乙型肝炎病毒感染者脂肪间充质干细胞分离培养及生物学特性

Isolation, culture and biological characteristics of adipose-derived mesenchymal stem cells from patients with hepatitis B virus infection

背景:慢性乙型肝炎病毒感染影响骨髓间充质干细胞的生物学特性,而脂肪来源间充质干细胞因其取材安全、创伤小、易纯化、增殖快等优点而备受关注。目的:建立乙型肝炎病毒感染者脂肪间充质干细胞的体外分离培养方法,观察其生物学特性。方法:胶原酶消化结合贴壁培养法分离培养乙型肝炎肝硬化患者皮下脂肪组织间充质干细胞,MTT法检测细胞生长曲线,流式细胞仪检测细胞表型,体外检测其诱导分化为脂肪细胞和成骨细胞的能力。结果与结论:110例乙型肝炎病毒感染者脂肪间充质干细胞全部培养成功,脂肪间充质干细胞的原代培养时间为(8.3±1.2)d,生长曲线均呈"S"形,培养三四天进入对数生长期,第7天进入平台期。2第3代脂肪间充质干细胞高表达CD29,CD166,HLA-ABC和CD44,不表达或低表达CD34和HLA-DR。3脂肪间充质干细胞在成脂肪诱导培养基培养下分化为脂肪细胞,油红O染色阳性,在成骨诱导培养基培养下分化为成骨细胞,碱性磷酸酶染色阳性。以上结果显示胶原酶消化结合贴壁培养法可以高效分离乙型肝炎病毒感染者脂肪间充质干细胞,其增殖迅速,在短期内即可获得大量的细胞。

BACKGROUND： Chronic hepatitis B virus infection can impact the biological characteristics of bone marrow mesenchymal stem cells. Adipose-derived mesenchymal stem cells have gained more and more attention due to their high safety, little invasiveness, easy purification and rapid proliferation. OBJECTIVE： To establish the isolation and culture method of adipose-derived mesenchymal stem cells from patients with hepatitis B virus infection in vitro, and to observe the biological characteristics of cells. METHODS： Adipose-derived mesenchymal stem cells were isolated from the subcutaneous fat of hepatitis B virus infection patients by collagenase digestion and adherent method. Growth curve of adipose-derived mesenchymal stem cells were detected by MTT method and cell phenotypes were detected by flow cytometry. The adipogenic and osteogenic differentiation potential of adipose-derived mesenchymal stem cells were detected in vitro. RESULTS AND CONCLUSION：（1） Adipose-derived mesenchymal stem cells from 10 patients with hepatitis B virus infection were all isolated and cultured successfully. The primary passage time of adipose-derived mesenchymal stem cells was（8.3±1.2） days. The growth curve of cells was ＂S＂ shaped. Cells came into a logarithmic phase at days 3-4, and came into platform at day 7.（3） Passage 3 adipose-derived mesenchymalstem cells highly expressed CD29, CD166, HLA-ABC and CD44, but did not express or lowly expressed CD34 and HLA-DR.（3） Adipose-derived mesenchymal stem cells differentiated into adipocytes after adipogenic induction, and differentiated cells were positive for oil red O staining; after osteogenic induction, adipose-derived mesenchymal stem cells differentiated into osteoblasts that were positive for alkaline phosphatase staining. These findings indicate that the collagenase digestion and adherent method can be used to effectively isolate adipose-derived mesenchymal stem cells from patients with hepatitis B virus infection, and the cell proliferation is rapid so that a large number of cells can be obtained in the short term.