There were 106 cases of CAP patients,of which 77 cases completed treatment,including 39 cases in the experimental group and 38 cases in the control group. There were significant differences in the clinical efficacy and bacterial clearance rate between the two groups,with the clinical efficacy of 89.7% and 68.4%(P < 0.01),the bacterial clearance rate of 87.9% and 54.5%(P < 0.05),respectively. Conclusion The clinical efficacy of drug resistant spectrum sensitive antibiotics in the treatment of CAP in Kunming was better than that of IDSA/ATS. Clinicians should pay attention to the characteristics and composition of resistance of common pathogenic bacteria in our country during the study and reference from foreign guideline,and adjust the therapeutic regimen according to the changes of the local drug resistance monitoring data rather than copy the recommended treatment plan by foreign countries.