摘要
目的:目前公认肾上腺素能β1受体含量和环磷酸腺苷水平在心肌细胞处于低氧状态时会发生改变,然而诱导分化的骨髓间充质干细胞经慢性低氧作用后能否表现与心肌细胞相同的特性还未知,故检测其β1受体mRNA表达及第二信使环磷酸腺苷活性变化。方法:实验于2006-02/2007-04在华中科技大学同济医学院附属协和医院心研所实验室完成。①动物:清洁级成年昆明小鼠10只,新生1d龄昆明小鼠40只,均购自华中科技大学同济医学院实验动物中心,实验过程中对动物的处置符合动物伦理学标准。②实验方法:成年小鼠麻醉后于肱骨及胫骨骨折处吸取适量骨髓,加入含15%胎牛血清、100U/mL青霉素G、80U/mL链霉素的IMDM培养基,贴壁法分离培养骨髓间充质干细胞。将新生鼠处死迅速取心脏,用Ⅱ型胶原酶消化离心,将生长较好的第1、2代心肌细胞制成1×109L-1细胞裂解液,体外模拟心肌微环境诱导分化骨髓间充质干细胞。设立2组,空白对照组仅加入IMDM培养基,实验组加入IMDM培养基和心肌细胞裂解液,隔天换液,继续培养1周后又分为4个亚组:常氧组,常氧不加药物作用72h;低氧组,用1%氧浓度作用72h;异丙肾上腺素组,用1×10-5mmol/L异丙肾上腺素作用72h;低氧+异丙肾上腺素组,用1%低氧和1×10-5mmol/L异丙肾上腺素联合作用72h。③实验评估:RT-PCR法检测骨髓间充质干细胞β1受体mRNA水平,放射免疫法测定骨髓间充质干细胞环磷酸腺苷的活性。结果:①骨髓间充质干细胞β1受体mRNA水平的表达:空白对照组不表达β1受体mRNA。作用72h后,常氧组表达β1受体mRNA,低氧组β1受体mRNA水平升高100%,异丙肾上腺素组、低氧+异丙肾上腺素组β1受体mRNA水平均降低40%。②骨髓间充质干细胞环磷酸腺苷活性检测:与常氧组比较,低氧组环磷酸腺苷活性无明显变化(P>0.05);异丙肾上腺素组环磷酸腺苷活性下降35%(P<0.05);低氧+异丙肾上腺素组与异丙肾上腺素组水平相当(P>0.05)。结论:①采用心肌细胞裂解液体外模拟心肌微环境诱导分化的骨髓间充质干细胞表达β1受体。②单纯低氧作用可明显升高骨髓间充质干细胞β1受体mRNA水平,环磷酸腺苷活性无变化。经异丙肾上腺素作用后,低氧环境无法显著改变β1受体mRNA水平和环磷酸腺苷活性。
AIM： It is well recognized that the levels of β1-adrenergic receptor and cyclic adenosine monophosphate （c-AMP） are altered during myocardial hypoxia. However, there are no data regarding regulation of β1-adrenergic receptors in myocardial-like marrow mesenchymal stem cells （MSCs） exposed to chronic hypoxia. We measured β1-adrenergic receptors at the mRNA level and the second messenger c-AMP activity. METHODS： The experiment was completed in the laboratory of Cardiology Institute, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from February 2006 to April 2007.（1）Ten clean Kuming adult mice and 40 newborn Kuming mice with one-day old were purchased from Tongji Medical College Test Animal Center. Animal dealing was obeyed by the ethical standard in the process.（2）After adult mice were anaesthetized, appropriate amount of bone marrow was absorbed from the fracture lesions of humerus and tibia. The IMDM culture medium containing 15% fetal bovine serum, 100 U/mL penicillin G and 80 U/mL streptomycin was added to separate the MSCs stuck closely to wall from cultured bone marrow cells. The newborn mice were put to death, and the hearts were promptly gained, which were then digested with type collagen Ⅱ enzyme and centrifugated. Myocardial cell lysate （1×10^9 L^-1） was made from fairly good 1^st and 2^nd generations of cardiac cells. MSCs were induced into differentiation in myocardial cell lysate＇s microenvironment in vitro and divided into 2 groups： blank control group was added with the IMDM culture medium only, while the experimental group with the IMDM culture medium and myocardial cell lysate, which was changed every two days. Four subgroups were set up after 1 week： normoxic group, MSCs were cultured for 72 hours; hypoxia group, MSCs were cultured in 1% oxygen for 72 hours; isoproterenol group, MSCs were cultured in 1×10^-5 mmol/L isoproterenol for 72 hours; hypoxia and isoproterenol group, MSCs were cultured in 1% oxygen and 1×10^-5 mmol/L isoproterenol for 72 hours.（3）RT-PCR was used to determine β 1 acceptor mRNA expression and cAMP was measured by radioimmunoassay in cultured MSCs. RESULTS： （1） β1 acceptor mRNA expression in MSCs： β1 acceptor mRNA did not express in the control group. After 72 hours, mice in the normoxic group expressed β1 acceptor mRNA, and β 1 acceptor mRNA expression in the hypoxia group increased 100% while that in isoproterenol group and hypoxia and isoproterenol group both decreased 40%.（2）cAMP activity in MSCs： Compared with normoxic group, cAMP activity in the hypoxia group did not change significantly （P 〉 0.05）; cAMP activity in the isoproterenol group decreased 35% （P 〈 0.05）; there was identical cAMP activity between hypoxia and isoproterenol group and isoproterenol group （P 〉 0.05）. CONCLUSION： （1）lnduced and differentiated MSCs by myocardial cell lysate, which simulates cardiac microenvironment ex vivo can express β 1 acceptor.（2）Hypoxia increases β 1-mRNA expression in the induced MSCs significantly, and has no effect on c-AMP activity. Hypoxia can not change β 1-mRNA expression and cAMP activity in the induced MSCs significantly after isoproterenol effect.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2301-2304,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
