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目的:通过研究人类免疫缺陷病毒1型(HIV-1)Tat蛋白对骨髓间充质干细胞(BMSC)造血支持功能的影响,进一步揭示HIV-1感染者造血损伤的机理。方法:原代培养BMSC,流式检测其表面标志,诱导分化鉴定其多向分化潜能;免疫磁珠分选造血干细胞(HSC),流式检测其纯度;HIV-1 Tat蛋白添加到培养基中培养20天的BMSC(BMSC_(Tat))与对照BMSC(BMSC_(Con))分别作为滋养层与HSC分6组进行共培养,随后计数各组造血细胞总数,诱导分化检测造血细胞集落形成能力;RT-PCR检测BMSC_(Tat)和BMSC_(Con)造血相关因子mRNA的表达强度,ELISA检测BMSC_(Tat)和BMSC_(Con)条件培养液中造血相关因子GM-CSF及IL-6的浓度。结果:经鉴定成功培养获得原代BMSC;免疫磁珠分选的HSC纯度可达95%以上;分6组共培养进行比较,以BMSC_(Tat)为滋养层培养的造血细胞总数及造血细胞形成的集落总数均明显少于以BMSC_(Con)为滋养层;BMSC_(Tat)的造血相关因子的mRNA的表达明显弱于BMSC_(Con),BMSC_(Tat)的条件培养液中GM-CSF和IL-6的浓度均明显低于BMSC_(Con)。结论:HIV-1 Tat蛋白对BMSC的造血支持功能有明显的抑制作用。
OBJECTIVE: To investigate the effect of HIV-1 Tat protein on hematopoietic function of bone marrow-derived mesenchymal stem cells (BMSCs) and further reveal the mechanism of hematopoietic damage in HIV-1 infected patients. Methods: BMSCs were cultured in primary culture, and their surface markers were detected by flow cytometry. Differentiation was induced to differentiate into multi-differentiation potential. Hematopoietic stem cells (HSCs) were sorted by immunomagnetic beads and their purity was detected by flow cytometry. Cultures of BMSC (BMSC_ (Tat)) and control BMSC (BMSC_ (Con)) cultured for 20 days were co-cultured with HSC in six groups as trophoblast respectively. Then the total number of hematopoietic cells in each group was counted and induced to differentiate to detect hematopoietic colony forming ability. The mRNA expression of hematopoietic related factors (BMSC_ (Tat) and BMSC_ (Con)) was detected by RT-PCR. The hematopoietic-related factors GM-CSF and IL-6 in BMSC_ (Tat) and BMSC_ (Con) Results: The primary cultured BMSCs were successfully obtained after being identified. The purity of HSC was over 95% by immunomagnetic bead sorting. The total number of hematopoietic cells and the hematopoietic cells The total number of colonies was significantly less than that of BMSC_ (Con). The mRNA expression of hematopoietic related factors of BMSC_ (Tat) was significantly weaker than that of BMSC_ (Con) and BMSC_ (Tat) -6 concentrations were significantly lower than BMSC_ (Con). Conclusion: The HIV-1 Tat protein significantly inhibits the hematopoietic function of BMSCs.