【作者】 曲燕；

【导师】 孙青；

【作者基本信息】 山东大学 ， 皮肤性病学（专业学位）， 2017， 博士

【摘要】 目的:比较10600nm CO2点阵激光照射和外用维A酸制剂对Wistar大鼠皮肤的组织学和超微结构改变。初步探讨10600nm CO2点阵激光和维A酸治疗效果的长效性和时效性,为临床实践提供参考。方法:选取45只雌性Wistar大鼠,随机分为9组,将背部皮肤作为实验观察区,进行脱毛后用十字形标记线将观察区分为四部分:近头端左侧为正常对照组(A区),近尾端左侧为维A酸组(B区),近尾端右侧为联合治疗组(C区),近头端右侧为点阵激光组(D区)。C区和D区于实验开始时照射10600 CO2点阵激光1次,参数设置:能量15mJ,能量密度5%,频率300Hz,图形为方形,10mm×10mm;激光照射完毕后B区和C区开始每日外擦0.025%维A酸乳膏,持续3周。分别在第3天、第1-8周分批处死9组大鼠,每组大鼠取背部4区皮肤组织,在组织切片镜下检测真皮厚度,采用羟脯氨酸试剂盒检测组织中羟脯氨酸含量,Real-time PCR法检测Ⅲ型前胶原mRNA的表达。3周时在透射电镜下观察大鼠皮肤的超微结构改变。结果:①自3周至8周,各治疗组(B、C、D)Wistar大鼠真皮厚度较正常对照组(A)增加(P<0.05); 2～4周点阵激光组(D)与维A酸组(B)比较真皮厚度明显增加,差别有统计学意义(P<0.05); 2～6周联合治疗组(C)比维A酸组(B)真皮厚度明显增加(P<0.05)。②第3天各治疗组(B、C、D)羟脯氨酸含量较正常组下调,1周时各治疗组(B、C、D)羟脯氨酸含量较正常组(A)升高并持续上调,联合治疗组(C)和点阵激光组(D)于第5周达最大值,维A酸组(B)于第6周达最大值;8周时联合治疗组(C)羟脯氨酸含量仍明显高于其他各组(P<0.05);点阵激光组(D)和维A酸组(B)羟脯氨酸含量于照射后1～4周差别有统计学意义(P<0.05);③3天～1周时点阵激光组(D)Ⅲ前胶原mRNA的表达较正常对照组(A)下调;1周后各治疗组(B、C、D)的Ⅲ前胶原mRNA的表达均较前上升,联合治疗组(C)上升最为明显,并且达峰值最快(3周达峰值,5.61±0.7);④.3周时观察各分区大鼠的皮肤超微结构改变,透射电镜下显示单位面积内各治疗组(B、C、D)胶原数量比正常对照组(A)明显增多,胶原纤维排列方式更加紧致密集。结论:维A酸制剂和10600nm CO2点阵激光均具有促进Wistar大鼠皮肤胶原增生和重构的作用,可维持起效至少8周;与维A酸制剂相比,10600nm(C02点阵激光的嫩肤作用起效迅速,维持时间较短;联合应用C02点阵激光和维A酸制剂能够产生良好的协同作用。目的:比较照射10600nm CO2点阵激光和外用维A酸制剂对Wistar大鼠皮肤组织中miR-29a、Akt、TGF-β的mRNA表达水平以及磷酸化的Akt和TGF-β蛋白的表达,从分子水平上进一步研究miR-29a/Akt/TGF-β信号通路诱导皮肤胶原合成的机制。方法:取10只雌性Wistar大鼠,将大鼠背部皮肤脱毛后,按十字方式将背部皮肤平均分为四份,并用苦味酸进行标记。近头端左侧为正常对照组(A区),近尾端左侧为维A酸组(B区),近尾端右侧为联合治疗组(C区),近头端右侧为点阵激光组(D区)。C区和D区于实验开始第1天照射10600 nm CO2点阵激光1次,参数设置:能量15mJ,能量密度5%,频率300Hz,图形为方形,10mm×10mm;激光照射完毕后于B区和C区每日外擦0.025%维A酸乳膏,持续3周。在第3周时处死大鼠,取每只大鼠背部4区皮肤组织,分别应用Western blot方法检测Wistar大鼠皮肤中磷酸化的Akt和TGF-β蛋白的表达,Real-time PCR方法检测Wistar大鼠皮肤组织中miR-29a、Akt、TGF-β的mRNA表达水平。结果:①Real-time PCR检测结果表明,各治疗组Wistar大鼠皮肤组织中Akt、TGF-β的mRNA表达水平均较正常对照组明显得到升高,miR-29a的mRNA表达水平则低于对照组。以联合治疗组的差异性最为显著(P<0.05)。②第3周时,各治疗组中大鼠皮肤组织磷酸化的Akt和TGF-β蛋白的表达水平明显高于对照组,联合治疗组的增加最为显著。结论:维A酸制剂和CO2点阵激光均具有激活miR-29a/Akt/TGF- β信号通路的作用;miR-29a/Akt/TGF-β信号通路可能在促进胶原合成的增生中发挥作用;联合应用CO2点阵激光和维A酸制剂对miR-29a/Akt/TGF- β信号通路的激活作用最为显著。更多还原

【Abstract】 Objective: To compare the effects of 10600nm CO2 fractional laser and topical retinoic acid formulation on the skin histological and ultrastructural changes of Wistar rats, to preliminary explore the long-term effectiveness and efficiency of 10600nm CO2 fractional laser and topical retinoic acid, and to provide a scientific treatment basis for clinical practice.Methods: Depilating dorsum skins of 45 female Wistar rats, randomly divided into nine groups, and divided the back skins into 4 groups: nearly the left side of the head is the normal control group (A); nearly the left side of the tail is the retinoic acid group (B); nearly the right side of the tail is the combination treatment group(C); nearly the right side of the head is the fractional laser group (D). Area C and D were irradiated with 10600nm CO2 fractional laser in the first week, parameter settings: micro pulse energy, 15mJ; density, 5%; frequency, 300Hz; with a figure of square,10mm×10mm area B and C were wiped with 0.025% retinoic acid cream daily after laser irradiation ,for 3 weeks. Specimens were collected on day 3 and in weeks 1-8 after CO2 fractional laser irradiation, and then histological detection,hydroxyproline kits, and Real-time PCR method were used for detection of the changes of dermis thickness, content of hydroxyproline and the expression level of type Ⅲ precollagen mRNA in the four regions of the back of rats. The ultrastructural changes of rat skin were observed by transmission electron microscope (TEM) at 3 weeks after irradiation.Results: ① 3 to 8 weeks later, the dermal thickness of the Wistar rats in the treatment groups (B, C, D) increased compared with the normal control group(A) (P< 0.05); 2 -4 weeks later,the dermal thickness of the fractional laser group (D)increased significantly compared with the retinoic acid group (B) , the difference was statistically significant (P <0.05); 2 ～6 weeks later,the dermal thickness of the combination treatment group (C) increased significantly compared with the retinoic acid group (B)(P < 0.05).② On the third day, the hydroxyproline content of the treatment groups (B, C, D) was lower than the normal control group(A); at 1 week , the hydroxyproline content of the treatment groups (B, C, D) was higher than the normal control group(A) and continued to rise, the maximum of the combination treatment group (C) and the fractional laser group (D) were at 5 weeks and the retinoic acid group (B) were at 6 weeks; at 8 weeks, the hydroxyproline content of the combination treatment group (C) was still significantly higher than other groups(P < 0.05); the difference of the hydroxyproline content of the fractional laser group(D) and the retinoic acid group (B) in 1 ～4 weeks after irradiation was statistically significant (P < 0.05); ③3 days to 1 weeks after irradiation, the expression of III collagen mRNA in the fractional laser group (D) were lower than the normal control group (A); after 1 weeks the III collagen mRNA expression of the treatment groups(B, C, D) were increased than before, especially the combination treatment group (C)which reached peak fastest (3 weeks up to peak,5.61 ± 0.7);④3 weeks later,when rat skin was observed by transmission electron microscopy ,a denser arrangement of collagen was found in the rat skin of the treatment groups (B, C, D) than that in the normal control group (A),and the number of collagen per unit was increased significantly.Conclusion: Collagen synthesisi and remodeling were accurred both topical retinoic acid and irradiate 10600nm CO2 fractional laser, and the effect could last for at least 8 weeks; Compared with retinoic acid preparations, the tender skin effect of 10600nm CO2 fractional laser work more quickly but maintain shorter;Combined use of CO2 fractional laser and retinoic acid preparations could produce synergy.Objective: To compare the expression level of miR-29a, Akt, TGF - β mRNA and the expression of phosphorylated Akt and TGF - β protein in Wistar rats’ skin with 10600nm CO2 fractional laser and topical retinoic acid formulation , further explore the possible molecular mechanisms of miR - 29a / Akt/TGF - β signaling pathways in inducing the skin collagen synthesis.Methods: The hair on the back of 10 female Wistar rats was removed and the back was divided equally to four regions with the cross-streaking method: nearly the left side of the head is the normal control group (A); nearly the left side of the tail is the retinoic acid group (B); nearly the right side of the tail is the combination treatment group (C); nearly the right side of the head is the fractional laser group (D). Area C and D were irradiated with 10600nm CO2 fractional laser in the first week,parameter settings: micro pulse energy, 15mJ; density, 5%; frequency, 300Hz;with a figure of square, 10mm×10mm; area B and C were wiped with 0.025%retinoic acid cream daily after laser irradiation,for 3 weeks.Specimens of 4 areas were collected in week 3 after CO2 fractional laser irradiation , respectively using Western blot method to detect he expression of phosphorylated Akt and TGF - βprotein and Real - time PCR method to detect the expression level of miR - 29a,Akt, TGF - β mRNA.Results:①At week 3 the expression level of Akt and TGF - β protein in rats skin tissue were significantly higher than the control group,the increase of the combination group was the most significant (P < 0.05). ②Real - time PCR detection results showed that up regulation of Akt and TGF-β mRNA expression and down regulation of miR-29a mRNA expression were observed by the treatment groups (B, C, and D); which the difference in the combination treatment group (C)was the most significant (P<0.05).Conclusion: Retinoic acid formulation and CO2 fractional laser both could the active miR-29a/Akt/TGF-P signal pathways; miR-29a/Akt/TGF-β signal pathways may play a certain role in the promotion of collagen synthesis and proliferation;combined use of CO2 fractional laser and retinoic acid preparations may produce the most significant role in the activation of miR-29a/Akt/TGF-β signal pathways.更多还原