红细胞裂解法分离及培养大鼠骨髓间充质干细胞

背景:骨髓间充质干细胞在骨髓中的含量非常少,而且与其他细胞特别是易与红细胞相混杂,因此在分离过程中需去除干扰,以获得尽可能多的高纯度的骨髓间充质干细胞。目的:采用红细胞裂解法分离培养大鼠骨髓间充质干细胞,同时进行生物学鉴定。设计:观察对比实验。单位:中国航天员科研训练中心航天细胞分子生物学实验室。材料:选用50只生后30d的雄性SD大鼠,体质量100g,SPF级,购自北京实验动物中心(合格证:SCXK(京)2002-0003)。DAB浓缩显色液(含过氧化氢,中杉公司),兔抗大鼠多克隆抗体(武汉博士德公司),FCS(PAA,Austria),LG-DMEM培养基(Sigma,USA)。方法:实验于2004-09/2005-09在中国航天员科研训练中心航天细胞分子生物学实验室完成。将大鼠脱臼处死,暴露骨髓腔,收集细胞悬液,采用全骨髓培养法对骨髓间充质干细胞分离与原代培养。①红细胞裂解实验:取A、B、C、D4管分别加入0.5mL过滤并充分吹打均匀后的骨髓冲洗液,分别对应加入红细胞裂解液、氯化氨2mL、磷酸盐缓冲生理盐水2mL和体积分数为0.04的乙酸溶液0.5mL,分别测量各组吸光度值及血红蛋白浓度,并观察细胞生长情况。②骨髓间充质干细胞生长曲线、倍增时间及表面标志分子表达情况的观察:根据公式:群体倍增时间(TD)=tlg2/(lgNt-lgN0)](N0和Nt分别代表接种后和培养t小时的细胞数),计算出细胞的群体倍增时间并描绘并分析骨髓间充质干细胞第2、4、6代细胞的生长曲线;采用亚甲基兰染色测定骨髓间充质干细胞增殖情况;采用免疫细胞化学染色检测骨髓间充质干细胞表面标志分子表达情况。主要观察指标:①大鼠骨髓间充质干细胞分离和培养的观察结果。②不同处理方法对红细胞的裂解效果及其对骨髓间充质干细胞生长的影响。③第2、4、6代细胞的生长曲线及细胞倍增时间。④大鼠骨髓间充质干细胞表面标志分子表达情况。结果:①大鼠骨髓间充质干细胞的分离和培养结果:原代培养48h后大部分细胞已贴壁,72h时贴壁细胞已开始分裂增殖。7￣8d后可见有明显集落形成,之后集落迅速增多,不断扩大,互相融合,14～16d时细胞克隆生长稠密。刚传代的细胞呈球形,很快沉降贴壁,少部分圆形细胞悬浮。贴壁细胞均匀分布,3～5d增殖迅速。虽然传代后细胞形态未变,但增殖速度明显增快,约6d长满培养瓶底。②不同处理方法对红细胞的裂解效果及其对骨髓间充质干细胞生长的影响:红细胞裂解液处理组,氯化氨处理组和4%乙酸处理组的血红蛋白浓度明显高于磷酸盐缓冲生理盐水处理组,差异有显著性(P<0.01)。③不同代骨髓间充质干细胞的生长曲线观察结果:第2,4,6代细胞生长曲线基本相同,经过一两天的潜伏适应期后进入对数生长期,第5天达顶点,以后进入平台期(在第5～7天)。第6代骨髓间充质干细胞的潜伏期表现不明显骨髓间充质干细胞的群体倍增时间平均约为34h。④不同代骨髓间充质干细胞的免疫表型鉴定:各代细胞CD44和CD106染色均呈阳性,表现为棕黄色颗粒沉淀,CD34染色呈阴性。结论:采用红细胞裂解液处理骨髓冲洗液后再行接种,能提高骨髓间充质干细胞的贴壁效率,同时不影响其贴壁后的生长,是一种可行的分离方法。细胞表面标记染色鉴定证明,本实验所分离细胞是骨髓间充质干细胞。

Isolation and culture of bone marrow mesenchymal stem cells by red blood cell lysis method

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are few in bone marrow, and they are easily mingled with other cells, especially red blood cells. Therefore, intervention needs to be depleted in the process of isolation of red blood cells so as to obtain highlypurified BMSCs as many as possible.OBJECTIVE: To isolate and culture BMSCs of rats with red blood cell lysis method, and perform biological identification.DESIGN: Observation and controlled trial.SETTING: Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts.MATERIALS: Fifty 30-day-old male SD rats, weighing about 100 g, of SPF degree, were purchased from Beijing Experimental Animal Center (License No. SCXK (Jing) 2002-0003) and involved in this trial. DAB condensed chromogen (hydrogen dioxide included, Zhongshan Company), rabbit anti-ret polyclonal antibody (Boster Co.,Ltd., Wuhan), fetal calf serum (PAA, Austria) and LG-DMEM medium (Sigma Company, USA) were used in this trial.METHODS: This trial was carried out in the Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts during September 2004 to September 2005. The rats were sacrificed by dislocation to expose bone marrow cavity. Cell suspension was collected. BMSCs were isolated and cultured primarily by whole bonemarrow culture method. ① Red blood cells lysis test: A, B, C and D 4 tubes were chosen and filled with 0.5 mL bonemarrow rinse solution which was filtered and fully beat upon. Then, red blood cell lysis buffer of 2 mL, ammonium chloride of 2 mL, phosphate buffer normal saline of 2 mL and 0.04 volume fraction acetic acid of 0.5 mL were correspondingly added into the 4 tubes. In each tube, absorbance and hemoglobin concentration were measured and cell growth was observed. ② Observation of growth curve, doubling time and surface marker molecule expression of BMSCs: Based on the formula, population doubling time (TD) =tlg2/(lgNt-lgN0)] (NO and Nt represented the cell number after inoculation and t hours after culture ,respectively), cell population doubling time was calculated and traced and growth curve of the 2nd, 4th and 6th generations of BMSCs were analyzed; The proliferation of BMSCs was measured by methylene blue staining method; Surface marker molecule expression of BMSCs was detected with immunocytochemical staining.MAIN OUTCOME MEASURES: ①Observation of isolation and culture of bone marrowmesenchymal stems of rats. ②Effect of different methods on the lysis of red blood cells and the growth of BMSCs. ③ The growth curve and cell doubling time of the 2nd, 4th and 6th generations of cells. ④Surface marker molecule expression of BMSCs of rats.RESULTS: ① Results of isolation and culture of BMSCs of rats: After 48-hour primary culture, most of the cells had adhered to the wall, and 72 hours later, division growth of the adherent cells presented. Seven to eight days later, cell colonies formed obviously, and then increased quickly, expanded incessantly and fused with each other. On 14 to 16 days, cell clones grew densely. Immediately generative cells presented ball-shape, subsided and adhered the wall verysoon. Some few round cells suspended. Adherent cells distributed evenly and proliferated quickly within 3 to 5 days. Although cell morphology of generative cells did not change after passage, cell proliferation was speeded up obviously and cells covered the bottom of the whole bottom on about 6 days. ② Effect of different methods on red blood cell lysis and the growth of BMSCs: hemoglobin concentration in the red blood cell lysate-treated group, ammonium chloride-treated group and 4％ acetic acid-treated group was significantly higher than that in the phosphate buffer normal saline-treated group, with significant difference (P ＜ 0.01). ③ Observation of growth curve of different generations of BMSCs: The growth curves of the 2nd, 4th and 6th generations of the cells were basically the same: latent period about 1 to 2 days, then logarithmic growth phase, peak on the 5th day and finally plateau phase (about on the 5th to 7th days). The latent period of the 6th generation of BMSCs was not obvious and the population doubling time of BMSCs was about 34 hours. ④ldentification of immunophenotype of different generations of BMSCs: Both CD44 and CD106 staining of each generation of cells were positive, presenting brown granule sediments, and CD34 staining was negative.CONCLUSION: Inoculation of red blood cells lysate-treated bone marrow rinse solution can boost the adherent rate of BMSCs, and does not influence its post-adherent growth, so it is a feasible separation method. Cell surface marker staining confirms that thecells isolated in this study are BMSCs.