稳定高效合成多巴胺的骨髓间充质干细胞系的创建和鉴定  

Establishment and characterization of bone marrow mesenchymal stem cell lines stably synthesizing high-level dopamine

作　　者：刘阳 常俊彦 王悦 杨盼 马彩云 刘高峰 郭俣 刘长青 王春景 LIU Yang;CHANG Junyan;WANG Yue;YANG Pan;MA Caiyun;LIU Gaofeng;GUO Yu;LIU Changqing;WANG Chunjing(School of Life Sciences,Bengbu Medical College,Bengbu 233000,Anhui,China;School of Laboratory Medicine,Bengbu Medical College,Bengbu 233000,Anhui,China)

机构地区：[1]蚌埠医学院生命科学学院,安徽蚌埠233000 [2]蚌埠医学院检验医学院,安徽蚌埠233000

出　　处：《生物工程学报》2023年第4期1773-1788,共16页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(81771381);安徽省高校自然科学基金项目(KJ2021ZD0085,KJ2021A0774,KJ2021A0784);安徽省重点研发计划(2022e07020030,2022e07020032);蚌埠医学院研究生科研创新计划项目(Byycx21050);国家级大学生创新创业训练项目资助(202110367043,202110367044,20210367058);蚌埠医学院厅重点科研平台开放课题(AHTT2022B001)。

摘　　要：创建能稳定合成多巴胺(dopamine,DA)递质的三转基因(tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1,TH/DDC/GCH1)的骨髓间充质干细胞系(bone marrow mesenchymal stem cells,BMSCs),移植至帕金森大鼠模型脑内,为帕金森病(Parkinson’s disease,PD)的临床治疗提供实验依据。通过三转基因的重组慢病毒创建能稳定合成并分泌DA递质的DA-BMSCs稳定转染细胞系,利用反转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)、蛋白免疫印迹(Western blotting)、免疫荧光等技术检测BMSCs中三转基因(TH/DDC/GCH1)的表达,采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和高效液相色谱法(high performance liquid chromatography,HPLC)检测DA的合成情况。染色体G-带法检测DA-BMSCs的遗传稳定性。将DA-BMSCs移植于6-羟基多巴胺(6-hydroxydopamine,6-OHDA)偏侧损毁帕金森大鼠模型右脑的前脑内侧束(medial forebrain bundle,MFB)区,检测其在PD大鼠脑内微环境中的存活、分化情况。阿扑吗啡(apomorphine,APO)诱导旋转实验检测DA-BMSCs移植后PD大鼠的运动障碍改善情况。DA-BMSCs细胞系中TH/DDC/GCH1三转基因均能够稳定高效表达,而正常BMSCs对照组中均不表达。三转基因感染组(DA-BMSCs)和LV-TH感染组细胞培养上清液中DA浓度极其显著高于BMSCs空白对照组(P<0.0001)。传代后的DA-BMSCs仍能稳定合成DA,并保持正常二倍体核型(94.5%)。DA-BMSCs移植PD大鼠脑内4周后,显著改善PD大鼠模型的运动障碍,在脑内微环境中大量存活,分化为TH+和GFAP+细胞,并显著上调脑移植区域的DA水平。本研究成功创建了能稳定分泌DA,在大鼠脑中大量存活并分化的三转基因DA-BMSCs细胞系,为DA-BMSCs工程化培养与移植治疗PD奠定基础。A triple-transgenic(tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1,TH/DDC/GCH1)bone marrow mesenchymal stem cell line(BMSCs)capable of stably synthesizing dopamine(DA)transmitters were established to provide experimental evidence for the clinical treatment of Parkinson’s disease(PD)by using this cell line.The DA-BMSCs cell line that could stably synthesize and secrete DA transmitters was established by using the triple transgenic recombinant lentivirus.The triple transgenes(TH/DDC/GCH1)expression in DA-BMSCs was detected using reverse transcription-polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Moreover,the secretion of DA was tested by enzyme-linked immunosorbent assay(ELISA)and high-performance liquid chromatography(HPLC).Chromosome G-banding analysis was used to detect the genetic stability of DA-BMSCs.Subsequently,the DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle(MFB)of Parkinson’s rat models to detect their survival and differentiation in the intracerebral microenvironment of PD rats.Apomorphine(APO)-induced rotation test was used to detect the improvement of motor dysfunction in PD rat models with cell transplantation.The TH,DDC and GCH1 were expressed stably and efficiently in the DA-BMSCs cell line,but not expressed in the normal rat BMSCs.The concentration of DA in the cell culture supernatant of the triple transgenic group(DA-BMSCs)and the LV-TH group was extremely significantly higher than that of the standard BMSCs control group(P<0.0001).After passage,DA-BMSCs stably produced DA.Karyotype G-banding analysis showed that the vast majority of DA-BMSCs maintained normal diploid karyotypes(94.5%).Moreover,after 4 weeks of transplantation into the brain of PD rats,DA-BMSCs significantly improved the movement disorder of PD rat models,survived in a large amount in the brain microenvironment,differentiated into TH-positive and GFAP-positive cells,and upregulated the DA level in the injured area of the brain.The triple-trans

关 键 词：帕金森病 大鼠间充质干细胞 酪氨酸羟化酶 多巴胺脱羧酶 GTP环化水解酶1 多巴胺 

分 类 号：Q813.11[生物学—生物工程] R742.5[医药卫生—神经病学与精神病学]