【作者】 陈琼玉；

【导师】 洪岸；

【作者基本信息】 暨南大学 ， 遗传学， 2006， 硕士

【摘要】 目的：探索小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cell，MSC)体外分离培养及诱导成骨分化。分析细胞向成骨方向分化过程中相关转录因子Osx的表达情况。并进一步深入研究Osx对成骨早期标记基因Ⅰ型胶(alphal Type Ⅰ Collagen，COLLa1)表达的调控机制。 方法：本实验联合应用密度梯度离心和贴壁筛选的方法从小鼠骨髓中分离出间充质干细胞，采用MTT比色法检测不同接种密度、不同浓度的胎牛血清对MSC生长的影响，用骨形成蛋白BMP2诱导MSC向成骨方向分化。通过RT-PCR和巢式PCR方法从小鼠成肌干细胞C2C12中扩增出Osx全长基因序列，并通过分子克隆手段将Osx装入质粒pCDNA3.1。用脂质体转染的方法将Osx基因转入细胞，分别采用生物化学、组织化学法检测成骨分化指标碱性磷酸酶ALP的表达，用RT-PCR方法检测Osx的表达。最后用电泳迁移试验(electrophoretic mobility shift assay，EMSA)检测Osx对成骨分化早期标记基因COLLa1的调节机制。 结果：以5x10~3个／cm~2的密度接种、加入10％胎牛血清有利于MSC增殖，以8μg／ml自制BMP2诱导MSC，于诱导后第六天能检测到较高的ALP活性，促使MSC向成骨方向分化。Osx转入MSC后，用G418能筛选出稳定表达的克隆，同时能检测到成骨分化的指标。EMSA结果显示Osx与成骨早期标记基因COLLa1启动子发生了特异性结合。 结论：成功分离出MSC，并初步优化了MSC体外培养条件，利用BMP2成功诱导MSC向成骨方向分化，证实了成骨转录因子Osx成骨分化过程中的调控作用，为进一步深入探讨成骨分化过程中的其它调控机制提供的方法学上的基础。更多还原

【Abstract】 Object: To explore a method of isolating and culturing mouse bone marrow mesenchymal stem cell(MSC) and the differentiation of MSC in vitro, analyze the expression of osteogensis association transcription factor Osx, and further study the regulatory mechanism of the early bone maker alphal Type I Collagen (COLLal) by Osx.Methods: We separated and obtained MSC from bone marrow of mouse by gradeient centrifugation in percoll. the effect of different cell density, different concentration of fetal bovine serum(FBS) on MSC proliferation was assessed by MTT assay. After amplified the osx gene through RT-PCR and net PCR, we constructed recombinant plasmid pCDNA3. 1- Osx and transfected to MSC, then biochemistry and histochemistry methods were used to detect ALP activity and the expression of Osx. Finaly, we used electrophoretic mobility shift assay(EMSA) to further study the regulatory mechanism of the early bone maker COLLal by Osx.Results: The best culture conditions for MSC in vitro was 5×10~3cells/cm~2 10%FBS. after induced with 8μg/ml BMP2, MSC differentiated into osteoblast, we can detect higher expression of ALP 6days after induced, after transfection of pCDNA3.1-Osx plasmid, G418-resistant cells were selected , and some of the bone maker was detected. The result of EMSA strongly show that Osx can interreact with the promoter of COLLal.Conclusion: It is a simple effective and pratical method to separate and obtain higher purity and activity of MSC from bone marrow by gradeient centrifugation in percoll. the best culture conditions for MSC was established, and MSC was successfully induced to osteoblast in vitro, further study confirm that Osx play an important role in the process of osteoblast differentiation.更多还原