WO2019100727A1 - Dna四面体在抑制阿尔兹海默细胞凋亡中的应用 - Google Patents

Dna四面体在抑制阿尔兹海默细胞凋亡中的应用 Download PDF

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WO2019100727A1
WO2019100727A1 PCT/CN2018/094562 CN2018094562W WO2019100727A1 WO 2019100727 A1 WO2019100727 A1 WO 2019100727A1 CN 2018094562 W CN2018094562 W CN 2018094562W WO 2019100727 A1 WO2019100727 A1 WO 2019100727A1
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cells
alzheimer
apoptosis
dna tetrahedron
inhibiting apoptosis
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林云锋
邵晓茹
蔡潇潇
马文娟
谢雪萍
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Sichuan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links

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  • the invention belongs to the technical field of apoptosis inhibition, and particularly relates to the application of a DNA tetrahedron in inhibiting apoptosis of Alzheimer's cells.
  • AD Alzheimer's disease
  • Its pathogenesis is characterized by mild cognitive dysfunction in the early stage of the disease, and then neuronal damage, which eventually evolves into dementia.
  • the pathogenesis of Alzheimer's disease is still unclear, and the broader impact is the beta-amyloid cascade hypothesis.
  • the hypothesis is that the deposition of A ⁇ in the brain is the central link of AD pathological changes, which can trigger a series of pathological processes, which further promote A ⁇ deposition, thus forming a cascade amplification reaction. Therefore, the formation, deposition and toxicity of anti-A ⁇ become the fundamental strategy for the treatment of AD.
  • studies on the neuroprotective effects of Alzheimer's cell model of certain drugs or materials have found that the drugs or materials used may have certain toxic effects on cells, ie, biocompatibility, biosafety, and biodegradability. Poor and other characteristics.
  • TDNs DNA tetrahedral nanomaterials
  • S1, S2, S3, S4 DNA single strands
  • TDNs have good biocompatibility, biosafety and biodegradability and are simple in preparation, high in yield and good in nuclease resistance.
  • the present invention provides a DNA tetrahedron for inhibiting apoptosis of Alzheimer's cells, which can effectively inhibit A ⁇ 25-35 in cells and enhance cell activity. Inhibition of apoptosis.
  • inhibition of Alzheimer's apoptosis by DNA tetrahedrons is achieved by modulating the expression level of a protein positively and negatively associated with apoptosis.
  • the process of DNA tetrahedron inhibiting apoptosis of Alzheimer's cells includes inhibiting the protein expression level of the apoptosis positive correlation protein Bax.
  • the process of DNA tetrahedron inhibiting Alzheimer's cell apoptosis includes inhibiting the protein expression level of the apoptosis positive correlation protein Caspase 3.
  • the process of inhibiting apoptosis of Alzheimer's cells by DNA tetrahedron includes inhibiting the protein expression level of cytochrome C, a positive correlation protein of apoptosis.
  • the process of DNA tetrahedron inhibiting apoptosis of Alzheimer's cells includes promoting the protein expression level of the apoptosis negative correlation protein Bcl-2.
  • the process of DNA tetrahedron inhibiting apoptosis of Alzheimer's cells includes inhibiting the expression levels of apoptosis-positive genes Bax and Caspase 3, and promoting the expression of apoptosis-related negative gene Bcl-2.
  • the concentration of the DNA tetrahedron in inhibiting apoptosis of Alzheimer's cells is 50 to 500 nM.
  • the concentration of the DNA tetrahedron in inhibiting apoptosis of Alzheimer's cells is 100 to 300 nM.
  • the concentration of the DNA tetrahedron in inhibiting apoptosis of Alzheimer's cells was 250 nM.
  • TDNs DNA tetrahedrons
  • Bax Bax
  • Caspase 3 cytochrome C protein
  • Bcl-2 Bcl-2
  • TDNs can also inhibit the formation of A ⁇ 25-35 in Alzheimer's cells, in order to reduce the deposition of A ⁇ 25-35 and enhance cell activity.
  • TDNs have good biocompatibility, biodegradability and low cytotoxicity, which can promote the proliferation and differentiation of nerve cells, and study the neuroprotective effect of TDNs on Alzheimer's cell model. And provide new ideas for the treatment of Alzheimer's disease.
  • FIG. 1 is a schematic representation of four single-stranded synthetic TDNs.
  • Figure 2 is a graph showing the results of 8% non-denaturing polyacrylamide gel electrophoresis (PAGE).
  • Figure 3 is a schematic diagram showing the results of transmission electron microscopy.
  • Figure 4 is a schematic diagram showing the results of atomic force microscopy.
  • Fig. 5 is a graph showing the results of examining the effect of the concentration of A ⁇ 25-35 on the activity of Alzheimer's cells.
  • Figure 6 is a graph showing the results of the effect of TDNs on the cell viability of Alzheimer's cell model.
  • Figure 7 is a graph showing the results of TDNs on the apoptosis of Alzheimer's cell model.
  • Figure 8 is a graph showing the results of detection of the effects of TDNs on apoptosis in Alzheimer's cell model.
  • Figure 9 is a graph showing the results of detecting the effect of TDNs on the cell cycle of Alzheimer's cells by flow cytometry.
  • Figure 10 is a graph showing the results of detection of inhibition of apoptosis of Alzheimer's cells by flow cytometry.
  • Figure 11 is a graph showing the results of detection of the expression of apoptosis-positive proteins Bax, Caspase3, cytochrome C, and apoptosis-related protein Bcl-2 in Alzheimer's cells under the action of TDNs.
  • Figure 12 is a graph showing the results of detection of the expression of apoptosis-positive genes Bax, Caspase 3, and apoptosis-related gene Bcl-2 in Alzheimer's cells under the action of TDNs.
  • Figure 13 is a graph showing the results of detection of the expression of apoptosis-related protein Bcl-2 in Alzheimer's cells under the action of TDNs.
  • TM buffer was prepared by 10 mM Tris-HCl and 50 mM MgCl 2 , and the pH was adjusted to 8.0, and then four ss DNA single strands (S1, S2, S3, S4) were prepared into 100 ⁇ L by ultra-micro quantitative method, each strip a system with a single chain concentration of 1 ⁇ M;
  • Polyacrylamide gel was prepared by using 4.2 mL of distilled water, 1.2 mL of 40% acrylamide, 0.6 mL of 10 ⁇ TAE, 60 ⁇ L of 10% Aps and 6 ⁇ L of TEMED;
  • the red circle is labeled as the synthesized tetrahedron, and the band of about 500 bp is the polymer formed during the synthesis of the tetrahedron, indicating that the DNA tetrahedron is successfully synthesized.
  • TDNs were identified by transmission electron microscopy and atomic force microscopy, respectively, and the results are shown in Figure 3 and Figure 4, respectively;
  • the shape of TDNs (marked by red triangles) is approximately triangular in shape under transmission electron microscopy, and the particle size is about 10 to 15 nM. As shown in Fig. 4, the TDNs have a particle size of about 10 to 15 nM, and transmission. The results of electron microscopy were consistent, which further indicated that the DNA tetrahedron was successfully synthesized.
  • the cultured cell suspension was divided into the control group and the experimental group.
  • the experimental group was added with different concentrations of A ⁇ 25-35 .
  • the control group was added with the same amount of serum-free DMEM medium, and then cultured in the incubator for 24 h (37 ° C). , 5% CO 2 ), after the end of the culture, add 10 ⁇ L of CCK-8 solution to each well, and avoid the production of air bubbles, and then incubate in the incubator for 1 to 4 hours (37 ° C, 5% CO 2 ).
  • the absorbance of each well was measured at 450 nm, and data processing was performed, and the results are shown in Fig. 5;
  • the concentrations of A ⁇ 25-35 were 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M and 100 ⁇ M, respectively.
  • the concentration of A ⁇ 25-35 was 25 ⁇ M, the cell activity was less than 70%, indicating that the modeling was successful.
  • the cultured cell suspension was divided into blank control group, TDNs group, AD cell model group and TDNs pretreated AD cell model group; among them, blank control group did not add A ⁇ 25-35 ; TDNs pretreated AD cells
  • the model group was pretreated with 250 nM TDNs for 6 h, then A ⁇ 25-35 was added at a concentration of 25 ⁇ M; the AD cell model group was pretreated with serum-free medium for 6 h, then A ⁇ 25-35 at a concentration of 25 ⁇ M was added; Groups, TDNs group, AD cell model group and TDNs pretreated AD cell model group were placed in the incubator for 24h (37 ° C, 5% CO 2 ).
  • the cell suspension was inoculated into a confocal dish, and grouped according to the step (2) in Example 3, and correspondingly treated, and then 24 hours later, each group of different medium was aspirated, and PBS was washed three times. , every 15 minutes;
  • Sheep serum was incubated for 1 hour at room temperature, washed with PBS three times each time;
  • the tetrahedral group had better spreading than the blank control group.
  • the cells in the AD cell model group showed obvious apoptosis, the number of cells decreased, the nucleus appeared to be lysed, and pyknosis.
  • the number of cells in the TDNs pretreated AD cell model group was reduced, the cell spreading was worse than that in the blank control group, but the nucleus was intact, and no obvious nuclear pyknosis occurred.
  • the apoptosis phenomenon was significantly lower than that of the AD cell model group.
  • step (2) grouping according to step (2) in Example 3, and performing corresponding treatment, and then inducing apoptosis, and setting up a negative control group, and collecting the cells;
  • the red fluorescent cells were collected by centrifugation with 0.25% trypsin in a 15 mL centrifuge tube (2000 rpm, 5 minutes), the supernatant was discarded, washed with PBS, and centrifuged (2000 rpm, 5 minutes). 500 ul of fixed cells were added to ice ethanol and allowed to stand overnight at 4 °C. The next day, PBS was added for centrifugation, the supernatant was discarded, washed with PBS, centrifuged, and the supernatant was discarded. Add 100 ⁇ L of RNase in a 37 ° C water bath for 30 minutes. Add 400 ⁇ L of PI stain and mix well, avoiding light at 4 ° C for 30 minutes. The cells were transferred to a flow tube, tested on the machine, and analyzed using WinMDI 2.9 and WinCycle 32 software. The results are shown in Figure 9;
  • the number of cells in the S phase (DNA synthesis phase) in the TDNs group increased significantly, indicating that TDNs changed the cell cycle of PC12 and promoted its value-added.
  • the TDNs pretreated AD cell model group had no obvious apoptotic peak, and the number of cells in the S phase (DNA synthesis phase) increased significantly, indicating that TDNs changed the cell cycle of the AD cell model.
  • step (2) grouping according to step (2) in embodiment 3, and performing corresponding processing
  • the proportion of apoptosis in the TDNs group was reduced compared to the blank control group. This indicates that TDNs have a certain inhibitory effect on apoptosis of PC12 cells.
  • the TDNs pretreated AD cell model group showed a significant decrease in the apoptotic ratio, indicating that TDNs significantly inhibited the apoptosis of the AD cell model.
  • step (2) grouping according to step (2) in Example 3, and using the whole protein extraction kit to extract proteins in the blank control group, the TDNs group, the AD cell model group, and the TDNs pretreated AD cell model group;
  • TDNs inhibit apoptosis of PC12 cells.
  • the expression of Bax, Caspase 3 and cytochrome C in the AD cell model group pretreated by TDNs decreased, and the expression of the negative correlation protein Bcl-2 increased. It is further indicated that TDNs have a significant inhibitory effect on the apoptosis phenomenon of AD cell model.
  • step (2) grouping according to step (2) in embodiment 3, and performing corresponding processing
  • step (2) grouping according to step (2) in embodiment 3, and performing corresponding processing
  • step (3) sucking the medium of step (2), washing 3 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;
  • Primary antibody Bcl-2 antibody was treated at 4 ° C overnight. On the next day, the temperature was rewarmed at 37 ° C for 0.5 hours, and the primary antibody was recovered and washed three times with PBS for 5 minutes each time. Fluorescent secondary antibody treatment, protected from light, 37 ° C, 1 hour, the secondary antibody was aspirated, washed three times with PBS for 5 minutes each time;
  • the expression of the apoptosis-related negative protein Bcl-2 was increased in the TDNs group compared with the blank control group, thereby further indicating that TDNs inhibited apoptosis of PC12 cells.
  • the TDNs pretreated AD cell model group increased the expression of apoptosis-related protein Bcl-2, which further indicated that TDNs had a significant inhibitory effect on the apoptosis of AD cell model.

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Abstract

一种DNA四面体在抑制阿尔兹海默细胞凋亡中的应用。其中，DNA四面体的四条单链序列如SEQ ID NO:1-4所示。该应用可有效的抑制细胞凋亡，提升细胞活性。

Description

DNA四面体在抑制阿尔兹海默细胞凋亡中的应用 技术领域
本发明属于细胞凋亡抑制技术领域，具体涉及一种DNA四面体在抑制阿尔兹海默细胞凋亡中的应用。
背景技术
阿尔兹海默病(AD)是常见的神经退行性疾病之一，具有年龄相关性，其发病特点为在疾病初期主要表现为轻微的认知功能障碍，而后出现神经元的损伤，最后演变为痴呆。阿尔兹海默病的发病机制目前尚不明确，影响较广的是β类淀粉样蛋白级联假说。该假说认为Aβ在脑内沉积是AD病理改变的中心环节，可引发一系列病理过程，这些病理过程又进一步促进Aβ沉积，从而形成级联式放大反应。因此，对抗Aβ的形成，沉积及毒性作用成为治疗AD的根本策略。目前关于某些药物或材料阿尔兹海默细胞模型神经保护作用的研究发现，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。
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具有广泛的应用前景。TDNs是由四条DNA单链(S1、S2、S3、S4)通过严格碱基互补配对原则形成的具有三维结构的DNA纳米材料。TDNs具有良好的生物相容性、生物安全性和生物可降解性且制备方法简单，产率较高，具有较好核酸酶耐受性。关于DNA四面体的研究虽多，但它对细胞各项生理活动影响的研究却很少，尤其是抑制细胞凋亡的研究更是没有。
发明内容
针对现有技术中的上述不足，本发明提供一种DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，该DNA四面体可有效的抑制细胞中的Aβ 25-35，提升细胞 活性，抑制细胞凋亡。
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四面体在抑制阿尔兹海默细胞凋亡中的应用，其中，DNA四面体的四条单链序列如SEQ ID NO:1-4所示。
进一步地，DNA四面体抑制阿尔兹海默细胞凋亡是通过调节与凋亡正负相关的蛋白的表达量来实现的。
进一步地，DNA四面体抑制阿尔兹海默细胞凋亡的过程包括抑制凋亡正相关蛋白Bax的蛋白表达量。
进一步地，DNA四面体抑制阿尔兹海默细胞凋亡的过程包括抑制凋亡正相关蛋白Caspase 3的蛋白表达量。
进一步地，DNA四面体抑制阿尔兹海默细胞凋亡的过程包括抑制凋亡正相关蛋白细胞色素C的蛋白表达量。
进一步地，DNA四面体抑制阿尔兹海默细胞凋亡的过程包括促进凋亡负相关蛋白Bcl-2的蛋白表达量。
进一步地，DNA四面体抑制阿尔兹海默细胞凋亡的过程分别包括抑制凋亡正相关基因Bax和Caspase 3的表达量，以及促进凋亡负相关基因Bcl-2的表达量。
进一步地，DNA四面体在抑制阿尔兹海默细胞凋亡过程中的浓度为50～500nM。
进一步地，DNA四面体在抑制阿尔兹海默细胞凋亡过程中的浓度为100～300nM。
进一步地，DNA四面体在抑制阿尔兹海默细胞凋亡过程中的浓度为250nM。
本发明的有益效果为：
1、DNA四面体(TDNs)通过抑制阿尔兹海默细胞中与凋亡正相关的Bax，Caspase 3，细胞色素C蛋白的表达量，促进凋亡负相关的Bcl-2的表达量，达到抑制细胞凋亡的目的，同时，TDNs还可抑制阿尔兹海默细胞内Aβ 25-35的形成，以降低Aβ 25-35的沉积量，提升细胞的活性。
2、TDNs具有良好的生物相容性，生物可降解性以及较低的细胞毒性，对于神经细胞的增殖分化具有一定的促进作用，研究TDNs对于阿尔兹海默细胞模型的神经保护作用，为预防和治疗阿尔兹海默病提供新的思路。
附图说明
图1为四条单链合成TDNs的示意图。
图2为8％非变性聚丙烯酰胺凝胶电泳(PAGE)鉴定结果图。
图3为透射电镜鉴定结果示意图。
图4为原子力显微镜鉴定结果示意图。
图5为Aβ 25-35的浓度对阿尔兹海默细胞活性影响的检测结果示意图。
图6为TDNs对阿尔兹海默细胞模型细胞活性影响的检测结果图。
图7为TDNs对阿尔兹海默细胞模型细胞凋亡影响的检测结果图。
图8为TDNs对阿尔兹海默细胞模型细胞凋亡影响的检测结果图。
图9为采用流式细胞术检测TDNs对阿尔兹海默细胞细胞周期影响的检测结果图。
图10为采用流式细胞术检测TDNs对阿尔兹海默细胞凋亡抑制作用的检测结果图。
图11为在TDNs作用下，阿尔兹海默细胞中凋亡正相关蛋白Bax、Caspase3、细胞色素C，以及凋亡负相关蛋白Bcl-2表达量的检测结果图。
图12为在TDNs作用下，阿尔兹海默细胞中凋亡正相关基因Bax、Caspase 3，以及凋亡负相关基因Bcl-2表达量的检测结果图。
图13为在TDNs作用下，阿尔兹海默细胞中凋亡负相关蛋白Bcl-2表达量的检测结果图。
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 TDNs的合成及鉴定
1、TDNs的合成
通过10mM Tris-HCl和50mM MgCl 2制备得到TM buffer，并将其pH调节至8.0，然后呈采用超微量定量法将四条ss DNA单链(S1、S2、S3、S4)制备为100μL，每条单链浓度为1μM的体系；
然后再涡旋，混匀，离心后置于PCR仪内，将温度迅速升高到95℃，并保持10min，再冷却至4℃，保持20min，即合成得到浓度约为1000nM的TDNs，其合成过程如图1所示；其中，四条DNA单链的具体序列如下：
S1：
5’-ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA-3’(SEQ ID NO:1)；
S2：
5’-ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG-3’(SEQ ID NO:2)；
S3：
5’-ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC-3’(SEQ ID NO:3)；
S4：
5’-ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG-3’(SEQ ID NO:4)。
2、TDNs的鉴定
(1)采用8％非变性的聚丙烯酰胺凝胶电泳(PAGE)进行鉴定
采用4.2mL蒸馏水、1.2mL 40％丙烯酰胺、0.6mL 10×TAE、60μL 10％Aps以及6μL 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，红色圆圈标注为所合成的四面体，500bp左右的条带为合成四面体过程中形成的多聚物，表明成功合成了DNA四面体。
(2)分别采用透射电镜和原子力显微镜对TDNs进行鉴定，其结果分别见图3和图4；
如图3所示，TDNs(红色三角形标注)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；如图4所示，TDNs粒径在10～15nM左右，与透射电镜结果吻合，进一步表明成功合成了DNA四面体。
实施例2 阿尔兹海默细胞模型的建立及验证
(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，实验组加入不同浓度的Aβ 25-35，对照组加入等量无血清的DMEM培养基，然后在孵箱中培养24h(37℃，5％CO 2)，培养结束后，向每孔中加入10μL的CCK-8溶液，并避免生产气泡，然后在孵箱中培养1～4h(37℃，5％CO 2)培育结束后，在450nm处检测每孔的吸光度，并进行数据处理，其结果见图5；
如图5所示，Aβ 25-35的浓度分别为6.25μM、12.5μM、25μM，50μM和100μM，当Aβ 25-35的浓度为25μM时，细胞活性低于70％，表明建模成功。
实施例3 TDNs对阿尔兹海默细胞模型细胞活性的影响
(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空白对照组、TDNs组、AD细胞模型组以及TDNs预处理的AD细胞模型组；其中，空白对照组不加Aβ 25-35；TDNs预处理的AD细胞模型组先用250nM的TDNs预处理6h，然后加入浓度为25μM的Aβ 25-35；AD细胞模型组用无血清培养基预处理6h，然后再加入浓度为25μM的Aβ 25-35；然后空白对照组、TDNs组、AD细胞模型组以及TDNs预处理的AD细胞模型组均置于孵箱中培养24h(37℃，5％CO 2)。
(3)在450nm处检测每孔的吸光度，并进行数据处理，其结果见图6；
如图6所示，加入四面体后，细胞有明显增殖，与前期结果一致，且经TDNs 预处理的TDNs+Aβ 25-35组，细胞活性较Aβ 25-35组有提高，表明TDNs可提高阿尔兹海默细胞的细胞活性。
实施例4 TDNs对阿尔兹海默细胞模型细胞凋亡的影响
1、采用免疫荧光技术检测TDNs对阿尔兹海默细胞模型细胞凋亡的影响
(1)将细胞悬浮液接种于共聚焦小皿中，并按实施例3中步骤(2)进行分组，并进行相应处理，然后24小时后吸去各组不同的培养基，PBS反复洗三遍，每遍15分钟；
(2)使用4％多聚甲醛在4℃固定15分钟，而后空气中放置5分钟，PBS洗三遍，每遍15分钟；
(3)0.5％tritonX-100试剂37℃打孔15分钟，PBS洗三遍，每遍15分钟；
(4)羊血清室温孵育1小时，PBS洗，每次三遍；
(5)孵育所要观察的一抗(抗nestin抗体)，4℃过夜；
(6)37℃复温30分钟，PBS洗三遍，每次15分钟；
(7)37℃孵育二抗1小时，PBS洗三遍，每次15分钟；
(8)细胞核染料DAPI处理10分钟，再用PBS清洗；
(9)用试剂鬼笔环肽进行细胞骨架染色10分钟，PBS洗；
(10)镜检，共聚焦显微镜观察细胞免疫荧光染色的情况，其结果见图7、图8；
如图7和图8所示，四面体组较空白对照组，细胞有更好的铺展。AD细胞模型组细胞出现明显凋亡，细胞数目减少，细胞核出现裂解，固缩。TDNs预处理的AD细胞模型组虽然细胞数目有所减少，细胞铺展较空白对照组差，但细胞核完整，未出现明显核固缩的现象，凋亡现象较AD细胞模型组有了大幅的降低。
2、采用流式细胞术检测细胞周期
(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)按实施例3中步骤(2)进行分组，并进行相应处理，然后诱导细胞凋亡，同时设立阴性对照组，并收集细胞；
(3)用PBS洗涤细胞一次，收集并调整细胞浓度为1×10 6/mL；然后将制备的单细胞悬液用体积分数为70％乙醇固定，4℃保存，染色前用PBS洗去固定液；
(4)向步骤(3)所得细胞悬液中加入RNase A，37℃，水浴30min，再加入PI染色混匀，于4℃下避光30min，然后上机检测，并记录激发波长为488nm处的红色荧光；
(5)用0.25％胰蛋白酶消化收集红色荧光处细胞于15mL离心管中(2000rpm、5分钟)，弃上清，PBS洗涤，离心(2000rpm、5分钟)。加入冰乙醇500ul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。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，使用WinMDI 2.9和WinCycle 32软件进行数据分析，其结果见图9；
如图9所示，与空白对照组相比，TDNs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PC12的细胞周期，促进其增值。TDNs预处理的AD细胞模型组较AD细胞模型组，无明显凋亡峰，且处于S期(DNA合成期)的细胞数目明显增加，说明TDNs改变了AD细胞模型的细胞周期。
3、采用流式细胞术检测TDNs对阿尔兹海默细胞凋亡的抑制作用
(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)按实施例3中步骤(2)进行分组，并进行相应处理；
(3)使用不含EDTA的胰蛋白酶，分别消化收集细胞于15mL离心管中(300g、5分钟)，弃上清，PBS洗涤，离心(300g、5分钟)。用400μL Annexin V结合液悬浮细胞，浓度大约为1×10 6cells/mL。在细胞悬液中加入5μL Annexin V-FITC染色液，轻轻混匀于4℃避光条件下孵育15分钟。加入5μL PI染色液，轻轻混匀于4℃避光条件下孵育5分钟。将细胞转移至流式管中，上机检测，数据分析，其结果见图10。
如图10所示，空白对照组相比，TDNs组中凋亡比例有所降低。说明TDNs对于PC12细胞的细胞凋亡具有一定抑制作用。TDNs预处理的AD细胞模型组较AD细胞模型组，凋亡比例明显降低，说明TDNs对于AD细胞模型的细胞凋亡现象具有明显的抑制作用。
实施例5 TDNs抑制阿尔兹海默细胞凋亡的机制
1、采用蛋白质印迹法检测TDNs抑制阿尔兹海默细胞凋亡的机制
(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)按实施例3中步骤(2)进行分组处理，并使用全蛋白提取试剂盒， 分别提取空白对照组、TDNs组、AD细胞模型组以及TDNs预处理的AD细胞模型组中的蛋白；
(3)然后再分别进行SDS-PAGE电泳，其具体过程为：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11。
如图11所示，与空白对照组相比，TDNs组中凋亡正相关蛋白Bax，Caspase 3，细胞色素C的蛋白表达量均有所下降，负相关蛋白Bcl-2的表达有所增加，从而，进一步说明TDNs抑制PC12细胞的凋亡。TDNs预处理的AD细胞模型组较AD细胞模型组，凋亡正相关蛋白Bax，Caspase 3，细胞色素C的蛋白表达量均有所下降，负相关蛋白Bcl-2的表达有所增加，从而，进一步说明TDNs对于AD细胞模型的细胞凋亡现象具有明显的抑制作用。
2、采用荧光定量PCR(Q-PCR)检测TDNs抑制阿尔兹海默细胞凋亡的机制
(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)按实施例3中步骤(2)进行分组，并进行相应处理；
(3)Q-PCR检测：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12；
如图12所示，与空白对照组相比，TDNs组中凋亡正相关基因Bax，Caspase  3的表达量均有所下降，负相关基因Bcl-2的表达有所增加，从而，进一步说明TDNs抑制PC12细胞的凋亡。TDNs预处理的AD细胞模型组较AD细胞模型组，凋亡正相关基因Bax，Caspase 3的表达量均有所下降，负相关基因Bcl-2的表达有所增加，从而，进一步说明TDNs对于AD细胞模型的细胞凋亡现象具有明显的抑制作用。
3、采用免疫荧光技术检测TDNs抑制阿尔兹海默细胞凋亡的机制
(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)，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)按实施例3中步骤(2)进行分组，并进行相应处理；
(3)然后吸去步骤(2)所述培养基，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Bcl-2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13。
如图13所示，与空白对照组相比，TDNs组中凋亡负相关蛋白Bcl-2的表达有所增加，从而，进一步说明TDNs抑制PC12细胞的凋亡。TDNs预处理的AD细 胞模型组较AD细胞模型组，凋亡负相关蛋白Bcl-2的表达有所增加，从而，进一步说明TDNs对于AD细胞模型的细胞凋亡现象具有明显的抑制作用。

Claims (10)

  1. DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，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四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体抑制阿尔兹海默细胞凋亡是通过调节与凋亡正负相关的蛋白的表达量来实现的。
  3. 根据权利要求2所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体抑制阿尔兹海默细胞凋亡的过程包括抑制凋亡正相关蛋白Bax的蛋白表达量。
  4. 根据权利要求2所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体抑制阿尔兹海默细胞凋亡的过程包括抑制凋亡正相关蛋白Caspase 3的蛋白表达量。
  5. 根据权利要求2所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体抑制阿尔兹海默细胞凋亡的过程包括抑制凋亡正相关蛋白细胞色素C的蛋白表达量。
  6. 根据权利要求2所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体抑制阿尔兹海默细胞凋亡的过程包括促进凋亡负相关蛋白Bcl-2的蛋白表达量。
  7. 根据权利要求2所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体抑制阿尔兹海默细胞凋亡的过程分别包括抑制凋亡正相关基因Bax和Caspase 3的表达量，以及促进凋亡负相关基因Bcl-2的表达量。
  8. 根据权利要求1～7任一项所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体在抑制阿尔兹海默细胞凋亡过程中的 浓度为50～500nM。
  9. 根据权利要求8所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体在抑制阿尔兹海默细胞凋亡过程中的浓度为100～300nM。
  10. 根据权利要求8所述的DNA四面体在抑制阿尔兹海默细胞凋亡中的应用，其特征在于，所述DNA四面体在抑制阿尔兹海默细胞凋亡过程中的浓度为250nM。
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