【摘要】：黑色素瘤起源于黑色素細胞,是發(fā)病率最低然而致死率卻最高的皮膚癌。由于傳統(tǒng)的治療方法如手術、化療和放射治療等不能解決黑色素瘤轉(zhuǎn)移和復發(fā)問題,因此靶向治療和免疫治療成為主要研究方向。目前,臨床應用的治療黑色素瘤藥物多為針對原癌基因如BRAF、MEK的抑制劑。但是長期服用抑制劑導致腫瘤的耐藥性和機體副作用使得黑色素瘤治療仍然存在局限性。因此,人們越來越關注從自然界尋找一些天然化合物應用于腫瘤治療中。脂肽是微生物的次生代謝產(chǎn)物,具有廣泛的生物活性,如消炎、殺菌、抗腫瘤等。表皮葡萄球菌是一類皮膚共生菌,我們實驗室從其發(fā)酵液中提取出一種脂肽,能夠誘導角質(zhì)形成細胞分泌抗菌肽,具有殺菌活性,而且其同分異構體具有抑制皮膚傷口過度炎癥,調(diào)節(jié)皮膚先天免疫應答的活性。本研究通過細胞周期流式分析和體外克隆形成實驗證明了表皮葡萄球菌來源的脂肽衍生物LP79能夠誘導黑色素瘤細胞B16F10停滯在細胞周期G1期,進而抑制其增殖。Transwell和傷口實驗表明LP79能夠顯著性抑制B16F10細胞的遷移。而且,我們通過構建實驗性腫瘤轉(zhuǎn)移模型,發(fā)現(xiàn)LP79抑制小鼠黑色素瘤的肺部遷移。進一步研究表明,脂肽LP79能通過調(diào)節(jié)雙重特異性磷酸酶MKP1進而抑制腫瘤細胞在上皮樣細胞轉(zhuǎn)化過程中的兩個重要蛋白——N-鈣黏蛋白和波形蛋白——的表達,從而抑制小鼠黑色素瘤的轉(zhuǎn)移。另外,脂肽LP79能夠通過誘導酪氨酸酶相關蛋白TRP1進而促進小鼠黑色素瘤細胞的分化。然而當TRP1基因沉默之后,LP79依然能夠抑制B16F10細胞的遷移,表明二者并無直接相關性。此外,TLR2作為脂肽的受體,并不參與脂肽LP79對黑色素瘤細胞B16F10的抑制遷移功能。綜上所述,本研究證明了表皮葡萄球菌來源的脂肽衍生物具有抑制黑色素瘤遷移的生物學活性。本研究進一步揭示了皮膚共生菌對宿主的益處,為治療黑色素瘤的新藥研發(fā)提出了獨特的視角,也為治療腫瘤提供了新的思路。
[Abstract]:Melanoma, originated from melanocytes, is the lowest incidence but the highest fatality rate of skin cancer. Because traditional treatment methods such as surgery chemotherapy and radiotherapy can not solve the problem of metastasis and recurrence of melanoma targeted therapy and immunotherapy have become the main research direction. At present, most of the drugs used in the treatment of melanoma are inhibitors of proto-oncogene such as BRAF,MEK. However, long-term use of inhibitors leads to tumor resistance and side effects, so melanoma treatment is still limited. As a result, more and more attention has been paid to finding natural compounds from nature for use in cancer therapy. Lipopeptide is the secondary metabolite of microorganism. It has a wide range of biological activities, such as anti-inflammatory, bactericidal, anti-tumor and so on. Staphylococcus epidermidis is a kind of symbiotic bacteria in skin. We extracted a lipopeptide from its fermentation broth, which can induce keratinocytes to secrete antibacterial peptides and have bactericidal activity. Moreover, the isomers can inhibit the excessive inflammation of skin wound and regulate the innate immune response of skin. In this study, we demonstrated that LP79, a lipopeptide derivative from Staphylococcus epidermidis, could induce B16F10 arrest in the G1 phase of melanoma cells by cell cycle flow analysis and clone formation in vitro. Transwell and wound experiments showed that LP79 could significantly inhibit the migration of B16F10 cells. Furthermore, we found that LP79 inhibits pulmonary migration of melanoma in mice by constructing experimental tumor metastasis model. Further studies showed that lipopeptide LP79 could inhibit the expression of two important proteins, N-cadherin and vimentin, during the transformation of epithelial-like cells by regulating the double specific phosphatase MKP1. Thus, the metastasis of melanoma in mice was inhibited. In addition, lipopeptide LP79 can promote the differentiation of mouse melanoma cells by inducing tyrosinase-associated protein TRP1. However, when TRP1 gene was silenced, LP79 could still inhibit the migration of B16F10 cells, indicating that there was no direct correlation between them. In addition, TLR2, as the receptor of lipopeptide, does not participate in the inhibitory effect of lipopeptide LP79 on B16F10 migration in melanoma cells. In conclusion, this study demonstrated that the lipopeptide derivatives derived from Staphylococcus epidermidis have the biological activity of inhibiting melanoma migration. This study further reveals the host benefits of skin symbiotic bacteria, provides a unique perspective for the research and development of new drugs for the treatment of melanoma, and also provides a new way of thinking for the treatment of tumors.
【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.5

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本文編號：2447866