载基因壳聚糖纳米粒的制作优化和表征

Objective To improve the preparation method of chitosan nanoparticle for gene probe.Methods The water soluble phosphonic chitosan (pCS) was synthesized, then mixed with gene probe of alpha fetoprotein with different molar concentrations to synthesize nanoparticles. The size and zeta potential of the nanoparticles were determined. The pH in gene solution was modulated and the rate of envelopment of pCS for the gene was examined. Fluorescence intensity of nanoparticles was analyzed by laser Raman spectroscopy. Results The modified method of nanoparticles was simpler, and the size of nanoparticle synthesized by modified method was (144.6±6.8) nm, comparable to the size as synthesized by conventional The size, zeta potential and rate of chitosan combined gene of nanoparticle produced by modified method were ( 102.6± 12.0) nm, ( 1.45 ± 1.75 ) mV, and (87.6 ± 3.5 )% respectively. The Raman spectra showed that the pCS could combine and envelop the gene probe. Conclusion The modified method for synthesizing chitosan nanoparticle is simple and feasible and pCS can envelop the gene probe.     

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.     

Influences of Organic Solvents on Particle Size and Drug-loading Efficiency for 5-Fluorouracil Poly（lactic acid） Nanoparticles

The objective of this study was to investigate the influences of organic solvents on particle size, drug content, loading efficiency and yield for 5-Fluorouracil Poly (lactic acid) nanoparticles . The 5-Fluorouracil was entrapped into poly(lactic acid)(PLA) nanoparticles using a water-in-oil-in-water solvent evaporation technique. During the preparation process, ethyl acetate and acetone were used as organic solvents since they are less toxic than the more commonly used dichloromethane. The effect of the three solvents on particle size, drug content, loading efficiency and yield of nanopartcles was compared. When the solvent of the oil phase was acetone, the highest drug content, smallest particle size and lowest yield were obtained for the PLA nanoparticles.     

Study on Tat Mediated Having Composite Magnetic Nanoparticles Targeting Function

This paper describes a new formulation of magnetic nanoparticles coated by a novel polymer matrix-O-Carboxylmethylated Chitosan （O-CMC） as a drug/gene carrier. The O-CMC magnetic nanoparticles were derivatized with a peptide sequence from the HIV-tat protein and transferrin to improve the translocational property and cellar uptake of the nanoparticles. To evaluate the O-MNPs-Tat-Tf as a drug carrier, Methotrexate （MTX） was incorporated as a model drug and MTX-loaded O-MNPs-Tat-Tf with an average diameter of 75 nm were prepared and characterized by TEM, AFM and VSM. The cytotoxicity of MTX-loaded O- MNPs-Tat-Tf was investigated with C6 cells. The results showed that the MTX-loaded O-MNPs-Tat-Tf retained significant antitumor toxicity.     

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.     

Entrapment and Sustained Release of Hydrophobic Drugs with Different Molecular Weights from PHBHHx-PEG Nanoparticles

Biodegradable polymeric nanoparticles are more and more frequently used in drug delivery systems, which represent one of the most rapidly developing areas. In our previous study, a novel natural hybrid polyester, polyethylene glycol 200 （PEG200） end-capped poly （3-hydroxybutyrate-co-3-hydroxyhcxanoate） （PHBHHx-PEG） was directly produced by Aeromonas hydrophila fermentation. In this study, the performance of the novel biodegradable PHBHHx-PEG copolyester as a sustained release carrier for hydrophobic drugs with different molecular weights and the in vitro sustained release profile were investigated. 5-Fluorouracil （5-Fu, Mw=130.1）, TGX221 （Mw=364.4）, and Rapamycin （RAP, Mw=914.2） were used as the model drugs. PHBHHx-PEG nanoparticles entrapped with 5-Fu, TGX221 and RAP were fabricated by a modified emulsification/solvent evaporation method, respectively. The average diameter of 5-Fu, TGX221, and RAP loaded PHBHHx-PEG nanoparticles was between 198.2-217.4 nm, and the entrapment efficiency of the three drugs was 62.5%, 93.4% and 91.9%, respectively. The in vitro release profiles of 5-Fu, TGX221 and RAP from PHBHHx-PEG nanoparticles were different. 5-Fu showed faster release rate and an obvious initial burst release phase. TGX221 and RAP were demonstrated to be released more slowly and steadily. The release percentages of 5-Fu, TGX221 and RAP were 97.7%, 85.1% and 74.7% after releasing for 72 h. PHBHHx-PEG is a kind of promising material as a carrier for the entrapment and delivery of hydrophobic drugs especially for those drugs with high molecular weight.     

MiRNA-429 suppresses the growth of gastric cancer cells in vitro

Micro-RNAs(miRNAs) have been found to be implicated in a very wide range of physiological processes.This study was aimed to investigate the regulation of miRNA-429(miR-429) in gastric cancer cells on cell proliferation and apoptosis.Quantitative PCR was employed to detect the expressions of miR-429 after eukaryotic expression plasmid of miR-429 and its inhibitor were transiently transfected into poorly differentiated human gastric can-cer cell line BGC823.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reduction as-says were used to examine proliferation ability.Apoptosis was analyzed by flow cytometry after transfection.The results showed that 48 h after transfection,overexpression of miR-429 reached maximum efficiency.Compared with mock transfection,miR-429 inhibited tumor cell proliferation significantly(P < 0.05) at 48 h and 72 h.of Overexpression of miR-429 promoted tumor cell apoptosis when compared with mock transfected cells(P < 0.05).On the contrary,miR-429 inhibitor promoted tumor cell proliferation and inhibited apoptosis when compared with controls(P < 0.05).Our results suggested that miRNA-429 may serve as a tumor suppressor during tumorigenesis of gastric cancer and may be a potential gastric cancer therapeutic target.     

构建HSA-PEI-pcDNA-Apoptin复合物及对乳腺癌细胞增殖的影响

BACKGROUND: Apoptin is known to induce apoptosis of more than 50 kinds of tumor cells. However, efficient systems are required to deliver apoptin to the cancer cells for clinical use. OBJECTIVE: To construct human serum albumin (HSA) and Apoptin complex and transfect it to cancer cells in vitro and in vivo so as to find an efficient approach for apoptin delivery. METHODS: Polyethylenimine was used to react with N-hydroxysuccinimide (NHS)-HSA solution to synthesized HSA-PEI, and then react with pcDNA-Apoptin to construct recombinant HSA-PEI-pcDNA-Apoptin. HSA-PEI-pcDNA-Apoptin complex was transformed into MCF-7 breast cancer cell lines. Then the expression of apoptin was detected by western blot assay and the cell proliferation was detected by MTT assay and flow cytometry. The MCF-7 breast cancer cell xenograft model was used to detect the in vivo performance of HSA-PEI-pcDNA-Apoptin by measuring the tumor volume at 4 weeks, with saline and HSA-PEI-pcDNA as controls. RESULTS AND CONCLUSION: (1) We successfully prepared and confirmed the construction of HSA-PEI-pcDNA-Apoptin complex, which was successfully transformed into MCF-7 cells. (2) MCF-7 cells could express apoptin in the HSA-PEI-pcDNA-Apoptin group at 24 hours, but neither HSA-PEI-pcDNA group nor blank control group could express apoptin. (3) MTT assay for cell viability showed that HSA-PEI-pcDNA-Apoptin group had significantly lower optical density value than that in the other two groups (P < 0.05). (4) The tumor volume in the HSA-PEI-pcDNA-Apoptin group was significantly less than that in the other two groups (P < 0.05). (5) These findings indicate that the HSA-PEI-pcDNA-Apoptin complex markedly inhibits tumor growth in vitro and in vivo. 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

Current perspectives of nanoparticles in medical and dental biomaterials

Nanotechnology is gaining tremendous impetus due to its capability of modulating metals into their nanosize, which drastically changes the chemical, physical and optical properties of metals. Nanoparticles have been introduced as materials with good potential to be extensively used in biological and medical applications. Nanoparticles are clusters of atoms in the size range of 1-100 nm. Inorganic nanoparticles and their nano-composites are applied as good antibacterial agents. Due to the outbreak of infectious diseases caused by different pathogenic bacteria and the development of antibiotic resistance, pharmaceutical companies and researchers are searching for new antibacterial agents. The metallic nanoparticles are the most promising as they show good antibacterial properties due to their large surface area to volume ratios, which draw growing interest from researchers due to increasing microbial resistance against metal ions, antibiotics and the development of resistant strains. Metallic nanoparticles can be used as effective growth inhibitors in various microorganisms and thereby are applicable to diverse medical devices. Nanotechnology discloses the use of elemental nanoparticles as active antibacterial ingredient for dental materials. In dentistry, both restorative materials and oral bacteria are believed to be responsible for restoration failure. Secondary caries is found to be the main reason to restoration failure. Secondary caries is primarily caused by invasion of plaque bacteria (acid-producing bacteria) such as Streptococcus mutans and lactobacilli in the presence of fermentable carbohydrates. To make long-lasting restorations, antibacterial materials should be made. The potential of nanoparticles to control the formation of biofilms within the oral cavity is also coming under in- creasing scrutiny. Possible uses of nanoparticles as topically applied agents within dental materials and the appli- cation of nanoparticles in the control of oral infections are also reviewed.     

Level of functioning of siblings and parents of probands of varying degrees of retardation

A further analysis of already published data supports the position that retardates of low ability level less frequently have retarded siblings, retarded parents, and parents low in occupational level than do retardates higher in ability level. The analysis supports the position that there are two types of retarded individuals, persons retarded as a result of gene or chromosomal anomalies, brain injury, etc., who more frequently occur in the lower-level retardate group, and persons whose retardation represents polygenic segregation, who more frequently occur in the higher-level group.     

Modes of Inheritance of Errors of Refraction

Eighteen families in which both parents had refractions within the range of +4·0 D to −4·0 D and axial lengths seen in emmetropia (22·3-26·0 mm) showed coefficients of correlation of the order 0·5 indicative of polygenic inheritance. Such coefficients were seen for axial length (0·407) and for the cornea (0·487), but not for the lens (which is known to be yoked to the axial length). No such coefficients were seen in 19 families in which one of the parents had axial length outside the emmetropic range (nine families with long axes and 10 with short axes).

The pattern of polygenic inheritance for emmetropia (completely correlated optical components) and errors of refraction up to 4·0 D (inadequately correlated components: correlation ametropia) follows that seen in stature and other measurable characters. In contrast the high refractive errors with their abnormal axial lengths (component ametropia) are—like the extremes in stature—pathological anomalies with monofactorial inheritance.