两种皮肤细胞对过氧化氢的不同反应及CD147在氧化应激所致人皮肤成纤维细胞衰老中的机制研究

发布时间：2018-03-29 15:27

本文选题：角质形成细胞　切入点：HaCaT　出处：《中南大学》2012年博士论文

【摘要】：第一部分：人皮肤原代角质形成细胞与HaCaT细胞对过氧化氢的不同反应目的：研究正常人皮肤原代角质形成细胞(NHEKs)与HaCaT细胞株对过氧化氢(H2O2)诱导的氧化应激的不同反应,为选择合适的皮肤细胞模型提供依据。方法：收集儿童包皮并分离培养原代角质形成细胞,复苏并培养HaCaT细胞,用150uM H2O2处理第2代人皮肤角质形成细胞和HaCaT细胞2小时后继续培养24小时,MTT法检测其细胞生长活性、比色法检测细胞凋亡相关酶caspase-3/7活性、流式细胞仪检测细胞周期中G1期细胞百分数、细胞凋亡百分数及细胞内ROS水平、WST法检测细胞内SOD酶活性水平、细胞衰老相关p-半乳糖苷酶试剂盒检测细胞中p-半乳糖苷酶阳性细胞百分数、Western blot检测衰老相关蛋白Klotho表达水平,并对相关数据进行统计分析。结果：经H2O2处理后NHEKs与HaCaT细胞活性均明显下降且呈浓度依赖和时间依赖关系,HaCaT细胞的抑制率高于NHEKs(p0.05), HaCaT细胞内ROS水平、G1期细胞百分数与细胞凋亡相关酶caspase-3/7活性均高于NHEKs细胞,而NHEKs细胞内SOD酶活性水平则高于HaCaT细胞(p0.05),H2O2处理后两种细胞衰老相关蛋白Klotho表达均下调,NHEKs与HaCaT细胞均均体积变大、圆顿等衰老表型,NHEKs细胞中SA-P-Gal阳性细胞百分数高于对照组(p0.05),而处理前后HaCaT细胞中均检测不到SA-P-Gal阳性细胞。结论：NHEKs比HaCaT细胞更能耐受H2O2诱导的氧化应激,细胞经H2O2诱导后,NHEKs既有衰老样的表型,也能表达衰老相关p-半乳糖苷酶,而HaCaT细胞有衰老样的表型,但不表达衰老相关p-半乳糖苷酶。第二部分CD147对氧化应激所致人皮肤成纤维细胞衰老的影响目的：观察CD147对H202所致人皮肤成纤维细胞衰老的影响。方法：收集儿童包皮并分离培养原代成纤维细胞,构建CD147shRNA慢病毒干扰载体,转染第二代成纤维细胞并进行鉴定,100uM H2O2处理转染前后细胞2h,MTT法分别检测诱导后12、24、36、48h细胞生长活性、比色法检测诱导后24h细胞凋亡相关酶caspase-3/7活性、细胞衰老相关p-半乳糖苷酶试剂盒检测诱导后24h细胞中p-半乳糖苷酶活性水平、流式细胞仪检测诱导后24h细胞生长周期中G1期细胞百分数及细胞凋亡率,并对相关数据进行统计分析。结果：观察到干扰组(NHSFs-CD147shRNA组)细胞体积较正常变大、扁平等细胞衰老的形态学表现,其生长活性受到轻度抑制,细胞衰老相关p-半乳糖苷酶、细胞G1期百分数、细胞凋亡相关酶caspase-3/7活性及细胞凋亡率均轻度升高。各细胞组经H202处理后,空载体病毒转染组(NHSFs-virus+H2O2组)细胞较前变大、扁平,少数有多角形等细胞衰老的形态学表现,其细胞生长活性受到中度抑制,细胞衰老相关p-半乳糖苷酶、细胞G1期百分数、凋亡相关酶caspase-3/7活性及细胞凋亡率均中度升高,而CD147干扰组(NHSFs-CD147shRNA+H2O2)细胞体积比对照组明显变大、扁平,并出现多角形等细胞衰老的形态学表现,其生长活性更明显受到抑制。结论：在正常和氧化应激条件下,CD147对成纤维细胞的衰老均有着一定的调控作用,CD147可能是调控人皮肤细胞衰老的重要分子。第三部分CD147对氧化应激所致人皮肤成纤维细胞衰老的保护机制研究目的：探讨CD147对氧化应激所致人皮肤成纤维细胞衰老的可能机制。方法：收集儿童包皮并分离培养原代成纤维细胞,构建CD147shRNA慢病毒载体并转染第二代成纤维细胞,100uM H2O2处理细胞诱导其衰老,流式细胞仪检测细胞内ROS水平、NBT法检测细胞内SOD活性、Western blot检测处理后细胞衰老相关蛋白Klotho表达水平,并对相关数据进行统计分析。结果：干扰成纤维细胞中CD147表达后,其细胞内荧光强度稍升高,NHSFs-CD147shRNA组细胞内荧光强度几何均值为7.30±0.58,NHSFs-virus组细胞内荧光强度几何均值为6.87±0.42(P0.05)；细胞经H2O2处理后,NHSFs-virus+H2O2组细胞内荧光强度几何均值为17.62±1.12,较NHSFs-virus组升高(P0.05),而NHSFs-CD147shRNA+H2O2组细胞内荧光强度几何均值28.91±1.33,较NHSFs-CD147shRNA组与NHSFs-virus+H2O2组明显升高(P0.05)。CD147干扰前后,细胞内SOD活性无明显变化,NHSFs-virus组细胞内SOD活性为0.28±0.06U, NHSFs-CD147shRNA组细胞内SOD活性为0.26±0.03U(P0.05)；细胞经H202处理后,NHSFs-virus+H2O2组细胞内SOD活性为2.18±0.17U,较对照组NHSFs-virus明显升高(P0.05),NHSFs-CD147shRNA+H2O2组细胞内SOD活性为0.88±0.06U,较对照组NHSFs-CD147shRNA轻度升高(P0.05),而NHSFS-Virus+H202组细胞内SOD活性较NHSFs-CD147shRNA+H202组高(P0.05)。细胞经H2O2处理后,NHSFs-virus+H202组细胞衰老相关蛋白Klotho的表达下调,而干扰细胞中CD147的表达后,H202处理前后均检测不到衰老相关蛋白Klotho的表达. 结论：在氧化应激条件下,CD147通过与皮肤成纤维细胞内增加的SOD一起加速对细胞内ROS的清除,减少ROS在细胞内的聚集,从而减轻细胞因氧化应激所致的损伤和衰老,该过程可能与Klotho蛋白相关,CD147可能通过调控细胞衰老相关蛋白Klotho表达而延缓细胞衰老。
[Abstract]:Part I : Different Responses of Human Skin Primary Keratinocyte to HaCaT Cells to Hydrogen Peroxide

Objective : To study the different responses of keratinocytes ( NHEKs ) and HaCaT cell lines to oxidative stress induced by hydrogen peroxide ( H2O2 ) in normal human skin .

Methods : The cultured primary keratinocytes were collected and cultured and cultured for 24 hours . The activity of caspase - 3 / 7 was detected by MTT assay . The activity of caspase - 3 / 7 was detected by MTT assay . The percentage of apoptotic cells , percentage of apoptotic cells and intracellular ROS level were detected by MTT assay . The expression level of p - galactosidase positive cells in the cells was detected by Western blot and the correlation data were analyzed statistically .

Results : The activity of NHEKs and HaCaT cells decreased significantly after H _ 2O _ 2 treatment , and the inhibition rate of HaCaT cells was higher than that of NHEKs ( P < 0.05 ) . The activity of caspase - 3 / 7 in HaCaT cells was higher than that in HaCaT cells ( P < 0.05 ) .

Conclusion : NHEKs can tolerate H2O2 - induced oxidative stress more than HaCaT cells . After H _ 2O _ 2 - induced oxidative stress , NHEKs have both aging - like phenotype and senescence - related p - galactosidase , while HaCaT cells have senescence - related phenotype , but do not express senescence - related p - galactosidase .

Effect of CD147 on aging of human skin fibroblasts induced by oxidative stress

Objective : To observe the effect of CD147 on aging of human skin fibroblasts induced by H202 .

Methods : The expression of caspase - 3 / 7 , caspase - 3 / 7 activity and apoptosis rate were detected by MTT assay . The activity of caspase - 3 / 7 and apoptosis rate were significantly increased in 24 hours after induction . The results showed that the cell growth activity was moderately inhibited , the cell senescence - related p - galactosidase , cell G1 phase percentage , apoptosis - related enzyme caspase - 3 / 7 activity and apoptosis rate were all moderately elevated .

Conclusion : CD147 may regulate the aging of fibroblasts under normal and oxidative stress conditions . CD147 may be an important molecule to regulate the aging of human skin cells .

The protective mechanism of CD147 on aging of human skin fibroblasts induced by oxidative stress

Objective : To investigate the possible mechanism of CD147 on aging of human skin fibroblasts induced by oxidative stress .

Methods : The primary fibroblasts were collected and isolated from children . CD147 shRNA lentivirus vector was constructed and the second generation fibroblasts were transfected . 100uM H2O2 treatment cells induced senescence . Flow cytometry was used to detect the level of ROS in the cells . The expression level of cell senescence - related protein Klotho was detected by Western blot , and the correlation data were analyzed statistically .

Results : After interfering with CD147 expression in fibroblasts , the intracellular fluorescence intensity was slightly increased , and the mean fluorescence intensity was 7.30 卤 0.58 in NHSFs - CD147 shRNA group , 6.87 卤 0.42 in NHSFs - virus group ( P0.05 ) .
Compared with NHSFs - CD147 shRNA and NHSFs - virus + H2O2 group , the activity of SOD in NHSFs - CD147 shRNA + H2O2 group increased significantly ( P0.05 ) , and the activity of SOD in NHSFs - CD147 shRNA + H2O2 group was 0.28 卤 0.06U and 0.26 卤 0.03 U ( P0.05 ) .
After treated by H202 , the activity of SOD in NHSFs - virus + H2O2 group was 2.18 卤 0.17 U , the activity of SOD in NHSFs - CD147 shRNA + H2O2 group was higher than that in control group ( P0.05 ) .

Conclusion : Under oxidative stress , CD147 accelerates the clearance of ROS in cells and reduces the accumulation of ROS in cells , thus reducing the damage and aging caused by oxidative stress , which may be associated with Klotho protein , which may delay cell senescence by regulating the expression of Klotho - related protein Klotho .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R751

【参考文献】