Objective To evaluate the feasibility of transfection of Sonic hedgehog gene (SHH)into bone marrow mesenchymal stem cells(BMMSC).Methods After the SHH gene was transfected into BMMSC by electroporation apparatus,the transfection rate was evaluated by fluorescence inverted microscope.The growth curves of untransfected and transfected BMMSC were drawn,respectively,to observe the influence of transfection on cells.The expression of SHH gene in the BMMSC was detected by PCR,RT-PCR,Western-blot analyses.Results Through fluorescence inverted microscope,the observed transfection rate was appropriately 30％,PCR showed a obvious increase of SHH expression in transfected cells than that in untransfected cells,and it is quantified by qPCR for appropriately 7 times.Western-blot further demonstrated that the SHH protein expression in transfected cells had a distinct increase.However,it was observed that the exponential phase of BMMSCSHH growth curve delayed.The growth curves of both overlap 12 days after transfection.Conclusions This electroporation method can transfect exogenous SHH gene into BMMSC sufficiently with the effective protein expression in BMMSCSHH.It is the foundation of further research of genetic therapy for ischemic heart disease.

Objective To investigate the survival and differentiated situation of transplanted mesenchymal stem cells ( MSCs) transfected with Sonic hedgehog (SHH) gene after chronic myocardial infarction (MI).Methods MSCs were obtained from bone marrow of limb bones by density gradient centrifugation combined with attachment culture method .SHH gene was cloned and eukaryotic expression plasmid pcDNA 3.1-SHH was constructed .Then nucleofector TM was used to transfer SHH gene into MSCs and the expres-sions of mRNA and protein of SHH gene were detected in vitro .Myocardial infarction model was established by ligating the left anterior descending branch .A total of 150μl (3 ×106 ) of MSCs transferred with SHH gene was injected into ischemic area at the fourth week after MI model was set up .Brdu immunohistochemical staining and electron microscope examination were used to detect the survival and differentiation of MSCs with SHH in vivo at the 1st, 2nd, 4th, and 8th week after MSCs SHH transplantation.Results It was con-firmed that SHH gene clone and the recombinant eukaryotic expression vector pcDNA 3.1-SHH construction were successful by gene se-quence analysis and double digestions with EcoR I and Bamh I .Reverse transcriptase polymerase chain reaction ( RT-PCR) and West-ern-blot demonstrated that the expression of SHH mRNA was significantly increased in transfected MSCs compared with untransfected MSCs.Brdu immunohistochemical staining confirmed MSCs with SHH were survived from one to eight weeks in MI area after transplan -tation.Electron microscope examination showed that MSCs had the trend to be differentiated into myocardiac -like cells in MI area at the 4th and 8th week after transplantation .Conclusions The expression of SHH mRNA was significantly increased while MSCs were transfected with SHH gene , and MSCs SHH could survive and differentiate in good condition in the transplanted recipients .Those data provide the experimental basis for further transgenic cell therapy research of ischemic heart disease .

The high-copy-number plasmid pcDNA3.1+ is unstable within S almonella typhimurium. A novel plasmid pmcDNA3.1+ was constructed by removin g the promoter sequence of ampicillin resistance gene (bla gene) in plasmid pcDNA3.1+. In contrast to pcDNA3.1+, pmcDNA3.1+ was stable within Salmonel la typhimurium SL7207 in LB medium with or without ampicillin. Further experi ments showed the ?-lactamase activity of Salmonella typhimurium SL7207(pmc DNA3.1+) was apparently lowered than that of Salmonella typhimurium SL7207( pcDNA3.1+) and the high ampicillin concentration was maintained longer in LB me dium culturing Salmonella typhimurium SL7207(pmcDNA3.1+). When mice were a dministered with Salmonella typhimurium SL7207(pmcDNA3.1+) intraperitoneall y, more than 95% of Salmonella cells separated from the spleen still harbore d the plasmid pmcDNA3.1+ 7 days later; but 99% of Salmonella cells lost the plasmid pcDNA3.1+ at day 3 in mice innoculated with Salmonella typhimurium SL7207(pcDNA3.1+). By lowering the expression of bla gene, the rapid deco mposition of ampicillin in LB medium was avoided and the metabolic pressure was relieved for the host cells. This method offers a solution for the problem of t he instability of high-copy-number plasmid within Salmonella typhimurium.

OBJECTIVEBy studying the process of reverse osmosis system for traditional Chinese medicine materials physicochemical parameters affecting the osmotic pressure of its relevance, new compound system reverse osmosis process design methods were explored.

METHODThree concentrations materials for high, middle and low were dubbed with Sini decoction as a model drug, and pretreated by 50 thousand relative molecular weight cut-off ultrafiltration membrane. The viscosity, turbidity, conductivity, salinity, TDS, pH value and osmotic pressure of each sample were determined after the reverse osmosis to study the physical and chemical parameters between their respective correlations with the osmotic pressure, and characterized by HPLC chromatograms showing changes before and after the main chemical composition of samples of reverse osmosis.

RESULTConductivity-osmotic pressure, salinity-osmotic pressure of the linear correlation coefficient, TDS-osmotic pressure between the three sets of parameters were 0.963 8, 0.932 7, 0.973 7, respectively. Reverse osmosis concentrate and its characteristic spectrum ultrafiltrate HPLC similarity were up to 0. 968 or more, except the low concentrations.

CONCLUSIONThere is a significant correlation between the three physicochemical parameters (conductivity, salinity, TDS) and osmotic pressure of each sample system, and there is also significant linear correlation between salinity, conductivity, TDS. The original chemical composition of Sini decoction material concentrate was completely remained after the process of reverse osmosis.

Chemical Phenomena ; Chemistry, Pharmaceutical ; methods ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Molecular Weight ; Osmotic Pressure ; Surface Properties ; Viscosity

BACKGROUND:Studies have shown that arthroscopic al-inside technology for meniscus repair has achieved good outcomes, and arthroscopic suture combined with sodium hyaluronate has an important role in the cartilage repair. OBJECTIVE:To observe the therapeutic effect of arthroscopic al-inside suture repair combined with sodium hyaluronate injection in the repair of discoid meniscus tears. METHODS:Twenty-two patients with discoid meniscus injury were subjected to arthroscopic al-inside suture repair combined with sodium hyaluronate injection. Al cases were confirmed to have attached edge relaxation or longitudinal crack in operation. After repair, effective rehabilitation training was done. RESULTS AND CONCLUSION:The operations for the 22 cases were al successful and there was no complication. Al patients were fol owed up for over 18 months. According to Ikenchi’s method (Lysholm knee score criterion), 11 cases were rated excel ent, 8 were rated good, and 3 were rated fair, with an excel ent/good rate of 86.36%. It is demonstrated that the al-inside suture repair under the guide of arthroscopy in discoid meniscus cases can remain the most appropriate anatomical structure of the meniscus that can play an effective function, and has the advantage of minimal invasion. Arthroscopic al-inside suture repair combined with intra-articular injection of sodium hyaluronate and standard rehabilitation training result in a lower complication rate and effectively promotes the meniscus repair.

Objective To explore a new model of medical humanity education in new situation. Methods Extensive survey was carried out among medical students,and discussion and consultation were hold to faculty and education administers.A new teaching model was established. Results A curriculum system with 1 or 2 core courses supported by some elective courses and social practice is under the process of implementation and achieved preliminary success.Conclusion The medical humanity education should pay attention to the development of connotation instead of pursuing the extension of quantity blindly,which means that students' school work must be eased by focusing on self-study and self-education.Medical students'life path with high-minded personality training should be strengthened.

Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.
Acyltransferases ; chemistry ; genetics ; metabolism ; Amino Acid Sequence ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Isatis ; chemistry ; classification ; enzymology ; genetics ; Models, Molecular ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Quinic Acid ; metabolism ; Sequence Alignment ; Shikimic Acid ; metabolism

Objective To compare four methods of monitoring vascularization of tissue engineered bone in the rhesus so as to find our the best. Methods Twenty-five lower limbs of 13 rhesuses were used in this study to make models of tibial diaphyseal defect of 20mm which were to be fixed with an AO reconstruction plate of 7 holes. The monkeys were randomly divided into five groups according to defect filling materials: group A:?-tricalcium phosphate (?-TCP) and bone marrow stromal cells (BMSCs) and blood vessel bundles; group B:?-TCP and blood vessel bundles; group C:?-TCP and BMSCs; group D:?-TCP; group E: blank. Perfusion weighted MR imaging (PWMR), X-ray, radionuclide imaging and histological examinations were carried out at weeks 4, 8, 12 postop- eratively. The maximum slope rates of the single intensity-time curve (SS_(max)) and values of baseline (Sl_(?))were calculated at the same time points. Transmittances of the X-ray films were assessed. Ratios between isotope counts in region of interest (ROI) were calculated. Chinese ink perfusion and calculation of blood vessel areas were done for histological examinations, Results Compared with other groups, the SS_(max) in group A was the highest at weeks 4, 8, 12 postoperatively. In group A, the SS_(max) at week eight was significantly higher than that at week four (P= 0. 003), and the SS_(max) and transmittance of X-ray were negatively related at week 12 after operation (rs=-0. 892, P=0. 042), but the SS_(max) and blood vessel area were positively related (rs=0. 894, P=0.041)Conclusions PWMR can be a sensitive, quantitative, noninvasive and non-radiant method to monitor vascularization of tissue engineered bone, because SS_(max) of the single intensity-time curve of PWMR can reflect the most accurately the process of vascularization of tissue engineered bone.

Objective:To evaluate the clinical efficacy of SKy bone expander system in percutaneous kyphoplasty for treat- ment of osteoporotie verterbral compression fracture.Methods:Twenty-two patients(aged 62-90 years,32 vertebrae)under- went percutaneous kyphoplasty using SKy bone expander system.The bone cement was injected into the collapsed vertebrae. The vasual analogue scale(VAS)and complications were recorded during follow up.Results:The operations were successful in all patients via unilateral or bilateral approach.The operation time ranged from 30 to 120 min.The mean volume of cement in- jected into each vertebra body was(4.8?1.1)ml,ranged from 3.1 to 6.8 ml.Extravertebral leakage of bone cement was ob- served in two vertebrae with no symptoms.All patients had their pain relieved;the VAS was 7.6?0.8 before operation,3.5?0.5 one day after operation,2.8?0.6 one week after operation,and 2.4?0.6 one month after operation,with significant difference found between preoperation and postoperation(P

The chemical constituents from Euphorbia lunulata was investigated in this paper. Fourteen compounds were isolated and purified by column chromatographies on silica gel and preparative HPLC. Their structures were identified by physiochemical properties and NMR data analysis as lupeol (1), euphol (2), cassipourol(3) , 24-methylenecycloartan-3beta-ol (4), 24-hydroperoxycycloart-25-en-3beta-ol (5), 25-hydroperoxycycloart-23-en-3beta-ol (6), betulin (7), uvaol (8), (23E) -25-methoxycycloart-23-en-3beta-ol (9), (23E) -cycloart-23,25-dien-3beta-ol (10), 24-methylenecycloartan-3beta, 28-diol (11), salicinolide (12), 2alpha, 3beta, 5alpha, 9alpha, 15beta-pentaacetoxy-11,12-epoxy-7beta, 8alpha-diisobutyryloxyjatropha-6 (17) -en-14-one (13) and 3beta, 5alpha, 15beta-triacetoxy-7beta-isobutyryloxy-9alpha-nicotinoyloxyjatropha-6 (17), 11(E)-dien-14-one (14). Among them, compounds 1-11 were isolated from E. lunulata for the first time.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Euphorbia ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Stereoisomerism