非诱导条件下犬骨髓基质干细胞的生物特性

背景种子细胞的研究是组织工程众多研究领域中最重要的基础环节,骨髓基质干细胞以其诸多优点,成为理想的骨组织工程的种子细胞.目的观察骨髓基质干细胞在体外培养的生长特点和非诱导条件下的成骨特性.设计单一样本实验.单位福建医科大学附属口腔医院种植中心.材料实验于2003-05/12在福建医科大学附属口腔医院中心实验室完成.骨髓基质干细胞来自4只1岁龄的杂交犬的原代～第3代传代细胞.方法无菌条件下在杂交犬两侧股骨大转子部做骨髓穿刺,抽取5 mL肝素抗凝的骨髓,加入到含有50 mL含双抗Dulbecco's改良的Eagle's培养液的离心管中分离单个核细胞,进行首次提纯获取骨髓基质干细胞单细胞,置培养箱中培养传代,培养48 h后,吸除培养液,以后每3天换液1次.继续传代.采用倒置相差显微镜及苏木精-伊红染色观察骨髓基质干细胞形态;每天进行细胞计数,测定倍增时间,绘生长曲线;用钙钴法检测碱性磷酸酶;用茜素红法染色观察钙化结节生长情况.主要观察指标①犬骨髓基质干细胞光镜结构.②犬骨髓基质干细胞生长曲线.③成骨分化指标碱性磷酸酶及钙化结节的观察.结果①形态学观察表明,骨髓基质干细胞贴壁细胞呈集落生长,有成纤维样细胞外观,未加入成骨诱导剂,细胞形态发生变化.②碱性磷酸酶表达原代细胞呈强阳性,第1代细胞呈阳性,第2,3代细胞呈弱阳性.③钙沉积出现,原代细胞染色强于传代细胞.结论①骨髓基质干细胞在体外培养能大量扩增,具有自然向成骨细胞分化的能力,是骨组织工程理想的种子细胞.②3代内扩增的骨髓基质干细胞有成骨活性,但原代细胞传代活性优于传代后细胞.

Biological characteristics of canine bone marrow mesenchymal stem cells in vitro under non-inducing conditions

BACKGROUND: Research on seed cells is the most important aspect in the filed of tissue-engineering research, and because of their various ad vantages, bone marrow mesenchymal stem cells (BMSCs) have been taken as the ideal bone tissue-engineering seed cells. OBJECTIVE: To observes the growth characteristics of in vitro cultured BMSCs and their osteogenetic characteristics under non-inducing conditions. DESIGN: A single sample experiment.SETTING: Implanting Center of Stomatological Hospital of Fujian Medical University MATERIALS: This experiment was conducted at the Central Laboratory of Stomatological Hospital of Fujian Medical University, between May and December 2003. BMSCs are derived from the primary passage to the 3rd passage of in vitro cultured cells from 4 one-year old dogs. METHODS: Under the aseptic conditions, bone marrow puncture was made at bilateral femur trochanter and 5 mL of heparin anticoagulated bone marrow was obtained. Then it was placed in 50 mL of anti-Dulbecco's modified Eagle's culture medium in centrifuge tube for monocyte isolation. The BMSCs single cells were primarily isolated and placed in culture box for subculture. After 48 hours, culture medium was removed and the medium was cha1ged once every 3 days. Then subculture was carried on continuously to observe BMSCs in morphology under inverted thephase contrast microscope with the assistance of HE staining. The number of cells was counted daily to calculate doubling time and to draw a growth curve. Alkaline phosphatase was detected by using calcium-cobalt method,and chinalizarin staining was used to detect the growth state of calcification tubercle. MAIN UTCOME MEASURES: ①Optical microscopic structure of dog BMSCs; ② The growth curve of dog BMSCs; ③Observation of osteogenetic index- alkaline phosphatase and calcification tubercle. RESULTS: ① Morphological observation indicated that BMSCs were adherent to the walls, clonogenic and appearing fibroblastic phenotype, and they presented morphological changes without exposing to osteogenetic inducer. ② The expression of Alkaline phosphatase in primary cells was stronger, and it was strong in the 1st passage cell, but weak in the 2nd and 3rd passage cells. ③Calcium deposition was observed, which was stronger in primary cells than in subcultured cells. CONCLUSION: ①BMSCs massively proliferated during in vitro culture,capable of differentiating into osteoblasts and considered as the optimal seed cells for bone tissue-engineering reconstruction. ② BMSCs derived from the 3rd passage has osteogenic activity, but the osteogenic activity of the primary cultured cells was stronger than thatr of the subcultured cells.