琼脂糖水凝胶多孔阵列在构建体外肺癌仿生系统的应用

  肺癌具有高发病率和高病死率，在人类健康问题中较为突出。现代医疗技术有段提供许多治疗方法包括手术、放疗、化疗、靶向治疗等。但是临床上可供选择的化疗药物与靶向药物种类繁多，与病人可承受的用药风险形成一对矛盾，所以需要开发适合的体外肿瘤仿生模型用于药物筛选。

  本研究通过三方面的条件进行调控，对仿生培养的条件组合进行探索，包括营养条件：10%FBS和5%血小板裂解液（hPL）、氧气条件：正常氧气含量和2%氧气含量、二维培养和三维培养。在三维培养中利用1%琼脂糖溶液制备三维多空阵列结构的多孔阵列，实现三维培养和软性基材两方面的仿生。

  随后探索出了在不同条件组合中基因表达的差异，蛋白标志物表达的差异。探索体外构建非小细胞肺癌细胞球用于药物筛选的最佳条件。证明了5% hPL培养基、2%低氧条件的细胞球培养条件下可以促使A549细胞中更高比例的细胞表达出癌症干细胞的特征蛋白，其中包括CD133、CD44、EpCAM；并且在进一步的顺铂、紫杉醇、厄洛替尼和长春瑞滨的药物抗性测试中证实了该培养条件下的细胞具有更高的药物抗性，与体内肺腺癌实体瘤中细胞的异质性和存在药物抗性相类似，并进行免疫缺陷小鼠体内成瘤性实验、传代性实验和肿瘤微环境pH变化实验。未来将改进动物成瘤实验，引入多细胞共培养体系，最终用于肿瘤类器官体外培养，进而用于新药开发、高通量病人药物筛选。

    Lung cancer is a prominent part of human health problems because of its high incidence rate and high mortality. Modern medical technology provides many treatment methods, including surgery, radiotherapy, chemotherapy, targeted therapy and so on. However, there are many kinds of chemotherapeutic drugs and targeted drugs that can be selected clinically, which forms a pair of contradictions with the drug risk that patients can bear.

         In this study, the combination of conditions for bionic culture was explored through the regulation of three aspects, including nutritional conditions: 10%FBS and 5% platelet lysate (hPL), oxygen conditions: normal oxygen content and 2% oxygen content, two-dimensional culture and three-dimensional culture. In three-dimensional culture, 1% agarose solution was used to prepare 3D multi-space array microporous plates to realize the bionics of 3D culture and soft substrate.

         Subsequently, differences in gene expression and protein marker expression were explored in different combinations of conditions. To explore the optimal conditions of cell spheres of non-small cell lung cancer for drug screening in vitro.It is proved that the condition of 5% hPL medium and 2% hypoxia for tumor spheroid can promote a higher proportion of A549 cells to express the characteristic protein of cancer stem cells, and the further drug resistance test confirmed that the cells under this culture condition have higher drug resistance, which is similar to the heterogeneity and drug resistance of cells in lung adenocarcinoma solid tumors in vivo. This research result can be used in the development of new drugs and the culture and amplification of patients before drug screening in the future.

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