Objective:To study the relation between conserved motif of sphingosine 1-phosphate receptor type 1(S1P1) and FTY720-induced internalization.Methods:With HA-S1P1(WT)-Myc-EGFP-N1 fusion vector as template,HA-S1P1(R142N)-Myc-EGFP-N1 fusion vector was constructed by overlap PCR.The conserved ERY motif of wild type S1P1 was mutated into ENY.The recombinant vectors were confirmed by sequencing,then they were transfected into HEK293 cells by Polyfect.The transfected HEK293 cells were selected with G418.Cells were incubated for 3,6,12 hours in the absence or presence of 100 nmol/L FTY720,then S1P1 gene expression was analyzed by fluorescence microscopy.Results:Sequencing confirmed HA-S1P1(R142N)-Myc-EGFP-N1 vectors was successfully constructed.S1P1(WT) protein and S1P1(R142N) protein were expressed on the stably transfected-HEK293 cell surface.FTY720 induced S1P1(WT) internalization,but not S1P1(R142N).Conclusion:FTY720-induced S1P1 internalization is related with conserved ERY motif.

Objective:To construct eukaryotic expressing vector of PcDNA3.1-rhGM-CSF and to investigate its effects of suppressing growth and inducing differentiation in K562 cells.Methods:The eukaryotic expressing vector PcDNA3.1-hGM-CSF was constructed by means of PCR and T-A clone techniques as well as directional cloning techniques.It was affirmed by the restriction map and DNA sequence analysis,and then transfected into K562 cells.It was observed that the target gene of the recombinant vector was expressed and exerted in K562 cells 72 h later,RT-PCR was used to affirm the expression of rhGM-CSF in K562 cells;Cell morphological,cell proliferation assay and immunohistochemistry were used to observe the effect of PcDNA3.1-rhGM-CSF on the growth and differentiation of K562 cells.Results:①The recombinant eukaryotic expressing vector PcDNA3.1-GM-CSF was constructed successfully;②Recombinant GM-CSF was expressed in the transfected K562 cells and the transfected K562 cells could differentiate into monocyte and macrophage cells;③ The proliferation of K562 cells was suppressed.Conclusion:The recombinant eukaryotic expression vector PcDNA3.1-rhGM-CSF was constructed successfully;Transfected K562 cell could differentiate into monocyte and macrophage cell.

Objective:To investigate the application value of optical coherence tomography (OCT) and intravascular ultrasound (IVUS) in evaluating flow diverter (FD) apposition and endothelialization in aneurysm animal models, and analyze the effect of incomplete stent apposition (ISA) on aneurysm lumen healing and stent endothelialization.Methods:Lateral common carotid artery aneurysm models in swines were established by surgical method and then FD was implanted. Immediately after surgery, OCT and IVUS were used to evaluate the locations and degrees of ISA, and difference between these 2 methods in evaluating FD apposition was compared. DSA was performed at 12 weeks after surgery to evaluate the aneurysm occlusion (Kamran grading) and stent patency. OCT and IVUS were used again to observe the stent endothelial situation; by comparing with histopathologic results, effect of ISA on aneurysm healing and stent endothelialization was analyzed.Results:Lateral common carotid artery aneurysm models in 6 swines were established, and 6 Tubridge FDs were successfully implanted. Compared with IVUS (3 stents, 4 locus), OCT could detect more ISA (6 stents, 14 locus); and the vascular diameter change area (7 locus), aneurysm neck area (4 locus) and the head and tail of FD (3 locus) were the main sites of FD malapposition; average distance between stent wire and vessel wall was (560.14±101.48) μm. At 12 weeks after surgery, DSA showed that 1 patient had a little residual contrast agent at the aneurysm neck (Kamran grading 3), and the remaining 5 had complete aneurysm occlusion (Kamran grading 4). One FD had moderate lumen stenosis, and the other 5 FDs had lumen patency. OCT indicated mostly disappeared acute ISA; ISA proportion decreased to 21.4 % (3/14), including 2 in the aneurysm neck and 1 in the partial stent. Histopathological results showed bare stent woven silk, without obvious endothelial coverage; in one FD with luminal stenosis, intimal hyperplasia was mainly composed of vascular smooth muscle cells.Conclusion:In carotid artery aneurysm model with FD implantation, OCT can detect more ISA than IVUS; most acute ISA have good outcome at 12 th week of follow-up, while severe ISA can cause delayed FD endothelialization and delayed aneurysm occlusion.