表达PEDF的人胎盘间充质干细胞抑制人脐静脉内皮细胞增殖和迁移

PEDF expressed by human placenta mesenchymal stem cells inhibits human umbilical vein endothelial cell proliferation and migration

目的探讨携带色素上皮衍生因子(pigment epithelium-derived factor,PEDF)基因的腺病毒转染胎盘间充质干细胞(PMSCs)后对人脐静脉内皮细胞增殖和迁移的抑制作用。方法取足月产胎儿的胎盘组织,分离、培养PMSCs,用流式细胞术鉴定其表面标记。用携带PEDF基因的腺病毒(Ad-PEDF)转染PMSCs,并用Western blot和ELISA检测转染了Ad-PEDF的PMSCs细胞(PMSCs-PEDF)培养上清中PEDF的表达。MTT测定PMSCs-PEDF表达的PEDF对HUVECs增殖的影响。Transwell实验检测PMSCs-PEDF所表达的PEDF蛋白对HUVECs迁移的抑制作用。结果用流式细胞术对分离的PMSCs进行免疫表型分析,结果显示,CD44、CD73、CD90、CD105表达呈阳性,而CD34、CD45表达呈阴性。Western blot结果显示,重组腺病毒成功将PEDF基因传送至PMSCs细胞内并产生分泌性的PEDF蛋白。ELISA结果显示PMSCs-PEDF可并分泌高水平的PEDF至培养基中[(65.2±4.9)ng/ml]。MTT结果显示,PMSCs-PEDF培养基上清在稀释比例为1∶2时对HUVECs抑制率为56.3%±8.7%,随着稀释比例的增加抑制率逐渐降低。而PMSCs及PMSCs-null的培养基上清则对HUVECs抑制作用很弱。Transwell实验结果显示,与PMSCs组(208.8±14.7)及PMSCs-null组(199.0±20.7)相比,PMSCs-PEDF组(79.4±14.5)可显著抑制HUVECs迁移(均P<0.05)。结论 PMSCs-PEDF可在体外高效表达PEDF蛋白,并抑制HUVECs细胞的增殖和迁移。

Objective To explore the inhibitive effect of human placenta mesenchymal stem cells（ PMSCs） transfected by recombinant adenoviruses loaded with pigment epithelium-derived factor（ PEDF） gene on the human umbilical vein endothelial cell（ HUVECs） proliferation and migration. Methods PMSCs were isolated from full-term pregnancy human placenta by the method of enzyme digestion.The cells were cultured in vitro. The phenotype characteristics of PMSCs were analyzed by flow cytometry. The PMSCs were transfected with recombinant adenovirus-loaded PEDF gene（ Ad-PEDF）,and then the expression of PEDF was detected by Western blot and ELISA. MTT assay was performed to observe the inhibitive effect of PMSCs-PEDF on HUVECs proliferation,and Transwell assay was used to determine the inhibitive effect of PEDF expressed by Ad-PEDF-infected PMSCs（ PMSCs-PEDF） on HUVECs. Results Flow cytometry showed that isolated PMSCs exhibited classical immunophenotype of MSCs,including positive CD44,CD73,CD90,CD105,but negative CD34,CD45. Western blot results showed that recombinant adenovirus successfully transfered the PEDF gene into PMSCs and produced secretory PEDF protein. ELISA results showed that PMSCs-PEDF secreted high level of PEDF into the medium[（65. 2 ±4. 9） ng / ml]. MTT assay showed that PMSCs-PEDF inhibited HUVEC proliferation by 56. 3% ± 8. 7% at a 1 ∶ 2 dilution concentration,and the inhibitory rate decreased with the increase of dilution concentration in a dose-dependent manner. The medium from PMSCs or PMSCs-null had very weak inhibitory effect on the proliferation of HUVECs. The transwell assay demonstrated that PMSCs-PEDF significantly reduced HUVECs migration compared with PMSCs or PMSCs-null（79. 4 ± 14. 5 vs 208. 8 ± 14. 7,199. 0 ± 20. 7,P〈0. 05）.Conclusion PEDF is highly expressed by PMSCs-PEDF and can inhibit HUVECs proliferation and migration.