【作者】 曹亮；

【导师】 蔡冬青；

【作者基本信息】 暨南大学 ， 生物医学工程， 2009， 硕士

【摘要】 目的：心脏微血管内皮细胞(CMECs)的增殖和移行在心肌梗塞后血管新生过程中扮演重要的角色。脑源性神经营养因子(BDNF)能维持内皮细胞的存活,是一种重要的血管再生因子。BDNF对CMECs功能的作用机制特别是促血管新生的机理目前所知甚少。因此本研究对BDNF在CMECs增殖、移行、衰老和体内促血管新生的作用进行研究。方法与结果：(1)使用RT-PCR和荧光免疫组化证明：经分离和培养的CMECs稳定表达内皮细胞标记物CD31,并且能摄入Dil-LDL;(2)使用核型分析证明：体外培养CMECs至第42代时细胞核型仍维持正常；(3)使用生长曲线结合流式细胞技术证明：经BDNF处理的CMECs倍增时间减少,处于分裂期的细胞数量增加；(4)经不同浓度BDNF处理的CMECs均促进细胞移行,并与剂量相关性,最佳作用剂量为70ng/ml;(5)与衰老相关的beta半乳糖苷酶染色证明：P6、P9、P14、P22和P45的阳性率分别为36%、48%、75%、83%和94%,β-gal阳性细胞随着细胞传代代数的增加而增加；(6)细胞的端粒长度分析证明：P8和P44端粒长度分别为17.2kb和11.9kb,随着细胞代数的增加而缩短,平均每传代一次下降147bp；(7)随着细胞传代代数的增加,细胞倍增时间增加,处于细胞分裂期的细胞比率下降,细胞移行能力降低；(8)经BDNF处理人源性内皮细胞株,人脐静脉内皮细胞株(HUVEC)和人内皮细胞株(EC304),细胞倍增时间减少,处于细胞分裂期的细胞比率上升,细胞移行能力增强；(9)体内直接心肌注射BDNF两天后,BDNF处理组无明显促进血管密度增加；(10)使用大鼠心肌梗塞模型,于结扎-LAD30min后向心肌注射BDNF,14天后注射BDNF实验组梗塞区和边缘区的新生血管密度增加,但梗死面积无明显减少。结论：(1) CMECs存在复制性衰老,并且随着代数的增加,细胞的增殖能力和细胞移行能力下降；(2) BDNF促进CMECs的增殖；(3) BDNF促进CMECs的移行,且呈浓度依赖；(4) BDNF体外同样促进人源性内皮细胞株,人脐静脉内皮细胞株(HUVEC)和人内皮细胞株(EC304)的增殖和移行；(5)正常状态下,心肌内注射BDNF无明显诱导血管新生的作用；(6)心肌梗死后于心肌注射BDNF能诱导梗死区域和边缘区域血管新生,但对梗死面积无明显减少的作用。更多还原

【Abstract】 Objectives:Cardiac microvascular endothelial cells (CMECs) play a key role in cardiac angiogenesis upon the MI. Brain derived neurotrophic factor (BDNF) promotes endothelial cell survival and induces angiogenesis in ischemic tissues. The mechanism regarding to BDNF-induced angiogenesis in CMECs is still unknown. The present study is therefore design to investigate the effects of BDNF in proliferation and migration of CMECs.Methods and Results:The RT-PCR revealed that the isolated cells (CMECs) expressed CD31, while the immunofluorescent staining for CD31 confirmed that more than 95% of the isolated CMECs were CD31 staining positive. Dil-LDL assay demonstrated that more than 95% of the isolated CMECs were Dil-LDL positive. The karyotype of the isolated CMECs remained normal at the passage of 42. BDNF could increase proliferation of CMECs according to the results from growth curve, cell doubling time and flow cytometry. In addition, BDNF could promote the migration of CMECs. The effect was dosage dependent. To investigate the replicative senescence of CMECs, senescence associatedβ-galactosidase (SA-β-gal) staining was applied. The results revealed that SA-β-gal positive CMECs in P6, P9, P14, P22 and P45 was 36%,48%, 75%,83% and 94% respectively. Further more, passage-related increase of SA-β-gal positive cells was found in consecutive culture of CMECs. Telomere length assay also documented that the telomere length was 17.2kb in passage 8, while it was 11.9kb in P44. The rate of shorten of telomere was 147bp per passage. It was also found that BDNF could increase the proliferation and migration of HUVEC and EC304 cell lines. It was found that BDNF could not induce angiogenesis by myocardium injection in physical situation. While BDNF treatment after MI was able to increase angiogenesis in infarcted site and border site. However, there is no significant difference in infarcted area between BDNF treated group and PBS control.Conclusions:(1) CMECs were experiencing replicative senescence with the passage-related decrease of proliferation and migration of CMECs. (2) BDNF could increase the proliferation of CMECs. (3) BDNF could promote the migration of CMECs. The effect was dosage dependent. (4) BDNF could promote the proliferation and migration of HUVEC and EC304 cell lines. (5) BDNF could not induce angiogenesis by myocardium injection in physical situation. (6) BDNF treatment after MI was able to increase angiogenesis in infarcted site and border site. However, there is no statistical significant in infarcted area difference between BDNF treated group and PBS control.更多还原