三七总皂苷对视神经节细胞谷氨酸兴奋毒性的保护作用

发布时间：2018-03-06 09:12

本文选题：三七总皂苷　切入点：视网膜神经节细胞　出处：《南华大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的观察三七总皂苷（total panax notoginseng saponins，tPNS）对谷氨酸（glutamate，Glu）所致的视神经节细胞兴奋毒性的保护作用，并初步探讨其作用机制，从而为其用于青光眼的治疗提供理论依据。方法体外培养视神经节细胞系RGC-5，根据分组给予不同的处理。其中空白对照组加入等量培养基，Glu兴奋模型组加入1mM Glu刺激24h，，阳性对照组在模型组的基础上加入10μM MK-801，观察组在Glu基础上加入100～500μg/mltPNS。24h后，通过钙黄绿素-乙酰甲酯染色法观察RGC-5的活性改变情况，流式细胞术检测RGC-5细胞凋亡。同时，采用RT-PCR检测细胞处理后Thy-1的表达情况，激光共聚焦法检测细胞内Ca~(2+)的水平，Western blot检测caspase-3表达，硝酸还原酶法检测一氧化氮(nitric oxide，NO)的产生情况。结果 1. Calcein-AM染色结果显示， Glu作用RGC-5细胞24h后，使其活细胞数明显降低（P0.01），而同时加入100～500μg/ml tPNS共孵育，能以剂量依赖性方式增加活细胞数。当tPNS浓度为500μg/ml时，RGC-5活细胞数比Glu组增加了112.3%； 2. RT-PCR结果显示，Glu处理能使Thy-1mRNA水平降低。tPNS和MK-801干预后，Thy-1的mRNA表达水平较Glu组明显上调，且tPNS和Thy-1的表达呈一定的剂量依赖性； 3.流式细胞术检测结果显示，与对照组相比，Glu能明显诱导RGC-5细胞，100～500μg/ml tPNS以及MK-801干预后凋亡细胞减少，呈一定的剂量-效应关系； 4.正常对照组几乎未检测到caspase-3的活性亚基（17-kDa），Glu处理后，17-kDa活性亚基明显增多。不同浓度tPNS处理后，caspase-3活性亚基量显著降低； 5. RGC-5细胞经Glu处理24h后，细胞内Ca~(2+)浓度显著增高。当给予tPNS处理后，胞内Ca~(2+)浓度随着tPNS剂量的增高而降低； 6. Glu组NO含量明显高于阴性对照组(P＜0.01)；tPNS处理后，NO含量随剂量的增加而降低。当tPNS浓度为500μg/ml时，NO含量达（0.9241±0.025）μM，与Glu组相比， P0.05。结论 1．tPNS有效保护Glu兴奋所致的细胞凋亡； 2．tPNS对Glu兴奋毒性的细胞保护作用可能是通过抑制胞内Ca~(2+)浓度、NO的产生以及降低caspase-3的活性而实现的。
[Abstract]:Purpose. To observe the protective effect of total panax notoginseng saponinstPNSs on the excitotoxicity of optic ganglion cells induced by glutamate glutamate, and to explore the mechanism of its action, so as to provide a theoretical basis for the treatment of glaucoma. Method. The optic ganglion cell line RGC-5 was cultured in vitro and treated with different groups. The blank control group was stimulated with 1 mm Glu for 24 h, and the positive control group was treated with 10 渭 M MK-801 on the basis of the model group. The observation group added 100 渭 g / ml PNS.100 渭 g / ml on the basis of Glu for 24 hours. The changes of RGC-5 activity, apoptosis of RGC-5 cells were detected by flow cytometry, and the expression of Thy-1 was detected by RT-PCR. The level of Ca~(2 in cells was detected by laser confocal method. The expression of caspase-3 was detected by Western blot, and the production of nitric oxide nitric oxide (no) was detected by nitrate reductase method. Results. 1. The results of Calcein-AM staining showed that the number of living cells of RGC-5 cells treated with Glu for 24 hours was significantly decreased, and the number of living cells was increased in a dose-dependent manner by adding 100 渭 g / ml tPNS. When the concentration of tPNS was 500 渭 g / ml, the number of living cells of RGC-5 was increased by 112.3than that of Glu group. 2. The results of RT-PCR showed that the level of Thy-1mRNA decreased. TPNS and MK-801 increased the expression of Thy-1 mRNA compared with Glu group, and the expression of tPNS and Thy-1 was dose-dependent. 3. The results of flow cytometry showed that Glu could induce the apoptosis of RGC-5 cells in a dose-dependent manner after the intervention of MK-801 and 100 渭 g / ml tPNS in a dose-dependent manner. 4. In the normal control group, the activity subunit of caspase-3 was almost not detected. The activity subunit of 17-kDa was significantly increased after treatment with tPNS, but the activity subunit of caspase-3 was significantly decreased after treatment with different concentrations of tPNS. 5. After Glu treatment for 24 h, the intracellular Ca~(2) concentration of RGC-5 cells increased significantly, and when treated with tPNS, the intracellular Ca~(2) concentration decreased with the increase of tPNS dose. 6. The content of no in Glu group was significantly higher than that in the negative control group (P < 0.01). The content of no in Glu group decreased with the increase of the dose. When the concentration of tPNS was 500 渭 g / ml, the content of no was 0.9241 卤0.025 渭 M, compared with that of Glu group, P 0.05. Conclusion. 1. TPNS could effectively protect the apoptosis induced by Glu stimulation. 2. The cytoprotective effect of tPNS on Glu excitotoxicity may be achieved by inhibiting the production of nitric oxide (no) at intracellular Ca~(2) and decreasing the activity of caspase-3.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R775

【参考文献】