摘要
目的探讨长脉宽1064 nm Nd∶YAG激光照射兔耳软骨后,导致兔耳软骨重塑的分子作用机制。方法将33只兔按照随机数表法分为6组,分别用于以下实验:筛选激光能量(6只),观察激光照射术后即刻(6只)、1周(6只)、3周(6只)、6周(6只)兔耳软骨组织的变化,以及转录组测序和样本验证(3只)。采用自身对照研究,左耳为不做处理,为正常对照侧;右耳进行激光照射(照射侧)。(1)激光能量筛选:使用长脉宽1064 nm Nd∶YAG激光以不同能量密度(50、60、70、80、90、100 J/cm^(2))照射6只兔耳右侧软骨后,取双侧厚度一致、相同规格、去除皮肤及软骨膜的软骨,进行组织学切片HE染色,观察软骨细胞的变化,确定最适宜塑形的激光能量密度(简称最适能量密度)。(2)兔耳软骨组织学观察:采用最适能量密度的激光照射24只兔右耳,分别于术后即刻及1、3、6周取材双侧软骨进行组织学观察(HE、Masson和天狼星红染色)。(3)转录组测序和样本验证:采用最适能量密度的激光照射3只兔右耳,照射后6 h内取下兔耳右侧软骨组织进行混合,设为激光照射组;取左耳相同部位、等体积的软骨组织,设为空白对照组,进行转录组RNA测序,并采用定量聚合酶链式反应(qPCR)和蛋白质印迹法检测相关基因和蛋白的表达。结果(1)经50~100 J/cm^(2)不同能量密度的激光照射后,HE染色显示,激光能量密度为80 J/cm^(2)时能使软骨细胞有改变但未发生空泡变形及凝固性坏死,损伤程度适宜。(2)80 J/cm^(2)的激光照射后,组织学观察显示,术后即刻出现激光辐射带,辐射带上软骨细胞整体形态拉长,呈梭形细胞样改变,基质染色深,折光明显,Ⅱ型胶原相对增多,1周时辐射带变浅,细胞大小较前恢复;3周到6周时辐射带周围基质染色又逐渐加深,整体形态又拉长。(3)转录组RNA测序发现:激光照射组与空白对照组相比,有198个差异表达基因(70个基因上调、128个基因下调)。通过进一步筛选和研究,激光照射组中CREB3L2基因表达上调。qPCR结果显示,激光照射组中CREB3L2 mRNA相对表达量明显高于空白对照组,差异有统计学意义(P<0.01);蛋白质印迹法检测显示,CREB3L2蛋白相对表达量高于空白对照组,且具有时间依赖性,差异有统计学意义(P<0.01)。结论长脉宽1064 nm Nd∶YAG激光照射兔耳软骨后,上调了CREB3L2基因的表达,引起软骨细胞重排及增殖,导致耳软骨形态和生物力学发生改变。
Objective To investigate the molecular mechanism of rabbit ear cartilage remodeling induced by 1064 nm Nd∶YAG laser irradiation.Methods Thirty-three rabbits were divided into 6 groups according to a random number table,and were used in the following experiments respectively:screening the optimal laser energy(6 rabbits),observing the changes of ear cartilage tissue immediately(6 rabbits),1 week(6 rabbits),3 weeks(6 rabbits),and 6 weeks after irridiation(6 rabbits),as well as transcriptome sequencing and verification(3 rabbits).The left ears of rabbits were used as untreated self-controls,while the right ears were irradiated.(1)Laser energy screening:the right ear cartilages of 6 rabbits were irradiated with 1064 nm Nd∶YAG laser at different energy densities(50,60,70,80,90,100 J/cm^(2)).Cartilages without skin and perichondrium were cut into the same thickness and size for HE staining,in order to observe the changes of chondrocytes and to determine the optimal laser energy density for shaping(hereinafter referred to as the optimal energy density).(2)Histological observation of rabbit ear cartilage:the right ears of 24 rabbits were irradiated with laser under the optimal energy density.Bilateral cartilage samples were collected immediately after surgery and at 1,3 and 6 weeks after surgery for histological observation(HE,Masson and Sirius red staining).(3)Transcriptome sequencing and sample verification:the right ears of 3 rabbits were irradiated with laser under the optimal energy density,and the cartilages were harvested and mixed within 6 hours after irradiation,which was set as the laser irradiation group.Cartilages of the same region and size on the left ears were set as blank control group.Transcriptome RNA sequencing was performed,and the expression levels of related genes and proteins were detected by quantitative polymerase chain reaction(qPCR)and Western blotting.Results(1)After laser irradiation with different energy density of 50-100 J/cm^(2),HE staining showed that the chondrocytes could be changed at the laser energy density of 80 J/cm^(2),without causing vacuolar deformation and coagulation necrosis,indicating that the damage degree was suitable.(2)After 80 J/cm^(2)laser irradiation,histological observation revealed that there was an irradiated zone immediately after the operation.The overall morphology of chondrocytes in the radiation zone was elongated and exhibited spindle cell-like changes,with deep matrix staining and obvious refraction,as well as a relative increase of typeⅡcollagen.At 1 week after irradiation,the radiation zone became shallower and the cell size recovered.From 3 weeks to 6 weeks,the matrix staining around the radiation zone gradually deepened and the overall cellular morphology was stretched again.(3)Transcriptome RNA sequencing revealed that there were 198 differentially expressed genes(70 up-regulated and 128 down-regulated)in the laser irradiated group compared with the blank control group.Through further screening and study,CREB3L2 gene expression was up-regulated in the laser irradiation group.qPCR results showed that the relative expression level of CREB3L2 mRNA in laser irradiation group was significantly higher than that in the blank control group,the difference was statistically significant(P<0.01).Western blotting showed that the relative expression level of CREB3L2 protein was higher than that of control group in a time-dependent manner,and the difference was statistically significant(P<0.01).Conclusions After irradiation with long-pulsed 1064 nm Nd∶YAG laser,the expression level of CREB3L2 gene is up-regulated,which then induced chondrocyte rearrangement and proliferation,resulting in morphological and biomechanical changes of ear cartilage.
作者
王虹顺
古琴妹
周婕
周伟豪
齐向东
Wang Hongshun;Gu Qinmei;Zhou Jie;Zhou Weihao;Qi Xiangdong(Department of Plastic Surgery,Zhujiang Hospital,Southern Medical University,Guangzhou 510280,China)
出处
《中华整形外科杂志》
CSCD
2022年第4期438-448,共11页
Chinese Journal of Plastic Surgery
基金
广州市科技计划项目(201903010001)。
关键词
耳软骨
激光
固体
Nd∶YAG激光
生物力学
重塑
耳畸形
Ear cartilage
Lasers,solid-state
Nd∶YAG laser
Biomechanics
Remodeling
Ear malformation
作者简介
通信作者:齐向东,Email:create_beauty@hotmail.com。
