胸腺免疫抑制五肽（TIPP）的抗癌活性研究

发布时间：2018-01-18 14:25

本文关键词：胸腺免疫抑制五肽（TIPP）的抗癌活性研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的癌症,是一类细胞异常生长且不受控制,并且可以渗透或扩散到身体其他部位的疾病。癌症被认为是世界上主要的死亡原因。在2008年全球约有1270万例癌症病例和760万例癌症死亡,而在2012,全球约有1410万例新癌症病例和820万例癌症死亡。因此,研究有效且毒性低的抗肿瘤药物一直是药物研究的热点。我们实验室从20世纪80年代开始研究胸腺免疫抑制提取物(TISE)。研究发现,TISE在体内体外均能显著的抑制淋巴细胞增殖以及免疫和过敏反应。此外,TISE可以显著抑制小鼠迟发型超敏反应和大鼠被动皮肤过敏反应,并且能明显延长大鼠移植皮肤存活时间,对卵蛋白诱导的豚鼠哮喘的发生也有显著的抑制作用。为深入研究免疫抑制活性和机制,对小牛胸腺来源的TISE通过微球菌核酸酶酶解和反相高效液相色谱法分离,得到了一种新的序列为Ala-Glu-Trp-Cys-Pro的五肽(分子量为604.68),并命名为胸腺免疫抑制肽(thymic immunosuppressive pentapeptide,TIPP)。廉倩倩等证明了TIPP能通过阻断MAPK激酶/NF-κB信号通路从而有效抑制过敏性小鼠的过敏和炎症反应,他们也证实了 TIPP可以靶向抑制MEK/ERK/NF-κB信号通路进而抑制IgE刺激的RBL-2H3细胞的活化,同时研究发现,TIPP对人类髓性白血病细胞K562增殖有显著的抑制作用。鉴于TIPP的广泛的药理学作用,我们决定研究其抗癌活性。这是对TIPP的抗癌活性的首次研究。基于对K562细胞系的抑制作用和其作用机制,我们假设TIPP也可能同样具有抗癌活性并且可以用于癌症治疗。为了证实这一假定,我们首先研究了 TIPP在细胞水平的抗癌活性,然后评价了其在肿瘤动物模型体内的治疗作用,最后检测了它对正常器官的影响。本课题的研究结果可能会提供一种癌症治疗的新方法,也为TIPP的进一步研究与开发奠定基础。研究方法1 TIPP体外抗肿瘤活性研究用MCF-7及K562癌细胞系对TIPP的细胞毒性及癌细胞增殖抑制活性进行了评价。MCF-7及K562培养至所需要的数量,然后接种于96孔板。细胞在经过五种不同浓度的TIPP给药48 h后,加入5 mg/ml MTT(Sigma)溶液20 μl继续孵育4 h。离心后,甲佨蓝沉淀溶解于DMSO 150 μl。用酶标仪检测该DMSO溶液在570 nm处的吸光值。2 TIPP体内抗肿瘤活性研究五周龄雌性Bablc/c裸鼠(无特定病原体的)皮下接种慢性粒细胞白血病细胞系K562细胞和乳腺癌MCF-7细胞。裸鼠的平均肿瘤体积达到100 mm3时,将接种K562裸鼠随机分为3组,将接种MCF-7裸鼠随机分为6组,每组7只。对于K562荷瘤小鼠模型,第1组经尾静脉注射PBS,第2组注射TIPP 25 mg/kg(溶解于0.1 ml PBS),第3组注射放线菌素D 0.1 mg/kg。对于乳腺癌模型,总共6组分别注射PBS、TIPP 25 mg/kg、TIPP 25 mg/kg联合放线菌素D 0.1 mg/kg、TIPP 25 mg/kg联合紫杉醇7.5 mg/kg、紫杉醇7.5 mg/kg和放线菌素D。给药3周,每3天测量肿瘤体积。研究结果1 TIPP具有体外抗肿瘤活性增殖实验表明,TIPP与肿瘤细胞一起孵育48 h后可显著地以剂量依赖的方式抑制K562和MCF-7细胞的增殖。TIPP还可以显著抑制两种肿瘤细胞系的存活率。TIPP对于两种不同的肿瘤细胞株抑制活性表明了 TIPP的体外有抗肿瘤活性。2 TIPP体内抗肿瘤活性体内试验结果表明,TIPP可明显抑制K562和MCF-7肿瘤的生长,体内试验结果与体外细胞增殖实验结果完全吻合。TIPP对K562瘤的抑制率为26%,而对MCF-7瘤的抑制率达到54%。在乳腺癌模型中,TIPP也分别与另外两种有效的抗癌药物放线菌素D和紫杉醇联合给药,TIPP联合放线菌素D组的抑瘤率为58%,TIPP联合紫杉醇组的抑瘤率为74%。3 TIPP诱导肿瘤凋亡对乳腺癌小鼠的肝脏和肿瘤组织进行HE染色观察TIPP的安全性及对肿瘤的促凋亡能力。结果表明,TIPP和紫杉醇无论单独给药还是联合给药,都对肝组织无明显损害,放线菌素-D无论单独还是联合TIPP给药都发现有轻微的肝毒性;不管TIPP和紫杉醇单独还是联合给药都可以显著的诱导乳腺肿瘤组织细胞凋亡,放线菌素-D无论单独还是联合TIPP给药都引起轻度的肿瘤组织坏死。上述表明TIPP单独或与紫杉醇联合给药是一种安全且能诱导细胞凋亡而抑制肿瘤生长的治疗方案。结论及意义(1)对TIPP体外抗癌活性进行了研究。TIPP显著抑制K562和MCF-7细胞的增殖,并且有剂量和时间依赖性。(2)首次对TIPP在慢性粒细胞白血病和乳腺癌小鼠模型的体内抗肿瘤作用进行了研究。TIPP有效抑制慢性粒细胞白血病和乳腺癌小鼠肿瘤的生长。(3)对肝脏和乳腺肿瘤组织进行了 HE染色和免疫组化检测。HE染色结果显示TIPP通过诱导细胞凋亡来抑制乳腺癌。(4)对于正常组织如肝脏的HE染色结果表明,TIPP是安全的药物,对肝脏无损伤。(5)首次揭示了 TIPP(与其他抗癌药物)联合治法对于乳腺癌的治疗作用。体内结果表明,TIPP联合紫杉醇给药组的抑瘤率(74%)比TIPP单独给药组的(54%)高。
[Abstract]:The purpose of the study is a kind of cancer, abnormal cell growth, which is not under control, and can penetrate or spread to other parts of the body disease. Cancer is considered to be the leading cause of death in the world. In 2008, there are around 12 million 700 thousand cases of cancer and 7 million 600 thousand cancer deaths, while in 2012, there are around 14 million 100 thousand new cases of cancer cases and 8 million 200 thousand cancer deaths. Therefore, the study of effective and low toxicity antitumor drugs has been a hot spot in drug research. Our laboratory began to study the Thymic Immunosuppressive Extract (TISE) from 1980s. The study found that TISE could significantly inhibit proliferation of lymphocytes and immune and allergic reactions in vivo and in vitro. In addition, TISE can significantly suppression of delayed hypersensitivity in mice and rat passive cutaneous allergic reaction, and can prolong the survival time of transplanted rat skin, ovalbumin induced asthma in guinea pigs. There was also a significant inhibitory effect. For the further research on the immunosuppressive activity and mechanism of calf thymus derived TISE separation chromatography by micrococcal nuclease hydrolysis and reversed-phase high performance liquid, obtained a new sequence of five peptides of Ala-Glu-Trp-Cys-Pro (molecular weight 604.68), and named the Thymic Immunosuppressive peptide (Thymic Immunosuppressive pentapeptide, TIPP). TIPP can prove that the low effects by blocking the allergic and inflammatory reaction of MAPK /NF- kinase B pathway to suppress allergic mice, they also confirmed that TIPP can inhibit MEK/ERK/NF- activation of B signaling pathway and inhibit IgE stimulated RBL-2H3 cells, the study found that TIPP has significant inhibitory effect on the proliferation of human myeloid leukemia cells K562. In view of the extensive pharmacological effect of TIPP, we decided to study the anticancer activity of TIPP. This is the anti For the first time on cancer activity. Inhibition of K562 cell line and its mechanism based on, we hypothesized that TIPP may also have anti-cancer activity and can be used for the treatment of cancer. In order to confirm this hypothesis, we first study the anticancer activity of TIPP at the cellular level, and evaluate its role in the treatment of tumor animal model. Finally, detection of its effects on normal organs. The results of this study may provide a new method for cancer treatment, also laid the foundation for the further research and development of TIPP. The number of research methods 1 TIPP antitumor activity in vitro by MCF-7 and K562 cancer cell lines to TIPP cytotoxicity and proliferation of tumor cells the inhibitory activity was evaluated by.MCF-7 and K562 to the training needs, and then inoculated in 96 well plates. After five cells in different concentrations of TIPP after treatment for 48 h, adding 5 mg/ml MTT (Sigma) The solution of 20 l to 4 h. incubation after centrifugation, a Bao blue precipitate was dissolved in DMSO 150 l. enzyme Standard Test of the DMSO solution at 570 nm absorbance on antitumor activity of.2 TIPP in the five week old female Bablc/c nude mice (SPF) subcutaneous inoculation of chronic myeloid leukemia cells K562 cells and human breast cancer cell line MCF-7. The average tumor volume reached 100 mm3, the inoculation of K562 mice were randomly divided into 3 groups, inoculated MCF-7 mice were randomly divided into 6 groups, 7 rats in each group. The K562 tumor bearing mice model first group by intravenous injection of PBS, the second group were injected with TIPP 25 mg/kg (dissolved in 0.1 ml, third PBS) group was injected with actinomycin D for 0.1 mg/kg. breast cancer model, a total of 6 groups were injected with PBS, TIPP 25 mg/kg, TIPP 25 mg/kg combined with actinomycin D 0.1 mg/kg, TIPP 25 mg/kg 7.5 mg/kg combined with paclitaxel, paclitaxel and actinomycin mg/kg 7.5 administration of D. 3 weeks, every 3 days, tumor volume was measured. The results showed that 1 TIPP has anti-tumor activity in vitro proliferation assay, TIPP and tumor cells after 48 h incubation significantly in a dose dependent manner inhibited K562 cell proliferation and MCF-7.TIPP can also inhibit two kinds of tumor cells survival rate of.TIPP for two different cancer cell lines in vitro inhibitory activity showed that TIPP.2 TIPP has antitumor activity in vivo antitumor activity in vivo test results show that TIPP can significantly inhibit K562 and MCF-7 tumor growth in vivo, the test results are coincident with the experimental results of in vitro cell proliferation inhibition of.TIPP on K562 tumor rate was 26%, and the inhibition of MCF-7 the tumor rate of 54%. in breast cancer model in TIPP respectively, and other two kinds of effective anticancer drug actinomycin D and paclitaxel combined with administration of TIPP combined with actinomycin D group the inhibition rate was 58%, TIPP Paclitaxel group the inhibition rate of 74%.3 TIPP induced tumor apoptosis in liver of mice breast cancer and tumor tissues were stained with HE to observe the TIPP safety and the tumor apoptosis ability. The results showed that both TIPP and paclitaxel administered alone or combined with medication, have no obvious damage to liver tissue, put actinomycin -D either alone or combined with TIPP drugs had liver toxicity was mild; no matter TIPP and paclitaxel alone or in combination to drugs can induce breast cancer cell apoptosis significantly, actinomycin -D either alone or in combination with TIPP administration caused mild tumor necrosis. These indicate that TIPP alone or with paclitaxel combined drug therapy is a safe and can induce cell apoptosis and inhibit the growth of tumor treatment. Conclusion and significance (1) of TIPP were studied in vitro antitumor activity of.TIPP significantly inhibited K562 and MCF-7 cells The proliferation, and a dose and time dependence. (2) for the first time on TIPP in chronic myeloid leukemia and breast cancer mouse model in vivo antitumor effect of.TIPP inhibit chronic myeloid leukemia and breast cancer tumor growth in mice. (3) on the liver and mammary gland tumor tissues were stained by HE and immunohistochemistry.HE staining showed that the apoptosis induced by TIPP inhibition of breast cancer. (4) in normal tissues such as liver HE staining results showed that TIPP is a safe drug, no damage to the liver. (5) revealed for the first time TIPP (with other anti-cancer drugs) for treatment of breast cancer combined therapy the in vivo results showed that TIPP combined with paclitaxel administered group inhibition rate (74%) than TIPP monotherapy group (54%).

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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