海藻酸钠载体中成年兔关节软骨细胞生长情况

目的:软骨细胞单层培养条件下增殖较快,但极易失分化,在生物载体材料构成的立体环境中培养,则能较好的维持表型。实验以海藻酸钠为载体,观察兔关节软骨细表型及增殖情况。方法:实验于2005-09/2006-12在山西医科大学第二医院骨科实验室完成。①实验动物和材料:6月龄新西兰大白兔24只,雌雄各半。实验过程中对动物处置符合动物伦理学要求。海藻酸钠由青岛明月海藻公司提供。②实验方法:以培养液配制的0.4% Pronease酶、0.025%Ⅱ型胶原酶顺序消化兔软骨分离细胞,以4×109 L-1海藻酸钠的浓度制成细胞悬液。③实验评估:倒置显微镜观察细胞在海藻酸钠中的形态及增殖情况,检测细胞收获效率和存活率。苏木精-伊红染色和AB-PAS染色观察软骨细胞的生长情况及评价软骨分泌胶原的情况。免疫组织化学定性观察Ⅱ型胶原的含量变化及有无Ⅰ型胶原的产生。Alcian blue染色法测定细胞盘中蛋白多糖的含量变化。结果:①软骨经两步酶消化软骨基质逐步解离和降解,细胞被完全分离,消化分离的软骨细胞总数达到5×106,细胞成活率达95.5%。②软骨细胞与海藻酸钠复合后体外培养,细胞生长旺盛、增殖活跃,增殖成株状或岛状,株状细胞团周围有类似的软骨陷窝形成,细胞排列密集,核圆形。③Ⅱ型胶原免疫组化和AB-PAS染色均呈阳性,细胞盘中蛋白多糖含量随培养时间延长逐渐增加。结论:海藻酸钠凝胶能与软骨细胞完全嵌合,是一种良好的软骨组织工程材料。     

胶原-壳聚糖支架与软骨细胞的生长

背景：目前软骨支架材料的种类比较多,但还没有一种材料能完全符合软骨修复的要求。目的：观察在混合材料胶原-壳聚糖支架中软骨细胞的生长情况。方法：采用冷冻干燥法将质量分数为2%胶原与3%壳聚糖混合制备胶原-壳聚糖多孔支架。将分离培养的第2代兔软骨细胞接种到胶原-壳聚糖支架上,对照组将软骨细胞接种到无支架的培养板中。观察支架的孔隙率、吸水性及内部形态结构,MTT法检测软骨细胞在支架上的增殖情况,组织切片苏木精-伊红染色,扫描电镜观察细胞在支架的生长、贴附情况,RT-PCR检测细胞支架复合物蛋白聚糖和Ⅱ型胶原mRNA表达情况。结果与结论：胶原-壳聚糖支架的吸水性为（80.0±0.55）%,孔隙率为（88.5±1.5）%,孔径为100～150μm,复合细胞培养2周后,细胞增殖活力高,软骨细胞分泌的蛋白聚糖和Ⅱ型胶原mRNA表达明显高于对照组。说明质量分数为2%胶原与3%壳聚糖的混合支架适合软骨细胞生长和快速增殖,是一种良好的修复和重建软骨载体。     

胶原-壳聚糖支架与软骨细胞的生长

背景:目前软骨支架材料的种类比较多,但还没有一种材料能完全符合软骨修复的要求。目的:观察在混合材料胶原-壳聚糖支架中软骨细胞的生长情况。方法:采用冷冻干燥法将质量分数为2%胶原与3%壳聚糖混合制备胶原-壳聚糖多孔支架。将分离培养的第2代兔软骨细胞接种到胶原-壳聚糖支架上,对照组将软骨细胞接种到无支架的培养板中。观察支架的孔隙率、吸水性及内部形态结构,MTT法检测软骨细胞在支架上的增殖情况,组织切片苏木精-伊红染色,扫描电镜观察细胞在支架的生长、贴附情况,RT-PCR检测细胞支架复合物蛋白聚糖和Ⅱ型胶原mRNA表达情况。结果与结论:胶原-壳聚糖支架的吸水性为(80.0±0.55)%,孔隙率为(88.5±1.5)%,孔径为100～150μm,复合细胞培养2周后,细胞增殖活力高,软骨细胞分泌的蛋白聚糖和Ⅱ型胶原mRNA表达明显高于对照组。说明质量分数为2%胶原与3%壳聚糖的混合支架适合软骨细胞生长和快速增殖,是一种良好的修复和重建软骨载体。     

关节软骨缺损修复的基因治疗实验 人胰岛素样生长因子Ⅰ基因转染软骨细胞及其在软骨细胞中的表达

目的：探讨胰岛素样生长因子Ⅰ基因转染人软骨细胞的可能性及其在软骨细胞中的表达，并初步判断转基因细胞的生物学功能变化。方法：实验于2002-07／2003-06在中山大学附属第二医院医学研究中心完成。①构建pcDNA3.1-胰岛素样生长因子Ⅰ／GS质粒。将构建质粒在大肠杆菌DH10B中大量扩增后，抽提纯化质粒，并对质粒进行测序鉴定。②将鉴定的质粒用阳离子脂质体转染人关节软骨细胞，通过逆转录-聚合酶链反应、Westren-blot等方法检测胰岛素样生长因子Ⅰ基因在人关节软骨细胞中的表达。③分别将同代软骨细胞与转基因细胞的培养液、软骨细胞与转基因细胞裂解液和二甲基亚甲蓝共孵育后，检测各混合液的吸光度，以此判断同代软骨细胞、转基因细胞、软骨细胞培养液、转基因细胞培养液中的蛋白多糖含量。结果：①质粒鉴定结果：质粒抽提纯化后电泳可见3条DNA条带，符合质粒的电泳图谱；质粒的测序结果和胰岛素样生长因子Ⅰ基因序列完全吻合。②胰岛素样生长因子Ⅰ基因在软骨细胞中的表达：质粒转染软骨细胞后，反转录-聚合酶链反应能见到298bp的胰岛素样生长因子ⅠmRNA片断的表达；Western-blot可见7．6ku的胰岛素样生长因子Ⅰ蛋白表达条带。③转胰岛素样生长因子Ⅰ基因对软骨细胞分泌蛋白多糖的影响：转基因软骨细胞裂解液和同代的软骨细胞裂解液的蛋白多糖含量分别是（5．23&;#177;0．62）mg／L和（2．47&;#177;0．31）mg／L．两组比较，差异有显著性意义（t=2．88，P〈0．05）；转基因软骨细胞培养液和同代非转染软骨细胞培养液中的蛋白多糖含量分别为（9．92&;#177;1．04）mg／L和（4．56&;#177;0．51）mg／L，两组比较，差异有显著性意义（t=6．47，P〈0．05）。结论：胰岛素样生长因子Ⅰ基因转染人关节软骨细胞后能在软骨细胞中表达，并能促进软骨细胞分泌软骨基质蛋白多糖，具有促DNA合成和维持软骨细胞表型稳定的能力。转胰岛素样生长因子Ⅰ基因软骨细胞的研究为体外软骨组织工程和关节软骨病的基因治疗提供了直接的实验依据。     

反转录病毒载体介导转化生长因子β_1基因体外转染兔膝关节软骨细胞的表达

背景:将功能基因片段整合到基因载体内,再将基因载体转染入靶细胞中或转染入关节腔内,通过转基因的靶细胞持续大量分泌功能基因产物,在一个较长的时期内保持局部治疗浓度,可修复关节软骨损伤.目的:以重组反转录病毒载体介导转化生长因子β_1体外转染兔膝关节软骨细胞,观察其表达情况及其对软骨细胞生物学性状的影响.方法:采用胰酶消化法分离培养兔软骨细胞.载体经PLNCX_2 Hind Ⅲ/Not Ⅰ双酶切、去磷酸化后,与载体pDsRed_2双酶切得到大的部分多克隆位点和RFP基因连接,构建PLNCX_2-RFP.转化生长因子β_1基因从PGEMT-TGF中扩增,经双酶切后与PLNCX_2-RFP连接,构建PLNCX_2-TGFβ_1-RFP.包装反转录病毒载体,并检测病毒上清滴度.将培养的兔膝关节软骨细胞分组转染:对照组(不做任何转染)、转染PLNCX_2组、转染PLNCX_2-TGFβ_1-RFP组,持续筛选2周,观察细胞变化.收集稳定转染的细胞上清液,以NO检测试剂盒检测基因转染对软骨细胞的影响,以ELISA法检测细胞培养上清液中人转化生长因子β_1表达.结果与结论:重组基因PLNCX_2-TGFβ_1-RFP经酶切鉴定测序TGFβ_1、RFP、序列均正确,表明构建成功预期的真核表达载体PLNCX_2-TGFβ_1-RFP.转染到包装细胞并筛选培养后,病毒滴度为1×10~6CFU.稳定转染软骨细胞后可观察到红色荧光,证明转染成功.持续筛选2周,散在贴壁细胞形成阳性克隆,逐渐弥漫融合,并有细胞簇出现,双核多见,细胞增生活跃.转染PLNCX_2-TGFβ_1-RFP组NO浓度高于对照组、转染PLNCX_2组(P<0.05),对照组、转染PLNCX_2组间差异无显著性意义.对照组与转染PLNCX_2组均无转化生长因子β_1表达,转染PLNCX_2-TGFβ_1-RFP组转化生长因子β_1质量浓度为(28.08±3.73)ng/L.提示反转录病毒载体PLNCX_2介导的人转化生长因子β_1能有效转染到兔膝关节软骨细胞并获得稳定表达,同时转染后的软骨细胞增生活跃.     

关节软骨缺损修复的基因治疗实验人胰岛素样生长因子Ⅰ基因转染软骨细胞及其在软骨细胞中的表达

目的探讨胰岛素样生长因子Ⅰ基因转染人软骨细胞的可能性及其在软骨细胞中的表达,并初步判断转基因细胞的生物学功能变化.方法实验于2002-07/2003-06在中山大学附属第二医院医学研究中心完成.①构建pcDNA3.1-胰岛素样生长因子I/GS质粒,将构建质粒在大肠杆菌DH10B中大量扩增后,抽提纯化质粒,并对质粒进行测序鉴定.②将鉴定的质粒用阳离子脂质体转染人关节软骨细胞,通过逆转录-聚合酶链反应、Westren-blot等方法检测胰岛素样生长因子Ⅰ基因在人关节软骨细胞中的表达.③分别将同代软骨细胞与转基因细胞的培养液、软骨细胞与转基因细胞裂解液和二甲基亚甲蓝共孵育后,检测各混合液的吸光度,以此判断同代软骨细胞、转基因细胞、软骨细胞培养液、转基因细胞培养液中的蛋白多糖含量.结果①质粒鉴定结果质粒抽提纯化后电泳可见3条DNA条带,符合质粒的电泳图谱;质粒的测序结果和胰岛素样生长因子Ⅰ基因序列完全吻合.②胰岛素样生长因子Ⅰ基因在软骨细胞中的表达质粒转染软骨细胞后,反转录-聚合酶链反应能见到298 bp的胰岛素样生长因子ⅠmRNA片断的表达;Western-blot可见7.6 ku的胰岛素样生长因子Ⅰ蛋白表达条带.③转胰岛素样生长因子Ⅰ基因对软骨细胞分泌蛋白多糖的影响转基因软骨细胞裂解液和同代的软骨细胞裂解液的蛋白多糖含量分别是(5.23±0.62)mg/L和(2.47±0.31)mg/L,两组比较,差异有显著性意义(t=2.88,P＜0.05);转基因软骨细胞培养液和同代非转染软骨细胞培养液中的蛋白多糖含量分别为(9.92±1.04)mg/L和(4.56±0.51)mg/L,两组比较,差异有显著性意义(t=6.47,P＜0.05).结论胰岛素样生长因子Ⅰ基因转染人关节软骨细胞后能在软骨细胞中表达,并能促进软骨细胞分泌软骨基质蛋白多糖,具有促DNA合成和维持软骨细胞表型稳定的能力.转胰岛素样生长因子Ⅰ基因软骨细胞的研究为体外软骨组织工程和关节软骨病的基因治疗提供了直接的实验依据.     

关节软骨缺损修复的基因治疗实验——人胰岛素样生长因子I基因转染软骨细胞及其在软骨细胞中的表达

目的:探讨胰岛素样生长因子I基因转染人软骨细胞的可能性及其在软骨细胞中的表达,并初步判断转基因细胞的生物学功能变化。方法:实验于2002-07/2003-06在中山大学附属第二医院医学研究中心完成。①构建pcDNA3.1-胰岛素样生长因子I/GS质粒,将构建质粒在大肠杆菌DH10B中大量扩增后,抽提纯化质粒,并对质粒进行测序鉴定。②将鉴定的质粒用阳离子脂质体转染人关节软骨细胞,通过逆转录-聚合酶链反应、Westren-blot等方法检测胰岛素样生长因子I基因在人关节软骨细胞中的表达。③分别将同代软骨细胞与转基因细胞的培养液、软骨细胞与转基因细胞裂解液和二甲基亚甲蓝共孵育后,检测各混合液的吸光度,以此判断同代软骨细胞、转基因细胞、软骨细胞培养液、转基因细胞培养液中的蛋白多糖含量。结果:①质粒鉴定结果:质粒抽提纯化后电泳可见3条DNA条带,符合质粒的电泳图谱;质粒的测序结果和胰岛素样生长因子I基因序列完全吻合。②胰岛素样生长因子I基因在软骨细胞中的表达:质粒转染软骨细胞后,反转录-聚合酶链反应能见到298bp的胰岛素样生长因子ImRNA片断的表达;Western-blot可见7.6ku的胰岛素样生长因子I蛋白表达条带。③转胰岛素样生长因子I基因对软骨细胞分泌蛋白多糖的影响:转基因软骨细胞裂解液和同代的软骨细胞裂解液的蛋白多糖含量分别是(5.23±0.62)mg/L和(2.47±0.31)mg/L,两组比较,差异有显著性意义(t=2.88,P<0.05);转基因软骨细胞培养液和同代非转染软骨细胞培养液中的蛋白多糖含量分别为(9.92±1.04)mg/L和(4.56±0.51)mg/L,两组比较,差异有显著性意义(t=6.47,P<0.05)。结论:胰岛素样生长因子I基因转染人关节软骨细胞后能在软骨细胞中表达,并能促进软骨细胞分泌软骨基质蛋白多糖,具有促DNA合成和维持软骨细胞表型稳定的能力。转胰岛素样生长因子I基因软骨细胞的研究为体外软骨组织工程和关节软骨病的基因治疗提供了直接的实验依据。     

藻酸钠复合自体软骨细胞修复猪关节软骨缺损的近期效果

目的:采用藻酸钠自体软骨细胞混合物修复猪的关节软骨缺损,观察近期成骨效应。方法:实验于2004-03/2005-04在中山大学附属第二医院实验中心完成。3个月龄的小型猪8只,取其关节软骨进行细胞原代培养,获取原代细胞后,分别进行免疫组织化学法检测细胞Ⅱ型胶原表达。然后对8只小型猪的32个关节进行动物关节缺损的模型建立,每个关节共造3个人工缺损,选择关节左右随机对照。将已经培养好的软骨细胞与已经配制好并消毒的藻酸钠混合,移植到猪的关节软骨缺损处。每只猪关节内注射3个实验点,1个用藻酸钠/细胞混合液为实验组,1个单纯支架材料为单纯材料对照组,1个缺损做空白对照组,模型中缺损区均为关节负重区,胶原纤维绿色(亮绿复染)。修复所用细胞为自体软骨体外培养后,进行移植修复,采用3代体外培养软骨细胞,均匀混和藻酸钠后,行自体移植。8周后取实验关节进行大体观察、苏木精-伊红染色、Masson染色、免疫组织化学法染色观察及O’driscoll组织学评分。结果:纳入小型猪8只,除其中1例因手术切断伸直肌腱而使猪关节活动障碍,其余均未见手术后关节活动障碍,所有手术无术后感染,无关节内感染,无动物死亡。移植的软骨细胞在藻酸钠载体中生长良好,形成透明软骨。根据O’driscoll组织学评分标准,实验组32个猪关节缺损修复的组织学评分为(20.25±1.64)分,高于单纯材料对照组的(7.46±1.29)分及空白对照组的(6.00±2.09)分。免疫组织化学染色可见修复软骨以表达Ⅱ型胶原为主,无Ⅰ型胶原。结论:藻酸钠复合自体软骨细胞作为软骨移植的替代物是可行的,远期效果还有待进一步研究。     

藻酸钙微载体培养胎儿关节软骨细胞

目的观察胎儿关节软骨细胞在藻酸钙微载体培养中的特征,探讨用藻酸钙微载体培养胎儿关节软骨细胞的优缺点。方法用藻酸钙微载体和体外单层培养法培养胎儿关节软骨细胞,观察细胞形态学变化,测定细胞数量变化及其生长曲线,观察细胞增殖;借助免疫组织化学的方法了解细胞II型胶原的合成情况。结果体外单层培养的胎儿关节软骨细胞传至第5代已出现明显的反分化,多数细胞变为成纤维细胞状,合成II型胶原的能力明显减弱;而用藻酸钙微载体培养的软骨细胞3个月后仍保持有旺盛的合成Ⅱ型胶原的能力。结论用藻酸钙微载体培养胎儿关节软骨细胞可较长时间保持其表型及合成基质能力,防止反分化的发生。     

降钙素基因相关肽表达载体的构建

背景:为研究降钙素基因相关肽基因治疗在骨质疏松中的应用,首先要建立高效的降钙素基因相关肽基因载体。目的:构建降钙素基因相关肽真核表达载体pBaBb-puro-CGRP。方法:设计引物并通过PCR扩增出降钙素基因相关肽,酶切后用T4DNA连接酶将其与pBaBb-puro进行连接,经过转化、阳性克隆筛选后,进行酶切和测序鉴定。结果与结论:经PCR扩增获得了含430bp的降钙素基因相关肽基因编码序列,经过酶切、连接、转化后,挑选10个菌落进行PCR检测,筛查到8个菌落含有重组质粒,进一步行酶切和测序鉴定,结果与理论预期完全相符。提示实验成功构建了pBaBb-puro-CGRP真核表达载体。     

壳聚糖季铵盐作为基因递送载体的初步研究

背景:非病毒载体因其安全性好而在基因治疗领域赢得了广泛的关注,各种各样的非病毒载体处在不断的研究中,壳聚糖季铵盐作为一种新的生物材料,同样也具有作为基因递送载体的潜质.目的:考察壳聚糖季铵盐体外基因转染活性,寻求一种新的非病毒基因载体递送系统.设计、时间及单位:对比观察实验,于2008-04/12在浙江省医学科学院生物工程所完成.材料:质粒pcDNA3.1-EGFP为浙江省医学科学院生物工程实验室保存.以3-氯-2-羟丙基三甲基氯化铵作为改性剂制备壳聚糖季铵盐,用复凝聚法制备载基因纳米粒.方法:凝胶阻滞实验分析壳聚糖季铵盐包裹质粒的能力,DNase Ⅰ的保护实验分析载基因纳米粒的抵抗核酸酶降解的能力,体外基因转染实验评价纳米粒的体外转染活性,用倒置荧光显微镜观察和流式细胞仪测定转染结果.通过考察转染液中有无牛血清和递送不同剂量的基因对转染效率的影响,寻求本递送系统较好的转染条件.另外,用MTT法测定壳聚糖季铵盐的细胞毒性.主要观察指标:壳聚糖季铵盐和pcDNA 3.1-EGFP以何种比例结合形成的纳米粒、转染液中有无牛血清,质粒的量为多少时对人胚肾T细胞的转染效率足最高的;不同浓度的壳聚糖季铵盐对细胞生长的影响.结果:壳聚糖季钱盐纳米粒能转入人胚肾T细胞,虽然转染效率略逊丁聚乙烯亚胺,但是细胞毒性明显小于聚乙烯亚胺.壳聚糖季铵盐纳米粒转染细胞72 h后效率较高,经综合分析,当pcDNA质量为2 μg,壳聚糖季铵盐和pcDNA以质量比为5结合形成的纳米粒,在无血清条件下对人胚肾T细胞进行转染,转染效率足最高的.结论:壳聚糖季铵盐纳米粒能将基因递送到细胞内,并且报告基因能在细胞内表达.因此,壳聚糖季铵盐用做基因递送的载体系统值得进一步的研究.     

壳聚糖载体介导关节内基因转移：转染效率与基因产品的关系

目的:分析壳聚糖-DNA超微颗粒在关节内的转基因效应。方法:实验于2005-09/2006-06在上海交通大学医学院健康科学研究所骨科细胞与分子生物学实验室完成。实验材料:①模型制备:采用切断内侧副韧带,切除内侧半月板的方法制备骨关节炎兔模型。②基因产品:白细胞介素(interleukin,IL)1Ra基因、IL-10基因。实验分组:15只新西兰兔按随机数字表法分为3组:①空载体对照组(n=3),造模后5d两侧膝关节关节腔注射400μL壳聚糖-PcDNA3.1溶液,共3次,每48h1次。②IL-1Ra基因治疗组和IL-10基因治疗组,每组6只,造模后5d对照侧膝关节关节腔分别注射20μg裸DNA(PcDNA3.1-IL-1Ra或PcDNA3.1-IL-10),实验侧膝关节关节腔注射400μL壳聚糖-DNA超微颗粒(含20μgIL-1Ra或IL-10),注射次数及间隔时间同空载体对照组。实验评估:①采用酶联免疫吸附分析及免疫组织化学检测IL-1Ra和IL-10基因的表达和分布。②苏木精-伊红染色和甲苯胺蓝染色观察骨关节炎软骨组织学变化。结果:纳入新西兰兔15只,均进入结果分析。①IL-1Ra和IL-10基因在关节滑液中的表达:空载体对照组及IL-1Ra基因治疗组对照侧膝关节滑液中未检测到IL-1Ra表达,实验侧于第1次基因注射后7,14d检测到IL-1Ra表达。IL-10基因治疗组对照侧和实验侧均未检测到IL-10表达。②IL-1Ra基因在兔膝关节的分布:IL-1Ra基因治疗组兔软骨表层和中间层部分细胞内表达IL-1Ra,至少持续到第1次基因注射后14d。在滑膜组织中未观察到明显的IL-1Ra表达。③兔骨关节炎软骨组织学变化:空载体对照组呈早期骨性关节炎的典型性改变。苏木精-伊红染色显示软骨细胞坏死,蛋白多糖甲苯胺蓝染色不均一,软骨表层和中间层大部分区域失染,软骨细胞簇聚区域其周围深染。IL-1Ra基因治疗组在软骨损坏方面明显减轻,甲苯胺蓝部分失染。结论:①壳聚糖-DNA超微颗粒的转染效率与基因产品有关。②将IL-1Ra用关节腔直接注射壳聚糖-DNA超微颗粒的方法直接转移入关节腔能一定程度上减轻骨性关节炎的进程。     

叶酸聚乙二醇接枝支化聚乙烯亚胺纳米载体转染螺旋神经节细胞

背景：新型阳离子纳米材料叶酸聚乙二醇接枝支化聚乙烯亚胺易于合成和改性,稳定性好,结构及性能容易调控,已被作为基因治疗转染载体应用于多种疾病的研究。 目的：观察叶酸聚乙二醇接枝支化聚乙烯亚胺转染体外培养小鼠内耳螺旋神经节细胞的可行性及特点。 方法：分别以叶酸聚乙二醇接枝支化聚乙烯亚胺（实验组）、阳离子脂质体Lipofectamine^TM 2000（对照组）作为载体,制备携带增强型绿色荧光蛋白的转染载体系统。将昆明小鼠螺旋神经节细胞分别培养于含实验组和对照组转染载体系统的DMEM/F-12培养基中,培养24 h后,MTT法观察载体系统的细胞毒性,倒置荧光显微镜观察细胞形态及绿色荧光蛋白表达强度,流式细胞仪检测细胞转染率。 结果与结论：两种载体分别转染后,螺旋神经节细胞形态均未发生明显改变,但两组载体对螺旋神经节细胞均有一定的毒性,实验组载体毒性较小且转染效率较高（P〈0.05）。结果说明叶酸聚乙二醇接枝支化聚乙烯亚胺基因转染体外螺旋神经节细胞的转染效率及细胞毒性均优于传统的阳离子脂质体。     

Functionalized organic nanotubes as tubular nonviral gene transfer vector

Tubular nanomaterials are expected to represent a novel nonviral gene transfer vectors due to the unique morphology and potential biological functionalities. Here we rationally constructed functionalized organic nanotubes (ONTs) for gene delivery through the co-assembly of bipolar glycolipid, arginine-lipid and PEG-lipid. The arginine- and PEG-functionalized ONTs efficiently formed complexes with plasmid DNA without aggregation, and protect DNA from enzymatic degradation; while the arginine-functionalized ONTs aggregated with DNA as large bundles. Long ONTs exceeding 1 μm in length was rarely taken up into the cells, while those with a length of 400-800 nm could effectively deliver plasmid DNA into cells and induce high transgene expression of green fluorescense protein. This study demonstrated the usefulness of functionalized ONT in gene delivery, and the functionalized ONT represents a novel type of tubular nonviral gene transfer vector.     

Vaccinia as a vector for gene delivery

Gene therapy is a promising approach, yet so far it has shown limited effectiveness in many clinical trials, mainly due to insufficient gene transduction. Recombinant vaccinia virus (rVV) has been well developed as a gene delivery vector, initially for protein expression in mammalian cells. rVV has been further developed to express antigens in vivo in generating immunity for protection against specific infectious diseases and cancer. rVVs, as non-replicating viral vectors, have been demonstrated for their great potential as vaccines, for their diminished cytopathic effects, high levels of protein expression and strong immunogenicity, and they are relatively safe in animals and in human patients. A number of clinical trials using rVVs as vaccines have shown promising results for treating infectious diseases and cancer. In the last few years, due to its exceptional ability to replicate in tumour cells, the Western Reserve strain vaccinia has been explored as a replicating oncolytic virus for cancer virotherapy. As more is learned about the functions of viral gene products in controlling the mammalian cell cycle and in disabling cellular defence mechanisms, specific viral functions can be augmented or eliminated to enhance antitumour efficacy and improve tumour cell targeting. General mechanisms by which this oncolytic virus achieves the antitumour efficacy and specificity are reviewed. Specifically, the deletion of the viral genes for thymidine kinase and vaccinia growth factor resulted in a vaccinia mutant with enhanced tumour targeting activity and fully retaining its efficiency of replication in cancer cells. Other potential strategies for improving this vector for gene delivery will also be discussed in this review.     

Persistence of non-viral vector mediated RPE65 expression: Case for viability as a gene transfer therapy for RPE-based diseases

Mutations in the retinal pigment epithelium (RPE) gene RPE65 are associated with multiple blinding diseases including Leber's Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65^−/− model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65^−/− mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are non-toxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy.     

Non-invasive gamma camera imaging of gene transfer using an adenoviral vector encoding an epitope-tagged receptor as a reporter

A model epitope-tagged receptor was constructed by fusing the hemagglutinin (HA) sequence on the extracellular N-terminus of the human somatostatin receptor subtype 2 (hSSTr2) gene. This construct was placed in an adenoviral (Ad-HAhSSTr2) vector. This study evaluated Ad-HAhSSTr2 in vitro and in vivo using FACS, fluorescent microscopy, radioactive binding assays, and gamma camera imaging techniques. Infection of A-427 non-small cell lung cancer cells with Ad-HAhSSTr2 or Ad-hSSTr2 resulted in similar expression of hSSTr2 by FACS analysis and binding assays using a (99m)Tc-labeled somatostatin analogue ((99m)Tc-P2045). HAhSSTr2 expression in A-427 cells was specific for infection with Ad-HAhSSTr2. FITC-labeled anti-HA antibody (FITC-HA) confirmed surface expression in live A-427 cells and the absence of internalization. Gamma camera imaging and gamma counter analysis of normal mice showed significantly greater (P<0.05) liver uptake of (99m)Tc-labeled anti-HA antibody ((99m)Tc-anti-HA) in mice injected i.v. 48 h earlier with Ad-HAhSSTr2 (53.6+/-6.9% ID/g) as compared to mice similarly injected with Ad-hSSTr2 (9.0+/-1.3% ID/g). In a mouse tumor model, imaging detected increased tumor localization of (99m)Tc-anti-HA due to direct intratumor injection Ad-HAhSSTr2. Gamma counter analysis confirmed significantly greater (P<0.05) uptake of (99m)Tc-anti-HA in tumors injected with Ad-HAhSSTr2 (12.5+/-4.1% ID/g) as compared to Ad-hSSTr2-infected tumors (5.1+/-1.5% ID/g). These studies demonstrate the feasibility of using an epitope-tagged reporter receptor for non-invasively imaging gene transfer.     

壳聚糖作为药物载体及基因载体的临床应用前景

甲壳素是存在于自然界中惟一能被生物降解的阳离子高分子材料,近年研究表明,它在调节机体免疫功能,降低血脂、血糖、血压,保护胃肠道等方面发挥着巨大的作用.壳聚糖是甲壳素的N-脱乙酰基衍生物,具有组织相容性好、生物学活性多样、可生物降解、无毒性、易于吸收等特点.壳聚糖作为药物载体能提高药物吸收,稳定药物成分,增加药物靶向性,增强药物缓释;它作为基因载体对DNA有一定的保护,能提高基因的表达时间.在药物载体、基因载体等研究领域壳聚糖将具有广泛的应用前景.     

Human JC virus-like particles as a gene delivery vector

Introduction: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy.

Areas covered: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed.

Expert opinion: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.     

Human JC virus-like particles as a gene delivery vector

INTRODUCTION: As a viral gene delivery vector, the recombinant JC virus-like particles (VLPs) can be easily generated in large quantities and at low cost. Exogenous genes of interest can be packaged by the VLP without the involvement of viral genetic material and then delivered into any tissue susceptible to JC virus (JCV) to allow gene transduction. Therefore, it should be possible in the future to develop a gene delivery vector using the human JC VLPs that will allow gene therapy. AREAS COVERED: Development of a gene delivery vector using the polyomavirus VLPs is reviewed in this article. The advantages and disadvantages of using JC VLP for gene delivery are discussed. EXPERT OPINION: Human JC VLPs are readily produced and can be engineered with ease; they allow specific targeting without the presence of any viral genetic material. For therapeutic purposes, gene(s) of interest or other compounds can be packaged into the VLP and delivered to JCV-susceptible cells at high efficiency.