用于治疗系统性红斑狼疮的脐带间充质干细胞的产品质量控制的研究

Quality Control of Umbilical Cord Mesenchymal Stem Cells for the Treatment of Systemic Lupus Erythematosus

摘要: 目的：分离培养符合用于治疗系统性红斑狼疮的产业化的脐带间充质干细胞(UC-MSC)，并进行脐带间充质干细胞产品质量控制的研究。方法：从脐带中通过贴壁法获得间充质干细胞，传代培养后，采用无血清培养体系扩增培养UC-MSC。通过流式细胞术和诱导分化技术，检测其中干细胞含量、细胞总数、细胞活率，并取样进行细菌检测、内毒素检测、支原体检测，满足质量标准方能应用。选取BALB/c小鼠，进行pristane造模，将BALB/c小鼠随机分为对照组、模型组和治疗组，对照组和模型组给予等量的生理盐水；治疗组给予UC-MSC (4 × 105 cells/ml)，0.2 ml。进行抗双链DNA (ds-DNA)抗体、系统性红斑狼疮活动度评分(SLEDAI)和常规血液检查。通过裸鼠致瘤性实验、毒性试验等评估脐带间充质干细胞产品的安全性。结果：经过技术改造升级，无血清培养的UC-MSC贴壁生长，主要为典型的成纤维样细胞形态，可传15代以上，第5代MSCs高表达CD73、CD105、CD90、CD44；低表达CD34、CD45和HLA-DR；在不同的诱导条件下，脐带MSCs可被诱导分化为成骨细胞和软骨细胞。治疗组UC-MSC给药30天，抗ds-DNA抗体水平明显降低，与对照组相比差异有显著性意义，疾病活动度评分明显降低，蛋白尿、血肌酐、尿素氮等指标得到改善，与模型组比较差异均具有统计学意义(P < 0.05)。结论：本研究建立了工业化生产UC-MSC的质量管理体系，验证了UC-MSC治疗系统性红斑狼疮的安全性及有效性，为后续开展UC-MSC治疗系统性红斑狼疮的临床试验开展提供理论依据和技术支持。

Abstract: Objective: To isolate and cultivate umbilical cord mesenchymal stem cells (UC-MSC) suitable for the treatment of systemic lupus erythematosus (SLE). Methods: MSCs were obtained from umbilical cord by adherent method. After subculture, UC-MSC was amplified by serum-free culture system. Through flow cytometry and induced differentiation technology, stem cell content, total number of cells and cell viability were detected, and samples were taken for bacterial detection, endotoxin detection and mycoplasma detection to meet the quality standards before application. To establish sound technical procedures and quality standards for the production, packaging, storage and transportation of umbilical cord mesenchymal stem cells; complete the quality control system plan, BALB/C mice were selected and pristane was used for modeling. BALB/C mice were randomly di-vided into control group, model group and treatment group. The treatment group was given UC-MSC (4 × 105 cells/ml), 0.2 ml. Antidouble-stranded DNA (ds-DNA) antibodies, systemic lupus erythem-atous Activity score (SLEDAI), and routine blood tests were performed. The safety of umbilical cord mesenchymal stem cells was evaluated by tumorigenicity test and toxicity test in nude mice. Re-sults and Conclusion: After technical modification and upgrading, the serum-free culture of UC-MSCs grew adherently, mainly in the form of typical fibroblasts, which could be transmitted over 15 gen-erations. The fifth generation MSCs showed high expression of CD73, CD105, CD90, CD44 and low expression of CD34, CD45 and HLA-DR. Under different induction conditions, umbilical cord MSCs could be induced to differentiate into osteoblasts and chondrocytes. After 30 days of UC-MSC ad-ministration, the level of anti-DS -DNA antibody in the treatment group was significantly reduced, with significant differences compared with the control group. The disease activity score was signifi-cantly reduced, and indicators such as proteinuria, serum creatinine and urea nitrogen were im-proved, with statistically significant differences compared with the model group (P < 0.05). This study established a quality management system for the industrial production of UC-MSC, verified the safety and effectiveness of UC-MSC in the treatment of systemic lupus erythematosus, and pro-vided theoretical basis and technical support for subsequent clinical trials of UC-MSC in the treat-ment of systemic lupus erythematosus.