發(fā)布時(shí)間：2018-03-29 15:27

  本文選題：角質(zhì)形成細(xì)胞　切入點(diǎn)：HaCaT　出處：《中南大學(xué)》2012年博士論文

【摘要】：第一部分：人皮膚原代角質(zhì)形成細(xì)胞與HaCaT細(xì)胞對過氧化氫的不同反應(yīng) 目的：研究正常人皮膚原代角質(zhì)形成細(xì)胞(NHEKs)與HaCaT細(xì)胞株對過氧化氫(H2O2)誘導(dǎo)的氧化應(yīng)激的不同反應(yīng),為選擇合適的皮膚細(xì)胞模型提供依據(jù)。 方法：收集兒童包皮并分離培養(yǎng)原代角質(zhì)形成細(xì)胞,復(fù)蘇并培養(yǎng)HaCaT細(xì)胞,用150uM H2O2處理第2代人皮膚角質(zhì)形成細(xì)胞和HaCaT細(xì)胞2小時(shí)后繼續(xù)培養(yǎng)24小時(shí),MTT法檢測其細(xì)胞生長活性、比色法檢測細(xì)胞凋亡相關(guān)酶caspase-3/7活性、流式細(xì)胞儀檢測細(xì)胞周期中G1期細(xì)胞百分?jǐn)?shù)、細(xì)胞凋亡百分?jǐn)?shù)及細(xì)胞內(nèi)ROS水平、WST法檢測細(xì)胞內(nèi)SOD酶活性水平、細(xì)胞衰老相關(guān)p-半乳糖苷酶試劑盒檢測細(xì)胞中p-半乳糖苷酶陽性細(xì)胞百分?jǐn)?shù)、Western blot檢測衰老相關(guān)蛋白Klotho表達(dá)水平,并對相關(guān)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。 結(jié)果：經(jīng)H2O2處理后NHEKs與HaCaT細(xì)胞活性均明顯下降且呈濃度依賴和時(shí)間依賴關(guān)系,HaCaT細(xì)胞的抑制率高于NHEKs(p0.05), HaCaT細(xì)胞內(nèi)ROS水平、G1期細(xì)胞百分?jǐn)?shù)與細(xì)胞凋亡相關(guān)酶caspase-3/7活性均高于NHEKs細(xì)胞,而NHEKs細(xì)胞內(nèi)SOD酶活性水平則高于HaCaT細(xì)胞(p0.05),H2O2處理后兩種細(xì)胞衰老相關(guān)蛋白Klotho表達(dá)均下調(diào),NHEKs與HaCaT細(xì)胞均均體積變大、圓頓等衰老表型,NHEKs細(xì)胞中SA-P-Gal陽性細(xì)胞百分?jǐn)?shù)高于對照組(p0.05),而處理前后HaCaT細(xì)胞中均檢測不到SA-P-Gal陽性細(xì)胞。 結(jié)論：NHEKs比HaCaT細(xì)胞更能耐受H2O2誘導(dǎo)的氧化應(yīng)激,細(xì)胞經(jīng)H2O2誘導(dǎo)后,NHEKs既有衰老樣的表型,也能表達(dá)衰老相關(guān)p-半乳糖苷酶,而HaCaT細(xì)胞有衰老樣的表型,但不表達(dá)衰老相關(guān)p-半乳糖苷酶。 第二部分CD147對氧化應(yīng)激所致人皮膚成纖維細(xì)胞衰老的影響 目的：觀察CD147對H202所致人皮膚成纖維細(xì)胞衰老的影響。 方法：收集兒童包皮并分離培養(yǎng)原代成纖維細(xì)胞,構(gòu)建CD147shRNA慢病毒干擾載體,轉(zhuǎn)染第二代成纖維細(xì)胞并進(jìn)行鑒定,100uM H2O2處理轉(zhuǎn)染前后細(xì)胞2h,MTT法分別檢測誘導(dǎo)后12、24、36、48h細(xì)胞生長活性、比色法檢測誘導(dǎo)后24h細(xì)胞凋亡相關(guān)酶caspase-3/7活性、細(xì)胞衰老相關(guān)p-半乳糖苷酶試劑盒檢測誘導(dǎo)后24h細(xì)胞中p-半乳糖苷酶活性水平、流式細(xì)胞儀檢測誘導(dǎo)后24h細(xì)胞生長周期中G1期細(xì)胞百分?jǐn)?shù)及細(xì)胞凋亡率,并對相關(guān)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。結(jié)果：觀察到干擾組(NHSFs-CD147shRNA組)細(xì)胞體積較正常變大、扁平等細(xì)胞衰老的形態(tài)學(xué)表現(xiàn),其生長活性受到輕度抑制,細(xì)胞衰老相關(guān)p-半乳糖苷酶、細(xì)胞G1期百分?jǐn)?shù)、細(xì)胞凋亡相關(guān)酶caspase-3/7活性及細(xì)胞凋亡率均輕度升高。各細(xì)胞組經(jīng)H202處理后,空載體病毒轉(zhuǎn)染組(NHSFs-virus+H2O2組)細(xì)胞較前變大、扁平,少數(shù)有多角形等細(xì)胞衰老的形態(tài)學(xué)表現(xiàn),其細(xì)胞生長活性受到中度抑制,細(xì)胞衰老相關(guān)p-半乳糖苷酶、細(xì)胞G1期百分?jǐn)?shù)、凋亡相關(guān)酶caspase-3/7活性及細(xì)胞凋亡率均中度升高,而CD147干擾組(NHSFs-CD147shRNA+H2O2)細(xì)胞體積比對照組明顯變大、扁平,并出現(xiàn)多角形等細(xì)胞衰老的形態(tài)學(xué)表現(xiàn),其生長活性更明顯受到抑制。 結(jié)論：在正常和氧化應(yīng)激條件下,CD147對成纖維細(xì)胞的衰老均有著一定的調(diào)控作用,CD147可能是調(diào)控人皮膚細(xì)胞衰老的重要分子。 第三部分CD147對氧化應(yīng)激所致人皮膚成纖維細(xì)胞衰老的保護(hù)機(jī)制研究 目的：探討CD147對氧化應(yīng)激所致人皮膚成纖維細(xì)胞衰老的可能機(jī)制。 方法：收集兒童包皮并分離培養(yǎng)原代成纖維細(xì)胞,構(gòu)建CD147shRNA慢病毒載體并轉(zhuǎn)染第二代成纖維細(xì)胞,100uM H2O2處理細(xì)胞誘導(dǎo)其衰老,流式細(xì)胞儀檢測細(xì)胞內(nèi)ROS水平、NBT法檢測細(xì)胞內(nèi)SOD活性、Western blot檢測處理后細(xì)胞衰老相關(guān)蛋白Klotho表達(dá)水平,并對相關(guān)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。 結(jié)果：干擾成纖維細(xì)胞中CD147表達(dá)后,其細(xì)胞內(nèi)熒光強(qiáng)度稍升高,NHSFs-CD147shRNA組細(xì)胞內(nèi)熒光強(qiáng)度幾何均值為7.30±0.58,NHSFs-virus組細(xì)胞內(nèi)熒光強(qiáng)度幾何均值為6.87±0.42(P0.05)；細(xì)胞經(jīng)H2O2處理后,NHSFs-virus+H2O2組細(xì)胞內(nèi)熒光強(qiáng)度幾何均值為17.62±1.12,較NHSFs-virus組升高(P0.05),而NHSFs-CD147shRNA+H2O2組細(xì)胞內(nèi)熒光強(qiáng)度幾何均值28.91±1.33,較NHSFs-CD147shRNA組與NHSFs-virus+H2O2組明顯升高(P0.05)。CD147干擾前后,細(xì)胞內(nèi)SOD活性無明顯變化,NHSFs-virus組細(xì)胞內(nèi)SOD活性為0.28±0.06U, NHSFs-CD147shRNA組細(xì)胞內(nèi)SOD活性為0.26±0.03U(P0.05)；細(xì)胞經(jīng)H202處理后,NHSFs-virus+H2O2組細(xì)胞內(nèi)SOD活性為2.18±0.17U,較對照組NHSFs-virus明顯升高(P0.05),NHSFs-CD147shRNA+H2O2組細(xì)胞內(nèi)SOD活性為0.88±0.06U,較對照組NHSFs-CD147shRNA輕度升高(P0.05),而NHSFS-Virus+H202組細(xì)胞內(nèi)SOD活性較NHSFs-CD147shRNA+H202組高(P0.05)。細(xì)胞經(jīng)H2O2處理后,NHSFs-virus+H202組細(xì)胞衰老相關(guān)蛋白Klotho的表達(dá)下調(diào),而干擾細(xì)胞中CD147的表達(dá)后,H202處理前后均檢測不到衰老相關(guān)蛋白Klotho的表達(dá). 結(jié)論：在氧化應(yīng)激條件下,CD147通過與皮膚成纖維細(xì)胞內(nèi)增加的SOD一起加速對細(xì)胞內(nèi)ROS的清除,減少ROS在細(xì)胞內(nèi)的聚集,從而減輕細(xì)胞因氧化應(yīng)激所致的損傷和衰老,該過程可能與Klotho蛋白相關(guān),CD147可能通過調(diào)控細(xì)胞衰老相關(guān)蛋白Klotho表達(dá)而延緩細(xì)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 鹵 0.58 in NHSFs - CD147 shRNA group , 6.87 鹵 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 鹵 0.06U and 0.26 鹵 0.03 U ( P0.05 ) .
After treated by H202 , the activity of SOD in NHSFs - virus + H2O2 group was 2.18 鹵 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 .

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R751

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