基于TRAIL受体热稳定的亲和性定量检测

Affinity Detection and Quantification of Receptors of TRAIL Based on Their Thermostability

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。

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-1and Capan-2 were detected.Boiling treated recombinant protein death receptor 5（DR5）,named DR5-AP,as well as pancreatic cancer cells lines AsPC-1and Capan-2were 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-1and Capan-2could 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-1and Capan-2could be recognized and bound by AP-TRAIL after treated at 100℃,the readings were 2.210±0.393 and 2.027±0.019.ConclusionThe TRAIL receptors are thermostable and this may provide a better diagnosis and prognosis of cancer as well as personalize cancer therapy.