表达肝细胞生长因子的人羊水干细胞培养上清对大鼠神经元突起生长的影响  

The effects of culture supernatant of human amniotic fluid stem cells expressing hepatocyte growth factor on neurite growth in rats

作　　者：李雪芳 郭润发 张立新 钱佳羽[1] 宋豪林 张志军 Li Xuefang;Guo Runfa;Zhang Lixin;Qian Jiayu;Song Haolin;Zhang Zhijun(Department of Human Anatomy,School of Medicine,Nantong University,Nantong 226001;Affiliated Taizhou People’s Hospital of Nanjing Medical University,Taizhou 225300,China)

机构地区：[1]南通大学医学院人体解剖学系,南通226001 [2]南京医科大学附属泰州市人民医院,泰州225300

出　　处：《神经解剖学杂志》2022年第6期611-617,共7页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(31970938,81571070);江苏省自然科学基金(BK20191448)。

摘　　要：目的:研究过表达肝细胞生长因子(HGF)的人羊水干细胞(hAFSCs)上清对大鼠背根节(DRG)神经突起生长的影响。方法:从人羊水中收集贴壁细胞,通过形态学、核型分析、细胞表面标记分子等手段鉴定hAFSCs。将hAFSCs通过慢病毒感染得到过表达HGF的HGF-hAFSCs,收集hAFSCs、HGF-hAFSCs培养上清液离心过滤后,用于DRG组织块的体外培养,比较hAFSCs上清液组(hAFSCs)、HGF-hAFSCs上清液组(HGF-hAFSCs)和单纯培养液对照组(Control)对DRG神经突起生长的影响。结果:从人羊水中获得的细胞为hAFSCs,其核型正常,表达CD29、CD73、CD90和CD105,不表达CD34和CD45。慢病毒感染72 h后,感染效率约为80%;经嘌呤霉素筛选,获得过表达HGF的hAFSCs。实时荧光定量PCR结果显示,HGF-hAFSCs组与hAFSCs组的细胞相比,HGF mRNA表达有显著性增加(P<0.001);ELISA结果显示,HGF-hAFSCs上清液中HGF的浓度为3.44 ng/ml,而hAFSCs组上清液未检测到,两组有显著性差异(P<0.001)。DRG组织内神经突起长度测量显示,hAFSCs组突起为(251.60±7.94)μm,对照组突起为(52.74±6.77)μm,而HGF-hAFSCs组突起较前两组都显著增加(P<0.001),为(306.57±10.65)μm。结论:利用hAFSCs过表达HGF有助于DRG神经的突起生长,为神经损伤提供了一种新的治疗方案。Objective:To explore the effects of culture supernatant of human amniotic fluid stem cells expressing hepatocyte growth factor on neurite growth of dorsal root ganglionic neurons in rats.Methods:Adherent cells were collected from amniotic fluid,and hAFSCs were examined by morphological analysis,karyotype analysis,and cell surface marker molecules.The hAFSCs were transfected with lentivirus to obtain HGF-hAFSCs over-expressing HGF.The culture supernatant of hAFSCs and HGF-hAFSCs was collected and centrifuged.Then we cultured dorsal root ganglion tissue mass with the culture supernatant in vitro,and compared with the growth of dorsal root ganglionic neurons among hAFSCs culture supernatant group(hAFSCs),HGF-hAFSCs culture supernatant(HGF-hAFSCs)and pure culture medium(control).Results:The cells collected from amniotic fluid were hAFSCs with normal karyotype,expressing CD29,CD73,CD90,and CD105,but not CD34 and CD45.Seventy-two hours later by lentiviral infection,the fluorescence rate was about 80%,and over-expression of HGF in hAFSCs was screened by puromycin.The results of real-time RT-PCR showed that the expression of HGF mRNA was significantly increased in HGF-hAFSCs group,compared with hAFSCs group(P<0.001).ELISA results showed that the concentration of HGF in the culture supernatant of HGF-hAFSCs group was 3.44 ng/ml,but HGF was not detected in hAFSCs group with this ELISA kit.Compared with the hAFSCs group(251.60±7.94)μm and the control group(52.74±6.77)μm,the average length of processes in the HGF-hAFSCs group was the longest(306.57±10.65)μm(P<0.001).Conclusion:These results indicate that the culture supernant of hAFSCs expressing HGF can promote neurites regeneration of dorsal root ganglionic neurons,which provides a new therapeutic strategy for nerve injury.

关 键 词：肝细胞生长因子 人羊水干细胞 背根节 神经突起 大鼠 

分 类 号：Q95-33[生物学—动物学]