兔骨髓基质干细胞诱导为骨髓源成骨细胞的时间及功能特征

Time of bone marrow stroma cells induced into bone marrow derived osteoblasts and the functional characteristics in rabbits

目的:观察经体外培养、扩增后的兔骨髓基质干细胞,应用诱导剂促使其转化为功能活跃的成骨细胞的功能及其时间特征。方法:实验于2004-03/10在为哈尔滨医科大学第一临床医学院实验中心进行。实验动物选择36只日本大耳白兔,均由胫骨髓腔抽取骨髓,每只抽取量为2.0mL,抗凝,将其加入淋巴细胞分离液,密度梯度离心,取有核细胞层,加入DMEM培养液进行贴壁培养、传代,取生长状态良好的第3代骨髓基质干细胞,以适量的地塞米松、β-甘油磷酸钠和抗坏血酸作为诱导剂进行诱导,使其向成骨细胞转化,并通过倒置相差显微镜、透射电镜分别对细胞的形态、细胞的内部显微结构进行观察,并应用反转录-聚合酶链反应方法检测其分泌的碱性磷酸酶和Ⅰ型胶原。结果:原代骨髓基质干细胞于24～48h开始贴壁,细胞形态为长梭形,核小,呈椭圆形,核浆比例小,三四天细胞集落形成,逐渐增大,2周时集落边缘融合,细胞呈单层旋涡状分布,形态均一。传代培养的骨髓基质干细胞于24h内开始贴壁,均匀生长,体积大,无集落形成,细胞排列规则。骨髓基质干细胞在加入诱导剂3d后其形态逐渐由长梭形转变为短梭形,1周后变为多角形或鳞片形,其胞体、细胞核和核浆比例逐渐增大;诱导的细胞细胞器不发达,为低分化细胞,突起内见丰富的粗面内质网;在诱导1周后,细胞开始分泌碱性磷酸酶、Ⅰ型胶原。碱性磷酸酶扩增产物长度为408bp,Ⅰ型胶原扩增产物长度为206bp。结论:兔骨髓基质干细胞在加入诱导剂1周后,细胞开始分泌碱性磷酸酶和Ⅰ型胶原,并形成钙结节,细胞功能状态良好,说明诱导1周后骨髓基质干细胞已成功向成骨细胞转化,并形成为功能活跃的骨髓源成骨细胞,是与支架材料构建组织工程骨的最佳时机。

AIM： To observe the bone marrow stroma cells （BMSCs） cultured and amplified in vitro, and observe the function of osteoblast transformed from BMSCs and the time characteristics. METHODS： The experiment was carried out in the laboratory center of the First Clinical Medical College of Haerbin Medical University from March to October 2004. Thirty-six Japanese big-ear rabbits were selected. Bone marrow （2.0 mL） was extracted from each rabbit from the medullary cavity of tibia, and then anticoagulated and added into the separating medium of lymphocytes. After density gradient centrifugation, cells were taken from karyocyte layer in DMEM by adherent culture and passage to obtain the third generation BMSCs. Sufficient dexamethasone, β sodium glycerophosphate and vitamin C were added as inductors to induce BMSC to osteoblasts. The form and inside microstructure of the cells were observed under inverted phase contrast microscope and transmission electron microscope, and the alkaline phosphatase and type Ⅰ collagen secreted by osteoblasts were detected with reverse transcription-polymerase chain reaction （RT-PCR）. RESULTS： The primary BMCs began to adhere at 24-48 hours, the forms were long fusiform, nuclear was small and oval-shape, the proportion of karyoplasms was small, and the cell colony formed at 3-4 days, then gradually amplified, and the colony margin fused at 2 weeks, the cells were single-whirlpool distributed, and the form was uniform. The suboultured BMCs began to adhere within 24 hours, grew evenly, volume was great, no colony formed, and the cells distributed regularly. At 3 days after added inductor, the form turned from long fusiform to short fusiform gradually, and changed to polygon or squamelliform after 1 week, and the cell body and cell nucleolus enlarged, karyoplasmic ratio increased also.The induced cells were poorly differentiated and cellular organs did not become prosperous with rough endoplasmic reticulum observed in cell process; Alkali phosphatase and type Ⅰ collagen were secreted after one week. The lengths of the amplified products of alkali phosphatase and type Ⅰ collagen were 408 bp and 206 bp respectively. CONCLUSION： At 1 week after added inductor, the cells began to secrete alkali phosphatase and type Ⅰ collagen, and the cellular function was good, indicating that bone marrow stroma cells are successfully induced into osteoblast with active function by one week, and formed bone marrow derived osteoblasts with active function, and it is the best occasion for cage material constructed tissue engineering bone.