Objective To evaluate effects of 4-hydroxyphenyl retinamide (4-HPR) in different vehicles on the proliferation and apoptosis of human keloid fibroblasts (HKFs).Methods A film-ultrasonic dispersion method was used to prepare 4-HPR liposome solution and 4-HPR microbubbles.Primary HKFs were in vitro treated with the 4-HPR liposome solution at different concentrations of 0-80 mg/L for 6-48 hours,and the proliferative activity of HKFs was evaluated by methyl thiazolyl tetrazolium (MTT) assay.Some other HKFs were divided into 3 experimental groups to be treated with 15 mg/L 4-HPR solution (4-HPR solution group),15 mg/L 4-HPR liposome solution (4-HPR liposome solution group) and 15 mg/L 4-HPR microbubbles (4-HPR microbubble group),respectively,and each group was divided into ultrasonic-treated and-untreated subgroups.HKFs without treatment served as control group.After 24-hour treatment,MTT assay was conducted to evaluate the proliferative activity of HKFs in the above groups,flow cytometry to detect apoptosis of HKFs in all groups except the 4-HPR solution group.Results The 4-HPR liposome solution and 4-HPR microbubbles were successfully prepared.MTT assay showed inhibitory effects of 4-HPR liposome solution at concentrations of 1-80 mg/L on the proliferation of HKFs,and the proliferation inhibition rate was positively associated with the drug concentrations (r =0.633,P ＜ 0.01).After the ultrasonic treatment,inhibitory effects on the proliferation of HKFs significantly differed among the 4-HPR microbubble group,4-HPR solution group and 4-HPR liposome solution group (P ＜ 0.01).The 4-HPR liposome solution group and the 4-HPR microbubble group both showed significantly increased apoptosis rates (21.81％ ± 3.73％,39.79％ ± 1.61％,respectively) compared with the control group (6.18％ ± 0.61％,both P ＜ 0.01).Conclusion The 4-HPR microbubbles are successfully prepared,and 4-HPR in different vehicles all can promote HKF apoptosis and suppress HKF proliferation,among which,4-HPR microbubbles in combination with ultrasonic treatment have stronger inhibitory effects than the 4-HPR liposome solution.

Objective To investigate the association of serum ferritin and hemoglobin with the pathogenesis of primary restless legs syndrome(RLS). Methods Thirty-five patients with primary restless legs syndrome and twenty insomnia controls were included in this study. The level of serum ferritin and hemoglobin were measured, and the data have been analyzed using t-test. Results The level of serum ferritin in the primary RLS patients (89.77μg/L (SD:48.52)) was significantly lower than in the contruls (123.36 μg/L (SD:35.06)) (t=-2.713,P <0.01), whereas the level of hemoglobin have no statistical difference between the RLS group (142.77 g/L (SD: 11.79)) and the control group (139.05 g/L (SD: 12.33)) (t = 1.108, P > 0.05). Conclu-sions The decrease of serum ferritin may be a risk factor of primary restless legs syndrome, but the level of hemo-globin is not associated with primary restless legs syndrome.

Objective:To provide appropriate test liquid preparation methods for antibacterial or water-insoluble drugs, and estab-lish a convenient and effective method for microbial limit test. Methods:Using function of polysorbate 80 as neutralization, emulsifica-tion, solubilization and microbial protection, We carried out the liquid preparations were prepared and microbial limit test validation was carried out for 11 kinds of hospital drugs. Results:The bacteria recovery rate and the control bacteria test were in accordance with the requirement of pharmacopoeia. Conclusion:Polysorbate 80 can be used in the microbial limit test of the drugs.

Objective To investigate characteristics of memory-guided saccade in Parkinson' s disease (PD),and to evaluate the application of memory-guided saccade in diagnosing PD.Methods Fiftythree subjects with early- or mid- stage PD were chosen as PD group,meanwhile,36 age-matched healthy subjects were chosen as control group.Memory-guided saccade test and event-related potential P300 were performed in all subjects,and results of the two groups were compared; furthermore,results of subgroups comprised of 29 patients with early-stage PD were analyzed.Results In comparison with control group,memory-guided saccade in PD group showed decreased velocity and primary gain,prolonged latency,extremely increased incidence of unwanted saccade and multi-step saccade ( U =124.000,37.000;both P ＜0.01 ),such abnormalities has already stood out even in subgroup comprised of early-stage PD patients.Meanwhile,latency of event-related potential P300 in PD group was prolonged compared with control group ((384.76 ± 34.48) ms vs (352.42 ± 24.99) ms,t =- 4.791,P ＜ 0.01 ).Multi-step saccade measurement demonstrated excellent sensitivity (96.2％ ) and specificity (94.4％) in the ability to discriminate PD patients from controls.Conclusion Memory-guided saccade in PD patients shows highly abnormal which may reflect the impairment of pontine saccade pathway and the dysfunction of frontal lobe.Memory-guided saccade test may be a useful examination in assisting the diagnosis of PD.

Objective:To explore the effects and mechanism of liraglutide on nitrogen monoxide (NO) release in human umbilical vein endothelial cells in the state of hypoxia and high glucose.Methods:A model of hypoxia and high glucose was established by using isolation and culture of primary human umbilical vein endothelial cells (HUVECs) in vitro.HUVECs were incubated with liraglutide and/or exendin (9-39) for 4 h.The metabolic ability of cells was detected by MTT assay,the activity of lactate dehydrogenase (LDH) was measured by a colorimetric method,and the levels of extracellular NO were measured by a nitrate reductive enzymatic method.The endothelial nitric oxide synthase (eNOS) mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR).Results:Compared with the model group,liraglutide could significantly increase cell metabolic ability,reduce LDH release,increase NO release and eNOS mRNA expression (P<0.05 or P<0.01).The above effects of liraglutide were partly inhibited by glucagon like peptide-1 (GLP-1) receptor antagonist exendin (9-39)(P<0.05).Conclusion:Liraglutide can improve endothelial relaxation function in HUVECs in the state of hypoxia and high glucose in vitro.The effect might be related to up-regulating eNOS mRNA expression and promoting NO release.

Objective:To develop an HPLC method for determining the total ginkgo flavonoid in self-emulsifying drug delivery sys-tem. Methods:Effective chromatographic separation was achieved using a phenomenex C18 column (250 mm × 4. 6 mm, 5 μm) with a mobile phase composed of methanol and water (0.4% phosphoric acid) with the ratio of 50 ∶50 (v/v). The mobile phase was pumped using an isocratic HPLC system at a flow rate of 1. 0 ml·min-1 , the detection wavelength was 360 nm and the column temper-ature was 30 ℃. Results:The three components in the total ginkgo flavonoid were well separated by the proposed method. The linear relationship between the peak area and the concentration was promising within the range of 2. 0-40. 0 μg·ml-1 for quercetin, 3. 0-60. 0 μg·ml-1 for kaempferide and 2. 0-40. 0 μg·ml-1 for isorhamnetin. The mean recovery of quercetin, kaempferide and isorham-netin was 98. 4%, 99. 7% and 100. 5% with RSD of 0. 92%,0. 62% and 1. 24% (n=9), respectively. Conclusion:The method is specific and stable in the determination of total ginkgo flavonoid in self-emulsifying drug delivery system.

Objective:To prepare total ginkgo flavonoid self-emulsifying drug delivery system(TGF-SMDDS)and estimate its char-acteristics in vitro. Methods:The formula of TGF-SMDDS was optimized based on the solubility tests,formula compatibility and microe-mulsion area in the pseudo ternary phase diagram. The appearance,morphology,particle size,zeta potential and in vitro dissolution of TGF-SMDDS were investigated. Results:The formula was composed of oleoyl macrogolglycerides as the oil phase,Tween-80 as the sur-factant and XCF as the co-surfactant. The ratio of oil phase,surfactant and co-surfactant was 10 ∶ 6 ∶ 4. The drug loading was 10. 0 mg· g -1 . After mixed with water,TGF-SMDDS was formed a clear and transparent microemulsion with homogeneous small spheres as seen un-der a transmission electron microscope. The particle size and zeta potential of TGF-SMDDS was(87. 4 ±26. 7)nm and( -13. 1 ±1. 5) mV,respectively. The accumulative dissolution of TGF-SMDDS in pH1. 2 hydrochloric acid solution was(96. 1 ±4. 8)% in 45 min. Con-clusion:The TGF-SMDDS can significantly enhance the dissolution of TGF in vitro,which may be a potential effective preparation for TGF.

Objective:To investigate the Migration ability toward human pancreatic carcinoma cell line and human colon carcinoma cell line with difference HSP 70 plasma membrane expression .Methods: CD3-CD56+NK cells were obtained from human peripheral blood mononuclear(PBMC)in stem cell growth medium SCGM,2μg/ml TKD was added to the medium on 10th day,the ac-tivating receptor CD94/NKG2C expression levels on NK cells was detected with FAC after 4 days.The human pancreatic carcinoma cell line Colo357 and the human colon carcinoma cell line CW 2 were separated into Colo+and CW2+with high HSP70 expression and Colo-and CW2-with low HSP70 expression;Migration assays of NK to the four difference cell lines were performed in a Transwell cell culture system.The cytolytic activity of TKD-activated NK cells against the four subline with HSP 70 expression on their cell surface was analyzed by MTT assay.Results:Flow cytometry analysis showed that CD 3-CD56+NK cells could expanded after 2 weeks in SCGM medium,and the largest percentage of NK cell was (92.50 ±1.25 )%.CD94 expression levels on NK cells increased obviously after TKD inducement the cell surface HSP 70 expression of Colo+, Colo-were ( 78.2 ±2.2 )% and ( 27.3 ±1.2 )% separately , the cell surface HSP70 expression of CW2+,CW2-were (91.1±2.5)%and (18.2±1.0)%separately after FACS;the Migration of NK cells toward Colo+was (68.6±2.8)%,higher than the migration toward Colo-with (22.8±1.5)%;the Migration of NK cells toward CW2+was(73.5±2.7)%,higher than the migration toward CW2-with (18.2±1.3)%;the cytolytic activity of NK against Colo +was(61.2± 3.0)%compared to (24.5 ±1.5)%against Colo-when the ratio of effector cells and target cell was 20 ∶1,the cytolytic activity of NK against CW2+was (63.8±3.2)%compared to (22.4±1.8)% against CW2-when the ratio of effector cells and target cell was 20∶1.Conclusion:TKD-activated NK cells are highly efficient cytolytic effector cells which have stronger significant migration toward HSP70-positive tumor target cells on their cell surface in vitro .

Objective:To establish an HPLC method for the simultaneous determination of eprosartan/hydrochlorothiazide tablets. Methods:Isocratic separation was achieved on a Phenomenex C18 column(250 mm × 4. 6 mm, 5 μm) using the mobile phase com-posed of 0. 5% formic acid-acetonitrile(60∶40, pH 2. 80). The flow rate was 1. 0 ml·min-1, the detection wavelength was 272nm, the column temperature was 30℃ and the injection volume was 20μl. Results:The linearity between peak area and concentration was observed within the range of 60. 0-1 200. 0 mg·L-1(r=0. 999 9) for eprosartan and 1. 25-25. 00 mg·L-1(r=0. 999 9) for hydro-chlorothiazide. The mean recovery of eprosartan and hydrochlorothiazide was 100. 02%(RSD=0. 35%, n=9) and 97. 93%(RSD=1. 54%, n=9), respectively. Conclusion:The method is simple, sensitive and accurate, and can be applied in the determination of eprosartan/hydrochlorothiazide tablets.

Objective:To prepare and optimize candesartan cilexetil tablets,and study the stability preliminarily. Methods:The formula was optimized by Box-Behnken experiment design,the ratio of lactose to pregelatinized starch( X1 ),the amount of disintegrant ( X2 ,%)and the amount of lubricant( X3 ,%)were selected as the independent variables,and weight difference( Y1 ,%),friability ( Y2 ,%),disintegration time( Y3 ,%)and candesartan cilexetil dissolution( Y4 ,%)were the dependent variables. The release rate of candesartan cilexetil tablets and the reference tablets were compared by similarity factor( f2 value). Preliminary stability was studied by high-temperature test,high-humidity test and illumination test. Results:The optimal formula of the tablets was as follows:the ratio of lactose to pregelatinized starch was 7:1,the amount of disintegrant was 5. 5%,and the amount of lubricant was 0. 5%. The f2 for the candesartan cilexetil tablets and the reference tablets in different dissolution meda was 60. 62,73. 34,66. 95 and 68. 60,respec-tively. Conclusion:The formula design is reasonable,the preparation process is feasible and the quality can be controlled.