本文選題：三七總皂苷　切入點：視網(wǎng)膜神經(jīng)節(jié)細(xì)胞　出處：《南華大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 觀察三七總皂苷（total panax notoginseng saponins，tPNS）對谷氨酸（glutamate，Glu）所致的視神經(jīng)節(jié)細(xì)胞興奮毒性的保護(hù)作用，并初步探討其作用機(jī)制，從而為其用于青光眼的治療提供理論依據(jù)。 方法 體外培養(yǎng)視神經(jīng)節(jié)細(xì)胞系RGC-5，根據(jù)分組給予不同的處理。其中空白對照組加入等量培養(yǎng)基，Glu興奮模型組加入1mM Glu刺激24h，，陽性對照組在模型組的基礎(chǔ)上加入10μM MK-801，觀察組在Glu基礎(chǔ)上加入100～500μg/mltPNS。24h后，通過鈣黃綠素-乙酰甲酯染色法觀察RGC-5的活性改變情況，流式細(xì)胞術(shù)檢測RGC-5細(xì)胞凋亡。同時，采用RT-PCR檢測細(xì)胞處理后Thy-1的表達(dá)情況，激光共聚焦法檢測細(xì)胞內(nèi)Ca~(2+)的水平，Western blot檢測caspase-3表達(dá)，硝酸還原酶法檢測一氧化氮(nitric oxide，NO)的產(chǎn)生情況。 結(jié)果 1. Calcein-AM染色結(jié)果顯示， Glu作用RGC-5細(xì)胞24h后，使其活細(xì)胞數(shù)明顯降低（P0.01），而同時加入100～500μg/ml tPNS共孵育，能以劑量依賴性方式增加活細(xì)胞數(shù)。當(dāng)tPNS濃度為500μg/ml時，RGC-5活細(xì)胞數(shù)比Glu組增加了112.3%； 2. RT-PCR結(jié)果顯示，Glu處理能使Thy-1mRNA水平降低。tPNS和MK-801干預(yù)后，Thy-1的mRNA表達(dá)水平較Glu組明顯上調(diào)，且tPNS和Thy-1的表達(dá)呈一定的劑量依賴性； 3.流式細(xì)胞術(shù)檢測結(jié)果顯示，與對照組相比，Glu能明顯誘導(dǎo)RGC-5細(xì)胞，100～500μg/ml tPNS以及MK-801干預(yù)后凋亡細(xì)胞減少，呈一定的劑量-效應(yīng)關(guān)系； 4.正常對照組幾乎未檢測到caspase-3的活性亞基（17-kDa），Glu處理后，17-kDa活性亞基明顯增多。不同濃度tPNS處理后，caspase-3活性亞基量顯著降低； 5. RGC-5細(xì)胞經(jīng)Glu處理24h后，細(xì)胞內(nèi)Ca~(2+)濃度顯著增高。當(dāng)給予tPNS處理后，胞內(nèi)Ca~(2+)濃度隨著tPNS劑量的增高而降低； 6. Glu組NO含量明顯高于陰性對照組(P＜0.01)；tPNS處理后，NO含量隨劑量的增加而降低。當(dāng)tPNS濃度為500μg/ml時，NO含量達(dá)（0.9241±0.025）μM，與Glu組相比， P0.05。 結(jié)論 1．tPNS有效保護(hù)Glu興奮所致的細(xì)胞凋亡； 2．tPNS對Glu興奮毒性的細(xì)胞保護(hù)作用可能是通過抑制胞內(nèi)Ca~(2+)濃度、NO的產(chǎn)生以及降低caspase-3的活性而實現(xiàn)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 鹵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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R775

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