组氨酸修饰的聚乙二醇-聚谷氨酸-聚乙烯亚胺作为基因载体的体外性能研究

为了对组氨酸修饰的阳离子聚合物载体进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。采用自由基聚合法合成聚乙二醇-聚谷氨酸-聚乙烯亚胺-组氨酸(PEG-b-PLG-g-PEIsHISs,GGIS)聚阳离子载体,用1H-NMR表征载体材料结构;通过凝胶电泳实验研究载体对质粒DNA的压缩能力,粒径分析仪测定载体结合DNA后在不同N/P比条件下的粒径及表面电荷;用MTT法测定载体在HEK293T,Hela,BEL7402和A549等细胞株上的细胞毒性,考察GGIS体外细胞转染效率。结果显示在N/P≤30时,载体材料的平均粒径在100~200 nm,表面电荷在10~30 m V,适合细胞吞噬。体外细胞毒性表明,GGIS的细胞毒性较PEI 25K低。GGIS具有较强的DNA缩合能力,且有较好的体外基因转染能力。因此,GGIS有较好的体外基因转染能力,能够携带报告基因在体外有效表达,是具有应用前景的非病毒性基因药物载体。     

聚乳酸-聚乙烯亚胺共聚物的制备、表征及其在多西紫杉醇传输中的应用研究

为了获得一种毒性低、可降解的药物载体,以Sn(Oct)2为催化剂,以1,4-丁二醇和丙交酯为原料,使用开环聚合的方法合成了聚乳酸(PLA),用异氟尔酮二异氰酸酯(IPDI)偶联小分子量的聚乙烯亚胺(2000Da),合成了阳离子嵌段共聚物聚乳酸-聚乙烯亚胺共聚物(PLA-PEI,PEA)。采用乳化溶剂挥发法,以脂溶性的PEA为基本材料,制备了疏水性核装载疏水性药物多西紫杉醇(DTX),壳为亲水性聚乙烯亚胺的阳离子纳米粒,并对PEA/DTX纳米粒的性能进行了表征和考察,包括粒度分布、形貌、电位、载药量、包封率、体外释放和细胞摄取。     

新一代阳离子聚合物转染试剂(梭华-Sofast) 转染效果研究

采用DNA延滞实验研究新一代阳离子聚合物梭华 Sofast转染试剂与DNA的结合能力,以不同报告基因(绿色荧光蛋白基因、β 半乳糖苷酶基因和荧光素酶基因),分析比较梭华 Sofast与阳离子脂质体Lipofectamine2000和阳离子聚合物JetPEI、Superfect转染HEK293细胞转染效率和细胞毒性,研究梭华 Sofast转染试剂转染效率.实验结果表明梭华 Sofast与DNA有很强的结合能力;以绿色荧光蛋白基因为报告基因,梭华 Sofast转染率最高,约60%左右,而且在单个细胞中表达的GFP量多;转染细胞荧光素酶活性检测结果表明,当梭华 Sofast与DNA质量比为16时,转染率最高,高于109RLU/mg蛋白,分别是JetPEI,Superfect和Lipofectamine2000转染细胞最高活性的1.6,2.3和5倍;梭华 Sofast转染β 半乳糖苷酶的染色结果显示95%以上的阳性细胞转染率;比较基因转染效率最高时的细胞毒性,在最佳转染剂量时,几种试剂的细胞存活率均在83%以上,而梭华 Sofast为94.23%.在目前市场上销售的转染试剂对HUV EC细胞的转染效果均不理想情况下,梭华 Sofast仍具一定的转染效率,阳性细胞约占20%.研究结果表明梭华 Sofast是一种高转染效率,低细胞毒性的转染试剂.     

线性与分枝状聚乙烯亚胺介导基因体外转染的比较与研究

聚乙烯亚胺(PEI)是一类新兴的阳离子多聚物非病毒载体,可缩聚DNA分子形成颗粒并转入真核细胞进行表达.本文选用分子量750 ku分枝状PEI与分子量25 ku线性PEI转染增强型绿色荧光蛋白(EGFP)报告基因于HeLa细胞,并对这两种PEI的转染效率进行比较.利用MTT法检测比较线性PEI与分枝状PEI的细胞毒性.通过凝胶阻滞实验比较两种PEI结合DNA能力.最后利用肝素介导释放实验比较两者在形成PEI/DNA复合物后释放DNA的能力.研究结果表明:线性PEI转染效率优于分枝状PEI,且随PEI/DNA质量比的增加而升高,而分枝状PEI则相反;两种PEI细胞毒性在一定范围内相近;分枝状PEI结合DNA能力略强于线性PEI;但是分枝状PEI释放DNA的能力远小于线性PEI.这可能导致DNA在细胞内无法从PEI/DNA复合体上释放出来,从而影响转录的正常进行,最终导致分枝状PEI转染效率下降.     

聚乙二醇-聚乙烯亚胺共聚物的制备及其表征

用亲水的聚乙二醇对聚乙烯亚胺进行改性，制备适用于基因转染的非病毒类载体。以异佛尔酮二异氰酸酯活化聚乙二醇，再与聚乙烯亚胺反应，两步法合成了聚乙二醇-聚乙烯亚胺（PEG—PEI）嵌段共聚物，分别用IR、^1H NMR、GPC、DSC列共聚物进行了表征。在IR谱图上可见mPEG—NCO中异氰酸基、及PEG—PEI中脲基的特征峰；根据^1H NMR谱图计算表明，此聚合反应为可控反应，通过调节PEG与PEI的投料比例可控制共聚物组成及相对分子质量；GPC曲线上共聚物为一单峰，与PEG和PEI均聚物峰位置不同，表明产物是PEGPE共聚物，DSC分析也表明共聚物Tm。较均聚物均有不同程度的下降，这是PEG和PEI两种嵌段相互缠结的结果。因此证明成功合成了PEG—PEI共聚物。     

以芳香亚胺为连接剂的聚乙烯亚胺衍生物的合成及体外活性研究

通过有机合成的方法合成新型聚阳离子载体聚五乙烯六胺对苯二甲醛-1,4-二亚胺(PEHA-TPAI),产物结构经核磁共振氢谱(1H NMR)和傅里叶红外光谱(FT-IR)确定系目标产物。采用琼脂糖凝胶电泳实验观察PEHATPAI包裹小干扰RNA(siRNA)的能力,并用粒度仪和Zeta电位仪对PEHA-TPAI/siRNA复合物进行表征。结果表明:PEHA-TPAI和siRNA复合后可形成稳定且粒径均一的纳米颗粒,在PEHA-TPAI/siRNA复合物质量比为10∶1的条件下,颗粒粒径为(129.90±1.02)nm,Zeta电位为(21.1±0.54)mV。体外细胞毒性实验和转染实验的结果表明:PEHA-TPAI的细胞毒性显著低于商业化转染试剂聚乙烯亚胺(PEI,分子量为25 000)(P0.001)且具有良好的转染效率,有作为低毒性、高转染效率的基因输送载体的潜力。     

聚乙二醇-聚乙烯亚胺负载超顺磁纳米Fe3O4的合成及其基因转染应用

采用单甲基醚聚乙二醇改性聚乙烯亚胺,得到水溶性接枝共聚物聚乙二醇接枝支化聚乙烯亚胺(mPEG-g-PEI),并利用离子交换法制备了PEG-PEI-SPIO. 同时,还合成了mal-PEG-COOH,并用其制备了多功能负载SPIO的抗体-聚乙二醇-聚乙烯亚胺. 负载SPIO的抗体-聚乙二醇-聚乙烯亚胺与pDNA复合后形成的复合物粒径约为105 nm,体外实验结果显示T细胞能特异性的吸收抗体-聚乙二醇-聚乙烯亚胺负载的SPIO,靶向组在基因转染实验中有较好的效果,可用磁共振手段进行实时无创观测.     

高分子聚合物对两种细胞转染效率的研究

比较纳米材料和阳离子多聚物转染试剂携带增强绿色荧光蛋白基因对C2C12细胞和DF-1细胞的转染效率,筛选适合的基因载体。采用Entranster TM-D纳米材料和3种阳离子多聚物(Superfect、Fect、Neofect)转染试剂介导p CDH-EGFP-puro质粒分别转染C2C12细胞和DF-1细胞,24 h后用实时荧光定量核酸扩增检测系统检测转染细胞中增强绿色荧光蛋白基因的mRNA表达水平,48 h后观察并计数阳性细胞率。结果发现4种试剂分别转染的两种细胞中均有增强绿色荧光蛋白基因的表达。C2C12细胞用阳离子多聚物转染的效率略高于纳米材料转染,其中Superfect的转染效率可达到30%;DF-1细胞用纳米材料和阳离子多聚物转染的效率都高,其中Fect的转染效率达到50%。因此,阳离子多聚物Superfect用于C2C12细胞的体外转染,Fect转染试剂用于DF-1细胞的体外转染,都表现出较高效的转染效率     

一种新颖的阳离子非病毒基因载体 Chitosan-g-PEI-g-PEG-OH 的性能研究

研究了新型阳离子聚合物Chitosan-g-PEI-g-PEG-OH的性能，重点考察其粒度，基因转染效率与细胞毒性，探讨了其作为基因载体的可能性.通过动态光散射仪（DSL）、透射电镜（TEM）观察了Chitosan-g-PEI-g-PEG-OH与DNA自组装形成的颗粒形态及粒径，Chitosan-g-PEI-g-PEG-OH可复合DNA形成粒径160～210 nm的纳米复合物，适合进入细胞.使用MTT比色法分析Chitosan-g-PEI-g-PEG-OH的毒性并与PEI，PEI-g-PEG-OH比较.选用增强型绿色荧光蛋白(EGFP) 转染Hela细胞，应用流式细胞术检测转染效率.新型阳离子多聚物Chitosan-g-PEI-g-PEG-OH在提高基因转染效率的同时降低了其细胞毒性，有望成为基因转移的有效载体.     

基于低分子量PEI的不同疏水链修饰的梳状低聚物基因载体

为了探究基于低分子量聚乙烯亚胺(polyethylenimine, PEI)基因载体的转染效率,通过Michael加成反应将PEI 600 Da接枝于含有疏水链的生物可降解聚酯上形成系列梳状聚合物,并将之应用于基因载体;利用核磁氢谱和凝胶渗透色谱对聚合物的化学结构与分子量进行了测定,此外,还利用凝胶电泳实验和绿色荧光蛋白实验研究了聚合物与DNA的结合能力及其复合物的转染性能。结果表明：本文方法成功合成了系列低聚物,并对DNA表现出较好的包裹能力;油酸修饰后的低聚物与DNA复合物在质量比为6.4时转染效果与PEI 25 kDa相当。可见,油酸修饰后的脂质体低聚物有作为非病毒基因载体的前景。     

供精个体和异种动物精浆对小鼠精子介导转染外源基因效率影响的研究

目的探讨供精个体和异种动物精浆对小鼠精子转染外源DNA效率的影响,以探索精子作为外源基因的载体建立转基因小鼠的影响因素。方法从性成熟小鼠的输精管及部分附睾管中取出精子,用DIG免疫组化方法检测和比较精予转染效率,因素为供精个体和异种动物精浆。结果不同供精个体间转染效率存在一定差异,但有的个体问差异极显著（P〈0．01）,而有的个体间差异不显著：共孵育体系中异种动物精浆可显著降低转染效率（实验组与对照组分别为（8．6±1．7）％VS（51．3±5.3）％,P〈0．01）。结论小鼠供精个体和异种动物精浆可影响小鼠精予转染外源DNA的效率。     

Enhancement of transfection efficiency for HeLa cells via incorporating arginine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and 13C NMR. Arg-CS/DNA polye- lectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candi- date as a safe and efficient vector for gene delivery and transfection.     

Enhancement of transfection efficiency for HeLa cells via incorporating arsinine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan （Arg-CS）. Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and ^13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering （DLS） and atomic force microscopy （AFM）. The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2：1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.     

壳寡糖纳米载体细胞内递送EGFR脱氧核酶的研究

设计靶向EGFR mRNA的脱氧核酶(EGFR DRz),以壳寡糖(COS)为材料,建立了一种有效的纳米基因细胞内传递体系,并研究其介导的靶向EGFR的脱氧核酶在Hela细胞内的生物学效应.流式结果表明COS-EGFR DRz复合体转染效率为88.7%,与脂质体转染试剂的89.7%相比无显著差异.半定量RT-PCR结果显示,经壳寡糖纳米载体递送的EGFR DRz能有效地靶向切割Hela细胞内的EGFR mRNA,使其表达下降.进一步的流式分析显示细胞被阻滞在G0～G1期,并且出现凋亡现象,其中COS组的凋亡率为19.3%,大于对照组脂质体的凋亡率13.0%.研究表明,COS较脂质体有相似的转染效率和更低的毒性,是一种潜在的、有效的脱氧核酶递送载体.     

复合环境离子强度对BDCP/DNA的粒径与转染效率的影响

为了探讨组装环境对基因载体/DN复合物的粒径和转染效果的影响,在三种不同离子浓度溶液体系(PBS, 5% 葡萄糖溶液, H₂O)中测量BDCP（biodegradable cationic polymer,一种生物可降解的阳离子聚合物）/DNA复合物粒径和结合力,进行了体外转染试验和毒性试验.结果显示,PBS最适合组装转染复合物,可取得更好的稳定性、最高的转染效率和较低细胞毒性、低溶血率;在5%葡萄糖溶液和水中组装的BDCP/质粒复合物结合力较弱,转染效率比较低.得出结论,BDCP/DNA粒径、结合力和基因转染效率受组装体系的离子强度影响.     

Apoptosis of transgenic cloned and recloned bovine blastocysts

Apoptosis plays an important role in preimplantation embryonic development. Investigating mechanisms of apoptosis can provide useful information for obtaining high-quality embryos and help to improve cloning efficiency. Here, we investigated the incidence of blastomere apoptosis in transgenic blastocysts generated by somatic cell nuclear transfer (SCNT) and recloning using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. Transgenic recloned embryos were the second generation SCNT embryos derived from the somatic cells of a transgenic SCNT calf. The blastocyst rate of transgenic SCNT embryos was lower than that of nontransgenic SCNT embryos. The incidence of apoptosis in transgenic SCNT embryos was higher than that of nontransgenic SCNT embryos. The blastocyst rate and the incidence of apoptosis in transgenic recloned embryos were similar to nontransgenic SCNT embryos. The process of donor cell transfection and drug selection may decrease the developmental capacity of transgenic SCNT embryos. Serial cloning did not influence the developmental capacity of transgenic recloned embryos.     

Intracellular siRNA delivery with novel spermine based surfactants

The design and development of safe and effective multifunctional siRNA delivery systems are critical for clinical application of RNAi therapeutics. Here we evaluated eight new spermine-based surfactant multifunctional carriers for siRNA delivery. These carriers complexed with siRNA forming stable compact nanoparticles with sizes around 100 nm. The multifunctional carriers mediated higher intracellular siRNA transfection than Lipofectamine-2000. The siRNA nanoparticles of the multifunctional carriers exhibited low cytotoxicity as shown by MTT assay. Three of the eight multifunctional carriers showed higher silencing efficiency than Lipofectamine-2000 in both U87-Luc cells and CHO-GFP cells. SKAHCO showed the highest siRNA delivery efficiency among the carriers. It resulted in 84.6±5.5% silencing of luciferase activity in U87-Luc cells, much higher than that (62.8± 3.4%) from Lipofectamine-2000. In conclusion, the spermine based multifunctional carriers are promising for highly efficient intracellular siRNA delivery.     

原核显微注射产生转基因绵羊胚胎

体外采集绵羊卵丘卵母细胞复合体,成熟培养24 h,经过体外受精培养17 h,比较高速离心对胚胎发育的影响;高速离心可以使黑色脂滴甩到一边从而使受精卵原核清晰可见.然后将绵羊乳腺特异表达人肝细胞再生增强因子和真核细胞表达增强绿色荧光蛋白(Enhanced Green fluorescence protein,EGFP)的载体DNA显微注射于绵羊受精卵雄原核中,并将异构胚在SOF液中发育培养.结果表明:高速离心组囊胚率低于对照组,但是没有显著性差异(P》0.05);显微注射外源基因2天后在激光共聚焦显微镜下可见荧光胚胎;PCR检测5个荧光胚胎均可见特异性条带.在原核显微注射生产转基因胚胎中,绿色荧光蛋白可作为标记基因进行早期胚胎筛选,为提高转基因动物移植效率奠定实验基础.     

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro

Objective: To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. Methods: We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. Results: The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. Conclusion: With a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.