摘要
目的:建立克隆化兔骨髓基质干细胞系,为组织缺损修复研究寻找理想的种子细胞提供新的途径。方法:实验于2004-09/2005-12在浙江省医学科学院生物工程研究所完成。①取3个月龄新西兰白兔,无菌抽取骨髓液,经密度梯度离心和贴壁培养,获得原代培养骨髓基质干细胞。用有限稀释法进行克隆化培养,选择单细胞克隆增殖细胞,进行扩大培养和液氮冻存保种。②取对数生长期第2,15和32代克隆化骨髓基质干细胞,用四甲基偶氮唑盐法测定其吸光值(A值),绘制生长曲线。③取第4和32代对数生长期骨髓基质干细胞,按常规方法制作染色体标本,进行染色体分析。④取第5代对数生长期骨髓基质干细胞,用成骨诱导试验进行体外分化潜能鉴定。⑤取第5代对数生长期的骨髓基质干细胞,按常规方法测定最适细胞接种密度。结果:①克隆化兔骨髓基质干细胞系的建立结果:获得一株克隆化兔骨髓基质干细胞系,其细胞形态为均一的长梭形,体外连续传代培养至32代,细胞仍保持旺盛的增殖能力。经-196℃冻存复苏后仍保持良好的生长状态。②生长曲线:克隆化骨髓基质干细胞的第2,15和32代细胞生长曲线基本相同,细胞增殖速度没有明显的下降趋势。③染色体分析:第4和32代克隆化兔骨髓基质干细胞的染色体众数均为44条,结构未见异常。显示该细胞在体外经过32代培养,仍保持稳定的二倍体核型。④最适接种密度:第5代对数生长期骨髓基质干细胞,经常规方法测定,以1×103/cm2的接种密度为最适接种密度。⑤体外分化潜能:第6代克隆化骨髓基质干细胞,在向成骨细胞诱导条件下,4,8,12碱性磷酸酶的表达均显著高于对照组[(375.41±13.17)比(239.38±7.33)nkat/L,(503.77±13.34)比(249.22±5.17)nkat/L,(605.95±10.84)比(298.73±8.17)nkat/L,P<0.01];茜素红染色显示有钙化结节形成,对照组为阴性。表明克隆化兔骨髓基质干细胞具有体外诱导分化潜能。结论:采用克隆化分离技术,成功获得了单细胞克隆的具有旺盛增殖能力和分化潜能,遗传性能稳定的兔骨髓基质干细胞系。
AIM： To establish cloned rabbit bone marrow mesenchymal stem cell （MSC） line, and provide new pathway for searching ideal seed cells of tissue defect repairing. METHODS： The experiment was carried out in the Bioengineering Institute of Zhejiang Academy of Medical Sciences from September 2004 to December 2005. ①The New Zealand rabbits of 3 months old were adopted in this study. MSC were isolated from rabbit bone marrow by density gradient centrifugation and their preferential adherence to the culture dish. Then the cells of the primary culture were cloned by limiting dilution assay and a cell line was established following expanding culture and liquid nitrogen cryopreservation. ②Cloned MSC of passages 2, 15 and 32 at logarithmic phase were selected to assess the absorbance （A value） by MTY method and draw growth curve. ③Cloned MSC of passages 4 and 32 at logarithmic phase were selected to establish chromosome samples routinely and conduct chromosome analysis. ④ Cloned MSC of passage 5 at logarithmic phase were selected to identify in vitro differentiation potential by ossify induction test and detect the optimal inoculating density. RESULTS： ①Establishment of cloned rabbit MSC： After 32 passages of in vitro consecutive culture, the cell line was still able to proliferate vigorously in uniform long-fusiform shape, and remained good growth after -196 ℃ cryopreservation.②Growth curve： Cloned MSC of passages 2, 15 and 32 presented similar growth curve, and proliferative velocity was not obviously decreased. ③Chromosome analysis： The modal numbers of cloned MSC of passages 4 and 32 were 44 pieces, without changes of their structure, suggesting that both its original diploid karyotype was kept after 32 passages of in vitro culture. ④The optimal inoculating density of passage 5 MSC at logarithmic phase was defined as 1×10^3/cm^2. ⑤ Differentiation potentials： Passage 6 cloned MSC expressed more alkaline phosphatase than control group and the osteoblast induction assay was positive [（375.41±13.17）/（239.38±7.33） nkat/L, （503.77±13.34）/ （249.22±5.17） nkat/L, （605.95±10.84）/（298.73±8.17） nkat/L, P 〈 0.01]. Alizarin bordeaux staining displayed the formation of calcificated nodes, and control group was negative for the staining. All the evidences showed that cloned rabbit MSC possessed in vitro differentiation potential. CONCLUSION： After cloned separation, the established MSC line presents high proliferation and differentiation potentials and is genetically stable.
出处
《中国临床康复》
CSCD
北大核心
2006年第45期1-3,I0003,共4页
Chinese Journal of Clinical Rehabilitation
