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李大伟 高广州 李欣 孙涛 摘要
解放军海军总医院急救部ICU 北京 100048 目的:观察肠道的缺血再灌注损伤刺激对骨髓间充质干细胞(B M-M S C s)旁分泌作用的影响,以证实
B M-M S C s在肠道修复中的旁分泌作用。方法:分离、培养、鉴定W i s t a r雄性大鼠的骨髓间充质干细胞,制
备大鼠肠道缺血再灌注模型,分别提取肠道缺血再灌注模型大鼠、正常大鼠的肠上皮黏膜提取物,与B M-
M S C s共同培养;分别在培养的第0小时、第24小时留取条件培养基,E L I S A检测不同细胞条件培养基以及
普通培养基中T G F-α、V E G F、F G F、I G F-1的浓度。结果:T G F-α、V E G F、F G F、I G F-1在含有正常肠上皮
提取物的条件培养基中的浓度分别为84.05±29.58、51.01±14.91、84.87±30.60、65.44±23.93;在
含有缺血再灌注肠上皮提取物的培养基中的浓度分别为120.04±43.31、70.82±25.33、96.81±51.70、
81.62±22.08,均明显高于普通培养基中的浓度(P<0.05)。其中T G F-α、V E G F的浓度明显高于含有正常
肠上皮提取物的培养基(P<0.05)。结论:BM-MSCs可以旁分泌TGF-α、VEGF、FGF、IGF-1等生长因子;而
在缺血再灌注损伤的微环境下,BM-MSCs对部分生长因子的分泌功能得到增强。
关键词:间充质干细胞;缺血再灌注损伤;旁分泌;小肠上皮细胞
责任作者与联系方式:孙涛,E-mail: davidleeee@sina.com
肠道缺血再灌注损伤对骨髓间充质干细胞
旁分泌作用的影响
Impact of Intestinal Ischemia/reperfusion Injuries on Paracrine Action of BM-MSCs
Da-wei Li, Guang-zhou Gao, Xin Li, Tao Sun
Department of ICU, Navy General Hospital, Beijing, 100048
Abstract
Objective: The paracrine patterns of BM-MSCs in intestinal ischemia/reperfusion injuries were investigated.
Methods: BMSCs were isolated, cultured, characterized, and stained. The rat intestine IRI model was developed. Intestinal epithelial mucosal
extracts were obtained from IRI rat or normal rat. The extracts were added into the culture medium, and BM-MSCs were cultured with the conditioned
medium respectively. The medium was collected at different time points. The levels of TGF-α, VEGF, FGF and IGF-1 in the medium were assayed by
ELISA.
Result: The contents of all the growth factors detected in conditioned medium with intestinal extracts were significantly higher than in normal
medium(P<0.05). The levels of TGF-α, VEGF in culture medium containing IRI epithelium extracts were significantly higher than that in the medium
containing normal intestinal epithelium extracts(P<0.05).
Conclusion: BM-MSCs can secret TGF-α, VEGF, FGF, and IGF-1 in the presence of intestinal extracts from IRI rat in the culture. These factors
may stimulate the differentiation and proliferation of intestinal epithelial cells through paracrine mechanism.
Key Words: Mesenchymal stem cell; Ischemia/reperfusion injuries; Paracrine; Intestinal epithelial cells
Corresponding Author: Tao Sun, E-mail: davidleeee@sina.com
骨髓来源的间充质干细胞(b o n e m a r r o w-d e r i v e d Wistar成年雄性大鼠,体重约180~200g,均购自解放军
mesenchymal stem cells,BM-MSCs)是一种存在于骨髓内 军事医学科学院实验动物中心,饲养繁殖解放军第二军医大
的具有多向分化潜能的成体干细胞。由于B M-M S C s具有易于 学海军临床医学院实验动物中心SPF级动物实验室,实验过程
获得、多向分化、高度增殖的特点,成为近年来器官损伤修 中对动物的处置符合1988年国家科委《实验动物管理条例》
复的重要手段。在动物试验已经证实同种异体间充质干细胞 的规定。
(mesenchymal stem cells,MSCs)移植可定植于胃肠道,
并参与肠道损伤的修复[1,2]。对于MSCs参与受损组织修复的具 2.设备和试剂
体的机制,目前认为有直接分化和旁分泌两种方式[3-9],但仍 超净工作台(上海恒字医用器械厂),37℃恒温、
缺乏进一步的研究证实。本研究试图模拟肠道缺血再灌注损 5%C O2细胞培养箱(美国T h e r m o F o r m a公司),倒置生物光
伤的病理生理环境,观察肠道的缺血再灌注刺激对BM-MSCs旁 学显微镜(Nikon公司),正置显微镜及照相系统(Olympus
分泌的影响,以证实BM-MSCs在肠道修复中的旁分泌作用。 公司),流式细胞仪(Beckman Coulter 公司),常温台
式离心机(H e r a e u s s e p a t e c h公司)等。特级胎牛血清
一、材料与方法 (Invitrogen 公司),DMEM/F12培养基(Thermo公司),胰
1.材料 蛋白酶-E D T A消化液、青链霉素混合液、P B S磷酸盐缓冲溶液
(Solarbio公司),大鼠成纤维生长因子(FGF)、大鼠血管
Laboratory and Clinical Investigation 53 FAM 2013 Jan/Feb Vol.20 Issue 1
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