辐照的血管内皮对造血干细胞迁徙的影响

Study on the relation among protein kinase C activity,adhesion molecules expression of irradiated vascular endothelial cells and transmigration of haemopoietic stem cells

目的通过对小剂量γ射线辐照的血管内皮细胞的蛋白激酶C(PKC)活性、黏附分子表达及造血干细胞跨内皮迁徙率的测定,研究辐照的血管内皮对造血干细胞跨内皮迁徙能力的影响。方法1～5Gyγ射线辐照人脐静脉内皮细胞(HUVECs);用[γ32P]ATP掺入外源性底物测定PKC活性;用ELISA法测定黏附分子VCAM1、PECAM1、CD44的表达;用双室培养法测定造血干细胞跨HUVECs的迁徙能力。结果3Gy以下γ射线辐照后的HUVECs膜PKC活性逐渐升高,浆PKC活性逐渐降低,尤以2、3Gy辐照的HUVECs变化最为显著(P<0.01),而高于3Gy辐照的HUVECs的膜、浆PKC活性呈下降趋势。3Gy以下辐照的HUVECs的VCAM1、PECAM1、CD44的表达量也显著升高(P<0.01)。造血干细胞通过γ射线辐照后的HUVECs的数量显著增加(P<0.01),但加入针对上述黏附分子的单克隆抗体后,能显著降低造血干细胞跨内皮能力。PKC活性与VCAM1、PECAM1、CD44的表达呈正相关关系。结论适当小剂量的γ射线辐照血管内皮细胞,能活化其PKC、增加PKC活性,并进而上调黏附分子VCAM1、PECAM1、CD44的表达,有利于造血干细胞的跨内皮迁徙。

Objective To study the relation among protein kinase C （PKC）, adhesion molecules expression of vascular endothelial cells irradiated by small dose of γ-rays and transmigration of haemopoietic stem cells. Methods Human umbilical vein endothelial cells （HUVECs） were irradiated with 1-5 Gy γ-rays.The PKC activity of irradiated cells was measured by the incorporation of [γ-^32P] ATP into exogenous substrate. The expression of adhesion molecules VCAM-1, PECAM-1, CD44 in irradiated HUVECs was detected by ELISA and transmigration of CD34 ^＋ haemopoietic stem cells across the irradiated HUVECs was determined by the transwell migration assay. Results PKC in HUVECs was activated by less than 3 Gy γ-irradiation, resulting in membranous PKC activity increasing and cytosolic PKC activity decreasing gradually. The dominant change occurred in HUVECs irradiated by 2-3 Gy γ-rays （ P 〈 0.01 ）. There was a decreasing trend of membranous and cytosolic activity PKC in HUVECs irradiated by more than 3 Gy γ-rays. The expressions of VACM-1 ,PECAM-1 and CD44 in HUVECs irradiated by less than 3 Gy γ-rays were significantly increased compared with the results of the control group（ P 〈 0.01 ）.The numbers of CD34 ^＋ haemopoietic stem cells migrating across the irradiated HUVECs were much higher than those in the control group （ P 〈 0.01 ） ,but monoclonal antibodies to VACM-1 ,PEACM- 1 ,CD44 could depress the transmigration activity. There was a positive relation beween the PKC activity and the expression of VCAM-1, PECAM-1 and CD44 respectively. Conclusions Optimal small dose of γ-rays could activate PKC, enhance PKC activity of irradiated vascular endothelial cells, and up-regulate the adhesion molecules expression of VACM-1, PECAM-1 and CD44, resulting in promoting the transendothelial migration of haemopoietic stem cells.