WO2019204989A1 - 一种cd4阳性细胞特异的dna核酸适体及其嵌合体 - Google Patents

一种cd4阳性细胞特异的dna核酸适体及其嵌合体 Download PDF

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WO2019204989A1
WO2019204989A1 PCT/CN2018/084284 CN2018084284W WO2019204989A1 WO 2019204989 A1 WO2019204989 A1 WO 2019204989A1 CN 2018084284 W CN2018084284 W CN 2018084284W WO 2019204989 A1 WO2019204989 A1 WO 2019204989A1
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aptamer
chimera
sirna
tumor
tams
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PCT/CN2018/084284
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French (fr)
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姚燕丹
宋尔卫
张明霞
李铨
黄松音
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中山大学孙逸仙纪念医院
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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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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers

Definitions

  • the present invention belongs to the field of biotechnology, and in particular to a DNA nucleic acid aptamer.
  • breast cancer is a systemic disease
  • systemic treatment programs have received increasing attention.
  • breast cancer that has undergone systemic metastasis remains an intractable disease.
  • These cases are often insensitive to a variety of chemotherapy and radiotherapy regimens.
  • Molecular targeted therapy came into being.
  • Molecular targeted therapy is designed to target specific tumor growth sites, and can directly find tumor cells to exert anti-tumor effects without excessive load on surrounding normal tissues. This method has gradually become a trend of anti-tumor therapy, and treatment The discovery of targets is the primary solution to targeted therapy.
  • the cytokine CCL18 is a member of the C-C membrane cytokine receptor family.
  • the CCL18 factor secreted by TAMs can induce breast cancer cell invasion and metastasis by activating the PI3K/Akt pathway to induce EMT in breast cancer cells.
  • EMT epithelial-mesenchymal transition
  • RNA intervention RNA intervention
  • the former such as the monoclonal antibody Trastuzumab, also known as herceptin
  • Herceptin monotherapy has been able to prevent Her2-positive advanced breast cancer cases from entering clinical remission by blocking receptor proteins. Maintained for 18 months.
  • Herceptin since the monoclonal antibody can only block the synthesized protein on the cell surface and can not completely prevent its synthesis, and the distribution of HER-2 protein is not completely specific, Herceptin has many toxic side effects as a macromolecular compound, and its effect is not enough. Specific and complete.
  • RNA interference is a powerful weapon for suppressing gene expression. Andrew Z. Fire and Craig C. Mello reported in 1998 that they discovered the phenomenon of Ribonucleic Acid Interference (RNAi) and won the Nobel Prize in Medicine in 2006. In 2001, Tuchl et al. introduced a 19-23 base pair, synthetically synthesized exogenous (Small Interfering RNA, siRNA) into a mammalian cell to induce an RNAi effect that specifically inhibits the expression of a complementary sequence gene. After the report was published, it set off a wave of research on RNAi. Since then, siRNA has not only been used as a tool to explore the function of cellular genes, but also more attractive is the use of siRNA to inhibit the expression of disease-causing genes, and to develop new gene drugs for the treatment of various diseases, especially malignant tumors.
  • siRNA synthetically synthesized exogenous exogenous
  • the effect of siRNA silencing gene expression is ten to hundreds times stronger, and its potential to inhibit disease-causing gene therapy is far greater than the traditional inverse. Genetic tools. Therefore, in combination with the above, the RNAi of the CCL18 gene is expected to break through the unsatisfactory gene suppression effect of the conventional antisense oligonucleotide and ribozyme, and become a novel gene drug for treating breast cancer.
  • RNAi successfully inhibits the expression of oncogenes such as k-ras and cyclin E, the expression of tumor anti-apoptotic gene BCL-2 and tumor resistance genes.
  • oncogenes such as k-ras and cyclin E
  • mdr1 effectively reduces the proliferation of cancer cells and increases their sensitivity to chemotherapeutic drugs.
  • RNAi silencing Her2 gene expression also successfully inhibited proliferation of breast cancer cells cultured in vitro.
  • RNAi anti-tumor experiments are directly transfected or transduced into RNAi in tumor cells cultured in vitro or directly injected into RNAi in nude mice transplanted tumor tissues, although these experiments have achieved some success, but the distance is clinically The real use of RNAi to treat tumors is still far apart.
  • RNAi small molecule RNA carriers
  • a protein molecule such as an antibody, conjugated to an siRNA molecule enters a cell upon binding to a cell or target organ surface antigen molecule, thereby causing the siRNA to exert a gene interference effect.
  • the advantage of this delivery system is that it has binding specificity by means of antigen-antibody molecule binding, but the antigen is non-specific.
  • the immunogenicity of the macromolecular protein in the body and the permeability of the internal environment barrier lead to drug consumption and toxicity. The reaction, as well as the particularity of production, result in high costs.
  • nanomaterials a hot spot in the research of drug delivery systems is nanomaterials.
  • Polymer nanomaterials can passively or actively target tumor tissue to deliver drugs through physical and chemical properties. Relative proteins are more easily absorbed through physiological barriers, and the side effects are relatively small.
  • biosafety factors there are still stable factors and biosafety factors in the research of nanomaterials, and the cost is high.
  • nucleic acid aptamer a small class of single-stranded oligonucleotides typically less than 200 bases in length. Aptamer can naturally fold into a spatial structure through its own sequence characteristics, thus having a high degree of binding ability to specific molecules.
  • nucleic acid fragments exist in nature and can also be screened by exponential enrichment ligand system evolution (SELEX). Because of its small molecular weight, it is easy to internalize into the cells by binding to the target protein, which also has a dual role, which can be recognized and assisted in drug internalization, and such small molecular nucleic acid fragments can be easily obtained after screening. It is simple and fast, easy to carry out chemical modification and multi-function, good tissue penetration, less immunogenicity and less toxic side effects.
  • nucleic acid aptamers the main obstacle to the application of nucleic acid aptamers is to screen for aptamers with specific binding of target proteins.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide a nucleic acid aptamer and an siRNA chimer thereof which are safe and efficient and have specific binding to CD4-positive tumor-associated macrophages and anti-cancer siRNA drugs.
  • a nucleic acid aptamer-siRNA chimera is a nucleic acid sequence formed by the above-described nucleic acid aptamer and small molecule RNA.
  • the small molecule RNA is a CCL18 siRNA having the sequence: 5'-ACAAGUUGGUACCAACAAATT-3'; as shown in SEQ NO. 5'-UUUGUUGGUACCAACUUGUGC-3'; as shown in SEQ NO.
  • the pharmaceutical vector constructed by the present invention capable of efficiently binding to a nucleic acid aptamer of CD4-positive cells has both a bidirectional function of targeting CD4-positive cells and carrying anti-cancer siRNA drugs.
  • 1.1 CD4 DNA aptamer, aptamer-linked siRNA sense strand intermediate and siRNA antisense strand were separately synthesized by the company (TAKARA, Gima). The same concentration of the intermediate and the siRNA antisense strand were mixed, added to the annealing buffer, slowly annealed to 25 ° C in a 90 ° C water bath, and stored at minus 80 ° C.
  • Tumor-associated macrophages are induced in vitro
  • Single nuclear macrophages were isolated from healthy human peripheral blood by gradient centrifugation and cultured in adherent growth.
  • TAMs a cy3 fluorophore-labeled CD4 nucleic acid aptamer was added at a final concentration of 10 nM.
  • TAMs were treated with an equal amount of plaques of prostate-specific membrane antigen PSMA (also labeled as cy3) as control one, MDA-MB-231 cells were treated with CD4 aptamer as control 2, and liposome was transfected with fluorescent labeling.
  • PSMA prostate-specific membrane antigen
  • siRNA double-stranded TAMs were used as control three.
  • TAMs or MDA-MB-231 cells were seeded on a small slide, and the cells were adhered to the above treatment. After 24 hours, the supernatant was removed, excess aptamer or siRNA was washed away with PBS, and fixed in 4% paraformaldehyde for 15 minutes. Wash three times with PBS, add 0.5% TritonX-100 to rupture the membrane for 10 minutes, and wash three times with PBS.
  • 3.4 5% BSA was blocked for 30 minutes, and human CD4 primary antibody was incubated overnight in a 4 °C wet box and washed three times with PBS. Fluorescent secondary antibody was added to incubate at room temperature for 2 hours in the dark, and washed three times with PBS. 2.8.4.3 DAPI was stained at room temperature for 15 minutes and washed three times with PBS. Anti-fluorescence quenching tablets were added dropwise to the slides, and the sheets were inverted and sealed. Keep away from light. The localization of aptamers or siRNA was observed by laser confocal microscopy.
  • a cy3 fluorophore-labeled CD4 aptamer at a final concentration of between 10 nM and 100 nM was added and cultured for 24 hours. The medium was removed and the TAMs were trypsinized. Wash the excess aptamer with PBS. The supernatant was discarded by centrifugation at 300 g for 5 minutes, and the cells were resuspended by adding 100 ⁇ l of PBS, and 2 ⁇ l of anti-human CD4 fluorescent antibody was added.
  • the precipitate was centrifuged as above, and the excess antibody was removed by washing with PBS, and then the supernatant was centrifuged and resuspended in 200 ⁇ l of PBS.
  • cy3 fluorophore-labeled CD4 aptamers were added at a final concentration of 10 nM, and the treated macrophages were collected at 6, 12, 24, 36, and 48 hours, and the antibody was incubated as above.
  • Flow cytometry was used to detect the uptake of TAMs by aptamers at different times.
  • the constructed chimera inhibits the synthesis and secretion of ccl18
  • a chimera with a final concentration of 20 nM was added, the CD4 aptamer was a no-load control, the chimera of the aptamer-linked GFP protein siRNA was used as a negative control, and the liposome was transfected as a positive control.
  • 1 ml of trizol lysing cells were added to each well to collect mRNA; the cell protein and cell supernatant were collected for about 48-60 hours.
  • the supernatant was discarded to obtain a precipitate, i.e., mRNA, and after air drying, the precipitate was dissolved in DEPC water, and the mRNA concentration was measured and stored at minus 80 °C.
  • the reagents and instruments in the process remain enzyme-free.
  • Each group of mRNA was reverse transcribed into cDNA by about 500 ng, and then semi-quantitative real-time fluorescence quantitative nucleic acid amplification assay was used to detect the mRNA expression level of ccl18 in each group, with GAPDH as a reference.
  • the band corresponding to ccl18 and internal reference gapdh was cut out, and 5% of the TBST was incubated with 1.5% milk at room temperature for 1.5 hours; 1:1000 with ccl18 primary antibody was incubated overnight at 4 °C shaker.
  • the TBST was washed 3 times, incubated for 2 hours at room temperature in the secondary antibody, and the rinsing step was repeated, and the exposure hydraulic sheet was exposed on the strip to expose.
  • the previously treated TAMs were co-cultured with 231 cells after 48 hours, and 20,000 231 cells were seeded in the upper chamber of 8 micron pore size, and the lower layer of the upper membrane was pre-plated with 40 ug/L of FN gel at 4 ° C overnight. .
  • the following chambers were cell-free normal complete medium as a blank control. After co-cultivation for about 6 hours, the upper chamber was taken out, fixed in 4% paraformaldehyde for 15 minutes, and then stained with crystal violet. Finally, under the light microscope, the average number of cells in 10 fields under the microscope was taken at 200 times, and the migration ability of 231 cells in different groups was compared.
  • matrigel Approximately 50 microliters of matrigel was placed in the co-cultured upper chamber, which was first diluted to 20% in serum-free DMEM medium. It was allowed to stand in a 37 ° C incubator for about 30 minutes to solidify. The upper layer of the upper chamber is pre-paved with FN glue. The remaining steps were the same as the migration experiment, and the co-cultivation time was about 16 hours. When the cells in the high-power field of the blank control group passed through, the experiment could be terminated. The upper chamber was taken out and immersed in 4% paraformaldehyde for 15 minutes, and crystal violet stained. High magnification observations were used to count stained cells.
  • the chimera was 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, IL-12 and IFN were detected by RT-QPCR.
  • the present invention has the following beneficial effects:
  • the CD4 DNA aptamer-CCL18 siRNA chimera constructed by the present invention has a bidirectional function of binding to CD4-positive tumor-associated macrophages and drugs carrying anti-cancer siRNA;
  • the CD4 DNA aptamer-CCL18 siRNA chimera constructed by the present invention successfully introduces an anti-cancer siRNA drug into tumor-associated macrophages, and develops a non-viral vector tool to accelerate the application of RNAi technology in clinical practice.
  • Figure 1 (top) is a CD4 DNA nucleic acid aptamer
  • Figure 1 is a predicted secondary structure prediction map of CCL18 siRNA
  • Figure 1 (bottom) is the electropherogram, the strip position is correct
  • Figure 2 is a diagram showing the constructed chimeric specific binding to CD4 positive cells
  • Figure 3 shows the expression of CD4 chimera knockdown of tumor-associated macrophage CCL18: mRNA (top left), protein (bottom left), factor secretion (right);
  • Figure 4 shows inhibition of breast cancer cell invasion (A) and migration (B) in a constructed CD4 chimera in vitro;
  • Figure 5 shows that the constructed CD4 chimera does not cause high expression of inflammation-related factors IL-6, IL-10, IL-12, IFN in tumor-associated macrophages (top); chimera does not cause tumor-associated macrophage toxicity (under).
  • Example 1 Construction and validation of CD4 nucleic acid aptamer aptamer and CCL18 siRNA-ligated chimeric chimera
  • 1.1 CD4 DNA aptamer, aptamer-linked siRNA sense strand intermediate and siRNA antisense strand were separately synthesized by the company (TAKARA, Gima). The same concentration of the intermediate and the siRNA antisense strand were mixed, added to the annealing buffer, slowly annealed to 25 ° C in a 90 ° C water bath, and stored at minus 80 ° C.
  • TAMs a cy3 fluorophore-labeled CD4 nucleic acid aptamer was added at a final concentration of 10 nM.
  • TAMs were treated with an equal amount of plaques of prostate-specific membrane antigen PSMA (also labeled as cy3) as control one, MDA-MB-231 cells were treated with CD4 aptamer as control 2, and liposome was transfected with fluorescent labeling.
  • PSMA prostate-specific membrane antigen
  • siRNA double-stranded TAMs were used as control three.
  • TAMs or MDA-MB-231 cells were seeded on a small slide, and the cells were adhered to the above treatment. After 24 hours, the supernatant was removed, excess aptamer or siRNA was washed away with PBS, and fixed in 4% paraformaldehyde for 15 minutes. Wash three times with PBS, add 0.5% TritonX-100 to rupture the membrane for 10 minutes, and wash three times with PBS.
  • a cy3 fluorophore-labeled CD4 aptamer at a final concentration of between 10 nM and 100 nM was added and cultured for 24 hours. The medium was removed and the TAMs were trypsinized. Wash the excess aptamer with PBS. The supernatant was discarded by centrifugation at 300 g for 5 minutes, and the cells were resuspended by adding 100 ⁇ l of PBS, and 2 ⁇ l of anti-human CD4 fluorescent antibody was added.
  • the precipitate was centrifuged as above, and the excess antibody was removed by washing with PBS, and then the supernatant was centrifuged and resuspended in 200 ⁇ l of PBS.
  • cy3 fluorophore-labeled CD4 aptamers were added at a final concentration of 10 nM, and the treated macrophages were collected at 6, 12, 24, 36, and 48 hours, respectively, and the antibody was incubated as above.
  • Flow cytometry was used to detect the uptake of TAMs by aptamers at different times.
  • chimeras labeled with Cy3 were incubated with TAMs.
  • the same method was used to construct a chimera in which the siRNA of CD4 aptamer and GFP protein was ligated as a negative control, and the aptamer of prostate specific antigen, ie, PSMA, was used as a control for binding ability, and CD8 + T lymphocytes were used as a control, and the abscissa was a Cy3 signal.
  • TAMs ingesting chimeras are detected with a fluorescent signal and gradually shift to the right along the abscissa as the intake increases.
  • DNA and RNA CD4 aptamer can detect the fluorescence signal after 24 hours of treatment of macrophages, the difference between the two is not obvious, and can reach about 89% of the degree of binding.
  • aptamer does not fluoresce CD8 T lymphocytes
  • PSMA aptamer with the same fluorophore does not allow macrophages to be fluorescent. This suggests that CD4 aptamer can selectively bind to TAMs and that this binding is specific for CD4 molecules.
  • DNA aptamer has good high affinity and specific binding ability, similar to RNA aptamer, and DAsiC can transport small RNA into tumor-associated macrophages.
  • the concentration was fixed at 20 nM, and the treatment time was from 1 hour to 48 hours. With the increase of time, the macrophage intake of the chimera was detected by flow detection, and the proportion of macrophage ingestion was also increased, and it was basically at 24 hours. Saturated.
  • Figure 2 is a diagram showing the constructed chimeric specific binding to CD4 positive cells.
  • Example 3 The constructed chimera inhibits the synthesis and secretion of ccl18
  • a chimera with a final concentration of 20 nM was added, the CD4 aptamer was a no-load control, the chimera of the aptamer-linked GFP protein siRNA was used as a negative control, and the liposome was transfected as a positive control.
  • 1 ml of trizol lysed cells were added to each well to collect mRNA; the cell protein and cell supernatant were collected by culture for about 48-60 hours.
  • the supernatant was discarded to obtain a precipitate, i.e., mRNA, and after air drying, the precipitate was dissolved in DEPC water, and the mRNA concentration was measured and stored at minus 80 °C.
  • the reagents and instruments in the process remain enzyme-free.
  • Each group of mRNA was reverse transcribed into cDNA by about 500 ng, and then semi-quantitative real-time fluorescence quantitative nucleic acid amplification assay was used to detect the mRNA expression level of ccl18 in each group, with GAPDH as a reference.
  • the band corresponding to ccl18 and internal reference gapdh was cut out, and 5% of the TBST was incubated with 1.5% milk at room temperature for 1.5 hours; 1:1000 with ccl18 primary antibody was incubated overnight at 4 °C shaker.
  • the TBST was washed 3 times, incubated for 2 hours at room temperature in the secondary antibody, and the rinsing step was repeated, and the exposure hydraulic sheet was exposed on the strip to expose.
  • the chimeric of the CCL 18 siRNA was incubated with TAMs for about 24 hours, and the mRNA level of CCL 18 was detected by Q-PCR.
  • CCL18 mRNA was significantly lower than that of the untreated group, and this knockdown effect was specific, and the siRNA-containing siRNA chimera (NC) or the empty aptamer group had almost no effect on its expression.
  • the above ability to knock down mRNA increased with increasing concentration of chimeras added to the incubation and was substantially saturated at 20 nM.
  • FIG. 3 shows the expression of CD4 chimera knockdown of tumor-associated macrophage CCL18: mRNA (top left), protein (bottom left), factor secretion (right);
  • Example 4 Chimera inhibits the ability of TAMs to promote migration and invasion of breast cancer cells
  • TAMs were subjected to transwell experiments with breast cancer cell MDA-MB-231 cells (hereinafter abbreviated as 231 cells) (Fig. 4A). It can be seen that the 231 cells not co-cultured with TAMs have significantly increased migration ability compared with TAMs co-culture; while the chimeric-treated TAMs are co-cultured with 231 cells, the migration ability of 231 cells is significantly decreased ( The number of cells was reduced by nearly 4 fold, which was similar to the transfection control group, while the control treatment group showed no significant change. This demonstrates that chimeras can inhibit the migration of breast cancer cells by interfering with the expression of CCL18 by TAMs.
  • the chimera was 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, IL-12 and IFN were detected by RT-QPCR.
  • the constructed CD4 chimera did not cause high expression of the inflammation-related factors IL-6, IL-10, IL-12, IFN of tumor-associated macrophages.
  • the LDH assay was performed on the TAMs supernatant treated with the chimera at 1, 6, 12, 24, 36, and 48 hours, and it was found that the chimera did not significantly cause LDH release from the cells, indicating that the chimera did not cause toxicity of TAMs.
  • For chimeric-treated TAMs we examined changes in the mRNA levels of the cell-associated factors IL-6, IL-10, IL-12, and IFN, and found that chimeras did not induce elevation of these inflammatory factors in TAMs. Expression indicates that the chimera does not cause the inflammatory response of TAMs. As shown in Figure 5 (bottom), the chimera did not cause tumor-associated macrophage toxicity.

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Abstract

提供了一种CD4阳性细胞特异的DNA核酸适体及其嵌合体。结合具有细胞导向功能的核酸适体技术和RNAi技术，构建CD4阳性细胞特异的DNA核酸适体，以携带抗癌的siRNA选择性导入CD4阳性的肿瘤相关巨噬细胞。在体外肿瘤相关巨噬细胞和乳腺癌细胞培养系统中检测CD4阳性细胞特异的DNA核酸适体作为载体导向工具输送抗癌siRNA的功能和抗瘤效应。提供了基于导向性RNAi的新型基因抗癌药物，同时为RNAi靶向导入目标细胞的研发提供新思路。

Description

一种CD4阳性细胞特异的DNA核酸适体及其嵌合体 技术领域
本发明属于生物技术领域，具体地说，涉及一种DNA核酸适体。
背景技术
我国乳腺癌的发病率有上升的趋势，特别是晚期转移乳腺癌的病例增多。目前治疗乳腺癌仍然是以手术为主的综合治疗。自从提出了“乳腺癌是全身性疾病”的概念以后，全身治疗的方案越来越受到重视。但是，已经发生全身转移的乳腺癌仍然属于难治之症，这些病例往往对多种化疗和放疗方案都不敏感，各种治疗手段除了稍能延长寿命，并没有突破性进展。分子靶向治疗应运而生。分子靶向治疗针对特定的肿瘤生发位点设计药物，能够直接找到肿瘤细胞发挥抗肿瘤效应，而不会对周围正常组织产生过多负荷，这种方式也逐渐成为抗肿瘤治疗的趋势，而治疗靶点的发现则是靶向治疗的首要解决问题。
研究发现，在乳腺癌的微环境中，肿瘤相关巨噬细胞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。研究发现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以通过抑制CCL18因子的分泌被打破。
目前通过靶向治疗乳腺癌的途径主要有单克隆抗体、酪氨酸激酶抑制剂以及RNA干预(RNAi)等。前者如单抗Trastuzumab，又称herceptin，数年来多个中心大宗病例的临床应用结果证明，Herceptin单药治疗能通过阻断受体蛋白起作用使Her2阳性的晚期乳腺癌病例进入临床缓解期，并维持18个月之久。但由于单抗只能阻断细胞表面已合成的蛋白而不能彻底阻止其合成，且HER-2蛋白的分布非完全特异性，Herceptin作为大分子化合物的使用也存在较多毒副作用，其作用不够特异、完全。因此，要进一步扩展治疗乳腺癌的临床成果，有必要发展毒性低，又能较广谱的针对多类型的乳腺癌的有效方法。其他如传统的反义基因方法由于抑制基因表达的效果较弱，因此不能满足临床应用的需求。
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(Small Interfering 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。该报道发表后，掀起了研究RNAi的热潮。从此，siRNA不仅被用作探讨细胞基因功能的工具，而且更吸引人的是应用siRNA抑制致病基因的表达，开托出治疗 各类疾病，特别是恶性肿瘤的新型基因药物。
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，其抑制致病基因治疗疾病的潜力也远远大于传统的逆基因工具。因此，结合前述，CCL18基因的RNAi有希望突破以往反义寡核苷酸和核糖酶不尽人意的基因抑制效果，成为治疗乳腺癌的新型基因药物。
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。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(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。Aptamer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。
目前核酸适配体的应用主要障碍是筛选得到具有靶蛋白特异结合的适配体。
发明内容
本发明的目的在于克服现有技术的不足，提供一种安全高效且具有特异结合CD4阳性肿瘤相关巨噬细胞和携带抗癌siRNA药物的核酸适体及其siRNA嵌合体。
为了实现上述目的，本发明采用如下技术方案：
一种CD4阳性细胞特异的DNA核酸适体，其核苷酸序列为TGACGTCCTTAGAATTGCGCATTCCTCACACAGGATCTT。如SEQ NO.1所示。
一种核酸适体-siRNA嵌合体，由上述的核酸适体与小分子RNA形成的核酸序列组合。所述小分子RNA为CCL18 siRNA，其序列为：5′-ACAAGUUGGUACCAACAAATT-3′；如SEQ NO.2所示。5′-UUUGUUGGUACCAACUUGUGC-3′；如SEQ NO.3所示。
本发明构建的能高效特异结合CD4阳性细胞的核酸适体的药物载体，同时具有靶向CD4阳性细胞和携带抗癌siRNA药物的双向功能。
CD4核酸适配体aptamer和CCL18 siRNA连接的嵌合体chimera的构建及其作为抗癌siRNA的药物载体的应用方案：
1.构建以及验证CD4核酸适配体aptamer和CCL18 siRNA连接的嵌合体chimera
1.1 CD4 DNA aptamer，aptamer连接siRNA正义链的中间体以及siRNA反义链分别由公司(TAKARA,吉玛)合成提供。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CD4核酸适体，中间体，siRNA反义链，嵌合体分别加入loadingbuffer，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。
2.肿瘤相关巨噬细胞体外诱导
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。
乳腺癌细胞MDA-MB-231细胞铺展密度达到75％左右时，更换新鲜培养基，再培养24小时，吸取全部上清，3000rpm，4℃离心15分钟后得到上清。用含有30％上清的完全培养基培养分离的巨噬细胞，诱导约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。
3.嵌合体的摄取率验证
3.1在TAMs中，加入终浓度为10nM的cy3荧光基团标记的CD4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，以CD4核酸适体处理MDA-MB-231细胞作为对照二，脂质体转染荧光标记的siRNA双链转染TAMs作为对照三。
3.2一同处理24小时后收集处理的细胞，一遍PBS洗去多余的处理试剂，300g离心5分钟，弃上清，加入200微升PBS重悬，用流式细胞分析仪分析细胞摄取适体或siRNA的情况。
3.3免疫荧光检测：以上处理前，将TAMs或MDA-MB-231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。
3.4 5％BSA封闭30分钟，人CD4一抗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。2.8.4.3 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。
3.5在TAMs中，加入终浓度10nM-100nM之间的cy3荧光基团标记的CD4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CD4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。
3.6在TAMs中，加入终浓度为10nM的cy3荧光基团标记的CD4核酸适体，在6、12、24、36、48小时时分别收集处理的巨噬细胞，抗体孵育同上。用用流式细胞分析仪检测TAMs对适体不同时间的摄取情况。
4.构建的嵌合体抑制ccl18的合成与分泌
4.1在TAMs中，加入终浓度20nM的嵌合体，CD4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。
4.2 RT-QPCR
4.2.1将含有trizol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。
4.2.2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。
4.3 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，在条带上滴加曝光液压片曝光。
4.4 ELISA：上述操作得到的上清，在包被了ccl18 capture antibody的96孔板中室温孵育2小时，PBST洗3遍，加入ccl18 detective antibody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。
5.嵌合体抑制乳腺癌MDA-MB-231细胞的迁移侵袭和黏附能力
5.1迁移
将前述处理的TAMs经48小时后与231细胞共培养，将20000个231细胞种在8微米孔径的上室里，上室的滤膜下层预先用40ug/L的FN胶铺好在4℃过夜。以下室为无细胞的普通完全培养基为空白对照。共培养约6小时，将上室取出，在4％多聚甲醛中固定15分钟，然后用结晶紫染色。最后在光镜下观察，取200倍镜下随机10个视野的细胞数取均值，比较不同组的231细胞迁移能力。
5.2侵袭
在共培养的上室内层铺约50微升matrigel，后者先用无血清的DMEM培养基稀释成20％。在37℃培养箱中放置约30分钟凝固。上室下层预先铺好FN胶。其余步骤同迁移实验，共培养时间约在16小时，当空白对照组高倍视野下有细胞穿过时可以终止实验。将上室取出浸泡在4％多聚甲醛中15分钟，结晶紫染色。高倍镜观察计数染色细胞。
6.嵌合体的安全性检测
6.1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、IL-12、IFN的mRNA表达水平。
6.2嵌合体对肿瘤相关巨噬细胞的毒性检测
6.2.1将嵌合体、核酸适体、慢病毒与TAMs共孵育，在1、3、6、12、24、36、48小时时各吸取约100微升上清，在上清中加入50微升LDH的底物混合液，37℃避光孵育30分钟，取出加入终止缓冲液，用酶标仪检测570nm的吸光度。以1％triton-X破膜细胞的上清作为阳性对照，纯培养基作为阴性对照。
6.2.2对于检测的值减去阴性对照组的值，与时间点作折线图，比较LDH的释放力从而比较加入试剂对肿瘤相关巨噬细胞的毒性反应。
与现有技术相比，本发明具有如下有益效果：
1.本发明构建的CD4 DNA aptamer–CCL18 siRNA嵌合体具有结合CD4阳性肿瘤相关巨噬细胞和携带抗癌siRNA药物的双向功能；
2.本发明构建的CD4 DNA aptamer–CCL18 siRNA嵌合体成功把抗癌siRNA药物定向导入肿瘤相关巨噬细胞中，开发了非病毒载体工具，加速RNAi技术应用于临床。
3.此嵌合体的成功开发为发展新型的抗癌基因药物奠定坚实的基础，将为治疗晚期乳腺癌提供有效的武器。
附图说明
图1(上)为CD4 DNA核酸适体；
图1(中)为与CCL18 siRNA连接的嵌合体二级结构预测图；
图1(下)为电泳图，条带位置正确；
图2为构建的嵌合体特异结合CD4阳性细胞图；
图3为构建的CD4嵌合体敲低肿瘤相关巨噬细胞CCL18的表达：mRNA(左上)，蛋白(左下)，因子分泌(右)；
图4为构建的CD4嵌合体体外实验中抑制乳腺癌细胞侵袭(A)及迁移(B)；
图5为构建的CD4嵌合体不引起肿瘤相关巨噬细胞的炎症相关因子IL-6、IL-10、IL-12、IFN的高表达(上)；嵌合体不引起肿瘤相关巨噬细胞毒性反应(下)。
具体实施方式
以下实施例可以使本领域技术人员全面的理解和实现本发明。
实施例1.构建以及验证CD4核酸适配体aptamer和CCL18 siRNA连接的嵌合体chimera
1.1 CD4 DNA aptamer，aptamer连接siRNA正义链的中间体以及siRNA反义链分别由公司(TAKARA,吉玛)合成提供。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CD4核酸适体，中间体，siRNA反义链，嵌合体分别加入loadingbuffer，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aptamer全长的二级结构，可发现CD4 aptamer具有两个茎环结构如图1(上)，与siRNA连接后的嵌合体形态如图1(中)。在二级结构上，CD4 aptamer与siRNA连接后的嵌合体chimera也能保持其茎环结构。在8％非变性聚丙烯酰胺凝胶中，如图1(下)可见，aptamer、aptamer连接siRNA正义链的中间体和嵌合体的长度分别是39，61，83个碱基，在凝胶上的位置对应marker的位置基本正确。
实施例2.嵌合体的摄取率验证
2.1在TAMs中，加入终浓度为10nM的cy3荧光基团标记的CD4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，以CD4核酸适体处理MDA-MB-231细胞作为对照二，脂质体转染荧光标记的siRNA双链转染TAMs作为对照三。
2.2一同处理24小时后收集处理的细胞，一遍PBS洗去多余的处理试剂，300g离心5分钟，弃上清，加入200微升PBS重悬，用流式细胞分析仪分析细胞摄取适体或siRNA的情况。
2.3免疫荧光检测：以上处理前，将TAMs或MDA-MB-231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。
2.4 5％BSA封闭30分钟，人CD4一抗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。
2.5 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。
2.6在TAMs中，加入终浓度10nM-100nM之间的cy3荧光基团标记的CD4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CD4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。
2.7在TAMs中，加入终浓度为10nM的cy3荧光基团标记的CD4核酸适体，在6、12、24、36、48小时时分别收集处理的巨噬细胞，抗体孵育同上。用用流式细胞分析仪检测TAMs对适体不同时间的摄取情况。
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CD4 aptamer与GFP蛋白的siRNA连接的嵌合体作为阴性对照，针对前列腺特异抗原的aptamer即PSMA作为结合能力的对照，以及用CD8 +T淋巴细胞做对照，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。DNA和RNA的CD4 aptamer处理巨噬细胞24小时后都能检测到荧光信号，二者相比差异不明显，并可以达到89％左右的结合度。但aptamer不能使CD8 T淋巴细胞标记 上荧光，而带有相同荧光基团的PSMA aptamer也不能让巨噬细胞带有荧光。这说明CD4 aptamer可以选择性与TAMs结合，并且这种结合是CD4分子特异的。
激光共聚焦荧光显微镜下观察aptamer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CD4抗体标记细胞膜。在DNA和RNA的CD4 aptamer处理的巨噬细胞内都可以看到带红光的aptamer，而PSMA aptamer处理的巨噬细胞内则不能看到红光，CD4 aptamer处理的CD8 T淋巴细胞也不能看到这种情况。综上说明DNA aptamer具有良好的高亲和性和特异结合力，与RNA aptamer相似，并且DAsiC能够将小分子RNA输送进入肿瘤相关巨噬细胞内。
随着加入嵌合体的浓度从5nM到100nM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40nM浓度处基本达到饱和，即90％左右。
以20nM浓度固定，处理时间从1小时到48小时，随着时间的递增，流式检测巨噬细胞对嵌合体的摄入，发现巨噬细胞摄入的比例也在上升，并在24小时基本达到饱和状态。
图2为构建的嵌合体特异结合CD4阳性细胞图。
实施例3.构建的嵌合体抑制ccl18的合成与分泌
3.1在TAMs中，加入终浓度20nM的嵌合体，CD4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，在条带上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CCL 18 capture antibody的96孔板中室温孵育2小时，PBST洗3遍，加入CCL 18  detective antibody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CCL 18 siRNA的嵌合体与TAMs孵育约24小时后，CCL 18的mRNA水平通过Q-PCR检测。CCL18 mRNA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aptamer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。
通过western blot实验可以发现，构建的嵌合体可以抑制TAMs的CCL 18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。
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。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。图3为构建的CD4嵌合体敲低肿瘤相关巨噬细胞CCL18的表达：mRNA(左上)，蛋白(左下)，因子分泌(右)；
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(一下简称231细胞)进行transwell实验(图4 A)。图上可见，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4 B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
5.1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、IL-12、IFN的mRNA表达水平。如图5(上)所示，构建的CD4嵌合体不引起肿瘤相关巨噬细胞的炎症相关因子IL-6、IL-10、IL-12、IFN的高表达。
5.2嵌合体对肿瘤相关巨噬细胞的毒性检测
5.2.1将嵌合体、核酸适体、慢病毒与TAMs共孵育，在1、3、6、12、24、36、48小时时各吸取约100微升上清，在上清中加入50微升LDH的底物混合液，37℃避光孵育30分钟，取出加入终止缓冲液，用酶标仪检测570nm的吸光度。以1％triton-X破膜细胞的上清作为阳性对照，纯培养基作为阴性对照。
5.2.2对于检测的值减去阴性对照组的值，与时间点作折线图， 比较LDH的释放力从而比较加入试剂对肿瘤相关巨噬细胞的毒性反应。
在在1、6、12、24、36、48小时对处理了嵌合体的TAMs上清进行LDH检测，发现嵌合体并没有明显引起细胞的LDH释放，说明嵌合体不会引起TAMs的毒性反应。对于嵌合体处理的TAMs，我们检测了细胞的验证相关因子IL-6、IL-10、IL-12、IFN的mRNA水平变化，发现嵌合体并不会诱导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。如图5(下)所示，嵌合体不引起肿瘤相关巨噬细胞毒性反应。
Figure PCTCN2018084284-appb-000001
Figure PCTCN2018084284-appb-000002
Figure PCTCN2018084284-appb-000003

Claims (4)

  1. 一种CD4阳性细胞特异的DNA核酸适体，其特征在于，其核苷酸序列为TGACGTCCTTAGAATTGCGCATTCCTCACACAGGATCTT。
  2. 一种核酸适体-siRNA嵌合体，其特征在于，由权利要求1所述的核酸适体与小分子RNA形成的核酸序列组合。
  3. 如权利要求2所述的核酸适体-siRNA嵌合体，其特征在于，所述小分子RNA为CCL18 siRNA，其序列为：
    5′-ACAAGUUGGUACCAACAAATT-3′
    5′-UUUGUUGGUACCAACUUGUGC-3′。
  4. 权利要求2所述嵌合体在制备用于抑制乳腺癌MDA-MB-231细胞迁移侵袭试剂中的应用。
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