基于TRAIL受体热稳定的亲和性定量检测

T echniques and Methods Affinity Detection and Quantification of Receptors of TRAIL Based on Their Thermostability

中文关键词:  肿瘤治疗 TRAIL受体 热稳定性 AP标签

英文关键词:Cancer therapy Receptors of TRAIL Thermostability AP-tag

中文摘要:

      目的 基于肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand，TRAIL）受体的热稳定性建立其在肿瘤中定量检测的新途径。 方法 检测胰腺癌细胞AsPC-1和Capan-2內源碱性磷酸酶（alkaline phosphatase，AP）的活性和变性温度。利用AP标记的TRAIL（AP-TRAIL）分别孵育转膜后的死亡受体5（death receptor 5，DR5）融合蛋白（DR5-AP）和经100℃沸水蒸煮去除内源AP活性的胰腺癌细胞系AsPC-1和Capan-2，再与AP的底物Reagent S和Reagent A进行颜色反应，进行细胞受体定量检测和细胞原位受体结合试验。 结果 胰腺癌细胞系AsPC-1和Capan-2的内源AP在65 ℃水浴下不能完全失活，阻碍了对TRAIL受体的检测，在沸水蒸煮后其AP活性显著下降，而DR5-AP在沸水中具有热稳定性。AP-TRAIL能够识别并结合高温蒸煮后的胰腺癌细胞AsPC-1和Capan-2表面的受体，AP-TRAIL孵育的读数为2.210±0.393和2.027±0.019 。结论 TRAIL受体具有热稳定的性质，它的发现能够为更好的肿瘤诊断、预测及个性化治疗提供新方法。

英文摘要:

      Objective To develop a way for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors quantification in cancer via its’ thermostability. Methods Endougenous alkaline phosphatase (AP) activity and denaturation temperature of pancreatic cancer cell lines AsPC-1 and Capan-2 were detected. Boiling treated recombinant protein death receptor 5 (DR5) ,named DR5-AP, as well as pancreatic cancer cells lines AsPC-1 and Capan-2 were incubated with AP-tagged TRAIL (AP-TRAIL), and then reacted with Reagent A and Reagent S, the substrate of AP, to quantitive and in site detection of the receptor. Results The endougenous AP activity of pancreatic cancer cells lines AsPC-1 and Capan-2 could not be totally inactivated by incubated at 65 ℃, thus inhibited the detection of TRAIL receptor, but the activity was dramatically decreased after treated with boiling water, whereas the DR5-AP was thermal stable. The surface receptor of AsPC-1 and Capan-2 could be recognized and bound by AP-TRAIL after treated at 100 ℃, the readings were 2.210±0.393 and 2.027±0.019. Conclusion The TRAIL receptors are thermostable and this may provide a better diagnosis and prognosis of cancer as well as personalize cancer therapy.