TPO基因修饰的基质细胞对巨核系细胞的定向扩增作用

Regulation of megakaryocytopoiesis by TPO gene-modified stromal cells

目的 研究TPO基因修饰的基质细胞对CD34+ 造血细胞向巨核系细胞定向增殖分化的影响。方法 将TPOcDNA构建到pBabe pura中 ,通过“乒乓转染法”获得产高滴度逆转录病毒载体的包装细胞 ;利用所获的病毒上清转染基质细胞HFCL ,用Northernblot检测细胞中TPO基因的表达 ,并利用TPO依赖株检测上清中的TPO活性 ;以未转基因的HFCL为对照 ,将CD34+ 造血细胞在TPO基因修饰的HFCL支持下扩增 ,用流式细胞仪检测扩增细胞中表型为CD34+ CD41+ 和表型为CD41+ CD6 1+ 细胞的比例。结果 基质细胞HFCL能够有效表达外源性TPO基因 ,在HFCL TPO培养上清中有 0 75ng ml水平的TPO活性 ;并且在HFCL TPO支持下 ,CD34+ 造血细胞经过 7d扩增后 ,CD34+ CD41+ 细胞的比例为 (13 7± 2 0 ) % ,HFCL为 (6 5± 1 8) % (P <0 0 1) ;CD41+ CD6 1+ 细胞的比例为 (11 9± 0 7) % ,略高于HFCL的 (10 6± 1 5 ) % (P >0 0 5 )。结论 基质细胞能够有效表达外源性TPO基因和其蛋白 ,而且TPO基因修饰的基质细胞能显著促进CD34+ CD41+ 巨核祖细胞的扩增 ,但对较成熟的CD41+ CD6 1+

Objective\ To study the effect of TPO gene\|modified stromal cells on stimulating megakaryocytopoiesis. Methods\ Retroviral vectors carrying TPO gene were constructed and used to transfect the stromal cell line HFCL by using a “Ping\|pang” technique.The expression of TPO gene was detected by Northern blot and dependent cell line TD\|3.By contrast with expansions supported by non\|modified stromal cells,CD34 + hematopoietic cells from cord blood were expanded on gene\|modified stromal cells for 7 days.Finally,percentages of CD34 +CD41 + cells and CD41 +CD61 + cells were analyzed by flow cytometry. Results\ The stromal cell line HFCL could express the TPO gene transduced by using the retroviral vectors,and the activity of TPO in supernatant of the tranduced HFCL cells was 0 75 ng/ml.The percentages of CD34 +CD41 + megakaryocyte\|committed progenitors in the presence of TPO gene\|modified HFCL cells(13 7%±2 0%) were higher than those in the nontransduced HFCL cells (6 5%±1 8%),and there was no difference statistically on the percentages of the CD41 +CD61 + cells between the cultures supported by transduced and non\|tranduced stromal cells. Conclusion\ Stromal cell line HFCL could express the TPO with biological activities after transfected by retroviral vectors,and enhance the growth of CD34 +CD41 + megakaryocyte\|committed progenitor cells.\