CN101829367A - 一种基因传递系统的三维纳米支架及其制备与应用 - Google Patents
一种基因传递系统的三维纳米支架及其制备与应用 Download PDFInfo
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Abstract
一种基因传递系统的三维纳米支架，它是由胶原、壳聚糖以及纤维粘结蛋白组成的三维纳米支架，其特征是：它包含有载有基因的磷酸钙复合纳米粒，它是一种疏松多孔的三维纳米支架，所述的胶原、壳聚糖、纤维粘结蛋白和戊二醛的质量比为：1∶1∶0.0005∶0.0124～1∶100∶0.08∶0.24，所述的纤维粘结蛋白为含有天门冬氨酸(Arg-Gly-Asp，RGD)序列的纤维粘结蛋白，所述的载有质粒的磷酸钙复合纳米粒是球形的、粒径小于50nm的载有治疗基因的磷酸钙纳米粒，其含量为三维纳米支架总质量的0.01％～1％。本发明的基因传递系统的三维纳米支架在3-15天内，蛋白表达水平维持在10ng/ml左右，说明嵌合有载有基因的磷酸钙复合纳米粒的三维纳米支架具有明显的长效、缓释特征。
Description
技术领域
本发明涉及三维非病毒基因传递系统，具体地说，涉及三维纳米支架、种子细胞的培养及其基因转染。
背景技术
基于组织工程三维支架的非病毒基因传递系统(3 dimensional scaffolds genedelivery system，3DS-GDS)已经成为非病毒型GDS研究的又一重要方向。种子细胞，三维支架及生长因子是组织工程研究的三大要素，三维支架和生长因子构成细胞赖以生存的微环境。细胞培养过程中，生长因子往往直接加入培养液中，由于生长因子半衰期短，易被稀释和降解，这种“外加法”需要反复大剂量给药，繁琐且价格非常昂贵。通过转基因技术将生长因子基因导入种子细胞后，可使细胞自身表达生长因子，这种“内加法”无疑是有利于组织的修复与再生。传统的基因传递技术，无论是病毒型还是非病毒型，一般只是瞬时转染，难以实现生长因子持续高效的表达。为此，人们尝试在三维支架中包嵌DNA，以便基因随着支架的逐步降解缓慢释放，持续转染细胞(参见：StorrieH，Mooney DJ.Sustained delivery of plasmid DNA from polymeric scaffolds fortissue engineering.Adc Drug Deliv Rev.，2006，58(4)：500-514；Tabata Y.Regenrative inductive therapy based on DDS technology of protein and gene.JDrug Target，2006，14(7)：483-495)。目前可用于基因缓释的支架材料有(参见：CaptioRM，Sepctor M.Collagen scaffolds for nonviral IGF-1 gene delivery in articularcartilage tissue engineering[J].Gene Ther.2007，14(9)：721-32；Yuanyuan W，Li L，Shengrong G.Characterization of biodegradable and cytocompatiblenano-hydroxyapatite/poly caprolactone porous scaffolds in degradation in vitro[J].Polymer Degradation and Stability，2009，1-7)：细胞外基质(Extracellularmatrix，ECM)、天然或合成的高分子及其共聚物等，其中ECM(参见：SepidehHeydarkhan-Hagvall，Katja Schenke-Layland，Andrew P.Dhanasopon，et al.Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovasculartissue engineering[J].Biomaterials，2008，29(19)：2907-2914)是由邻近细胞分泌的蛋白纤维、蛋白聚糖和非纤维蛋白等成分通过化学和物理交联组成的具有三维结构的网状结构，为组织和细胞的生长提供支撑和弹性，与细胞特别亲合、易于模拟体内环境。Capito等将胰岛素样生长因子的基因与脂质体转染试剂复合后，与ECM(胶原-氨基聚糖)三维支架交联，在两周培养期内仅释放出约1％，连续表达的胰岛素样生长因子可显著促进组织的再生(参见：Captio RM，Sepctor M.Collagen scaffolds for nonviralIGF-1 gene delivery in articular cartilage tissue engineering[J].Gene Ther.2007，14(9)：721-32)。
发明内容
本发明的一个目的是提供一种基于组织工程三维支架的非病毒基因传递系统及其在非病毒基因传递系统中的应用。
本发明的技术方案如下：
一种基因传递系统的三维纳米支架，它是由胶原、壳聚糖以及纤维粘结蛋白(Fibronectin，FN)组成的三维纳米支架，其特征是：它包含有载有基因的磷酸钙复合纳米粒，它是一种疏松多孔的三维纳米支架，所述的胶原、壳聚糖、纤维粘结蛋白和交联剂戊二醛的质量比为：1∶1∶0.0005∶0.0124～1∶100∶0.08∶0.24，所述的纤维粘结蛋白为含有天门冬氨酸(Arg-Gly-Asp，RGD)序列的纤维粘结蛋白，所述的载有基因的磷酸钙复合纳米粒是球形的、粒径小于50nm的载有治疗基因的磷酸钙纳米粒，其含量为三维纳米支架总质量的0.01％-1％。
一种制备上述的基因传递系统的三维纳米支架的方法，它是在1％胶原醋酸溶液0.1～1ml中加入1％壳聚糖醋酸溶液0.1～1ml，加入0.25％戊二醛50μL～1ml交联，室温过夜，冷冻干燥，缓冲液(PBS)洗涤三次，细胞培养基(Dulbecco’s Modified EagleMedia，DMEM)浸泡过夜后再冷冻干燥，用PBS洗涤，加入载有治疗基因的磷酸钙纳米粒0.6μg～60μg和纤维粘结蛋白(FN)0.05μg～8μg，冷冻干燥得到基因传递系统的三维纳米支架。
本发明的基因传递系统的三维纳米支架在非病毒基因传递系统中的应用。
实验表明：应用本发明的基因传递系统的三维纳米支架的非病毒基因传递系统具有高效缓释的特性，其所负载的基因的表达可达到外加因子的水平。
本发明的有益之处是：本发明是在三维支架中加入如含有RGD序列的FN及磷酸钙-TGFβ1复合纳米粒，形成新型三维非病毒纳米基因传递系统，这种新型的传递系统具有高效缓释的特性，其所负载的基因的表达可达到外加因子的水平。为非病毒基因传递及组织工程修复方面提供了新的思路。
附图说明
图1自制载有治疗基因磷酸钙纳米粒的电镜图
图2为胶原-壳聚糖-FN三维支架扫描电镜图(不含载基因的磷酸钙纳米粒)。
图3为含有载基因磷酸钙纳米粒的三维支架扫描电镜图(箭头所示为复合纳米粒)。
图4为三维非病毒纳米基因传递系统接种细胞的扫描电镜图(箭头所示为细胞)。
图5为ELISA法测定三维纳米支架与游离DNA+支架在不同时间(3-15天)细胞蛋白表达浓度。(*P＞0.05，**P＜0.05，***P＜0.01)。
图6为ELISA法测定三维纳米支架与市售转染试剂脂质体lipfectamineTM2000 3-15天细胞表达的目的蛋白结果图(*P＞0.05，**P＜0.05，***P＜0.01)。
具体实施方式
本发明通过实施例作进一步的说明。
实施例1：载有治疗基因的磷酸钙纳米粒的制备
(1)制备：将0.1M Igepal CO-520溶于25ml环己烷，加入1.36M氯化钙，搅拌混匀形成微乳1；将0.1M Igepal CO-520，50μlTis-HCL(pH 7.4)溶于25ml环己烷，加入磷酸氢二钠和TGF-β1质粒，加入磷酸氢二钠的质量为3.4785μg，磷酸氢二钠与质粒的质量比为0.1∶1～10∶1，搅拌形成微乳2，将微乳2缓慢滴加到微乳1中，搅拌10min，形成载有基因的磷酸钙复合纳米粒的乳液。
(2)载有基因的磷酸钙复合纳米粒的分离：
(a)层析柱的预处理：
取90g硅球加入含有0.336mL氨基丙基三乙氧基硅烷(aminopropyltriethoxysilane，APS)，1.5mL冰乙酸，7.5μL双蒸水的150mL乙醇溶液中，搅拌过夜，70℃烘干备用；
(b)载有基因的磷酸钙复合纳米粒的分离：
将上述制得的载有基因的磷酸钙复合纳米粒乳液过硅胶层析柱，先用无水乙醇洗脱环己烷及游离的质粒和盐，然后用浓度为5×10-4mol/L的NaCl的70％乙醇溶液洗脱载基因的磷酸钙复合纳米粒；
(c)载有基因的磷酸钙复合纳米粒乙醇液的浓缩
将步骤2所得的含有载有基因的磷酸钙复合纳米粒的乙醇液在37℃旋转减压蒸发6h除去乙醇，浓缩液置于12KD的透析袋中，置于磷酸盐缓冲液中，4℃透析过夜，即得到载有基因的磷酸钙复合纳米粒。得到的载有基因的磷酸钙复合纳米粒的电镜照片见图1。
实施例2：一种作为对比的三维支架的制备
取1％胶原醋酸溶液0.1ml加入1％壳聚糖溶液0.1ml，冷冻干燥，加入0.25％戊二醛50μL交联，室温过夜，冷冻干燥，PBS洗涤三次，冷冻干燥，加入纤维粘结蛋白(FN)1μg，得到不含载有治疗基因的磷酸钙纳米粒的三维支架，其电镜照片见图2。
实施例3：本发明的三维纳米支架的制备
取1％胶原醋酸溶液0.1ml加入1％壳聚糖溶液0.1ml，冷冻干燥，加入0.25％戊二醛50μL交联，室温过夜，冷冻干燥，PBS洗涤三次，20min/每次，细胞培养基(Dulbecco’sModified Eagle Media，DMEM)浸泡过夜；PBS洗涤三次，20min/每次，加入实施例1制得的载有基因的磷酸钙复合纳米粒1μg和FN1μg，冷冻干燥得到本发明的三维纳米支架，其电镜照片见图3。
实施例4：本发明的三维纳米支架的制备
取1％胶原醋酸溶液0.1ml加入1％壳聚糖溶液10ml，冷冻干燥，加入0.25％戊二醛1ml交联，室温过夜，冷冻干燥，PBS洗涤三次，20min/每次，细胞培养基(Dulbecco’sModified Eagle Media，DMEM)浸泡过夜；PBS洗涤三次，20min/每次，加入实施例1制得的载有基因的磷酸钙复合纳米粒60μg和FN8μg，冷冻干燥得到本发明的三维纳米支架，其形貌和性能同实施例3制得的三维纳米支架。
实施例5：大鼠间质干细胞(mesenchymal stem cells，MSC)的培养
将SD大鼠拉颈处死，体积分数为75％乙醇浸泡3～5min，无菌条件下取出胫骨和股骨；将其两端干骺端切除，显露骨髓腔，用无菌注射器吸取适量PBS彻底冲洗骨髓腔；反复吹打冲出的骨髓，使骨髓细胞充分分散；所获骨髓单细胞悬液沿管壁缓慢滴加于预加的Percoll分离液(相对体积质量1.073)的离心管中，骨髓单细胞悬液与分离液的比例为1∶1；2000rpm，离心20min，吸取中间云雾状细胞层，用PBS洗涤3次；重新悬起细胞，加入完全培养基(含体积分数为10％胎牛血清的DMEM培养基)，置于培养瓶中，37℃体积分数为5％的CO2培养箱中培养。
实施例6：三维纳米支架的体外转染
(1)取适量实施例3制得的三维纳米支架置于24孔板中，将MSC按2×104/mL接种于支架上，加入完全培养基至500μL，置37℃、5％CO2培养箱中培养；分别在3、6、9、12、15d收集上清培养液于-20℃保存，细胞继续用完全培养基进行培养。接种有细胞的三维支架电镜照片见图4。
(2)对照组一：取适量的三维支架置于24孔板中，将MSC按2×104/mL接种于支架上，加入完全培养基至500μL，置37℃、5％CO2培养箱中培养；24h，吸弃前一天铺板的培养液，加入游离质粒以及无血清的DMEM培养液至500μL，置37℃、5％CO2培养箱中培养；分别在3、6、9、12、15d收集上清培养液于-20℃保存，细胞继续用完全培养基进行培养。
(3)对照组二：取适量的三维支架置于24孔板中，将MSC按2×104/mL接种于支架上，加入完全培养基至500μL，置37℃、5％CO2培养箱中培养；24h，吸弃前一天铺板的培养液，用PBS洗涤后，加入市售转染试剂脂质体lipfectamineTM2000以及无血清的DMEM培养液至500μL，继续培养6h；换用完全培养基继续培养。分别在3、6、9、12、15d收集上清培养液于-20℃保存，细胞继续用完全培养基进行培养。
实施例7：体外转染细胞的转染效率测定
收集细胞培养物上清，离心，加到酶标板中。分别设空白孔、标准孔、待测样品孔。除空白孔外，余孔分别加标准溶液或待测样品100ul，注意不要有气泡，轻轻混匀，酶标板加上盖，37℃反应120分钟。弃去液体，甩干，不用洗涤。每孔加检测溶液A工作液100ul，37℃，60分钟。洗板3次，350ul/每孔，甩干。每孔加检测溶液B工作液100ul，37℃，60分钟，洗板5次，甩干。依序每孔加底物溶液90ul，37℃避光显色30分钟(此时肉眼可见标准品的前3-4孔有明显的梯度兰色，后3-4孔梯度不明显)。依序每孔加终止溶液50ul，终止反应(此时兰色立转黄色)。用酶联免疫检测仪在450nm波长测量各孔的光密度(OD值)，测定目的蛋白表达量，其结果见图5和图6。
三维纳米支架在3-15天内，蛋白表达水平维持在10ng/ml左右，显著高于对照组一(游离DNA+三维支架)(P＜0.01)。从第6天开始，对照组二(市售转染试剂脂质体lipfectamineTM2000+三维支架)的蛋白表达水平远低于三维纳米支架(P＜0.05或P＜0.01)，说明嵌合有载有基因的磷酸钙复合纳米粒的三维纳米支架具有明显的长效、缓释特征。
Claims (3)
1.一种基因传递系统的三维纳米支架，它是由胶原、壳聚糖以及纤维粘结蛋白(Fibronectin，FN)组成的三维纳米支架，其特征是：它包含有载有基因的磷酸钙复合纳米粒，它是一种疏松多孔的三维纳米支架，所述的胶原、壳聚糖、纤维粘结蛋白和戊二醛的质量比为：1∶1∶0.0005∶0.0124～1∶100∶0.08∶0.24，所述的纤维粘结蛋白为含有天门冬氨酸序列的纤维粘结蛋白，所述的载有质粒的磷酸钙复合纳米粒是球形的、粒径小于50nm的载有治疗基因的磷酸钙纳米粒，其含量为三维纳米支架总质量的0.01％～1％。
2.一种制备权利要求1所述的基因传递系统的三维纳米支架的方法，其特征是：它是将质量百分浓度为1％的胶原醋酸溶液加入壳聚糖，使壳聚糖的质量百分浓度达到1％，冷冻干燥，加入0.25％，50μL戊二醛交联，室温过夜后再冷冻干燥，用缓冲液洗涤，加入载有治疗基因的磷酸钙纳米粒0.6μg～60μg和纤维粘结蛋白0.05μg～8μg，冷冻干燥得到基因传递系统的三维纳米支架。
3.根据权利要求1所述的基因传递系统的三维纳米支架在非病毒基因传递系统中的应用。
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| CN102154350A (zh) * | 2010-12-30 | 2011-08-17 | 江苏大学 | 阳离子化枸杞多糖纳米粒基因传递系统及其制法 |
| CN102949750A (zh) * | 2012-11-15 | 2013-03-06 | 中国人民解放军第三军医大学 | 双层电纺仿生骨膜及其制备 |
| CN110312788A (zh) * | 2016-06-21 | 2019-10-08 | 江苏大学 | 一种基于三维体系的细胞重编程方法 |
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| US20050025807A1 (en) * | 2003-07-31 | 2005-02-03 | National Institute Of Advanced Industrial Science And Technology | Cured porous calcium phosphate material and uses thereof |
| CN1820790A (zh) * | 2006-03-13 | 2006-08-23 | 西北大学 | 一种制备可生物降解的组织工程用支架材料的方法 |
| CN101062430A (zh) * | 2007-04-25 | 2007-10-31 | 韩春茂 | 胶原－壳聚糖/纤维蛋白胶不对称支架及其制备方法和应用 |
| CN101445806A (zh) * | 2008-12-31 | 2009-06-03 | 江苏大学 | 钙离子化介孔二氧化硅纳米粒基因传递系统及其制法 |
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| US20050025807A1 (en) * | 2003-07-31 | 2005-02-03 | National Institute Of Advanced Industrial Science And Technology | Cured porous calcium phosphate material and uses thereof |
| CN1820790A (zh) * | 2006-03-13 | 2006-08-23 | 西北大学 | 一种制备可生物降解的组织工程用支架材料的方法 |
| CN101062430A (zh) * | 2007-04-25 | 2007-10-31 | 韩春茂 | 胶原－壳聚糖/纤维蛋白胶不对称支架及其制备方法和应用 |
| CN101445806A (zh) * | 2008-12-31 | 2009-06-03 | 江苏大学 | 钙离子化介孔二氧化硅纳米粒基因传递系统及其制法 |
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| CN102154350A (zh) * | 2010-12-30 | 2011-08-17 | 江苏大学 | 阳离子化枸杞多糖纳米粒基因传递系统及其制法 |
| CN102949750A (zh) * | 2012-11-15 | 2013-03-06 | 中国人民解放军第三军医大学 | 双层电纺仿生骨膜及其制备 |
| CN102949750B (zh) * | 2012-11-15 | 2014-11-05 | 中国人民解放军第三军医大学 | 双层电纺仿生骨膜及其制备 |
| CN110312788A (zh) * | 2016-06-21 | 2019-10-08 | 江苏大学 | 一种基于三维体系的细胞重编程方法 |
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