普萘洛尔及异丙肾上腺素对体外培养婴儿血管瘤内皮细胞影响的研究

发布时间：2018-05-18 06:43

本文选题：婴儿血管瘤 + 普萘洛尔　；参考：《新疆医科大学》2014年博士论文

【摘要】：目的：1）研究普萘洛尔及异丙肾上腺素在体外对婴儿血管瘤内皮细胞生长曲线的影响，从而进一步探索研究β-肾上腺素受体在婴儿血管瘤发病机制中的作用；2）研究普萘洛尔及异丙肾上腺素在体外对人血管内皮细胞生长因子，人血管内皮细胞生长因子-2，人碱性成纤维细胞生长因子；3）研究普萘洛尔及异丙肾上腺素在体外对血管瘤内皮细胞表面β2-肾上腺素能受体表达的影响；4）研究普萘洛尔及异丙肾上腺素在体外对血管瘤内皮细胞凋亡的影响。方法将体外培养的2～3代增殖期婴儿血管瘤内皮细胞分为三大组，分别为空白组、普萘洛尔组及异丙肾上腺素组，其中后两组再分别给予该药物的高、中、低浓度，作用一定时间后采用MTT法分别测定各组细胞的生长曲线。研究两种药物对细胞生长曲线的影响。在上述研究的基础上将培养的第2～3代增殖期婴儿血管瘤内皮细胞分为若干组，每组分别使用含有不同浓度的普萘洛尔和异丙肾上腺素的培养基及空白培养基作用一定时间（24～48小时）后使用ELISA的方法测定细胞培养上清液中人血管内皮细胞生长因子，人血管内皮细胞生长因子-2，人碱性成纤维细胞生长因子的浓度及人β2-肾上腺素能受体的表达并与空白组对照，从而研究两种药物对上述细胞因子及受体的影响。随后将培养的第2～3代血管瘤内皮细胞分为若干组，每组分别使用含有不同浓度的普萘洛尔和异丙肾上腺素的培养基及空白培养基作用一定时间（24～48小时），使用流式细胞仪分别测定两种药物作用前后的血管瘤内皮细胞的凋亡率并与空白组对照，以探讨普萘洛尔和异丙肾上腺素两种药物对该细胞凋亡的影响。结果：一定浓度的普萘洛尔作用于体外培养的血管瘤内皮细胞一定时间后发现，，24～48小时组，0.16%DMSO组及其余三种浓度对于细胞平均吸光度的影响无明显差别（P＞0.05）但在72～96小时其吸光度不全相同，进行两两比较后发现空白组、0.16%DMSO组及低浓度组（10、15ug/ml组）无明显差异，而20ug/ml组与空白组有差异（P＜0.05）。一定浓度的异丙肾上腺素作用于体外培养的血管瘤内皮细胞一定时间后发现，24～72小时内各组的吸光度值无明显差异（P＞0.05），但在96小时时发现，空白组与其余各组含有药物的培养基相比，吸光度值不全相同（P＜0.05），两两比较后发现浓度越高，测得的吸光度值越高（P＜0.05）。同时使用一定浓度的普萘洛尔及异丙肾上腺素作用于体外培养的血管瘤内皮细胞一定时间后，测定细胞培养上清液中人血管内皮细胞生长因子，人血管内皮细胞生长因子-2，人碱性成纤维细胞生长因子的浓度及人β-2肾上腺素能受体的表达，结果发现使用普萘洛尔后人碱性成纤维细胞生长因子及人血管内皮细胞生长因子-2的浓度下降，人血管内皮细胞生长因子在24小时时各组浓度无差异，在作用48小时时有所下降，而人β2-肾上腺素能受体的表达是下降的。一定浓度的异丙肾上腺素作用一定时间后发现其作用与普萘洛尔相反。最后使用流式细胞仪检测两种药物作用于血管瘤内皮细胞后的凋亡率的变化发现，普萘洛尔作用24小时时，高浓度组（20μg/ml）的凋亡率较对照组及低浓度组是明显升高的（P＜0.05）；48小时时中浓度组（10μg/ml）的细胞凋亡率较空白组也是升高的（P＜0.05），而高浓度组（20μg/ml）的凋亡率较其他各浓度组细胞的凋亡率也是升高的（P＜0.05）。异丙肾上腺素作用组与之相反，作用24小时时中高浓度组细胞较空白及低浓度组的细胞凋亡率是下降的，而作用48小时时高浓度组的细胞凋亡率较空白和中低浓度组的细胞凋亡率是下降的。结论：一定浓度普萘洛尔作用于增殖期婴儿血管瘤内皮细胞一定时间后能够抑制细胞生长，而异丙肾上腺素则反之；β-肾上腺素受体在婴儿血管瘤发病中有重要作用。同时一定浓度普萘洛尔作用于体外培养的血管瘤内皮细胞，对人血管内皮细胞生长因子，人血管内皮细胞生长因子-2，人碱性成纤维细胞生长因子的浓度及血管瘤内皮细胞表面β2-肾上腺素能受体的表达是起抑制作用的，而异丙肾上腺素的作用是与之拮抗的。最后普萘洛尔及异丙肾上腺素对于体外培养的血管瘤内皮细胞的凋亡率的影响的作用也是相互拮抗的。因此，β-肾上腺素能受体在血管瘤的发生发展过程中是起到重要作用的，它有可能是通过对相关细胞因子以及对细胞凋亡的影响而起作用的。
[Abstract]:Objective: 1) to study the effect of propranolol and isoproterenol on the growth curve of the endothelial cell of infantile hemangioma in vitro, and to further explore the role of beta adrenergic receptor in the pathogenesis of infantile hemangioma; 2) the study of propranolol and isoproterenol on human vascular endothelial growth factor, human blood in vitro The effect of propranolol and isoproterenol on the expression of beta 2- adrenergic receptor on the surface of hemangioma endothelial cells in vitro; 4) to study the effect of propranolol and isoproterenol on the apoptosis of endothelial cells of hemangioma in vitro. Methods the method will be used in vitro. Methods: the effect of propranolol and isoproterenol on the apoptosis of endothelial cells in vitro of hemangioma was studied. Methods the method will be used in vitro. The cultured endothelial cells of the 2~3 generation proliferative phase of infantile hemangioma were divided into three groups: blank group, propranolol group and isoproterenol group, and the latter two groups were given high, middle and low concentration of the drug respectively. After a certain time, the growth curves of each group were measured by MTT method. The growth curve of two kinds of drugs on cell growth was studied. On the basis of the above study, the endothelial cells of the second to 3 generation proliferative phase of infantile hemangioma were divided into several groups. Each group used the medium of propranolol and isoproterenol with different concentrations and the blank medium for a certain time (24~48 hours) to determine the cell culture supernatant by using ELISA method. Human vascular endothelial growth factor (human vascular endothelial cell growth factor), human vascular endothelial growth factor -2, human basic fibroblast growth factor (bFGF) and human beta 2- adrenergic receptor (adrrenergic receptor) expression were compared with the blank group. The effects of two drugs on the above cytokines and receptors were studied. Then the cultured endothelial cells of second to 3 generations of hemangioma were cultured. The effects of propranolol and isoproterenol on the apoptosis rate of the endothelial cells of hemangioma before and after the two drugs were measured and compared with that of the blank group, and propranolol and isopropane were discussed. The effect of two adrenaline on the apoptosis of the cells. Results: a certain concentration of propranolol was found in the cultured hemangioma endothelial cells for a certain time. The effect of the 24~48 hour group, the 0.16%DMSO group and the other three concentrations on the average absorbance of the cells had no significant difference (P > 0.05), but the absorbance was 72~96 hours at 72~96 hours. The difference was found in the 22 comparison group. There was no significant difference in the group 0.16%DMSO and the low concentration group (10,15ug/ml group), but there was a difference between the group 20ug/ml and the blank group (P < 0.05). The concentration of isoproterenol in the cultured endothelial cells of the hemangioma in vitro was found, and the absorbance value of each group was not obvious within 24~72 hours. The difference (P > 0.05) was found at 96 hours, but at 96 hours, the absorbance value of the blank group was not all the same (P < 0.05). The higher the concentration was, the higher the measured absorbency (P < 0.05). At the same time, a certain concentration of pralanolol and isoproterenol were used in the cultured hemangioma. After a certain time, the human vascular endothelial growth factor (human vascular endothelial growth factor), human vascular endothelial growth factor -2, human basic fibroblast growth factor (bFGF) and human beta -2 adrenergic receptor expression were measured in the cell culture supernatant. The results were found in the use of basic fibroblast growth factor and human vascular endothelium after propranolol. The concentration of cell growth factor -2 decreased, and the concentration of human vascular endothelial cell growth factor was not different at 24 hours, but decreased at 48 hours, but the expression of human beta 2- adrenergic receptor was decreased. The apoptosis rate of two kinds of drugs was detected after the action of two drugs on the endothelial cells of hemangioma. The apoptosis rate of the high concentration group (20 mu) was significantly higher than that of the control group and the low concentration group at 24 hours (P < 0.05), and the apoptosis rate of the medium concentration group (10 u g/ml) at 48 hours was also higher than that in the blank group (P < 0. 5), the apoptosis rate of the high concentration group (20 g/ml) was also higher than that of the other concentration groups (P < 0.05). In contrast to the isoproterenol group, the apoptosis rate of the medium and high concentration group was lower than that in the blank and low concentration group at 24 hours, but the apoptosis rate of the high concentration group was less than that of the high concentration group at 48 hours. The apoptosis rate of the cells in the middle and low concentration groups was decreased. Conclusion: a certain concentration of propranolol can inhibit the growth of the endothelial cells in the proliferating infantile hemangioma after a certain time, while isoproterenol is the opposite; the beta adrenergic receptor plays an important role in the pathogenesis of infantile hemangioma. The endothelial cells of hemangioma used in vitro were inhibited by human vascular endothelial growth factor, human vascular endothelial growth factor -2, human basic fibroblast growth factor and the expression of beta 2- adrenergic receptor on the surface of hemangioma endothelial cells. The effect of propranolol and isoproterenol on the apoptosis rate of endothelial cells in cultured hemangioma is antagonistic to each other. Therefore, the beta adrenergic receptor plays an important role in the development of hemangioma, and it may be through the effects on the cytokine and apoptosis of the cells. It works.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R969.4

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