Querol S, Cancelas J A, Amat L, Capmany G, Garcia J
Cryobiology and Cell Therapy Department and Barcelona Cord Blood Bank, Institut de Recerca Oncològica (I.R.O.), Hospital Universitari Sant Joan de Déu, Barcelona, Spain.
Haematologica. 1999 Jun;84(6):493-8.
Granulocytic colony-stimulating factor (G-CSF) is a cytokine widely used for several purposes such as stem cell mobilization, treatment of neutropenia or in vitro cultures. Recombinant human G-CSF (rh-G-CSF) is available in two forms: a non-glycosylated (E. Coli-derived), and a glycosylated CHO-derived rhG-CSF. As previously shown, glycosylation gives a higher degree of homology between the recombinant and the wild human G-CSF molecule. This study analyses the role of glycosylation in expansion cultures comparing the biological effects of the two forms of G-CSF.
CD34+ cells from nine cord blood samples were positively selected (median purity 84%) and cultured in the presence of 50 ng/mL of stem cell factor and 1, 10 or 100 ng/mL of glycosylated rh-G-CSF (GG) or a deglycosylated form (DG). After 5 days of a static, serum-dependent culture fed on day 0, nucleated cells (NC), CD34+ cells and colony-forming units were evaluated and compared using the paired Student's t-test.
For all concentrations tested, GG was able to generate more NC and progenitors than DG was able to (p<0.05). This effect was mainly observed in CFU-GM colonies, and in CFU-Mix, and indeed no influence was detected in terms of BFU-E expansion. The presence of GG in culture causes the generation of more mature granulocytic cells, assessed by the expression of CD11b/CD15 on CD13+ population, than the presence of DG. In order to check the role of the molecule's stability in this difference, the effect of daily supplementation was tested. Continuous presence of cytokines using either form of G-CSF (daily feeding) significantly increased the rate of expansion, but again GG produced higher generation than its DG counterpart.
Our results suggest that the stability of the G-CSF molecule has a predominant effect on the higher biological activity found. A glycosylated form of G-CSF is recommended for in vitro cultures using serum-dependent conditions.
粒细胞集落刺激因子（G-CSF）是一种细胞因子，广泛应用于多种用途，如干细胞动员、中性粒细胞减少症的治疗或体外培养。重组人G-CSF（rh-G-CSF）有两种形式：非糖基化的（大肠杆菌来源）和糖基化的中国仓鼠卵巢细胞（CHO）来源的rhG-CSF。如先前所示，糖基化使重组人G-CSF分子与野生型人G-CSF分子之间具有更高的同源性。本研究通过比较两种形式的G-CSF的生物学效应，分析糖基化在扩增培养中的作用。
从9份脐血样本中阳性选择CD34+细胞（中位纯度84%），并在含有50 ng/mL干细胞因子和1、10或100 ng/mL糖基化rh-G-CSF（GG）或去糖基化形式（DG）的条件下培养。在第0天进行静态、血清依赖性培养5天后，使用配对学生t检验评估并比较有核细胞（NC）、CD34+细胞和集落形成单位。
对于所有测试浓度，GG比DG能够产生更多的NC和祖细胞（p<0.05）。这种效应主要在CFU-GM集落和CFU-Mix中观察到，实际上在BFU-E扩增方面未检测到影响。与DG相比，培养中GG的存在导致通过CD13+群体上CD11b/CD15的表达评估的更成熟粒细胞的产生。为了检查分子稳定性在这种差异中的作用，测试了每日补充的效果。使用任何一种形式的G-CSF持续存在细胞因子（每日投喂）显著提高了扩增率，但GG再次比其DG对应物产生更高的产量。
我们的结果表明，G-CSF分子的稳定性对所发现的更高生物学活性具有主要影响。对于使用血清依赖性条件的体外培养，推荐使用糖基化形式的G-CSF。
