本文關(guān)鍵詞：交聯(lián)型雞蛋白的制備及其在卵巢癌細(xì)胞3D培養(yǎng)中的應(yīng)用　出處：《東南大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 交聯(lián)雞蛋白 京尼平 戊二醛 甲基乙烯基醚馬來酸交替共聚物 3D細(xì)胞培養(yǎng)

【摘要】：卵巢癌已成為當(dāng)前個人及社會的沉重負(fù)擔(dān),其重要原因是很難預(yù)防,或在早期診斷時大部分患者仍可被治愈。卵巢癌的復(fù)發(fā)和轉(zhuǎn)移是導(dǎo)致大約90%的女性患者死亡的主要原因。關(guān)于卵巢癌發(fā)病機(jī)制的研究有所進(jìn)展,但仍不明確,困難之處在于缺乏全面的腫瘤進(jìn)展模型。癌癥是與機(jī)體的衰老密切關(guān)聯(lián)的疾病。隨著機(jī)體的衰老,細(xì)胞外基質(zhì)(ECM)微環(huán)境可以顯著促進(jìn)上皮細(xì)胞的惡性變化。根據(jù)衰老的交聯(lián)理論學(xué)說,蛋白質(zhì)和其它分子,例如核酸等通過共價交叉連接形成更大的分子,從而干擾到正常細(xì)胞的功能。交聯(lián)反應(yīng)發(fā)生的范圍涉及核酸脫氧核糖核酸(DNA)和細(xì)胞外的膠原纖維成分。雞蛋白含有細(xì)胞生長發(fā)育所需要的很多營養(yǎng)和信號成分,有良好的生物相容性,價格低廉,易于獲取。近年來,國外的科研工作者將天然雞蛋白分別用于不同細(xì)胞進(jìn)行培養(yǎng),取得了和人工基底膜、膠原等生物材料一樣好的效果。但天然雞蛋白強(qiáng)度弱,韌性低、耐水性差,易分散和降解。交聯(lián)不僅可以模擬衰老的細(xì)胞外基質(zhì),還可以增加雞蛋白的力學(xué)強(qiáng)度和延緩降解。本研究通過對雞蛋白培養(yǎng)基采用不同的化學(xué)交聯(lián),調(diào)控模擬體內(nèi)不同硬度梯度、多孔性、孔徑大小等微環(huán)境對卵巢癌細(xì)胞增殖分化、侵襲行為的影響,跟蹤細(xì)胞的生長乃至發(fā)展形成腫瘤的過程,為闡明卵巢癌的形成機(jī)制、篩選腫瘤藥物、預(yù)防和治療奠定基礎(chǔ)。研究內(nèi)容概括如下:(1)京尼平交聯(lián)雞蛋白的制備及其模擬衰老細(xì)胞外基質(zhì)作為卵巢癌細(xì)胞3D培養(yǎng)支架的應(yīng)用。京尼平(genipin,GP)作為天然產(chǎn)物,含有較低的生物學(xué)毒性,是很好的蛋白質(zhì)交聯(lián)劑。本研究采用京尼平交聯(lián)的不同形貌結(jié)構(gòu)和力學(xué)強(qiáng)度的雞蛋白模擬衰老的細(xì)胞外基質(zhì)作為培養(yǎng)支架,重點(diǎn)研究多樣化的形貌和力學(xué)強(qiáng)度改變對卵巢癌細(xì)胞的增殖和聚集行為的影響。研究表明,交聯(lián)雞蛋白的硬度和多孔性增加,能夠促進(jìn)卵巢癌細(xì)胞的增殖和聚集。因此,交聯(lián)的雞蛋白支架是研究衰老導(dǎo)致的細(xì)胞外基質(zhì)交聯(lián)、癌細(xì)胞增殖和癌癥的發(fā)展的合適模型。為防止癌細(xì)胞的浸潤和轉(zhuǎn)移,應(yīng)該關(guān)注可以防止衰老導(dǎo)致的細(xì)胞外基質(zhì)退行性變化的藥物作為篩選對象。(2)京尼平交聯(lián)的雞蛋白對卵巢癌細(xì)胞聚集生長和耐藥影響的機(jī)制初步探索。通過構(gòu)建卵巢癌SKOV-3細(xì)胞的3D培養(yǎng)模型,模擬宿主體內(nèi)腫瘤細(xì)胞生長的微環(huán)境,探討了卵巢癌細(xì)胞的表型形成、細(xì)胞聚集分布、CD44/ABCG2分子表達(dá)、以及細(xì)胞黏附分子E-cadherin/N-cadherin的分子表達(dá)。在考慮微環(huán)境中各影響因素作用的基礎(chǔ)上,推測多細(xì)胞耐藥性(MCR)與卵巢癌腫瘤干細(xì)胞特性之間的可能聯(lián)系。(3)戊二醛交聯(lián)的雞蛋白的制備及其模擬衰老細(xì)胞外基質(zhì)對卵巢癌細(xì)胞分布和聚集行為的影響。戊二醛(glutaraldehyde,GA)是最先使用于明膠的交聯(lián)劑。它水溶性好,具有雙官能團(tuán),價格相對低廉。此外,它在低濃度可以完全交聯(lián)明膠,可以改善其親水性和力學(xué)性能。戊二醛與蛋白質(zhì)的交聯(lián)反應(yīng)高效、產(chǎn)物穩(wěn)定。卵巢癌SKOV-3細(xì)胞在3D培養(yǎng)條件下,容易增殖聚集、成團(tuán)。通過制備不同濃度戊二醛交聯(lián)的雞蛋白培養(yǎng)支架,改變支架材料的力學(xué)強(qiáng)度和孔徑大小,模擬不同材料微環(huán)境對卵巢癌細(xì)胞增殖、分化、浸潤和轉(zhuǎn)移行為的影響,更好地明確腫瘤的生物學(xué)特性和腫瘤細(xì)胞生長、形成、發(fā)展的關(guān)鍵過程。(4)P(MVE-alt-MA)交聯(lián)的雞蛋白制備及其模擬衰老細(xì)胞外基質(zhì)對卵巢癌細(xì)胞增殖和轉(zhuǎn)移的影響。甲基乙烯基醚馬來酸交替共聚物(P(MVE-alt-MA))由于無毒副作用,可用于生物醫(yī)學(xué)組織工程中大孔支架聚合物高分子材料的交聯(lián)改性。當(dāng)孔隙率大于90%,會有較大的有利于細(xì)胞粘附的特定表面。均勻分布和相互連通的孔隙結(jié)構(gòu),允許細(xì)胞在支架材料里形成網(wǎng)絡(luò)分布,細(xì)胞因子和營養(yǎng)也能夠遍布整個支架材料內(nèi)部。制備的P(MVE-alt-MA)交聯(lián)雞蛋白支架材料細(xì)胞相容性高,有合適的力學(xué)強(qiáng)度、可塑性、合適的3D空間結(jié)構(gòu),能夠支持細(xì)胞持續(xù)生長。這些交聯(lián)的雞蛋白可以提供模擬衰老的細(xì)胞外基質(zhì)所要求的細(xì)胞外力學(xué)微環(huán)境,揭示了衰老和腫瘤進(jìn)程之間的關(guān)系。研究旨在模擬衰老所帶來的微環(huán)境細(xì)胞外基質(zhì)中大孔結(jié)構(gòu)的形成和基質(zhì)硬度的變化,評估了其對卵巢癌SKOV-3細(xì)胞的浸潤和轉(zhuǎn)移的影響。同時提出了針對細(xì)胞外基質(zhì)抗衰老和癌癥治療的一致性,二者并不矛盾,對于腫瘤的臨床預(yù)防治療具有重要的參考價值。
[Abstract]:Ovarian cancer has become a heavy burden on the individual and society, the important reason is that it is very difficult to prevent, or in the early diagnosis of most patients can be cured. The recurrence and metastasis of ovarian cancer is the leading cause of death in women with about 90%. Some research progress on Mechanism of ovarian cancer, but it is still not clear. The difficulty lies in the lack of progress in the overall tumor model. Cancer is closely associated with aging disease. With the aging of the body, the extracellular matrix (ECM) micro environment can significantly promote the malignant transformation of epithelial cells. According to the theory of crosslinking of senescence, proteins and other molecules, such as nucleic acid molecules formed by covalent cross connect the larger, thereby interfering with the function of normal cells. The range of crosslinking reaction involving nucleic acid deoxyribonucleic acid (DNA) collagen fibers and extracellular protein containing chicken. Cell growth to a lot of nutrition and signal components, compatibility, good biocompatibility and low price, easy to obtain. In recent years, foreign research workers will be natural chicken protein respectively for different cells were cultured, and made artificial basement membrane, collagen and other biological materials as well. But the natural chicken protein weak strength, low toughness, poor water resistance, easy dispersion and degradation of extracellular matrix crosslinking can not only simulate the aging, but also can increase the mechanical strength of chicken protein and delay the degradation. The research base by chemical crosslinking different culture of chicken protein, simulation in different hardness gradient, porosity, pore size etc. the microenvironment of the ovarian cancer cell proliferation and differentiation, invasion behavior, tumor cell growth and the formation process of tracking development, to elucidate the formation mechanism of ovarian cancer, tumor drug screening, prevention To lay the foundation and treatment. The contents are summarized as follows: (1) genipin chicken protein preparation and aging simulation of extracellular matrix as a scaffold to ovarian cancer cell 3D. The application of genipin (genipin, GP) as a natural product, containing biological low toxicity, is a protein crosslinking agent as well. This study used the extracellular matrix structure and mechanical strength of different morphologies of genipin crosslinked chicken protein to simulate aging as a scaffold, focusing on morphology and mechanical strength of various changes on the proliferation of ovarian cancer cells and the effects of aggregation behavior. The results show that the hardness of chicken protein cross-linking and porous, can promote ovarian the proliferation of cancer cells and aggregation. Therefore, chicken protein scaffolds cross-linked extracellular cross-linking on aging caused by the development of suitable model for cancer cell proliferation and cancer. In order to prevent the infiltration of cancer cells And transfer, the drug should be the extracellular matrix degeneration can prevent the aging caused by attention as screening objects. (2) explore the mechanism of genipin crosslinked chicken protein aggregation growth and drug resistance of ovarian cancer cell culture model. Through the construction of ovarian cancer cell SKOV-3 3D, micro environment simulation host tumor cell growth and discusses the formation phenotype of ovarian cancer cells, cell aggregation distribution, expression of CD44/ABCG2 molecules, and molecular cell adhesion molecule E-cadherin/N-cadherin expression. In consideration of the effect of the influence of micro environment, that multicellular drug resistance (MCR) stem cell characteristics and the possible link between ovarian cancer (3) glutaraldehyde. Chicken protein preparation and aging simulation of extracellular matrix on the distribution of ovarian cancer cells and aggregation behavior. The effects of glutaraldehyde (glutaraldehyde, GA) is the first. A crosslinking agent for gelatin. It has good water solubility, with two functional groups, the price is relatively low. In addition, it can be completely crosslinked gelatin at low concentrations, can improve the hydrophilicity and mechanical properties. The crosslinking reaction of glutaraldehyde and protein efficient, stable products. Ovarian cancer SKOV-3 cells in 3D culture condition, easy to proliferate and into the group. Through the preparation of different concentrations of glutaraldehyde chicken protein scaffold, changing the mechanical strength and pore size of scaffold materials, simulation of different material microenvironment on the proliferation of ovarian cancer cell differentiation, invasion and metastasis influence behavior, a better understanding of the biological characteristics of the tumor growth, and tumor cell formation, the key process of development. (4) P (MVE-alt-MA) cross-linked chicken protein preparation and aging simulation of extracellular matrix on the proliferation and metastasis of ovarian cancer cells. The effects of methyl vinyl ether maleic acid copolymer (P (alternating MVE-a Lt-MA)) due to non-toxic side effects, can be used for Macroporous Crosslinked biomedical tissue engineering scaffold polymer material modification. When the porosity is greater than 90%, there will be a larger specific surface for cell adhesion. Uniformly distributed and interconnected pore structure, allowing cells to form network distribution in the scaffold, cell factor and also can spread the internal nutrient scaffold. The prepared P (MVE-alt-MA) cross-linking of chicken protein scaffolds biocompatibility, mechanical strength, good plasticity, 3D space structure, to support the continued growth of extracellular matrix cells. These crosslinked chicken proteins can provide the required simulation of aging the extracellular mechanical microenvironment, reveals the relationship between aging and tumor progression. The research aims to simulate the aging of micro pore structure environment in extracellular matrix formation and hard matrix The change of degree has evaluated its effect on the invasion and metastasis of ovarian cancer SKOV-3 cells. Meanwhile, the consistency of anti-aging and cancer treatment for extracellular matrix has been put forward. The two are not contradictory. It has important reference value for clinical prevention and treatment of tumor.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R737.31;R943

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1 郭振超;交聯(lián)型雞蛋白的制備及其在卵巢癌細(xì)胞3D培養(yǎng)中的應(yīng)用[D];東南大學(xué);2017年

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