CN110917397A - 基于病毒载体投递MicroRNA与骨粉的复合材料及其制备方法 - Google Patents

基于病毒载体投递MicroRNA与骨粉的复合材料及其制备方法 Download PDF

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CN110917397A
CN110917397A CN201811096957.5A CN201811096957A CN110917397A CN 110917397 A CN110917397 A CN 110917397A CN 201811096957 A CN201811096957 A CN 201811096957A CN 110917397 A CN110917397 A CN 110917397A
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microrna
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赵瑾
李学平
韩星
战琦
白钰
侯信
原续波
杨贤金
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Abstract

本发明提供基于病毒载体投递MicroRNA与骨粉的复合材料及其制备方法，MicroRNA病毒载体复合体与骨粉进行混合，经真空低温冻干，得到基于病毒载体投递MicroRNA与骨粉的复合材料。与现有技术相比，本发明选用microRNA与病毒类载体复合物与Bio‑Oss骨粉相结合，填补了临床上单独使用骨粉带来的缺陷，赋予骨粉骨生成性和诱导性，而且microRNA在牙槽骨愈合治疗上的应用病不多见，既可提高牙槽骨的成骨质量，又可缩短成骨时间，给临床带来又一种治疗牙槽骨萎缩的手段。

Description

基于病毒载体投递MicroRNA与骨粉的复合材料及其制备方法
技术领域
本发明涉及生物医用组织工程材料技术领域，更具体地说涉及一种基于病毒载体投递MicroRNA与骨粉的复合材料及其制备方法。
背景技术
随着物质生活水平的提高，人类的饮食逐渐变得多样化。由龋齿、牙龈炎、牙周炎等口腔疾病，骨质疏松以及糖尿病等带来的并发症，往往会造成牙齿松动甚至脱落。为解决该问题，目前医学上多采用可摘义齿、烤瓷牙技术或牙齿种植术对牙列进行填补修复。人工种植牙将种植体植入牙槽骨中充当牙根，不仅增加了假牙的固定性，不易磨损邻近牙齿，还能更好的恢复口腔咀嚼功能，美观舒适，故而口腔种植技术逐渐受到人们关注。而由于口腔疾病引起的牙槽嵴萎缩，致使牙槽骨骨量不足，往往会使种植牙的植入难度加大甚至无法实施。因此，为有效植入种植牙，牙槽骨骨量不足便成为我们亟待解决的问题。
现今医学上广泛应用
Figure BDA0001805786210000011
骨粉对牙槽窝进行填充。
Figure BDA0001805786210000012
骨粉是经灭菌的孔隙率75％至80％且晶体颗粒大小约10μm的脱蛋白牛骨材料，其保持了多孔天然骨的无机结构，具有与人体牙槽骨相似的物理化学结构和抗压强度，且生物相容性较好。刘慧颖等对4只杂种犬双侧下颌牙槽窝内进行
Figure BDA0001805786210000013
骨粉填充，而后覆盖钛膜，实验发现拔牙窝内填塞
Figure BDA0001805786210000014
骨粉可对拔牙创处的牙槽骨产生较好的引导作用，并使得剩余牙槽嵴的吸收量减少。然而，一些研究表明，其成骨细胞增殖和分化情况低于自体骨，且不能完全再吸收。由此说明
Figure BDA0001805786210000015
骨粉缺乏活性因子，使其缺乏骨生成性和骨诱导性，因而限制了其在临床上的大量应用。
MicroRNA作为近些年新发现的一种可特异性调控基因表达的非编码小分子RNA，具有高效性、低毒性等显著优势，因而在骨组织工程中备受关注。MicroRNA，又称微小RNA，长度约为22个核苷酸，参与机体包括干细胞增殖分化在内的大部分生物过程。MicroRNA通过结合靶mRNA的非翻译区特异性识别靶mRNA，控制mRNA及相应蛋白质的水平，以调控靶细胞的增殖分化过程。因此，MicroRNA作为可调节多个靶基因的内源性治疗分子，在调节干细胞自我更新和分化过程中起着极为重要的作用。
但是MicroRNA链段带负电、半衰期短，无法自主透过细胞膜渗透进入细胞内，同时容易被细胞内的溶酶体降解。为使microRNA能够有效导入靶细胞内部并稳定存在，同时能够有效表达和发挥作用，我们需要选择合适的载体来进行投递。常用载体有两种：病毒载体及非病毒载体。
病毒载体作为目前临床上应用较多的基因载体，包括逆转录病毒、腺病毒、慢病毒等。病毒载体的作用机理是将病毒基因组中的致病基因去除，保留其可携带基因组进入机体细胞的功能，并将目的基因组装进病毒载体的基因组内。经过目的基因修饰的病毒载体失去了自我复制的功能，同时减少了病毒载体的毒性，但进入人体细胞后，病毒载体负载的目的基因可进行表达，以使靶细胞向成骨细胞分化。病毒载体能够较为稳定的整合进靶细胞内、表达基因较为持久、装载基因容量较大且转染率较高。
金丹以逆转录病毒为载体，对兔的骨髓基质干细胞(BMSc)进行hBMP基因的转染，并检测相应骨形态发生蛋白的表达。组织学结果表明使用逆转录病毒介导hBMP基因转染后的BMSc中碱性磷酸酶的含量明显增加，说明该种方法可以诱导BMSc向成骨细胞分化。Chang等以腺病毒为载体，将血小板衍生生长因子(AdPDGF-B)基因递送进大鼠牙槽骨骨缺损区，组织学结果表明，AdPDGF-B被高效地递送至局部的骨缺损处，且并未发现邻近器官受损。马雷从实验犬体内获得了骨髓间充质干细胞(BMSCs)，而后以经bFGF修饰后的慢病毒转染诱导第三代BMSCs，体外观察该载体对BMSCs的转染效率及生物学活性，与此同时进行了体内实验，观察利用bFGF-慢病毒载体修饰的骨髓间充质干细胞向成骨细胞分化的情况。组织学检测结果表明，以慢病毒为载体递送的bFGF基因在BMSCs中得到了较高程度的表达，同时细胞存活率较高。动物实验也证明了该种载体在生物体内未出现免疫、炎症等不良反应。
发明内容
本发明克服了现有技术中的不足，提供了一种基于病毒载体投递MicroRNA与骨粉的复合材料及其制备方法，以
Figure BDA0001805786210000021
骨粉为支架材料，保持了多孔的天然骨无机结构，有着与人牙槽骨相似的物化结构和力学性能，且生物相容性较好，添加以病毒为载体的microRNA-21用于促进牙槽骨部位骨的快速再生，既利用了已应用于临床的骨支架的位点保留功能，又弥补了无机骨的无活性的缺点。
本发明的目的通过下述技术方案予以实现。
基于病毒载体投递MicroRNA与骨粉的复合材料，MicroRNA病毒载体复合体与骨粉进行混合，经真空低温冻干，得到基于病毒载体投递MicroRNA与骨粉的复合材料；其中，骨粉与MicroRNA病毒载体复合体溶液的体积比为(1-100)：1。
MicroRNA为MicroRNA-21、MicroRNA-29b或者MicroRNA-26a。
病毒载体采用能够投递microRNA的病毒类载体，如逆转录病毒、腺病毒、慢病毒等。
骨粉为具有占位及支架作用的骨粉，如Bio-oss骨粉，直径为0.25-1mm。
骨粉与MicroRNA病毒载体复合体溶液的体积比为(40-60)：1。
真空低温冻干的时长为10-15h。
基于病毒载体投递MicroRNA与骨粉的复合材料的制备方法，将携带microRNA的病毒载体加入900-1200μL的水中稀释，将上述溶液逐滴加入到骨粉颗粒中混合均匀，滴加速率为3-5滴/min，经真空低温冻干，即得到基于病毒载体投递MicroRNA与骨粉的复合材料，将上述材料储存于-90--70℃环境下备用。
水为去氧无RNA酶的双蒸水。
MicroRNA为MicroRNA-21、MicroRNA-29b或者MicroRNA-26a。
病毒载体采用能够投递microRNA的病毒类载体，如逆转录病毒、腺病毒、慢病毒等。
骨粉为具有占位及支架作用的骨粉，如Bio-oss骨粉，直径为0.25-1mm。
骨粉与MicroRNA病毒载体复合体溶液的体积比为(1-100)：1，优选(40-60)：1。
真空低温冻干的时长为10-15h。
本发明的有益效果为：与现有技术相比，本发明选用microRNA与病毒类载体复合物与Bio-Oss骨粉相结合，填补了临床上单独使用骨粉带来的缺陷，赋予骨粉骨生成性和诱导性，而且microRNA在牙槽骨愈合治疗上的应用病不多见，既可提高牙槽骨的成骨质量，又可缩短成骨时间，给临床带来又一种治疗牙槽骨萎缩的手段。
附图说明
图1是用荧光显微镜观察的以病毒为载体的microRNA转染间充质干细胞的荧光图；
图2是以病毒为载体的microRNA及其与骨粉复合后的细胞毒性测试图；
图3是以病毒为载体的microRNA与骨粉复合后刺激细胞后，钙钴法染色观察碱性磷酸酶活性图；
图4是以病毒为载体的microRNA与骨粉复合材料促成骨图。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的说明。
除非特殊说明，溶液即为水溶液，如MicroRNA模拟物溶液为MicroRNA模拟物水溶液。实施例中使用的药品为市购药品，MicroRNA-21、29b、26a，病毒载体均购自上海吉玛制药技术有限公司，骨粉为Bio-oss骨粉，购自瑞斯美口腔医疗器械(北京)有限公司。
实施例1
制备microRNA病毒类载体复合物与骨粉的复合材料，MicroRNA选用microRNA-21，与直径为1mm的Bio-oss骨粉，将Bio-oss骨粉与microRNA-21病毒类载体复合物溶液稀释液按体积比为50:1进行混合，冻干12h后取出备用(采用冻干常采用的低温即可)，将上述材料储存于-80℃环境下备用。
制备MicroRNA病毒类载体复合物—骨粉复合材料及其细胞层次成骨诱导效果的验证
选取实施例1制备的microRNA-21病毒类载体复合物与骨粉的复合材料。microRNA-21病毒类载体复合物具有诱导干细胞向成骨分化的能力，但是为了在临床应用中使其具有更好的治疗效果，我们将microRNA-21病毒类载体复合物溶液与Bio-oss骨粉复合，形成既为成骨留有空间又有骨诱导效果的复合材料。图一是microRNA-21病毒类载体复合物转染细胞的荧光显微图，用DAPI将干细胞的细胞核染成蓝色，microRNA-21用FAM荧光标记成黄绿色，microRNA-21病毒类载体复合物与干细胞共孵育4-12h。由图片我们可以看到，microRNA-21病毒类载体复合物可以高效率地转染干细胞。图二是microRNA-21病毒类载体复合物与骨粉的复合材料与脐带血间充质干细胞共培养后的细胞活性分析。选用最常用的MTT法检测细胞活性，由图我们可以看到实验组和空白对照组的细胞活性都在85％以上，说明本发明制备的复合材料细胞毒性很低。图三是microRNA-21病毒类载体复合物与骨粉的复合材料刺激脐带血间充质干细胞7天后，检测细胞中碱性磷酸酶活性的图片，由南京建成生物技术有限公司成产的改良钙钴法碱性磷酸酶试剂盒处理后的细胞，碱性磷酸酶活性高的区域被染成黑色块状或条状，显然microRNA-21病毒类载体复合物与骨粉的复合材料组碱性磷酸酶活性要比空白对照组高很多。
制备MicroRNA病毒类载体复合物—骨粉复合材料动物学实验表征成骨情况
选取实施例1制备的microRNA-21病毒类载体复合物与骨粉的复合材料。建立体内研究纳米微囊促进牙槽骨愈合的动物模型，先将大白兔的下切齿拔出，填入成功制备的miRNA-21病毒类载体复合物与骨粉颗粒的复合物，缝合后分笼喂养2周、4周、8周后取材进行后续的表征。由铂悦仪器(上海)有限公司生产的micro-CT机测定离体下颌骨的牙槽窝处新生骨占本体骨的比例，如下表所示，本发明中制备的复合材料在促进骨再生方面尤其是牙槽骨快速愈合方面，有出色的效果。之后我们进行了硬组织切片实验，图四是2周是骨粉组(左)与复合物组(右)新生骨的对比图，从图中可以看出复合物组新生骨成骨量更多，治疗效果更好。
时间 2周 4周 8周
新生骨占比(％) 35％ 53％ 72％
依照发明内容和上述实施例的记载，变更MicroRNA的种类，以及各个病毒类载体的种类进行制备，均可实现基于病毒载体投递MicroRNA与骨粉的复合材料且性质基本一致。
实施例2
制备microRNA病毒类载体复合物与骨粉的复合材料，MicroRNA选用microRNA-29b，与直径为0.25mm的Bio-oss骨粉，将Bio-oss骨粉与microRNA-29b病毒类载体复合物溶液稀释液按体积比为40:1进行混合，冻干10h后取出备用(采用冻干常采用的低温即可)，将上述材料储存于-70℃环境下备用。
实施例3
制备microRNA病毒类载体复合物与骨粉的复合材料，MicroRNA选用microRNA-26a，与直径为0.5mm的Bio-oss骨粉，将Bio-oss骨粉与microRNA-26a病毒类载体复合物溶液稀释液按体积比为60:1进行混合，冻干15h后取出备用(采用冻干常采用的低温即可)，将上述材料储存于-90℃环境下备用。
实施例4
制备microRNA病毒类载体复合物与骨粉的复合材料，MicroRNA选用microRNA-21，与直径为0.75mm的Bio-oss骨粉，将Bio-oss骨粉与microRNA-21病毒类载体复合物溶液稀释液按体积比为100:1进行混合，冻干11h后取出备用(采用冻干常采用的低温即可)，将上述材料储存于-85℃环境下备用。
以上对本发明做了示例性的描述，应该说明的是，在不脱离本发明的核心的情况下，任何简单的变形、修改或者其他本领域技术人员能够不花费创造性劳动的等同替换均落入本发明的保护范围。

Claims (10)

1.基于病毒载体投递MicroRNA与骨粉的复合材料，其特征在于：MicroRNA病毒载体复合体与骨粉进行混合，经真空低温冻干，得到基于病毒载体投递MicroRNA与骨粉的复合材料；其中，骨粉与MicroRNA病毒载体复合体溶液的体积比为(1-100)：1。
2.根据权利要求1所述的基于病毒载体投递MicroRNA与骨粉的复合材料，其特征在于：MicroRNA为MicroRNA-21、MicroRNA-29b或者MicroRNA-26a；病毒载体采用能够投递microRNA的病毒类载体，如逆转录病毒、腺病毒、慢病毒等；骨粉为具有占位及支架作用的骨粉，如Bio-oss骨粉，直径为0.25-1mm。
3.根据权利要求1所述的基于病毒载体投递MicroRNA与骨粉的复合材料，其特征在于：骨粉与MicroRNA病毒载体复合体溶液的体积比为(40-60)：1。
4.根据权利要求1所述的基于病毒载体投递MicroRNA与骨粉的复合材料，其特征在于：真空低温冻干的时长为10-15h。
5.制备如权利要求1-4任一所述的基于病毒载体投递MicroRNA与骨粉的复合材料的方法，其特征在于：将携带microRNA的病毒载体加入900-1200μL的水中稀释，将上述溶液逐滴加入到骨粉颗粒中混合均匀，滴加速率为3-5滴/min，经真空低温冻干，即得到基于病毒载体投递MicroRNA与骨粉的复合材料，将上述材料储存于-90--70℃环境下备用，其中，骨粉与MicroRNA病毒载体复合体溶液的体积比为(1-100)：1。
6.根据权利要求5所述的基于病毒载体投递MicroRNA与骨粉的复合材料的制备方法，其特征在于：水为去氧无RNA酶的双蒸水；MicroRNA为MicroRNA-21、MicroRNA-29b或者MicroRNA-26a；病毒载体采用能够投递microRNA的病毒类载体，如逆转录病毒、腺病毒、慢病毒等；骨粉为具有占位及支架作用的骨粉，如Bio-oss骨粉，直径为0.25-1mm。
7.根据权利要求5所述的基于病毒载体投递MicroRNA与骨粉的复合材料的制备方法，其特征在于：骨粉与MicroRNA病毒载体复合体溶液的体积比为(40-60)：1。
8.根据权利要求5所述的基于病毒载体投递MicroRNA与骨粉的复合材料的制备方法，其特征在于：真空低温冻干的时长为10-15h。
9.如权利要求1-4任一所述的基于病毒载体投递MicroRNA与骨粉的复合材料在制备治疗骨愈合药物中的应用。
10.根据权利要求9所述的应用，其特征在于：基于病毒载体投递MicroRNA与骨粉的复合材料能够持续释放活性因子—以病毒类载体投递的MicroRNA；赋予骨粉骨生成性和诱导性，既可提高牙槽骨的成骨质量，又可缩短成骨时间。
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