骨髓基质细胞和CD34+的造血祖细胞间的交流由connexin43型通道连接调节

hxf_86

Intercellular communication between bone marrow stromal cells and CD34+ haematopoietic progenitor cells is mediated by connexin 43-type gap junctions
2 y6 v. g. s5 u& h骨髓基质细胞和CD34+的造血祖细胞间的交流由connexin43型通道连接调节2 Y1 m* [. @. Z3 N; U: a
4 H, p8 v' N) `
摘要：% M. j/ ?$ V6 `5 q' d4 L3 N* r
关于造血祖细胞（HPC）和基质细胞间是否存在功能性的通道连接，一直存在着争议。原始的CD34+的HPC与人类骨髓基质细胞系L87/4共培养。利用具有高度敏感性的全细胞钳技术，可以发现大多数的（91%）CD34+的HPC与L87/4间有电流交流。更重要的是，）CD34+的HPC依附在L87/4后的1小时内可以观察到有效的偶联。通过对比，由L87/4组成的同类细胞间有显著更高的电偶联。单通道导电率分析发现了一种形式，以connexin43型通道连接为特征，对于同类和不同类的细胞都是这样的。这个Cx表型通过Cx-43特异性的单克隆抗体流式细胞仪分析和RT-PCR的验证。最后，染料转染实验利用降落伞技术使得染料扩散而不影响浆膜成分，这个实验是对电生理实验的补充。总的来说，我们的数据表明，功能性的Cx-43型通道连接存在于基质细胞和未成熟的造血祖细胞中，可能提供造血过程中一个重要的调节通道。
8 g4 {* I# k! k# H8 g2 ]' I2 M& A- Z; [( g1 [' f* i
Summary. The existence of functional gap junctions between haematopoietic progenitor cells (HPCs) and stromal
8 }& y+ C2 |/ f4 Ucells of the haematopoietic microenvironment in the human system is a controversial issue. Primary CD341 HPCs( k/ \5 l! h" P7 |4 `1 X) d+ K* y3 B
isolated from leukapheresis products were co-incubated with the human fibroblastoid bone marrow stromal cell. |! M+ D# G+ V& G3 }
line L87/4 in short-term liquid culture. Using the highly sensitive double whole-cell patch-clamp technique, we
' F2 Q. [  i7 h! ?5 H  y8 a! pfound that the majority (91%) of CD341 HPCs are electrically coupled to L87/4 cells. Importantly, efficient, {* J5 Z% ?$ L* Q
coupling was observed within 1 h of the attachment ofCD341 HPCs to plastic adherent L87/4 cells. By comparison,& ~9 }4 ?% r% |
homologous cell pairs formed by L87/4 cells exhibited a significantly higher electric coupling. Analysis of singlechannel conductances revealed an electric profile characteristic of connexin 43 (Cx43)-type gap junctions for both homologous and heterologous cell pairs. The Cx phenotype was confirmed using Cx43-specific monoclonal antibodies in a flow cytometric assay and reverse transcription polymerase chain reaction (RT-PCR) for the detection of Cx43 mRNA. Finally, the electrophysiological studies were complemented by dye-transfer experiments using the recently described `parachute' technique that allows the monitoring of dye diffusion without disruption of the plasma membrane. Taken together, our data indicate that functional Cx43-type gap junctions exist between stromal cells and immature HPCs and, thus, may provide an important regulatory pathway in haematopoiesis.
; r  l* N4 }, {8 S