【摘要】：胰腺癌是死亡率較高的一種癌癥,患病后平均存活時間只有6個月,5年生存率只有3%-5%。70%-95%的胰腺癌組織中都存在KRAS基因突變,胰腺癌腫瘤細(xì)胞所處的組織環(huán)境血管化程度低,營養(yǎng)較為貧瘠。已有研究報道證明,胰腺癌細(xì)胞可通過KRAS突變所誘導(dǎo)的巨胞飲作用攝取胞外蛋白質(zhì),以維持腫瘤細(xì)胞的生存和增殖,相應(yīng)地這種細(xì)胞較少依賴網(wǎng)格蛋白介導(dǎo)的內(nèi)吞途徑內(nèi)化胞外物質(zhì)。近年來納米抗腫瘤藥物載體的研究逐漸受到了廣泛的關(guān)注,針對于不同腫瘤細(xì)胞設(shè)計不同的納米抗腫瘤藥物載體也是研究的重點之一,本研究致力于KRAS突變胰腺癌細(xì)胞表面巨胞飲結(jié)構(gòu)的確認(rèn),巨胞飲結(jié)構(gòu)特點和發(fā)生位置的分析,巨胞飲組成的分析以及介孔二氧化硅抗腫瘤藥物載體入胞特征等研究,為達(dá)到以上研究目標(biāo),本論文按如下方案實施:1、合成帶有兩端酶切位點的HSA基因全序列,連接到pPIC9K質(zhì)粒中,并轉(zhuǎn)入畢赤酵母菌株rHSA GS115,使HSA基因融入酵母基因組中,又通過表達(dá)、鑒定及搖瓶培養(yǎng)、蛋白純化濃縮等步驟,得到具有一定濃度和純度的重組人血清白蛋白,連接到紅色熒光分子Alexa Fluor 568上,獲得了本研究所需要的重要工具蛋白rHSA和rHSA-Alexa Fluor 568。2、利用含有十四烷酰佛波醇乙酸酯(TPA)的培養(yǎng)液孵育細(xì)胞,刺激細(xì)胞產(chǎn)生巨胞飲,觀察到細(xì)胞表面出現(xiàn)大量“杯”狀結(jié)構(gòu),分別給予細(xì)胞帶有綠色熒光的巨胞飲標(biāo)志物Dextran Alexa Fluor488,以及已被確認(rèn)是通過巨胞飲進(jìn)入細(xì)胞的白蛋白(rHSA-Alexa Fluor 568),都觀察到了“杯”狀結(jié)構(gòu)和它們的共定位,在同時含有Dextran Alexa Fluor488、Dextran Alexa Fluor488兩種培養(yǎng)液孵育細(xì)胞后我們也觀察到了二者的共定位,確認(rèn)了“杯”狀結(jié)構(gòu)為巨胞飲在細(xì)胞表面的瞬時狀態(tài),經(jīng)過進(jìn)一步的研究獲得了巨胞飲結(jié)構(gòu)的尺寸信息和大致形態(tài),為方便理解,給出了巨胞飲的三種形態(tài)示意圖。3、利用已知的板狀偽足中微絲和細(xì)胞膜之間的關(guān)系作為參照,分析形成巨胞飲的膜皺褶的組成成分,通過超高分辨率顯微鏡所拍攝的圖片,能夠清楚地觀察到微絲和細(xì)胞膜之間的位置關(guān)系,通過iMaris軟件的擬合后所形成的模型,觀察到具有一定直徑和深度的巨胞飲“杯”狀結(jié)構(gòu),通過動態(tài)拍攝,觀察到經(jīng)血清饑餓處理的胰腺癌細(xì)胞突變株KRASG12CMIA PaCa-2細(xì)胞,在加入白蛋白后細(xì)胞表面“杯”狀結(jié)構(gòu)隨著時間發(fā)生明顯變化,微絲在此期間變化明顯,隨著時間的變化貫穿狀的微絲逐漸消失,呈現(xiàn)散在狀態(tài),隨后貫穿狀微絲又重新出現(xiàn)。4、利用本研究中建立的方法觀察到人臍靜脈內(nèi)皮細(xì)胞(HUVEC)中巨胞飲標(biāo)志物Dextran-568(紅色熒光)與單壁碳納米管(綠色熒光)的共定位,確定在HUVEC細(xì)胞中單壁碳納米管通過巨胞飲內(nèi)化,驗證了本論文中所建立的納米材料入胞試驗系統(tǒng)所得結(jié)果的可信度和可靠性,從而對不同尺寸的介孔二氧化硅納米顆粒的入胞方式進(jìn)行分析,得出圓形介孔二氧化硅納米顆粒作為抗腫瘤藥物載體的直徑不應(yīng)超過400nm的結(jié)論。本研究中結(jié)構(gòu)照明顯微技術(shù)為我們的實驗奠定了基礎(chǔ),利用基于結(jié)構(gòu)照明顯微技術(shù)的超高分辨率顯微鏡,本研究建立了一種實時觀察細(xì)胞表面巨胞飲的方法,對KRAS突變胰腺癌細(xì)胞KRASG12CMIA PaCa-2表面的巨胞飲結(jié)構(gòu)進(jìn)行了表征,并實現(xiàn)了對介孔二氧化硅納米顆粒入胞方式的研究,為胰腺癌的納米靶向給藥載體設(shè)計提供可借鑒的細(xì)胞生物學(xué)數(shù)據(jù)基礎(chǔ)。
[Abstract]:Pancreatic cancer is a kind of cancer with higher mortality. The average survival time after the disease is only 6 months, and the 5-year survival rate is only 3% -5%. In 70%-95% of the pancreatic cancer tissues, the KRAS gene mutation is present, the degree of vascularization of the pancreatic cancer cells is low, and the nutrition is poor. It has been reported that pancreatic cancer cells can take the extracellular protein by the giant cell-drinking induced by the KRAS mutation to maintain the survival and proliferation of the tumor cells, and accordingly the cells are less dependent on the endocytosis-mediated endocytosis to internalize the extracellular substance. In recent years, the research of nanometer anti-tumor drug carrier has been widely concerned, and it is also one of the key points of the study to design different nanometer anti-tumor drug carriers for different tumor cells, and this study is devoted to the confirmation of KRAS mutant pancreatic cancer cell surface giant cell drink structure, In order to achieve the above research objectives, the whole sequence of HSA gene with both ends is synthesized by the following scheme: 1. the recombinant human serum albumin with a certain concentration and purity is obtained through the steps of connecting to the pPIC9K plasmid and transferring into the Pichia pastoris strain rHSA GS115, The important tool proteins rHSA and rHSA-Alexa Fluor 568.2, which were required by the study, were connected to the red fluorescent molecule Alexa Fluor 568. The cells were incubated with a culture solution containing tetradecanol acetate (TPA) to stimulate the cells to produce a giant cell drink, and a large number of cell surfaces were observed "Cup" The cell-like structure, the macrocytosis marker Dextran Alexa Fluor 488 with green fluorescence, and the albumin (rHSA-Alexa Fluor 568), which have been confirmed to enter the cells through the giant cell, were observed. "Cup" After incubation of the cells with both Dextran Alexa Fluori488 and Dextran Alexa Fluor488, we also observed a co-location of the two, and confirmed the 鈥淐up "The shape structure is the transient state of the giant cell drink on the surface of the cell, and the size information and the general form of the giant cell drink structure are obtained through further research, and the three forms of the giant cell drink are given for the convenience of understanding. 3, by using the relation between the micro-filament and the cell membrane in the known plate-like pseudo-foot as a reference, the composition component of the film fold forming the giant cell drink is analyzed, and the positional relationship between the micro-filament and the cell membrane can be clearly observed through the picture taken by the super-high-resolution microscope, With the model formed by the fitting of iMaris software, a giant cell drink with a certain diameter and depth was observed.鈥，

本文編號：2352033