干細(xì)胞條件培養(yǎng)液對(duì)RGCs內(nèi)質(zhì)網(wǎng)應(yīng)激的保護(hù)作用及機(jī)制
發(fā)布時(shí)間：2018-10-21 08:02
【摘要】：目的： 1.建立大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞系(retinal ganglion cell5, R.GC-5)的內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress,ERS)模型。 2.探討干細(xì)胞條件培養(yǎng)液(conditioned medium,CM)對(duì)RGC-5的ERS是否具有保護(hù)作用。 3.通過(guò)檢測(cè)ERS相關(guān)標(biāo)志物的變化,探討CM發(fā)揮保護(hù)作用的可能機(jī)制。 方法： 將RGC-5以1×105個(gè)/孔或2×105個(gè)/孔接種于12孔或6孔培養(yǎng)板內(nèi),分為對(duì)照組、衣霉素組和治療組。對(duì)照組：空白對(duì)照組(ND組)為以完全培養(yǎng)基培養(yǎng)的RGC-5,干細(xì)胞條件培養(yǎng)液組(NM組)為以完全培養(yǎng)基和CM培養(yǎng)的RGC-5；衣霉素組：RGC-5接種48小時(shí)后,分別以濃度為1μg/ml(1D組)、2μg/ml(2D組)和4μg/ml(4D組)衣霉素干預(yù),以建立RGC-5細(xì)胞的ERS模型,衣霉素干預(yù)后同時(shí)加完全培養(yǎng)基共培養(yǎng)；治療組：RGC-5細(xì)胞在不同濃度衣霉素干預(yù)的同時(shí)加入等量的CM共培養(yǎng)(分別為1M組、2M組和4M組)。對(duì)照組、衣霉素組和治療組在衣霉素誘導(dǎo)后24小時(shí)行Hoechst33342/PI雙熒光活染法檢測(cè)細(xì)胞凋亡率；采用qRT-PCR法檢測(cè)各組在衣霉素誘導(dǎo)后3、6、9、12、24小時(shí)PERK、XBP1、ATF6、CHOP的mRNA表達(dá)；采用Western Blot法檢測(cè)PERK、XBP1、ATF6、CHOP的蛋白表達(dá)。 結(jié)果： 1、在同一濃度衣霉素誘導(dǎo)下,治療組RGC-5的細(xì)胞凋亡率明顯低于衣霉素組(P0.05)。 2、在1μg/mL,衣霉素誘導(dǎo)RGC-5損傷后9和12小時(shí),衣霉素組ATF6、XBP1和CHOP mRNA表達(dá)水平均較治療組下調(diào),且誘導(dǎo)后9小時(shí)兩組間ATF6mRNA表達(dá)水平的差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；兩組間PERK mRNA表達(dá)水平無(wú)明顯區(qū)別。 3、在4μg/mL,衣霉素誘導(dǎo)RGC-5損傷后,治療組PERK、ATF6、 CHOP、XBP1mRNA表達(dá)水平較衣霉素組依次下調(diào),且均于誘導(dǎo)后12小時(shí)兩組間差距最明顯；其中誘導(dǎo)后12小時(shí)兩組間XBP1mRNA表達(dá)水平的差異具有統(tǒng)計(jì)學(xué)意義(P0.05) 結(jié)論： 1、衣霉素可成功建立RGC-5的ERS模型。 2、CM可對(duì)ERS狀態(tài)下的RGC-5有一定的保護(hù)作用。 3、低濃度衣霉素誘導(dǎo)RGC-5發(fā)生未折疊蛋白反應(yīng)時(shí),在誘導(dǎo)后9小時(shí),CM可通過(guò)ATF6通路增強(qiáng)細(xì)胞的未折疊蛋白反應(yīng)而起到保護(hù)作用。 4、高濃度衣霉素誘導(dǎo)RGC-5發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的凋亡時(shí),在誘導(dǎo)后12小時(shí),CM可通過(guò)XBP1通路減輕ERS而起到保護(hù)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R774
本文編號(hào)：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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R774
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相關(guān)期刊論文 前2條
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