【摘要】：皮膚惡性黑素瘤(cutaneous malignant melanoma, CMM)是惡性程度最高的皮膚腫瘤,近年來(lái)CMM發(fā)病率在世界范圍內(nèi)以3%-7%比例逐年上升,具有進(jìn)展速度快、易于轉(zhuǎn)移、放化療不敏感、治療后易復(fù)發(fā)和預(yù)后差等特點(diǎn)。侵襲和轉(zhuǎn)移是影響該疾病患者生存的首要因素,是治療的最大障礙,也是造成該疾病臨床預(yù)后極差的關(guān)鍵因素,目前CMM的侵襲和轉(zhuǎn)移機(jī)制尚未完全闡明。細(xì)胞骨架重組是細(xì)胞遷移和侵襲所必需的,Rho GTPases家族是細(xì)胞骨架的關(guān)鍵調(diào)控因子,在腫瘤發(fā)生發(fā)展多個(gè)步驟和過(guò)程均可發(fā)揮重要作用。因此,我們以M14、A375和MV3三株人黑素瘤細(xì)胞和人黑色素細(xì)胞(Melanocyte, MC)為研究對(duì)象,探討Rho GTPases家族不同成員的表達(dá)及與細(xì)胞骨架、細(xì)胞遷移、侵襲的關(guān)系,然后選取該家族中在腫瘤細(xì)胞系及MC中表達(dá)差異最明顯的RhoD為進(jìn)一步研究的目標(biāo),構(gòu)建慢病毒介導(dǎo)的過(guò)表達(dá)RhoD的A375細(xì)胞株,探討RhoD過(guò)表達(dá)對(duì)A375細(xì)胞株細(xì)胞骨架、遷移、侵襲等生物學(xué)功能的影響及可能的機(jī)制。第一部分Rho GTPases家族在黑素瘤細(xì)胞骨架和遷移運(yùn)動(dòng)中的作用及分子機(jī)制目的：黑素瘤遷移和侵襲轉(zhuǎn)移是影響患者生存的關(guān)鍵因素之一,細(xì)胞骨架重組是細(xì)胞遷移和侵襲所必需的,Rho GTPases家族是細(xì)胞骨架關(guān)鍵調(diào)控因子。本研究擬探討Rho GTPases家族不同成員在黑素瘤細(xì)胞中的表達(dá),觀察細(xì)胞骨架的異同,揭示Rho GTPases家族通過(guò)調(diào)節(jié)細(xì)胞骨架在黑素瘤遷移和侵襲中發(fā)揮的作用和分子機(jī)制。方法：1、霍夫曼顯微鏡下觀察M14、A375和MV3三株人黑素瘤細(xì)胞與人黑色素細(xì)胞(Melanocyte, MC)形態(tài)學(xué)上的差異；羅丹明標(biāo)記的鬼筆環(huán)肽染色,在超高分辨率顯微鏡下觀察以肌動(dòng)蛋白絲為主要組成成分的絲狀偽足、板狀偽足、應(yīng)力纖維和粘著斑在四種細(xì)胞中的差異。2、Transwell遷移實(shí)驗(yàn)檢測(cè)M14、A375和MV3和MC四種細(xì)胞的遷移能力。3、實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(Quantitative Real-time Polymerase Chain Reaction, QPCR)檢測(cè)Rho GTPases家族各成員的mRNA轉(zhuǎn)錄水平；免疫印跡法(western blotting, WB)檢測(cè)RhoD、Diaph2(diaphanous related formin2)、絲切蛋白Cofilin和磷酸化絲切蛋白(P-Cofilin)蛋白的表達(dá)。結(jié)果：1、M14、A375和MV3和MC四種細(xì)胞絲狀偽足、板狀偽足、應(yīng)力纖維和粘著斑具有明顯差異。MC無(wú)應(yīng)力纖維和粘著斑,M14、A375、MV3三種黑素瘤細(xì)胞雖然形態(tài)學(xué)不同,但均含有應(yīng)力纖維和粘著斑；MV3應(yīng)力纖維的數(shù)量少于M14和A375,但較后兩者粗；四種細(xì)胞都具有極細(xì)且短的絲狀偽足。2、Transwell遷移實(shí)驗(yàn)結(jié)果顯示M14和A375細(xì)胞跨膜遷移能力類似,遷移率高；MV3細(xì)胞遷移率低于M14和A375,MC無(wú)跨膜遷移能力。3、Rho GTPases家族的不同成員在四種細(xì)胞中的QPCR結(jié)果各不相同,并且呈現(xiàn)與細(xì)胞骨架免疫熒光相對(duì)應(yīng)的變化；WB結(jié)果顯示Rho GTPases家族成員之一的RhoD在M14.A375.MV3中基本不表達(dá),與QPCR水平變化一致,其下游效應(yīng)因子Diaph2亦出現(xiàn)了相對(duì)應(yīng)的變化；肌動(dòng)蛋白絲重要調(diào)控因子cofilin蛋白雖沒(méi)有顯著變化,但是其磷酸化水平P-Cofilin在黑素瘤細(xì)胞中明顯高于黑色素細(xì)胞。結(jié)論：Rho GTPases通過(guò)調(diào)節(jié)以肌動(dòng)蛋白絲為主要組成成分的絲狀偽足、板狀偽足、應(yīng)力纖維、粘著斑進(jìn)而影響黑素瘤細(xì)胞遷移和侵襲；黑色素和黑素瘤細(xì)胞骨架和細(xì)胞遷移運(yùn)動(dòng)是不同Rho GTPases家族成員精準(zhǔn)調(diào)節(jié)的結(jié)果。第二部分慢病毒介導(dǎo)的過(guò)表達(dá)RhoD人黑素瘤A375細(xì)胞株的構(gòu)建及鑒定目的：構(gòu)建人RhoD基因的慢病毒載體并進(jìn)行慢病毒包裝和鑒定,體外轉(zhuǎn)染人黑素瘤細(xì)胞A375,使其過(guò)表達(dá)RhoD蛋白,為后續(xù)研究RhoD在黑素瘤中的作用奠定基礎(chǔ)。方法：1、利用Gateway技術(shù)構(gòu)建攜帶增強(qiáng)型綠色熒光蛋白EGFP的RhoD慢病毒載體,經(jīng)PCR及基因測(cè)序鑒定后,與輔助質(zhì)粒pLV/helper-SL3、pLV/helper-SL4及pLV/helper-SL5混合采用脂質(zhì)體法制備DNA-Lipofectamine2000復(fù)合物,并共同轉(zhuǎn)染293FT細(xì)胞進(jìn)行慢病毒包裝,產(chǎn)生相應(yīng)慢病毒顆粒,通過(guò)定量PCR方法測(cè)定病毒滴度。2、利用包裝好的慢病毒轉(zhuǎn)染人黑素瘤A375細(xì)胞,建立RhoD過(guò)表達(dá)的人黑素瘤A375細(xì)胞株,熒光顯微鏡下觀察熒光表達(dá)情況,流式細(xì)胞儀檢測(cè)轉(zhuǎn)染效率；實(shí)驗(yàn)分為A375(未處理對(duì)照組)、A375-EGFP(不含目的基因的空病毒對(duì)照組)和A375-RhoD(含RhoD基因的病毒組)三組,采用實(shí)時(shí)熒光定量PCR (QPCR)及免疫印記法(Western blot, WB)驗(yàn)證RhoD在A375-RhoD組細(xì)胞中的過(guò)表達(dá)。結(jié)果： 1、 通過(guò)PCR、 基因測(cè)序證實(shí), 慢病毒表達(dá)載體pLV[Exp]-EGFP/Neo-CMVhRhoD構(gòu)建成功。與輔助質(zhì)粒共轉(zhuǎn)染293FT細(xì)胞包裝出具高效感染力的慢病毒,經(jīng)測(cè)定病毒滴度為(5.13±2)×108TU/ml。2、轉(zhuǎn)染A375細(xì)胞后,在細(xì)胞內(nèi)及細(xì)胞膜表面都有明顯的綠色熒光表達(dá),流式檢測(cè)轉(zhuǎn)染效率大于80%；A375-RhoD組RhoD mRNA及蛋白水平均較A375-EGFP組和A375組細(xì)胞中明顯增高(P0.05)。結(jié)論：成功構(gòu)建RhoD慢病毒表達(dá)載體,包裝出具高效感染力的慢病毒顆粒并成功轉(zhuǎn)染人黑素瘤A375細(xì)胞,為進(jìn)一步研究RhoD在黑素瘤中的作用提供實(shí)驗(yàn)基礎(chǔ)。第三部分過(guò)表達(dá)RhoD對(duì)黑素瘤細(xì)胞A375細(xì)胞骨架和遷移侵襲的影響及作用機(jī)制的探討目的：探討RhoD對(duì)人黑素瘤細(xì)胞株A375細(xì)胞骨架、遷移和侵襲能力等生物學(xué)行為的影響及其作用機(jī)制。方法：采用羅丹明-鬼筆環(huán)肽染色觀察細(xì)胞骨架、Transwell小室實(shí)驗(yàn)檢測(cè)細(xì)胞遷移及侵襲能力,流式細(xì)胞技術(shù)檢測(cè)細(xì)胞周期,觀察空白對(duì)照組A375、陰性對(duì)照慢病毒載體轉(zhuǎn)染組A375-EGFP及過(guò)表達(dá)RhoD慢病毒載體轉(zhuǎn)染組A375-RhoD三組細(xì)胞的體外生物學(xué)行為改變；采用Western blot免疫印跡法比較三組細(xì)胞中Diaph2、 cofilin和P-cofilin的表達(dá)情況,分析RhoD影響黑素瘤細(xì)胞骨架和運(yùn)動(dòng)、侵襲功能的可能機(jī)制。結(jié)果：1、細(xì)胞骨架染色顯示,與A375和A375-EGFP組細(xì)胞對(duì)比,A375-RhoD組細(xì)胞體積增大,應(yīng)力纖維變細(xì),軟弱無(wú)力,粘著斑增多；絲狀偽足形成增加；皺褶緣和板狀偽足無(wú)明顯變化。2、Transwell小室遷移實(shí)驗(yàn)顯示：A375、A375-EGFP和A375-RhoD組平均每視野穿膜細(xì)胞數(shù)分別為(149.67±11.93)、(152.67±11.23)和(72.67.25±5.03),RhoD過(guò)表達(dá)可以顯著抑制黑素瘤細(xì)胞A375的跨膜運(yùn)動(dòng)和遷移能力(P0.05); Transwell小室人工基底膜侵襲試驗(yàn)顯示：A375、A375-EGFP和A375-RhoD組平均每視野穿膜細(xì)胞數(shù)分別為(83±7)、(78.33±12.34)和(9±1), RhoD過(guò)表達(dá)可以顯著抑制黑素瘤細(xì)胞A375的侵襲能力(P0.05)。3、流式細(xì)胞技術(shù)分析細(xì)胞周期結(jié)果表明：A375、A375-EGFP和A375-RhoD三組細(xì)胞間G1期、S期和G2期細(xì)胞所占百分比無(wú)統(tǒng)計(jì)學(xué)差異(P=0.685、0.138和P=0.410),過(guò)表達(dá)RhoD和陰性對(duì)照慢病毒載體對(duì)細(xì)胞周期均無(wú)影響。5、WB免疫印跡結(jié)果顯示,A375-RhoD細(xì)胞可在RhoD的刺激下激活下游信號(hào)效應(yīng)分子Diaph2,促進(jìn)其表達(dá),而對(duì)照組未能激活。RhoD過(guò)表達(dá)和陰性對(duì)照慢病毒載體對(duì)cofilin和P-cofilin均無(wú)影響。結(jié)論：RhoD在黑素瘤A375細(xì)胞的細(xì)胞骨架以及運(yùn)動(dòng)和遷移侵襲中發(fā)揮一定作用,有可能成為黑素瘤治療中的靶向目標(biāo)。
[Abstract]:Cutaneous malignant melanoma (CMM) is the most malignant skin tumor. In recent years, the incidence of CMM in the world is increasing year by year in the proportion of 3%-7%. It has the characteristics of rapid progress, easy transfer, insensitivity to radiotherapy and chemotherapy, easy to relapse after treatment and poor prognosis after treatment. Invasion and metastasis are the factors affecting the survival of the disease patients. The most important factor, the biggest obstacle to treatment, is the key factor in the poor clinical prognosis of the disease. At present, the mechanism of invasion and metastasis of CMM has not been fully elucidated. The cytoskeleton recombination is essential for cell migration and invasion. The Rho GTPases family is the key regulating factor of the cytoskeleton, and many steps and processes in the development of the tumor. Therefore, we take M14, A375 and MV3 three human melanoma cells and human melanocytes (Melanocyte, MC) as research objects to investigate the expression of different members of the Rho GTPases family and the relationship with cytoskeleton, cell migration and invasion, and then select the most distinct R expression in the family of the tumor cell lines and MC. HoD is the goal of further research to construct a A375 cell line of RhoD mediated by lentivirus, and to explore the effects of RhoD overexpression on the biological functions of cytoskeleton, migration and invasion of A375 cell lines and the possible mechanisms. The first part of the Rho GTPases family in the cytoskeleton and migration of melanoma and the molecular mechanism of the molecular mechanism: Black The migration and invasion of vegetarian tumor are one of the key factors that affect the survival of the patients. The cytoskeleton recombination is necessary for cell migration and invasion. The Rho GTPases family is a key regulatory factor in the cytoskeleton. This study intends to explore the expression of the different members of the Rho GTPases family in the melanoma cells, observe the similarities and differences of cytoskeleton, and reveal the Rho GTPases The role and molecular mechanism of the family by regulating the cytoskeleton in the migration and invasion of melanoma. Methods: 1, the morphological differences between M14, A375 and MV3 three melanoma cells and human melanocytes (Melanocyte, MC) were observed under the Hoffman microscope; the Luo Danming labeled phallus cytosine staining was observed under the ultrahigh resolution microscope. The difference between the actin filaments as the main components of the filamentous pseudo foot, the plate-like pseudo foot, the stress fiber and the plaque in the four cells was detected by.2. The migration tests of the four cells of M14, A375 and MV3 and MC were detected by the Transwell migration test, and the real-time fluorescence quantitative polymerase chain reaction (Quantitative Real-time Polymerase Chain Reaction) was detected. The mRNA transcriptional level of the members of the Rho GTPases family; Western blotting (WB) for the detection of RhoD, Diaph2 (diaphanous related formin2), the expression of filamentin Cofilin and phosphorylated silk cut proteins. Results: 1. The three melanoma cells of.MC, M14, A375, and MV3 were different in morphology, but they all contained stress fibers and adhesion spots, and the number of MV3 stress fibers was less than M14 and A375, but the latter two were coarsely. The four cells all had very thin and short filamentous pseudfoot.2, Transwell migration experimental results showed M14 and A375 cells. The transmembrane migration ability was similar and the migration rate was high; the migration rate of MV3 cells was lower than that of M14 and A375, and the migration ability of MC without transmembrane was.3. The QPCR results of the different members of the Rho GTPases family were different in the four cells, and the corresponding changes in the cytoskeleton immunofluorescence were presented; WB results showed that RhoD in the Rho GTPases family members was MV3 was basically not expressed, consistent with the change of QPCR level, and its downstream effect factor Diaph2 also changed. Although the important regulatory factor of actin filament cofilin protein did not change significantly, the phosphorylation level P-Cofilin was obviously higher in melanoma cells than black vegetarian cells. Conclusion: Rho GTPases is regulated by muscle movement. The main components of the filaments are filamentous pseudo foot, plate-like pseudo foot, stress fiber, and adhesion spots that affect the migration and invasion of melanoma cells; the cytoskeleton and cell migration of melanoma and melanoma are the result of the precise regulation of Rho GTPases family members. The second part of lentivirus mediated overexpression of RhoD human melanoma A375 cells Construction and identification of the plant: construct the lentivirus vector of human RhoD gene and carry out lentivirus packaging and identification, transfect human melanoma cell A375 in vitro, make it overexpress RhoD protein, and lay the foundation for the follow-up study of the role of RhoD in melanoma. Method: 1, using Gateway technique to construct RhoD with enhanced green fluorescent protein EGFP. The lentivirus vector was identified by PCR and gene sequencing, and DNA-Lipofectamine2000 complex was prepared by liposomes mixed with auxiliary plasmid pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5, and 293FT cells were transfected together to package lentivirus, produce corresponding lentivirus particles, determine the virus titer.2 by quantitative PCR, and use packaging. A good lentivirus was transfected into human melanoma A375 cells to establish a human melanoma A375 cell line overexpressed by RhoD, the fluorescence expression was observed under the fluorescence microscope, and the transfection efficiency was detected by flow cytometry. The experiment was divided into A375 (untreated control group), A375-EGFP (free virus control group without target gene) and A375-RhoD (containing RhoD gene virus group). Three groups, using real time fluorescence quantitative PCR (QPCR) and immune imprint (Western blot, WB) to verify the overexpression of RhoD in the A375-RhoD group cells. Results: 1, through PCR, gene sequencing confirmed that the Lentivirus Expression Vector pLV[Exp]-EGFP/Neo-CMVhRhoD construction was successful. The slow disease of the high effective infection force was issued with the adjuvant plasmids co transfected 293FT cells. The virus titer was (5.13 + 2) x 108TU/ml.2. After transfection of A375 cells, there was a clear green fluorescence expression on both cell and cell membrane surface, and the transfection efficiency of flow detection was more than 80%. The level of RhoD mRNA and protein in group A375-RhoD was significantly higher than that in A375-EGFP and A375 groups (P0.05). Conclusion: RhoD lentivirus was successfully constructed. The expression vector, packed with highly effective lentivirus particles and successfully transfected human melanoma A375 cells, provided an experimental basis for further study of the role of RhoD in melanoma. The third part overexpressed the effect of RhoD on the A375 cytoskeleton and migration and invasion of melanoma cells and the mechanism to explore the effect of RhoD on human melanin. The effect and mechanism of biological behavior such as cytoskeleton, migration and invasion ability of tumor cell line A375. Methods: the cytoskeleton was observed by Luo Danming phallus cyclin staining, cell migration and invasion ability was detected by Transwell chamber test, cell cycle was detected by flow cytometry, A375 in blank control group and negative control lentivirus were observed. The biological behavior changes in the transfected group of A375-EGFP and RhoD lentivirus vector transfected group A375-RhoD three were compared, and the expression of Diaph2, cofilin and P-cofilin in the three groups was compared by Western blot immunoblotting, and the possible mechanism of RhoD affecting the bone frame and movement of melanoma cells and the possible mechanism of invasion was analyzed. 1, cytoskeleton staining showed that, compared with A375 and A375-EGFP groups, the cell volume of the A375-RhoD group increased, the stress fiber became thinner, the soft weak, the adhesion plaque increased, the formation of filamentous pseudo foot increased, the folds margin and the plate like pseudo foot had no obvious changes of.2, and the Transwell chamber migration experiment showed that A375, A375-EGFP, and A375-RhoD groups averaged every film in each field of vision. The number of cells was (149.67 + 11.93), (152.67 + 11.23) and (72.67.25 + 5.03). The overexpression of RhoD could significantly inhibit the transmembrane movement and migration of A375 in melanoma cells (P0.05). The invasion test of artificial basement membrane in the Transwell compartment showed that the average number of membrane cells in each field of A375-EGFP and A375-RhoD was (83 + 7), respectively (78.33 + 12.34). And (9 + 1), RhoD overexpression could significantly inhibit the invasiveness of the melanoma cell A375 (P0.05).3. Flow cytometry analysis of cell cycle results showed that A375, A375-EGFP and A375-RhoD three groups had G1 phase, and there was no statistical difference in the percentage of S and G2 cells (P =0.685,0.138 and negative), over expression and negative control lentivirus The carrier had no effect on the cell cycle of.5, and the WB immunoblotting showed that A375-RhoD cells could activate the downstream signal effector Diaph2 and promote its expression under the stimulation of RhoD, while the control group failed to activate.RhoD overexpression and negative control lentivirus vector with no influence on cofilin and P-cofilin. Conclusion: RhoD is in the thinner A375 cell of melanoma. Cytoskeleton may play a role in motility, migration and invasion, and may become a target in melanoma therapy.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.5

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7 陸原;翁翊;李清;何雯;金麗;陳達(dá)燦;y⒐，

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