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BACKGROUND: Recombinant adeno-associated virus 2(rAAV-2) has attracted considerable attention due to its nonpathogenic nature in contrast to other viral vectors such as adenoviral and retroviral vectors in gene therapy attempts.OBJECTIVE: To explore rAAV-2 transduction to bone marrow mesenchymalstem cell(BMSC) in vitro and evaluate the possibility of using rAAV-2 as a vector for gene therapy of acute myelogenous leukemia(AML).DESIGN: An open experiment with cells as the observational subjects.SETTING: Department of Hematology, Nanfang Hospital, Southern Medical University.MATERIALS: The experiment was conducted in the Department of Hematology, Nanfang Hospital, Southern Medical University from February to July 2004. We used passages 3 to 5 BMSCs derived from six de novo AML patients and four healthy volunteers in this study.METHODS: BMSC was isolated from 6 to 10 mL of bone marrow aspirates obtained from the iliac crests of the patients who had been diagnosed as having de novo AML and from those of healthy volunteers. The acquired BMSC was infected by rAAV-2 which contained enhanced green fluorescent protein (rAAV-2-eGFP) at different multiplicity of infection(MOI) (MOI = 1 × 102,1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107) . Then we observed through phase contrast fluorescent microscope and flow cytometer to evaluate green fluorescent protein(GFP) expression 10 to 14 days after transduction. GFP expression was observed as the rAAV-2-eGFP transduced BMSC cultured in vitro. We also observed the in vitro gene expression profile of GFP in rAAV-2-eGFP transduced BMSC which was selected by neomycin ( G418). First, we confirmed GFP expression in BMSC through phase contrast fluorescent microscope, then on flow cytometer to detect the percentage of GFP expression.MAIN OUTCOME MEASURES: The efficiency of rAAV-2-eGFP transduction to BMSC. GFP expression was observed through phase contrast fluorescent microscope and flow cytometer at different time points after transduction.rAAV-2-eGFP to BMSC derived from normal volunteers and AML patients had no significant differences. GFP began to express 10 to 14 days after transduction, and the transduction efficiency ranged from 0. 3% to 1.4%. By changing infection condition, we could not make a higher transduction efficiency( P ＞ 0.05) . One round infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was ( 1. 030 ± 0. 034) %, 3 rounds of infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was (1. 140 ±0. 036)%, and coinfected by LipofectAMINE was (1. 380 ± 0. 054)%. However, 293 cell line which was the package cell of rAAV-2 could be efficiently transduced by AML patients transduced by rAAV-2-eGFP at MOI = 1 × 105: The percentage of GFP expression cell gradually decreased from 1.14% at day 12 after transduction to 0. 6% as cell passaged from 2 to 3, and maintained at a level of 0. 5% to 0. 6% later on till 61 days after transduction. After selected by neomycin(G418) 1 month later, rAAV-2-eGFP transduced BMSCs could maintain a long-term GFP expression at a level of 6.0% in vitro without significant decay within 100 days of observation period after transduction.CONCLUSION: The advantages of rAAV-2 mediated gene transduction lie in safety, no immune response to the host, and long-term expression maintained by the target gene. rAAV-2 and BMSC can be used for in vitro gene therapy, and as a systemic gene delivery system, it might be an alternative for systemic gene therapy in the future.

Objective To investigate the outcome between endoscopically assisted and routine anterior transposition of the ulnar nerve for treatment of cubital tunnel syndrome.Methods From Februray 2008 to June 2010, forty-four patients with cubital tunnel syndrome were treated with routine anterior subcutaneous transposition (routine group,28 cases) and endoscopically assisted anterior subcutaneous transposition (endoscope group,16 cases).The operate time,drug administration,scar and postoperative hospital stay were compared.The patients were followed 1-12 month postoperatively,postoperative time back to work and function of ulner nerve were recorded.Results The results of endoscope group were as follows: operative time was (67.20 ± 19.69)min; postoperative scar length was (1.5％ ± 0.58) cm; rate of administration of anodyne was 6.3％; postoperative hospital stay was (2.4％ ± 1.42) days; postoperative time back to work,(14.6 ± 4.69)days; the results of open surgery group were as follows:operative time (62.8％ ± 11.06) min; postoperative scar length was (8.7％ ± 1.42) cm; rate of administration of anodyne was 42.8％; postoperative hospital stay was (5.7％ ± 2.53) days; postoperative time back to work was (29.40 ± 8.75) days; all differences of the results were significant between two groups (P ＜ 0.05).According to function of ulner nerve scoring system,one year postoperatively, excellent or good results were 82.14％ in routine group and 81.25％ in endoscope group,no significant difference between two groups (P ＞ 0.05). Conclusion Compared with routine anterior transposition of the ulnar nerve,endoscopically assisted anterior transposition has the following advantages: smaller incision and less tissue damage,less postoperative pain and sooner returning to work.And similar outcome was achieved from the two group.

OBJECTIVEStudy the effect of self-microemulsifying drug delivery system(SMEDDS) on the solubility and absorption of tanshinones to guide the selection of composition of tanshinone SMEDDS.

METHODThe solubility of tanshinones in the solution of SMEDDS was determined by UV-spectrometer and the absorption of tanshinone SMEDDS was determined by HPLC as the detection method.

RESULTThe solubility of tanshinones in solution of SMEDDS was 10 times in water and 2.5 times in micelle solution. The solubility of tanshinones in solution of SMEDDS was increased with the increasing of oil (MCT) in composition of tanshinone SMEDDS. The absorption constants (Ka) in SMEDDS and micelle solution was 0.479 h(-1) and 0.326 h(-1) respectively, and the absorption half life (t1/2) was 1.44 h and 2.12 h respectively. The absorption was increased with the oil increasing in composition of tanshinone SMEDDS.

CONCLUSIONSMEDDS can increase the solubility and absorption of tanshinones significantly and the increasing of oil content (MCT) in SMEDDS composition promote the dissolution and absorption of tanshinones.

Animals ; Diterpenes, Abietane ; Drug Delivery Systems ; methods ; Emulsions ; Intestinal Absorption ; drug effects ; Male ; Phenanthrenes ; chemistry ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Solubility

OBJECTIVETo investigate the expression pattern of resistin (RSTN) in skeletal muscle tissue and its influence on glycometabolism in rats with traumatic brain injury (TBI).

METHODSSeventy-eight SD rats were randomly divided into traumatic group (n=36), RSTN group (n=36) and sham operation group (n=6). Fluid percussion TBI model was developed in traumatic and RSTN groups and the latter received additional 1 mg RSTN antibody treatment for each rat. At respectively 12 h, 24 h, 72 h, 1 w, 2 w, and 4 w after operation, venous blood was collected and the right hind leg skeletal muscle tissue was sampled. We used real-time PCR to determine mRNA expression of RSTN in skeletal muscles, western blot to determine RSTN protein expression and ELISA to assess serum insulin as well as fasting blood glucose (FBG) levels. Calculation of the quantitative insulin sensitivity check index (Q value) was also conducted. The above mentioned indicators and their correction were statistically analyzed.

RESULTSCompared with sham operation group, the RSTN expression in the skeletal muscle as well as serum insulin and FBG levels revealed significant elevation (P<0.05), and reduced Q value (P<0.05) in traumatic group. Single factor linear correlation analysis showed a significant negative correlation between RSTN expression and Q values (P<0.001) in traumatic group.

CONCLUSIONThe expression of RSTN has been greatly increased in the muscular tissue of TBI rats and it was closely related to the index of glycometabolism. RSTN may play an important role in the process of insulin resistance after TBI.

Animals ; Brain Injuries ; metabolism ; Glucose ; metabolism ; Insulin Resistance ; Male ; Muscle, Skeletal ; chemistry ; metabolism ; Rats, Sprague-Dawley ; Resistin ; analysis

Objective: To study the effect of nonthermal argon atmospheric pressure plasma (NTAPP) treatment on the bonding strength between dentine and self-etch adhesive and the wettability of dentine surfaces under different treatment times. Methods: The plasma jet was operated at an input power of 9 W. Argon was used as the operating gas at a flow rate of 5 L/min. The dentin surface was exposed to the plasma jet (n=6) for various times (0, 5, 10, 15, 20 s). After a one-step self-etch adhesive (S3 Bond) was applied to the treated dentine surface, microtensile bonding specimens were made, and the microtensile bonding strength was tested. Then, the dentine surface contact angles were measured after NTAPP treatment for 5, 10, 15, and 20 s with the same gas flow rate and input power described above. Results: Along with the NTAPP treatment time, the dentin immediate bonding strength was significantly increased. The 15 s group showed significantly elevated bonding strength (31.82±2.80 MPa) in contrast to the other groups. The contact angles of each experimental group significantly decreased compared with the contact angles of the negative control group (75.57°±1.45°). The contact angles decreased the most to 33.56°±2.14° with NTAPP treatment for 15 s, and its wettability was the highest. Conclusion NTAPP treatment can significantly increase the wettability of the dentin surface and improve the adhesive strength of the adhesive interface with self-etching adhesive, which is also related to the treatment time.