干细胞条件培养液对RGCs内质网应激的保护作用及机制
发布时间：2018-10-21 08:02
【摘要】：目的： 1.建立大鼠视网膜神经节细胞系(retinal ganglion cell5, R.GC-5)的内质网应激(endoplasmic reticulum stress,ERS)模型。 2.探讨干细胞条件培养液(conditioned medium,CM)对RGC-5的ERS是否具有保护作用。 3.通过检测ERS相关标志物的变化,探讨CM发挥保护作用的可能机制。 方法： 将RGC-5以1×105个/孔或2×105个/孔接种于12孔或6孔培养板内,分为对照组、衣霉素组和治疗组。对照组：空白对照组(ND组)为以完全培养基培养的RGC-5,干细胞条件培养液组(NM组)为以完全培养基和CM培养的RGC-5；衣霉素组：RGC-5接种48小时后,分别以浓度为1μg/ml(1D组)、2μg/ml(2D组)和4μg/ml(4D组)衣霉素干预,以建立RGC-5细胞的ERS模型,衣霉素干预后同时加完全培养基共培养；治疗组：RGC-5细胞在不同浓度衣霉素干预的同时加入等量的CM共培养(分别为1M组、2M组和4M组)。对照组、衣霉素组和治疗组在衣霉素诱导后24小时行Hoechst33342/PI双荧光活染法检测细胞凋亡率；采用qRT-PCR法检测各组在衣霉素诱导后3、6、9、12、24小时PERK、XBP1、ATF6、CHOP的mRNA表达；采用Western Blot法检测PERK、XBP1、ATF6、CHOP的蛋白表达。 结果： 1、在同一浓度衣霉素诱导下,治疗组RGC-5的细胞凋亡率明显低于衣霉素组(P0.05)。 2、在1μg/mL,衣霉素诱导RGC-5损伤后9和12小时,衣霉素组ATF6、XBP1和CHOP mRNA表达水平均较治疗组下调,且诱导后9小时两组间ATF6mRNA表达水平的差异具有统计学意义(P0.05)；两组间PERK mRNA表达水平无明显区别。 3、在4μg/mL,衣霉素诱导RGC-5损伤后,治疗组PERK、ATF6、 CHOP、XBP1mRNA表达水平较衣霉素组依次下调,且均于诱导后12小时两组间差距最明显；其中诱导后12小时两组间XBP1mRNA表达水平的差异具有统计学意义(P0.05) 结论： 1、衣霉素可成功建立RGC-5的ERS模型。 2、CM可对ERS状态下的RGC-5有一定的保护作用。 3、低浓度衣霉素诱导RGC-5发生未折叠蛋白反应时,在诱导后9小时,CM可通过ATF6通路增强细胞的未折叠蛋白反应而起到保护作用。 4、高浓度衣霉素诱导RGC-5发生内质网应激介导的凋亡时,在诱导后12小时,CM可通过XBP1通路减轻ERS而起到保护作用。
[Abstract]:Objective: 1. The endoplasmic reticulum stress (endoplasmic reticulum stress,ERS) model of rat retinal ganglion cell line (retinal ganglion cell5, R.GC-5 was established. 2. To investigate the protective effect of stem cell conditioned medium (conditioned medium,CM) on ERS of RGC-5. 3. 3. By detecting the changes of ERS related markers, the possible mechanism of protective effect of CM was discussed. Methods: RGC-5 was inoculated with 1 脳 105 / well or 2 脳 105 / well into 12 or 6 hole culture plates and divided into control group, chlortetracycline group and treatment group. Control group: the blank control group (ND group) was RGC-5, stem cell conditioned medium group (NM group) cultured in full medium and RGC-5; chlamycin group cultured in CM culture medium: RGC-5 inoculated 48 hours later. The ERS model of RGC-5 cells was established by the intervention of 1 渭 g/ml (1D), 2 渭 g/ml (2D) and 4 渭 g/ml (4D) respectively. In the treatment group, RGC-5 cells were co-cultured with CM at different concentrations (1 M group, 2 M group and 4 M group). In the control group, the control group and the treatment group, the apoptotic rate was detected by Hoechst33342/PI double fluorescent staining 24 hours after the induction of chlamycin, the mRNA expression of PERK,XBP1,ATF6,CHOP was detected by qRT-PCR assay, and the protein expression of PERK,XBP1,ATF6,CHOP was detected by Western Blot assay. Results: 1. The apoptotic rate of RGC-5 in the treatment group was significantly lower than that in the chlortetracycline group (P0.05) at the same concentration of chlortetracycline (P0.05). At 1 渭 g / mL, 9 and 12 hours after RGC-5 injury induced by chlortetracycline. The expression levels of ATF6,XBP1 and CHOP mRNA in the chlortetracycline group were lower than those in the treatment group, and there was a significant difference in the ATF6mRNA expression between the two groups at 9 hours after induction (P0.05). There was no significant difference in the expression of PERK mRNA between the two groups. 3. At 4 渭 g 路mL ~ (-1), the expression of PERK,ATF6, CHOP,XBP1mRNA in the treatment group was lower than that in the control group, and the most obvious difference was between the two groups at 12 hours after induction. There was significant difference in the expression of XBP1mRNA between the two groups 12 hours after induction (P0.05) conclusion: 1. The ERS model of RGC-5 can be successfully established by chlortetracycline. 3, when low concentration of chlortetracycline induces unfolded protein reaction in RGC-5, At 9 hours after induction, CM could enhance the unfolded protein response of cells through ATF6 pathway. 4. When high concentration of itamycin induced endoplasmic reticulum stress-mediated apoptosis in RGC-5, At 12 hours after induction, CM could attenuate ERS through XBP1 pathway and play a protective role.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R774
本文编号：2284475
[Abstract]:Objective: 1. The endoplasmic reticulum stress (endoplasmic reticulum stress,ERS) model of rat retinal ganglion cell line (retinal ganglion cell5, R.GC-5 was established. 2. To investigate the protective effect of stem cell conditioned medium (conditioned medium,CM) on ERS of RGC-5. 3. 3. By detecting the changes of ERS related markers, the possible mechanism of protective effect of CM was discussed. Methods: RGC-5 was inoculated with 1 脳 105 / well or 2 脳 105 / well into 12 or 6 hole culture plates and divided into control group, chlortetracycline group and treatment group. Control group: the blank control group (ND group) was RGC-5, stem cell conditioned medium group (NM group) cultured in full medium and RGC-5; chlamycin group cultured in CM culture medium: RGC-5 inoculated 48 hours later. The ERS model of RGC-5 cells was established by the intervention of 1 渭 g/ml (1D), 2 渭 g/ml (2D) and 4 渭 g/ml (4D) respectively. In the treatment group, RGC-5 cells were co-cultured with CM at different concentrations (1 M group, 2 M group and 4 M group). In the control group, the control group and the treatment group, the apoptotic rate was detected by Hoechst33342/PI double fluorescent staining 24 hours after the induction of chlamycin, the mRNA expression of PERK,XBP1,ATF6,CHOP was detected by qRT-PCR assay, and the protein expression of PERK,XBP1,ATF6,CHOP was detected by Western Blot assay. Results: 1. The apoptotic rate of RGC-5 in the treatment group was significantly lower than that in the chlortetracycline group (P0.05) at the same concentration of chlortetracycline (P0.05). At 1 渭 g / mL, 9 and 12 hours after RGC-5 injury induced by chlortetracycline. The expression levels of ATF6,XBP1 and CHOP mRNA in the chlortetracycline group were lower than those in the treatment group, and there was a significant difference in the ATF6mRNA expression between the two groups at 9 hours after induction (P0.05). There was no significant difference in the expression of PERK mRNA between the two groups. 3. At 4 渭 g 路mL ~ (-1), the expression of PERK,ATF6, CHOP,XBP1mRNA in the treatment group was lower than that in the control group, and the most obvious difference was between the two groups at 12 hours after induction. There was significant difference in the expression of XBP1mRNA between the two groups 12 hours after induction (P0.05) conclusion: 1. The ERS model of RGC-5 can be successfully established by chlortetracycline. 3, when low concentration of chlortetracycline induces unfolded protein reaction in RGC-5, At 9 hours after induction, CM could enhance the unfolded protein response of cells through ATF6 pathway. 4. When high concentration of itamycin induced endoplasmic reticulum stress-mediated apoptosis in RGC-5, At 12 hours after induction, CM could attenuate ERS through XBP1 pathway and play a protective role.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R774
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