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医学论文范文:体外非接触共培养诱导骨髓间充质干细胞向肺泡上皮细胞的分化
【摘要】 目的 建立体外非接触共培养模型，诱导小鼠骨髓间充质干细胞(MSCs)向肺泡上皮细胞的分化。方法 分三组，每组6例。对照组：小鼠MSCs单独培养组;实验组A：正常肺组织单细胞悬液+MSCs;实验组B：损伤肺组织单细胞悬液+MSCs。共同培养8d，激光共聚焦和RT-PCR检测共培养体系下室中的SP-C和AQP5的表达情况。结果 对照组和实验组A都只检测到AQP5;而实验组B可同时检测到AQP5和SP-C。实验组B的AQP5 mRNA的表达量较对照组明显增多(P<0.01)，而与实验组A比较，其AQP5 mRNA的表达量也有统计学差异(P<0.01)。但是，实验组A与对照组比较则无明显统计学差异(P>0.05)。结论 本实验室成功建立了体外非接触诱导小鼠MSCs分化为肺泡上皮细胞的实验模型。
【关键词】 骨髓间充质干细胞;肺泡上皮细胞;激光共聚焦
Establishment of co-culture model in vitro to induce bone marrow mesenchymal stem cells differentiate into lung epithelial cellsWANG Yan， YANG Zhi-jun, HE Xi-yu, FENG Zhi-chunDepartment of Pediatrics, Southern Medical University Affiliated Zhujiang Hospital,Guangzhou 510080; Department of Neonatology, Beijing Military General Hospital Affiliated Bayi Childrens Hospital, Beijing 100700, ChinaABSTRACT: Objective To establish the co-culture model in vitro and induce bone marrow mesenchymal stem cells (MSCs) to differentiate into lung alveolar epithelial cells. Methods Each group had 6 samples, control group was MSCs alone; Group A was the MSCs cultured with the cells from normal lung; and Group B was the MSCs with the cells from injuried lung. Each group was cultured for 8 days and the two markers of lung alveolar epithelial cells including AQP5 and SP-C were tested by laser confocal microscopy and RT-PCR. Results Only AQP5 was detected in the control group and Group A, both AQP5 and SP-C were detected in Group B, the AQP5 mRNA expression in Group B was significantly increased compared with that in the control group(P<0.01). The AQP5 mRNA expression in Group B was also significantly increased compared with that in Group A (P<0.01). But there was no significant difference in AQP5 mRNA expression between Group A and control group. Conclusion We have successfully established the co-culture model in vitro to induce bone marrow mesenchymal stem cells to differentiate into lung epithelial cells医学全.在线www.med126.com.
KEY WORDS: mesenchymal stem cells; lung alveolar epithelial cell; laser confocal microscopy
骨髓间充质干细胞(mesenchymal stem cells, MSCs)是一种具有多分化潜能的干细胞，在不同的培养条件下可分化为一系列的细胞类型。肺损伤体内实验研究表明，肺损伤小鼠移植MSCs后，MSCs可转化为肺泡Ⅱ型(type Ⅱ alveolar epithelial cell, AECⅡ)和Ⅰ型上皮细胞(type Ⅰ alveolar epithelial cell, AECI)等，以修复损伤肺组织[1]。ROJAS等[2]研究发现，在体外损伤肺组织分泌的因子可以诱导MSCs增殖并向损伤肺迁移，而正常肺组织细胞并没有此功能。POPOV等[3]采用MSCs与肺上皮细胞(A549)非接触共同培养，成功诱导MSCs向肺上皮细胞分化。本研究采用MSCs与肺组织单细胞悬液非接触共培养模型，观察能否在体外利用损伤肺组织诱导MSCs向肺泡上皮细胞分化,为进一步研究骨髓源性的间充质干细胞治疗肺损伤奠定基础。
1 材料与方法
1.1 材料
出生4周的健康昆明小鼠，体重为10.0～11.5g，共25只，均为雄性(中国医学科学院实验动物中心提供)。DMEM完全培养基和标准胎牛血清(GIBCO公司，美国)，胰蛋白酶(Sigma公司，美国)。Taq DNA聚合酶(TaKaRa公司，日本)，RT-PCR试剂盒(QIAGEN，德国)。 倒置显微镜(TE2000, Nikon公司，日本)，高分辨率成像系统(DXM1200C，尼康，日本)，激光共聚焦显微镜(C1-SHS，尼康公司，日本)，二氧化碳细胞培养箱(SKP-02B，湖北省黄石市恒丰医疗公司)，24孔板(Hanging Cell Culture Insert PET 0.4μm，MILLPORE公司，德国)。
1.2 小鼠骨髓间充质干细胞(MSCs)的体外分离、扩增和鉴定
