【摘要】：背景：近年來黑色素瘤的發(fā)病率呈逐年上升，已成為所有惡性腫瘤中發(fā)病率增長最快的腫瘤。惡性黑色素瘤早期經(jīng)外科擴(kuò)大切除術(shù)后，95%可得到治愈。但是早期惡性黑色素瘤的癥狀與體征不明顯，，常被忽視，患者就診時(shí)往往已進(jìn)入腫瘤的快速生長期，或已發(fā)生血液和淋巴轉(zhuǎn)移，此時(shí)手術(shù)已不能達(dá)到無瘤狀態(tài)，則只能進(jìn)行全身治療。目前，MM相對(duì)敏感的化療藥物有達(dá)卡巴嗪、替莫唑胺、鉑類等，然而其單藥客觀有效率均低于20%。達(dá)卡巴嗪是臨床中治療惡性黑色素瘤的主導(dǎo)藥物，是美國FDA批準(zhǔn)的治療轉(zhuǎn)移性惡性黑色素瘤的標(biāo)準(zhǔn)藥物，也被視為臨床試驗(yàn)中評(píng)估新藥療效的基準(zhǔn)。因此，尋找一種有效的抗惡性黑色素瘤藥物及治療靶點(diǎn)，或有效的藥物聯(lián)合方案勢(shì)在必行。 FLNa是肌動(dòng)蛋白結(jié)合蛋白，在多種細(xì)胞中廣泛表達(dá)，其可參與細(xì)胞膜受體轉(zhuǎn)位、信號(hào)轉(zhuǎn)導(dǎo)及基因轉(zhuǎn)錄調(diào)節(jié)等多種功能，與細(xì)胞的黏附、增殖、遷移、侵襲、腫瘤發(fā)生等過程密切相關(guān)。相關(guān)研究表明：FLNa與DNA修復(fù)蛋白BRCA2有關(guān)，當(dāng)FLNa缺乏時(shí)，修復(fù)蛋白BRCA2表達(dá)減弱，DNA損傷的修復(fù)能力下降，導(dǎo)致細(xì)胞的DNA損傷嚴(yán)重，因此沉默F(xiàn)LNa的表達(dá)可增強(qiáng)細(xì)胞對(duì)DNA損傷的敏感性。因此本研究選用DTIC、VP-16等與DNA損傷有關(guān)的化療藥物作用于M2/A7細(xì)胞，研究藥物對(duì)細(xì)胞的作用與FLNa的表達(dá)水平的關(guān)系，為黑色素瘤的個(gè)體化治療尋找新的藥物及新的治療靶點(diǎn)。 目的：檢測(cè)FLNa的表達(dá)對(duì)于黑色素瘤M2/A7細(xì)胞的影響 方法：本研究首先對(duì)于黑色素瘤M2/A7細(xì)胞系的FLNa的表達(dá)情況進(jìn)行驗(yàn)證，之后應(yīng)用MTT法檢測(cè)DTIC、VP-16對(duì)于黑色素瘤M2/A7細(xì)胞系（M2-FLNa陰性表達(dá)，A7-FLNa陽性表達(dá)）的抑制率。同時(shí)用流式細(xì)胞術(shù)檢測(cè)DTIC、VP-16是否能誘導(dǎo)黑色素瘤M2/A7細(xì)胞系的凋亡。最后采用單細(xì)胞瓊脂糖凝膠電泳法檢測(cè)DTIC、VP-16對(duì)于黑色素瘤M2/A7細(xì)胞系的DNA損傷。 結(jié)果：（1）DTIC及VP-16對(duì)FLNa陰性表達(dá)的M2細(xì)胞的抑制明顯強(qiáng)于陽性表達(dá)的A7細(xì)胞。 （2）DTIC及VP-16可誘導(dǎo)M2及A7細(xì)胞的凋亡，但對(duì)FLNa陰性表達(dá)的M2細(xì)胞的凋亡作用較陽性表達(dá)的A7細(xì)胞明顯。 （3）DTIC及VP-16可引起M2及A7細(xì)胞DNA損傷，但對(duì)FLNa陰性表達(dá)的M2細(xì)胞DNA損傷作用更強(qiáng)。 結(jié)論：（1）DTIC及VP-16可誘導(dǎo)M2及A7細(xì)胞的凋亡，且相同濃度的藥物對(duì)FLNa陰性表達(dá)的M2細(xì)胞的凋亡作用更明顯。 （2）DTIC及VP-16可引起M2及A7細(xì)胞DNA損傷，且相同濃度的藥物對(duì)FLNa陰性表達(dá)的M2細(xì)胞DNA損傷作用更強(qiáng)。 （3）FLNa陰性表達(dá)的M2細(xì)胞對(duì)于DTIC及VP-16更加敏感。
[Abstract]:Background: in recent years, the incidence of melanoma has increased year by year, and has become the fastest growing of all malignant tumors. 95% of malignant melanoma can be cured after early surgical excision. However, the symptoms and signs of early malignant melanoma are not obvious, are often ignored, the patient often has entered the rapid growth stage of the tumor, or has occurred blood and lymphatic metastasis, at this time, the operation can no longer reach a tumor-free state. Then can only carry on the whole body treatment. At present, the relatively sensitive chemotherapeutic drugs of MM are dacarbazide, temozolamide, platinum, etc. However, the objective effective rate of single drug is lower than 20%. Dacamaxine is the leading drug in the treatment of malignant melanoma. It is the standard drug approved by FDA for the treatment of metastatic malignant melanoma. It is also regarded as the benchmark for evaluating the efficacy of new drugs in clinical trials. Therefore, it is imperative to find an effective anti-malignant melanoma drug and therapeutic target, or an effective combination of drugs. FLNa is an actin binding protein widely expressed in many kinds of cells. It can participate in cell membrane receptor translocation, signal transduction, gene transcription regulation and other functions, adhesion, proliferation, migration, invasion and so on. Tumorigenesis and other processes are closely related. Related studies have shown that FLNa is related to DNA repair protein BRCA2. When FLNa is deficient, the expression of repair protein BRCA2 weakens, and the repair ability of DNA damage decreases, which leads to the serious DNA damage of cells. Therefore, silencing the expression of FLNa can enhance the sensitivity of cells to DNA damage. In this study, DTIC,VP-16 and other chemotherapeutic drugs related to DNA damage were selected to act on M2/A7 cells, and to study the relationship between the effect of drugs on cells and the expression level of FLNa. To find new drugs and new therapeutic targets for individualized treatment of melanoma. Objective: to detect the effect of FLNa expression on melanoma M2/A7 cells. In this study, the expression of FLNa in melanoma M2/A7 cell line was verified firstly, and then DTIC, was detected by MTT assay. Inhibitory rate of VP-16 on melanoma M2/A7 cell line (M2-FLNa negative expression, A7-FLNa positive expression). At the same time, flow cytometry was used to detect whether DTIC,VP-16 could induce apoptosis of melanoma M2/A7 cell line. Finally, single cell agarose gel electrophoresis was used to detect the DNA damage of melanoma M2/A7 cell line induced by DTIC,VP-16. Results: (1) the inhibition of FLNa negative M2 cells by DTIC and VP-16 was significantly stronger than that of positive A7 cells. (2) DTIC and VP-16 could induce the apoptosis of M2 and A7 cells, but the apoptosis of M2 cells with negative FLNa expression was more obvious than that of A7 cells with positive expression. (3) DTIC and VP-16 could induce DNA damage in M2 and A7 cells, but DNA damage was stronger in M2 cells with negative FLNa expression. Conclusion: (1) DTIC and VP-16 can induce the apoptosis of M2 and A7 cells, and the same concentration of drugs can induce the apoptosis of M2 cells with negative expression of FLNa. (2) DTIC and VP-16 could induce DNA damage in M2 and A7 cells, and the same concentration of drugs had stronger effect on DNA damage of M2 cells with negative FLNa expression. (3) M 2 cells with negative FLNa expression were more sensitive to DTIC and VP-16.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.5

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