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

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<0.000 1)。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。

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

Abstract:

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<0.000 1). 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-transgenic DA-BMSCs cell line that stably produced DA, survived in large numbers, and differentiated in the rat brain was successfully established, laying a foundation for the treatment of PD using engineered culture and transplantation of DA-BMSCs.

Key words:Parkinson’s disease (PD);rat mesenchymal stem cells;tyrosine hydroxylase;dopamine decarboxylase;GTP cyclohydrolase 1;dopamine