荧光三维共培养研究整合素αvβ3受体拮抗剂对非小细胞肺癌辐射敏感性的影响及机制

Lung cancer remains one of the major causes of cancer-related deaths worldwide, and among all of the diseases, non-small-cell lung cancer (NSCLC) accounts for more than 85% and most of them are diagnosed at an advanced stage. Radiotherapy, alone or in combination with surgery or chemotherapy, plays a critical role in treatment of NSCLC. However, the therapeutic outcomes are not fully satisfactory in many cases. With unexpected radiation responses during radiotherapy, (intrinsic/acquired) radioresistance is considered as a main factor which restricts the efficacy of radiation treatment for NSCLC. Reversing the radioresistance is the key to enhance radiotherapy effect in NSCLC. The purpose of the project is to study the enhanced radiation sensitivity in NSCLC by antagonist of integrin αVβ3. Molecular biology techniques such as fluorescent three-dimensional culture, flow cytometry, microarray, quantitative PCR, immunoblotting techniques and immunohistochemical techniques are applied in the research. Firstly, tumor cells and microvascular endothelial cells are transfected with different fluorescence, respectively. Then tumor cells and microvascular endothelial cells are fluorescent three-dimensional (3D) co-cultured on the laminin-rich extracellular matrix (lrECM), which mimics the crucial tumor growth microenvironment in vivo. The angiogenesis, vascular mimicry and autophagy, pre and post ionizing radiation, are determined in both radiosensitive (H460) and radioresistance (A549) tumor cells growth in vitro. The different expression levels of integrin αvβ3 between H460 and A549 are compared. All the data are summarized to evaluate the effect and mechanisms of integrin αvβ3 induced NSCLC radiation tolerance. Collected clinical pathological biopsy samples are used to verify the role. Based on these results, fluorescent three-dimensional co-culture tumor spheroids model and tumor-bearing mouse models are used to verify the enhanced radiotherapy anti-tumor effect and mechanism in non-small cell lung cancer by integrin αvβ3 antagonists RGD2. The anti-angiogenesis, against vascular mimicry, as well as induction of autophagy roles by RGD2 will be assessed to clarify the mechanism.

提高肿瘤组织的辐射敏感性是非小细胞肺癌（NSCLC）放射治疗的研究热点。我们预实验发现整合素αvβ3受体异常表达与NSCLC辐射抵抗有关。本研究以整合素αvβ3受体为靶点，利用荧光三维共培养技术，从经典的血管新生，以及全新的血管生成拟态及肿瘤细胞自噬等三个角度在细胞水平探讨高强度表达的αvβ3受体在NSCLC辐射抵抗形成中的作用及机理；通过比较不同放疗效果NSCLC患者放疗前，以及放疗抵抗的NSCLC患者放疗前后病理标本中整合素αvβ3受体的表达情况，从临床角度验证。选用新颖的亲和力更强的精氨酸-甘氨酸-天冬氨酸环肽二聚体（RGD2）作为整合素αvβ3受体拮抗剂，通过荧光三维共培养肿瘤球体模型体外实验和小鼠移植瘤模型体内实验，研究整合素αvβ3拮抗剂RGD2对逆转辐射抵抗，增强NSCLC辐射敏感性的作用及机制，为提高临床NSCLC放疗疗效提供新的思路和解决办法。

本研究以整合素αvβ3受体为靶点，利用荧光三维共培养技术，从经典的血管新生，以及全新的血管生成拟态及肿瘤细胞自噬等三个角度在细胞水平探讨高强度表达的αvβ3受体在NSCLC辐射抵抗形成中的作用及机理。研究结果表明，整合素αvβ3受体的异常表达与NSCLC辐射抵抗有关；整合素αvβ3受体拮抗剂通过抑制血管新生增强NSCLC的辐射敏感性；整合素αvβ3受体拮抗剂通过抑制血管生成拟态增强NSCLC的辐射敏感性。. 本研究选用新颖的亲和力更强的精氨酸-甘氨酸-天冬氨酸环肽二聚体（RGD2）作为整合素αvβ3受体拮抗剂，通过荧光三维共培养肿瘤球体模型体外实验和小鼠移植瘤模型体内实验，研究整合素αvβ3拮抗剂RGD2对逆转辐射抵抗，增强NSCLC辐射敏感性的作用及机制，结果表明，在0-40uM范围内，单独应用RGD2对A549生长的抑制作用无统计学差异。 2 Gy联合30 uM RGD2用药组与单独应用4Gy照射的G2/M期阻滞效率相当；4 Gy联合30 uM RGD2用药组G2/M期阻滞阻滞效率与单独应用8 Gy照射的G2/M期阻滞效率相当，放疗联合应用αvβ3拮抗剂RGD2，可发挥协同效应，增强NSCLC辐射敏感性。. 99mTc -Galacto-RGD2 A549荷瘤小鼠体内生物分布及显像结果表明，99mTc -Galacto-RGD2可特异性地识别肿瘤细胞表面的αvβ3受体的表达，且该结合特性可被RGD2特异性地阻断。荷瘤小鼠的治疗不同时间点u-SPECT/CT显像结果表明，αvβ3受体为靶点的RGD2可以逆转辐射抵抗，提高肿瘤组织的辐射敏感性。. 综上所述，整合素αvβ3拮抗剂RGD2可增强非小细胞肺癌的辐射敏感性，为提高临床NSCLC放疗疗效提供了新的思路和解决办法。

内容获取失败，请点击重试