WO2019100726A1 - Dna四面体在促进神经干细胞增殖分化过程中的应用 - Google Patents

Dna四面体在促进神经干细胞增殖分化过程中的应用 Download PDF

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WO2019100726A1
WO2019100726A1 PCT/CN2018/094561 CN2018094561W WO2019100726A1 WO 2019100726 A1 WO2019100726 A1 WO 2019100726A1 CN 2018094561 W CN2018094561 W CN 2018094561W WO 2019100726 A1 WO2019100726 A1 WO 2019100726A1
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stem cells
neural stem
differentiation
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林云锋
马文娟
蔡潇潇
李谦顺
赵丹
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Sichuan University
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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  • the invention belongs to the technical field of cell proliferation and differentiation, and particularly relates to the application of a DNA tetrahedron in promoting the proliferation and differentiation of neural stem cells.
  • Mouse neural stem cells which are derived from the ATCC cell bank, are one of the better models for studying the nervous system in vitro.
  • the NE-4C cell line can maintain the characteristics of neural stem cells under certain circumstances, but can differentiate into mature neurons or glial cells after specific induction treatment.
  • the main direction is to study the effects of drugs or materials on the proliferation and differentiation of neural stem cells, such as the proliferation and differentiation of prostaglandin neural stem cells; and neural stem cells in some central degenerative diseases.
  • Cellular therapeutic effects such as the therapeutic role of neural stem cells in animal models of Alzheimer's disease.
  • TDNs DNA tetrahedral nanomaterials
  • S1, S2, S3, S4 The base sequences of four ss DNAs (S1, S2, S3, S4) strictly follow the "base complementary pairing". The principle is then precisely and subtly designed.
  • TDNs The synthesis method of TDNs is simple, the yield is high, and the tolerance to specific or non-specific nucleases is better than that of ordinary linear DNA, and it has good biocompatibility, biosafety and biodegradability.
  • TDNs there are many studies on TDNs, there are few studies on the effects of various physiological activities on cells, especially the research on promoting cell proliferation and differentiation.
  • the present invention provides a DNA tetrahedron for promoting proliferation and differentiation of neural neural stem cells by affecting Wnt/ ⁇ -catenin and Notch signaling pathway-related genes and proteins. Expression changes, respectively, to promote the proliferation and differentiation of neural stem cells.
  • DNA tetrahedron in promoting proliferation and differentiation of neural stem cells, D, wherein the four single-stranded sequences of DNA tetrahedra are as shown in SEQ ID NO: 1-4.
  • DNA tetrahedron promotes proliferation of neural stem cells by activating Wnt/ ⁇ -catenin signaling pathway; DNA tetrahedron promotes differentiation of neural stem cells by inhibiting Notch signaling pathway.
  • the process of promoting neural stem cell proliferation includes promoting the expression of ⁇ -catenin, Lef-1, and Cyclin-D proteins.
  • the process of promoting neural stem cell proliferation includes promoting the expression of the ⁇ -catenin, Lef-1, and Cyclin-D genes.
  • the process of promoting differentiation of neural stem cells includes reducing the expression of Notch-1, Hes-1 and Hes-5 proteins.
  • the process of promoting differentiation of neural stem cells comprises promoting expression of a ⁇ -III-Tubulin protein.
  • the processes of promoting differentiation of neural stem cells include inhibition of expression of Notch-1, Hes-1 and Hes-5 genes, respectively, and promotion of expression of ⁇ -III-Tubulin gene.
  • the concentration of the DNA tetrahedron in promoting proliferation and differentiation of the neural stem cells is 50 to 500 nM.
  • the concentration of the DNA tetrahedron in promoting proliferation and differentiation of the neural stem cells is 100 to 300 nM.
  • the concentration of the DNA tetrahedron in promoting proliferation and differentiation of neural stem cells was 250 nM.
  • TDNs have good biocompatibility, biosafety and biodegradability, they can effectively solve the problem of poor biological performance of traditional drugs or materials.
  • DNA tetrahedron (TDNs) nanomaterials promote the proliferation and differentiation of neural stem cells, and at the same time solve the problems of slower proliferation of neural stem cells and slower differentiation and maturation, which lays a certain foundation for subsequent in vivo neural stem cell therapy experiments. Research basis.
  • DNA tetrahedrons can promote the proliferation and differentiation process by regulating Wnt/ ⁇ -catenin and Notch signaling pathways, respectively, by promoting the expression of ⁇ -catenin, Lef-1 and Cyclin-D genes and proteins. Promote the proliferation of mouse neural stem cells; by reducing the expression of Notch-1, Hes-1 and Hes-5 proteins, and promoting the expression of ⁇ -III-Tubulin genes and proteins, the purpose of promoting differentiation of mouse neural stem cells can be achieved.
  • FIG. 1 is a schematic representation of four single-stranded synthetic TDNs.
  • FIG. 2 is a schematic diagram showing the results of TDNs polyacrylamide gel electrophoresis.
  • Figure 3 is a schematic illustration of the results of transmission electron microscopy identification.
  • Fig. 4 is a diagram showing the results of identifying the undifferentiated state of mouse neural stem cells by immunofluorescence technique.
  • Figure 5 is a graphical representation of the results of detection of TDNS uptake by mouse neural stem cells using fluorescent tracing techniques.
  • Figure 6 is a graphical representation of the results of the effect of TDNs concentration on the proliferation of mouse neural stem cells.
  • Figure 7 is a graph showing the results of cell cycle changes of mouse neural stem cells under the action of TDNs by flow cytometry.
  • Fig. 8 (a), (b) and (c) are schematic diagrams showing the detection of the expression levels of ⁇ -catenin, Lef-1 and Cyclin-D genes and proteins in mouse neural stem cells under the action of TDNs.
  • Fig. 9 (a), (b) and (c) are schematic diagrams showing the detection of ⁇ -III-Tubulin gene and protein expression in mouse neural stem cells under the action of TDNs.
  • Figures 10 and 11 are diagrams showing the results of detecting the expression level of ⁇ -III-Tubulin protein by immunofluorescence technique.
  • Fig. 12 (a), (b) and (c) are schematic diagrams showing the detection of the expression levels of Notch-1, Hes-1, Hes-5 genes and proteins in mouse neural stem cells under the action of TDNs.
  • TDNs DNA Tetrahedrons
  • Polyacrylamide gel was prepared by using 40% acrylamide, 10 ⁇ TAE, 10% APS solution, distilled water and TEMED;
  • the four single-stranded S1, S2, S3 and S4 sizes of TDNs are about 60 bp, 50 bp, 50 bp and 50 bp, respectively, and the size of TDNs is about 210 bp.
  • TDNs were identified by transmission electron microscopy. The results are shown in Fig. 3. As shown in Fig. 3, the shape of TDNs (triangular) is approximately triangular in shape under transmission electron microscopy, and the particle size is about 10-15 nM. The circle is labeled as a polymer.
  • the cell suspension was inoculated into a confocal dish and placed in an incubator for 24 hours.
  • the medium containing DMEM + 10% serum + 1% double antibody was aspirated, and washed three times with PBS for 5 minutes each time;
  • Triton-100 treatment for 20-25 minutes, aspirate Triton-100, wash 3 times with PBS for 5 minutes each time;
  • the sheep serum was treated for 1 hour, the sheep serum was aspirated, and the PBS was washed 3 times for 5 minutes each time;
  • Phalloidin treatment protected from light, 10-30 minutes, aspirate phalloidin, wash 3 times in PBS for 5 minutes each time;
  • DAPI treatment protected from light, 10 minutes, aspirate DAPI, wash 3 times with PBS for 5 minutes each time.
  • 10% glycerol seal protected from light, stored at 4 ° C. Check on the machine.
  • Fig. 4 The detection results are shown in Fig. 4. As shown in Fig. 4, the mouse neural stem cells showed that the nestin antibody was positive, indicating that the cells were still in an undifferentiated state, and could be used for subsequent proliferation and differentiation experiments.
  • the cultured cell suspension was divided into a control group and an experimental group.
  • the concentration of 250 nM was added to the control group, and the Cy-5-modified DNA single-stranded S1 was added.
  • the experimental group was added at a concentration of 250 nM and modified with Cy-5.
  • the TDNs were cultured in an incubator for 6 hours (37 ° C, 5% CO 2 ).
  • single-stranded S1 is less taken up by neural stem cells; neural stem cells take up more TDNs, and most of the TDNs that enter the cells accumulate in the cytoplasm of the cells and enter the nucleus less.
  • the cultured cell suspension was divided into a control group and an experimental group, and TDNs were added to the experimental group, and an equal amount of PBS was added to the control group, followed by incubation in an incubator for 24 hours (37 ° C, 5% CO 2 ). ).
  • TDNs As shown in Fig. 6, when the concentration of TDNs was 62.5 nM, 125 nM, and 250 nM, the proliferation process of mouse neural stem cells in the experimental group was promoted to a certain extent by TDNs, and 250 nM was the most. Good concentration indicates that TDNs have the effect of promoting the proliferation of mouse neural stem cells.
  • the cultured cell suspension was divided into a control group and an experimental group, and TDNs were added to the experimental group, and an equal amount of PBS was added to the control group, followed by incubation in an incubator for 24 hours (37 ° C, 5% CO 2 ). ).
  • control cells and the experimental group cells were separately digested with 0.25% trypsin, placed in a 15 ml centrifuge tube (2000 rpm, 5 minutes), the supernatant was discarded, washed with PBS, centrifuged (2000 rpm, 5 minutes), and then added with ice. 500 ⁇ l of ethanol was fixed, and the cells were fixed at 4 ° C overnight. The next day, PBS was added to centrifuge, the supernatant was discarded, washed with PBS, centrifuged, and the supernatant was discarded. Then, 100 ⁇ l of RNase was added, and a 37 ° C water bath was added for 30 minutes. 400 ⁇ l of PI was added and mixed. Protected from light at 4 ° C for 30 minutes. The cells were transferred to a flow tube, detected by the machine, and analyzed by data. The results are shown in Fig. 7.
  • the number of cells in the S phase (DNA synthesis phase) in the experimental group increased significantly, indicating that TDNs changed the cell cycle of neural stem cells and promoted its proliferation.
  • the three proteins in the Wnt/ ⁇ -catenin signaling pathway associated with the neural stem stem cell proliferation process in the experimental group were ⁇ -catenin, respectively.
  • Lef-1 and Cyclin-D the expression levels of three proteins and corresponding control genes were increased by ⁇ -catenin, Lef-1 and Cyclin-D, further indicating that TDNs promoted the proliferation of neural stem cells.
  • the suspension cells were divided into control group and experimental group, and TDNs at a concentration of 250 nM were added to the experimental group.
  • the same amount of PBS was added to the control group, and then cultured in an incubator for 24 hours (37 ° C, 5%). CO 2 ), then the gene extraction kit was used to extract the control and experimental group genes, respectively, and then the stable cDNA was obtained by the high purity total RNA rapid extraction kit and the reverse transcription kit.
  • the three proteins in the Wnt/ ⁇ -catenin signaling pathway associated with neural stem cell proliferation in the experimental group were ⁇ -catenin, Lef-1, and Cyclin-, respectively.
  • D the expression levels of the three proteins and the corresponding control genes ⁇ -catenin, Lef-1 and Cyclin-D were increased, further indicating that TDNs promoted the proliferation of neural stem cells.
  • the expression levels of the differentiation-related protein ( ⁇ -III-Tubulin) in the experimental group were higher than those in the control group.
  • the expression levels of Notch-1, Hes-1 and Hes-5 were decreased in the three proteins related to the Notch signaling pathway in the experimental group, indicating that TDNs can promote the differentiation of mouse neural stem cells. mature.
  • (1) Inoculate the cell suspension (100 ⁇ l/well) in a 6-well plate, place the plate in an incubator for 24 hours (37 ° C, 5% CO 2 ), and then the component is DMEM+
  • the serum concentration in the medium of 10% serum + 1% double antibody was reduced from 10% to 6%, cultured in the incubator for 6 hours (37 ° C, 5% CO 2 ), and then the serum concentration in the medium was 6 % was lowered to 0 and incubated for 1 hour in the incubator (37 ° C, 5% CO 2 ).
  • the expression level of the differentiation-related gene ( ⁇ -III-Tubulin) in the experimental group was higher, and compared with the control group,
  • the three proteins on the Notch signaling pathway in the differentiation group were Notch-1, Hes-1 and Hes-5, and the expression levels of the corresponding genes Notch-1, Hes-1 and Hes-5 were decreased.
  • TDNs can promote the differentiation and maturation of mouse neural stem cells.
  • the cell suspension obtained in the step (1) is cultured in a medium containing DMEM/F-12+1% double antibody + 1% B27, and the cell suspension is divided into a control group and an experimental group, and each day. At the same time, the medium was changed; TDNs at a concentration of 250 nM were added to the experimental group, and an equal amount of PBS was added to the control group for 1 day and 7 days.

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Abstract

一种DNA四面体在促进神经干细胞增殖分化过程中的应用。其中，DNA四面体的四条单链序列如SEQ ID NO:1-4所示。该应用可有效的促进神经干细胞的增殖和分化。

Description

[根据细则26改正10.09.2018]　DNA四面体在促进神经干细胞增殖分化过程中的应用 技术领域
本发明属于细胞增殖分化技术领域，具体涉及一种DNA四面体在促进神经干细胞增殖分化过程中的应用。
背景技术
小鼠神经干细胞(NE-4C)，该细胞系来源于ATCC细胞库，是一种较好的体外研究神经系统的模型之一。NE-4C细胞系在一定的环境下可以维持神经干细胞的特点，但是在特定诱导处理后，可分化为成熟的神经元或者神经胶质细胞。目前，在关于神经干细胞的研究中，主要方向是研究药物或者材料对神经干细胞增殖以及分化的影响，比如前列腺素神经干细胞增殖及分化过程中的影响；以及神经干细胞在一些中枢退行性病变中的细胞治疗作用，比如神经干细胞在阿尔茨海默症动物模型中的治疗作用。
DNA四面体纳米材料(TDNs)是一种新型的DNA纳米材料，目前在生物医学领域有着十分广泛的研究和巨大的潜在运用前景。目前的研究中发现，增殖分化研究过程中所使用的药物或者材料可能对细胞一定的毒性作用，即具有生物相容性、生物安全性、生物可降解性较差等特点。TDNs是由四条ss DNA单链在特定的条件下自组装形成的具有三维结构的DNA纳米材料，四条ss DNA(S1、S2、S3、S4)的碱基序列是严格遵循了“碱基互补配对原则”进而精确、巧妙地设计出来的。TDNs合成方法简便、产率较高，对特异性或非特异性核酸酶的耐受性均较普通的线性DNA好，且具有良好的生物相容性、生物安全性和生物可降解性。此外，关于TDNs的研究虽多，但它对细胞各项生理活动影响的研究却很少，尤其是促进细胞增殖分化过程的研究较少。
发明内容
针对现有技术中的上述不足，本发明提供一种DNA四面体在促进神经神经干细胞增殖分化过程中的应用，该DNA四面体通过影响Wnt/β-catenin和Notch信号通路相关的基因和蛋白的表达变化，分别达到促进神经干细胞增殖分化的目的。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
DNA四面体在促进神经干细胞增殖分化过程中的应用，D其中，DNA四面体的四条单链序列如SEQ ID NO:1-4所示。
进一步地，DNA四面体促进神经干细胞的增殖是通过激活Wnt/β-catenin信号通路发挥作用的；DNA四面体促进神经干细胞的分化是通过抑制Notch信号通路发挥作用的。
进一步地，促进神经干细胞增殖的过程包括促进β-catenin、Lef-1和Cyclin-D蛋白的表达。
进一步地，促进神经干细胞增殖的过程包括促进β-catenin、Lef-1和Cyclin-D基因的表达。
进一步地，促进神经干细胞分化的过程包括降低Notch-1、Hes-1和Hes-5蛋白的表达。
进一步地，促进神经干细胞分化的过程包括促进β-III-Tubulin蛋白的表达。
进一步地，促进神经干细胞分化的过程分别包括抑制Notch-1、Hes-1和Hes-5基因的表达，以及促进β-III-Tubulin基因的表达。
进一步地，DNA四面体在促进神经干细胞增殖分化过程中的浓度为50～500nM。
进一步地，DNA四面体在促进神经干细胞增殖分化过程中的浓度为100～300nM。
进一步地，DNA四面体在促进神经干细胞增殖分化过程中的浓度为250nM。
本发明的有益效果为：
1、由于TDNs具有良好的生物相容性、生物安全性及生物可降解性，因此，可有效地解决传统药物或者材料的生物性能较差的问题。DNA四面体(TDNs)纳米材料在神经干细胞增殖及分化过程中起到促进，同时一定程度上解决了神经干细胞增殖较慢，分化成熟速度较慢等问题，为后续的体内神经干细胞治疗实验奠定一定的研究基础。
2、DNA四面体(TDNs)通过调控Wnt/β-catenin和Notch信号通路，分别促进其增殖和分化过程，即通过促进β-catenin、Lef-1和Cyclin-D基因、蛋白的表达，可达到促进小鼠神经干细胞增殖的目的；通过降低Notch-1、Hes-1和Hes-5蛋白的表达，以及促进β-III-Tubulin基因、蛋白的表达，可达到促进小鼠神经干细胞分化的目的。
附图说明
图1为四条单链合成TDNs的示意图。
图2为TDNs聚丙烯酰胺凝胶电泳结果示意图。
图3为透射电镜鉴定结果的示意图。
图4为采用免疫荧光技术对小鼠神经干细胞的未分化状态进行鉴定的结果示意图。
图5为采用荧光示踪技术对小鼠神经干细胞对于TDNS摄取量进行检测的结果示意图。
图6为TDNs浓度对小鼠神经干细胞增殖影响的检测结果示意图。
图7为采用流式细胞术检测小鼠神经干细胞在TDNs作用下细胞周期变化的结果示意图。
图8(a)、(b)(c)为在TDNs作用下小鼠神经干细胞中β-catenin、Lef-1和Cyclin-D基因、蛋白表达量的检测示意图。
图9(a)、(b)(c)为在TDNs作用下小鼠神经干细胞中测β-III-Tubulin基因、蛋白表达量的检测示意图。
图10、11为采用免疫荧光技术检测β-III-Tubulin蛋白表达量的结果示意图。
图12(a)、(b)、(c)为在TDNs作用下小鼠神经干细胞中Notch-1、Hes-1、Hes-5基因、蛋白表达量的检测示意图。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1 DNA四面体(TDNs)的合成及鉴定
1、TDNs的合成
将等浓度的4条ss DNA单链(S1、S2、S3、S4)加入到含有100μl的TM buffer(10mM Tris-HCl，50mM MgCl2，pH 8.0)的200μl EP管中，将反应液加热到95℃维持10min，然后快速降温到4℃，并保持20min，得到TDNs，合成过程如图1所示；其中，四条DNA单链的具体序列如下：
S1：
Figure PCTCN2018094561-appb-000001
S2：
Figure PCTCN2018094561-appb-000002
S3：
Figure PCTCN2018094561-appb-000003
S4：
Figure PCTCN2018094561-appb-000004
2、TDNs的鉴定
(1)聚丙烯酰胺凝胶电泳鉴定
采用40％丙烯酰胺、10×TAE、10％APS溶液、蒸馏水和TEMED制备得到聚丙烯酰胺凝胶；
然后取1μL 6×loading buffer和5μL制备得到的TDNs混合均匀，分别将其和marker加入对应的电泳槽中，冰浴、恒压100V，电泳60min；然后采用浓度比为1:50的GelRed和蒸馏水，在避光条件下，摇床处理15～25min，然后曝光，再进行检测，其结果见图2。
如图2所示，TDNs的四条单链S1、S2、S3和S4大小分别在60bp、50bp、50bp和50bp左右，TDNs的大小约在210bp左右。
(2)透射电镜鉴定
采用透射电镜对TDNs进行鉴定，其结果见图3；如图3所示，TDNs(三角形标注)的形状在透射电镜下呈近似三角形形状，粒径大小约在10～15nM左右。圆圈标注的为多聚物。
实施例2 采用免疫荧光技术对小鼠神经干细胞的鉴定
采用免疫荧光技术对小鼠神经干细胞进行鉴定，一次包括以下步骤：
将细胞悬浮液接种于共聚焦小皿中，放置于孵箱中培养24小时。吸去组分为DMEM+10％血清+1％双抗的培养基，PBS洗3次，每次5分钟；
4％多聚甲醛固定25分钟后，吸去多聚甲醛，PBS洗3次，每次5分钟；
0.5％Triton-100处理20-25分钟，吸去Triton-100，PBS洗3次，每次5分钟；
羊血清处理1小时，吸去羊血清，PBS洗3次，每次5分钟；
一抗(抗nestin抗体)处理，4℃，过夜。第二天，37℃复温0.5小时，回收一抗，PBS洗3次，每次5分钟。携带荧光的二抗处理，避光，37℃，1小时，吸去二抗，PBS洗3次，每次5分钟；
鬼笔环肽处理，避光，10-30分钟，吸去鬼笔环肽，PBS洗3次，每次5分钟；
DAPI处理，避光，10分钟，吸去DAPI，PBS洗3次，每次5分钟。10％甘油封样，避光，4℃保存。上机检测。
检测结果见图4，如图4所示，小鼠神经干细胞显示nestin抗体阳性，表示细胞仍处于未分化的状态，可用于进行后续的增殖分化实验。
实施例3 采用荧光示踪技术检测小鼠神经干细胞对TDNs的摄取量
(1)在共聚焦小皿中接种细胞悬液(100μl/孔)，将培养板在孵箱中预培养24小时(37℃，5％CO 2)，然后组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，再将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)将培养的细胞悬液分为对照组和实验组，在对照组中加入浓度为250nM，采用Cy-5修饰的DNA单链S1，实验组中加入浓度为250nM，采用Cy-5 修饰的TDNs，分别于孵箱中培养6小时(37℃，5％CO 2)。
(3)分别吸去实验组和对照组的培养基，PBS洗3次，每次5分钟。再用4％多聚甲醛固定25分钟后，吸去多聚甲醛，PBS洗3次，每次5分钟，然后鬼笔环肽处理，避光，10-30分钟，吸去鬼笔环肽，PBS洗3次，每次5分钟；再采用DAPI处理，避光，10分钟，吸去DAPI，PBS洗3次，每次5分钟；最后用10％甘油封样，避光，4℃保存。上机检测，其检测结果见图5。
如图5所示，单链S1被神经干细胞摄取的较少；神经干细胞对TDNs摄取的较多，且进入细胞的TDNs大部分聚集在细胞的胞浆内，进入细胞核的较少。
实施例4 TDNs促进小鼠神经干细胞的增殖及检测
1、增殖
(1)在96孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)将培养的细胞悬液分为对照组和实验组，并在实验组中加入TDNs，对照组中加入等量的PBS，然后于孵箱中培养24小时(37℃，5％CO 2)。
(3)分别向实验组和对照组中加入CCK-8溶液(10μl/孔)，然后于孵箱中孵育1～4h(37℃，5％CO 2)，再在450nm处检测每孔的吸光度，其结果见图6。
如图6所示，当TDNs浓度为62.5nM、125nM、250nM时，与对照组相比，实验组中小鼠神经干细胞的增殖过程，在一定程度上都受到了TDNs的促进作用，且250nM是最佳浓度，表明TDNs具有促进小鼠神经干细胞增殖的作用。
2、采用流式细胞术检测细胞增殖
(1)在25ml培养瓶中接种细胞悬液，将培养瓶置于孵箱中预培养24小时 (37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)将培养的细胞悬液分为对照组和实验组，并在实验组中加入TDNs，对照组中加入等量的PBS，然后于孵箱中培养24小时(37℃，5％CO 2)。
(3)使用0.25％胰蛋白酶分别消化收集对照组细胞和实验组细胞，置于15ml离心管中(2000rpm、5分钟)，弃上清，PBS洗涤，离心(2000rpm、5分钟)，再加入冰乙醇500μl固定细胞，4℃过夜，第二天加入PBS离心，弃上清，再加入PBS洗涤，离心，弃上清，然后加入100μl RNase，37℃水浴，30分钟，加入400μl PI染色混匀，4℃避光，30分钟。将细胞转移至流式管中，上机检测，并进行数据分析，其结果见图7。
如图7所示，与对照组相比，实验组中处于S期(DNA合成期)的细胞数目明显增加，说明TDNs改变了神经干细胞的细胞周期，具有促进其增殖的作用。
实施例5 TDNs促进小鼠神经干细胞增殖的机制
1、采用蛋白质印迹法检测TDNs促进小鼠神经干细胞增殖的机制
(1)在6孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)采用组分为DMEM+1％双抗的培养基对步骤(1)所得细胞悬液进行培养，将细胞悬液分为对照组和实验组，且每天同时更换培养基；并在实验组中加入浓度为250nM的TDNs，对照组中加入等量的PBS，然后分别在培养24 小时后，使用全蛋白提取试剂盒分别提取对照组和实验组蛋白。
(3)对步骤(2)所得对照组和实验组蛋白分别进行SDS-PAGE电泳，其具体过程如下：灌胶→上样→电泳→转膜→封闭液摇动封闭1小时→一抗4℃过夜→回收一抗，TBST洗涤3次，每次5-10分钟→二抗，1小时→弃二抗，TBST洗涤3次，每次5-10分钟→曝光，检测并进行数据处理，其结果见图8(a)和图8(b)。
如图8(a)和图8(b)所示，与对照组相比，实验组中与神经干干细胞增殖过程相关的Wnt/β-catenin信号通路上的三个蛋白，分别是β-catenin、Lef-1和Cyclin-D，三种蛋白及相应控制基因的表达量β-catenin、Lef-1和Cyclin-D均有所增加，进一步说明TDNs促进了神经干细胞的增殖。
2、采用荧光定量PCR(Q-PCR)检测TDNs促进小鼠神经干细胞增殖的机制
(1)在6孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)将悬浮液细胞分为对照组和实验组，并在实验组中加入浓度为250nM的TDNs，对照组中加入等量的PBS，然后于孵箱中培养24小时(37℃，5％CO 2)，然后使用基因提取试剂盒，分别提取对照组和实验组基因，然后再通过高纯总RNA快速提取试剂盒及逆转录试剂盒获得稳定的cDNA。
(3)Q-PCR：每孔加入20μl的反应体系(2μl cDNA、10μl SYBR、0.8μl引物Forward、0.8μl引物Reserve、6.4μl ddH 2O)，上机检测，并进行数据处理，其结果见图8(c)。
如图8(c)所示，与对照组相比，实验组中与神经干细胞增殖过程相关的Wnt/β-catenin信号通路上的三个蛋白，分别是β-catenin、Lef-1和Cyclin-D，三种蛋白及相应控制基因β-catenin、Lef-1和Cyclin-D的表达量均有所增加，进一步说明TDNs促进了神经干细胞的增殖。
实施例6 TDNs促进小鼠神经干细胞分化及其分化机制的检测
1、采用蛋白质印迹法检测TDNs促进小鼠神经干细胞分化的现象及其机制
(1)在6孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)采用组分为DMEM/F-12+1％双抗+1％B27的培养基对步骤(1)所得细胞悬液进行培养，并将细胞悬液分为对照组和实验组，且每天同时更换培养基；并在实验组中加入浓度为250nM的TDNs，对照组中加入等量的PBS，然后分别在培养1天、3天和7天后，使用全蛋白提取试剂盒分别提取对照组和实验组蛋白。
(3)对步骤(2)所得对照组和实验组蛋白分别进行SDS-PAGE电泳，其具体过程如下：灌胶→上样→电泳→转膜→封闭液摇动封闭1小时→一抗4℃过夜→回收一抗，TBST洗涤3次，每次5-10分钟→二抗，1小时→弃二抗，TBST洗涤3次，每次5-10分钟→曝光，检测并进行数据处理，其结果见图9(a)、图9(b)、图12(a)和图12(b)。
如图9(a)、图9(b)、图12(a)和图12(b)所示，实验组中分化相关的目的蛋白(β-III-Tubulin)的表达量均高于对照组，且与对照组相比，实验组中分化相关Notch信号通路上的三个蛋白，分别是Notch-1、Hes-1、Hes-5的表达 量降低，表明TDNs能够促进小鼠神经干细胞的分化成熟。
2、采用荧光定量PCR(Q-PCR)检测TDNs促进小鼠神经干细胞分化的现象及机制
(1)(1)在6孔板中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)采用组分为DMEM/F-12+1％双抗+1％B27的培养基对步骤(1)所得细胞悬液进行培养，并将细胞悬液分为对照组和实验组，且每天同时更换培养基；并在实验组中加入浓度为250nM的TDNs，对照组中加入等量的PBS，然后分别在培养1天、3天和7天后，使用基因提取试剂盒，分别提取对照组和实验组基因，然后再通过高纯总RNA快速提取试剂盒及逆转录试剂盒获得稳定的cDNA。
(3)Q-PCR：每孔加入20μl的反应体系(2μl cDNA、10μl SYBR、0.8μl引物Forward、0.8μl引物Reserve、6.4μl ddH 2O)，上机检测，并进行数据处理，其结果见图9(c)和图12(c)。
如图9(c)和图12(c)所示，与对照组相比，实验组中分化相关的目的基因(β-III-Tubulin)的表达量均较高，且与对照组相比，实验组中分化相关Notch信号通路上的三个蛋白，分别是Notch-1、Hes-1、Hes-5，其所对应的基因Notch-1、Hes-1、Hes-5的表达量降低，表明TDNs能够促进小鼠神经干细胞的分化成熟。
3、免疫荧光技术
(1)在共聚焦小皿中接种细胞悬液(100μl/孔)，将培养板置于孵箱中预培养24小时(37℃，5％CO 2)，再将组分为DMEM+10％血清+1％双抗的培养基中 的血清浓度由10％降到6％，于孵箱中培养6小时(37℃，5％CO 2)，然后将培养基中的血清浓度由6％降到0，于孵箱中培养1小时(37℃，5％CO 2)。
(2)采用组分为DMEM/F-12+1％双抗+1％B27的培养基对步骤(1)所得细胞悬液进行培养，将细胞悬液分为对照组和实验组，且每天同时更换培养基；并在实验组中加入浓度为250nM的TDNs，对照组中加入等量的PBS，培养1天、7天。
(3)分别吸去实验组和对照组的培养基，PBS洗3次，每次5分钟。再用4％多聚甲醛固定25分钟后，吸去多聚甲醛，PBS洗3次，每次5分钟，0.5％Triton-100处理20-25分钟，吸去Triton-100，PBS洗3次，每次5分钟。羊血清处理1小时，吸去羊血清，PBS洗3次，每次5分钟。一抗β-III-Tubulin抗体处理，4℃，过夜。第二天，37℃复温0.5小时，回收一抗，PBS洗3次，每次5分钟。携带荧光的二抗处理，避光，37℃，1小时，吸去二抗，PBS洗3次，每次5分钟。DAPI处理，避光，10分钟，吸去DAPI，PBS洗3次，每次5分钟。10％甘油封样，避光，4℃保存。上机检测。其结果见图10和图11。
如图10和图11所示，于1天和7天后，与对照组相比，实验组中的荧光强度(β-III-Tubulin)较高，且细胞的形态较好。

Claims (10)

  1. DNA四面体在促进神经干细胞增殖分化过程中的应用，其中，DNA四面体的四条单链序列如SEQ ID NO:1-4所示。
  2. 根据权利要求1所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，所述DNA四面体促进神经干细胞的增殖是通过激活Wnt/β-catenin信号通路发挥作用的；所述DNA四面体促进神经干细胞的分化是通过抑制Notch信号通路发挥作用的。
  3. 根据权利要求2所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，促进神经干细胞增殖的过程包括促进β-catenin、Lef-1和Cyclin-D蛋白的表达。
  4. 根据权利要求2所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，促进神经干细胞增殖的过程包括促进β-catenin、Lef-1和Cyclin-D基因的表达。
  5. 根据权利要求2所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，促进神经干细胞分化的过程包括降低Notch-1、Hes-1和Hes-5蛋白的表达。
  6. 根据权利要求2所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，促进神经干细胞分化的过程包括促进β-III-Tubulin蛋白的表达。
  7. 根据权利要求2所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，促进神经干细胞分化的过程分别包括抑制Notch-1、Hes-1和Hes-5基因的表达，以及促进β-III-Tubulin基因的表达。
  8. 根据权利要求2所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，所述DNA四面体在促进神经干细胞增殖分化过程中的浓度 为50～500nM。
  9. 根据权利要求8所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，所述DNA四面体在促进神经干细胞增殖分化过程中的浓度为100～300nM。
  10. 根据权利要求8所述的DNA四面体在促进神经干细胞增殖分化过程中的应用，其特征在于，所述DNA四面体在促进神经干细胞增殖分化过程中的浓度为250nM。
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