本文選題：層層組裝 + 三維培養(yǎng)��； 參考：《南方醫(yī)科大學(xué)》2015年碩士論文

【摘要】：研究背景腫瘤是指機體細(xì)胞在各種致癌因素作用下發(fā)生基因突變,導(dǎo)致組織失去機體對其生長的正常調(diào)控,進(jìn)而經(jīng)過一系列不可控的異�？寺≡鲋乘纬傻漠惓Ｐ律M織。腫瘤包括良性和惡性兩大類。腫瘤,特別是惡性腫瘤(癌癥)嚴(yán)重危害人類的生命健康。而且隨著人類壽命的不斷增長,癌癥的發(fā)病率和死亡率也逐漸上升,成為全世界最受關(guān)注的健康問題之一。腫瘤微環(huán)境是腫瘤細(xì)胞在其發(fā)生發(fā)展過程中所處的內(nèi)環(huán)境,由腫瘤細(xì)胞本身、間質(zhì)細(xì)胞、微血管、組織液及少量浸潤細(xì)胞等共同組成。腫瘤的發(fā)生發(fā)展始終伴隨著腫瘤細(xì)胞同其周圍微環(huán)境的相互作用。相關(guān)研究已揭示,早在實體瘤血管發(fā)生之前,因腫瘤細(xì)胞迅速增殖而造成的缺氧環(huán)境可能導(dǎo)致腫瘤進(jìn)展和更差的臨床結(jié)局。而在血管形成后,腫瘤局部長期的缺血缺氧環(huán)境所造成的瘤體中央壞死也被看作是侵襲性癌的共同特征。體外模擬腫瘤細(xì)胞微環(huán)境,可以為更進(jìn)一步研究腫瘤細(xì)胞癌變機理、增殖、分化及臨床藥物篩選提供更好的研究方法。迄今為止,在絕大多數(shù)研究中,腫瘤細(xì)胞的培養(yǎng)均是采用平面培養(yǎng)皿的二維(2D)培養(yǎng)模型和動物在體培養(yǎng)模型。在2D培養(yǎng)模型中,腫瘤細(xì)胞所處的環(huán)境顯然與在體的實體瘤微環(huán)境截然不同,腫瘤細(xì)胞缺乏細(xì)胞外基質(zhì)(extracellular matrix, ECM),無法同腫瘤微環(huán)境中的多種因素發(fā)生交互作用,導(dǎo)致細(xì)胞與細(xì)胞、細(xì)胞與細(xì)胞外基質(zhì)之間相互聯(lián)系所致的細(xì)胞形態(tài)改變、細(xì)胞生長、增殖與分化等變化被忽略。相比于2D培養(yǎng)模型,在三維(3D)培養(yǎng)條件下的腫瘤組織中存在不同表型的腫瘤細(xì)胞,包括增殖細(xì)胞、非增殖細(xì)胞以及壞死細(xì)胞等,這使培養(yǎng)的腫瘤細(xì)胞能夠表現(xiàn)出更接近于在體腫瘤細(xì)胞的特性。另外,通過在裸鼠體內(nèi)注射腫瘤細(xì)胞或者移植腫瘤組織的方法建立的動物腫瘤模型已經(jīng)得到大多數(shù)學(xué)者的認(rèn)可,這也是目前進(jìn)行腫瘤研究的金標(biāo)準(zhǔn)。然而,動物腫瘤模型有一定的局限性,注射到裸鼠體內(nèi)的腫瘤細(xì)胞會受到體內(nèi)諸多因素的影響,體內(nèi)的腫瘤細(xì)胞難免會發(fā)生不可控制的基因及相關(guān)蛋白表達(dá)的改變。因此,我們有必要探索一種更好的方法來替代2D培養(yǎng)系統(tǒng)和動物模型。3D體外組織的構(gòu)建可以用來彌補二維培養(yǎng)模型的不足,為我們研究腫瘤及其相關(guān)機制提供了一種比動物模型更快捷、經(jīng)濟的可靠選擇。隨著近年來組織工程學(xué)的迅猛發(fā)展,基于3D培養(yǎng)的組織工程腫瘤技術(shù)進(jìn)展迅速,呈現(xiàn)出多樣化的發(fā)展趨勢。然而,這其中的大部分方法都難以控制瘤體結(jié)構(gòu)以及細(xì)胞與細(xì)胞之間、細(xì)胞與細(xì)胞外基質(zhì)間的相互作用。例如,多細(xì)胞腫瘤懸浮球(multicellular tumor suspension, MCTs)由多種單個細(xì)胞組成,但是細(xì)胞與細(xì)胞之間無細(xì)胞外基質(zhì),因而預(yù)想的細(xì)胞與基質(zhì)之間的相互作用缺失。另一方面,目前的3D模型大多依賴于天然的和人工合成的高分子聚合物。天然分子雖然具有與ECM相似的特性,能夠盡可能的模擬腫瘤微環(huán)境,但由于其往往存在殘存的信號分子及不定量的雜質(zhì),導(dǎo)致使用天然材料的實驗無法做到完全可控和絕對嚴(yán)謹(jǐn)。人工聚合高分子材料雖然避免了上述缺陷,但由于其微纖維間的孔徑往往能達(dá)到細(xì)胞直徑的千倍級甚至萬倍級,從而導(dǎo)致有效成分的過快擴散和迅速丟失。據(jù)報道,膠原凝膠硬度低,降解速度快,而且其許多特性受提取過程的影響,從而限制了它們的應(yīng)用。因此,從該領(lǐng)域發(fā)展的角度來看,我們可以將從納米級或微納米級到宏觀水平的各種因素均應(yīng)考慮在體外構(gòu)建的三維組織模型中。納米級的細(xì)胞外基質(zhì)存在諸多優(yōu)點。首先,ECM本身就是由多種納米級高分子纖維組織組成；其次,納米級細(xì)胞外基質(zhì)有較高的表面積和體積比,能夠促使細(xì)胞之間的聯(lián)系,并且促進(jìn)生物活性分子的運輸；再者,細(xì)胞膜和細(xì)胞遷移相關(guān)的細(xì)胞骨架均屬于納米級。因此,納米級ECM可以促進(jìn)細(xì)胞與細(xì)胞之間,細(xì)胞與細(xì)胞微環(huán)境之間的相互作用,在腫瘤發(fā)生發(fā)展、診斷及臨床藥物篩選的研究中具有明顯的優(yōu)勢。尋找合適的生物材料,并通過便捷的方法構(gòu)建三維培養(yǎng)腫瘤模型,以達(dá)到模擬體內(nèi)真實腫瘤微環(huán)境的目的,是本研究的重點。明膠和海藻酸鈉作為天然生物材料,無毒、可降解,并且具有良好的親水性、細(xì)胞親和性及細(xì)胞生物相容性。在組織工程支架中,明膠/海藻酸鈉溶液作為一種軟支架材料,可以與細(xì)胞混合,實現(xiàn)細(xì)胞的立體培養(yǎng)。這種支架材料的優(yōu)勢在于不但在表面而且在支架內(nèi)部也可以含有細(xì)胞,從而為多種細(xì)胞的空間合理分布提供了可能。殼聚糖是甲殼素脫乙酞化產(chǎn)物,屬于聚陽離子多糖(Pka=6.3)物質(zhì),殼聚糖中的N-乙酞基毗喃與細(xì)胞外基質(zhì)中的糖胺聚糖(GAGs)組成類似,且具有生物活性、可生物降解。本研究選用A型明膠、海藻酸鈉及中分子量殼聚糖,通過層層組裝和聚合的方法,構(gòu)建一種體外三維多細(xì)胞腫瘤球體模型來模擬腫瘤細(xì)胞微環(huán)境,為研究乳腺癌的癌變機理、癌細(xì)胞轉(zhuǎn)移以及藥物臨床前評價等方面提供一種更加靈活簡便的方法。上皮間質(zhì)轉(zhuǎn)化(Epithelial Mesenchymal Transition, EMT)是指本身具有極性的上皮細(xì)胞在某些因素的刺激下,上皮特性減少、間質(zhì)特性增多、細(xì)胞基質(zhì)粘附消失和細(xì)胞骨架重塑,從而導(dǎo)致上皮細(xì)胞極性消失,細(xì)胞運動能力增加,獲得浸潤性和遷移能力。在上皮來源的惡性腫瘤中,EMT是腫瘤細(xì)胞獲得浸潤和遷移能力的重要途徑。盡管EMT的發(fā)生同腫瘤微環(huán)境密切相關(guān),但腫瘤微環(huán)境誘導(dǎo)腫瘤細(xì)胞發(fā)生EMT的確切機制仍不清楚。本研究在已構(gòu)建的三維腫瘤模型的平臺上比較研究二維培養(yǎng)體系與三維培養(yǎng)體系中乳腺癌細(xì)胞EMT相關(guān)蛋白的表達(dá)差異,如CD47、N-Cadherin等。在體外三維培養(yǎng)體系下模擬體內(nèi)乳腺癌N-Cadherin表達(dá)增多觸發(fā)EMT促進(jìn)腫瘤細(xì)胞浸潤和遷移的臨床特點,對我們構(gòu)建的三維腫瘤模型進(jìn)行驗證。目的：1.構(gòu)建用于基礎(chǔ)研究以及藥物篩選的體外組織工程化腫瘤三維模型,并對該模型的形貌、表面電荷、生物相容性等性質(zhì)進(jìn)行研究；2.體外所構(gòu)建的三維人源性乳腺癌腫瘤模型的體外驗證。方法：1.細(xì)胞培養(yǎng)購入人乳腺癌細(xì)胞株MDA-MB-231,復(fù)蘇后傳代,將其培養(yǎng)在含有10%胎牛血清,0.1%青霉素和鏈霉素的RPMI 1640培養(yǎng)基中,于37℃,含5%C02的培養(yǎng)箱中常規(guī)培養(yǎng)。每天換液,待細(xì)胞融合率達(dá)80%時,用含0.02% EDTA的0.25%的胰蛋白酶液消化并傳代。待細(xì)胞量足夠,取足量細(xì)胞用于層層組裝。2.對單個細(xì)胞進(jìn)行(明膠-海藻酸鈉)3-殼聚糖的層層組裝及多細(xì)胞腫瘤球體的形成設(shè)計合成與細(xì)胞相容性良好的納米級細(xì)胞外基質(zhì)成分,以促進(jìn)細(xì)胞與細(xì)胞之間,細(xì)胞與細(xì)胞微環(huán)境之間的相互作用。該納米級細(xì)胞外基質(zhì)成分包括A型明膠、海藻酸鈉,需分別帶有正電荷和負(fù)電荷,因此細(xì)胞外基質(zhì)成分依次層層包裹于細(xì)胞表面。細(xì)胞經(jīng)胰酶消化后,計數(shù)1×107,于離心機中,1000rpm/min,離心5min,棄去上清液,用7ml 0.1% (w/v)明膠溶液輕輕重懸收集的細(xì)胞后,常溫放置10min;接著于離心機中,1000rpm/min,離心5min,棄去明膠溶液,此時明膠層粘附于單個細(xì)胞表面。然后,用7m1預(yù)熱的0.1%(w/v)海藻酸鈉溶液輕輕重懸包裹有明膠層的細(xì)胞團(tuán),則帶有陰離子的聚合電解質(zhì)層粘附于明膠層上,常溫孵育10min后,離心棄去海藻酸鈉溶液；接著將等體積的A型明膠溶液(陽離子聚合電解質(zhì)溶液)加入包裹有明膠和海藻酸鈉的細(xì)胞團(tuán)。此過程通過依次在細(xì)胞表面包裹帶有相反電荷的聚合電解質(zhì)三個循環(huán)。最后,用7m1預(yù)熱的0.2%(w/v) PH 6.7的中分子量殼聚糖溶液(陽離子聚合電解質(zhì))使最外層均粘附有陰離子聚合電解質(zhì)(海藻酸鈉)的單個腫瘤細(xì)胞之間發(fā)生聚合反應(yīng),數(shù)分鐘后,1000rpm/min,離心10min,多細(xì)胞腫瘤球形成,棄去殼聚糖溶液,用預(yù)熱的含10%FBS的RPMI 1640培養(yǎng)基輕輕重懸,然后將多細(xì)胞腫瘤球轉(zhuǎn)移至一個新的六孔板中,加入RPMI 1640培養(yǎng)基,置于5%CO2,37℃培養(yǎng)箱中培養(yǎng),培養(yǎng)基每天更換。3.組織工程化人源性乳腺癌三維模型的表征及生物相容性研究組織工程化人源性乳腺癌三維模型構(gòu)建成功后,一般情況下,需要對其表面形貌、所帶電荷、生物相容性等性質(zhì)進(jìn)行研究。本實驗中分別采用光學(xué)顯微鏡、激光粒度分析儀、掃描電鏡、熒光顯微鏡、活死染色等儀器和技術(shù)對其性質(zhì)進(jìn)行測量和分析。4.體外所構(gòu)建三維人源性乳腺癌腫瘤模型的體外驗證實驗分為三組,3Di,3Dm和2D,稱腫瘤球中的細(xì)胞為3Dis；腫瘤球培養(yǎng)過程中,觀察發(fā)現(xiàn)其培養(yǎng)皿底板有細(xì)胞遷移出來并貼壁生長,稱遷移出來的細(xì)胞為3Dms；稱二維培養(yǎng)的細(xì)胞為2D。分別培養(yǎng)四天后,提取總蛋白,Western blotting檢測p-ERK, ERK, CD47蛋白的表達(dá)；取出多細(xì)胞腫瘤球,PBS洗三次,4%多聚甲醛固定24小時,30%蔗糖溶液固定1周后,冰凍切片,放于37℃干燥箱中烤24小時,免疫熒光檢測E-Cadherin, N-Cadherin蛋白的表達(dá)。5.統(tǒng)計學(xué)分析以上實驗均設(shè)有陰性對照實驗,每次實驗至少重復(fù)3次。統(tǒng)計分析采用SPSS13.0統(tǒng)計軟件完成,結(jié)果用均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)來表示。組間比較采用單因素方差分析(One-way ANOVA),方差齊性的組間比較采用Tukey法,方差不齊的組間比較采用Dunnett's T3法,p0.05認(rèn)為差異有統(tǒng)計學(xué)意義。結(jié)果：1.成功構(gòu)建體外組織工程化人源性乳腺癌三維模型。通過A型明膠與海藻酸鈉靜電作用層層包裹于細(xì)胞三個循環(huán),得到單個腫瘤細(xì)胞層層組裝的模型；然后加入中分子量殼聚糖溶液,使層層組裝的單個細(xì)胞間發(fā)生聚合,形成多細(xì)胞腫瘤球,即組織工程化人源性乳腺癌腫瘤模型。2.光學(xué)顯微鏡下顯示人源性乳腺癌三維模型呈團(tuán)塊狀,內(nèi)部細(xì)胞(3Dis)形態(tài)規(guī)則,呈圓形,輪廓清晰,連接緊密；隨著細(xì)胞培養(yǎng)時間的延長,一些腫瘤細(xì)胞從腫瘤球體遷出,遷移出的腫瘤細(xì)胞(3Dmms)形態(tài)規(guī)則,輪廓清晰,貼壁生長,狀態(tài)良好。3.用FITC標(biāo)記明膠,在熒光顯微鏡下單純(明膠-海藻酸鈉)3層層組裝組以及(明膠-海藻酸鈉)3層層組裝后用殼聚糖聚合組均顯示：每個細(xì)胞周圍均有一層綠色熒光。這說明層層組裝的納米膜成功包裹于單個細(xì)胞。4.激光粒度分析儀測試結(jié)果顯示,細(xì)胞表面電荷隨著帶相反電荷聚合電解質(zhì)的輪流加入,呈現(xiàn)曲折變化,表明細(xì)胞表面層層組裝上了納米材料。5.掃描電鏡觀察3D體外腫瘤模型的形態(tài)顯示：多細(xì)胞腫瘤球具有三維立體結(jié)構(gòu),細(xì)胞生長良好,細(xì)胞表面成功包裹了納米材料,保持了細(xì)胞外基質(zhì)的完整性；細(xì)胞與細(xì)胞之間并非緊密連接,有較大孔隙,有利于水分,營養(yǎng)物質(zhì)及氧氣滲透,形成適于腫瘤細(xì)胞生長的三維立體環(huán)境。6.對多細(xì)胞腫瘤球進(jìn)行活死染色,熒光顯微鏡下觀察示,整個腫瘤球呈綠色(活細(xì)胞),幾乎沒有紅色(死細(xì)胞)出現(xiàn)。證明納米材料生物相容性良好,成功構(gòu)建了便于基礎(chǔ)研究以及藥物篩選的三維人源性乳腺癌模型。7. Western blotting和免疫熒光驗證了此基于單個細(xì)胞層層組裝構(gòu)建的三維多細(xì)胞腫瘤球體模型內(nèi)的微環(huán)境可通過N-Cadherin的過表達(dá)來觸發(fā)EMT。Western blotting結(jié)果顯示3Dm和2D組p-ERK和ERK的表達(dá)均明顯高于3Di組；而3Di組CD47表達(dá)明顯高于3Dmm和2D組。免疫熒光結(jié)果顯示2D組呈現(xiàn)E-Cadherin過表達(dá),但無N-Cadherin表達(dá)；而3Di組呈現(xiàn)高表達(dá)的E-Cadherin和N-Cadherin; 3Dm組則呈現(xiàn)E-Cadherin高表達(dá),N-Cadherin低表達(dá)。結(jié)論：在本研究中,我們用超薄基質(zhì)材料層層包裹單個腫瘤細(xì)胞的方法構(gòu)建了一個三維多細(xì)胞腫瘤球體模型來模擬在體實體瘤的微環(huán)境,并對該模型的形貌及表面電荷進(jìn)行一系列表征,通過活死染色驗證了模型中細(xì)胞的活力。還在該三維模型的平臺上進(jìn)一步比較研究了二維培養(yǎng)體系與三維培養(yǎng)體系中乳腺癌細(xì)胞EMT相關(guān)蛋白的表達(dá)差異,并且該模型在體外三維培養(yǎng)體系下很好地模擬了體內(nèi)乳腺癌N-Cadherin表達(dá)增多觸發(fā)EMT促進(jìn)腫瘤細(xì)胞浸潤和遷移的臨床特點,對我們構(gòu)建的三維腫瘤模型進(jìn)行驗證。
[Abstract]:Background tumor is a kind of malignant tumor, including two types of benign and malignant tumors, including benign and malignant tumors, especially malignant tumors (cancer). With the growth of human life, the incidence and mortality of cancer are increasing, and it has become one of the most concerned health problems in the world. The tumor microenvironment is the internal environment of the tumor cells in the process of its development, including the tumor cells, interstitial cells, microvessels, tissue fluids and less. The development of the tumor is always accompanied by the interaction between the tumor cells and the surrounding microenvironment. The related research has revealed that the hypoxia environment caused by the rapid proliferation of tumor cells may lead to tumor progression and worse clinical outcome before the tumor angiogenesis. The central necrosis of the tumor caused by the ischemic anoxia environment in the tumor bureau is also seen as a common feature of invasive cancer. In vitro simulation of the tumor cell microenvironment can provide better research methods for further research on the mechanism, proliferation, differentiation and clinical drug screening of tumor cells. The culture of tumor cells is a two-dimensional (2D) culture model and an animal model in vivo. In the 2D culture model, the environment of the tumor cells is obviously different from the solid tumor microenvironment in the body. The tumor cells lack the extracellular matrix (extracellular matrix, ECM), which can not be associated with various factors in the microenvironment of the tumor. The interaction caused cell morphology changes, cell growth, proliferation and differentiation caused by the interaction between cells and cells, cells and extracellular matrix. Compared to the 2D culture model, tumor cells with different phenotypes, including proliferating cells and non proliferative cells, were found in the tumor tissues under the condition of three-dimensional (3D) culture. In addition, the animal tumor model established by injecting tumor cells in nude mice or transplanted tumor tissue in nude mice has been recognized by most scholars, which is also the gold standard for cancer research. The animal tumor model has some limitations. The tumor cells injected into the nude mice will be affected by many factors in the body. The tumor cells in the body are unavoidable to change the changes in the expression of the incontrollable genes and related proteins. Therefore, it is necessary to explore a better formula to replace the 2D culture system and the animal model.3D in vitro The construction of the organization can be used to compensate for the deficiency of the two-dimensional culture model. It provides a more efficient and reliable choice for the study of tumor and its related mechanisms. With the rapid development of the tissue engineering in recent years, the technology of tissue engineering tumor based on 3D has developed rapidly and presents a variety of development trends. Most of these methods are difficult to control the structure of the tumor body and the interaction between cells and cells, cells and extracellular matrix. For example, multicellular tumor suspension (MCTs) is composed of a variety of single cells, but the cells and cells have no extracellular matrix, thus preconceived cells and bases. On the other hand, most of the current 3D models depend on natural and synthetic polymers. Although natural molecules have similar characteristics to ECM, they can simulate tumor microenvironment as much as possible, but they often exist in the presence of residual signal molecules and indeterminate impurities, resulting in the use of natural materials. The artificial polymer materials can not be completely controlled and absolutely rigorous. Although the artificial polymer materials can avoid the above defects, the pore size of the microfibers can often reach the 1000 or even ten thousand times of the diameter of the cells, which leads to the rapid diffusion and rapid loss of the effective components. It is reported that the hardness of the collagen gel is low and the degradation rate is fast. And many of its characteristics are affected by the extraction process, thus limiting their applications. Therefore, from the perspective of development in this field, we can consider the various factors from nanoscale or micro nanoscale to macro level in the three-dimensional tissue model constructed in vitro. There are many advantages of the nanoscale extracellular matrix. First, ECM Ben The body is made up of a variety of nanoscale polymer fibers. Secondly, the nanoscale extracellular matrix has a high surface area and volume ratio, which can promote the connection between cells and promote the transport of bioactive molecules. Furthermore, the cell membrane and cell migration related bone shelves are all nanoscale. Therefore, nanoscale ECM can be promoted. The interaction between cell and cell, cell and cell microenvironment has obvious advantages in the study of tumor development, diagnosis and clinical drug screening. Finding the appropriate biomaterials and constructing a three-dimensional tumor model through a convenient method can be used to simulate the real tumor microenvironment in the body. Gelatin and sodium alginate, as natural biomaterials, are non-toxic, biodegradable, and have good hydrophilic, cellular affinity and cellular biocompatibility. In tissue engineering scaffolds, gelatin / sodium alginate solution can be used as a kind of soft scaffold material and can be mixed with cells to realize cell stereoscopic culture. The potential is that it is possible not only on the surface but also in the inside of the scaffold, thus providing the possible spatial distribution of a variety of cells. Chitosan is the product of chitin deglyphthalide, a polycationic polysaccharide (Pka=6.3) substance, and the N- phthalide in chitosan is similar to the glycosaminoglycan (GAGs) in the extracellular matrix. In this study, A gelatin, sodium alginate and medium molecular weight chitosan were used in this study to construct a three-dimensional multi cell tumor cell model in vitro to simulate the microenvironment of tumor cells in vitro, to study the carcinogenesis, metastasis of cancer cells and the pre clinical evaluation of drugs. A more flexible and simple method. Epithelial Mesenchymal Transition (EMT) refers to the epithelial cells in which their polar epithelial cells are stimulated by some factors, the decrease of epithelial properties, the increase of interstitial properties, the disappearance of cell matrix adhesion and the remodeling of cytoskeleton, resulting in the disappearance of the epithelial cell polarity and the cell movement. EMT is an important approach to the invasion and migration of tumor cells in epithelial cancer cells. Although the occurrence of EMT is closely related to the tumor microenvironment, the exact mechanism of the tumor cells to induce EMT is still unclear. On the platform of the model, the difference in the expression of EMT related proteins in breast cancer cells in the two-dimensional culture system and the three-dimensional culture system, such as CD47, N-Cadherin and so on, is used to simulate the clinical characteristics of the increase of N-Cadherin expression in breast cancer in vivo to trigger the EMT to promote the invasion and migration of the tumor cells, and to build the three-dimensional swelling of the tumor cells. The tumor model was verified. Objective: 1. to construct an in vitro three-dimensional model of tissue engineered tumor for basic research and drug screening, and to study the morphology, surface charge, biocompatibility and other properties of the model. 2. in vitro validation of the three-dimensional human breast cancer model in vitro. Method: 1. cell culture buyers. The breast cancer cell line, MDA-MB-231, was transmitted after resuscitation, and was cultured in the RPMI 1640 medium containing 10% fetal bovine serum, 0.1% penicillin and streptomycin. It was cultured in a incubator containing 5%C02 at 37 degrees C. A daily liquid was changed, when the cell fusion rate was 80%, it was digested and passaged with 0.25% trypsin containing 0.02% EDTA. Sufficient cells are used to assemble layers of.2. to assemble a single cell (gelatin - sodium alginate) 3- chitosan and the formation of a multicellular tumor sphere to synthesize the nanoscale extracellular matrix components with good cellular compatibility to promote the interaction between cells and cells and the microcellular environment. The nanoscale cells The external matrix components include A gelatin, sodium alginate, with positive and negative charges, so the extracellular matrix components are sequentially coated on the surface of the cell. After trypsin digestion, the cells are counted 1 x 107, in centrifuge, 1000rpm/min, centrifuge 5min, supernatant, and 7ml 0.1% (w/v) gelatin solution. At the temperature of 10min, then in the centrifuge, 1000rpm/min, centrifuge 5min, discarded gelatin solution, gelatin layer adhered to the surface of a single cell at this time. Then, with 7m1 preheated 0.1% (w/v) sodium alginate solution to gently overhang the cell mass of the gelatin layer, then the polyelectrolyte layer with anions adhered to the gelatin layer, after incubating 10min at normal temperature. The solution of sodium alginate was abandoned; then the equal volume A gelatin solution (cationic polyelectrolyte solution) was added to the cell masses wrapped with gelatin and sodium alginate. This process was carried out by three cycles of polyelectrolytes with opposite charges on the cell surface. Finally, the molecular weight of the middle molecular weight of 0.2% (w/v) PH 6.7 was preheated with 7m1. The glucose solution (cationic polyelectrolyte) polymerized the single tumor cells with the most outer layer adhered to the anionic polyelectrolyte (sodium alginate). After a few minutes, 1000rpm/min, centrifugation, 10min, multicellular tumor balls were formed, the chitosan solution was abandoned, and the preheated 10%FBS containing RPMI 1640 medium was gently suspended and then more thin. The cell tumor ball was transferred to a new six hole plate, the RPMI 1640 medium was added, and the culture medium was placed in the 5%CO2,37 centigrade incubator. The culture medium changed every day to replace the.3. tissue engineering human derived breast cancer three-dimensional model and the biocompatibility study. Study on the properties of surface morphology, charge and biocompatibility. In this experiment, optical microscopy, laser particle size analyzer, scanning electron microscopy, fluorescence microscopy, live death staining and other instruments and techniques were used to measure and analyze the properties of.4. in vitro. The three groups, 3Di, 3Dm and 2D, called the cells in the tumor ball 3Dis. In the process of tumor ball culture, it was observed that the cell plates were migrated and adhered to the wall in the culture plate. The cells migrated were 3Dms, and the two dimensional cultured cells were cultured for four days, respectively, to extract the total protein, and the Western blotting detected the tables of p-ERK, ERK, CD47 protein. Three times of PBS washing, 4% polyoxymethylene fixed for 24 hours, 30% sucrose solution for 24 hours, 30% sucrose solution fixed for 1 weeks, frozen section for 24 hours at 37 centigrade drying box, immunofluorescence detection E-Cadherin, and the expression of N-Cadherin protein in the above experiments all had negative control experiments, each experiment repeated 3 times at least. The analysis was completed with SPSS13.0 statistical software, and the results were expressed with mean standard deviation (Mean + SD). Single factor analysis of variance (One-way ANOVA) was used among groups. Tukey method was used to compare the homogeneity of variance. Dunnett's T3 method was used for the comparison of variance between groups. The difference was statistically significant. Results: 1. a successful construction of an in vitro group was made. A three-dimensional model of human derived human breast cancer is built. Through the three cycles of A gelatin and sodium alginate electrostatic action, a model of a single tumor cell is assembled by a single tumor cell. Then a medium molecular weight chitosan solution is added to make the aggregation of the individual cells of the layers assembled to form a multicellular tumor ball, that is, tissue engineering. Human breast cancer tumor model.2. under optical microscope showed that the three-dimensional model of human breast cancer was lump shaped and the internal cell (3Dis) morphology.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R73-35

【共引文獻(xiàn)】

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