Li Y, Douglas S D, Ho W
Department of Pediatrics, University of Pennsylvania Medical School, Philadelphia 19104, USA.
J Hematother Stem Cell Res. 2000 Aug;9(4):445-52. doi: 10.1089/152581600419107.
We studied the expression of Substance P (SP) and its receptor in an established human stem cell line (TF-1) and primary stem cells derived from human placental cord blood (HPCB). Using reverse transcriptase polymerase chain reaction (RT-PCR) assay, SP mRNA is detected in both TF-1 cells and HPCB stem cells. Among the alpha, beta, gamma, and delta transcripts of the SP gene, only the beta, gamma, and delta transcripts are detectable in these cells. These RT-PCR-amplified transcripts are confirmed by Southern blot assay using a specific SP probe. Sequence analysis of the RT-PCR-amplified products transcribed from mRNA extracted from the HPCB stem cells also confirmed that these transcripts are identical to those found in human neurons. At the protein level, TF-1 cells produced endogenous SP as determined by an enzyme-linked immunosorbent assay (EIA). Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released SP from TF-1 cells. In addition, using RT nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (NK-1R, the receptor for SP) in both TF-1 cells and HPCB stem cells, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human stem cells express SP and its receptor support the notion that SP is biologically involved in the hematopoietic regulating network.
我们研究了P物质（SP）及其受体在已建立的人干细胞系（TF-1）和源自人胎盘脐带血（HPCB）的原代干细胞中的表达。使用逆转录聚合酶链反应（RT-PCR）检测法，在TF-1细胞和HPCB干细胞中均检测到SP mRNA。在SP基因的α、β、γ和δ转录本中，这些细胞中仅可检测到β、γ和δ转录本。使用特异性SP探针的Southern印迹检测法证实了这些RT-PCR扩增的转录本。对从HPCB干细胞提取的mRNA转录的RT-PCR扩增产物进行的序列分析也证实，这些转录本与在人神经元中发现的转录本相同。在蛋白质水平上，通过酶联免疫吸附测定（EIA）确定TF-1细胞产生内源性SP。辣椒素，一种香草基脂肪酸酰胺（辣椒成分），可从TF-1细胞中释放SP。此外，使用RT巢式-PCR分析，我们在TF-1细胞和HPCB干细胞中均鉴定出神经激肽-1受体（NK-1R，SP的受体）的mRNA存在，这通过Southern印迹和DNA测序分析得到证实。人干细胞表达SP及其受体这一发现支持了SP在造血调节网络中具有生物学作用这一观点。
