Objective To establish a mini-column centrifugation-HPLC method to determine the entrapment efficiency of levodopa-loaded PEGylated-solid lipid nanoparticles.Methods A dextran gel(Sephadex G-50) mini-column centrifugation was employed to separate the free drug from solid lipid nanoparticles.The content of levodopa was qualified by HPLC.Results Under the applied chromatographic condition,the excipients had no influence on the determination of levodopa.A calibrated linear of levodopa concentration was within 10.54-527.00 μg·mL-1.The recoveries of high,medium and low concentrations of levodopa were 99.13%,99.51% and 99.04%(RSD were 1.25%,1.91% and 1.71%), respectively.The free levodopa was well separated from solid lipid nanoparticles by using mini-column centrifugation.The addition of blank solid lipid nanoparticles recovery was 98.84% with RSD of 0.80%(n=3).The average adsorption rates of the three concentrations of free levodopa were 100.00%,98.75% and 98.56%(RSD were 0.00%,0.19% and 0.18%,n=3),respectively.The adsorption rate of the physical mixtures of three different concentrations of drugs and empty PEGylated solid lipid nanoparticles were 99.68%,98.46% and 99.21%(RSD were 1.52%,0.23% and 0.21%),respectively.Conclusion The method was simple,accurate and reproducible,which can be used for determination of the entrapment efficiency of levodopa-loaded PEGylated-solid lipid nanoparticles.

Objective To construct a lentiviral vector over-expressing β-catenin gene by multisite Gateway technology and confirm its effect.Methods By using multisite Gateway clone technique,the entry clone of pDown-Ctnnb1 was constructed using BP recombination reaction.Then,LR recombination reaction was performed among pUp-EF1A,pDown-Ctnnb1,pTail-IRES/DsRed-Express2 and pLV.Des3d.P/puro to generate an expression clone of pLV.EX3d.P/puro-EF1A＞Ctnnb1 ＞IRES/DsRed-Express2.In each step,PCR and sequencing analysis were used to verify the constructions.When it was verified that plasmids were transfected into 293T cells,PT-PCR was performed to determine the mRNA level of β-catenin gene.Results Both PCR and sequencing analysis revealed that β-catenin over-expression gene was inserted into the target site and the insertion sequence was perfectly corrected.The RT-PCR results showed that the expression of β-catenin gene was significantly upregulated.Conclusion The lenvivirus-mediate β-catenin over-expression gene was successfully constructed..

Objective To explore the characteristics of resting myocardial perfusion of type 2 diabetes mellitus(T2DM) by real-time myocardial contrast echocardiography(RT-MCE). Methods Thirteen patients with T2DM and 12 in control were enrolled in the study. RT-MCE was performed using a continuous infusion of SonoVue with vena mediana in elbow. Images of left ventricle filled with contrast were acquired from apical 4-, 2-chamber and long axis views with real-time myocardial contrast mode of GE Vivid 7 Dimension system. All above images were captured in continuous 3 cardiac cycles before "flash" ,and then 15 cardiac cycles after "flash". All clips were stored for off-line analysis. Results In quiescent condition, there were significant decrease on indices of A,k and A × k in patients compared with control group (A:6.46 ±1.60 vs 6.81 ± 1.53, P ＜0.05;k:1.04 ± 0.39 vs 1.28 ± 0.31*, P ＜0.01;A× k:6.55 ± 2.72 vs 8.78 ±3.16, P ＜ 0.01, respectively). Conclusions RT-MCE could evaluate the early change of myocardial perfusion in T2DM patients and offers important reference for clinical diagnosis and treatment.

ObjectiveTo assess the effectiveness and prognosis of different radical surgery for Hirschsprung disease (HD).MethodsThe bowel function of HD patients undergoing the anus modified Soave operation (84 cases,modified Soave group),modified Swenson operation (60 cases,modified Swenson group),modified Duhamel operation (76 cases,modified Duhamel group) was followed up by 3,6 months and 2 years after surgery.Long-term bowel function,clinical type,removal length,anorectal manometry,barium enema were analyzed and compared among three groups.ResultsThe occurrence rates of bowel dysfunction 3,6 months and 2 years after surgery in modified Soave group[17.9％(15/84),7.1％(6/84),4.8％ (4/84)] were significantly lower than those in modified Swenson group[41.7％ (25/60),21.7％(13/60),18.3％(11/60) ] and modified Duhamel group [ 36.8％ (28/76),18.4％ (14/76),13.2％(10/76) ].There was significant difference between modified Soave group and modified Swenson group,modified Duhamel group(P＜ 0.05 ).There was no significant difference between modified Swenson group and modified Duhamel group (P ＞ 0.05).When the removal length ≤35 cm,the occurrence rate of bowel dysfunction after surgery in modified Soave group [ 18.7％ (14/75)] was lower than that in improved Swenson group [ 39.5％ ( 17/43 ) ] and modified Duhamel group [ 34.4％ (21/61 ) ].There was significant difference between modified Soave group and modified Swenson group,modified Duhamel group (P ＜ 0.05).There was no significant difference between modified Swenson group and modified Duhamel group (P ＞ 0.05).When the removal length ＞ 35 cm,there was no significant difference in the occurrence rate of bowel dysfunction after surgery among three groups (P ＞ 0.05 ).The occurrence rates of bowel dysfunction in short-segment type and common type in modified Soave group was lower than those in modified Swenson group and modified Duhamel group.There was significant difference between modified Soave group and modified Swenson group,modified Duhamel group(P ＜ 0.05).There was no significant difference between modified Swenson group and modified Duhamel group (P＞ 0.05).The anorectal angle 2 years after surgery in modified Soave group [(93.67 ± 10.50)° ] was less than that in modified Swenson group [(110.20 ± 11.88)° ] and modified Duhamel group [(106.33 ± 12.21)° ].There was significant difference (P ＜0.05).ConclusionThe complication and trauma are significantly lower in the anus modified Soave operation than the modified Swenson operation and modified Duhamel operation,but the choice of surgery should be strictly controlled according to the anal HD treatment indications.

Objective To explore the clinical value of color Doppler flow imaging(CDFI) combined with quantitative analysis technique to evaluate renal flow perflusion. Methods Twenty patients with different renal diseases matched with healthy control subjects were examined with Philips iU22 CDFI system. CDFI system and Qlab quantitative technique were used to measure renal restrictive index(RI), vascularization index(VI), flow index(FI), vascularization-flow index (VFI). The patients all received renal single-photon emission computed tomography (SPECT) examination. Results The RI of 32 kidney with lower flow perfusion in SPECT were higher than in the contol group (P <0.01). VI,FI, VFI were lower than those in control(VI, VFI, P <0.01 ;FI, P <0.05). Conelusious The renal blood flow perfusion of patients with renal diseases are lower than that of healthy people. It has high clinical value using CDFI combined with quantitative analysis technique to evaluate the renal perfusion of the patients with renal diseases.

Objective To investigate the expression of macrophage migration inhibition factor (MIF) and cell cycle regulating factor Cyclin D1 in hepatocellular carcinoma tissue and the interaction between MIF and Cyclin D1 in hepatocellular carcinoma cell cycle controlling. Methods Using quantitative real-time PCR and Western blotting to detect mRNA and protein expression of MIF and Cyelin DI in HCC tissues and tumor adjacent tissues. Specific small interfering RNA(siRNA) targeting MIF gene was transfccted at doses of 50 nmol/L and 100 nmoL/L into HCC cell lines of PLC and HepG2 with lipofeetamine 2000 methods to knockdown the expression of M1F gene and to investigare the the interaction between M1F and Cyclin D1. Results MIF and Cyclin D1 protein and mRNA were overexpressed in HCC tumor tissues. The relative expression of MIF,Cyclin D1 protein and mRNA were 0.825±0.13,0.843± 0.104 and 7.31±1.85 folds、4.27±1.05 folds, compared with the tumor adjacent tissues (FMIF= 15.5, P<0.01;FCyclin D1=87.5,P <0.01). In MIF siRNA treated PLC and HepG2 cells, MIF mRNA down regulation 71.2%±7.2%, 87.4%±2.9% ,74.3%±8.9% and 88.4%±4.6% respectively (FPLC = 315.5 ,P < 0.01 ; FHepG2= 201.2 P < 0.01). While MIF protein expression were significandy reduced to 0.33±0.03,0.11±0.02, 0.81±0.08 and 0.36±0.02 in a dose-dependent manner (FPLC= 43.9, P <0.01 ;FHepG2 = 133.4 P <0.01). Cyclin D1 mRNA was significantly down-regnlated in MIF siRNA treated PLC and HepG2 cell lines when compared with control group(P <0.01). In 50 nmol/L and 100 nmol/L groups, Cyclin DI mRNA levels were respectively decreased by 68.2%±3% and 78.1%±1.4% in PLC cell, 65.8%±4.7% and 77.3%±2.6% in HepG2 cell (FPLC= 1569, P < 0.01 ; FHepG2= 480.4, P <0.01). Compared with control groups, Cyclin D1 protein levels significantly reduced to 0.28±0.06、0.15±0.03 and 0.44 ±0.04、0.13±0.02 in the PLC and HepG2 after M IF siRNA treatment(FPLC= 35.5, P < 0.01 ; FHepG2 = 114.7, P < 0.01). Conclusions MIF and Cyclin D1 mRNA and protein were overexpressed in HCC tumor tissues and participated in tumor cell cycle regulation. MIF may up-regnlate the expression of Cyclin DI via ERK signalling and precipitate in carcinogenesis of hepatocellular carcinoma.

Objective To assess the value of quantitative color Doppler sonography in patients with systemic lupus erythematosus (SLE). Methods The right renal in 21 patients with SLE and 22 normal subjects were examined with Philips IU22 ultrasonic diagnostic device. Resistence index (RI) of interlobar artery and interlobular artery were measured with color Doppler sonography, vascularity index (VI), flow index (FI) and vascularization flow index (VFI) which reflecting the renal cortical blood flow were calculated with quantitated analysis software. All the parameters between two groups were compared and analyzed. Results The color signals of renal cortical blood flow in patients with SLE were inferior to those of the normal group, especially in the area near the edge of renal. No statistical difference of RI was found between the two groups (P>0.05), while VI, FI and VFI in SLE group were significantly lower than those of the normal group (VI, VFI, P<0.01, FI, P=0.01). Conclusion Color Doppler sonography combined with blood flow quantitative technique provide a helpful, sensitive and convenient tool to detecte the renal changes in SLE patients.

Objective To explore the difference in ocular biologically effective ultraviolet irradiance in two areas with different altitudes ,Xichang and Shaoxing,and provide a reference basis for ocular UV protection. Methods A self?designed rotating mannequin and dual?detector spectrome?ter were used to monitor the intensity of ocular exposure to UV irradiation under clear skies in Xichang and Shaoxing. Monitoring data were pro?cessed and analyzed with AvaSoft 7.4 USB2 software and OriginPro 8.0 software. Results The diurnal variations of ocular biologically effective ul?traviolet irradiance exhibited bimodal distribution in Xichang and Shaoxing. The maximum UVBEcorn,UVBEconj,and UVBElens of Xichang were about 1.7 times,2 times,and 1.8 times that of Shaoxing,respectively. Under the same solar elevation angle,the biologically effective ultraviolet irra?diance of the cornea,conjunctiva,and lens in Xichang were higher than those of Shaoxing. Conclusion The diurnal variations of ocular biologi?cally effective ultraviolet irradiance exhibit bimodal distribution in areas at two different altitudes. Under the same solar elevation angle ,the biologi?cally effective ultraviolet irradiance of the cornea,conjunctiva,and lens of Xichang are significantly higher than those of Shaoxing.

Objective To observe the rotation of subendocardium and subepidium by two-dimensional speckle tracking imaging(2D-STI),and to evaluate its performance in diastolic heart failure patients(DHF)with a normal left ventricular ejection fraction. MethodsNinety-seven consecutive clinically stable patients were enrolled in this study [41 healthy controls,36 with diastolic heart failure,20 with systolic heart failure (SHF)]. High frame rate dynamic two-dimensional images were recorded at the left ventricular short-axis view,including basal, papillary muscle and apical planes. Subendocardial and subepicardial global rotation were measured using Q-lab 7.0 software offline. Results ① In all the subjects, the rotation of the subendocardium was obviously greater than that of subepicardium. ②As seen from the apex,left ventricular subendocardium and subepicardium performed a wringing motion with a clockwise rotation at the base and countclockwise rotation at the apex. ③In the apical plane, subendocardial rotation was significantly lower in both heart failure groups than in controls,and was depressed to a larger extent in SHF patients than in those with DHF. Subepicardial rotation was no significant difference between the DHF group and the control group, though it was significantly lower in patients with SHF. ④At the base, the rotation of subendocardium and subepicardium were not different between DHF and control groups, but it was significantly reduced in patients with SHF. Conclusions The subendocardial rotation is reduced, but subepicardial rotation is normal in DHF patients. On the other hand, in patients with SHF, subendocardial and subepicardial rotation are both reduced. The left ventricular systolic properties are impaired in DHF patients.

Objective To summarize the characteristic of muscle MRI of lower limbs in patients with GNE myopathy and to explore the correlation between the fatty degenerative score of muscle MRI and clinical phenotype.Methods This was a prospective study. Seventeen patients with genetically confirmed GNE myopathy,having lower limb muscle MRI test and completed clinical and laboratory data.The degree of fatty degeneration in 18 muscles of lower limbs in each patient was grading.According to the GM-W score, these patients were divided into two groups.GM-W score≤3 were divided into mild group(n=8)and GM-W score≥ 4 were divided into severe group(n=9). Kruskal-Wallis test was used to compare the fatty degenerative score in different muscles of the thigh and the calf level;Mann-Whitney U test was used to compare score of the same muscle between mild and severe group;Spearman rank correlation test was used to analysis the relationship between fatty degenerative score and the course of disease (year), GM-W score, creatinine kinase (IU/L), respectively.Results At the thigh level, the most severely involved muscle of GNE myopathy was semi-tendinosusand adductor, followed by semi-membranous, biceps femoris and gracilis. There was no statistically significant difference in the fatty degenerative score of the above-mentioned muscles (P=0.058). At the calf level, the most severely involved muscle was medial of soleus which score was 4.0(3.0, 4.0), followed by tibialis anterior,extensor digitorum longus and lateral of soleus. There was no significant difference of the above (P=0.259).The fatty degenerative score showed difference between the mild and severe group at sartorius and adductor(P<0.05).At the calf level,the fatty degenerative score in peroneus longus, medial of soleus, lateral of soleus, medial of gastrocnemius and lateral of gastrocnemius showed difference between groups(P<0.05).The total score of fatty degenerative of Lower limb muscles was positively correlated with GM-W score(r=0.730, P<0.05). There were positive correlations between the score of fatty degenerative of the sartorius,peroneal longus,lateral of soleus,medial of gastrocnemius, lateral of gastrocnemius and the GM-W scores( r=0.630,0.845,0.569,0.591,0.640, 0.659,P<0.05).The total score of fatty degenerative of Lower limb muscles was not correlated with the level of creatine kinase(P=0.582), course of disease(P=0.601) and age of onset(P=0.850). Conclusions GNE myopathy in the thigh level within the adductor muscle and posterior muscle involvement, calf level to the tibial anterior muscle early involvement. The total score of fatty degenerative of lower limb muscles is positively correlated with GM-W score,but not correlated with the level of creatine kinase,course of disease and age of onset.