MAPK信号传导通路的调控对皮肤光老化的保护作用研究
发布时间：2018-09-17 07:29
【摘要】：目的研究UVA照射体外培养的皮肤成纤维细胞后,细胞形态变化及相关细胞因子变化,检测MAPK信号传导的始动受体EGFR的表达情况,及MAPK信号途径中的下游基因c-jun,c-fos的表达变化。通过筛选出高效特异的c-junsiRNA后,对UVA照射后的皮肤成纤维细胞进行转染,探讨下调MAPK下游的基因c-jun对皮肤光老化中的MMPs,胶原的影响。 方法：1.取年轻成人包皮成纤维细胞培养传代,用第5代细胞做实验对象,进行细胞分组,采用不同计量的UVA(5 J/cm2、10J/cm2、20J/cm2)照射,设立对照组(0J/cm2),建立成纤维细胞光老化模型,培养24h后,光镜及电镜下观察细胞形态,RT-PCR及酶联免疫法(ELISA)测定照射前后体外培养的皮肤成纤维细胞的Ⅰ、Ⅲ型前胶原mRNA表达及蛋白表达。MTT法测定细胞活性。2.采用UVA(5 J/cm210J/cm2,15J/cm2)照射,设立对照组,照射后24小时用realtimePCR检测MAPK信号传导途径下游基因c-jun,c-fos,及终末基因MMP-1,MMP-3的mRNA表达情况。酶联免疫法(ELISA)测定细胞上清液中的MMP-1,MMP-3表达,weasternblog检测c-jun, c-fos的表达。3.针对前期实验中c-jun随照射剂量依赖性增高,设计5条c-junsiRNA, realtimePCR,及westembolg从基因层面和蛋白层面对其筛选,筛选出沉默效率最高的c-junsiRAN,并优化转染条件。4。以15J/cm2辐射剂量照射体外培养的成纤维细胞,2小时后转染c-junsiRNA,转染后24小时,realtimePCR检测MMP-1、MMP-3 mRNA,Ⅰ、Ⅲ型前胶原mRNA,48小时后酶联免疫法(ELISA)检测细胞上清液中MMP-1、MMP-3,Ⅰ、Ⅲ型前胶原含量。5,以(5 J/cm2、10J/cm2、20J/cm2)照射成纤维细胞,设立对照,检测MAPK信号传导通路的始动受体EGFR的表达情况,检测TGF-β1、IGF-1、KGF、VEGF几个细胞因子在照射前后的变化. 结果：1.随着UVA照射剂量增加,成纤维细胞MTT的OD值下降,与对照组比较,UVA 10 J/cm2、UVA 20 J/cm2照射组OD值明显下降,有显著性差异(p0.01)。Ⅰ型(?)mRNA RT-PCR产物积光吸光度随着UVA照射剂量增加而降低,UVA 10 J/cm2、UVA20J/cm2照射组降低明显(p0.05,P0.01),UVA 20 J/cm2组抑制胶原合成作用更加明显,与对照组及其它两组比较,有非常显著性差异(P0.01)。Ⅲ型胶原含量和mRNA表达,与对照组比较,UVA 20 J/cm2照射组明显降低,有非常显著性差异(P0.01)。细胞上清液中Ⅰ型胶原含量随着UVA照射剂量增加而降低,呈剂量依赖关系。UVA 10J/cm2、UVA 20J/cm2照射组,Ⅰ型胶原含量明显降低,与对照组比较(P0.05, P0.01),说明成纤维细胞分别给予UVA 10J/cm2、UVA 20J/cm2照射剂量,均可以抑制胶原合成,且剂量UVA 20 J/cm2组抑制胶原合成作用更加明显,与对照组及其它两组比较,有非常显著性差异(P0.01)；而5 J/cm2剂量组与对照组比较,无显著性差异(P0.05)。Ⅲ型胶原含量,与对照组比较,UVA 20 J/cm2照射组Ⅲ型胶原含量明显降低,有非常显著性差异(P0.01),说明成纤维细胞在UVA 20J/cm2照射剂量后,能明显抑制胶原合成,而UVA 5 J/cm2、UVA 10 J/cm2剂量组与对照组比较,Ⅲ型胶原含量无显著性差异(P0.05)。2.随着UVA照射剂量增加,成纤维细胞c.junmRNA表达增加,与对照比较有显著差异,(p0.01)。c-fosmRNA各组无差异(P0.05)。MMP-1、MMP-3随着照射剂量增加mRNA表达也增加,有显著差异。蛋白表达也呈现出与基因表达的一致性。C-jun随着照射剂量增加而增加,各组间差异p0.01,而c-fos表达无明显变化(P0.05)。3,设计出5条c-junsiRNA,经realtimePCR,及westembolg从基因层面和蛋白层面共同对其筛选出沉默效率最高的c-junsiRAN,沉默效率大于80%。序列为：GCAUUCUUGUCACAAUAAATT。4,以15J/cm2辐射剂量照射体外培养的成纤维细胞,2小时后转染c-junsiRNA,转染后24小时,MMP-1、MMP-3 mRNA,与对照组(转染阴性序列组)相比mRNA表达下调,其中MMP-1(P0.01)、MMP-3(P0.05)。Ⅰ、Ⅲ型前胶原mRNA,相比对照组(转染阴性序列组)Ⅰ型前胶原(P0.01),Ⅲ型前胶原(P0.05)。48小时后酶联免疫法(ELISA)检测细胞上清液中MMP-1、MMP-3,Ⅰ、Ⅲ型前胶原含量也呈现出与mRNA表达一致的结果MMP-1(P0.01)、MMP-3(P0.05)、Ⅰ胶原(P0.01)、Ⅲ型胶原(P0.05)。5.UVA照射体外培养的皮肤成纤维细胞EGFRmRNA与蛋白表达呈现一致性,均随照射剂量增高而增高,5 J/cm2(p0.05),10J/cm2、20J/cm2(P0.01)。UVA照射体外培养的皮肤成纤维细胞导致TGF-β1、IGF-1、KGF分泌下降, UVA 10 J/cm2、UVA 20 J/cm2照射组明显下降,与对照组比较,有显著性差异(P0.01)。VEGF分泌升高,UVA 20 J/cm2照射组与对照组比较,有非常显著性差异(P0.01)。 结论：1.UVA照射体外培养的皮肤成纤维细胞,导致细胞衰老,增殖活性下降；对Ⅰ型、Ⅲ型胶原合成有抑制作用。2.UVA照射体外培养的皮肤成纤维细胞激活MAPK信号传导通路,其下游基因c-jun上调明显,c-fos变化不大,其终末基因MMP-1、MMP-3均上调。3.经realtimePCR,及westernbolg从基因层面和蛋白层面共同对5条c-junsiRNA筛选,筛选出沉默效率最高的c-junsiRAN,沉默效率大于80%。序列为：GCAUUCUUGUCACAAUAAATT,名称为JUN-h-825。4.UVA照射体外培养的成纤维细胞2小时后,经c-junsiRNA转染后的成纤维细胞,一定程度上MMP-1,MMP-3可以逆转下调,Ⅰ型、Ⅲ型胶原合成减少的趋势也得到减轻。5.UVA照射后体外培养的皮肤成纤维细胞,EGFR表达明显增加,细胞上清液中的的细胞因子,TGF-β1、IGF-1.KGF分泌下降；VEGF分泌升高。
[Abstract]:Objective To study the morphological changes of skin fibroblasts cultured in vitro after UVA irradiation and the changes of related cytokines, detect the expression of EGFR, the initiating receptor of MAPK signal transduction, and the expression of c-jun, c-fos, the downstream genes of MAPK signal pathway. Fiber cells were transfected to investigate the effects of down-regulation of c-jun gene downstream of MAPK on MMPs and collagen in skin photoaging.
METHODS: 1. Young adult prepuce fibroblasts were cultured and subcultured. The 5th generation cells were divided into different groups. The control group (0J/cm2) was irradiated with different doses of UVA (5J/cm2, 10J/cm2, 20J/cm2), and the photoaging model of fibroblasts was established. The morphology, RT-PCR and ELISA were observed under light and electron microscopy 24 hours after culture. METHODS: The mRNA expression and protein expression of type I and type III procollagen in cultured skin fibroblasts were measured by ELISA before and after irradiation. The cell viability was measured by MTT assay. 2. UVA (5J/cm210J/cm2, 15J/cm2) was used to irradiate the skin fibroblasts. The control group was set up and the downstream genes of MAPK signal transduction pathway, c-jun, c-fos and terminal gene, MMP-1, were detected by realtime PCR 24 hours after irradiation. MMP-3 mRNA expression. The expression of MMP-1 and MMP-3 in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of c-jun and c-fos was detected by weasternblog. 3. Five c-junsiRNA, realtime PCR, and westembolg were designed to screen the c-Jun from gene and protein levels. The highest rate of c-junsiRAN was obtained, and the optimal transfection conditions were optimized. 4. The cultured fibroblasts were irradiated with 15J/cm2 radiation, then transfected with c-junsiRNA 2 hours later. MMP-1, MMP-3 mRNA, type I and type III procollagen mRNA were detected by realtime PCR 24 hours after transfection, and MMP-1, MMP-3, type I and type III procollagen mRNA were detected by enzyme-linked immunoassay (ELISA) 48 hours later. Content. 5. Fibroblasts were irradiated with (5J/cm 2, 10J/cm 2, 20J/cm 2) to detect the expression of EGFR, the initiating receptor of MAPK signal transduction pathway, and the changes of TGF-beta 1, IGF-1, KGF and VEGF before and after irradiation.
Results: 1. With the increase of UVA dose, the OD value of MTT of fibroblasts decreased. Compared with the control group, the OD value of UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.01). The cumulative absorbance of type I (?) mRNA RT-PCR products decreased with the increase of UVA dose, and UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.05, P 0.05). Compared with the control group and the other two groups, the content of collagen type III and the expression of mRNA were significantly lower in the UVA 20 J/cm 2 irradiation group (P 0.01). Compared with the control group (P 0.05, P 0.01), the content of type I collagen in UVA 10J/cm2 and UVA 20J/cm2 irradiation groups decreased significantly, indicating that fibroblasts irradiated with UVA 10J/cm2 and UVA 20J/cm2 could inhibit collagen synthesis, and the effect of UVA 20J/cm2 irradiation on collagen synthesis was more obvious than that of control group (P 0.05, P 0.01). There was a significant difference between the control group and the other two groups (P 0.01), but there was no significant difference between the 5 J / cm 2 dose group and the control group (P 0.05). The content of collagen type III in the UVA 20 J / cm 2 irradiation group was significantly lower than that in the control group (P 0.01), indicating that fibroblasts were significantly lower than that in the UVA 20 J / cm 2 irradiation group (P 0.01). Compared with the control group, the content of collagen type III was not significantly different (P 0.05). 2. With the increase of UVA dose, the expression of C. Jun mRNA in fibroblasts increased significantly (P 0.01). There was no significant difference in the expression of C - fos mRNA between the groups (P 0.05). MMP - 1 and MMP - 3 with the increase of UVA dose. The expression of C-jun increased with the increase of irradiation dose, but the expression of c-fos did not change significantly (P 0.05). 3. Five c-junsiRNA were designed and screened for silencing by realtime PCR and westembolg at both gene and protein levels. The most efficient c-junsiRAN, the silencing efficiency was more than 80%. The sequence was GCAUUCUUGUCACA AAAATT.4, irradiated with 15J/cm 2 radiation dose, transfected with c-junsiRNA 2 hours later, 24 hours after transfection, MMP-1, MMP-3 mRNA expression was down-regulated compared with the control group (transfection negative sequence group), MMP-1 (P 0.01), MMP-3 (P 0.05). MMP-1, MMP-3, type I and type III procollagen in supernatant were detected by enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. MMP-1 (P 0.01), MMP-3 (P 0.05), type I collagen (P 0.01), type III collagen (P 0.05) and type I procollagen (P 0.05) were also detected by ELISA. 5. The expression of EGFR mRNA and protein in cultured skin fibroblasts irradiated by UVA was consistent with that in vitro. Both EGFR mRNA and protein increased with the increase of irradiation dose. 5 J/cm2 (p0.05), 10 J/cm2, 20 J/cm2 (P 0.01). Cultured skin fibroblasts irradiated by UVA decreased the secretion of TGF-beta 1, IGF-1, KGF, UVA 10 J/cm2 and UVA 20 J/cm2, and decreased significantly compared with the control group. Compared with the control group, there was a significant difference (P 0.01). The secretion of VEGF was elevated. There was a significant difference (P 0.01) between the UVA 20 J/cm 2 irradiation group and the control group.
Conclusion: 1. UVA irradiation of cultured skin fibroblasts can induce cell senescence and decrease proliferation activity, and inhibit the synthesis of collagen type I and type III. 2. UVA irradiation of cultured skin fibroblasts can activate MAPK signal transduction pathway. The downstream genes c-Jun are up-regulated and c-fos is not changed, and the terminal genes MMP-1 and MMP-3 are both up-regulated. Five c-junsiRANs were screened by realtime PCR and Western bolg at the gene and protein levels. The highest silencing efficiency of c-junsiRAN was screened out. The sequence was GCAU UCU UCA CAA UAAATT, named JUN-h-825.4. After 2 hours of UVA irradiation, fibroblasts were transfected with c-junsiRNA. Vitamin cells, to a certain extent, MMP-1 and MMP-3 can reverse the downregulation, and the decrease of collagen synthesis of type I and type III can also be alleviated. 5. After UVA irradiation, the expression of EGFR in cultured skin fibroblasts increased significantly, the secretion of cytokines, TGF-beta 1 and IGF-1.KGF in cell supernatant decreased, and the secretion of VEGF increased.
【学位授予单位】：昆明医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.1
本文编号：2245165
[Abstract]:Objective To study the morphological changes of skin fibroblasts cultured in vitro after UVA irradiation and the changes of related cytokines, detect the expression of EGFR, the initiating receptor of MAPK signal transduction, and the expression of c-jun, c-fos, the downstream genes of MAPK signal pathway. Fiber cells were transfected to investigate the effects of down-regulation of c-jun gene downstream of MAPK on MMPs and collagen in skin photoaging.
METHODS: 1. Young adult prepuce fibroblasts were cultured and subcultured. The 5th generation cells were divided into different groups. The control group (0J/cm2) was irradiated with different doses of UVA (5J/cm2, 10J/cm2, 20J/cm2), and the photoaging model of fibroblasts was established. The morphology, RT-PCR and ELISA were observed under light and electron microscopy 24 hours after culture. METHODS: The mRNA expression and protein expression of type I and type III procollagen in cultured skin fibroblasts were measured by ELISA before and after irradiation. The cell viability was measured by MTT assay. 2. UVA (5J/cm210J/cm2, 15J/cm2) was used to irradiate the skin fibroblasts. The control group was set up and the downstream genes of MAPK signal transduction pathway, c-jun, c-fos and terminal gene, MMP-1, were detected by realtime PCR 24 hours after irradiation. MMP-3 mRNA expression. The expression of MMP-1 and MMP-3 in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of c-jun and c-fos was detected by weasternblog. 3. Five c-junsiRNA, realtime PCR, and westembolg were designed to screen the c-Jun from gene and protein levels. The highest rate of c-junsiRAN was obtained, and the optimal transfection conditions were optimized. 4. The cultured fibroblasts were irradiated with 15J/cm2 radiation, then transfected with c-junsiRNA 2 hours later. MMP-1, MMP-3 mRNA, type I and type III procollagen mRNA were detected by realtime PCR 24 hours after transfection, and MMP-1, MMP-3, type I and type III procollagen mRNA were detected by enzyme-linked immunoassay (ELISA) 48 hours later. Content. 5. Fibroblasts were irradiated with (5J/cm 2, 10J/cm 2, 20J/cm 2) to detect the expression of EGFR, the initiating receptor of MAPK signal transduction pathway, and the changes of TGF-beta 1, IGF-1, KGF and VEGF before and after irradiation.
Results: 1. With the increase of UVA dose, the OD value of MTT of fibroblasts decreased. Compared with the control group, the OD value of UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.01). The cumulative absorbance of type I (?) mRNA RT-PCR products decreased with the increase of UVA dose, and UVA 10 J / cm2 and UVA 20 J / cm2 irradiation groups decreased significantly (p0.05, P 0.05). Compared with the control group and the other two groups, the content of collagen type III and the expression of mRNA were significantly lower in the UVA 20 J/cm 2 irradiation group (P 0.01). Compared with the control group (P 0.05, P 0.01), the content of type I collagen in UVA 10J/cm2 and UVA 20J/cm2 irradiation groups decreased significantly, indicating that fibroblasts irradiated with UVA 10J/cm2 and UVA 20J/cm2 could inhibit collagen synthesis, and the effect of UVA 20J/cm2 irradiation on collagen synthesis was more obvious than that of control group (P 0.05, P 0.01). There was a significant difference between the control group and the other two groups (P 0.01), but there was no significant difference between the 5 J / cm 2 dose group and the control group (P 0.05). The content of collagen type III in the UVA 20 J / cm 2 irradiation group was significantly lower than that in the control group (P 0.01), indicating that fibroblasts were significantly lower than that in the UVA 20 J / cm 2 irradiation group (P 0.01). Compared with the control group, the content of collagen type III was not significantly different (P 0.05). 2. With the increase of UVA dose, the expression of C. Jun mRNA in fibroblasts increased significantly (P 0.01). There was no significant difference in the expression of C - fos mRNA between the groups (P 0.05). MMP - 1 and MMP - 3 with the increase of UVA dose. The expression of C-jun increased with the increase of irradiation dose, but the expression of c-fos did not change significantly (P 0.05). 3. Five c-junsiRNA were designed and screened for silencing by realtime PCR and westembolg at both gene and protein levels. The most efficient c-junsiRAN, the silencing efficiency was more than 80%. The sequence was GCAUUCUUGUCACA AAAATT.4, irradiated with 15J/cm 2 radiation dose, transfected with c-junsiRNA 2 hours later, 24 hours after transfection, MMP-1, MMP-3 mRNA expression was down-regulated compared with the control group (transfection negative sequence group), MMP-1 (P 0.01), MMP-3 (P 0.05). MMP-1, MMP-3, type I and type III procollagen in supernatant were detected by enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. MMP-1 (P 0.01), MMP-3 (P 0.05), type I collagen (P 0.01), type III collagen (P 0.05) and type I procollagen (P 0.05) were also detected by ELISA. 5. The expression of EGFR mRNA and protein in cultured skin fibroblasts irradiated by UVA was consistent with that in vitro. Both EGFR mRNA and protein increased with the increase of irradiation dose. 5 J/cm2 (p0.05), 10 J/cm2, 20 J/cm2 (P 0.01). Cultured skin fibroblasts irradiated by UVA decreased the secretion of TGF-beta 1, IGF-1, KGF, UVA 10 J/cm2 and UVA 20 J/cm2, and decreased significantly compared with the control group. Compared with the control group, there was a significant difference (P 0.01). The secretion of VEGF was elevated. There was a significant difference (P 0.01) between the UVA 20 J/cm 2 irradiation group and the control group.
Conclusion: 1. UVA irradiation of cultured skin fibroblasts can induce cell senescence and decrease proliferation activity, and inhibit the synthesis of collagen type I and type III. 2. UVA irradiation of cultured skin fibroblasts can activate MAPK signal transduction pathway. The downstream genes c-Jun are up-regulated and c-fos is not changed, and the terminal genes MMP-1 and MMP-3 are both up-regulated. Five c-junsiRANs were screened by realtime PCR and Western bolg at the gene and protein levels. The highest silencing efficiency of c-junsiRAN was screened out. The sequence was GCAU UCU UCA CAA UAAATT, named JUN-h-825.4. After 2 hours of UVA irradiation, fibroblasts were transfected with c-junsiRNA. Vitamin cells, to a certain extent, MMP-1 and MMP-3 can reverse the downregulation, and the decrease of collagen synthesis of type I and type III can also be alleviated. 5. After UVA irradiation, the expression of EGFR in cultured skin fibroblasts increased significantly, the secretion of cytokines, TGF-beta 1 and IGF-1.KGF in cell supernatant decreased, and the secretion of VEGF increased.
【学位授予单位】：昆明医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R758.1
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