[Objective]To study the transfection and expression of green fluorescent protein gene in rat chondrocytes by constructing a combination lentiviral vector containing green fluorescent protein gene.[Method]293T cells were co-transfected with the vector plasmids pLentiLox 3.7,the packaging plasmids pRSV-REV,pMDL g/p RRE and the envelope plasmids VSV-G to produce lentiviral vector particles.Rat chondrocytes were isolated,cultured and infected by the lentiviral vector particles.The expression of reporter gene GFP was observed under fluorescent microscope.[Result]At 96 hours after transfection,a strong expression of GFP could be found in 293T cells.The viral titer was 3.0?103ifu/?l.GFP expression was observed in about 80% of the rat chondrocytes.[Conclusion]A combination lentiviral vector containing green fluorescent protein gene was successfully established and it could infect rat chondrocytes efficiently,which will provide a basis for gene modification of chondrocytes through RNA interference.

Objective To evaluate the methods and efficacy of microsurgical treatment for the premalignant and malignant lesions of the conjunctiva. Methods Forty-seven patients witn premalignant and malignant lesions of the conjuncitva were managed by microsurgical, these patients include 12 melanoma, 26 squamous cell carcinoma, 6 Bowen’ diseases, 3 primary acquired melanosis. The surgical method differs with limbal tumors, extralimbal tumors, and primary acquired melanosis. Results In all 47 patients, the tumor was completely removed in in one procedure. After follow-up for 4~60 months(mean 17 months), these were no tumor recurrence. Conclusion It is effective methods that premalignant and malignant lesions of the conjunctiva are managed by microsurgical resection, alcohol application, and supplemental cryotherapy. Careful propeirativa clinical evaluation of patient with a conjunctiva neoplasm is important in making the correct diagnosis and planning the surgical approach.

BACKGROUND: The repair of articular cartilage defect is always a problem that is dedicatedly solved by doctors of orthopaedics. Autologous perichondrium, periosteum or allografting of cartilage have been used previously; however, the source of the donor is limited, the fixation is difficult as well as the occurrence of endochondrial ossification and delamination between the inferior cartilage and reparative cartilage, etc. Type Ⅱ collagen, the main component of cartilage matrix, has certain effects in the repair of articular cartilage defect.OBJECTIVE: To investigate the effects of type Ⅱ collagen sponge filling on the repair of articular cartilage defect.DESIGN: A randomized controlled trial.SETTING and MATERIALS: The study was conducted in the Guangzhou Institute of Traumatic Surgery. Materials were 24 adult male purebred New Zealand Rabbits(48 knees), ordinary grade, with a body mass of (2.29 ±0. 25) kg. Animals were fed with standard feeding in separate cage.INTERVENTIONS: A full-thickness defect in articular cartilage was made on the femoral trochlear surface by a drill of 5 mm in diameter and 3 mm in depth. Rabbits were allocated into filling group(type Ⅱ collagen sponge was grafted into left keen joint defect) and control group(right knee joint defect site was set as control) according to random number table.MAIN OUTCOME MEASURES: Gross morphological and histological observation of the defect repair in each dual week within 12 weeks after operation.RESULTS: During 10 - 12 weeks, in cuntrol group: The defect area was repaired by white and soft tissue that had no resistance to press. The repaired tissue was still lower than the surrounding articular surface with clear boundary. By histological observation, it was found that the defect was repaired by the mechanism similar to inflammatory reaction and the defect is ultimately filled by the hyperplasia of hyaline degenerative fibrous tissues. In filling group: the defect was repaired by semi-transparent, smooth, textured tissues with polish that had resistance to press as well as elasticity. The repaired tissue was almost similar to the shape of the surrounding cartilage,difficult to be distinguished. After histological observation, it was found that there was no inflammatory reaction, but active hyperplasia of inner bonetissue and cartilage tissues; a lot of osteoid tissues and trabeculation were found. Newlborn cartilage was fused with surrounding cartilage tissue and connected with surrounding tissues. Type Ⅱ collagen had significantly promoting effects in the repair of articular cartilage defect, and the repaired cartilage was close to normal cartilage. CONCLUSION: The self-made high purity type Ⅱ collagen sponge has favorable promoting effects in the repair of articular cartilage defect with good histocompatibility but without obvious toxic side effect.

Objective: To construct the eurokaryotic expression vector of KCNE1 gene and express recombinant KCNE1 in HEK293 cells.Methods:Human KCNE1 gene fragment was amplified from human placenta total RNA by RT-PCR and cloned into the vector of pCR2.1 TOPO by means of T-A cloning.KCNE1 cDNA was obtained from pCR2.1-KCNE1 by restriction enzyme digestion and inserted into the same restriction site of pEGFP-N1.Thus pEGFP-N1-KCNE1 was constructed and transfected into HEK293 cells with Effectene transfection reagent.Results:The eukaryotic expression vector pEGFP-N1-KCNE1 was successfully constructed by gene cloning and recombinant method and expressed in HEK293 cells.Conclusion:The cloning of KCNE1 gene and the construction and expression of its eukaryotic expression vector may shed some light on further functional study of KCNE1 gene.

Objective To compare the cardiac function effect of right ventricular septum(RVS) pacing with that of right ventricular apex(RVA) pacing.Methods One hundred and six patients with indication of dual chamber pacemaker implantation were divide into two groups randomly.In each patient,influence of different pacing site to LVEF and pacing parameter were examined and left ventricular eject fractions were compared.Results All patients' operation were successful,LVEF of RVS group compared with that of RVA showed a significant difference.Conclusion The cardiac function are significantly different between right ventricular septum pacing group and right ventricular apex group.

Objective To investigate the feasibility and methodology of active fixation lead on patients under right ventricular outflow tract septum(RVOTS) pacing.Methods Fifty DDD pacemaker patients were enrolled 31 male,23 female,50~86 years old,mean age 67.7?8.6.Ventricular active fixation lead was implanted in the right ventricular apex(RVA) and RVOTS successively and pacing parameter was tested.Results The success rate of RVOTS active fixation lead implantation was 98.15%.Mean lead threshold was 0.73?0.12 V.Pacing QRS duration show a significant difference between RVOTS pacing and RVA pacing,130.45?18.24 and 153.11?20.10,respectively(P

Objective To examine the stability of high-density lipoprotein cholesterol (HDL-C)during serum incubation at different temperature and time periods.Methods Ten healthy volunteers (4 males and 6 females,aged 24 to 59 years)from Beijing Hospital were recruited in May 2015.Fasting venous blood samples were collected and centrifuged to separate the sera.Serum samples were incubated at 4℃ for 24 h,25℃for 0,1,8 and 24 h (with or without an LCAT inhibitor).Serum to-tal cholesterol (TC),total free cholesterol (TFC)HDL-C and HDL-FC were measured by the HPLC Method.Results HDL-FC and HDL-C changed -6.91% and -2.17% during serum incubation at 4℃for 24h.TFC,HDL-FC and HDL-C changed significantly (averaged -13.70%,-25.88% and -1.53% respectively)during serum incubation at 25℃ for 24 h,in which the decrease of TFC and HDL-FC were inhibited by the addition of the LCAT inhibitor.The decrease of HDL-C was even higher in the presence of the LCAT inhibitor.Conclusion Serum TFC,HDL-C and HDL-FC levels changed during serum incubations,which were caused by the LCAT and CETP activities and the transfer of cholesterol among lipoproteins. For accurate measurement of serum HDL-C,prolonged serum storage should be avoided in clinical laboratories.

Objective To determine the prognosis and staging non small cell lung cancer (NSCLC) that extends across the fissure into adjacent lobe after surgery.Methods 3752 patients with histopathologically confirmed non small cell lung cancer (NSCLC) received surgical reeessetion from January,1997 to April,2007.Among them,163 patients have a tumor invasion beyond fissure.After matching by pathologic TNM staging (7th),326 patients whose tumor defined in a single lobe were eligible for analysis.Results Histopatholngic staging of matched patients was I a:10 patiens(6.1％ ),I b:79 patients (48.5％),Ⅱa:5 patients (3.1％ ),111:44 patients (27.0％) and Ⅲa:25 patients( 15.3％ ).5 years survival in patients with stage 1 tumors crossing the interlobar fissure was 51％,while in patients not cross the interlobar fissure was 63％ ( P ＜0.05 ).There was no difference in survival for tumors stage Ⅱa and above with regard to importance of interlobar extension.The T2 tumor extending across a lung fissure had a reduction in survival compared with T2 tumor not cross the lung fissure and similar to the T3 tumor without the fissure invasion.Conclusion Our results suggest that TNM staging should be modified for tumor extends the fissure into adjacent lobe.

To investigate the effects of 2-(4-methoxycarbonyl-2-tetradecyloxyphenyl)carbamoylbenzoic acid (CX09040) on protecting pancreatic β cells, the β cell dysfunction model mice were induced by injection of alloxan into the caudal vein of ICR mice, and were treated with compound CX09040. Liraglutide was used as the positive control drug. The amount and the size of islets observed in pathological sections were calculated to evaluate the β cell mass; the glucose stimulated insulin secretion (GSIS) test was applied to estimate the β cell secretary function; the oral glucose tolerance test (OGTT) was taken to observe the glucose metabolism in mice; the expressions of protein in pancreas were detected by Western blotting. The effects on the target protein tyrosine phosphatase 1B (PTP1B) were assessed by the PTP1B activities of both recombinant protein and the intracellular enzyme, and by the PTP1B expression in the pancreas of mice, separately. As the results, with the treatment of CX09040 in alloxan-induced β cell dysfunction mice, the islet amount (P<0.05) and size (P<0.05) increased significantly, the changes of serum insulin in GSIS (P<0.01) and the values of acute insulin response (AIR, P<0.01) were enhanced, compared to those in model group; the impaired glucose tolerance was also ameliorated by CX09040 with the decrease of the values of area under curve (AUC, P<0.01). The activation of the signaling pathways related to β cell proliferation was enhanced by increasing the levels of p-Akt/Akt (P<0.01), p-FoxO1/FoxOl (P<0.001) and PDX-1 (P<0.01). The effects of CX09040 on PTP1B were observed by inhibiting the recombinant hPTP1B activity with IC50 value of 2.78x 10(-7) mol.L-1, reducing the intracellular PTP1B activity of 72.8% (P<0.001), suppressing the PTP1B expression (P<0.001) and up-regulating p-IRβ/IRβ (P<0.01) in pancreas of the β cell dysfunction mice, separately. In conclusion, compound CX09040 showed significant protection effects against the dysfunction of β cell of mice by enlarging the pancreatic β cell mass and increasing the glucose-induced insulin secretion; its major mechanism may be the inhibition on target PTP1B and the succedent up-regulation of β cell proliferation.

Objective To develop a solid phase extraction and Folin-Ciocalteu method for the measurement of total polyphenols in urine samples.Methods From a group of individuals attending an annual physical examination at Beijing hospital, 123 healthy volunteers (52 males and 71 females, ranging in age from 18 to 81 years ) were recruited during the period from December 2013 to April 2014.Urine samples were stored in 0.5%HCl at -80 ℃.For analysis, samples were applied to the Plexa PAX solid phase extraction cartridge, to purity the polyphenols through washing, evaporating and reconstituting.Total polyphenols were measured by Folin-Ciocalteu colorimetric assay, calculated by gallic acid standard curve, and corrected by urine creatinine concentrations.The relationship between total polyphenols and fruits and vegetable intake and cardiovascular disease risk factors were analyzed. Results Gallic acid standard solution and urine samples were stable in 0.5% HCl for 48 h at RT and 7days at 4 ℃, respectively.The PAX cartridge effectively eliminated the possible interfere materials in urine and had better recovery for most of the polyphenol types.The inter assay and total CVs for the measurement of total polyphenols were 2.7%-3.8% and 2.4%-4.6 %, respectively.Total polyphenol concentrations of 123 healthy subjects were 114.13(82.97-146.70) mg GAE/g Crea.Total polyphenol levels positively correlated with both HDL-C (r=0.194, P=0.032) and apoAI (r=0.312,P<0.001), and negatively correlated with serum uric acid levels(r=-0.220,P=0.014).Conclusions We established a measurement of total polyphenols in urine samples using solid phase extraction and Folin-Ciocalteu method.This simple, precise method reliable and may be use to assess dietary polyphenols intake.