WO2017219232A1 - 一种基于三维体系的细胞重编程方法 - Google Patents

一种基于三维体系的细胞重编程方法 Download PDF

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WO2017219232A1
WO2017219232A1 PCT/CN2016/086536 CN2016086536W WO2017219232A1 WO 2017219232 A1 WO2017219232 A1 WO 2017219232A1 CN 2016086536 W CN2016086536 W CN 2016086536W WO 2017219232 A1 WO2017219232 A1 WO 2017219232A1
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scaffold
dimensional
polysaccharide
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calcium phosphate
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徐希明
邓纹纹
余青桐
余江南
曹霞
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Jiangsu University
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  • the invention relates to the field of biotechnology and tissue engineering, relates to a gene delivery system, and in particular to the establishment of a cell reprogramming method based on a three-dimensional system.
  • Biomimetic Collagen nanofibrous materials for bone tissue engineering [J].Advanced Engineering Materials,2010,12(9):B451-B466;Han,J.,Chen,L.,Luo,G.,Dai,B.,Wang,X. , Dai, J. Three-dimensional culture may promote cell reprogramming [J]. Organogenesis, 2013, 9(2): 118-120.].
  • the extracellular matrix typically includes collagen and laminin interlaced with the heparan sulfate proteoglycan.
  • laminin and heparan sulfate proteoglycans are distributed on the cell surface at the stage of two cells; while expression of type IV collagen and fibronectin can be detected in the cell cluster stage of blastocysts.
  • ECMs have multiple functions for cell growth, differentiation, and maintenance of tissue morphology. Cells interact with ECM via surface receptors (such as integrins), acting as a mechanism for mechanical riveting and cell membrane signaling.
  • surface receptors such as integrins
  • the invention establishes a novel and efficient non-viral three-dimensional gene delivery system, and is first applied to the induction of iPSCs in a three-dimensional environment.
  • the invention relates to a cationized Pleurotus eryngii polysaccharide, a calcium phosphate nanoparticle and a three-dimensional collagen scaffold, and the cationized Pleurotus eryngii polysaccharide modified calcium phosphate nanoparticle by a reverse microemulsion method, further improving the gene carrying of the non-viral vector Capacity; the polysaccharide-calcium phosphate-loaded gene nanoparticles were fused with a three-dimensional collagen scaffold to form a three-dimensional induction system of iPSCs.
  • the invention applies the three-dimensional system to the reprogramming research of human umbilical cord stem cells, evaluates the cell transfection efficiency and the cell reprogramming effect, and sets the two-dimensional gene delivery system under the same conditions as a positive control, and the experimental results show that the three-dimensional system indicates the three-dimensional system.
  • the gene delivery system is significantly superior to the two-dimensional gene delivery system in gene transfection efficiency; in addition, human-derived iPSCs induced by three-dimensional system appear iPSCs cell spheres on the 4th day after cell inoculation, and then gradually form embryoid body cells. ball.
  • HE staining, laser confocal microscopy, immunohistochemistry, karyotype analysis, and intratumoral tumors were identified, indicating that human iPSCs induced by three-dimensional system have the potential of whole germ layer differentiation similar to embryonic stem cells.
  • the present invention employs a chemical modification method to provide a highly efficient and non-toxic three-dimensional gene delivery system for reprogramming human umbilical cord stem cells (HUMSCs) into human induced pluripotent stem cells (hiPSCs).
  • HUMSCs human umbilical cord stem cells
  • hiPSCs human induced pluripotent stem cells
  • a cell reprogramming method based on a three-dimensional system specifically comprising the following steps:
  • Step 4 Expression of the Yamanaka factor in a three-dimensional system:
  • Step 5 Induction and amplification of iPSCs in a three-dimensional system:
  • Collagen scaffolds prepared from natural I collagen provide a safe and stable three-dimensional culture environment for HUMSCs.
  • Polysaccharide-calcium phosphate-loaded gene nanoparticles have good gene carrying capacity and are evenly distributed in three-dimensional scaffolds for HUMSCs to iPSCs.
  • Targeted reprogramming provides ample source of foreign genes and is capable of long-term release.
  • the expression of exogenous genes in the three-dimensional system was significantly higher than that of the two-dimensional system by RT-PCR.
  • the results of iPSCs showed that the human iPSCs formed in the three-dimensional system have the ability to differentiate into the whole germ layer of embryonic stem cells. Stable amplification in vitro, passage to more than 20 generations, and can maintain a normal karyotype. This method provides a new three-dimensional environment for the induction of iPSCs, and provides a theoretical basis for the application of iPSCs from cell level to tissue level.
  • Figure 1 is a flow diagram of cell reprogramming in a three dimensional system.
  • Figure 2 is a scanning electron micrograph of a three-dimensional gene-loaded nanoparticle-collagen scaffold.
  • Figure 3 shows the morphological changes of HUMSCs in a three-dimensional gene-loaded nanoparticle-collagen scaffold under an optical microscope.
  • Figure 4 is a three-dimensional gene-loaded nanoparticle-collagen scaffold and HE staining of iPSCs therein.
  • Figure 5 shows the immunohistochemistry of iPSCs.
  • Figure 6 is a diagram of cell karyotype
  • Figure 7 shows the pluripotency of iPSCs by immunofluorescence staining.
  • Figure 8 shows the degree of methylation of the Oct4 and Nanog promoters, wherein hESCs are human embryonic stem cells.
  • Figure 9 shows the differentiation of the three germ layers of iPSCs cell spheres in vivo, where: g represents the stroma structure, which is the characteristic of endoderm, b and c represent the bone tissue and cartilage tissue, respectively, which is the characteristic of mesoderm differentiation, and n represents the neural tube. Germ marking.
  • cationic polysaccharide D-Sp LB medium (homemade); type I-collagen, type IV collagenase, agarose, ethidium bromide, L-glutamine, MINI26-1KT, biotinylated IgG (American sigma); trypsin, Tris base, ampicillin, streptomycin, DAB display kit (Biyuntian Biotechnology Research Institute); basic fibroblast growth factor (bFGF) (PeproTech, USA); plasmid extraction kit (Promega, USA); calcium chloride (National Pharmaceutical Group Chemical Reagent Co., Ltd.); MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide), PicoGreen dsDNA quantification Kit (Invitrogen, USA); basal medium DMEM, basal medium DMEM/F12, knockout DME, fetal bovine serum (FBS), knockout serum replacement (GIBCO, USA); Oligo
  • DH5 ⁇ glycerol bacteria Najing Jitian Biological
  • human umbilical cord mesenchymal stem cells Jiangsu Beike Biotechnology Co., Ltd.
  • mouse embryonic fibroblasts Shanghai Chinese Academy of Sciences stem cell bank
  • PEI Fat Diabetes/Severe Combined Immunodeficiency Mice (Center for Comparative Medicine, Yangzhou University)
  • plasmid non-viral plasmid OCT4, SOX2, KLF4 and CMYC (Guangzhou Fueneng Gene Co., Ltd.)
  • OCT4 upstream primer TGGAGGTGAT GGGTTAGG 18
  • OCT4 downstream primer CATCAAACTA CCCTATCATA ACC 23
  • NANOG upstream primer GTGAATGAAA GAGGAAAATG GAG 23
  • NANOG downstream primer AATAACCCAC CCCTATAATC C 21
  • PBS solution take NaCl 8.00g, KCl 0.20g, Na 2 HPO 4 .12H2O 3.5g, KH 2 PO 4 0.2g, add 800mL double distilled water, adjust the pH to 7.4, dilute to 1000mL, dispense, Autoclaved, stored at 4 ° C.
  • Knockout DMEM medium was sequentially added with 20% serum substitute (Knockout SR), 2 mmol/L L-glutamine, 0.1 mmol/L ⁇ -mercaptoethanol, 1% non-essential amino acid, 100 U/ mL qing-streptomycin solution, 4 ng / mL human recombinant basic fibroblast growth factor, mix, placed in a refrigerator at 4 ° C for use.
  • Kerckout DMEM medium was sequentially added with 20% serum substitute (Knockout SR), 2 mmol/L L-glutamine, 0.1 mmol/L ⁇ -mercaptoethanol, 1% non-essential amino acid, 100 U/ mL qing-streptomycin solution, 4 ng / mL human recombinant basic fibroblast growth factor, mix, placed in a refrigerator at 4 ° C for use.
  • Example 1 Preparation of a three-dimensional collagen scaffold:
  • Igepal CO-520 was dissolved in cyclohexane to prepare an Igepal CO-520 29% Igepal CO-520/cyclohexane mixture;
  • microemulsion A Take a clean conical flask, add 25mL Igepal CO-520/cyclohexane mixture, then 650 ⁇ L 0.01M calcium chloride solution and 0.8mg cationic polysaccharide D-Sp, magnetically stirred Under the conditions, together with the addition to the net conical flask, continue to stir for 2min, forming microemulsion A;
  • microemulsion B Take another clean conical flask, add 25mL Igepal CO-520/cyclohexane mixture, then add 650 ⁇ L of 0.06M disodium hydrogen phosphate and plasmid mixed solution 10 ⁇ L (including Oct4, Sox2) Klf4, c-Myc each 2.5 ⁇ g) together into the net conical flask, continue to stir for 2min, forming microemulsion B;
  • microemulsion A was added dropwise to the microemulsion B while stirring, until the whole system was transparent and clear, and the polysaccharide-calcium phosphate hybrid nanoparticle microemulsion was obtained;
  • each well containing the inoculated cell scaffold was added DMEM medium containing 10% FBS, and continued to culture for 4 h;
  • paraffin wax After the paraffin wax is completely immersed in the stent, it is embedded: first prepare the container (such as folding a small carton), pour the melted paraffin, quickly pick up the saturated paraffin tissue block, and cool it into a block. Yes;
  • the embedded wax block is fixed on a microtome and cut into thin slices, generally 5 to 8 ⁇ m thick.
  • the cut slices tend to wrinkle, and should be placed in heated water to be flattened, then attached to a glass slide, and dried in a 45 ° C incubator.
  • Sections were subjected to conventional dewaxing and hydration, and a part of the sections were used for alkaline phosphatase (AP) staining;
  • AP alkaline phosphatase
  • colchicine treatment add 24 ⁇ L to the cell culture flask 3 h before terminating the cell culture Colchicine, the final concentration of 0.8 ⁇ g / mL, and then returned to the incubator for 72h;
  • hypotonic treatment add 1mL 0.075mol / L KCl hypotonic solution to the centrifuge tube, gently pipet with a straw, so that the cells are evenly suspended in the hypotonic solution, add 6mL 0.075mol / L KCl hypotonic solution, Placed in a constant temperature water bath at 37 ° C, allowed to stand for 20 min, so that the cells swell and chromosomes dispersed;
  • Dropping tablets Add about 1 mL of the appropriate amount of fixative solution to the sediment, gently pipe the suspension cells with a pipette, and pipette the cell suspension. Drip 2 to 3 drops of water at a certain height and pre-soak clean. On the slide, immediately blow it away in one direction with the mouth, dry it several times on the alcohol, dry it, and mark each piece;
  • Dyeing Place the carrier piece with the cell suspension in the dyeing tank, so that there is a certain gap between the piece and the piece. Dip the diluted Giemsa dyeing solution into the dyeing tank, dye it for 20 minutes, take it out and rinse it with tap water. dry;
  • Genomic DNA extraction and quality inspection genomic DNA extraction and OD were performed using a special gDNA extraction kit, three cell samples (human umbilical cord mesenchymal stem cells, iPSCs induced by the experiment, and human embryonic stem cell line HN4). Concentration and agarose gel electrophoresis were determined.
  • Methylation treatment of qualified DNA 500 ng to 2 ⁇ g of gDNA was subjected to methylation treatment using a specialized methylation kit in strict accordance with the instructions to obtain 10 ⁇ L of methylated DNA.
  • Specific methylation procedure Add 20 ⁇ L of gDNA (500 ng to 2 ⁇ g) to 130 ⁇ L of CT solution (a reagent in the kit), mix gently, and operate according to the following procedure: 98 ° C, 10 min; 64 ° C, 2.5 h; 4 ° C, hold.
  • PCR amplification and recovery and purification PCR amplification was carried out using methylated DNA as a template.
  • the first round of PCR system (total volume 25 ⁇ L):
  • the first round of PCR amplification procedures 95 ° C for 3 min; 95 ° C for 30 sec, 53 ° C for 30 sec, 72 ° C for 30 sec, for 40 cycles; followed by 72 ° C for 5 min, the end.
  • the second round of PCR amplification procedures 95 ° C for 3 min; 95 ° C for 30 sec, 53 ° C for 30 sec, 72 ° C for 30 sec, 40 cycles; followed by 72 ° C for 5 min, the end.
  • the PCR product was recovered and purified: the second round of the PCR product was subjected to gel recovery and purification, and the concentration was determined by OD.
  • TA cloning The PCR purified product was cloned into T vector by T vector kit, transformed with DH5 ⁇ competent cells, plated, and cultured at 37 ° C overnight (see the T vector kit for specific procedures)
  • Sequencing analysis of the sequencing results First, the sequence alignment software was used for sequencing analysis, and then methylation analysis software was used to analyze the methylation analysis of the correct sequencing results, and the information of the methylation number of the CpG island was counted. As can be seen from Fig. 8, demethylation was evident in the OCT4 and NANOG promoter regions of iPSCs, and the degree of methylation was similar to that of the positive control human embryonic stem cells, but significantly different from the original human umbilical cord mesenchymal stem cells. This result further demonstrates the successful reprogramming of human umbilical cord mesenchymal stem cells in a three-dimensional gene-loaded nanoparticle-collagen scaffold.
  • iPSCs were digested and centrifuged, a cell suspension of 5 ⁇ 10 6 /mL was prepared and inoculated into the abdomen of a 5-week-old male NOD-SCID nude mouse, and then tumor growth was observed. After 5 to 8 weeks, the mice were sacrificed and the tumors were removed for HE staining analysis.
  • iPSCs cell spheres can successfully form teratomas in immunodeficient NOD-SCID nude mice, and teratomas show significant trigeminal differentiation, respectively, with endoderm glandular tissue, mesoderm Tissues such as bone and cartilage tissues and neural tubes of the ectoderm are representative. This result fully demonstrates that the three-dimensional gene-loaded nanoparticle-collagen scaffold can be successfully applied to cell reprogramming.

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Abstract

一种基于三维体系的细胞重编程方法，以阳离子杏鲍菇多糖D-Sp、磷酸钙以及编码Yamanaka因子的4种质粒为原料，采用反相微乳法制备了多糖-磷酸钙混杂纳米粒并吸附到空白胶原支架内表面，得到三维载基因纳米粒-胶原支架；将人脐带间充质干细胞接种于三维载基因纳米粒-胶原支架进行重编程，形成诱导多能干细胞细胞球。

Description

一种基于三维体系的细胞重编程方法 技术领域
本发明涉及生物技术和组织工程领域，涉及基因传递系统，具体涉及一种基于三维体系的细胞重编程方法的建立。
发明背景
通常，科学家们在二维(2D)体系中进行多能性干细胞的自我更新以及多向分化潜能的研究。但科学研究发现，与2D环境相比，三维(3D)体系更能够模拟体内组织器官天然的微环境。在体内胚胎发育的过程中，细胞不仅有细胞之间的相互作用，还有细胞与细胞外基质(ECM)之间的作用[参考：Zheng,W.,Zhang,W.,Jiang,X.Biomimetic collagen nanofibrous materials for bone tissue engineering[J].Advanced Engineering Materials,2010,12(9):B451-B466；Han,J.,Chen,L.,Luo,G.,Dai,B.,Wang,X.,Dai,J.Three-dimensional culture may promote cell reprogramming[J].Organogenesis,2013,9(2):118-120.]。细胞外基质通常包括胶原蛋白以及与硫酸类肝素蛋白多糖相交错的层粘连蛋白。例如，在小鼠胚胎中，在两个细胞的阶段，层粘连蛋白与硫酸类肝素蛋白多糖分布于细胞表面；而在胚泡内细胞团阶段，能够检测到IV型胶原和纤维连接蛋白的表达。这些ECM对于细胞生长、分化以及组织形态的保持具有多重功能。细胞通过表面受体(如整合素)与ECM进行接触，起到对细胞机械铆定和细胞膜信号传导的作用。越来越多的学者将兴趣转移到了模拟体内3D环境的生物材料的开发以及对ECM在调节干细胞行为等方面的研究[参考：Wang,K.,Kievit,F.M.,Florczyk,S.J.,Stephen,Z.R.,Zhang,M.3d porous chitosan–alginate scaffolds as an in vitro model for evaluating nanoparticle-mediated tumor targeting and gene delivery to prostate cancer[J].Biomacromolecules,2015,16(10):3362-3372.]。目前，已经有许多研究集中于3D环境中多能性干细胞的扩增、定向分化以及体内移植[参见：Leisten,I.,Kramann,R.,Ventura Ferreira,M.S.,Bovi,M.,Neuss,S.,Ziegler,P.,Wagner,W.,Knüchel,R.,Schneider,R.K.3d co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche[J].Biomaterials,2012,33(6):1736-1747；Wang,X.,Liu,S.,Zhao,Q.,Li,N.,Zhang,H.,Zhang,X.,Lei,X.,Zhao,H.,Deng,Z.,Qiao,J.Three-dimensional hydrogel scaffolds facilitate in vitro self-renewal of human skin-derived precursors[J].Acta Biomaterialia,2014,10(7):3177–3187；Elangovan, S.,D'Mello,S.R.,Hong,L.,Ross,R.D.,Allamargot,C.,Dawson,D.V.,Stanford,C.M.,Johnson,G.K.,Sumner,D.R.,Salem,A.K.The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor[J].Biomaterials,2014,35(2):737-747.]。但是，迄今还没有文献进行报道在3D环境中进行多能性干细胞的诱导生成。本发明建立了一种全新的、高效的非病毒三维基因传递系统，并首次应用于三维环境下iPSCs的诱导。
发明内容
本发明涉及阳离子化杏鲍菇多糖，磷酸钙纳米粒以及三维胶原支架三部分，通过反向微乳法以阳离子化杏鲍菇多糖修饰磷酸钙纳米粒，进一步提高其作为非病毒载体的基因携载能力；将多糖-磷酸钙载基因纳米粒与三维胶原支架融合，形成了iPSCs的三维诱导体系。本发明将该三维体系应用于人源脐带干细胞的重编程研究，对细胞转染效率、细胞重编程效果进行评价，同时设置相同条件下的二维基因传递系统作为阳性对照，实验结果表明该三维基因传递系统在基因转染效率方面显著优于二维基因传递系统；此外，通过三维体系诱导生成的人来源iPSCs自细胞接种后第4天开始出现iPSCs细胞球，此后逐渐形成拟胚体状细胞球。经HE染色，激光共聚焦，免疫组化，染色体核型分析，体内崎胎瘤等手段鉴定，表明经三维体系诱导生成的人源iPSCs具有类似于胚胎干细胞的全胚层分化潜能。
本发明采用了化学修饰的方法，提供了一种将人源脐带干细胞(HUMSCs)定向重编程为人源诱导多能干细胞(hiPSCs)的高效无毒的三维基因传递系统。
本发明的技术方案如下：
一种基于三维体系的细胞重编程方法，具体包括如下步骤：
步骤1.胶原支架的制备：
取适量2～5％胶原溶液于支架制备的聚四氟乙烯模具中(68×38×5mm3)，先在4℃放置0.5～1h，再迅速放在-80℃冰箱预冻3h以上,然后冻干；于烘箱内热固化8～10h之后，用无水乙醇浸泡24h，再以10～100％浓度梯度的乙醇溶液洗涤，双蒸水洗至无醇味；再次冻干，切成大小为5×5×3mm3的小块，于超净台进行紫外灭菌备用；
步骤2.多糖磷酸钙纳米粒的制备：
配制体积分数为20～30％的Igepal CO-520/环己烷混合液并以此为溶剂，以0.01 ～0.05M氯化钙溶液和0.8～1.5mg阳离子多糖D-Sp，0.03～0.08M磷酸氢二钠和质粒混合溶液10μL(含Oct4、Sox2、Klf4、c-Myc各2.5μg)为原料，通过反相微乳法，得到多糖-磷酸钙混杂纳米粒微乳；再经柱层析，旋转蒸发及透析等步骤，得到多糖-磷酸钙混杂纳米粒溶液；
步骤3.三维载基因纳米粒‐胶原支架的制备：
取5×5×3mm3的支架样品放入24孔板的板孔中以无血清DMEM培养基使支架充分润湿；吸除多余的培养基；调整多糖-磷酸钙混杂纳米粒的浓度，使每100μL纳米粒溶液中含0.8～1.5μg总质粒；向每一块润湿之后5×5×3mm3的支架上滴加100μL多糖-磷酸钙混杂纳米粒溶液后冷冻干燥，得到三维载基因纳米粒-胶原支架；
步骤4.Yamanaka因子在三维体系中的表达：
取5×5×3mm3三维载基因纳米粒-胶原支架并以无血清DMED浸润；将200μL HUMSCs细胞悬液(5～8×104个细胞/支架)加入浸润后的支架，37℃放置0.5～2h；取出支架，以10％FBS的DMEM培养基继续培养2～6h，通过RT-PCR对四种外源基因的表达量进行评价，设置多糖-磷酸钙基因纳米粒转染组为阳性对照，未经处理的HUMSCs为阴性对照；
步骤5.三维体系中的iPSCs诱导和扩增：
取5×5×3mm3三维载基因纳米粒-胶原支架并以无血清DMED浸润；将200μL HUMSCs细胞悬液(5～8×104个细胞/支架)加入浸润后的支架，在完全培养基中培养3～5天后，换成人胚胎干细胞培养基，每2～3天换液一次，以使多能性干细胞扩增。
有益效果
以天然I胶原为原料制备的胶原支架为HUMSCs提供了安全稳定的三维培养环境，多糖‐磷酸钙载基因纳米粒具有良好的基因携载能力，且均匀分布于三维支架中，为HUMSCs向iPSCs的定向重编程提供了充足的外源基因来源并能够长效释放。经RT‐PCR评价，三维体系中外源基因的表达量显著高于二维体系；经iPSCs相关鉴定，结果表明，三维体系中形成的人源iPSCs具有类似于胚胎干细胞的全胚层分化能力，且能够于体外稳定扩增、传代至20代以上，并能保持正常的染色体核型。该方法为iPSCs的诱导提供了全新的三维环境，为iPSCs的应用由细胞水平向组织水平过渡提供了理论依据。
附图说明
图1为三维体系中细胞重编程的流程图。
图2为三维载基因纳米粒‐胶原支架的扫描电镜图。
图3为光学显微镜下HUMSCs在三维载基因纳米粒‐胶原支架中的形态变化。
图4为三维载基因纳米粒‐胶原支架及其中iPSCs的HE染色。
图5为iPSCs免疫组化。
图6为细胞染色体核型图
图7为免疫荧光染色鉴定iPSCs的多能性。
图8为Oct4和Nanog启动子的甲基化程度，其中hESCs为人胚胎干细胞。
图9为iPSCs细胞球在体内的三胚层分化，其中：g代表纤体组织，为内胚层特征，b和c分别代表骨组织和软骨组织，为中胚层分化特征，n代表神经管，为外胚层标记。
具体实施方式
一下实施例所采用的材料和仪器：
1材料和试剂：阳离子多糖D-Sp、LB培养基(自制)；I型-胶原、IV型胶原酶、琼脂糖、溴化乙锭、L-谷氨酰胺、MINI26-1KT、生物素化IgG(美国sigma)；胰蛋白酶、Tris碱、氨苄青霉素、链霉素、DAB显示试剂盒(碧云天生物技术研究所)；碱性成纤维生长因子(bFGF)(美国PeproTech)；质粒抽提试剂盒(美国Promega)；氯化钙(国药集团化学试剂有限公司)；MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四唑溴盐)、PicoGreen dsDNA定量试剂盒(美国Invitrogen)；基础培养基DMEM、基础培养基DMEM/F12、knockout DME、胎牛血清(FBS)、knockout血清替代品(美国GIBCO)；Oligo(诺百设计，美国Invitrogen合成)；T载体试剂盒(D101B，TAKARA)；DH5α感受态细胞(CD201-02，全式金)；抗生素平板(诺百生物自配)；双蒸水(诺百生物自配)；动植物基因组DNA提取试剂盒(AP-MN-MS-GDNA-250，Axygen)；全血基因组DNA提取试剂盒(AP-MN-BL-GDNA-250，Axygen)；甲基化试剂盒(D5005，ZYMO)；Platinum Taq DNA Polymerase(10966034，美国invitrogen)；PCR产物纯化试剂盒(AP-GX-250，Axygen)；100mM dNTPs(18427013，美国invitrogen)
2菌株、细胞株和小鼠：DH5α甘油菌(南京基天生物)；人脐带间充质干细胞(江苏省北科生物科技有限公司)；小鼠胚胎成纤维细胞(上海中科院干细胞库)；非PEI胖糖尿病/重度联合免疫缺陷小鼠(NOD-SCID)(扬州大学比较医学中心)
3质粒：非病毒质粒OCT4、SOX2、KLF4和CMYC(广州复能基因有限公司)
4抗体：Anti-OCT4、anti-SSEA-3、anti-SSEA-4、anti-TRA-1-81、anti-NANOG、羊抗兔IgG-Cy3(以上抗体由美国Abcam公司提供。)
5引物：
甲基化测定引物序列：
OCT4上游引物：TGGAGGTGAT GGGTTAGG 18
OCT4下游引物：CATCAAACTA CCCTATCATA ACC 23
NANOG上游引物：GTGAATGAAA GAGGAAAATG GAG 23
NANOG下游引物：AATAACCCAC CCCTATAATC C 21
(以上引物由上海生物工程有限公司提供)
6溶液配制：
(1)2％胶原溶液：称取2.00g I胶原蛋白，加入100mL 1％醋酸溶液，于60℃水浴加热，使其完全溶解，得到半透明的溶液，经0.22μm滤膜的滤器过滤，4℃保存，备用。
(2)PBS溶液：取NaCl 8.00g,KCl 0.20g,Na2HPO4.12H2O 3.5g,KH2PO4 0.2g,加入800mL双蒸水，调pH至7.4，定容至1000mL，分装，高压灭菌，4℃保存。
(3)完全培养基：低糖DMEM，加入10％FBS和100U/mL青‐链霉素溶液
(4)人胚胎干细胞培养基：Knockout DMEM培养基中依次加入20％血清代替物(Knockout SR)，2mmol/L左旋谷氨酰胺，0.1mmol/Lβ‐巯基乙醇，1％非必须氨基酸，100U/mL青‐链霉素溶液，4ng/mL人类重组碱性成纤维细胞生长因子，混匀，放置4℃冰箱备用。
7仪器：发射扫描电镜(JSM-7001F，日本电子)；CO2细胞培养箱(美国Thermo)；超净工作台(SW-CJ-2F，苏州净化设备公司)；普通离心机(美国Eppendrof)；超速低温离心机(德国Heraeus)；定轨摇床(上海第一医学院仪器厂)；共焦激光扫描显微镜(德国Leica)；垂直净化工作台(SW-CJ-2D，汇金净化)，高速台式离心机(Neofuge13，Heal Force)，震荡恒温培养箱(THZ-98，华美生化)，PCR仪(T100TM，Bio-Rad)，电泳以及成像系统(Tanon1600，天能)，序列分析仪(3730xl，Applied
Figure PCTCN2016086536-appb-000001
)
实施例1：三维胶原支架的制备：
(1)取适量2％胶原溶液于支架制备的聚四氟乙烯模具中(68×38×5mm3)，先在4℃放置0.5h，再迅速放在-80℃冰箱预冻3h以上,然后冻干；
(2)于120℃烘箱内热固化10h；
(3)之后，用无水乙醇浸泡24h，再以95％，85％，75％，65％，55％，30％，15％浓度梯度的乙醇溶液洗涤，最后用大量双蒸水洗涤，直至无醇味；
(4)再次冻干，切成大小为5×5×3mm3的小块，于超净台进行紫外灭菌3h，备用。热固化之后的过程均保证无菌操作。
实施例2：多糖‐磷酸钙混杂纳米粒的制备
(1)将Igepal CO-520溶解于环己烷中制备Igepal CO-520体积分数为29％的Igepal CO-520/环己烷混合液；
(2)微乳A的制备：取一干净锥形瓶，加入25mL Igepal CO-520/环己烷混合液，然后将650μL 0.01M氯化钙溶液和0.8mg阳离子多糖D-Sp，在磁力搅拌的条件下，一起加入到净锥形瓶，继续搅拌2min，形成微乳A；
(3)微乳B的制备：另取一干净锥形瓶，加入25mL Igepal CO-520/环己烷混合液，然后将650μL 0.06M磷酸氢二钠和质粒混合溶液10μL(含Oct4、Sox2、Klf4、c-Myc各2.5μg)一起加入到净锥形瓶，继续搅拌2min，形成微乳B；
(4)在4℃条件下，将微乳A逐滴加入到微乳B，边加边搅拌，直至整个体系透明澄清，得到多糖-磷酸钙混杂纳米粒微乳；
(5)加5mL pH＝7.0的无水乙醇对多糖-磷酸钙混杂纳米粒微乳进行稀释；
(6)然后上硅胶色谱柱，先用无水乙醇洗脱环己烷及游离的DNA，然后用含有5×10-4mM NaCl的70％乙醇溶液洗脱多糖-磷酸钙混杂纳米粒，分别收集不同时间点的洗脱夜，HPLC法测定其DNA含量；
(7)将含有多糖-磷酸钙混杂纳米粒的乙醇溶液在37℃旋转蒸发6h除去乙醇，浓缩液置于12kDa的透析袋中，pH7.4的PBS中4℃过夜，即得到多糖-磷酸钙混杂纳米粒溶液。
实施例3：三维载基因纳米粒-胶原支架的制备
(1)取5×5×3mm3的支架样品放入24孔板的板孔中，每孔加入0.5mL无血清DMEM培养基，让支架充分润湿；
(2)0.5h之后，吸除多余的培养基；
(3)调整多糖-磷酸钙混杂纳米粒的浓度，使每100μL纳米粒溶液中含0.8μg总质粒；
(4)向每一块润湿之后5×5×3mm3的支架上滴加100μL多糖-磷酸钙混杂纳米 粒溶液，必须缓慢、少量多次的滴加，使纳米粒溶液与支架充分接触、吸附；
(5)然后，冷冻干燥，得到三维载基因纳米粒-胶原支架。
(6)于扫描电镜下观察三维载基因纳米粒-胶原支架的形态。由图2可以看出，三维载基因纳米粒-胶原支架仍然呈现疏松多孔的内部结构，孔径在200～400μm范围内。而且，从右边的放大图可以明显看出多糖-磷酸钙混杂纳米粒吸附到了胶原支架的内表面(如箭头所指示的位置)。
实施例4：HE染色
(1)每一块5×5×3mm3的三维载基因纳米粒-胶原支架用200μL无血清DMEM培养基润湿；
(2)制备人脐带间充质干细胞的细胞悬液，将200μL细胞悬液(5×104个细胞/支架)少量多次滴加到润湿的支架上；
(3)接种细胞的支架于37℃、5％CO2的培养箱中放置0.5h；
(4)然后，每个放有接种细胞支架的孔中加入含10％FBS的DMEM培养基，继续培养4h；
(5)将种植细胞后第4、8、12天的支架经4％多聚甲醛固定24h后，用低浓度到高浓度梯度的乙醇脱水，逐渐脱去支架中的水份；
(6)再将支架置于既溶于酒精，又溶于石蜡的透明剂二甲苯中透明，以二甲苯替换出支架的中乙醇；
(7)将已透明的支架置于已溶化的石蜡中，放入溶蜡箱保温；
(8)待石蜡完全浸入支架后进行包埋：先制备好容器(如折叠一小纸盒)，倒入已融化的石蜡，迅速夹取已浸透的石蜡组织块放入其中，冷却凝固成块即可；
(9)将包埋好的蜡块固定于切片机上，切成薄片，一般为5～8μm厚。切下的薄片往往皱褶，要放到加热的水中烫平，再贴到载玻片上，放45℃恒温箱中烘干。
(10)用二甲苯脱去切片中的石蜡，再经由高浓度到低浓度酒精脱蜡，最后放入蒸馏水中进行水化；
(11)苏木素染色5min，水洗；之后，于盐酸及氨水中分色各30s，流水冲洗1h后入蒸馏水片刻；
(12)放入70％和90％酒精中脱水各10min；
(13)再入酒精伊红染色2～3min，水洗；
(14)染色后的切片经纯酒精脱水，再经二甲苯使切片透明；
(15)将已透明的切片滴上加拿大树胶，盖上盖玻片封固，镜下观察支架及细胞 形态。图3显示：支架中的细胞球呈规整的球形或椭球形；图4显示：细胞质被伊红(eosin)染成红色，而细胞核被苏木素(hematoxylin)染色成蓝色，显示出类似于人胚胎干的细胞核/质比
实施例5免疫组化：
(1)参照实施例4中(5～9)制备石蜡包埋切片；
(2)切片进行常规脱蜡、水合，一部分切片用于碱性磷酸酶(AP)染色；
(3)另一部分水合之后的切片，加入3％H2O2，室温5～10min灭活内源性酶，再用蒸馏水洗3次
(4)热修复抗原：将切片浸入0.01M枸橼酸盐缓冲液(pH＝6.0),电炉加热至沸腾后断电，间隔5～10min后，重复1～2次，冷却后，用PBS(pH＝7.4)洗涤1～2次；
(5)将5％BSA封闭液滴加于切片上，室温放置20min，甩去多余液体，不洗；
(6)在切片上滴加适当稀释的一抗(SSEA3,Tra‐1‐81,Oct4和Nanog)，4℃过夜，后用PBS洗涤三次，2min/次；
(7)加生物素化IgG溶液，于27℃反应20min，后用PBS洗涤3次，2min/次；
(8)使用DAB显示试剂盒进行显色：取1mL蒸馏水，加试剂盒中A，B，C试剂各一滴，混匀后加至切片。室温显色，镜下控制反应时间，蒸馏水洗涤；脱水，透明，封片；显微镜观察并拍照；由图5可以明显看出：三维载基因纳米粒-胶原支架中的细胞球呈现碱性磷酸酶(AP)阳性表达。而且，随着时间推移，人胚胎干细胞标记因子SEA3、TRA-1-81、OCT4和NANOG逐渐呈现强阳性表达。在第8天的时候，所有的检测的多能性因子都呈现阳性表达，表示细胞向多能性状态的转变。相比之下，空白胶原支架中的细胞没有任何免疫染色反应。上述结果证明了三维载基因纳米粒-胶原支架中的细胞球是多能性细胞重编程的产物：iPSCs；而且，与二维体系相比(病毒诱导体细胞重编程所需的平均时间为3周)，本发明中三维非病毒体系中的重编程所需的时间缩短为8天左右，大大加速了细胞重编程的进程。
实施例6：核型分析
操作过程：
(1)秋水仙素处理：在终止细胞培养前3h向细胞培养瓶中分别加入24μL 秋水仙素，使得最终浓度达0.8μg/mL，再将其放回培养箱内培养满72h；
(2)收集细胞：取出细胞，用吸管充分吹打瓶壁，使贴壁的细胞冲散均匀，转入离心管内做好标记，1500rpm离心5min，去上清；
(3)低渗处理：向离心管中加入1mL 0.075mol/L KCl低渗溶液，用吸管轻轻吹打，使细胞均匀悬浮于低渗溶液中，补加6mL 0.075mol/L KCl低渗溶液，置于37℃恒温水浴中，静置20min，使细胞膨胀、染色体分散；
(4)预固定：向离心管中加入新配的卡诺固定液1mL，轻轻吹打均匀，室温放置20min，1500rpm离心5min，去上清；
(5)固定：沿离心管壁加入新配的卡诺固定液6mL，吹打均匀，室温放置20min，1500rpm离心5min，去上清；
(6)滴片：向沉淀物中加入新配之的适量固定液1mL左右，用吸管轻轻吹打悬浮细胞，吸取细胞悬液，在一定高度垂直滴2～3滴于冰水预浸泡的干净载玻片上，立即用口朝一个方向吹散，在酒精上过几次，干燥，并将每张片子做好标记；
(7)染色：将滴有细胞悬液的载波片放于染缸，使片与片之间有一定的缝隙，将稀释的Giemsa染液倒在染缸中，染色20min，取出后用自来水冲洗，烘干；
(8)镜检：在低倍镜找到处于中期分裂相的细胞，然后转至高倍镜下观察，并把分散度好的细胞拍下来。由图6可以看出，三维支架中释放的细胞球在二维环境中扩增的第1代和第15代细胞染色体核型正常，与对照人脐带干细胞的染色体核型一致，说明三维支架中诱导生成的iPSCs核型正常，没有发生染色体突变，证明了所述方法的安全性良好。
实施例7：免疫荧光
对在二维环境中扩增到不同代数(第2、10、20以及25代)的iPSCs进行免疫荧光染色，一抗为anti-SSEA3、anti-TRA-1-81、anti-OCT4和anti-NANOG，二抗为FITC-IgG和Cy3-IgG。由图7可以看出：载基因纳米粒-胶原支架中诱导生成的iPSCs在二维环境扩增的过程中，不同代数(第2、10、20以及25代)的细胞仍然保持各多能性因子的阳性表达，证明了本实验诱导生成的iPSCs能够稳定传代。
实施例8：启动子甲基化检测
(1)基因组DNA提取和质检：利用专门gDNA提取试剂盒，3个细胞样本(人脐带间充质干细胞、本实验诱导的iPSCs以及人胚胎干细胞株HN4)进行基因组DNA的提取，并进行OD浓度和琼脂糖凝胶电泳测定。
(2)合格DNA的甲基化处理：利用专门甲基化试剂盒，严格按说明书操作，将500ng-2μg的gDNA进行甲基化处理，获得10μL的甲基化后的DNA。具体的甲基化程序：添加20μL gDNA(500ng～2μg)到130μL的CT溶液(试剂盒中的一种试剂)中，轻微混匀，按照如下程序进行操作：98℃，10min；64℃，2.5h；4℃，保持。
(3)将上述反应产物在纯化柱上进行一系列的纯化和富集，10μL双蒸水洗脱收集获得甲基化的DNA；
(4)PCR扩增以及回收纯化：以甲基化后的DNA为模板，进行PCR扩增。
第一轮PCR体系(总体积25μL)：
Figure PCTCN2016086536-appb-000002
第一轮PCR扩增程序：95℃3min；95℃30sec，53℃30sec，72℃30sec，进行40个循环；接着72℃5min，结束。
第二轮PCR体系(总体积50μL)：
Figure PCTCN2016086536-appb-000003
Figure PCTCN2016086536-appb-000004
第二轮PCR扩增程序：95℃3min；95℃30sec，53℃30sec，72℃30sec，进行40个循环；接着72℃5min，结束。
PCR产物回收纯化：将上述第二轮PCR产物进行凝胶回收纯化，OD测定浓度。
(5)TA克隆：利用T载体试剂盒，将PCR纯化产物克隆至T载体中，用DH5α感受态细胞转化，涂板，于37℃、过夜倒置培养(具体流程见T载体试剂盒说明书)
(6)单克隆菌液测序：挑取有效单克隆(避开卫星菌落)，进行摇菌培养，并进行PCR菌液检定，阳性单克隆送测。
(7)测序结果比对分析：首先利用序列比对软件进行测序分析，然后用甲基化分析软件对正确测序结果进行甲基化分析，并统计CpG岛的甲基化数量等信息。由图8可以看出，iPSCs的OCT4和NANOG启动子区域明显出现了去甲基化，其甲基化程度和阳性对照人胚胎干细胞相似，而与初始的人脐带间充质干细胞具有明显差异。该结果进一步证明了人脐带间充质干细胞在三维载基因纳米粒-胶原支架中重编程成功。
实施例9：体内畸胎瘤实验
iPSCs消化离心后，制成5×106个/mL的细胞悬液，接种到5周龄的雄性NOD-SCID裸鼠的腹部皮下，之后观察是否有肿瘤生长。5～8周后，处死小鼠，摘除肿瘤进行HE染色分析。从图9可以看出，iPSCs细胞球在免疫缺陷的NOD-SCID裸鼠体内能够成功形成畸胎瘤，而且畸胎瘤显示出明显的三胚层分化，分别以内胚层的腺体组织，中胚层的骨和软骨组织以及外胚层的神经管等组织为代表。该结果充分证明了三维载基因纳米粒-胶原支架能够成功应用于细胞重编程。

Claims (1)

  1. 一种基于三维体系的细胞重编程方法，其特征是具体包括如下步骤：
    步骤1.胶原支架的制备：取2～5％胶原溶液于支架制备的聚四氟乙烯模具中(68×38×5mm3)，先在4℃放置0.5～1h，再迅速放在-80℃冰箱预冻3h以上,然后冻干；于烘箱内热固化8～10h之后，用无水乙醇浸泡24h，再以10～100％浓度梯度的乙醇溶液洗涤，双蒸水洗至无醇味；再次冻干，切成大小为5×5×3mm3的小块，于超净台进行紫外灭菌备用；
    步骤2.多糖磷酸钙纳米粒的制备：
    配制体积分数为20～30％的Igepal CO-520/环己烷混合液并以此为溶剂，以0.01～0.05M氯化钙溶液和0.8～1.5mg阳离子多糖D-Sp，0.03～0.08M磷酸氢二钠和含Oct4、Sox2、Klf4与c-Myc各2.5μg质粒的质粒混合溶液10μL为原料，通过反相微乳法，得到多糖-磷酸钙混杂纳米粒微乳；再经柱层析，旋转蒸发及透析，得到多糖-磷酸钙混杂纳米粒溶液；
    步骤3.三维载基因纳米粒‐胶原支架的制备：
    取5×5×3mm3的支架样品放入24孔板的板孔中以无血清DMEM培养基使支架充分润湿；吸除多余的培养基；调整多糖-磷酸钙混杂纳米粒的浓度，使每100μL纳米粒溶液中含0.8～1.5μg总质粒；向每一块润湿之后5×5×3mm3的支架上滴加100μL多糖-磷酸钙混杂纳米粒溶液后冷冻干燥，得到三维载基因纳米粒-胶原支架；
    步骤4.Yamanaka因子在三维体系中的表达：
    取5×5×3mm3三维载基因纳米粒-胶原支架并以无血清DMED浸润；将200μLHUMSCs细胞悬液(5～8×104个细胞/支架)加入浸润后的支架，37℃放置0.5～2h；取出支架，以10％FBS的DMEM培养基继续培养2～6h，通过RT-PCR对四种外源基因的表达量进行评价，设置多糖-磷酸钙基因纳米粒转染组为阳性对照，未经处理的HUMSCs为阴性对照；
    步骤5.三维体系中的iPSCs诱导和扩增：
    取5×5×3mm3三维载基因纳米粒-胶原支架并以无血清DMED浸润；将200μLHUMSCs细胞悬液(5～8×104个细胞/支架)加入浸润后的支架，在完全培养基中培养3～5天后，换成人胚胎干细胞培养基，每2～3天换液一次，以使多能性干细胞扩增。
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