切应力对内皮祖细胞t-PA基因表达和小径人工血管移植通畅率的影响

Effect of Shear Stress on t-PA mRNA Expression in Human Endothelial Progenitor Cells and Transplantation Patency of Small Diameter Artificial Vessels

【目的】研究切应力对内皮祖细胞组织纤溶酶原激活物(tissue-type plasminogen activator,t-PA)基因表达和种植内皮祖细胞小径聚氨酯人工血管移植通畅率的影响。【方法】诱导健康成人外周血的单个核细胞分化成为内皮祖细胞。种植内皮祖细胞到小径人工血管表面后,分为静态组、低切应力组(5dyn/cm2)、中切应力组(15dyn/cm2)和高切应力组(25dyn/cm2)4种不同处理组,用酶联免疫吸附法测定不同时间点培养液中t-PA的水平,并用荧光定量RT-PCR分析内皮祖细胞t-PA的基因表达。19只家犬随机分为2组,实验组10只用中切应力处理后内皮祖细胞种植的小径聚氨酯人工血管移植犬颈总动脉,对照组9只用未经切应力处理内皮祖细胞种植的人工血管移植。移植术后2个月,比较2组小径人工血管的移植通畅率。【结果】外周血单个核细胞分化成为内皮祖细胞,倒置荧光显微镜下呈典形的"纺锤样"梭形细胞,ac-LDL吞噬及lectin抗体荧光标记双阳性,FLK-1和VWF免疫荧光抗体染色均为阳性。流体切应力增加内皮祖细胞t-PA的产生,并上调内皮祖细胞t-PA的基因表达。行移植术后2个月,实验组10只人工血管均获通畅,对照组通畅率为55.6%。【结论】切应力处理可上调内皮祖细胞t-PA的基因表达,并改善内皮祖细胞种植后小径人工血管的移植通畅率,这为解决临床小径人工血管通畅率低提供了新的策略。

[Objective] The study was to investigate the effect of shear stress on tissue-type plasminogen activator （t-PA） mRNA expression in human endothelial progenitor cells （EPCs） and transplantation patency of endothelial progenitor cells （EPCs）-lined small diameter polyurethane artificial vessels. [ Methods ] The peripheral blood mononuclear cells of healthy adult were induced into EPCs. Then EPCs were seeded on the small diameter artificial vessels, and then divided into four different experimental groups including stationary group, low-flow shear stress greup（5 dyn/cm^2）, medium-flow shear stress group （15 dyn/cm^2）and high-flow shear stress greup（25 dyn/cm^2）. The levels of t-PA from EPC culture medium were measured by enzyme-linked immunesorbent assay（ELISA）. Furthermore, the t-PA mRNA expression in human EPCs was analyzed by quantitative reverse transcriptase real- time polymerase chain reaction（PCR）. Nineteen domestic dogs were randomly divided into two groups. The canine carotid arteries of experiment group were transplanted with shear stress loaded EPC-fined small diameter polyurethane artificial vessels （n=10）, but those of control group were transplanted with non-shear stress loaded EPC-lined ones （n=9）. The transplantation patency of small diameter polyurethane artificial vessels in two groups was observed after the two months of operation. [Results] The peripheral blood mononuclear cells differentiated into EPCs. They presented typical ＂spindle-shaped＂ appearance, and were positively labeled by fluorescent acetylated- LDL, lectin, FLK-1, and VWF. Shear stress enhanced production of the t-PA of EPCs, and further upregulated the t-PA mRNA expression in human EPCs. After two months of the operation, the patency rate of small diameter artificial vessels of experiment group showed 100.0% ,and that of control group showed 55.6%. [Conclusions] Shear stress treatment contributes to upregulation of t-PA mRNA expression in human endothelial progenitor cells and improvement in transplantation patency of small diameter artificial vessels, which provides a novel strategy for clinical small diameter artificial vessels transplantation.