交联型鸡蛋白的制备及其在卵巢癌细胞3D培养中的应用

发布时间：2017-12-30 17:26

本文关键词：交联型鸡蛋白的制备及其在卵巢癌细胞3D培养中的应用　出处：《东南大学》2017年博士论文　论文类型：学位论文

【摘要】：卵巢癌已成为当前个人及社会的沉重负担,其重要原因是很难预防,或在早期诊断时大部分患者仍可被治愈。卵巢癌的复发和转移是导致大约90%的女性患者死亡的主要原因。关于卵巢癌发病机制的研究有所进展,但仍不明确,困难之处在于缺乏全面的肿瘤进展模型。癌症是与机体的衰老密切关联的疾病。随着机体的衰老,细胞外基质(ECM)微环境可以显著促进上皮细胞的恶性变化。根据衰老的交联理论学说,蛋白质和其它分子,例如核酸等通过共价交叉连接形成更大的分子,从而干扰到正常细胞的功能。交联反应发生的范围涉及核酸脱氧核糖核酸(DNA)和细胞外的胶原纤维成分。鸡蛋白含有细胞生长发育所需要的很多营养和信号成分,有良好的生物相容性,价格低廉,易于获取。近年来,国外的科研工作者将天然鸡蛋白分别用于不同细胞进行培养,取得了和人工基底膜、胶原等生物材料一样好的效果。但天然鸡蛋白强度弱,韧性低、耐水性差,易分散和降解。交联不仅可以模拟衰老的细胞外基质,还可以增加鸡蛋白的力学强度和延缓降解。本研究通过对鸡蛋白培养基采用不同的化学交联,调控模拟体内不同硬度梯度、多孔性、孔径大小等微环境对卵巢癌细胞增殖分化、侵袭行为的影响,跟踪细胞的生长乃至发展形成肿瘤的过程,为阐明卵巢癌的形成机制、筛选肿瘤药物、预防和治疗奠定基础。研究内容概括如下:(1)京尼平交联鸡蛋白的制备及其模拟衰老细胞外基质作为卵巢癌细胞3D培养支架的应用。京尼平(genipin,GP)作为天然产物,含有较低的生物学毒性,是很好的蛋白质交联剂。本研究采用京尼平交联的不同形貌结构和力学强度的鸡蛋白模拟衰老的细胞外基质作为培养支架,重点研究多样化的形貌和力学强度改变对卵巢癌细胞的增殖和聚集行为的影响。研究表明,交联鸡蛋白的硬度和多孔性增加,能够促进卵巢癌细胞的增殖和聚集。因此,交联的鸡蛋白支架是研究衰老导致的细胞外基质交联、癌细胞增殖和癌症的发展的合适模型。为防止癌细胞的浸润和转移,应该关注可以防止衰老导致的细胞外基质退行性变化的药物作为筛选对象。(2)京尼平交联的鸡蛋白对卵巢癌细胞聚集生长和耐药影响的机制初步探索。通过构建卵巢癌SKOV-3细胞的3D培养模型,模拟宿主体内肿瘤细胞生长的微环境,探讨了卵巢癌细胞的表型形成、细胞聚集分布、CD44/ABCG2分子表达、以及细胞黏附分子E-cadherin/N-cadherin的分子表达。在考虑微环境中各影响因素作用的基础上,推测多细胞耐药性(MCR)与卵巢癌肿瘤干细胞特性之间的可能联系。(3)戊二醛交联的鸡蛋白的制备及其模拟衰老细胞外基质对卵巢癌细胞分布和聚集行为的影响。戊二醛(glutaraldehyde,GA)是最先使用于明胶的交联剂。它水溶性好,具有双官能团,价格相对低廉。此外,它在低浓度可以完全交联明胶,可以改善其亲水性和力学性能。戊二醛与蛋白质的交联反应高效、产物稳定。卵巢癌SKOV-3细胞在3D培养条件下,容易增殖聚集、成团。通过制备不同浓度戊二醛交联的鸡蛋白培养支架,改变支架材料的力学强度和孔径大小,模拟不同材料微环境对卵巢癌细胞增殖、分化、浸润和转移行为的影响,更好地明确肿瘤的生物学特性和肿瘤细胞生长、形成、发展的关键过程。(4)P(MVE-alt-MA)交联的鸡蛋白制备及其模拟衰老细胞外基质对卵巢癌细胞增殖和转移的影响。甲基乙烯基醚马来酸交替共聚物(P(MVE-alt-MA))由于无毒副作用,可用于生物医学组织工程中大孔支架聚合物高分子材料的交联改性。当孔隙率大于90%,会有较大的有利于细胞粘附的特定表面。均匀分布和相互连通的孔隙结构,允许细胞在支架材料里形成网络分布,细胞因子和营养也能够遍布整个支架材料内部。制备的P(MVE-alt-MA)交联鸡蛋白支架材料细胞相容性高,有合适的力学强度、可塑性、合适的3D空间结构,能够支持细胞持续生长。这些交联的鸡蛋白可以提供模拟衰老的细胞外基质所要求的细胞外力学微环境,揭示了衰老和肿瘤进程之间的关系。研究旨在模拟衰老所带来的微环境细胞外基质中大孔结构的形成和基质硬度的变化,评估了其对卵巢癌SKOV-3细胞的浸润和转移的影响。同时提出了针对细胞外基质抗衰老和癌症治疗的一致性,二者并不矛盾,对于肿瘤的临床预防治疗具有重要的参考价值。
[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.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.31;R943

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