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骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

周小莹, 王绮如, 黄艳红, 程腊梅, 谭孟群

中南大学湘雅医学院生理学系血液生理研究室,中南大学生殖与干细胞工程研究所.河南,长沙 410078

摘要

该文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum--free murine bone marrow endothelial cell conditioned medium,mBMEC--CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC--CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright's Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[~(3)H]--TdR掺入量,观察mBMEC--CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC--CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF检测阳性。mBMEC--CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[~(3)H]--TdR掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[~(3)H]--TdR掺入量。外源加入IL--6、IL--11、SCF、GM--CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[~(3)H]--TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM--CSF、SCF、MSP--1、endothelin--2、thymosin #beta#10、connective tissue GF、PDGF--A chain、MIP--2#alpha#、PlGF、neutrophil activatingprotein ENA--78、INF--#gamma#、IL--1、IL--6、IL--13、IL--11、inhibin--#alpha#等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。

关键词： 骨髓内皮细胞; 无血清条件培养液; 增殖

Promoting effects of serum--free murine bone marrow endothelial cell conditioned medium on the growth of bone marrow endothelial cells

Zhou Xiaoying, Wang Yiru, Huang Yanhong, Cheng Lamei, Tan Mengqun

Experimental Hematology Laboratory, Department of Physiology, Xiangya School of Medicine；China

Abstract

To study the effects of murine bone marrow endothelial cell conditioned medium on the growth of bone marrow endothelial cells, serum-free murine bone marrow endothelial cell conditioned medium (mBMEC-CM) was collected and ultrafiltration was performed with a centriprep 10 cut off molecular weight 10kD. The retentate of mBMEC-CM (mw>10kD) and the filtrate of mBMEC-CM (mw<10kD) were obtained. The effect of bone marrow conditioned media, their components and exogenous cytokines on the formation of endothelial cell colonies were observed. The effect of bone marrow conditioned media, their components and exogenous cytokines on the proliferation of murine bone marrow endothelial cells were determined by [~(3)H]-thymidine incorporation.The method of hybridizing to the Atlas cDNA Array was used to determine the expression of cytokine mRNAs in bone marrow endothelial cells. The results obtained are as follows: vWF was expressed in bone marrow endothelial cells. The original mBMEC-CM and mw>10kD component of mBMEC-CM promoted the proliferation of bone marrow endothelial cell colonies and increased [~(3)H]-Thymidine incorporation of bone marrow endothelial cells. The mw<10kD component did not affect the production of endothelial cell colonies did not increase [~(3)H]-thymidine incorporation of endothelial cells. Six cytokines( IL-6, IL-11, SCF, GM-CSF, VEGF, bFGF) promoted the proliferation of bone marrow endothelial cell colonies. VEGF, bFGF and SCF increased [~(3)H]-thymidine incorporation of bone marrow endothelial cells. According to the results of the Atlas cDNA Array, GM-CSF,TGF-#beta#,BMP-2, bFGF, SCF, endothelin-2 ,thymosin#beta#10, MSP-1, connective tissue GF, PDGF-A Chain, MIP-2#alpha#，PlGF，neutrophil activating protein ENA-78，INF-r，IL-1，IL-6，IL-13，IL-11，inhibin-#alpha# mRNAs were expressed in endothelial cells. These results suggest that murine bone marrow endothelial cell conditioned medium promoted the proliferation of bone marrow endothelial cells.

Key words: Bone marrow endothelial cells;serum-free condition medium;Proliferation

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引用本文：

周小莹, 王绮如, 黄艳红, 程腊梅, 谭孟群. 骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用[J]. 生理学报 2005; 57 (2): .

Zhou Xiaoying, Wang Yiru, Huang Yanhong, Cheng Lamei, Tan Mengqun. Promoting effects of serum--free murine bone marrow endothelial cell conditioned medium on the growth of bone marrow endothelial cells. Acta Physiol Sin 2005; 57 (2): (in Chinese with English abstract).