Zarei Mahboobeh, Ferdosi-Shahandashti Elaheh, Badalzadeh Mohsen, Kardar Gholam Ali
Student Research Committee, Babol University of Medical Sciences, Babol, I.R. Iran.
Biomedical and Microbial Advanced Technologies (BMAT) Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, I.R. Iran.
Iran J Biotechnol. 2024 Apr 1;22(2):e3772. doi: 10.30498/ijb.2024.409915.3772. eCollection 2024 Apr.
Coagulation factor VIII (FVIII) is applied for spontaneous hemorrhaging inhibition and excessive bleeding after trauma in patients with hemophilia A. High-quality human recombinant factor VIII (rFVIII) has been produced relatively in large quantities in cultured mammalian cells. NS0 is one of the most common mammalian cell lines for therapeutic protein production. Production of rFVIII has increased due to low FVIII expression levels and rising demand for hemophilia A prophylactic treatment. Several methods have been developed to prevent cell cycle progression in mammalian cells for increased recombinant protein yields.
The aim of the study was to investigate the level of recombinant BDD-FVIII expression in NS0 mouse myeloma cells. Additionally, the study aimed to determine the effects of chemical drugs, Mitomycin C, Lovastatin, and Metformin on the secretion of FVIII through cell cycle arrest.
We cultured NS0 cells and transfected them with the 2 μg pcDNA3-hBDDFVIII plasmid by Lipofectamine 3000. The cells were treated with 10 μg.mL Mitomycin C, 20 μM Lovastatin, and 20 mM Metformin separately. After 24 and 48 hours, the samples were collected and, protein expression was analyzed using RT-PCR, Dot blot, and ELISA.
A higher protein expression level was observed in treated cells 24h and 48h after treatment with all three drugs. According to real-time PCR, Metformin treatment resulted in the highest expression level within 24 h (P=0.0026), followed by Mitomycin C treatment within 48 h (P=0.0030).
The NS0 cell line can be regarded as a suitable host for FVIII production. FVIII protein expression level was increased by using Lovastatin, Metformin, and Mitomycin C drugs. Further investigations are suggested, and the potential application of these drugs to increase recombinant protein yield can be used to produce therapeutic proteins in the industry.
凝血因子VIII（FVIII）用于抑制甲型血友病患者的自发性出血及创伤后的过度出血。高质量的人重组因子VIII（rFVIII）已在培养的哺乳动物细胞中相对大量生产。NS0是用于治疗性蛋白质生产的最常见哺乳动物细胞系之一。由于FVIII表达水平低以及对甲型血友病预防性治疗的需求不断增加，rFVIII的产量有所提高。已开发出几种方法来阻止哺乳动物细胞的细胞周期进程，以提高重组蛋白产量。
本研究旨在调查NS0小鼠骨髓瘤细胞中重组BDD-FVIII的表达水平。此外，该研究旨在确定化学药物丝裂霉素C、洛伐他汀和二甲双胍通过细胞周期阻滞对FVIII分泌的影响。
我们培养NS0细胞，并通过Lipofectamine 3000用2μg pcDNA3-hBDDFVIII质粒转染它们。细胞分别用10μg/mL丝裂霉素C、20μM洛伐他汀和20mM二甲双胍处理。24小时和48小时后，收集样本，并使用RT-PCR、斑点印迹和ELISA分析蛋白质表达。
在用所有三种药物处理后24小时和48小时，处理后的细胞中观察到更高的蛋白质表达水平。根据实时PCR，二甲双胍处理在24小时内导致最高表达水平（P = 0.0026），其次是48小时内的丝裂霉素C处理（P = 0.0030）。
NS0细胞系可被视为生产FVIII的合适宿主。使用洛伐他汀、二甲双胍和丝裂霉素C药物可提高FVIII蛋白表达水平。建议进一步研究，这些药物在提高重组蛋白产量方面的潜在应用可用于工业生产治疗性蛋白质。
