IGF-1对受损血管内皮细胞的影响及机制研究

发布时间：2018-03-01 14:17

本文关键词： IGF-1 内皮细胞雪旺细胞神经元共培养　出处：《重庆医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：观察胰岛素样生长因子-1 (Insulin-like Growth Factor-1, IGF-1)对受损血管内皮细胞(Vascular Endothelial Cell, VEC)增殖及存活的影响,并对其机制进行初步探讨。建立雪旺细胞、神经元、内皮细胞的共培养模型,为外周神经损伤研究打基础。方法：用不同浓度(10ng/ml、20ng/ml、40ng/ml、80ng/ml、160ng/ml)的IGF-1作用于血管内皮细胞48h后,通过MTT法检测细胞的生长情况。采用划痕实验观察IGF-1对受损内皮细胞增殖的影响,并用PI染色观察细胞情况,ELISA法进行VEGF含量测定。采用组织块贴壁法、机械消化法分别培养雪旺细胞及神经元原代细胞,内皮细胞采用细胞株,并用S-100、β-tubulin-Ⅲ、vWF分别对其进行免疫学鉴定。用NB、DMEM/F-12混合培养基,根据生长周期不同先后将雪旺细胞、神经元、内皮细胞接种在培养皿里,37℃培养7d后进行免疫荧光鉴定。结果：浓度为Ong/ml、20ng/ml、40ng/ml、80ng/ml的IGF-1对血管内皮细胞增殖都有不同程度的促进作用,浓度为40ng/ml、80ng/ml实验组与未加IGF-1的对照组比较,差异有统计学意义(P 0.05),160ng/ml的IGF-1对细胞的增殖作用下降,差异无统计学意义(P 0.05)。取40ng/ml的IGF-1作用于受损血管内皮细胞48h后,细胞增殖速度较对照组明显加快,PI染色显示实验组死亡细胞明显减少,实验组培养液上清VEGF含量较对照组高,差异均有统计学意义(P0.05)。激光共聚焦显微镜下可见雪旺细胞呈长梭形,胞核呈卵圆形或长圆形,“海鱼状”排列生长。神经元胞体较小,从胞体长出许多长而细的突起,细胞间突起可连接成网状。内皮细胞胞体多呈梭形、三角形,团簇存在,胞核清晰,胞浆丰富,细胞生长活跃,成“铺路石”排列生长。结论：MTT法观察细胞增殖程度时发现,10ng/ml、20ng/ml、40ng/ml、80ng/ml的IGF-1对血管内皮细胞的增殖都有不同程度的促进作用(P0.05),160ng/ml的IGF-1对细胞增殖作用下降,差异无统计学意义(P0.05),提示IGF-1在低剂量时对内皮细胞的增殖有促进作用,且表现出一定的量效关系,40ng/ml作用明显,但高剂量(160ng/ml) IGF-1却对细胞增殖有抑制作用,具体机制有待进一步探讨。根据IGF-1细胞增殖研究结果,进一步观察IGF-1对受损内皮细胞存活和增殖的影响。以40ng/ml作为实验组,在倒置显微镜下观察到同时相点实验组划痕空白宽度比对照组明显缩窄；DAPI/PI双标记观察死亡细胞数目,实验组死亡细胞数较对照组少；对其分泌的细胞生长因子VEGF测定,观察到实验组经过IGF-1作用后内皮细胞分泌VEGF明显高于对照组,提示IGF-1可能促进受损内皮细胞的存活、增殖和迁移,以修复内皮细胞的损伤,其机制与其抑制细胞死亡和分泌营养因子VEGF有关,至于该作用具体的信号通路机制尚需以后深入研究。激光共聚焦显微镜下显示雪旺细胞、神经元、内皮细胞模型清晰可见,雪旺细胞为蓝色染色细胞,细胞呈长梭形,胞核呈卵圆形或长圆形,海鱼状排列生长。神经元细胞为绿色染色,胞体较小,从胞体长出许多长而细的突起,细胞间突起可连接成网状。内皮细胞为红色染色细胞,胞体细胞多呈梭形、三角形,胞核清晰,胞浆丰富,细胞生长活跃,成“铺路石”排列生长,表明共培养模型构建成功。
[Abstract]:Objective: To observe the effect of insulin-like growth factor -1 (Insulin-like Growth Factor-1, IGF-1) on the damage of vascular endothelial cells (Vascular Endothelial Cell, VEC) proliferation and survival, and to investigate its possible mechanism. The establishment of Schwann cells, neurons, endothelial cell co culture model, laying the foundation for the study of peripheral nerve injury.
Methods: with different concentrations (10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 160ng/ml) IGF-1 effects on vascular endothelial cells after 48h, the growth of MTT cells detected by scratch test. The effect of IGF-1 on endothelial cell proliferation, and PI staining cells, ELISA method for determination of VEGF. Using tissue explant method, mechanical digestion method cultured Schwann cells and neurons in primary cells, endothelial cells and cell lines by using S-100, -tubulin-, beta III, vWF respectively on the immunological identification. With NB, DMEM/F-12 mixed medium, according to the different growth periods will have Schwann cells, neurons, endothelial cells inoculated in a culture dish, at 37 7d after culture were identified by immunofluorescence.
Results: the concentration of Ong/ml, 20ng/ml, 40ng/ml, 80ng/ml and IGF-1 have different effect on the proliferation of vascular endothelial cells, the concentration of 40ng/ml, compared to control group, 80ng/ml group and without IGF-1, the difference was statistically significant (P 0.05), decreased the proliferation of 160ng/ml IGF-1 cells, no significant difference the significance of 40ng/ml (P 0.05). The effects of IGF-1 on damaged vascular endothelial cells after 48h cell proliferation rate compared with the control group increased significantly, PI staining showed that the experimental group significantly reduced cell death in the experimental group, the supernatant VEGF content was higher than the control group, the differences were statistically significant (P0.05). Laser scanning confocal microscope the Schwann cells were fusiform, nuclei were ovoid or oblong, "fish shape arrangement growth. Neurons were small, growing from the cell bodies of many long and thin neurites, cell processes could be connected into the network. The cells of the skin cell are mostly spindle, triangular, cluster, clear, rich in cytoplasm and active in cell growth, and grow up as "pave stone".
Conclusion: MTT was used to observe the proliferation of 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, IGF-1 on the proliferation of vascular endothelial cells have different degrees of promotion (P0.05), decreased effect of 160ng/ml IGF-1 on cell proliferation, there was no statistically significant difference (P0.05), suggesting that IGF-1 at low doses on endothelial cells the proliferation promoting effect, and showed a dose-response relationship, the 40ng/ml effect is obvious, but the high dose (160ng/ml) IGF-1 has inhibitory effect on cell proliferation, the specific mechanism needs to be further discussed. According to the result of IGF-1 cell proliferation, further observe the effect of IGF-1 on endothelial cell survival and proliferation. Using 40ng/ml as the experimental group in, observed under the inverted microscope at the same time point blank width of the experimental group was shorter than the control group; DAPI/PI double marker to observe the number of dead cells, the number of dead cells in experimental group than in the control The group is less; the secretion of cell growth factor VEGF were observed in the experimental group after VEGF of endothelial cells after IGF-1 treatment was significantly higher than the control group, suggesting that IGF-1 may promote the survival of injured endothelial cells, proliferation and migration, in order to repair the injury of endothelial cells, and its mechanism is related to the inhibition of cell death and the secretion of neurotrophic factor VEGF as for the future, the role of specific signal transduction mechanism remains to be further studied. Laser confocal microscopy showed that Schwann cells, neurons, endothelial cell model is clearly visible, Schwann cells for blue stained cells, cells were fusiform, nuclei were ovoid or oblong, arranged in the form of fish growth. Neurons for green staining. Smaller cell body, growing from the cell bodies of many long and thin neurites, cell protrusions can be connected into the network. Endothelial cells were red stained cells, somatic cells were spindle shaped, triangular, cell The nucleus is clear, the cytoplasm is rich, the cell growth is active, and the "paving stone" is arranged and grown, which indicates that the co culture model is constructed successfully.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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