聚乙烯亚胺-聚乙二醇-siRNA纳米复合物的制备和理化性质的研究

Objective To investigate the preparation method and characteristics of polyethyleneimine- polyethylene glycal (PEI- PEG) siRNA nanocomplex to improve the cell transfection efficiency of siRNA. Methods PEI-PEG copolymer was synthesized as gene carrier and complexed with siRNA targeting to CD44v6 to form PEI-PEG-siRNA nanoparticle. The size and zeta potential was measured. The morphology of the complex was observed by scanning electron microscopy. The complex abilities of the nanocomplex were validated by gel retardation assay. And the transfection efficiency of nanocomplexes at different N/P ratios was measured by flow cytometry. Results The nanoparticles appeared spherical,uniform in size, and well-dispersed. When N/P ratio was over 10, the size of nanocomplex decreased as N/P value increased. Meanwhile, the zeta potential was positive and increased. At this time, siRNA was completely complexed with PEI-PEG, producing a fluorescence quenching phenomenon. The flow cytometry showed that the transfection efficiency of nanocomplexes was improved as N/P value increasied. When N/P was 30, the transfection efficiency was (75.6±9.2)%. Conclusion PEI-PEG may be a promised gene carrier and can provide evidences for related research of PEI-PEG-siRNA nanoparticles in animals and in vitro experiments.     

Evaluation of metal nanoparticles for drug delivery systems

Diminazene aceturate is a trypanocide with unwanted toxicity and limited efficacy.It was reasoned that conjugating diminazene aceturate to functionalized nanoparticle would lower untoward toxicity while improving selectivity and therapeutic efficacy.Silver and gold nanoparticles were evaluated for their capacities to serve as carriers for diminazene aceturate.The silver and gold nanoparticles were synthesized,functionalized and coupled to diminazene aceturate following established protocols.The nanoparticle conjugates were characterized.The free diminazene aceturate and drug conjugated nanoparticles were subsequently evaluated for cytotoxicity in vitro.The characterizations by transmission electron microscopy or UV/Vis spectroscopy revealed that conjugation of diminazene aceturate to silver or gold nanoparticles was successful.Evaluation for cytotoxic actions in vitro demonstrated no significance difference between free diminazene aceturate and the conjugates.Our data suggest that surface modified metal nanoparticles could be optimized for drug delivery systems.     

Induction of Thl-Type Immune Response by Chitosan Nanoparticles Containing Plasmid DNA Encoding House Dust Mite Allergen Der p 2 for Oral Vaccination in Mice

This study was to prepare the chitosan-pDer p 2 nanoparticles and to investigate the effect of chitosan-DNA nanoparticles on immune response in mice by oral delivery of chitosan-DNA nanoparticles. The nanoparticles were synthesized by complexing chitosan with plasmid DNA. The DNA was fully complexed into chitosan-DNA nanoparticles, suggesting a 100% encapsulation efficiency. Chitosan-DNA complex renders a significant protection of the plasmid. No effect on cell viability was observed in both cell types and average cell viability over 100% was obtained. Oral gene delivery with chitosan-DNA nanoparticles can generate a higher level expression of gene in vivo. Oral chitosan-pDer p 2 nanoparticles in BALB/c mice can induce IFN-γ in serum and prevent subsequent sensitization of Th2 cell-regulated specific IgE responses. The data indicate that the oral administration of chitosan-pDer p 2 nanoparticles results in the expression of Der p 2 in the epithelial cells of both stomach and small intestine and the induction of Th1-type immune response. Cellular ＆ Molecular Immunology.     

黏膜免疫壳聚糖-多胺转运蛋白D(PotD)DNA纳米微粒对小鼠鼻咽部肺炎链球菌定植的保护作用研究

Objective To prepare the chitosan-potD nanoparticles and to evaluate its protective efficacy against pneumococcal nasopharyngeal colonization. Methods potD gene was amplificated from pneumococcal genome and was inserted into pVAX1 expression vectors to construct pVAX1-potD recombinant plasmid which was then transfected into 293T cell using LipofectAMINE 2000 to analyze transient potD gene expression in vitro by RT-PCR and Western blot. Chitosan-potD nanoparticles were freshly prepared by coacervation methods at each time and the characterizations of the nanoparticles were then evaluated. BALB/c mice were immunized with chitosan-potD, naked potD DNA or pVAX1 for 4 times at two-week intervals. Anti-PotD IgG, IgG1 and IgG2a levels in serum and IgA levels in nasal washes, bronchoalveolar lavage fluids (BALF) and middle ear lavages(MEL) were detected by indirect enzyme-linked immunosorbent assay (ELISA). IL-17A, IL-4 and IFN-γ levels in splenocytes were determined by double sandwich ELISA. Mice were intrannsally challenged with Streptococcus pneumoniae ATCC6303, and Pneumococci were recovered from the nasopharyngeal niche at the fifth day after challenge. Results potD gene was successfully amplificated by PCR and the sequence was confimed to be consistent with that in the Genbank. The pVAX1-potD recombinant plasmid was successfully constructed and was expressed in eukaryocytes in vitro. The mean size and zeta potential of chitosan-potD nanoparticles was 430 nm and + 20.5 mv, respectively. Chitosan-potD nanoparticles were not digested by DNase Ⅰ , while naked potD DNA was completely digested. The levels of antibodies inculding IgG, IgG1, IgG2a, IgA and cytokines including IL-17A, IL-4 and IFN-γ were significantly higher in mice immunized with chitosan-potD nanoparticles than mice with naked potD or pVAX1 ( P <0.05) only. More importantly, much less Pneumococci were recovered from mice immunized with chitosan-potD nanoparticles than the other groups(P <0.05). Conclusion Chitosan-potD nanoparticles significantly enhanced the immunogenicity and protection efficacy of DNA vaccines by intranasal immunization and could be used as a potential mucosal vaccine to prevent pneumococcal infection.     

黏膜免疫壳聚糖-多胺转运蛋白D(PotD)DNA纳米微粒对小鼠鼻咽部肺炎链球菌定植的保护作用研究

Objective To prepare the chitosan-potD nanoparticles and to evaluate its protective efficacy against pneumococcal nasopharyngeal colonization. Methods potD gene was amplificated from pneumococcal genome and was inserted into pVAX1 expression vectors to construct pVAX1-potD recombinant plasmid which was then transfected into 293T cell using LipofectAMINE 2000 to analyze transient potD gene expression in vitro by RT-PCR and Western blot. Chitosan-potD nanoparticles were freshly prepared by coacervation methods at each time and the characterizations of the nanoparticles were then evaluated. BALB/c mice were immunized with chitosan-potD, naked potD DNA or pVAX1 for 4 times at two-week intervals. Anti-PotD IgG, IgG1 and IgG2a levels in serum and IgA levels in nasal washes, bronchoalveolar lavage fluids (BALF) and middle ear lavages(MEL) were detected by indirect enzyme-linked immunosorbent assay (ELISA). IL-17A, IL-4 and IFN-γ levels in splenocytes were determined by double sandwich ELISA. Mice were intrannsally challenged with Streptococcus pneumoniae ATCC6303, and Pneumococci were recovered from the nasopharyngeal niche at the fifth day after challenge. Results potD gene was successfully amplificated by PCR and the sequence was confimed to be consistent with that in the Genbank. The pVAX1-potD recombinant plasmid was successfully constructed and was expressed in eukaryocytes in vitro. The mean size and zeta potential of chitosan-potD nanoparticles was 430 nm and + 20.5 mv, respectively. Chitosan-potD nanoparticles were not digested by DNase Ⅰ , while naked potD DNA was completely digested. The levels of antibodies inculding IgG, IgG1, IgG2a, IgA and cytokines including IL-17A, IL-4 and IFN-γ were significantly higher in mice immunized with chitosan-potD nanoparticles than mice with naked potD or pVAX1 ( P <0.05) only. More importantly, much less Pneumococci were recovered from mice immunized with chitosan-potD nanoparticles than the other groups(P <0.05). Conclusion Chitosan-potD nanoparticles significantly enhanced the immunogenicity and protection efficacy of DNA vaccines by intranasal immunization and could be used as a potential mucosal vaccine to prevent pneumococcal infection.     

Preparation of Curcumin Chitosan Nanoparticles and Its Effect on Free Radical Injury in Mice with Mycoplasma Pneumoniae Pneumonia

Objective: Curcumin(Cur) and Chitosan(CS) were utilized as primary components for the production of curcumin chitosan nanoparticles. The impact of these nanoparticles on oxidative stress in mycoplasma pneumoniae-infected mice was assessed.Methods: The drug loading and entrapment efficiency of Cur-CS nanoparticles were determined for various feeding ratios, and the release profiles of Cur-CS nanoparticles in different release media were investigated using the dynamic membrane dialysis method.The ...     

大肠的应用解剖学

ObjectiveTo provide morphological data for the enema,pneumo-barium double contrast examination and endoscopy.MethodsThe length and perimeter of large intestine were measured on 30 adult cadaver specimens.The volumes of each sectional colon were calculated with the formula of cylinder volume.That of caecum was calculated with the formula of cone volme.The volume of free large intestine was also measured with immersion method.ResultsThe lengths of large intestine,caecum,colon and rectum in pelvic cavity were 126.9±27.4 cm,43.6±1.5 cm,113.3±25.0 cm and 9.2±3.3 cm respectively.The lengths of ascending,transverse,descending and sigmoid colons were 15.6±5.8 cm,40.3±8.5 cm,23.2±7.2 cm and 34.3±10.0 cm separately.The volume of large intestine was 850.1±411.2mL by calculation and 756.6±91.9mL by immersion method.ConclusionThese morphological data will benefit the enema,pneumo-barium double contrast examination and endoscopy in clinic.     

小粒径磁共振成像超敏感纳米对比剂的研制及初步动物实验

Objective To synthesize a small size and super-sensitive magnetic resonance imaging (MRI) nano-contrast agent for preliminary animal experiment and to explore the feasibility for its use in molecular imaging studies.Methods Polyethylene glycol (PEC) was used for ring-opening polymerization of ε-caprolactone(CL), and amphiphilic PEG-b-PCL block polymer was prepared by self-assembly technique.Single particle superparamagnetic iron oxide (SPIO) was loaded at the hydrophobic core of PEG-b-PCL to develop a nano-contrast agent.The particle diameter and SPIO-loading density were respectively determined by transmission electron microscope and atomic absorption spectroscopy.Echo multiplanar sequence was used for T2 value test, with transverse relaxation rate R2 computed.Meanwhile, magnetic properties were tested.After intravenous injection via caudal vein of Balb/c nude mice, the distributions and circulation time of this nano- particle were analyzed using MRI scanner at before injection and lh, 3h, 6 h, 12 h and 24 h after injection.Results The micelle diameter was around (38+3) nm and the content of Fe3O4 (wt%) was 37.0%.The transverse relaxation rate R2 was 110 Fe(mmol/L)-1 ·s-1.Superparamagnetism was observed in magnetic property test.After intravenous injection, the nano-particles were mainly distributed in the liver with a long circulation time.Signal intensity at 1 h decreased significantly when compared with that of before injection (120±32 vs 4986±0, P＜0.05) , and then decreased to (88±28) at 24 h (P＜0.05).Conclusion The nanometer micelle loaded with single SPIO particle demonstrates its potential as a powerful MRI contrast agent for molecular imaging studies because of its small size and high sensitivity.     

不明原因肺炎监测中SARS-CoV实时荧光RT-PCR检测方法的建立

Objective To develop and evaluate a real-time RT-PCR method for detecting SARS-CoV, based on TaqMan hybridization probe technology in order to provide a laboratory diagnosis for the examination of SARS-CoV infection during the surveillance of unexplained pneumonia. Methods Two pairs of primers and probes were designed to identify 1b and NP gene of SARS-CoV respectively. The two probes were 5'end labeled with FAM and 3'end labeled with TAMRA. The PCR reaction conditions were optimized according to the reaction conditions for detecting H5N1 which was set up by the National Influenza Center. Different concentration of plasmid DNA containing the target gene, 12 kinds of other respiratory viruses, mycoplasma pneumoniae and legionella pneumophilia were tested using this method to evaluate the specificity, sensitivity and reproducibility of the assay. Results All kinds of the viruses, mycoplasma pneumoniae and legionella pneumophilia tested were negative. The sensitivity of this assay for 1b gene was 10-9μg/ml DNA/reaction and for NP gene was 10 -7μg/ml DNA/reaction. The coefficients of variation (CV) value were 0.2% -0.9% during the reproducibility test. The whole process takes 2.5h including the extraction of RNA from the sample, and could be completed in the same machine, under the same condition with the detection of H5N1.Conclusion This real-time RT-PCR setting up based on TaqMan probe is a specific, rapid and sensitive method for detecting SARS-CoV. The establishment of this method will provide a strong support for quick examination of SARS-CoV infection during the unexplained pneumonia surveillance.     

应用多重连接依赖式探针扩增技术快速高通量诊断胎儿染色体非整倍体异常

Objective To assess the diagnostic value of multiplex ligation-dependent probe amplification (MLPA) for detection of common chromosome aneuploidy in amniotic fluid (AF) cells in order to obtain an accurate, rapid, cost-effective and high-throughput method in routine prenatal clinical practice.Methods The MLPA test was performed on 500 AF samples by using kit P095 and the results were obtained by using analysis software RH-MLPA-v511. The results were compared with that from fluorescence in situ hybridization (FISH) and traditional karyotyping (TK). The technical critical issues were analyzed in routine diagnostic application. Results The absolute specificity and sensitivity of the MLPA test to detect the aneuploidy were 100%. For the 500 AF samples, the success rate of the MLPA tests was 97%. Among them 92% were finished within three working days and 5% required more days for repeating. The test failure rate was 3%. The results confirmed that for the 38 detectable aneuploid samples,the probe reliability weighted mean ratio values were more than 4SD compared to normal diploids and the 2 suspected trisomy samples were more than 2SD. In this study, authors analyzed hybridization efficiencies of 8 probes for chromosome 21, and the presence of a trisomy was considered if at least 4 of the 8 probes gave probe ratio of ＞1.3. Conclusion The data suggested that MLPA is a rapid, simple and reliable method for large scale testing for aneuploidy of chromosomes 13, 18, 21, X, or Y in AF. The MLPA technology is complementary to AF culture and valuable for prenatal diagnosis.