AIMTo study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.

METHODSDipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.

RESULTSThe co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

CONCLUSIONCo-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.

Androstadienes ; pharmacology ; Animals ; Asialoglycoproteins ; pharmacology ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cell Survival ; Cells, Cultured ; Endocytosis ; drug effects ; Female ; Fetuins ; Flow Cytometry ; Hepatitis B Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Liposomes ; Microscopy, Fluorescence ; Nigericin ; pharmacology ; Oligonucleotides, Antisense ; chemistry ; genetics ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Wistar ; Sitosterols ; chemistry ; Transfection ; methods ; alpha-Fetoproteins ; pharmacology

AIMTo prepare fluorescein sodium (FS) cationic liposomes and investigate the influence of cationic lipid (DC-chol) and polyethylene glycol (PEG) with different molecule weight (MW) on cationic liposome incorporation efficiency, cellular delivery and fluidity of liposome membrane.

METHODSUsing FS as a model material for encapsulation, the liposomes were prepared and separated (by sephadex G-50 1 cm x 20 cm column), and the liposome incorporation efficiencies was measured. The interaction between the FS and cationic liposomes was investigated by measuring the change of fluorescent spectrum. The cellular uptake of different liposome forms by choosing HepG2 2.2.15 as an in vitro cell culture assay model, and the influence of PEG on the fluidity of liposome membrane with the technique of fluorescence polarization were investigated.

RESULTSCationic lipid and different PEGs showed great effects on increasing liposome incorporation efficiency (from 0.64% to 86.57%), cellular uptake (from 2.18% to 48.46%) and fluidity of liposome membrane. The effect of PEG was MW dependent, and with the increase of MW, the incorporation efficiency and transfection was improved, and the fluidity of liposome membrane increased.

CONCLUSIONAddition of cationic lipid and high MW PEG into cationic liposomes can enhance the cellular delivery and fluidity of cationic liposomes. Also, they can improve the incorporation efficiency of cationic liposomes.

Cholesterol ; analogs & derivatives ; pharmacology ; Drug Delivery Systems ; Fluorescein ; Hepatoblastoma ; pathology ; Humans ; Liposomes ; Liver Neoplasms ; pathology ; Membrane Fluidity ; drug effects ; Molecular Weight ; Polyethylene Glycols ; chemistry ; pharmacology ; Transfection ; methods ; Tumor Cells, Cultured

AIMTo construct a liposomal liver targeting delivery system by adding soybean-derived sterylglucoside (SG) to the cationic liposomes.

METHODSThe physico-chemical properties of SG modified cationic lipsomes were investigated using fluorescein sodium (FS) as a model drug, as well as the interaction of SG modified liposomes with HepG2 2. 2. 15 cells in the point of involvement of asialoglycoprotein receptor (ASGP-R) mediated transfection. Liver targeting of modified cationic liposomes were also investigated using liver perfusing technique, and hepatocytes and non-hepatocytes were separated and examined after perfusing.

RESULTSAll the formula yielded high incorporation efficiency (83.12% - 91.74%), small particle size (93.0 - 124.4 nm). The zeta potential of blank liposomes all showed positive values. The transfection efficiency of FS entrapped in SG-liposomes with HepG2 2.2. 15 was significantly higher than that of liposomes without modification. The transfection of SG-liposomes were reduced significantly by the 20/30 mmol galactose as a competitor of ASGP-R. All the cationic liposomes showed high level of liver uptake of FS. Compared with the uptake of non-hepatocytes of each respectively, only SG/Brij-35 liposomes showed difference in FS uptake by hepatocytes (P < 0.05).

CONCLUSIONIt showed that SG/Brij-35 modified cationic liposomes are potentially useful drug carrier to liver but may be affected by different modification.

Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cations ; pharmacokinetics ; Cell Line, Tumor ; Cholestenes ; administration & dosage ; pharmacokinetics ; Drug Delivery Systems ; Galactose ; pharmacology ; Humans ; Liposomes ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Male ; Particle Size ; Polyethylene Glycols ; administration & dosage ; pharmacokinetics ; Rats ; Transfection

Leaf senescence is often caused by water deficit and the chimeric gene P(SAG12)-IPT is an auto-regulated gene delaying leaf senescence. Using in vitro leaf discs culture system, the changes of contents of chlorophylls, carotenoids, soluble protein and thiobarbituric acid reactive substance (TBARS) and antioxidant enzymes activities were investigated during leaf senescence of P(SAGl2)-IPT modified gerbera induced by osmotic stress compared with the control plant (wild type). Leaf discs were incubated in 20%, 40% (w/v) polyethylene glycol (PEG) 6000 nutrient solution for 20 h under continuous light [130 micromol/(m(2) x s)]. The results showed that the contents of chlorophylls, carotenoids and soluble protein were decreased by osmotic stress with the decrease being more pronounced at 40% PEG, but that, at the same PEG concentration the decrease in the transgenic plants was significantly lower than that in the control plant. The activities of superoxide dismutase (SOD), catalases (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR) were stimulated by PEG treatment. However, the increases were higher in P(SAG12)-IPT transgenic plants than in the control plants, particularly at 40% PEG treatment. Lipid peroxidation (TBARS content) was increased by PEG treatment with the increase being much lower in transgenic plant than in the control plant. It could be concluded that the increases in the activities of antioxidant enzymes including SOD, CAT, APX, GPX and DHAR were responsible for the delay of leaf senescence induced by osmotic stress.
Alkyl and Aryl Transferases ; genetics ; Antioxidants ; metabolism ; Arabidopsis Proteins ; genetics ; Ascorbate Peroxidases ; Asteraceae ; genetics ; metabolism ; Carotenoids ; metabolism ; Catalase ; metabolism ; Chlorophyll ; metabolism ; Cysteine Endopeptidases ; genetics ; Genes, Bacterial ; Genes, Plant ; Lipid Peroxidation ; Osmotic Pressure ; Oxidoreductases ; metabolism ; Peroxidase ; metabolism ; Peroxidases ; metabolism ; Plant Leaves ; metabolism ; Plant Proteins ; metabolism ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Solubility ; Superoxide Dismutase ; metabolism

Soxhlet extraction is a common method of sample preparation. However, there has been no discussion about the efficiency of Soxhlet extraction from different batches and the factors that cause content fluctuation. In this study, Panax ginseng was selected as a model sample. Soxhlet extraction by means of a water bath, which has always been neglected, was identified as a novel key factor in the poor repeat-ability in different batches of Soxhlet extraction, as it can affect the siphon times and reflux time, which have been positively correlated with the ginsenoside contents. By substituting round bottom flasks in the same column, the relative standard deviation of the most fluctuated compound, ginsenoside Rb1, was decreased from 24.6% to 5.02%. Scanning electron microscopy analysis confirmed that the breakdown of the surface of the ginseng powder in the Soxhlet extraction led to a better dissolution of ginsenosides, indicating that chloroform may promote the extraction of ginsenosides by disrupting the cell structure. Moreover, 70% methanol was regarded as the better solvent for extracting the ginsenosides. Overall, this work offers a practical and effective protocol for improving the accuracy and repeatability of Soxhlet extraction methodology for ginsenosides and other analytes.

OBJECTIVE: To evaluate the long-term efficacy and safety of ultrasound-guided percutaneous nephrolithotomy (PCNL) in the treatment of patients with solitary kidney stones. METHODS: The clinical data of 22 patients with solitary kidney stones treated with PCNL in Peking University People's Hospital from September 2008 to June 2014, with the follow-up data of more than 5 years were analyzed retrospectively. Perioperative indicators, postoperative stone free rate (SFR) and incidence of complications were recorded. Ultrasonography was used to evaluate the long-term stones recurrence rate. Serum creatinine and estimated glomerular filtration rate (eGFR) were used to assess the long-term renal function. RESULTS: In this group of 22 patients, the average age was (50.3±11.8) years, with 10 cases of anatomic solitary kidneys, 12 functional solitary kidneys, and the median stone diameter was 1.65 (1.1-3.9) cm. All the patients had multiple stones, including 7 cases of staghorn stones. The median pre-operative serum creatinine was 104.5 (60.0-460.0) μmol/L, and the mean eGFR was (60.3±29.4) mL/min, showing no statistically significant difference compared with that before surgery. The mean operative time was (88.2±42.0) min, and there were 11 cases of single-channel and double-channel PCNL. The median serum creatinine on the first day after surgery was 102.0 (63.0-364.0) μmol/L, and the mean eGFR was (58.0±25.1) mL/min. The mean postoperative hospital stay was (8.7±5.2) days. In this group, 5 patients (22.7%) presented short-term complications, among which 4 patients presented postoperative infection and massive hemorrhage at the same time, which improved after conservative treatment, and 1 patient presented pleural injury and improved after closed thoracic drainage. Two patients (9.1%) developed long-term complications, and ureteral stricture occurred 3 months after operation, which improved after balloon dilatation. The median follow-up time was 6.2 (4.7-11.1) years. The median serum creatinine at the last follow-up was 104.0 (72.4-377.0) μmol/L, and the mean eGFR was (60.1±23.7) mL/min, showing no statistically significant difference compared with that before surgery. Renal function decreased in 6 patients (27.3%). Initial and final SFR were 72.7% and 100%, respectively. During the 6.2-year follow-up, 9 patients (40.9%) experienced recurrence of kidney stone. After stone recurrence, 13 lithotomy surgeries were performed, and the SFR by the latest follow-up was 63.6%. CONCLUSION This study had the longest follow-up time for patients with solitary kidney stones after PCNL reported at home and abroad. Ultrasound-guided standard PCNL was safe and effective in the treatment of solitary kidney stones. Long-term follow-up results showed that the recurrence rate of kidney stones was still high, but the long-term renal function was stable after operation, and some patients showed mild renal function decline.
Adult ; Humans ; Kidney Calculi ; Middle Aged ; Nephrolithotomy, Percutaneous ; Retrospective Studies ; Solitary Kidney/surgery* ; Treatment Outcome

The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7, Ad-K14 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5% Avertint and the vagina, mammary gland, ovaries and uterus were dissected and fixed in 3.75% paraformaldehyde overnight at 4 degrees C. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.
Adenoviridae ; genetics ; Animals ; Blotting, Western ; Cell Line ; Female ; Genital Diseases, Female ; pathology ; virology ; Genitalia, Female ; pathology ; virology ; Humans ; Immunohistochemistry ; Mammary Glands, Animal ; metabolism ; pathology ; Mice ; Mice, Nude ; Oncogene Proteins, Viral ; genetics ; metabolism ; Ovary ; metabolism ; pathology ; Papillomaviridae ; metabolism ; physiology ; Papillomavirus E7 Proteins ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; metabolism ; Uterus ; metabolism ; pathology ; Vagina ; metabolism ; pathology

Objective To determine the awareness, treatment and control of hypertension and its determinants in the elderly hypertensive patients of Dean nationality so as to explore the associated determinants. Methods This was a cross-sectional, population-based survey. A total of 939 persons aged 60 years and older sampled by stratified sampling method from the elderly population of Dean nationality were examined from July to September 2016. Meanwhile, the data of medical history and examination were collected. Results There were 353 elderly patients with hypertension in the 939 subjects. The awareness, treatment and control of hypertension in the elderly hypertensive patients (EHP), which was 47.3％, 36.5％and 22.1％respectively, were higher than the results of previous national study and had the upward trends with the increasing age (<0.05) . In comparison with the reference group, EHP living in middle economic level area had the higher awareness, treatment and control of hypertension (<0.05) . The results of covariate-adjusted multilevel logistic regression model indicated that there was a cross-level interaction effect between middle economic level area and dyslipidemia on the awareness and treatment of hypertension respectively, and a cross-level interaction effect between middle economic level area and low fat diet on the control of hypertension ( <0.05) . The individual level covariates, age (OR=1.3412, 1.3144, 95％ CI 1.0217-1.7609, 1.0030-1.7225) and diabetes ( OR=2.6735, 2.3078, 95％ CI 1.0721-6.6672, 1.0240-5.2007), had the positive association with the awareness and treatment of hypertension;age (OR=1.4740, 95％CI 1.0034-2.1654), non-drinking (OR=3.2056, 95％CI 1.5554-6.6066) and low salt diet (OR=3.6969, 95％CI 1.7996-7.5946) had the positive association with the control of hypertension. Conclusion The awareness, treatment and control of hypertension in the EHP of Dean nationality were higher than those of the national level, and the dominant factors were economic level, age, dyslipidemia, diabetes, non-drinking, low fat diet and low salt diet, etc.

Based on the thinking of integrative and holistic acupuncture and moxibustion, FU Wen -bin proposes to treat depression complicated with obsessive-compulsive disorder from perspective of heart and gallbladder. In clinical practice, the treatment pattern of "acupuncture at the top priority, followed by moxibustion, and consolidation at the end" is applied, acupuncture, refined moxibustion, intradermal needle and other therapies are comprehensively adopted, acupoints on the heart meridian, pericardium meridian and gallbladder meridian are selected or back-shu points and front-mu points of the heart, gallbladder and pericardium are matched, and the acupoints on the conception vessel and governor vessel and with the functions of tonifying kidney and regulating qi are added to calm the heart and tranquilize the mind, so as the symptoms of depression and obsessive-compulsive can be effectively relieved.
Depression/therapy* ; Gallbladder ; Humans ; Meridians ; Moxibustion ; Obsessive-Compulsive Disorder

Objective: To investigate the effect of berbamine hydrochloride on the absorption characteristics of berberine hydrochloride in different intestinal segments of rats in normal environment and high calcium environment. Method: Taking rat everted intestinal sac model,the content of berberine hydrochloride in absorbent solution of everted intestinal sac from different compatibility groups was determined by HPLC,and the uptake per unit area in different groups was analyzed by One-way ANOVA. Result: Compared with the normal J70 group(in normal environment,the concentration of berberine hydrochloride was 70 mg·L^-1) at the same time point,the uptake per unit area of the normal J70+Ver100 group(in normal environment,the concentration of berberine hydrochloride was 70 mg·L^-1,adding verapamil hydrochloride to a concentration of 100 mg·L^-1) was significantly increased in the ileum(P<0.05) at 120 min;the uptake per unit area values of the high calcium J70+A35 group(in high calcium environment,the concentration of berberine hydrochloride was 70 mg·L^-1,adding berbamine hydrochloride to a concentration of 35 mg·L^-1) were significantly increased in the duodenum(P<0.05) at 30,60,90 min;and the uptake per unit area values of the high calcium J70+A70 group(in high calcium environment,the concentration of berberine hydrochloride was 70 mg·L^-1,adding berbamine hydrochloride to a concentration of 70 mg·L^-1) were significantly increased in the ileum(P<0.05) at 30,90,120 min.By comparing with the normal group with the same mass concentration,the intestinal absorption of berberine hydrochloride was better in the high calcium J70+A35 group and the high calcium J70+A70 group. Conclusion: Berbamine hydrochloride can promote the absorption of berberine hydrochloride in intestine to a certain extent,especially in the high calcium environment.