血管活性肠肽对人脐血CD34^+细胞增殖分化的影响

Effect of vasoactive intestinal polypeptide on proliferation and differentiation of human CD34^+ cells derived from cord blood

目的探索血管活性肠肽(vasoactiveintestinalpolypeptide,VIP)对造血干细胞(hematopoieticstemcells,HSC)增殖分化的影响及其可能机制。方法采用免疫磁珠分选技术纯化人脐血CD34+细胞;VIP孵育人CD34+细胞后,通过体外集落形成、扩增培养了解细胞增殖;ELISA测定细胞内外TNF-α、TGF-β水平;流式细胞术检测细胞表面CD13、CD14、CD71、CD41、CD19、CD4/CD8、CD33、CD34等抗原;生物分子相互作用系统检测人CD34+细胞VIP受体;RT-PCR分析其受体亚型。结果在1×10-7~1×10-12mol/L浓度范围内,VIP抑制人CD34+细胞集落形成及扩增倍数,其细胞集落形成抑制与VIP浓度呈正相关(r=0·925,P<0·01)。VIP(1×10-8mol/L)作用于人CD34+细胞14d后,粒系(CD13+蓝色)及单核系(CD14+)细胞百分比均明显下降(P<0·05)。VIP增加人CD34+细胞的CD34抗原表达(P<0·05)。VIP作用后CD34+细胞内、外TNF-α及TGF-β1浓度显著增加(P<0·05);人脐血CD34+细胞表达VIPI型受体。结论VIP通过人脐血CD34+细胞上VIPI型受体介导,提高造血抑制因子TNF-α、TGF-β水平,抑制人CD34+细胞的增殖、维持其干细胞特性,阻止其向粒、单系细胞分化。

Objective To investigate the effect and mechanisms of vasoactive intestinal polypeptide （VIP） on proliferation and differentiation of hematopoietic stem cells （HSC）. Methods Mononuclear cell （MNC） derived from human cord blood was isolated by Ficoll, Then MACS assay was used to purify human CD34^＋ cells from MNC. After treated with VIP, colony forming unit （CFU） enumeration and amplification were done to assess the effect on proliferation of human CD34^＋ cells. TNF-α and TGF-β in human CD34^＋ cells or its supernatant were measured with ELISA. the antigens such as CD13, CDI4, CD71, CD41 , CD19, CD4/CD8, CD33,CD34 were identified by flow cytometry （FCM）. Biomolecular interaction analysis and reverse transcriptation polymerase chain reaction were used to quantify receptor for VIP（VIPR） and detect its subtype. Results VIP at concentration of 1 ×10^-12 to 1 ×10^- 7 mol/L inhibited the proliferation of human CD34^＋ cells. The inhibition of CFU forming bore a positive relation with the concentration of VIP （ r =0. 925, P 〈0. 01 ）. VIP（ 1 ×10^-8mol/L） decreased the ratio of granulocyte （CD13^＋ ） and monocytes（ CD14^＋ ）in human CD34^＋ cell population （ P 〈 0.05）. Expression of CD34 in human CD34^＋ cells was up-regulated by VIP （ P 〈 0. 05 ）. The concentration of TNF-α or TGF-β1 of human CD34^＋ cells treated with VIP was increased as compared to control group （P 〈0.05）. Human CD34^＋ cells expressed receptor for VIPR-1. Conclusion Mediated by VIPR-1 in human CD34^＋ cells, VIP inhibited the proliferation of human CD34^＋ cells due to increasing the intracellular concentrations of TNF-α and TGF-β, the inhibitive eytokines for hematopoiesis. VIP tended to keep characteristics of human CD34^＋ cells and inhibit the differentiation of human CD34^＋ cells to granuloeyte-monoeytes.