本文關(guān)鍵詞：胸腺免疫抑制五肽（TIPP）的抗癌活性研究　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胸腺免疫抑制五肽(TIPP) 癌癥 慢性粒細(xì)胞白血病 乳腺癌 抗腫瘤作用

【摘要】：研究目的癌癥,是一類細(xì)胞異常生長(zhǎng)且不受控制,并且可以滲透或擴(kuò)散到身體其他部位的疾病。癌癥被認(rèn)為是世界上主要的死亡原因。在2008年全球約有1270萬(wàn)例癌癥病例和760萬(wàn)例癌癥死亡,而在2012,全球約有1410萬(wàn)例新癌癥病例和820萬(wàn)例癌癥死亡。因此,研究有效且毒性低的抗腫瘤藥物一直是藥物研究的熱點(diǎn)。我們實(shí)驗(yàn)室從20世紀(jì)80年代開始研究胸腺免疫抑制提取物(TISE)。研究發(fā)現(xiàn),TISE在體內(nèi)體外均能顯著的抑制淋巴細(xì)胞增殖以及免疫和過(guò)敏反應(yīng)。此外,TISE可以顯著抑制小鼠遲發(fā)型超敏反應(yīng)和大鼠被動(dòng)皮膚過(guò)敏反應(yīng),并且能明顯延長(zhǎng)大鼠移植皮膚存活時(shí)間,對(duì)卵蛋白誘導(dǎo)的豚鼠哮喘的發(fā)生也有顯著的抑制作用。為深入研究免疫抑制活性和機(jī)制,對(duì)小牛胸腺來(lái)源的TISE通過(guò)微球菌核酸酶酶解和反相高效液相色譜法分離,得到了一種新的序列為Ala-Glu-Trp-Cys-Pro的五肽(分子量為604.68),并命名為胸腺免疫抑制肽(thymic immunosuppressive pentapeptide,TIPP)。廉倩倩等證明了TIPP能通過(guò)阻斷MAPK激酶/NF-κB信號(hào)通路從而有效抑制過(guò)敏性小鼠的過(guò)敏和炎癥反應(yīng),他們也證實(shí)了 TIPP可以靶向抑制MEK/ERK/NF-κB信號(hào)通路進(jìn)而抑制IgE刺激的RBL-2H3細(xì)胞的活化,同時(shí)研究發(fā)現(xiàn),TIPP對(duì)人類髓性白血病細(xì)胞K562增殖有顯著的抑制作用。鑒于TIPP的廣泛的藥理學(xué)作用,我們決定研究其抗癌活性。這是對(duì)TIPP的抗癌活性的首次研究�；趯�(duì)K562細(xì)胞系的抑制作用和其作用機(jī)制,我們假設(shè)TIPP也可能同樣具有抗癌活性并且可以用于癌癥治療。為了證實(shí)這一假定,我們首先研究了 TIPP在細(xì)胞水平的抗癌活性,然后評(píng)價(jià)了其在腫瘤動(dòng)物模型體內(nèi)的治療作用,最后檢測(cè)了它對(duì)正常器官的影響。本課題的研究結(jié)果可能會(huì)提供一種癌癥治療的新方法,也為TIPP的進(jìn)一步研究與開發(fā)奠定基礎(chǔ)。研究方法1 TIPP體外抗腫瘤活性研究用MCF-7及K562癌細(xì)胞系對(duì)TIPP的細(xì)胞毒性及癌細(xì)胞增殖抑制活性進(jìn)行了評(píng)價(jià)。MCF-7及K562培養(yǎng)至所需要的數(shù)量,然后接種于96孔板。細(xì)胞在經(jīng)過(guò)五種不同濃度的TIPP給藥48 h后,加入5 mg/ml MTT(Sigma)溶液20 μl繼續(xù)孵育4 h。離心后,甲佨藍(lán)沉淀溶解于DMSO 150 μl。用酶標(biāo)儀檢測(cè)該DMSO溶液在570 nm處的吸光值。2 TIPP體內(nèi)抗腫瘤活性研究五周齡雌性Bablc/c裸鼠(無(wú)特定病原體的)皮下接種慢性粒細(xì)胞白血病細(xì)胞系K562細(xì)胞和乳腺癌MCF-7細(xì)胞。裸鼠的平均腫瘤體積達(dá)到100 mm3時(shí),將接種K562裸鼠隨機(jī)分為3組,將接種MCF-7裸鼠隨機(jī)分為6組,每組7只。對(duì)于K562荷瘤小鼠模型,第1組經(jīng)尾靜脈注射PBS,第2組注射TIPP 25 mg/kg(溶解于0.1 ml PBS),第3組注射放線菌素D 0.1 mg/kg。對(duì)于乳腺癌模型,總共6組分別注射PBS、TIPP 25 mg/kg、TIPP 25 mg/kg聯(lián)合放線菌素D 0.1 mg/kg、TIPP 25 mg/kg聯(lián)合紫杉醇7.5 mg/kg、紫杉醇7.5 mg/kg和放線菌素D。給藥3周,每3天測(cè)量腫瘤體積。研究結(jié)果1 TIPP具有體外抗腫瘤活性增殖實(shí)驗(yàn)表明,TIPP與腫瘤細(xì)胞一起孵育48 h后可顯著地以劑量依賴的方式抑制K562和MCF-7細(xì)胞的增殖。TIPP還可以顯著抑制兩種腫瘤細(xì)胞系的存活率。TIPP對(duì)于兩種不同的腫瘤細(xì)胞株抑制活性表明了 TIPP的體外有抗腫瘤活性。2 TIPP體內(nèi)抗腫瘤活性體內(nèi)試驗(yàn)結(jié)果表明,TIPP可明顯抑制K562和MCF-7腫瘤的生長(zhǎng),體內(nèi)試驗(yàn)結(jié)果與體外細(xì)胞增殖實(shí)驗(yàn)結(jié)果完全吻合。TIPP對(duì)K562瘤的抑制率為26%,而對(duì)MCF-7瘤的抑制率達(dá)到54%。在乳腺癌模型中,TIPP也分別與另外兩種有效的抗癌藥物放線菌素D和紫杉醇聯(lián)合給藥,TIPP聯(lián)合放線菌素D組的抑瘤率為58%,TIPP聯(lián)合紫杉醇組的抑瘤率為74%。3 TIPP誘導(dǎo)腫瘤凋亡對(duì)乳腺癌小鼠的肝臟和腫瘤組織進(jìn)行HE染色觀察TIPP的安全性及對(duì)腫瘤的促凋亡能力。結(jié)果表明,TIPP和紫杉醇無(wú)論單獨(dú)給藥還是聯(lián)合給藥,都對(duì)肝組織無(wú)明顯損害,放線菌素-D無(wú)論單獨(dú)還是聯(lián)合TIPP給藥都發(fā)現(xiàn)有輕微的肝毒性;不管TIPP和紫杉醇單獨(dú)還是聯(lián)合給藥都可以顯著的誘導(dǎo)乳腺腫瘤組織細(xì)胞凋亡,放線菌素-D無(wú)論單獨(dú)還是聯(lián)合TIPP給藥都引起輕度的腫瘤組織壞死。上述表明TIPP單獨(dú)或與紫杉醇聯(lián)合給藥是一種安全且能誘導(dǎo)細(xì)胞凋亡而抑制腫瘤生長(zhǎng)的治療方案。結(jié)論及意義(1)對(duì)TIPP體外抗癌活性進(jìn)行了研究。TIPP顯著抑制K562和MCF-7細(xì)胞的增殖,并且有劑量和時(shí)間依賴性。(2)首次對(duì)TIPP在慢性粒細(xì)胞白血病和乳腺癌小鼠模型的體內(nèi)抗腫瘤作用進(jìn)行了研究。TIPP有效抑制慢性粒細(xì)胞白血病和乳腺癌小鼠腫瘤的生長(zhǎng)。(3)對(duì)肝臟和乳腺腫瘤組織進(jìn)行了 HE染色和免疫組化檢測(cè)。HE染色結(jié)果顯示TIPP通過(guò)誘導(dǎo)細(xì)胞凋亡來(lái)抑制乳腺癌。(4)對(duì)于正常組織如肝臟的HE染色結(jié)果表明,TIPP是安全的藥物,對(duì)肝臟無(wú)損傷。(5)首次揭示了 TIPP(與其他抗癌藥物)聯(lián)合治法對(duì)于乳腺癌的治療作用。體內(nèi)結(jié)果表明,TIPP聯(lián)合紫杉醇給藥組的抑瘤率(74%)比TIPP單獨(dú)給藥組的(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%).

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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