WO2022160374A1 - 一种靶向cd206阳性细胞的核酸适体及其应用 - Google Patents

一种靶向cd206阳性细胞的核酸适体及其应用 Download PDF

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WO2022160374A1
WO2022160374A1 PCT/CN2021/075671 CN2021075671W WO2022160374A1 WO 2022160374 A1 WO2022160374 A1 WO 2022160374A1 CN 2021075671 W CN2021075671 W CN 2021075671W WO 2022160374 A1 WO2022160374 A1 WO 2022160374A1
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chimera
nucleic acid
aptamer
acid aptamer
cells
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姚燕丹
宋尔卫
张明霞
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中山大学孙逸仙纪念医院
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  • the invention relates to a biomolecule drug carrier technology in the field of biomedicine, in particular to a nucleic acid aptamer targeting CD206 positive cells and its application.
  • RNA intervention is a powerful weapon for inhibiting gene expression.
  • RNAi ribonucleic acid interference
  • Tuchl et al. introduced an artificially synthesized exogenous silencing RNA (siRNA) of about 19-23 base pairs into mammalian cells, which could induce RNAi effects that specifically inhibit the expression of complementary sequences.
  • siRNA silencing RNA
  • RNA intervention was used to inhibit the expression of pathogenic genes, and a variety of new gene drugs were developed for the treatment of malignant tumors.
  • antisense oligonucleotides and ribozymes the effect of siRNA to silence gene expression is tens to hundreds of times more powerful.
  • siRNA silencing gene interference treatment of diseases lies in: first, finding therapeutic targets; second, finding targeted delivery vehicles.
  • TAMs tumor-associated macrophages
  • cytokine CCL18 secreted by them play an important role in the infiltration and metastasis of tumor cells.
  • the cytokine CCL18 is a member of the C-C membrane somatic cytokine receptor family.
  • the CCL18 factor secreted by TAMs can induce the EMT of breast cancer cells by activating the PI3K/Akt pathway, thereby promoting the invasion and metastasis of breast cancer cells.
  • tumor-associated macrophages can secrete CCL18 to induce epithelial-mesenchymal transition (EMT) in breast cancer cells, enhance the latter's invasion and metastasis.
  • EMT epithelial-mesenchymal transition
  • Tumor-associated macrophages have been found to have tumor-promoting effects not only in breast cancer, but also in more solid malignancies-related literature reports. Therefore, tumor-associated macrophages can be used as an important target for the treatment of breast cancer, and can even be developed into a common target for the treatment of various solid malignancies.
  • CD206 also known as mannose receptor, is a class of receptors that are mainly expressed on the cell membrane surface of macrophages and dendritic cells, especially on the surface of tumor-associated macrophages.
  • Receptor protein a member of the C-type lectin superfamily.
  • RNAi successfully inhibited the expression of oncogenes such as k-ras and cyclin E, the expression of tumor anti-apoptotic gene BCL-2 and tumor drug resistance gene.
  • mdr1 expression and effectively reduced cancer cell proliferation and increased its sensitivity to chemotherapeutic drugs.
  • RNAi silencing Her2 gene expression also successfully inhibited the proliferation of breast cancer cells in vitro.
  • RNAi anti-tumor is directly transfecting or transducing RNAi in tumor cells cultured in vitro or injecting RNAi directly into the transplanted tumor tissue of nude mice. Although these experiments have achieved certain success, they are far from clinical. The real use of RNAi to treat tumors is still a long way off.
  • RNAi the main obstacle to the application of RNAi is how to introduce specific RNAi into the cytoplasm of target cells in clinical application, especially into tumor cells that overexpress the target gene.
  • the delivery vehicle is the key to RNA intervention.
  • the more common small molecule RNA carriers are:
  • siRNA mainly includes antibodies and their fragments and short peptides and polypeptides, such as antibody-coupled siRNA delivery systems.
  • Protein molecules such as antibodies coupled to siRNA molecules enter cells when they bind to surface antigen molecules of cells or target organs, thereby enabling siRNA to exert gene interference effects.
  • the advantage of this drug delivery system is that it has binding specificity through the combination of antigen and antibody molecules, but there is antigen non-specificity.
  • the immunogenicity of macromolecular proteins in the body and the permeability of the internal environment barrier lead to drug consumption and toxic side effects.
  • the reaction, as well as the specificity of production lead to high costs.
  • nanomaterials are used in drug delivery system research.
  • Polymer nanomaterials can passively or actively target tumor tissues to deliver drugs through physical and chemical properties. Compared with proteins, they are easier to pass through the physiological barrier and be absorbed, and have relatively few toxic and side effects.
  • controlled-release nanomaterials which have safer and more controllable drug delivery effects.
  • stability factors and biosafety factors in the current research on nanomaterials and the cost is relatively high.
  • nucleic acid aptamers or nucleic acid aptamers which are small fragments of single-stranded oligonucleotides, generally within 200 bases in length, and can be naturally folded through their own sequence characteristics. , so that it has a high binding ability to specific molecules.
  • This nucleic acid fragment exists in nature and can also be screened by phylogenetic evolution of ligands by exponential enrichment (SELEX). Because of its small molecular weight, it is easy to be internalized into cells by binding to the target protein, which also makes it have a dual role, which can not only identify, but also assist in the internalization of drugs, and such small molecular nucleic acid fragments are easily obtained after screening and synthesis. It is relatively simple and fast, easy to carry out chemical modification and multifunctionalization, good tissue penetration, less immunogenicity, and less toxic and side effects.
  • the present invention provides a nucleic acid aptamer targeting CD206 positive cells, the sequence of which includes the following sequence: 5'-TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGA-3'.
  • the nucleic acid aptamer preferably has the two spatial secondary structures shown in FIG. 1 .
  • the CD206 positive cells include macrophages or dendritic cells.
  • the present invention further discloses its application in preparing a kit for detecting CD206 positive cells, a molecular probe and a targeted drug carrier.
  • the invention further discloses a chimera formed by carrying the above-mentioned nucleic acid aptamer with a small molecule RNA drug and targeting CD206 positive cells; and its application in the preparation of antitumor drugs.
  • a chimera that inhibits the secretion and synthesis of cytokine CCL18 and its application in the preparation of anti-breast cancer drugs are disclosed.
  • the chimera is formed by combining a nucleic acid aptamer with CCL18 siRNA. Its sequence is:
  • the preparation method of described chimera comprises the steps:
  • Synthesize nucleic acid aptamers connect the intermediates of RNA sense strands and RNA antisense strands;
  • the nucleic acid aptamer of the present invention can accurately identify CD206 positive cells, has excellent specific targeting and affinity, and can be used as a kit, molecular probe and targeted drug carrier for detecting CD206 positive cells.
  • the nucleic acid aptamer has a specific spatial structure and an appropriate molecular weight, and can effectively carry small-molecule RNA drugs such as siRNA to the target cells to achieve precise and efficient drug efficacy.
  • the chimera combined with CCL18siRNA can significantly inhibit the synthesis and secretion of CCL18 cytokines, greatly reduce the migration and invasion ability of breast cancer cells, and provide a new therapeutic approach for advanced breast cancer.
  • the nucleic acid aptamer is a non-viral vector, which helps to accelerate the clinical application of RNAi technology, has high biological safety, and can be synthesized artificially, with a simple synthesis process and large-scale production.
  • Fig. 1 is the secondary structure diagram of the nucleic acid aptamer of the embodiment of the present invention.
  • Fig. 2 is the secondary structure diagram of the chimera of the embodiment of the present invention.
  • Fig. 3 is the schematic diagram of chimera annealing synthesis according to the embodiment of the present invention.
  • Fig. 4 is the electrophoresis band diagram of the chimera of the embodiment of the present invention.
  • Figure 5 is a diagram showing the effect of the chimera specifically binding to CD206 positive cells according to an embodiment of the present invention.
  • FIG. 6 is a graph showing the effect of knocking down the expression of CCL18 in tumor-associated macrophages by chimeras according to an embodiment of the present invention
  • FIG. 7 is a graph showing the effect of chimera inhibiting the invasion and migration of breast cancer cells according to an embodiment of the present invention.
  • Fig. 8 is the effect diagram of chimera inflammation-related factor test according to the embodiment of the present invention.
  • Example 1 Screening of CD206 nucleic acid aptamer and construction of a chimera in which the nucleic acid aptamer was linked to CCL18 siRNA.
  • the CD206 nucleic acid aptamer was screened with human recombinant CD206 protein in the DNA library by exponential enrichment ligand system evolution technology (SELEX) to obtain 8 (S1-S8) nucleic acid fragments with high affinity and specificity. See Table 1. For laboratory verification and screening, refer to Table 2 and select the one with the best specificity and drug delivery (S5) to continue the subsequent experiments.
  • the S5 nucleic acid fragment obtained by screening was significantly different from the other seven nucleic acid fragments in sequence, secondary structure and molecular size. Experiments have shown that the specificity and drug delivery of the S5 nucleic acid fragment are significantly better than the other seven nucleic acid fragments. Only the experimental results of S5 will be recorded subsequently.
  • the resulting S5 sequence was: 5'-TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGA-3'.
  • the target cell aptamer has the affinity of recombinant human CD206 protein, and the target cell affinity is the binding specificity of aptamer to CD206 positive cells.
  • the CD206 nucleic acid aptamer, the intermediate of the nucleic acid aptamer connecting the sense strand of CCL18siRNA and the antisense strand of CCL18siRNA were synthesized and provided by Shanghai Gema Pharmaceutical Technology Co., Ltd. and Bao Bioengineering (Dalian) Co., Ltd.
  • the nucleic acid sequence is shown in the sequence table.
  • nucleic acid aptamer sequence is shown in SEQ ID NO.1; the CCL18siRNA sense strand sequence is shown in SEQ ID NO.2; the CCL18siRNA antisense strand is shown in SEQ ID NO.3; the intermediate sequence is shown in Shown in SEQ ID NO.4; the chimera sequence shown is shown in SEQ ID NO.5; the nucleic acid aptamer sequence of prostate specific membrane antigen (PSMA) is shown in SEQ ID NO.6.
  • PSMA prostate specific membrane antigen
  • the secondary structure of the full-length aptamer predicted in the secondary structure prediction software is shown in Figure 1. It can be found that the CD206 nucleic acid aptamer has two stem-loop structures, and the chimera shape after connecting with siRNA is shown in Figure 2. In terms of secondary structure, the chimera of CD206 nucleic acid aptamer and siRNA can also maintain its stem-loop structure.
  • Figure 3 shows the annealing and binding process of the chimera.
  • the lengths of the intermediate and chimera of siRNA, nucleic acid aptamer connected to the sense strand of siRNA are 21, 64, and 85 bases, respectively. The position corresponds to the position of the marker (marker) is basically correct.
  • TAMs tumor-associated macrophages
  • Mononuclear macrophages were isolated from the peripheral blood of healthy people by gradient centrifugation, and were grown and cultured adherently.
  • the medium containing IL4 factor at a concentration of 20ng/ml was induced for about 4-5 days, and the morphology of macrophages was observed under a microscope. It was found that the shape of macrophages changed from small circles to elongated needles, from scattered single to clusters. If the aggregation distribution is similar, the induction is successful.
  • TAMs add the CD206 nucleic acid aptamer labeled with Cy3 fluorophore at a final concentration of 10 nM.
  • TAMs were treated with an equal amount of prostate-specific membrane antigen (PSMA) aptamer (also labeled with Cy3) as control one, and TAMs were transfected with fluorescently-labeled siRNA double-strands transfected with liposomes as control two.
  • PSMA prostate-specific membrane antigen
  • the CD206 nucleic acid aptamer labeled with Cy3 fluorophore at final concentrations of 10, 20 and 50 nM was added to the TAMs, and incubated for 24 hours. The medium was removed and the TAMs were trypsinized. Wash off excess aptamer with PBS. Centrifuge at 300g for 5 minutes to discard the supernatant, add 100 ⁇ l of PBS to resuspend the cells, and add 2 ⁇ l of anti-human CD206 fluorescent antibody. After incubating at 4°C for 30 minutes, centrifuge as above to leave the pellet, add PBS to wash once to remove excess antibody, and then centrifuge to discard the supernatant and resuspend in 200 ⁇ l of PBS. Uptake rates were measured by flow cytometry.
  • Cy3-labeled chimeras were incubated with TAMs.
  • the CD206 nucleic acid aptamer constructed by the same method was transfected with liposomes as a positive control, and the nucleic acid aptamer for prostate specific antigen, namely PSMA, was used as a control for binding ability, and the abscissa was the Cy3 signal.
  • a fluorescent signal was detected and gradually shifted to the right along the abscissa with increasing uptake.
  • the CD206 nucleic acid aptamer could detect the fluorescent signal after 24 hours of treatment of macrophages, while the PSMA nucleic acid aptamer with the same fluorophore could not make macrophages fluorescent. This indicates that the CD206 aptamer can selectively bind to TAMs.
  • nucleic acid aptamers The binding of nucleic acid aptamers to cells was observed under a laser confocal fluorescence microscope. Cy3 shows red light under the microscope. If the cell takes up the nucleic acid aptamer, the red light can be detected in the cell, and the macrophages label the cell membrane with the green CD206 antibody. The aptamer with red light could be seen in macrophages treated with CD206 aptamer, but no red light could be seen in macrophages treated with PSMA aptamer. In conclusion, DNA aptamers have good high affinity and specific binding force, and chimeras can deliver small RNA molecules into tumor-associated macrophages. The results are shown in Part A of Figure 5 .
  • Example 3 Inhibitory effect of chimeras on the synthesis and secretion of CCL18 biological factors.
  • the chimera with a final concentration of 20nM to the TAMs the CD206 nucleic acid aptamer aptamer is the empty control, the chimera with the aptamer linked to GFP protein siRNA is the negative control, and the liposome transfection is used as the positive control.
  • the liposome transfection is used as the positive control.
  • Example 4 Chimeras inhibit the ability of TAMs to promote migration and invasion of breast cancer cells
  • transwell experiments were performed on TAMs and breast cancer cells MDA-MB-231 cells (hereinafter referred to as "231 cells").
  • the results are shown in Part A of Figure 7.
  • the migration ability of the latter was significantly increased; while the chimera-treated TAMs were co-cultured with 231 cells, 231 cells
  • the migratory ability of the cells was significantly decreased (the number of cells decreased by nearly 4-fold), which was similar to the transfection control group, while the control treatment group did not change significantly. This indicates that chimeras can inhibit the promotion of TAMs on breast cancer cell migration by interfering with the expression of CCL18 in TAMs.
  • the results of the invasion experiment are shown in part B of Figure 7.
  • the chimera also showed a similar effect to the ability to inhibit migration, and the number of cells also decreased by about 3 times, which indicates that the chimera can also inhibit breast cancer cells by inhibiting TAMs. Promotion of invasive ability.
  • the chimeras were incubated with TAMs for 24 hours, the mRNA of TAMs was collected in the same manner as above, and the mRNA expression levels of IL-6, IL-10, TGF-beta, CCL22 and IFN were detected by RT-QPCR.
  • the results are shown in Figure 8.
  • the mRNA expression levels of each group are basically the same. It can be seen that the addition of chimera has no significant difference in the expression of inflammatory factors compared with the control group, that is, the chimera does not induce the elevated expression of these types of inflammatory factors in TAMs. , indicating that the chimera does not cause inflammation in TAMs.

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Abstract

一种靶向CD206阳性细胞的核酸适体及其作为药物载体、检测试剂盒和分子探针等方面的应用。所述核酸适体可与CCL18 siRNA结合形成靶向CD206阳性细胞的抗肿瘤嵌合体，可明显抑制CCL18细胞因子的合成和分泌，大幅度降低乳腺癌细胞的迁移侵袭能力，为晚期乳腺癌提供新的治疗途径。所述核酸适体及其嵌合体具有优良的特异靶向性和亲和性，安全高效；可通过人工合成，规模化生产。

Description

一种靶向CD206阳性细胞的核酸适体及其应用 技术领域
本发明涉及生物医药领域的生物分子药物载体技术，具体涉及一种靶向CD206阳性细胞的核酸适体及其应用。
背景技术
RNA干预(RNAi)是抑制基因表达的强大武器。Andrew Z.Fire和Craig C.Mello两位科学家于1998年报道发现了核糖核酸干扰(Ribonucleic Acid Interference,RNAi)现象，并于2006年获得诺贝尔医学奖。2001年，Tuchl等把长度约为19-23碱基对、人工合成的外源性沉默RNA(siRNA)导入哺乳动物细胞内，能诱导出特异地抑制互补序列基因表达的RNAi效应。随后通过RNA干预抑制致病基因的表达，开拓出治疗包括恶性肿瘤在内的多种新型基因药物。与传统的抑制基因表达工具反义寡核苷酸和核糖酶比较，siRNA沉默基因表达的效应要强大数十倍至数百倍。
siRNA沉默基因干扰治疗疾病的关键在于：一、发现治疗靶点；二、找到靶向输送载体。
以乳腺癌为例，研究发现，在乳腺癌的微环境中，肿瘤相关巨噬细胞(以下简称“TAMs”)以及其分泌的细胞因子CCL18对肿瘤细胞的浸润转移有十分重要的作用。细胞因子CCL18是C-C膜体细胞因子受体家族的一员，TAMs分泌的CCL18因子可以通过激活PI3K/Akt通路诱导乳腺癌细胞的EMT从而促进乳腺癌细胞的侵袭与转移。和肿瘤相关巨噬细胞与乳腺癌细胞之间存在一种正反馈作用，它表现在肿瘤相关巨噬细胞可以分泌CCL18诱导乳腺癌细胞发生上皮-间充质转化(EMT)、加强后者侵袭和远处转移的能力；而乳腺癌细胞则同时大量分泌GM-CSF 促进肿瘤相关巨噬细胞继续分泌更多CCL18；这种正向促进作用可通过抑制CCL18因子的分泌被打破。肿瘤相关巨噬细胞不仅在乳腺癌，甚至在更多的实体恶性肿瘤相关文献报道中也被发现具有促肿瘤的作用。因此肿瘤相关巨噬细胞可作为治疗乳腺癌的一个重要靶标，甚至可发展为治疗多种实体恶性肿瘤的共同靶标。
CD206也叫甘露糖受体，是一类主要表达在巨噬细胞和树突状细胞的细胞膜表面，尤其在肿瘤相关巨噬细胞表面高表达、具有识别病原体、递呈抗原和保持内环境稳定的受体蛋白，属于C型凝集素超家族成员。以CD206作为靶向肿瘤相关巨噬细胞的靶点是可行的尝试。
在体外细胞培养或裸鼠移植肿瘤模型的实验中，有文献报道应用RNAi成功地抑制了癌基因如k-ras和cyclin E的表达，肿瘤抗凋亡基因BCL-2的表达和肿瘤耐药基因mdr1的表达，并有效地减少了癌细胞增殖，增加了其对化疗药物的敏感性。沉默Her2基因表达的RNAi也成功地抑制体外培养的乳腺癌细胞增殖。这些初步的实验充分证明了武装RNAi成为新一代抗肿瘤药物的潜力很大。但是，目前RNAi抗肿瘤的实验研究都是在体外培养的肿瘤细胞中直接转染或转导RNAi或在裸鼠移植肿瘤组织内直接注入RNAi，虽然这些实验获得一定的成功，但是距离在临床上真正使用RNAi治疗肿瘤还相差很远。
目前，RNAi应用的主要障碍在于如何在临床应用时把特异的RNAi导入目标细胞胞浆内起作用，特别是导入过量表达目标基因的肿瘤细胞内。输送载体是RNA干预的关键所在。较常见的小分子RNA载体有：
1.蛋白类
主要包括抗体及其片段和短肽和多肽，如抗体偶联siRNA递送体统。偶联siRNA分子的蛋白分子如抗体在与细胞或靶器官表面抗原分子结合时进入细胞从 而使siRNA发挥基因干扰作用。此递药系统的优势是通过抗原抗体分子结合的方式，具有结合特异性，但存在抗原非特异性，大分子蛋白在机体内的免疫原性及内环境屏障的透过率导致药物消耗及毒副反应，以及生产的特殊性导致费用高昂。
2.纳米材料
目前递药系统研究的一大热点即纳米材料，高分子纳米材料可通过物理化学性质被动或主动靶向肿瘤组织递送药物，相对蛋白类更容易穿过生理屏障被吸收，毒副作用相对较少，现也有研究可控释放的纳米材料，具有更安全可控的递药效果，但目前纳米材料的研究仍存在稳定因素及生物安全因素，且费用较高。
3.核酸类
主要指核酸适配体或核酸适体(aptamer)，这是一类小片段的单链寡聚核苷酸，长度一般在200个碱基以内，可通过自身的序列特点自然折叠形成的空间结构，从而与特定的分子具有高度的结合能力。这种核酸片段在自然界中存在，同时也可以通过指数富集配体系统进化技术(SELEX)筛选得到。因其分子量很小，易通过结合靶蛋白内化进入细胞，这也使其具有双重作用，既可识别，还有协助药物内化，并且这类小分子核酸片段筛选出来后很容易得到，合成较简单快速，易于进行化学修饰和多功能化，组织穿透性好，免疫原性也更小，具有较少的毒副反应。
如何筛选获得靶向性能好，又可与siRNA药物完美结合，协助药物内化的适配体，是目前siRNA分子靶向治疗药物有待解决的技术难题。
发明内容
由此，本发明提供了一种靶向CD206阳性细胞的核酸适体，其序列包括如下序列：5′-TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGA-3′。
该核酸适体优选图1所示的两种空间二级结构。
所述的CD206阳性细胞包括巨噬细胞或树突状细胞。
基于本核酸适体的靶向特异性，本发明进一步公开其在制备检测CD206阳性细胞的试剂盒、分子探针及靶向药物载体方面的应用。
并进一步公开由上述核酸适体搭载小分子RNA药物所形成，靶向CD206阳性细胞的嵌合体；及其在制备抗肿瘤药物中的应用。
同时，公开一种抑制细胞因子CCL18分泌合成的嵌合体及其在制备抗乳腺癌药物中的应用。该嵌合体由核酸适体搭载CCL18siRNA结合而成。其序列为：
5′-TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGAACAAGUUGGUACCAACAAATTUUUGUUGGUACCAACUUGUGC-3′。
该嵌合体在应用中优选图2所示的三种空间二级结构。
所述嵌合体的制备方法，包括如下步骤：
1、合成核酸适体，连接RNA正义链的中间体以及RNA反义链；
2、将相同浓度的中间体和RNA反义链混合，加入退火缓冲液，在90℃水浴锅中缓慢退火至25℃，得目标物。
经实验证明：本发明的核酸适体可精准识别CD206阳性细胞，具有优良的特异靶向性和亲和性；可作为检测CD206阳性细胞的试剂盒、分子探针和靶向药物载体。该核酸适体具有特定的空间结构及适当的分子量，可有效搭载siRNA等小分子RNA药物到达靶点细胞内部精准高效发挥药效。特别是与CCL18siRNA结合的嵌合体，可明显抑制CCL18细胞因子的合成和分泌，大幅度降低乳腺癌细胞迁移侵袭能力，为晚期乳腺癌提供新的治疗途径。该核酸适体是非病毒载体，有助于加速RNAi技术在临床上的应用，生物安全性高，且可通过人工合成，合成工 艺简单，可规模化生产。
附图说明
图1是本发明实施例核酸适体的二级结构图；
图2是本发明实施例嵌合体的二级结构图；
图3是本发明实施例嵌合体退火合成示意图；
图4是本发明实施例嵌合体的电泳条带图；
图5是本发明实施例嵌合体特异结合CD206阳性细胞效果图；
图6是本发明实施例嵌合体敲低肿瘤相关巨噬细胞CCL18表达的效果图；
图7是本发明实施例嵌合体抑制乳腺癌细胞侵袭及迁移的效果图；
图8是本发明实施例嵌合体炎症相关因子试验效果图；
具体实施方式
以下结合具体实施例对本发明作进一步详细的说明。
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，如未作特别说明，则均为市售产品。
除非另有定义或说明，本发明所使用的所有的技术或科学术语，及外文缩写或短语与本技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
实施例1.筛选CD206核酸适体，构建核酸适体与CCL18 siRNA连接的嵌合体。
筛选CD206核酸适体：
CD206核酸适体通过指数富集配体系统进化技术(SELEX)在DNA文库中与人重组CD206蛋白筛选得到8条(S1-S8)亲和性及特异性较高的核酸片段见表1， 通过实验室的验证筛选，参考表2后选取特异性及递药性最佳的一条(S5)继续后续实验。筛选得到的S5核酸片段在序列、二级结构和分子大小等方面均与其余7条核酸片段具有明显的区别。经实验证明，S5核酸片段的特异性及递药性明显优于其他7条核酸片段。后续只记录S5的实验结果。所得S5序列为：5′-TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGA-3′。
表1
编号 序列
S1 AAACCATGAGATAAATAGCACATGCAGTATGAGCGAGCGTTGCGA
S2 AAATGCATCTGTACGGCTGTACAGTATGAGCGAGCGTTGCGA
S3 TAGGCTAGCTTCTTTTGGTGTGCAGTATGAGCGAGCGTTGCGA
S4 GAAGGATGGATGCTTTGGTGTGTAGTATGAGCGAGCGTTGCGA
S5 TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGA
S6 AAGCTGCAAGTTTGGTGTGCAGTATGAGCGAGCGTTGCGA
S7 TAAGGGTTAGTTCTTTGGTGTGTAGTATGAGCGAGCGTTGCGA
S8 AGGATGTGTCTGTCTTTGAGTATGAGCGAGCGTTGCAGA
表2 aptamer与重组人CD206蛋白亲和性
序列 靶蛋白亲和性 靶细胞亲和性
S1 35％ 84.4％
S2 47％ 86％
S3 23％ /
S4 74％ 89.4％
S5 56％ 86％
S6 61％ 78.8％
S7 32％ /
S8 43％ 89％
注：靶细胞aptamer与重组人CD206蛋白亲和性，靶细胞亲和性是aptamer与CD206阳性细胞结合特异性。
构建核酸适体与CCL18 siRNA连接的嵌合体(chimera)：
1.1 CD206核酸适体，核酸适体连接CCL18siRNA正义链的中间体以及 CCL18siRNA反义链分别由上海吉玛制药技术有限公司、宝生物工程(大连)有限公司合成提供，其核酸序列见序列表。
序列表中，本核酸适体序列如SEQ ID NO.1所示；CCL18siRNA正义链序列如SEQ ID NO.2所示；CCL18siRNA反义链如SEQ ID NO.3所示；所述中间体序列如SEQ ID NO.4所示；所示嵌合体序列如SEQ ID NO.5所示；前列腺特异胞膜抗原(PSMA)的核酸适体序列如SEQ ID NO.6所示。
合成方法：
将相同浓度的中间体和siRNA反义链混合，加入退火缓冲液，在90℃水浴锅中缓慢退火至25℃，分装放于负80℃保存。
1.2将相同浓度的CD206核酸适体中间体，siRNA，嵌合体分别加入上样缓冲液(loading buffer)，在8％非变性PAGE胶中150V电压电泳10分钟，观察构建的嵌合体在电泳的位置。
在二级结构预测软件中预测核酸适体全长的二级结构如图1，可发现CD206核酸适体具有两个茎环结构，与siRNA连接后的嵌合体形态如图2。在二级结构上，CD206核酸适体与siRNA连接后的嵌合体也能保持其茎环结构。图3为嵌合体的退火结合过程。在8％非变性聚丙烯酰胺凝胶中，如图4可见，siRNA、核酸适体连接siRNA正义链的中间体和嵌合体的长度分别是21，64，85个碱基，在凝胶上的位置对应标注(marker)的位置基本正确。
实施例2.嵌合体的摄取率验证
肿瘤相关巨噬细胞(TAMs)体外诱导：
利用梯度离心从健康人外周血中分离得到单个核巨噬细胞，贴壁生长培养。用含有20ng/ml浓度的IL4因子的培养基诱导约4-5天，镜下观察巨噬细胞的 形态可发现巨噬细胞形状从小圆形变为拉长如针状，从散在单个分布到簇状聚集分布，则为诱导成功。
2.1在TAMs中，加入终浓度为10nM的Cy3荧光基团标记的CD206核酸适体。以等量的前列腺特异胞膜抗原(PSMA)的核酸适体(也作Cy3标记)处理TAMs作为对照一，脂质体转染荧光标记的siRNA双链转染TAMs作为对照二。
2.2免疫荧光检测：以上处理前，将TAMs细胞种在小玻片上，待细胞贴壁后作以上处理。24小时后将上清去除，PBS洗去多余的适体或siRNA，加入4％多聚甲醛固定15分钟。PBS洗三遍，加入0.5％TritonX-100破膜10分钟，PBS洗三遍。
2.2.1 5％BSA封闭30分钟，人CD206一抗4℃湿盒孵育过夜，PBS洗三遍。加入荧光二抗室温避光孵育2小时，PBS洗三遍。
2.2.2 DAPI室温核染15分钟，PBS洗三遍。在载玻片上滴加抗荧光淬灭封片剂，将片子倒扣封片。避光保存。用激光共聚焦显微镜观察适体或siRNA的定位情况。
2.3在TAMs中，加入终浓度为10nM的Cy3荧光基团标记的CD206核酸适体，在4、8、12、24小时时分别收集处理的巨噬细胞，抗体孵育同上。用上述相同方法免疫荧光法测TAMs对适体不同时间的摄取情况。
2.4在TAMs中，加入终浓度10、20和50nM的Cy3荧光基团标记的CD206核酸适体，培养24小时。去除培养基，胰酶消化TAMs。PBS洗去多余的核酸适体。300g 5分钟离心弃上清，加入100微升PBS重悬细胞，加入2微升抗人CD206荧光抗体。4℃孵育30分钟后同上条件离心留沉淀，加入PBS洗一遍去除多余的抗体，再离心弃上清后200微升PBS重悬。用流式检测摄取率。
为检测巨噬细胞对嵌合体的结合摄入情况，标记Cy3的嵌合体与TAMs孵育。将同样方法构建得到CD206核酸适体用脂质体转染作为阳性对照，针对前列腺特异抗原的核酸适体即PSMA作为结合能力的对照，以横坐标为Cy3信号，摄入嵌合体的TAMs会带有荧光信号被检测到，并随着摄入的增加而沿横坐标逐渐右移。CD206核酸适体处理巨噬细胞24小时后能检测到荧光信号，而带有相同荧光基团的PSMA核酸适体也不能让巨噬细胞带有荧光。这说明CD206核酸适体是可以选择性与TAMs结合的。
激光共聚焦荧光显微镜下观察核酸适体与细胞的结合情况。Cy3在镜下显示红光，若细胞摄入核酸适体，则可在细胞内检测到红光，巨噬细胞以绿色的CD206抗体标记细胞膜。在CD206核酸适体处理的巨噬细胞内都可以看到带红光的核酸适体，而PSMA核酸适体处理的巨噬细胞内则不能看到红光。综上说明DNA核酸适体具有良好的高亲和性和特异结合力并且嵌合体能够将小分子RNA输送进入肿瘤相关巨噬细胞内。结果见图5的A部分。
以20nM浓度固定，处理时间从1小时到24小时，随着时间的递增，免疫荧光检测巨噬细胞对嵌合体的摄入，发现巨噬细胞摄入的比例在上升，并在24小时基本达到饱和状态。结果见图5的B部分。
随着加入嵌合体的浓度从10nM到50nM的升高，处理24小时后收集的巨噬细胞进行流式检测发现检测到带荧光的巨噬细胞的比例在不断上升，并在20nM浓度处基本达到饱和，即90％左右。结果见图5的C部分。
实施例3.嵌合体对CCL18生物因子合成和分泌的抑制作用。
3.1在TAMs中，加入终浓度20nM的嵌合体，CD206核酸适体适体为空载对照，适体连接GFP蛋白siRNA的嵌合体为阴性对照，脂质体转染作为阳性对照。 培养24-36小时后每孔加入1毫升trizol试剂裂解细胞收集mRNA；培养48-60小时左右收集细胞蛋白以及细胞上清。
3.2 RT-QPCR
3.2.1将含有trizol试剂的细胞混合液加入200微升氯仿，剧烈震荡混匀后静置10分钟，4℃12000rpm离心15分钟；离心后吸取约400微升上层透明液相，加入1毫升异丙醇，轻轻混匀后静置5分钟，4℃12000rpm离心10分钟；离心后弃去上清，保留底部沉淀，加入1毫升70％乙醇，轻轻吹起沉淀后4℃12000rpm离心5分钟，弃去上清得到沉淀即mRNA，风干水分后用DEPC水溶解沉淀，测量mRNA浓度后负80℃保存。过程中的试剂以及器械保持无酶操作。
3.2.2每组mRNA取约500ng逆转录成cDNA，继而进行半定量实时荧光定量核酸扩增实验检测每组的CCL18的mRNA表达水平，以GAPDH作为参考。
3.3 Western Blot：将得到的蛋白利用BCA法在37℃水浴锅孵育30分钟后酶标仪检测562nm波长的吸光度计算浓度，加入含溴酚蓝的上样缓冲液(loading buffer)，95℃水浴锅煮5分钟后可放置于负80℃保存。将同量的蛋白分别在5％、10％的聚丙烯酰胺凝胶浓缩胶和分离胶中以70伏、120伏电泳继而将胶上的蛋白转到PVDF膜上。剪下CCL18和内参GAPDH对应的条带，TBST配的5％的牛奶室温孵育1.5小时封闭；1：1000配的CCL18一抗4℃摇床孵育过夜。TBST涮洗3遍，在二抗中室温孵育2小时，重复涮洗步骤，滴加曝光液压片曝光。
3.4 ELISA：上述操作得到的上清，在包被了CCL18捕获抗体(capture antibody，武汉伊莱瑞特生物科技股份有限公司，E-EL-H1270c)的96孔板中室温孵育2小时，PBST洗3遍，加入CCL18检测抗体(detective antibody，武汉伊莱瑞特生物科技股份有限公司，E-EL-H1270c)同样条件孵育2小时，重复 洗的步骤，加入底物20分钟，洗3遍，加入显色液。当标准品出现明显梯度显色时终止显色。在酶标仪上检测450nm吸光度，以570nm为参考。
结合CCL18 siRNA的嵌合体与TAMs孵育约24小时后，CCL18的mRNA水平通过Q-PCR检测。CCL18mRNA明显较未处理组降低，且这种敲低效果是特异的，连接GFP蛋白的siRNA的嵌合体(NC)或者空载核酸适体组则几乎对其表达不影响。上述对mRNA的敲低能力随着加入孵育的嵌合体浓度的上升有所增加，并可在20nM浓度达到基本饱和。结果见图6的A和C部分。
通过western blot实验可以发现，构建的嵌合体可以抑制TAMs的CCL18蛋白合成，同未处理组相比，TAMs的CCL18蛋白量减少了60％，而阴性对照组或空载组则无明显变化，结果见图6的D部分。
嵌合体与TAMs孵育48-60小时后取上清进行ELISA实验检测上清中的CCL18从而比较不同组间CCL18的分泌，可以发现嵌合体处理组的CCL18分泌明显降低，而同样对照组则基本不变，结果见图6的B部分。以上实验证明构建的嵌合体可以特异输送siRNA进入TAMs并达到敲低目的基因的作用。
实施例4.嵌合体抑制TAMs对乳腺癌细胞的促进迁移侵袭能力
4.1通过将嵌合体与TAMs共孵育48小时后，将TAMs与乳腺癌细胞MDA-MB-231细胞(以下简称“231细胞”)进行transwell实验。结果如图7的A部分所示，未与TAMs共培养的231细胞和与TAMs共培养的相比，后者明显迁移能力增高；而经过嵌合体处理的TAMs再与231细胞共培养，231细胞的迁移能力明显下降(细胞数减少接近4倍)，这与转染对照组相类似，而对照处理组则没有明显改变。这说明了嵌合体可以通过干扰TAMs的CCL18表达从而抑制TAMs对乳腺癌细胞迁移能力的促进作用。
4.2侵袭实验结果如图7的B部分所示，嵌合体也呈现了具有与抑制迁移能力相类似的效果，细胞数也有下降3倍左右，这说明了嵌合体也可以通过抑制TAMs对乳腺癌细胞侵袭能力的促进作用。
实施例5.嵌合体的安全性检测
嵌合体对肿瘤相关巨噬细胞验证因子的诱导
将嵌合体与TAMs共孵育24小时，同上述方式收取TAMs的mRNA，通过RT-QPCR检测IL-6、IL-10、TGF-beta、CCL22、IFN的mRNA表达水平。结果如图8，各组的mRNA表达水平基本一致，可见嵌合体的加入对炎性因子的表达和对照组无明显差异，即嵌合体并不会诱导TAMs的这几类炎症因子的升高表达，说明嵌合体不会引起TAMs炎症反应的发生。
以上所述实施例仅表达了本发明的几种实施方式，不能因此而理解为对发明专利范围的限制。在不脱离本发明构思的前提下，所做出若干变形和改进，都属于本发明的保护范围。

Claims (10)

  1. 一种靶向CD206阳性细胞的核酸适体，其特征在于，其序列包括如下序列：5′-TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGA-3′。
  2. 如权利要求1所述的核酸适体，其特征在于，所述核酸适体的二级结构包括以下两种：
    Figure PCTCN2021075671-appb-100001
    和/或
    Figure PCTCN2021075671-appb-100002
  3. 如权利要求1或2所述的核酸适体，其特征在于，所述的CD206阳性细胞包括CD206阳性巨噬细胞或树突状细胞。
  4. 权利要求1或2所述的核酸适体在制备检测CD206阳性细胞试剂盒或分子探针或靶向药物载体中的应用。
  5. 一种靶向CD206阳性细胞的嵌合体，其特征在于，所述嵌合体是由权利要求1或2或3所述的核酸适体和小分子RNA药物结合而成的核酸序列组合。
  6. 如权利要求5所述的嵌合体，其特征在于，所述RNA为CCL18siRNA，其序列为5′-ACAAGUUGGUACCAACAAATT-3′，5′-UUUGUUGGUACCAACUUGUGC-3′；
    所述嵌合体的序列为：5′-TAAGGCTTACTATTTTGGTGTGCAGTATGAGCGAGCGTTGCGAACAAGUUGGUACCAACAAATTUUUGUUGGUACCAACUUGUGC-3′。
  7. 如权利要求6所述的嵌合体，其特征在于，所述嵌合体的二级结构包括如下三种：
    Figure PCTCN2021075671-appb-100003
    和/或
    Figure PCTCN2021075671-appb-100004
    和/或
    Figure PCTCN2021075671-appb-100005
  8. 如权利要求6所述的嵌合体的制备方法，包括如下步骤：
    (1)合成所述核酸适体，连接RNA正义链的中间体以及RNA反义链；
    (2)将相同浓度的中间体和RNA反义链混合，加入退火缓冲液，在90℃水浴锅中缓慢退火至25℃，得目标物。
  9. 如权利要求5或6或7所述的嵌合体在制备抗肿瘤药物中的应用。
  10. 如权利要求9所述的应用，其特征在于，所述的抗肿瘤药物为抗乳腺癌药物。
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