OBJECTIVE: To establish effective and reliable methods for the determination of adenovirus and carmustine in anionic liposomes-mediated co-delivery system. METHODS: The complexes of co-delivery system were prepared by calcium-induced phase changing method. For carrying out the quantitative-PCR (Q-PCR) detection, the sequences of primer and probe were designed accoding to the hexon gene sequences of Ad5, and then the reaction system of Q-PCR was optimized to detect the loading rate of Ad5. The HPLC condition was also optimized to determine the drug loading of carmustine in the co-delivery system. RESULTS: In the co-delivery system, the loading rate of adenovirus detected by Q-PCR method was (23.2±1.8)% and that of carmustine was (55±2.8)%. CONCLUSION Both of Q-PCR and HPLC methods have been successfully used for the quantification of adenovirus and carmustine in anionic liposomes-mediated co-delivery system. These methods are simple, reliable and accurate, which can be used in other similar experiments.

Adult ; Herpes Zoster ; etiology ; Herpesvirus 3, Human ; genetics ; Humans ; Male ; Middle Aged ; Mutation

Objective To explore the value of SOD activity in diagnosis of central nervous system leukemia (CNSL )by detec-ting SOD activity of cerebrospinal fluid of patients with CNSL .Methods The cerebrospinal fluid of 55 patients from department of hematology of Nanfang hospital of southern medical university were collected from January 2008 to January 2009 ,in which 30 pa-tients suffered with central nervous system leukemia (CNSL group) ,the other 25 patients suffered with acute leucemia without im-paired central nervous system(control group) .The SOD activity of cerebrospinal fluid was detected by the xanthine oxidase meth-od ,while the routine test ,biochemistry test and cell smear of cerebrospinal fluid was detected .Results There were statistics differ-ence in the level of white cell and protein in cerebrospinal fluid between CNSL and control group (P<0 .05) ,but with no difference in the level of cerebrospinal fluid pressure ,glucose ,chlorine(P>0 .05) .There was statistics difference in the level of SOD activity between CNSL and control group(P<0 .05) .The white cell quantity and the protein level in cerebrospinal fluid had negative corre-lation with the activity of SOD ,(r=0 .871 ,P=0 .000 ;r=0 .518 ,P=0 .003) .The activity of SOD in the cerebrospinal fluid had sta-tistics difference before and after intrathecal chemotherapy (P<0 .05) .The activity of SOD in the cerebrospinal fluid whose under 45 year-old (755 .64 ± 345 .77) ,which was significant lower than that of the paitents whose equal with or above 45 year-old (1 420 .49 ± 307 .69)(P<0 .05) .Conclusion The changes of the SOD activity in the cerebrospinal fluid had relation with central nervous system leukemia ,and the SOD activity might be a auxiliary diagnosis index used in central nervous system leukemia by revi-sing age factor .

A novel transferrin modified non-viral gene delivery system Tf-PLPD was developed and the related characteristics was investigated. Blank procationic liposomes were prepared by film dispersion-filteration method. PLPD was prepared as follows by first mixing the plasmid DNA and protamine together, then the resulted polyplexes were incubated for 10 min at room temperature, followed by addition of preformed blank procationic liposomes. Transferrin was adsorbed at the surface of PLPD via electrostatic interactions to form Tf-PLPD. Central composite design (CCD) was employed to optimize the formulation. The HepG2 cells were transfected using lacZ as reporter gene and characteristics such as the morphology, the mean particle size, the zeta potential and the transfection efficiency in HepG2 cells were further investigated by different methods. The resulting PLPD had a regular spherical surface with an average size of (228. 9 +/- 8. 0) nm (polydispersity index, PDI = 0. 122 +/- 0. 02, n = 3) , a zeta potential of ( - 25. 08 +/-2. 50) mV (n = 3) and a transfection efficiency of (12. 18 +/- 3. 80) mU x mg(-1) (protein). The Tf-PLPD had an average size of (240 +/- 12) nm (polydispersity index, PDI = 0. 150 +/- 0. 03, n = 3), a zeta potential of ( - 24. 10 +/- 2. 50) mV ( n = 3) and a transfection efficiency of (24. 26 +/- 2. 60) mU x mg(-1) (protein) , 20 times greater than that of the naked plasmid DNA. The presence of serum didn' t affect the tansfection activity of PLPD or Tf-PLPD. Compared to one kind of cationic liposomes (liposome-protamine-DNA, LPD), the PLPD and Tf-PLPD had much less cytotoxicity to three hepatic cell lines (including HepG2, SMMC7721 and Chang' s normal hepatocyte). The results indicated that the Tf-PLPD is a perspective non-viral vector for gene delivery systems.
Cations ; chemistry ; Cell Line ; Cell Line, Tumor ; Cell Survival ; DNA ; chemistry ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Liposomes ; chemistry ; Liver Neoplasms ; genetics ; pathology ; Particle Size ; Plasmids ; chemistry ; genetics ; Protamines ; chemistry ; Transfection ; methods ; Transferrin ; chemistry ; genetics

This study is to report the preparation of complexes of Ad5 and anionic liposomes (AL-Ad5), the amplification of adenoviruses with enhanced green fluorescent protein (eGFP) reporter gene performed by HEK 293 cells, the adenoviral vectors purified by cesium chloride gradient centrifugation, and the titer of adenovirus determined by cytopathic effect (CPE) method, hexon capsid immunoassay and quantitative-PCR (Q-PCR), separately. The prescription and experiment conditions were optimized by central composite design (CCD). The complexes of Ad5 and AL-Ad5 were formulated by the calcium-induced phase change method. The morpholopy, particle size and zeta potential were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Additionally, the bicolourable fluoresce-labeled complexes (F(labeled)-AL-Ad5) were prepared and their intracellular location in MDCK cells was detected by confocal laser scanning microscopy (CLSM). The results indicate that the complexes of AL-Ad5 exhibited a uniform distribution with a particle size of 211 +/- 10 nm and a zeta potential of -41.2 +/- 2.2 mV. The result of CLSM demonstrates that the intracellular location of red fluoresce-labeled adenovirus was consistent with that of green fluoresce-labeled liposomes suggesting that the naked adenovirus was well encapsulated by the anionic liposomes in complexes of AL-Ad5.
Adenoviridae ; genetics ; ultrastructure ; Animals ; Anions ; Cytopathogenic Effect, Viral ; Dogs ; Drug Compounding ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; chemistry ; HEK293 Cells ; Humans ; Liposomes ; chemistry ; pharmacokinetics ; ultrastructure ; Madin Darby Canine Kidney Cells ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Particle Size ; Polymerase Chain Reaction ; methods ; Recombinant Fusion Proteins ; ultrastructure

WRKY transcription factor is one of the Zinc finger proteins which contains a highly conserved WRKY domain and is a family of the plant-specific transcription factor. The plasmid pET28a-SmWRKY1 harboring Salvia miltiorrhiza WRKY1 (SmWRKY1) gene was successfully transformed and expressed in Escherichia coli BL21 (DE3). The conditions on protein expression of SmWRKY1 in E. coli, including induction duration, temperature, IPTG concentration and the E. coli concentration were optimized. The results showed that the highest protein expression of SmWRKY1 was obtained at 24 hours after the E. coli was cultured in the presence of 0.2 mol x L(-1) IPTG at 20 degrees C with A600 values of 1.0-1.5. This recombinant histidine-tagged protein was expressed at 2.454 g x L(-1) as inclusion body, which was first extracted using urea, and then purified by Ni2+ affinity chromatography and identified by SDS-PAGE. The expression of SmWRKY1 in E. coli was further confirmed by western blotting analysis.
Blotting, Western ; Cloning, Molecular ; DNA-Binding Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; chemistry ; genetics ; metabolism ; Molecular Weight ; Plant Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Salvia miltiorrhiza ; genetics

OBJECTIVETo evaluate the effect of autologous bone marrow stem cell transplantation in the treatment of severe liver damage.

METHODSAutologous bone marrow (50 ml) was harvested from 6 patients aged 44 to 69 years admitted for severe liver damage. Human bone marrow stem cells (HMSCs) were isolated and transplanted in to the patients' liver. At l, 4, 8, and 12 weeks after the transplantation, the changes in ALT, ALB, Cr, TB, PT and the clinical symptoms of the patients were observed.

RESULTSThe transplantation of autologous bone marrow stem cells resulted in obvious improvement of the liver function. At 12 weeks after the transplantation, ALT was reduced from 98.4 IU/L to 41.5 IU/L, TB from 136.5 µmol/L to 78.4 µmol/L, Cr from 112.3 µmol/L to 72.1 µmol/L, and ALB rose from 23.3 g/L to 32.6 g/L. The survival of the patients was 100% at 12 weeks, but one patient died at 7 months after the transplantation. The symptoms of the patients were also alleviated after the transplantation. At 12 weeks after transplantation, 3 patients reported improved appetite, 3 showed recovery of physical strength, and 2 showed lessened abdominal swelling. No serious adverse complications in association with the transplantation were found in the in 4 patients available to the follow-up.

CONCLUSIONAutologous bone marrow stem cell transplantation can improve the liver function of patients with severe liver damage without causing serious complications.

Cells, Cultured ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Homocysteine ; pharmacology ; Humans ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Monocytes ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology

Nanoparticles have better applicability in the detection, treatment of cancer and various difficult diseases, but mononuclear phagocytosis system can seriously shorten the time of nanoparticles in vivo circulation, reduce the drug efficacy. The protein crown formed on the surface of the nanoparticle after entering the body can change its surface properties, interfere with the recognition of phagocytes, and thus affect its circulation time in vivo. This article outlines the general composition and formation process of protein crowns. It also summarizes the influence of the physical and chemical properties of nanoparticles, such as particle size, surface charge, hydrophilicity and surface materials on the formation of protein crowns. The protein crown affects the circulation of nanoparticles in vivo, mainly because the adsorbed opsonic protein promotes cell phagocytosis. Therefore, we also introduce the method of using protein crowns to promote the long circulation of nanoparticles in vivo. By designing appropriate physical and chemical properties, surface modification, and directed design of protein crowns, the adsorption of proteins on the surface of nanoparticles can be reduced. Therefore, it can reduce the clearance of nanoparticles in the mononuclear phagocytic system (mainly the phagocytes of the liver and spleen), and achieve the goal of long circulation of nanoparticles in the body.

Objective To evaluate the effect of the intermittent deferomamine(DF) therapy on relieving iron overload caused by transfusion in children with ? thalassemia.Methods Sixteen children who were finally diagnosed as ? thalassemia major were treated with deferomamine for 124 times totally to low the iron overload. The serum iron(SI), serum ferritin(SF) and urine ferritin were detected each time with radio-immunity technique and difference was compared before and after treatment. Meanwhile, weather DF involved children′s liver and renal function was observed in whole procedure.Results Iron overload exists in 16 cases of ? thalassemia major children by a long- term hypertransfusion therapy, with average level SI 33.69?6.72 mmol/L,SF 441.19? 54.70 ?g/L,urine ferritin 8.64?6.79 ?g/L. The difference was significant (paired-samples t test,t =6.173 P 0.05).Conclusion The study suggest that intermittent low-dose DF therapy is effective for iron overload caused by transfusion in ? thalassemia children, without apparent side effects.