大鼠富血小板血浆凝胶对过表达胶质细胞源性神经营养因子的自体脂肪间充质干细胞的作用  

Effect of rat platelet-rich plasma gel on autologous adipose-derived mesenchymal stem cells overexpressing glia-derived neurotrophic factor

作　　者：蔡维霞 郑朝 刘佳琦 刘洋 张婷 计鹏 田晨阳 Cai Weixia;Zheng Zhao;Liu Jiaqi;Liu Yang;Zhang Ting;Ji Peng;Tian Chenyang(Department of Burns and Cutaneous Surgery,Burn Center of PLA,the First Affiliated Hospital of Air Force Medical University,Xi′an 710032,China)

机构地区：[1]空军军医大学第一附属医院全军烧伤中心,烧伤与皮肤外科,西安710032

出　　处：《中华烧伤与创面修复杂志》2024年第12期1176-1183,共8页Chinese Journal of Burns And Wounds

基　　金：陕西省重点研发计划(2022SF-399);国家自然科学基金面上项目(81471879、82172208)。

摘　　要：目的探讨大鼠富血小板血浆(PRP)凝胶对过表达胶质细胞源性神经营养因子(GDNF)的自体脂肪间充质干细胞(ADSC)的作用。方法该研究为实验研究。取5只成年雄性SD大鼠,采用胶原酶消化法获取原代ADSC并成功鉴定。取第3代ADSC,按照随机数字表法(分组方法下同)分为感染空载腺病毒的阴性对照组和感染过表达GDNF腺病毒的过表达GDNF组。培养48 h后,大体观察细胞感染情况。另取5只成年雄性SD大鼠,采血后采用差速离心法获取PRP,制备成凝胶后采用扫描电子显微镜观察其微观结构。取第3代ADSC,加入PRP成胶前的混合液中,成胶后常规培养48 h,行苏木精-伊红染色和钙黄素/碘化丙啶染色观测细胞生长情况。将感染空载腺病毒的ADSC或感染过表达GDNF腺病毒的ADSC在PRP凝胶中进行常规培养。培养48 h后,采用钙黄素/碘化丙啶染色检测细胞生长情况;培养24、48、72 h及1、2、3、4周后,收集细胞培养基的上清液,采用酶标仪测定其吸光度值并计算GDNF含量,样本数为3;培养48 h后,采用免疫荧光法检测细胞表达施万细胞特异性标志物S100蛋白质的情况。结果培养48 h后,阴性对照组和过表达GDNF组中感染腺病毒的细胞占比均接近90%且生长状态良好。阴性对照组细胞正常生长;过表达GDNF组细胞的形态发生明显变化,80%~90%的细胞伸出2个或多个突起,且在细胞聚集的地方,突起交织成网。PRP凝胶呈三维网状结构,且孔径大小不一。培养48 h后,ADSC可以很好地附着于PRP凝胶,且活细胞占比达98%。培养48 h后,感染空载腺病毒的ADSC生长状态良好,且呈典型的ADSC样梭形生长;感染过表达GDNF腺病毒的ADSC生长状态良好,且多数细胞伸出2个或多个突起,突起聚集处交织成网。培养24、48、72 h及1、2、3、4周后,感染过表达GDNF腺病毒的ADSC培养基的上清液中GDNF含量分别为(90±10)、(133±15)、(150±10)、(137±15)、(132±18)、(120±10)Objective To investigate the effect of rat platelet-rich plasma(PRP)gel on autologous adipose-derived mesenchymal stem cells(ADSCs)overexpressing glial-derived neurotrophic factor(GDNF).Methods This study was an experimental study.Five adult male Sprague-Dawley rats were used,and the primary ADSCs were obtained by collagenase digestion,and then the cells were identified successfully.The 3 rd passage of ADSCs were obtained and divided into negative control group infected with unloaded adenovirus and overexpressing GDNF group infected with overexpressing GDNF adenovirus,according to random number table method(the grouping method was the same below).After 48 hours of culture,the infection of cells was observed.Five adult male Sprague-Dawley rats were used,and the PRP was obtained after collecting blood by differential centrifugation.PRP was prepared into a gel and its microstructure was observed by scanning electron microscope.The ADSCs of 3 rd passage were added into the PRP solution mixture and cultured for 48 hours after gelation.The cell growth was observed by hematoxylin-eosin staining and calcein/propyl iodide staining.ADSCs infected with unloaded adenovirus and ADSCs infected with overexpressing GDNF adenovirus were routinely cultured in PRP gel.After 48 hours of culture,the cell growth was detected by calcein/propyl iodide staining.After culture for 24,48,72 hours and 1,2,3,4 weeks,the supernatant of cell culture medium was collected,the absorbance value was determined by microplate analyzer,and the GDNF content was calculated,with the sample number of 3.After 48 hours of culture,the expression of S100 protein(a specific marker of Schwann cells)was detected by immunofluorescence assay.Results After 48 hours of culture,the proportions of cells infected with adenovirus in negative control group and overexpressing GDNF group were close to 90%,and the cell growth was good.The cells in negative control group grew normally.The morphology of the cells in overexpressing GDNF group changed significantly with 80%-90%o

关 键 词：周围神经 间质干细胞 胶质细胞源性神经营养因子 富血小板血浆 施万细胞 脂肪间充质干细胞 神经修复 

分 类 号：R622[医药卫生—整形外科]