Preparation of Superparamagnetic Dextran-coated Iron Oxide Nanoparticles used as a Novel Gene Carrier into Human Bladder Cancer Cells'

Objective: Application of magnetic nanoparticles as gene carrier in gene therapy has developed quickly. This study was designed to investigate the preparation of superparamagnetic dextran-coated iron oxide nanoparticles (SDION) and the feasibility of SDION used as a novel gene carrier for plasmid DNA in vitro. Methods: SDION were prepared by chemical coprecipitation and separated by gel filtration on Sephacryl S-300HR, characterized by TEM, laser scattering system and Vibrating Sample Magnetometer Signal Processor. The green fluorescent protein (pGFP-C2) plasmid DNA was used as target gene. SDION-pGFP-C2 conjugate compounds were produced by means of oxidoreduction reaction. The connection ratio of SDION and pGFP-C2 DNA was analyzed and evaluated by agarose electrophoresis and the concentration of pGFP-C2 in supernatant was measured. Using liposome as control, the transfection efficiency of SDION and liposome was respectively evaluated under fluorescence microscope in vitro. Results: The diameter of SDION ranges from 3 nm to 8 nm, the effective diameter was 59.2 nm and the saturation magnetization was 0.23 emu/g. After SDION were reasonably oxidized, SDION could connect with pGFP-C2 to a high degree. The transfection efficiency of SDION as gene carrier was higher than that of liposome. Conclusion: The successes in connecting SDION with pGFP-C2 plasmid by means of oxidoreduction reaction and in transferring pGFP-C2 gene into human bladder cancer BIU-87 cells in vitro provided the experimental evidence for the feasibility of SDION used as a novel gene carrier.

Delivering DNA into Plant Cell by Gene Carriers of ZnS Nanoparticles

The development of nanotechnology provides a new method for genetic engineering.However,the nanoparticles as gene carriers have been mainly used in the mammalian cells so far.We observed that ZnS nanoparticles modified with positively charged poly-L-lysine（PLL） successfully delivered GUS-encoding plasmid DNA into tobacco cells by means of ultrasound-assisted method.Polymerase chain reaction（PCR） detection,Southern blot analysis and GUS histochemical staining were carried out for the regenerated plants.The stable genetic modified plants mediated by ZnS nanoparticles can be obtained.This article demonstrates the great potential of nanoparticles as gene carrier in plant transformation and proves a novel approach for plant genetic decoration.

Enhancement of Immunological Activity of CpG ODN by Chitosan Gene Carrier

To investigate the enhancement of immunological activity of CpG ODN by chitosan gene carrier in mice, the effect of lymphocyte proliferation was detected in mice by using MTT, the levels of IgG and cytokines （IL-2 and IL-12） in serum were measured by ELISA and peripheral blood T lymphocyte subsets CD4^＋, CD8^＋ were analyzed by flow cytometry. Our results showed that spleen lymphocytes isolated from the CS-CpG ODN group of mice showed the strongest proliferation （SI =1.551）, and the levels of IgG, IL-2 and IL-12 in serum were higher than those of other groups. Compared with the immunization with CpG ODN, the immunization with CS-CpG ODN gene carrier was more efficient in up-regulating the percentage of CD4^＋T cells and the ratio of CD4^＋/CD8^＋ of mice, It was concluded that CS gene carrier of CpG ODN was much more effective in improving immunity of CpG ODN in mice.

Recent progress in cationic polymeric gene carriers for cancer therapy

In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety.In particular,the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy.Herein,this review focused on the development of cationic polymeric carriers for cancer therapy,including polyethylenimine(PEI),polyamidoamine(PAMAM) dendrimers,polylysine(PLL),chitosan and modified cationic polymers.And recent progresses in the development of novel polymeric carriers for gene delivery,such as targeted gene carriers,responsive gene carriers and multifunctional gene carriers,were summarized.Finally,the future perspectives in the development of novel polymeric carriers for delivery gene were presented.

Development of novel gene carrier using modified nano hydroxyapatite derived from equine bone for osteogenic differentiation of dental pulp stem cells

Hydroxyapatite(HA)is a representative substance that induces bone regeneration.Our research team extracted nanohydroxyapatite(EH)from natural resources,especially equine bones,and developed it as a molecular biological tool.Polyethylenimine(PEI)was used to coat the EH to develop a gene carrier.To verify that PEI is well coated in the EH,we first observed the morphology and dispersity of PEI-coated EH(pEH)by electron microscopy.The pEH particles were well distributed,while only the EH particles were not distributed and aggregated.Then,the existence of nitrogen elements of PEI on the surface of the pEH was confirmed by EDS,calcium concentration measurement and fourier transform infrared spectroscopy(FT-IR).Additionally,the pEH was confirmed to have a more positive charge than the 25 kD PEI by comparing the zeta potentials.As a result of pGL3 transfection,pEH was better able to transport genes to cells than 25 kD PEI.After verification as a gene carrier for pEH,we induced osteogenic differentiation of DPSCs by loading the BMP-2 gene in pEH(BMP-2/pEH)and delivering it to the cells.As a result,it was confirmed that osteogenic differentiation was promoted by showing that the expression of osteopontin(OPN),osteocalcin(OCN),and runt-related transcription factor 2(RUNX2)was significantly increased in the group treated with BMP-2/pEH.In conclusion,we have not only developed a novel nonviral gene carrier that is better performing and less toxic than 25 kD PEI by modifying natural HA(the agricultural byproduct)but also proved that bone differentiation can be effectively promoted by delivering BMP-2 with pEH to stem cells.

Exploring Quaternized Hydroxyethylcellulose as Potential Gene Carriers

Cationic polysaccharides have been receiving more attentions and used as nonviral gene delivery vectors. In this paper, quaternized hydroxyethylcellulose （QHEC） derivatives were studied as gene carriers for their efficient DNA binding abilities. All QHECs could form stable QHEC/DNA complexes and resist the degradation of DNase I. And the dynamic light scatter （DLS） results showed that all QHEC/DNA complexes could form compact particles. These QHEC/DNA complexes exhibited effective transfeetion abilities in comparison to the naked DNA. The cytotox- icities of QHEC and QHEC/DNA complexes were also evaluated in four cell lines which were relatively low com- pared with 25 kDa bPEI. All results indicated that these quaternized hydroxyethylcelluloses could be used as poten- tial gene delivery vectors.

Studies of poly-L-lysine-starch nanoparticle preparation and its application as gene carrier

Anion starch nanoparticle (StNP) with a diameter of 50 nm was prepared in wa- ter-in-oil microemulsion, with soluble starch as raw materials and POCl3 as crosslinking agent. PLL-StNP was prepared by linking poly-L-lysine (PLL) on the surface of StNP. At the same time, the size of PLL-StNP and its stability in aqueous solution were checked by AFM. The analysis of plasmid DNA binding, DNase I enzymatic degradation, toxicity and transfection were done. We discovered that PLL-StNP may be used as non-virus nanoparticle gene carrier. And we devel- oped the method of preparing PLL-StNP gene carrier and used it in cell transfection. As non-virus gene carrier, PLL-StNP has some advantages, such as large load of DNA, high transfection effi- ciency, low cell toxicity and biodegradability.

A novel gene carrier based on amino-modified silica nanoparticles

Uniform-sized amino-modified silica nanopar-ticles have been prepared by the controlled synchronous hydrolysis of tetraethoxysilane and N-(?amimoethyl)-- aminopropyltriethoxysilane in water nanodroplet of the wa-ter-in-oil microemulsion. These nanoparticles display posi-tive charge potential at definited pH. This is due to the pres-ence of amino groups on the surface of the nanoparticles. Nanoparticles-plasmid DNA complexes can easily form through electrostatical binding between the positive charges of the amino-modified silica nanoparticles and the negative charges of the plasmid DNA. The complexes can be also dis-sociated under alkaline pH or high ionic strength conditions. And enzymatic digestion of the plasmid DNA is almost in-hibited by these nanoparticles complexes. A novel non-viral gene carrier based on the amino-modified silica nanoparti-cles is proposed under the combination of nanotechnology, biotechnology and gene engineering technology. The plasmid DNA can successfully cross various systemic barriers to COS-7 cells as well as mediate high expression of Green Fluorescence Protein (GFP) gene in cells by use of this novel gene carrier.

Bioreducible crosslinked low molecular weight branched PEI-PBLG as an efficient gene carrier

Low molecular weight polyethylenimine-poly(gamma-benzyl L-glutamate) (PEI-PBLG) was crosslinked by N,N'-cystaminebisacrylamide (CBA) to get the polymer named as CBA-PEI-PBLG (CPP).CPP not only inherits PEI-PBLG's amphiphilic advantages,but also possesses reducible properties.CPP can complex with DNA to form nanoparticles.CPP/DNA complex particles were characterized by particle size and zeta potential analysis.The result showed that the complex particles have suitable size and surface charges for gene delivery.And gel retardation assay also prove CBA-PEI-PBLG has proper condensing ability for DNA.CPP has good reducible property,and also has good biocompatibility because of introducing PBLG segment.The cytotoxicity of CPP was evaluated using MTT assay,and the results showed CPP has lower cytotoxicity compared with PEI 25 K.The transfection properties were characterized in different cells by using plasmid DNA as a reporter.CPP showed higher transfection efficiencies and lower cytotoxicity in HeLa cells.This was attributed to bioreducible and biocompatibility properties of the CPP.These results suggested that CPP is a promising low-toxic,highly effective non-viral gene carrier.

Genomic Analysis of Mitochondrial Carrier Genes in the Bombyx mori

This study is performed to investigate the mitochondrial carrier gene family in silkworm genome. In total, 30 genes are identified and claded into eight well-conserved groups. Gene duplication contributes to the expansion and complexity of this family. Diverse expression patterns suggest their functional differentiation. Analyses of the sitespecific profiles reveal critical amino acid residues for functional divergence. This study highlights the molecular evolution of the mitochondrial carrier gene family in silkworm and may provide a starting point for further experimental verification.

Modulating the Oxidation Degree of Linear Polyethyleneimine for Preparation of Highly Efficient and Low-cytotoxicity Degradable Gene Delivery Carriers

Polyethyleneimine(PEI),as a widely used polymer material in the field of gene delivery,has been extensively studied for modification and shielding to reduce its cytotoxicity.However,research aimed at preparing degradable PEI is scarce.In this work,the hydrogen peroxide(H_(2)O_(2))oxidation method was used to introduce degradable amide groups in the PEI and a series of oxidized PEI22k(oxPEI22k)with different degrees of oxidation were synthesized by regulating the dosage of H_(2)O_(2).The relationship between the oxidation degree of oxPEI22k and the gene transfection efficiency of oxPEI22k was studied in detail,confirming that the oxPEI22k with oxidation degrees of 16.7%and 28.6%achieved improved transfection efficiency compared to unmodified PEI.These oxPEI22k also proved reduced cytotoxicity and improved degradability.Further,this strategy was extended to the synthesis of low-molecular-weight oxPEI1.8k.The oxPEI1.8k with suitable oxidation degree also achieved improved transfection efficiency and reduced cytotoxicity.In brief,this work provided high-efficiency and low-cytotoxicity degradable gene delivery carriers by regulating the oxidation degree of PEI,which was of great significance for promoting clinical applications of PEI.

小粒径Fe_(3)O_(4)-DMSA-PEI磁性纳米颗粒的制备及其基因负载能力研究

四氧化三铁(Fe_(3)O_(4))磁性纳米颗粒因其制备简单,在外加磁场作用下具有靶向性,并且表面易接枝等特性,可作为被动靶向载体应用于基因治疗领域。本研究采用溶剂热法制备纳米颗粒,并调控堆积生长时间,制得粒径在4~9 nm范围内可控的油相Fe_(3)O_(4)纳米颗粒;使用内消旋-2,3-二巯基丁二酸(DMSA)二次取代其表面的油酸分子,使其具备良好的水相分散性;通过酰胺化反应在其表面接枝支链型聚乙烯亚胺(PEI),最终得到Fe_(3)O_(4)-DMSA-PEI磁性纳米颗粒。研究发现,Fe_(3)O_(4)-DMSA-PEI磁性纳米颗粒的表面Zeta电位高达(52.50±1.94)mV,具有一定的超顺磁性(14.48 emu/g,1 emu/g=1 A·m^(2)/kg)。磁性纳米颗粒与质粒DNA的质量比为15:1时可完全阻滞DNA在凝胶上的电泳,装载量高达6.67%。本研究制备的Fe_(3)O_(4)-DMSA-PEI磁性纳米颗粒具有一定的基因负载能力,有望作为基因载体应用于基因转染领域。

BVDV非结构蛋白NS4B的结构分析及其蛋白表达与鉴定

NS4B是牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)的非结构蛋白,为研究BVDV在病毒侵染宿主细胞过程中的作用,对ns4b进行了结构分析、表达载体构建及蛋白表达。采用Prot-Param、TExpasy和SignalP-4.1等软件分析NS4B蛋白的氨基酸组成、疏水性和空间结构。根据Genebank上公布的BVDVns4b基因序列设计引物,利用PCR方法获得ns4b基因片段,连接到p3×Flag-CMV10表达载体上,构建重组质粒p3×Flag-CMV10-ns4b,经双酶切与测序鉴定正确后,将p3×FlagCMV10-ns4b转染到HEK-293T细胞中,利用Western blot检测NS4B蛋白的表达。结果表明BVDV NS4B蛋白分子量为38.4 kDa,不存在信号肽,有49个磷酸化位点和6个糖基化位点,是一种主要由α-螺旋和无规则卷曲结构组成的疏水性稳定蛋白。成功构建了重组质粒p3×Flag-CMV10-ns4b,并且p3×Flag-CMV10-ns4b在HEK-293T细胞中成功表达,为今后研究NS4B蛋白在BVDV感染宿主细胞及其免疫逃逸机制奠定了基础。

广西来宾市育龄人群珠蛋白生成障碍性贫血基因检测结果分析

目的探讨珠蛋白生成障碍性贫血基因在广西来宾市本地人群的携带率,为来宾市珠蛋白生成障碍性贫血防控工作提供理论依据。方法采用血细胞检测和血红蛋白电泳联合测定对2020年1月~2021年12月在广西来宾市四县一市一区妇幼医院门诊就诊的88152例样本进行珠蛋白生成障碍性贫血筛查,采用多重裂口PCR(gap polymerase chain reaction,Gap-PCR)和反向斑点杂交(reverse dot blot,RBD)等技术,对初筛阳性者进行常见型和罕见型基因检测,并对检测结果进行统计学分析。结果①22553例初筛呈阳性,其中8327例初筛阳性者接受基因诊断,共检出4944例珠蛋白生成障碍性贫血基因携带者,推导出该地区育龄人群总携带率为15.19%,其中α-为3200例(64.73%),β-为1424例(28.80%),αβ复合型为320例(6.47%)。②α-珠蛋白生成障碍性贫血基因携带者中,常见型为3168例(99.00%),罕见型32例(1.00%),共检出13种突变基因和34种基因型,基因型--^(SEA)/αα排在首位。③β-珠蛋白生成障碍性贫血基因携带者中,常见型为1411例(99.09%),罕见型13例(0.91%),共检出19种突变基因和25种基因型,以CD41-42(-CTTT)最为常见。④αβ复合型携带者中共检出53种不同基因型,排在首位的是--^(SEA)/ααβ^(CD41-42M)/β^(N)。⑤瑶族与汉族携带率相当,差异无统计学意义(χ^(2)=0.300,P=0.584),壮族与瑶族、壮族与汉族比较,差异具有统计学意义(χ^(2)=23.66,116.98,均P<0.001)。⑥象州县携带率最高(20.04%),合山市携带率最低(12.38%)。⑦女性携带率(16.56%)高于男性(13.32%),差异有统计学意义(χ^(2)=182.03,P<0.001)。结论来宾地区珠蛋白生成障碍性贫血变异基因类型复杂,该研究首次调查珠蛋白生成障碍性贫血基因在来宾地区人群中的携带率和基因突变谱,为该地区遗传咨询和产前诊断提供了有价值的基线数据。

广州市从化区育龄人群α-珠蛋白生成障碍性贫血筛查及基因鉴定结果分析

目的了解和分析广州市从化区育龄人群中α-珠蛋白生成障碍性贫血发病率及基因突变类型。方法应用血细胞分析和血红蛋白电泳对24083例育龄人群血样进行初筛,初筛阳性者采用跨越断裂点聚合酶链反应(GAP-PCR)和PCR反向点杂交技术检测α-珠蛋白变异基因,使用PCR反向点杂交方法检测β-珠蛋白17种常见突变基因。结果经基因鉴定共检出α-珠蛋白生成障碍性贫血基因异常者2596例,异常发生率10.78%。α-β复合基因突变者170例,复合发生率0.71%。在突变基因中,包括缺失型2550例,占98.23%;非缺失型46例,占1.77%。共含有14种基因突变类型,其中血红蛋白H(HbH)病5种,以--^(SEA)/-α^(3.7)为主;轻型4种,--^(SEA)/αα基因型达到了68.61%;静止型5种,占比最高的前两种基因型为-α^(3.7)/αα和-α^(4.2)/αα。αβ复合基因突变类型检出23种,检出率最高的前六种分别为--^(SEA)/β^(CD41-42),-α^(3.7)/β^(CD41-42),--^(SEA)/β^(654),--^(SEA)/-28,-α^(3.7)/β^(654)和-α^(3.7)/βCD17,占全部复合类型的75.27%。结论广州市从化区α-珠蛋白生成障碍性贫血基因异常发生率较高,基因突变类型和构成比具有自己的特点,是α-珠蛋白生成障碍性贫血一个较为特殊的区域。

脊髓性肌萎缩症携带者筛查及产前诊断结果分析

目的 探索SMA携带者筛查及产前诊断的临床意义。方法 运用实时荧光定量PCR技术进行SMN1基因7号外显子(E7)拷贝数缺失检测,并对结果为阳性的孕妇其配偶进行检测,夫妇均为携带者对胎儿行产前诊断。结果 2472例孕妇共检出SMN1基因E7纯合缺失1例,携带者34例,携带率约为14%。SMA患者女儿诊断为杂合缺失携带者。召回配偶18例,其中1对夫妇检出均为为SMN1基因E7杂合缺失,对其胎儿进行产前诊断未检测到明确致病变异。1例SMA先证者家庭再生育发现:先证者为SMN1基因E7纯合缺失,先证者母亲及父亲为SMN1基因杂合缺失,二胎进行SMA产前诊断,未检测到明确致病变异。结论 本研究通过开展孕妇携带者筛查,可为当地SMA的遗传咨询和产前诊断提供理论依据,并对高风险胎儿使用多技术平台进行产前诊断,可有效避免SMA患儿的出生,对出生缺陷防控具有重要临床指导意义。

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

APPLICATION OF GENETIC DEAFNESS GENE CHIP FOR DETECTION OF GENE MUTATION OF DEAFNESS IN PREGNANT WOMEN

Objective The study is to identify the carrier rate of common deafness mutation in Chinese pregnant women via detecting deafness gene mutations with gene chip. Methods The pregnant women in obstetric clinic without hearing impairment and hearing disorders family history were selected. The informed consent was signed. Peripheral blood was taken to extract genom- ic DNA. Application of genetic deafness gene chip for detecting 9 mutational hot spot of the most common 4 Chinese deafness genes, namely GJB2 （35delG, 176del16bp, 235delC, 299delAT）, GJB3 （C538T） ,SLC26A4 （ IVS72A〉G, A2168G） and mito- chondrial DNA 12S rRNA （A1555G, C1494T） . Further genetic testing were provided to the spouses and newborns of the screened carriers. Results Peripheral blood of 430 pregnant women were detected, detection of deafness gene mutation carri- ers in 24 cases（4.2%）, including 13 cases of the GJB2 heterozygous mutation, 3 cases of SLC26A4 heterozygous mutation, 1 cases of GJB3 heterozygous mutation, and 1 case of mitochondrial 12S rRNA mutation. 18 spouses and 17 newborns took further genetic tests, and 6 newborns inherited the mutation from their mother. Conclusion The common deafness genes muta- tion has a high carrier rate in pregnant women group, 235delC and IVS7-2A〉G heterozygous mutations are common.

Cloning and characterization of a stearoyl-ACP desaturase gene from Jatropha curcas

Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.

不同来源肠炎沙门菌耐药特征及遗传多样性特点的研究

目的分析福建地区腹泻病人和表观健康人群携带的肠炎沙门菌耐药特征及遗传多样性特点。方法收集福建地区腹泻患者和表观健康人群携带的肠炎沙门菌,采用K-B法进行药敏实验,PCR扩增毒力基因,多位点融数目亭联重复序列分析(multiple-locus variable-number tandem repeat analysis,MLVA)技术进行分子分型,并应用BioNumerics软件进行聚类分析。结果福建地区肠炎沙门菌除对CIP和NOR 100%敏感外,对其他8种药物(AMP、AMC-AC、CTX、CRO、CHL、NAL、SXT和TET)均有不同程度的耐药。腹泻患者分离株的多重耐药率(33.33%)高于表观健康人群分离株(15.22%)。10种毒力基因(sitC、hilA、sseL、sifA、mgtC、siiE、sopB、stn、spvB和pefA)均有检出,且其在腹泻患者和表观健康人群分离株中分布不同。毒力基因谱型54个(VP1~VP54),其中VP3只存在于表观健康人群分离株,且为其优势谱型,占比为13.04%;VP4和VP5只存在于腹泻患者分离株;VP4和VP3、VP1均为腹泻患者分离株中的优势谱型,占比均为11.90%。所有分离株共分为40个MLVA型(MT0001~MT0040)、2个主要基因簇(clusterⅠ和clusterⅡ),其中clusterⅡ包含的菌株均来自表观健康人群;clusterⅠ包含的菌株来自及腹泻患者和表观健康人群。结论福建地区肠炎沙门菌呈现遗传多态性,腹泻患者分离株的耐药性和致病力均高于表观健康人群分离株。