WO2020006922A1 - 一种合成肽sp4及其应用 - Google Patents

一种合成肽sp4及其应用 Download PDF

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WO2020006922A1
WO2020006922A1 PCT/CN2018/109931 CN2018109931W WO2020006922A1 WO 2020006922 A1 WO2020006922 A1 WO 2020006922A1 CN 2018109931 W CN2018109931 W CN 2018109931W WO 2020006922 A1 WO2020006922 A1 WO 2020006922A1
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tumor
synthetic peptide
cells
cell
group
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PCT/CN2018/109931
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French (fr)
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张万琴
李荫田
吉学文
赵丽美
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泰安市启航生物科技有限公司
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Priority to US16/762,470 priority Critical patent/US20210179664A1/en
Priority to EP18925139.0A priority patent/EP3696187A4/en
Priority to CA3079766A priority patent/CA3079766C/en
Priority to JP2020545841A priority patent/JP6934217B2/ja
Publication of WO2020006922A1 publication Critical patent/WO2020006922A1/zh
Priority to US17/656,347 priority patent/US11939399B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the technical field of biomedicine, and relates to a synthetic peptide sp4 (Synthetic Peptide-4) and its application, in particular to its application in the preparation of a new class of antitumor drugs.
  • Osteosarcoma is also known as osteosarcoma (the most common primary malignant tumor of bone, is a highly malignant mesenchymal tumor, which occurs in Adolescents and children under 21 years of age, osteosarcoma grows aggressively locally, grows rapidly, has a high degree of malignancy, is prone to metastasis, has poor limb salvage treatment effects, has a high disability rate and a high mortality rate, and is very effective in chemotherapy for metastases Important;
  • cytotoxic antitumor drugs are mainly used, Paclitaxel, Cisplatinum, Methotrexate (MTX), Adriamycin (ADM, Ifosfamide) , IFO) and other combined drugs, its toxic and side effects bring great harm and pain to patients.
  • Esophagus cancer is one of the common digestive system tumors, and also combined chemotherapy is used, its toxic and side effects are large.
  • Polypeptide compounds are a An important biologically active molecule.
  • the chemically synthesized peptide sp4 is a small peptide synthesized using peptide solid-phase synthesis technology. It is purified by high-performance liquid chromatography (HPLC), and analyzed by mass spectrometry and other biological technologies. Surgical treatment provides quantitative data for the preparation, structural confirmation, quality research, etc. of sp4. So far, no similar chemically synthesized peptides and their application in antitumor drugs have been seen in the prior art.
  • the invention discloses a chemically synthesized peptide sp4, the amino acid sequence of which is shown in SEQ ID No. 1. Compared with natural antitumor polypeptide, chemical synthesis can obtain a high-purity peptide monomer. No similar compounds have been seen in the prior art.
  • the present invention also protects the use of the synthetic peptide sp4 as described in any of (1) to (8): (1) preventing and / or treating tumors; (2) inhibiting tumor cell proliferation and / or growth and / Or invasion; (3) enhance anti-tumor immune response; (4) induce tumor cell differentiation; (5) prepare a class of anti-tumor drugs; (6) inhibit tumor telomerase activity; (7) regulate tumor cell cycle; (8) ) Preparation of products for regulating tumor cell cycle.
  • the inhibition of tumor telomerase activity refers to the application as a tumor telomerase inhibitor peptide.
  • the synthetic peptide sp4 has a strong inhibitory effect on telomerase activity in both tumor cell lines in vitro and tumor tissues in vivo.
  • the preparation of tumor cell cycle regulating products refers to the application as a tumor cell cycle regulating drug.
  • the synthetic peptide sp4 has a G1 phase blocking effect. Related to inhibition of telomerase activity.
  • the tumor described above is human osteosarcoma or human esophageal cancer.
  • the human esophageal cancer includes malignant tumors of esophageal squamous epithelium and columnar epithelium.
  • the invention also protects a biologically active molecular product whose active ingredient is the synthetic peptide sp4. Further, the only effective active ingredient of this product is the synthetic peptide sp4.
  • the synthetic peptide sp4 is used as an effective active ingredient in an antitumor drug to obtain a good application.
  • the target of sp4 antitumor effect is clear, and it can be used in the form of a single medication, that is, as the only effective active ingredient, the effect is significant.
  • Cytotoxic antitumor drugs are one of the important methods for the treatment of malignant tumors. In order to reduce the toxicity of cytotoxic antitumor drugs, a variety of combinations are often used.
  • the present invention provides a non-cytotoxic chemically synthesized peptide sp4, which is a single drug, has a significant curative effect, targets the telomerase activity of tumor cell DNA telomeres, and participates in cell cycle regulation.
  • the in vitro anti-tumor activity screening test (CCK-8 method)
  • CCK-8 method in the in vitro anti-tumor activity screening test (CCK-8 method)
  • the inhibition rate of sp4 on MG-63 was 93.84%
  • Eca-109 The inhibition rate was 73.58% with a clear dose-response relationship
  • the in vivo test is a decisive indicator for evaluating the effectiveness of the test substance on the killing or inhibitory effect of specific types of tumor cells.
  • the in vivo pharmacodynamic test of the present invention uses a nude mouse subcutaneous human cancer xenograft tumor model, and measures the tumor diameter every other day to dynamically observe the growth of the transplanted tumor.
  • the safety of the present invention is that the maximum tolerated dose is tested using an acute toxicity test. After intravenous injection of sp4 at a dose of 700 mg / kg BW to animals, no toxic reaction and death were seen within 24 hours. Within 2 weeks of continuous observation, No behavioral abnormalities and deaths were seen. In the in vivo pharmacodynamic test, the weight gain data of the tumor-bearing nude mice in each treatment group were not different from those in the control group (P> 0.05), and no death occurred, as shown in Figure 15 of the accompanying drawings. The maximum tolerated dose of sp4 is shown in Tables 2 and 3.
  • Telomeres are a special structure located at the end of chromosomes. Telomeres can maintain cell division. Telomerase can catalyze the replication and extension of telomere DNA, leading to infinite division and proliferation of cancer cells. Anti-telomerase activity is one of the important targets for anti-tumor therapy. Sp4 is effective for telomerase in tumor cell lines in vitro and tumor tissue in vivo. The activities have a significant inhibitory effect, and both have a significant dose-response relationship.
  • the cell cycle is the basic process of cell life activity. Flow cytometry was used to detect the cell cycle, and it was found that the cells were blocked in G1 phase after treatment with sp4, suggesting that sp4 may participate in the regulation of tumor cell cycle through the inhibition of telomerase activity. Inhibition of telomerase activity increases the inhibitory activity of cyclin-dependent kinase CDK inhibitors. CDK inhibitors compete with cyclin D1 to bind to CDK4, inhibit the increase of CDK4 activity, and prevent cells from entering the S phase from the G1 phase.
  • FIG. 1 Schematic diagram of the chemical synthesis of sp4
  • Figure 5 In vitro pharmacological effect of sp4;
  • Figure 5A Inhibition rate of human esophageal carcinoma Eca109 in vitro by sp4;
  • Figure 5B Inhibition rate of human osteosarcoma MG63 by sp4;
  • Figure 7 In vivo antitumor efficacy of sp4-inhibitory effect on tumor weight of tumor-bearing nude mice. Note: After different concentrations of sp4, compared with the model group, each group has statistically different changes. Among them, * represents P ⁇ 0.05; ** represents P ⁇ 0.01; *** represents P ⁇ 0.001;
  • FIG 8. Photo results of the anti-tumor drug efficacy evaluation test administered with sp4 via tail vein. The shooting conditions for measuring tumor volume in the figure are the same. The tumor volume in each group is shown in Figure 8 on a scale.
  • FIG. 9 Evaluation index of in vivo antitumor efficacy of sp4-relative tumor proliferation rate T / C (%); according to national FDA regulations, cytotoxic antitumor drugs, nude mice xenograft model pharmacodynamic evaluation criteria: T / C ( %)> 40% is invalid; T / C (%) ⁇ 40%, and P ⁇ 0.05 is effective after statistical processing.
  • the results showed that the sp4 drug appeared to be effective on the 19th day of treatment at low doses; the treatment was effective to start on the 15th day at medium doses; and the effective to start on the 7th day at high doses;
  • Figure 10 Comparison of in vivo antitumor efficacy between sp4 and the positive control paclitaxel-Comparison of tumor growth curves between sp4 and paclitaxel. There is no significant difference between the two (P> 0.05).
  • the vertical coordinate is tumor volume and the mold coordinate is Days of administration
  • Sp4 participates in HL-60 tumor cell cycle regulation; the effect of different doses of sp4 on HL-60 cell cycle A: control group, B: 200 ⁇ M, C: 100 ⁇ M, D: 50 ⁇ M, E: 25 ⁇ M;
  • Figure 15 Cell cycle changes of subcutaneous human osteosarcoma in nude mice after administration of sp4; Figure ** indicates that there is a significant difference between G0 / G1 phase of each group and model group; ## indicates G2 / M phase comparison of each group and model group, There are significant differences.
  • sp4 is composed of 7 kinds of SGAILFP amino acids, as shown in the following table and attached drawings 1A and B.
  • SP4 solid-phase chemical synthesis process preparation The use of peptide Fmoc solid-phase synthesis technology is a process of repeatedly adding amino acids, in the order from the C-terminus to the N-terminus of a known polypeptide amino acid sequence.
  • the experimental process is shown in Figure 2: First, the carboxyl group of the first amino acid at the C-terminus of the target polypeptide is covalently linked to a solid-phase carrier (resin), and then the amino group of this amino acid is used as the starting point for synthesis to occur with the carboxyl group of an adjacent amino acid. Acylation reactions form peptide bonds.
  • Cells with a proportion of more than 90% of live cells were taken for experiments.
  • Cell proliferation inhibition test uses cell digestion, counting, and making a cell suspension with a concentration of 1 ⁇ 10 5 cells / mL.
  • 100 ⁇ L of cell suspension (1 ⁇ 10 4 cells per well) is added to each well of a 96-well plate; 96 wells The plate was placed in a 37 ° C, 5% CO 2 incubator for 24 hours; 100 ⁇ L of the corresponding drug-containing culture medium was added to each well, and a negative control group, a vehicle control group, and a positive control group were set up in 5 duplicate wells; 96 The wells were incubated at 37 ° C for 72 hours in a 5% CO 2 incubator; 10 ⁇ L of CCK-8 solution was added to each well, and the plates were incubated in the incubator for 4 hours.
  • the OD value was measured at 450 nm using a microplate reader.
  • mice BALB / c nude mice
  • the MG-63 cell line with good recovery status was inoculated into a T75 cell culture flask and cultured at 37 ° C and 5% CO 2 .
  • the cells are collected by digestion and centrifugation, and the cell concentration is adjusted to 5 * 10 7 cells / mL with physiological saline, and nude mice are ready to be inoculated.
  • mice Take out the mice, wipe the inoculation area with alcohol (under the right forelimb armpit), inoculate 100 ⁇ L of cell suspension (about 5 * 10 6 cells / cell) in a biosafety cabinet, and inoculate 40 cells.
  • mice were randomly divided into 5 groups, namely the model group, the positive control group, the low-dose sp4 group, the middle-dose sp4 group, and the high-dose sp4 group, 6 rats per group (low-dose group: 4 mg / kgBW; medium The dose group was 8 mg / kg BW; the high-dose group was 16 mg / kg BW; the model group was injected with an equal volume of normal saline; the positive control was given paclitaxel at 10 mg / kg BW twice a week).
  • the tail vein injection was performed at 4 pm every day.
  • the model group and the model administration group were injected with saline and drugs respectively for 4 weeks, and the paclitaxel group were administered twice a week.
  • nude mice were sacrificed by cervical dislocation, and photos were recorded according to different groups; axillary tumors were stripped, and photos were recorded according to different groups, and the tumor weight was weighed.
  • tumors were stored at -80 ° C, 4% paraformaldehyde, and 2.5% glutaraldehyde, respectively.
  • tumor growth data statistically analyze tumor growth curve and relative tumor proliferation rate T / C%.
  • telomerase activity of MG-63 cells by sp4 Experimental method steps provided by Shanghai Meixuan Biotechnology Co., Ltd .:
  • the final primer concentration is 0.2 ⁇ M, which can get better results.
  • the primer concentration can be adjusted within the range of 0.1 to 1.0 ⁇ M.
  • ROX Reference Dye II (50 ⁇ ) has a lower concentration than ROX Reference Dye (50 ⁇ ).
  • 7500 Real-Time PCR System and 7500 Fast Real-Time PCR System please use ROX Reference Dye II (50 ⁇ ).
  • ABI PRISM 7300 Real-Time PCR System and Step OnePlusTM use ROX Reference Dye (50 ⁇ ).
  • the amount of DNA template added is usually less than 100ng. Because the copy number of the target gene contained in different kinds of DNA templates is different, if necessary, gradient dilution can be performed to determine the optimal amount of DNA template to be added. If you want to use this product for the 2nd step RT-PCR reaction of the second step PCR amplification reaction, the amount of RT reaction solution used as the DNA template in the first step should not exceed 10% of the total volume of the PCR reaction solution.
  • the final primer concentration is 0.2 ⁇ M, which can get better results.
  • the primer concentration can be adjusted within the range of 0.1 to 1.0 ⁇ M.
  • ROX Reference Dye II (50 ⁇ ) has a lower concentration than ROX Reference Dye (50 ⁇ ).
  • ROX Reference Dye II 50 ⁇
  • ABI PRISM 7300 Real-Time PCR System and Step One Plus TM use ROX Reference Dye (50 ⁇ ).
  • the amount of DNA template added is usually less than 100ng. Because the copy number of the target gene contained in different kinds of DNA templates is different, if necessary, gradient dilution can be performed to determine the optimal amount of DNA template to be added.
  • Propidium iodide is a fluorescent dye for double-stranded DNA. Propidium iodide and double-stranded DNA combine to produce fluorescence, and the intensity of fluorescence is proportional to the content of double-stranded DNA.
  • the DNA in the cell is After propidium iodide staining, the DNA content of the cells can be determined by flow cytometry, and then cell cycle analysis can be performed based on the distribution of DNA content.
  • the cell pellet was dissolved and suspended with 100 ⁇ L of RNase A at a concentration of 1 mg / mL, and the RNA in the cells was digested in a 37 ° C incubator;
  • NC (HL-60ONLY) 41.43 14.29 44.29 2.00 4.70 44.29 sp4 (200 ⁇ M) 66.54 9.69 23.76 1.93 5.33 23.76 sp4 (100 ⁇ M) 65.79 6.03 28.17 2.00 5.18 28.17 sp4 (50 ⁇ M) 64.76 14.23 21.01 1.89 5.89 21.01 sp4 (25 ⁇ M) 62.32 14.85 22.82 1.92 5.70 22.82
  • the tumor mass was washed 3 times with PBS buffer, and the tumor was cut into small pieces (1-2 mm) with ophthalmic surgical scissors, washed 3 times with PBS, and transferred to a 50 ml centrifuge tube.
  • Cell fixation add 1 ml of ice-bath pre-cooled 70% ethanol, gently mix by pipetting, fix at 4 ° C for 2 hours, centrifuge at 1000g for 3-5 minutes, and precipitate the cells. Aspirate the supernatant and add approximately 1 ml of ice-cold PBS to resuspend the cells. Centrifuge the cells again and gently bounce the bottom of the tube to disperse the cells appropriately.
  • Preparation of propidium iodide staining solution Prepare an appropriate amount of propidium iodide staining solution according to the number of samples to be tested; Note: The prepared propidium iodide staining solution can be stored at 4 ° C for a short period of time and should be used on the same day.
  • the G0 / G1 phase of the high-dose SP4 group increased significantly (P ⁇ 0.01), and the S and G2 / M phases decreased, and the G2 / M phase decreased significantly (P ⁇ 0.01).
  • the G0 / G1 phase of the SP4 low-dose group decreased significantly (P ⁇ 0.01), and the S and G2 / M phases increased, showing a dose-response relationship.
  • Test sample sp4;
  • mice tail vein injection mice tail vein injection
  • Dosing volume 0.5ml / time
  • Acute toxicity refers to the toxic reaction produced within a certain period of time after a single or multiple administration of a drug within 24 hours.
  • the narrow single dose toxicity study (Single dose toxicity study) examines the acute toxicity after a single administration of a test substance. This guideline refers to the broad single-dose toxicity study, which can be obtained by single or multiple doses within 24 hours;
  • Antibody name source Antibody manufacturers Article number PD-L1 rabbit Abcam Ab213480 CD47 rabbit proteintech 18470-1-AP
  • the data provided are the average optical density (IOD abbreviated as OD) and the positive area ratio (positive area / total area.) The results were analyzed using a positive index.
  • results Compared with the model group, the expression of CD47 in the sp4 group was significantly reduced; the expression of PD-L1 was significantly decreased in the SP4 group compared with the model group. This indicates that sp4 can significantly inhibit the high expression of CD47 and PD-L1 in tumor cells.
  • CD47 on the surface of tumor cells interacts with SIRPa on the surface of macrophages to send out immunosuppressive signals, thereby protecting tumor cells from macrophages.
  • PD-L1 on the surface of tumor cells binds to PD-1 molecules on immune cells and inhibits T cell activity. Therefore, sp4 can significantly inhibit the high expression of CD47 and PD-L1 in tumor cells, and enhance the immunity to clear tumor cells effect. Restore the ability of macrophages and T cells to recognize tumor cells (as shown in Figures 17, 18).
  • Hematoxylin, eosin stain, neutral gum seal Hematoxylin, eosin stain, neutral gum seal.
  • Nitro blue tetrazolium also known as Nitrotetrazolium (NBT)
  • NBT Nitrotetrazolium
  • Formulamazan Water-insoluble blue-black formazan
  • HL-60 cell line cultured in RPM11640 medium containing 15% fetal bovine serum at 37 ° C, 5% CO 2 saturated humidity. Take the logarithmic growth phase cells for experiments.
  • the 3000 ⁇ g / bottle of sp4 lyophilized powder was completely dissolved with 1.20 ml of ultrapure water (its concentration is 2000 ⁇ M, that is, 2502 ⁇ g / ml), which is equivalent to 10 times the concentration of the working fluid concentration of one group (200 ⁇ M, that is, 250.2 ⁇ g / ml) Times
  • Sample number NBT positive cell rate (%) Take multiple 1NS control 6.0 200 2.sp4 200 ⁇ M 80.5 200 3.sp4 100 ⁇ M 75.0 200 4.sp4 50 ⁇ M 73.0 200 5.sp4 25 ⁇ M 70.0 200

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Abstract

提供了一种氨基酸序列如SEQ ID No.1所示的合成肽sp4，其在抗肿瘤药效试验中作为唯一有效成分，对裸鼠人骨肉瘤MG-63的肿瘤体积和重量均具有抑制作用；所述合成肽sp4与阳性对照组紫杉醇的相对肿瘤增殖率T/C（%）比较无差异；静脉注射途径给药其最大耐受剂量为700mg/kgBW；在具有体内药效的同时，对肿瘤端粒酶具有抑制作用，对肿瘤细胞周期呈现G1期阻滞作用，对肿瘤PD-L1和CD47的高表达呈抑制作用。

Description

一种合成肽sp4及其应用 技术领域
本发明属于生物医药技术领域，涉及一种合成肽sp4(Synthetic Peptide-4)及其应用，尤其是在制备一类抗肿瘤新药中的应用。
背景技术
癌症正严重威胁着人类的健康和生命，骨肉瘤(osteosarcoma，OS)也称为成骨肉瘤(是一种最常见的原发性骨恶性肿瘤，是高度恶性的间叶组织肿瘤，好发于21岁以下的青少年和儿童，骨肉瘤在局部呈侵袭性生长，生长迅速、恶性度极高、易发生转移、保肢治疗效果差，具有致残率和死亡率高等特点，化疗治疗转移灶非常重要；目前临床上，主要釆用细胞毒类抗肿瘤药物，紫杉醇(Paclitaxel)、顺铂(Cisplatinum)、甲氨碟呤(Methotrexate，MTX)、阿霉素(Adriamycin，ADM、环磷酰胺(Ifosfamide，IFO)等联合用药，其毒副作用给病人带来极大伤害和痛苦。食管癌(esophagus cancer)是常见消化系统肿瘤之一，也多采用联合化疗，其毒副作用大。多肽类化合物是一类重要的生物活性分子。化学合成肽sp4是釆用多肽固相合成技术合成的小肽，经高效液相色谱(HPLC)纯化、质谱分析鉴定等生物技术处理，为sp4的药物制备、结构确证、质量研究等方面提供了量化资料。到目前为止，现有技术中，未见有类似的化学合成肽及其在抗肿瘤药物中的应用。
发明内容
本发明公开一种化学合成肽sp4，其氨基酸序列如SEQ ID NO.1所示，与天然抗肿瘤多肽比较，化学合成可获得高纯度的多肽单体。在现有技术中，未见有类似的化合物。
本发明还保护所述的合成肽sp4如下(1)至(8)中任一所述的应用：(1)预防和/或治疗肿瘤；(2)抑制肿瘤细胞的增殖和/或生长和/或侵袭；(3)增强抗肿瘤免疫反应；(4)诱导肿瘤细胞分化；(5)制备一类抗肿瘤药物；(6)抑制肿瘤端粒酶活性；(7)调控肿瘤细胞周期；(8)制备调控肿瘤细胞周期类产品。
进一步的，上文所述的合成肽sp4的应用(6)中，抑制肿瘤端粒酶活性，是指在作为肿瘤端粒酶抑制肽中的应用。所述合成肽sp4对体外肿瘤细胞株及体内肿瘤组织内的端粒酶活性均具有强的抑制作用。
进一步的，上文所述的合成肽sp4的应用(8)中，制备调控肿瘤细胞周期类产品，是指作为肿瘤细胞周期调控类药物中的应用，合成肽sp4具有G1期阻滞作用，可能与抑制端粒酶活性有关。
进一步的，上文所述的肿瘤为人骨肉瘤或人食管癌。所述的人食管癌包括食管鳞状上皮和柱状上皮的恶性肿瘤。
本发明还保护一种生物活性分子产品，它的活性成分为所述的合成肽sp4。进一步的，此 产品的唯一有效活性成分为所述的合成肽sp4。尤其是本发明实施例中利用所述的合成肽sp4在抗肿瘤药物中作为有效活性成分取得很好的应用。sp4抗肿瘤作用靶点明确，可以采用单一用药的形式使用，即作为唯一有效活性成分，疗效显著。
细胞毒类抗肿瘤药物是目前治疗恶性肿瘤的重要手段之一，为了降低细胞毒类抗肿瘤药物的毒性，多采用多种方式的联合用药。本发明提出了一种单一用药、疗效显著、靶向肿瘤细胞DNA端粒的端粒酶活性、参与细胞周期调控的非细胞毒类的化学合成肽sp4。对于临床前抗肿瘤有效性研究，除了探索受试物的作用强度和对不同类型肿瘤的敏感性外，还应特别关注受试物与阳性对照药物试验结果的比较。本发明首先在体外抗肿瘤活性筛选试验(CCK-8法)中发现sp4与MG-63和Eca-109细胞共培养72h后，sp4对MG-63的抑制率为93.84％，对Eca-109的抑制率是73.58％具有明显的剂量-效应关系；体内试验是评价受试物对特定类型肿瘤细胞杀伤或抑制作用有效性的决定性指标。本发明的体内药效试验采用裸鼠皮下人癌异种移植瘤模型，通过隔日测量瘤径的方法，动态观察移植瘤的生长情况。实验结束时称取瘤重。本发明发现sp4对裸鼠皮下人骨肉瘤MG-63的异种移植瘤的肿瘤体积和重量均具有明显抑制作用，具有明显剂量-效应和时间-效应关系；相对肿瘤增殖率T/C(％)＝10.27±1.87，、肿瘤生长抑制率为88.31％，经统计学处理表明sp4具有强的抗肿瘤作用。而且sp4与阳性对照组紫杉醇的相对肿瘤增殖率T/C(％)的比较无明显差异(P>0.05)，这是评价受试物sp4是否有必要进入临床试验的重要指标之一。本发明的安全性在于采用急性毒性试验的最大耐受剂量检测，给动物静脉注射剂量为700mg/kgBW的sp4后，在24小时内未见毒性反应和死亡，在连续观察的2周内，也未见行为异常和死亡。体内药效试验中，各治疗组荷瘤裸鼠体重增长数据均与对照组无差异(P>0.05)，也均未出现死亡现象，如附图15。sp4的最大耐受剂量检测如表2、3所示。
人早幼粒白血病细胞株HL-60在不同浓度sp4作用后，G0/G1期细胞表达量增加，S期细胞表达量减少，G2期细胞表达量减少，呈G1期阻滞，具有剂量-反应关系。在裸鼠皮下人骨肉瘤的异种移植瘤模型中，sp4的中、高剂量组和阳性对照紫杉醇组也均呈现明显G1期阻滞现象。
端粒是一种位于染色体末端的特殊结构，端粒能维持细胞分裂。端粒酶能催化端粒DNA复制和延伸，导致癌细胞无限分裂增殖，抗端粒酶活性是抗肿瘤治疗的重要靶点之一，sp4对体外肿瘤细胞株及体内肿瘤组织内的端粒酶活性均具有显著抑制作用、均具有明显的剂量-效应关系。
细胞周期是细胞生命活动的基本过程。用流式细胞仪检测细胞周期，发现经sp4处理后细胞被阻滞于G1期，提示sp4可能通过对端粒酶活性的抑制作用参与了对肿瘤细胞周期的调控。抑制端粒酶活性使细胞周期素依赖激酶CDK抑制剂的抑制活性增加。CDK抑制剂与细胞周期素D1竞争性结合CDK4，抑制CDK4活性增加，阻止细胞从G1期进入S期。
附图说明
图1. A、B sp4的氨基酸组成；
图2. sp4的化学合成过程示意图；
图3. sp4色谱结果；
图4. sp4质谱结果；
图5. sp4的体外药效图；图5A sp4对人食管癌Eca109体外增殖活性的抑制率；图5B sp4对人骨肉瘤MG63体外增殖活性的抑制率；
图6. sp4体内抗肿瘤药效-肿瘤生长曲线；
图7. sp4体内抗肿瘤药效-对荷瘤裸鼠肿瘤重量的抑制作用，注：不同浓度sp4后，相比较模型组，每组统计学差异变化。其中，*代表P<0.05；**代表P<0.01；***代表P<0.001；
图8. sp4尾静脉给药抗肿瘤药效评价实验的图片结果；图中测量肿瘤体积大小的拍摄条件相同，各组肿瘤体积大小均按比例尺呈现在图8中；
图9. sp4体内抗肿瘤药效评价指标-相对肿瘤增殖率T/C(％)；根据国家FDA规定，细胞毒类抗肿瘤药物，裸鼠移植瘤模型药效学评价标准：T/C(％)＞40％为无效；T/C(％)≤40％，并经统计学处理P＜0.05为有效。结果显示：sp4药物，低剂量时，治疗第19天开始表现为有效；中剂量时治疗第15天开始表现为有效；高剂量时，治疗第7天开始表现为有效；
图10. sp4与阳性对照紫杉醇的体内抗肿瘤药效比较-sp4和紫杉醇的肿瘤生长曲线比较，两者之间未见明显差异(P>0.05)，纵座标为肿瘤体积、模座标为给药天数；
图11. sp4与阳性对照紫杉醇的体内抗肿瘤药效评价指标-相对肿瘤增殖率T/C(％)比较，两者无明显差异；
图12. sp4对肿瘤细胞株MG-63端粒酶活性的抑制作用；**表示P＜0.01；***表示P＜0.001；具有剂量依赖关系；
图13. sp4对裸鼠人骨肉瘤MG-63体内肿瘤组织中端粒酶活性的抑制作用；**表示与MG-63细胞皮下瘤模型组比较，给药组的端粒酶活性变化，具有显著差异(P＜0.01)，而且呈剂量依赖关系；
图14. sp4参与HL-60肿瘤细胞周期调控；不同剂量sp4作用对HL-60细胞周期的影响A：对照组、B：200μM、C：100μM、D：50μM、E：25μM；
图15. sp4给药后裸鼠皮下人骨肉瘤细胞周期变化；图**表示各组和模型组G0/G1期比较，具有显著性差异；##表示各组和模型组G2/M期比较，具有显著性差异。
图16. sp4静脉给药4W各组荷瘤裸鼠体重变化；
图17. sp4明显抑制PD-L1在肿瘤细胞中的高表达；*表示相比较于模型组，具有差异(P值＜0.05)；**表示相比较于模型组，具有显著性差异(P值＜0.01)。
图18. sp4明显抑制CD47在肿瘤细胞中的高表达；*表示相比较于模型组，具有差异(P 值＜0.05)；**表示相比较于模型组，具有显著性差异(P值＜0.01)。
具体实施方式
以下实施例进一步解释本发明的内容，阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施，因此本发明不受下面公开的具体实施的限制。
实施例1
Sp4的氨基酸组成分析：
称取10.2mg试样，以6N HCl 7mL溶解，氮气保护110℃酸水解22小时，冷却后转移至10mL容量瓶，定容。取0.2mL用55℃氮气吹干，加入1mL蒸馏水再烘干，反复三次。用去离子水(0.02mol/L HCl)1.2mL充分溶解，混匀。用0.45μm滤膜过滤，进样20μL上机(日立L-8900氨基酸分析仪)测试
测式结果如下：
Figure PCTCN2018109931-appb-000001
sp4由SGAILFP 7种氨基酸组成，见下表和附图1A和B。
实施例2
sp4的固相化学合成、纯度检测及分子量确认：
sp4固相化学合成工艺制备：采用多肽Fmoc固相合成技术，是一个反复添加氨基酸的过程，顺序为从已知的多肽氨基酸序列的C端到N端。实验过程如图2所示：首先将目标多肽C端第一个氨基酸的羧基共价连接到固相载体(树脂)上，再以这一氨基酸的氨基为合成起点，与相邻氨基酸的羧基发生酰化反应，形成肽键。不断重复这一过程，直到目标多肽合成完毕再将目标多肽从树脂上切割下来，同时脱去侧链保护基。最后加入冰乙醚沉淀出粗肽，利用高效液相色谱进行分离纯化，然后使用质谱进行产物鉴定(附图2-4)。
实施例3
抗肿瘤体外筛选实验(CCK-8法)，本技术由南京欧际医药科技服务有限公司提供，釆用EnoGeneCell TM Counting Kit-8(CCK-8)细胞活力检测试剂盒
取活细胞比例达90％以上的细胞进行实验。细胞增殖抑制试验采用细胞消化、计数、制 成浓度为1×10 5个/mL的细胞悬液，96孔板中每孔加入100μL细胞悬液(每孔1×10 4个细胞)；96孔板置于37℃，5％CO 2培养箱中培养24小时；每孔加入100μL相应的含药物的培养基，同时设立阴性对照组，溶媒对照组，阳性对照组，每组5复孔；96孔板置于37℃，5％CO 2培养箱中培养72小时后；每孔加入10μL CCK-8溶液，将培养板在培养箱内孵育4小时，用酶标仪测定在450nm处OD值计算sp4对MG-63和Eca-109肿瘤细胞株的抑制率及IC 50值，评价标准以同一样品的不同浓度sp4对肿瘤细胞抑制率作图可得到剂量效应曲线(附图5)，然后采用Logit法计算半数有效浓度(IC 50值)。
实施例4
sp4尾静脉给药抗肿瘤体内药效评价试验，
1.细胞系和动物
细胞系：MG-63(人成骨肉瘤细胞)，上海美轩生物科技有限公司提供(MXC245)
动物：BALB/c裸鼠
2.细胞准备阶段
2-1.复苏状态良好的MG-63细胞系，接种到T75细胞培养瓶中，37℃、5％CO 2培养。
2-2.每隔2-3天换液一次，细胞愈合度达到80％左右1：3传代，大约需要20瓶左右。
2-3.待细胞传代培养数量够后，消化离心收集细胞，用生理盐水调整细胞浓度为5*10 7个/mL，准备接种裸鼠。
3.动物准备阶段
3-1. 4周左右的BALB/c裸鼠，适应性培养一周后，准备接种细胞。
3-2.无菌条件下，用1mL注射器吸取细胞悬液(期间需要反复摇晃，避免细胞沉降)。
3-3.取出小鼠，酒精擦拭接种部位(右前肢腋下)，在生物安全柜中接种100μL细胞悬液(约5*10 6个/只)，接种40只。
3-4.细胞接种完后，放回饲养，25℃，12h光照/无光照，食物、水照常给予。
3-5.接种完后五天左右，触摸接种部位，确认是否有小瘤块，若无，双倍细胞接种量补种细胞。
3-6.接种完第8天后，用游标卡尺测量移植瘤直径，计算肿瘤体积，待肿瘤体积生长至80-100mm 3时，分组给药。
4.动物给药及处理
4-1.随机将裸鼠分成5组，分别是模型组、阳性对照组、sp4低剂量组、sp4中剂量组、sp4高剂量组，每组6只(低剂量组：4mg/kgBW；中剂量组8mg/kgBW；高剂量组16mg/kgBW；模型组等体积生理盐水注射；阳性对照给予紫衫醇10mg/kgBW，一周给药2次)。
4-2.动物分组后，每日下午4点，进行尾静脉注射，模型组和模型给药组分别注射生理盐水和药物，持续给药4w，紫衫醇组，一周给药2次。
4-3.从分组开始，当天及每隔一天测量肿瘤大小及体重情况，TV＝0.5*a*b 2，a为长径b为短径。
4-4.实验结束后，颈椎脱臼法处死裸鼠，按照不同的分组拍照留记录；剥离腋下肿瘤，按照不同的分组拍照留记录，并称瘤重。
4-5.按照不同的后续实验要求，分别-80℃、4％多聚甲醛、2.5％戊二醛保存肿瘤。
4-6.根据肿瘤生长数据，统计分析肿瘤生长曲线及相对肿瘤增殖率T/C％等。
实验结果数据均采用X±SD表示(附图6-11)。
实施例5
sp4对MG-63细胞端粒酶活性检测：实验方法步骤由上海美轩生物科技公司提供：
1.细胞准备
1.1 细胞复苏
1.2 细胞传代，细胞长满后，进行传代培养
1.3 细胞铺板
2.总RNA的提取
3. RNA反转录成cDNA
4. QPCR反应
应用Applied Biosystems 7300的操作方法：
1.通常引物终浓度为0.2μM可以得到较好结果。反应性能较差时，可以在0.1～1.0μM范围内调整引物浓度。
2.ROX Reference Dye II(50×)比ROX Reference Dye(50×)浓度低，使用7500 Real-Time PCR System和7500 Fast Real-Time PCR System时，请使用ROX Reference Dye II(50×)。使用ABI PRISM 7300 Real-Time PCR System和Step One PlusTM时，用ROX Reference Dye(50×)。
3.在20μL反应体系中，DNA模板的添加量通常在100ng以下。因不同种类的DNA模板中含有的靶基因的拷贝数不同，必要时可进行梯度稀释，确定最佳的DNA模板添加量。如果欲使用本制品进行2 Step RT-PCR反应的第二步PCR扩增反应，第一步的RT反应液作为DNA模板时的添加量不要超过PCR反应液总体积的10％。
4.按照各仪器推荐体系进行反应液配制。
5.进行Real Time PCR反应。按两步法PCR扩增标准程序。(附图12)
实施例6
实体瘤端粒酶活性检测
1.样本准备：裸鼠体内抗肿瘤药效实验结束时取下的肿瘤样本
2.总RNA的提取
3.RNA反转录成cDNA
4.QPCR反应：应用Applied Biosystems 7500的操作方法
1)配制PCR反应液
通常引物终浓度为0.2μM可以得到较好结果。可以在0.1～1.0μM范围内调整引物浓度。
2)ROX Reference Dye II(50×)比ROX Reference Dye(50×)浓度低，使用7500 Real-Time PCR System和7500 Fast Real-Time PCR System时，使用ROX Reference Dye II(50×)。使用ABI PRISM 7300 Real-Time PCR System和Step One Plus  TM时，使用ROX Reference Dye(50×)。
3)在20μL反应体系中，DNA模板的添加量通常在100ng以下。因不同种类的DNA模板中含有的靶基因的拷贝数不同，必要时可进行梯度稀释，确定最佳的DNA模板添加量。
4)按照各仪器推荐体系进行反应液配制。
5)进行Real Time PCR反应：两步法按PCR扩增标准程序
6)实验结果分析。(附图13)
实施例7细胞周期检测
(A)HL-60细胞周期检测：
碘化丙啶(Propidium，简称PI是一种双链DNA的荧光染料。碘化丙啶和双链DNA结合后可以产生荧光，并且荧光强度和双链DNA的含量成正比。细胞内的DNA被碘化丙啶染色后，可以用流式细胞仪对细胞进行DNA含量测定，然后根据DNA含量的分布情况，可以进行细胞周期分析。
1.细胞培养
2.将培养好的不同组别的细胞用0.25％胰酶消化，使其成为单个细胞，充分吹打混匀细胞悬液并收集至流式专用管中；
3.1000g离心5min，吸弃上清，沉淀用300μL含有10％胎牛血清的PBS溶液悬浮，移入干净的1.5mL离心管内；
4.加入700μL无水乙醇，置于-20℃冰箱内固定细胞24h以上；
5.取出固定的样品，于3,000g离心细胞30sec，吸弃上清；
6.细胞沉淀用100μL浓度为1mg/mL的RNase A溶溶悬浮，在37℃培养箱内消化细胞内的RNA；
7.加入400μL浓度为50μg/mL的碘化丙啶(PI，Propidium lodide)溶液，避光染核10min。用流式细胞仪进行细胞DNA含量测定，确定细胞在各细胞周期所占比例；
表1.流式细胞仪检测人早幼粒白血病细胞株HL-60在不同浓度sp4作用后细胞周期(％)
对应样品 DipG1(％) DipG2(％) DipS(％) G2/G1 ％CV Total S-Phase(％)
NC(HL-60ONLY) 41.43 14.29 44.29 2.00 4.70 44.29
sp4(200μM) 66.54 9.69 23.76 1.93 5.33 23.76
sp4(100μM) 65.79 6.03 28.17 2.00 5.18 28.17
sp4(50μM) 64.76 14.23 21.01 1.89 5.89 21.01
sp4(25μM) 62.32 14.85 22.82 1.92 5.70 22.82
与对照组相比，sp4作用后HL-60G0/G1期细胞表达量明显增加，G2/M期细胞表达量减少，S期细胞表达量减少，呈G1期阻滞，且随着剂量增大G1期细胞的比例随之增加。这说明sp4能够阻止人早幼粒白血病细胞株HL-60细胞从G1期转变到S期，使细胞周期出现G1期阻滞，减少有丝分裂，从而抑制细胞增殖，且sp4剂量越大，效果越明显(附图14)
(B)裸鼠人骨肉瘤MG-63细胞周期检测:
1、制备实体瘤单细胞悬液
1.1 瘤块，用PBS缓冲液洗涤3次，用眼科手术剪将肿瘤剪成小块(1-2mm)，再用PBS洗涤3次，转移至50ml离心管中。
1.2 加入0.25％胰酶液，37度消化20-40min，每隔5min轻轻震荡一次，使细胞分离。
1.3 加入2-5ml含血清培养基，以终止胰酶消化作用。
1.4 静置2-3min，将悬液转移到新的离心管中，用200目尼龙网过滤悬液2次。
1.5 将过滤后的悬液1000rpm，离心5min，弃上清液。
1.6 加入PBS缓冲液5ml，再离心一次，弃上清液。
1.7 根据细胞量加入1-2ml培养液，细胞计数，备用
2.细胞周期检测
1)收集细胞培养液到一离心管内备用。用胰酶消化细胞，至细胞可以被轻轻用移液管或枪头吹打下来时，加入前面收集的细胞培养液，1000g左右离心3-5分钟，沉淀细胞。吸除上清，加入约1毫升冰浴预冷的PBS，重悬细胞，并转移到1.5毫升离心管内。再次离心沉淀细胞，吸除上清，轻轻弹击离心管底以适当分散细胞。
2)细胞固定：加入1毫升冰浴预冷70％乙醇，轻轻吹打混匀，4℃固定2小时，1000g离心3-5分钟，沉淀细胞。吸除上清，加入约1毫升冰浴预冷的PBS，重悬细胞。再次离心沉淀细胞，轻轻弹击离心管底以适当分散细胞。
3)碘化丙啶染色液的配制：根据待检测样品的数量配制适量的碘化丙啶染色液；注：配制好的碘化丙啶染色液短时间内可以4℃保存，宜当日使用。
4)染色：每管细胞样品中加入0.5毫升碘化丙啶染色液，缓慢并充分重悬细胞沉淀，37℃避光温浴30分钟。随后可以4℃或冰浴避光存放。染色完成后宜在24小时内完成流式检测。
5)流式检测和分析：用流式细胞仪在激发波长488nm波长处检测红色荧光，同时检测光散射情况。采用分析软件进行细胞DNA含量分析和光散射分析。
结果：相比较于模型组，SP4中剂量组G0/G1期显著增加(P＜0.01)，S期和G2/M期减少；
SP4高剂量组G0/G1期显著增加(P＜0.01)，S期和G2/M期降低，其中G2/M期显著降低(P＜0.01)。SP4低剂量组G0/G1期显著降低(P＜0.01)，S期和G2/M期增加，呈剂量-反应关系。(附图15)
实施例8动物急性毒性试验-最大耐受量检测
一.实验条件：GLP
1.受试动物：ICR种小鼠健康、成年、雄性，体重19-20g，n＝10；
2.受试样品：sp4；
3.受试样品配制方法；
4.给药途径：小鼠尾静脉注射；
5.给药容量：0.5ml/次；
6.给药次数，按FDA指导原则；
急性毒性(Acute toxicity)是指药物在单次或24小时内多次给予后一定时间内所产生的毒性反应。狭义的单次给药毒性研究(Single dose toxicity study)是考察单次给予受试物后所产生的急性毒性反应。本指导原则所指为广义的单次给药毒性研究，可采用单次或24小时内多次给药的方式获得；
二.最大耐受量检测：
即不引起受试动物死亡的最高剂量；先将可能不引起受试动物死亡的最高剂量的3个浓度的工作液配置好，给1只动物(1.5mL分3次)尾静脉注射；第1个剂量为1150mg/kgBW的工作液总量为1.5mL，24h内分3次给小鼠尾静脉注射；次日如果未见死亡，将余下的9只动物进行同样的尾静脉注射(总数n＝10)；如果第1只动物死亡则降低一个剂量。依次进行，结果发发现700mg/kgBW剂量组小鼠，在24h内，分3次给药过程中未出现毒性反应和死亡(见表2)。ICR小鼠尾静脉注射剂量为700mg/kg.BW的Sp4的工作液
表2. 700mg/kg.BW剂量组最大耐受量实验
Figure PCTCN2018109931-appb-000002
Figure PCTCN2018109931-appb-000003
继续观察，在给药后2周内，均未见毒性反应和死亡(见表3)
表3. 700mg/kg.BW剂量组存活小鼠14天体重变化
日期 No.1 No.2 No.3 No.4 No.5 No.6 No.7 No.8 No.9 No.10
2018.5.15 20.5 20.6 20.7 20.4 20.2 20.9 20.1 20 20.3 20.2
2018.5.16 20.5 21.3 21.2 20.8 20.4 21.5 20.4 20.5 20.5 20.7
2018.5.17 21.1 21.7 21.3 21.4 20.8 22.2 21.2 20.8 21.1 21.1
2018.5.18 21.5 22.1 21.9 21.7 21.5 22.7 21.9 21.1 21.9 21.5
2018.5.19 21.9 22.6 22.6 22.5 21.8 23 22.5 21.8 22.5 21.6
2018.5.20 22.3 22.8 22.8 22.8 22.4 23.7 22.9 22.5 22.8 22.8
2018.5.21 22.7 23.5 23.1 23.1 22.9 24.1 23.3 23.1 23.4 22.9
2018.5.22 23.3 24.1 23.5 23.6 23.2 24.8 23.8 23.4 23.8 23.5
2018.5.23 24.0 24.8 24.2 24.2 23.7 25.1 24.5 24.4 24.1 24.1
2018.5.24 24.7 25.2 24.7 24.7 23.9 25.6 25.1 24.8 24.6 24.7
2018.5.25 25.3 25.8 25.1 25.1 24.8 26.1 25.5 25.1 24.9 25.5
2018.5.26 25.8 26.3 25.9 25.6 25.2 26.5 25.9 25.5 25.6 25.9
2018.5.27 26.1 26.8 26.3 26.2 26.4 27.2 26.3 26 25.8 26.2
2018.5.28 27.2 27.3 26.9 26.8 26.8 27.7 26.8 26.4 26.6 27.1
2018.5.29 28.1 28.5 27.2 27.6 27.5 28.3 27.5 27.1 27.1 27.9
另外，在体内药效试验中，各治疗组荷瘤裸鼠体重增长数据均与对照组无差异(P>0.05)，也均未出现死亡现象。附图16
实施例9
sp4对PD-L1、CD47的免疫荧光试验
样本：裸鼠皮下人骨肉瘤MG-63异种移植瘤组织
2.抗体：
抗体名称 来源 抗体厂家 货号
PD-L1 兔 Abcam Ab213480
CD47 兔 proteintech 18470-1-AP
3.实验步骤：
3-1.组织切片、展片
3-2.组织脱蜡水化
3-3.抗原修复
将切片放入盛有柠檬酸盐缓冲液(0.01mol/L，pH6.0)的容器中，置微波炉中加热(微波3档)，使容器内液体温度保持在98度左右，并持续10-15min。取出容器，室温冷却20-30min。PBS(0.01M，pH7.4)洗5min×3次，抗原修复液为，热蒸汽修复20分钟，自然冷却至室温。
3-4.吸去PBS，5％BSA/0.01MPBS封闭30分钟，不洗，用吸水纸，从边缘吸去5％BSA封闭液。滴加稀释的抗体溶液(稀释液为用PBS配制的5％BSA)，空白对照组以PBS(0.01M，pH＝7.4)替代抗体，湿盒中，4度冰箱孵育过夜。
3-5.第二天将湿盒从冰箱拿出，室温放置15分钟复温。PBS(0.01M，pH＝7.4)，洗5min×5次。吸去多余的PBS，滴加荧光二抗，室温避光孵育30分钟。PBS洗5min×5次。
3-6.滴加DAPI避光孵育2分钟，显核，蓝色荧光。用PBS 1min×3次洗去DAPI。
3-7.最后用甘油封片，并在荧光显微镜下立即观察。
4.分析方法：
所提供的数据是平均光密度(IOD简称OD)和阳性面积比(阳性面积/总面积.)结果分析采用阳性指数进行分析。
阳性指数＝阳性面积比×OD
结果：sp4给药组相比较于模型组CD47的表达显著下降；SP4给药组相比较于模型组PD-L1表达显著下降。表明sp4能明显抑制CD47和PD-L1在肿瘤细胞的高表达。肿瘤细胞表面的CD47与巨噬细胞表面的SIRPa相互作用，发出免疫抑制信号，从而保护肿瘤细胞免受巨噬细胞呑噬。肿瘤细胞表面的PD-L1与位于免疫细胞上的PD-1分子结合后，抑制T细胞活性，因此，sp4能明显抑制CD47和PD-L1在肿瘤细胞的高表达，具有增强清除肿瘤细胞的免疫效应。恢复巨噬细胞和T细胞对肿瘤细胞的识别能力(如附图17、18)。
实施例10
诱导分化实验：
1. HE染色方法(悬浮细胞)
1)涂片与固定
2)染色
苏木精、伊红液染色、中性树胶封片。
3)HE染色(悬浮细胞)结果和分析
(20).通过显微镜油镜下拍照，Lecia Applaction Stiue图象系统采集分析
表4. SP4对HL-60(人急性粒细胞白血病)细胞成熟、幼稚细胞(％)
编号 成熟细胞(％) 幼稚细胞(％)
C(对照) 40.1 59.9
SP4 200μM 58.6 41.4
SP4 100μM 57.0 43.0
SP4 50μM 46.3 53.7
SP4 25μM 43.3 56.7
2.四氮唑蓝(NBT)还原反应
1)原理：硝基蓝四氮唑，又称四氮唑蓝(Nitrotetrazolium，NBT)，是一种水溶性的淡黄色活性染料，当其被嗜中性粒细胞的酶还原后，则变为非水溶性的蓝黑色甲臜(Formazan)颗粒，沉淀于胞浆内。
2)方法
①细胞培养：
细胞：HL-60细胞株，用含15％胎牛血清的RPM11640培养基37℃，5％CO 2飽和式湿度条件下培养。取对数生长期细胞进行实验。
②合成肽贮备液的配制：
将3000μg/瓶的sp4冻干粉用1.20ml超纯水彻底溶解(其浓度为2000μM，即2502μg/ml)，相当于其剂量1组的工作液浓度(200μM，即250.2μg/ml)的10倍；
表5. NBT阳性细胞率(％)
样品序号 NBT阳性细胞率(％) 拍照倍数
1NS对照 6.0 200
2.sp4 200μM 80.5 200
3.sp4 100μM 75.0 200
4.sp4 50μM 73.0 200
5.sp4 25μM 70.0 200
对于任何熟悉本领域的技术人员而言，在不脱离本发明技术方案范围情况下，都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰，或修改为等同变化的等效实施例。因此，凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰，均应仍属于本发明技术方案保护的范围内。
Figure PCTCN2018109931-appb-000004

Claims (6)

  1. 一种合成肽sp4，其氨基酸序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的合成肽sp4的如下(1)至(8)中任一所述的应用：(1)预防和/或治疗肿瘤；(2)抑制肿瘤细胞的增殖和/或生长和/或侵袭；(3)增强抗肿瘤免疫反应；(4)诱导肿瘤细胞分化；(5)制备一类抗肿瘤药物；(6)抑制肿瘤端粒酶活性；(7)调控肿瘤细胞周期；(8)制备调控肿瘤细胞周期类产品。
  3. 根据权利要求2所述的合成肽sp4的应用，其特征在于：(1)至(8)中任一所述的肿瘤为人骨肉瘤或人食管癌。
  4. 根据权利要求3所述的合成肽sp4的应用，其特征在于：所述的人食管癌包括食管鳞状上皮和柱状上皮的恶性肿瘤。
  5. 一种生物活性分子产品，它的活性成分为权利要求1所述的合成肽sp4。
  6. 一种生物活性分子产品，它的唯一有效活性成分为权利要求1所述的合成肽sp4。
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