低氧预处理骨髓间充质干细胞耐受缺血缺氧的能力

中国组织工程研究 第 17 卷 第 49 期 20131203 出版 Chinese Journal of Tissue Engineering Research December 3, 2013 Vol.17, No.49 doi:10.3969/j.issn.2095-4344.2013.49.003 Xu QY, Xia L, Wang JC. Hypoxic preconditioning effects on the ability of bone marrow mesenchymal stem cells tolerant to ischemia and hypoxia. Zhongguo Zuzhi Gongcheng Yanjiu. 2013;17(49): 8474-8480. P.O. Box 1200, Shenyang 110004 www.CRTER.org 8474 www.CRTER.org Xu Qiao-yan, Attending physician, Department of Respiratory Medicine, the Peoples Hospital of Laizhou, Yantai 261400, Shandong Province, China Corresponding author: Wang Ji-chang, M.D., Attending physician, Department of Plastic Surgery, the Second Hospital of Shandong University, Jinan 250033, Shandong Province, China Accepted: 2013-09-18 (201303132/WJ) Hypoxic preconditioning effects on the ability of bone marrow mesenchymal stem cells tolerant to ischemia and hypoxia* Xu Qiao-yan1, Xia Lin2, Wang Ji-chang3 1 Department of Respiratory Medicine, the Peoples Hospital of Laizhou, Yantai 261400, Shandong Province, China 2 Department of Plastic Surgery, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China 3 Department of Plastic Surgery, the Second Hospital of Shandong University, Jinan 250033, Shandong Province, China Abstract BACKGROUND: According to the previous studies, it is known that bone marrow mesenchymal stem cells live in the physiological environment of lower oxygen concentration compared with the common culture concentration (20%-21%). Hypoxia can promote bone marrow mesenchymal stem cells proliferation and maintain the characteristics of differentiation potential. OBJECTIVE: To understand the biological characteristics of bone marrow mesenchymal stem cells after hypoxic preconditioning, and search for in vitro basis for bone marrow mesenchymal stem cells therapy in vivo with hypoxic preconditioning strategy. METHODS: After passage and inoculation, bone marrow mesenchymal stem cells were cultured in the hypoxic incubator (1% O2, 5% CO2). Bone marrow mesenchymal stem cells cultured in the normoxic incubator (20% O2, 5% CO2) served as controls. RESULTS AND CONCLUSION: Compared with bone marrow mesenchymal stem cells cultured under normoxia (20%O2), bone marrow mesenchymal stem cells cultured under hypoxia (1% O2) began to display exponential growth phase after a longer incubation period. Hypoxia (1% O2) was not the lethal condition for bone marrow mesenchymal stem cells. Cell survival rates of both groups reduced over time, while those of bone marrow mesenchymal stem cells cultured under normoxia reduced faster (P 0.05; Table 1). Expression of VEGF mRNA and protein The expression of VEGF mRNA increased after bone marrow mesenchymal stem cells being cultured under hypoxia within 48 hours (Figure 3).The VEGF protein level was found significantly higher in the supernatant from the HpBMSCs group, (214.0520.24 ) pg/L, than that from the nBMSCs group, (126.5516.58) pg/L (P 0.05; Figure 5). DISCUSSION When compared with the atmosphere, it was found that bone marrow mesenchymal stem cells live in the hypoxic physical microenvironment. The physical oxygen concentrations varies from 2% to 8% according to previous studies23. As shown in our study, the growth curves of bone marrow mesenchymal stem cells under hypoxia and normoxia demonstrated that bone marrow mesenchymal stem cells growth under hypoxia was inhibited for a longer time after incubation, while they both entered the platform period after a 4-day logarithm multiplication period. We could deduce that 1% O2 is non-lethal condition for BMSCs and can be used for hypoxic preconditioning. In the present study, 1% O2 (48 hours) culture was selected for hypoxic preconditioning. After being transplanted to the injury sites, bone marrow mesenchymal stem cells live in the hypoxia/ischemic condition for a long time and a lot of cells die6-7.During cell therapy, the bone marrow mesenchymal stem cells were usually suspended and transplanted with serum- free culture medium. We applied the sealed serum-free DMEM (low glucose) to imitate the hypoxia/ischemic condition in vivo and study the survival rate of HpBMSCs. The results of the present study demonstrated that hypoxic preconditioning could improve the survival rate of bone marrow mesenchymal stem cells in the sealed serum-free DMEM (low glucose). Hypoxic preconditioning could be used to improve the therapy results by improving the survival rate of transplanted cell. However, hypoxic preconditioning could not change the trend of cell death under hypoxia/ischemic condition for a long time. It means that the transplanted cells need the restoration of oxygen and nutrient supply as soon as possible to survive further longer. It has been found that bone marrow mesenchymal stem cells can maintain their stemness under a certain low oxygen concentration5. We applied flow cytometry to examine the surface markers of bone marrow mesenchymal stem cells before and after hypoxic preconditioning. Because the hematopoietic stem cells cannot grow adherently, we did not select the anti-CD34 antibody. We preconditioned the bone marrow mesenchymal stem cells under hypoxia, which may introduce the bone marrow mesenchymal stem cells differentiated into vascular endothelial cells. We selected the anti-CD31 antibody to examine the trend. However, the results showed that the expression of CD29(+), CD90(+), CD31(-) and CD45(-) did not change greatly after hypoxic preconditioning. That means HpBMSCs did not differentiate into vascular endothelial cells, still maintained their stemness and could be used for cell therapy or seed cells of tissue engineering. The mechanism of hypoxic preconditioning on cell protection is mainly studied in the nervous system. There are some factors involved in the mechanism, such as VEGF, SOD, Bcl22 and HSP70. Figure 5 The change of hypoxia-inducible factor 1 (HIF-1) expression after hypoxic preconditioning aP 0.05, vs. 0 h group; bP 0.05, vs. 24 h group. Data are represented as the ratio of HIF-1/-actin. a ab 1.5 1.0 0.5 0 HIF-1/-actin 0 h 24 h 48 h 0 h 24 h 48 h HIF- -actin Figure 4 The change of vascular endothelial growth factor (VEGF) protein expression in supernatant after hypoxic preconditioning nBMSCs HpBMSCs aP 0.05, vs. HpBMSCs group. HpBMSCs: hypoxia preconditioned bone marrow mesenchymal stem cells; nBMSCs: normoxic bone marrow mesenchymal stem cells. a 250 200 150 100 50 0 The expression of VEGF in supern (pg/mL) Xu QY, et al. Hypoxic preconditioning effects on the ability of bone marrow mesenchymal stem cells ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 8479 www.CRTER.org However the primary factor mediating the response is hypoxia-inducible factor-1 (HIF-1), an oxygen-sensitive transcriptional activator. HIF-1 consists of a constitutively expressed subunit HIF-1 and an oxygen- regulated subunit HIF-1. The HIF-1 subunit is regulated by oxygen sensing and activated under hypoxia. The present study demonstrated that HIF-1 expression increased 24 hours after hypoxic preconditioning and maintained a high level until 48 hours. The expression of VEGF mRNA and protein increased, too. These results back the mechanism that HIF-1 activation promote VEGF expression under hypoxia for cell survivability. Some scholars have transfected the HIF-1 gene into endothelial progenitor cells and found that the proliferation, differentiation and migration of endothelial progenitor cells are increased. The transfected endothelial progenitor cells can promote angiogenesis in vivo when they are transplanted to the ischemic limb. We all know that gene transfection has many shortcomings, which restrict its clinical application. While hypoxic preconditioning is a simple and feasible method to improve bone marrow mesenchymal stem cells survival and promote the VEGF secretion, it may be used for cell therapy for ischemic diseases. In summary, culture under hypoxia (1% O2) does not produce a lethal effect on rat bone marrow mesenchymal stem cells and can be used for hypoxic preconditioning. Surface markers of bone marrow mesenchymal stem cells after hypoxic preconditioning under hypoxia (1% O2) cannot change greatly and maintain the characteristics of stemness. Hypoxic preconditioning can promote the ability of BMSCs to surviveunder hypoxia/ ischemic condition. The activation of the hypoxic response signal transduction pathway through HIF-1 may explain this. REFERENCES 1 Malgieri A, Kantzari E, Patrizi MP, et al. Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art. Int J Clin Exp Med. 2010;3(4):248-269. 2 Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation. 2003;75(3):389-397. 3 Li Y, McIntosh K, Chen J, et al. 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Ann NY Acad Sci. 2005;1049:1-8. 低氧预处理骨髓间充质干细胞耐受缺血缺氧的能力* 徐巧岩 1，夏 琳 2，王吉昌 3 (1山东省莱州市人民医院呼吸科，山东省烟台市 261400；2山东大学齐鲁医院整形科，山东省济南市 250012；3山东大学第二医院整形科，山东省济南市 250033) 徐巧岩，女，1976 年生，山东省莱州市人， 汉族，1999 年山东医科大学毕业，主治医 师。 通讯作者：王吉昌，博士，主治医师。山东 大学第二医院整形科，山东省济南市 250033 文章亮点： 1 实验特点即在于利用体积分数 1% 氧对大鼠骨髓间充质干细胞进行低氧预 处理，检测其对大鼠骨髓间充质干细胞 干性及低氧无血清条件下低氧预处理的 大鼠骨髓间充质干细胞存活率的变化。 2 实验首次证实体积分数 1%氧浓度 (处理 48 h)可以作为低氧预处理大鼠骨髓 间充质干细胞氧浓度；且预处理后其表 型未发生明显变化，能提高低氧无血清 条件下大鼠骨髓间充质干细胞存活率。 关键词： 干细胞；骨髓干细胞；骨髓间充质干细 胞；低氧预处理；低氧诱导因子 1；血 管内皮生长因子；大鼠；国家自然科学 基金 主题词： 缺氧；间质干细胞；干细胞移植；细胞 低氧；氧 基金资助： 国家自然科学基金项目青年项目 (30900309)* 摘要 背景：有研究表明骨髓间充质干细胞的 生理环境氧体积分数低于常规培养用的 20%-21%，适度低氧体积分数下，骨髓 间充质干细胞表现出促进生长增殖，保 持分化潜能的特性。 目的：观察低氧预处理后的大