SD大鼠骨髓间充质干细胞原代培养条件的选择

发布时间：2018-08-22 10:14

【摘要】：目的探讨分离及培养SD大鼠骨髓间充质干细胞(BMSCs)的最佳条件。方法选取不同周龄(2、3、4、8 w)的SD大鼠;采用全骨髓贴壁筛选法对不同的培养基(L-DMEM、DMEM/F12),血清体积分数(7.5%、10%、12.5%、15%、17.5%),首次换液时间(48、72、96 h),换液方法(全换液、半换液)分组进行培养,每日于倒置显微镜下观察细胞形态、数目的变化,细胞融合80%~90%时进行消化计数,记录达到传代要求的时间,取第三代细胞绘制BMSCs生长曲线,各组间免疫荧光检测CD34、CD44的表达率。结果 1第2~3周SD大鼠达到传代要求时BMSCs数目最多、所需时间最短,第8周BMSCs含量明显减少,难以形成集落生长,细胞融合不到50%;DMEM/F12培养基中的细胞贴壁早、活性强,L-DMEM培养基中的细胞生长缓慢,两组相比差异显著(P0.05);72 h全换液和48 h半换液组细胞数目多,二者相比无统计学意义(P0.05),24 h全换液细胞数目少,72h半换液杂细胞多,细胞呈全分布,二者增值速度慢,与前者相比差异显著(P0.05);体积分数越高细胞生长分化越快,时间越短,但各组间比较无统计学意义(P0.05),传代培养中高体积分数的细胞易老化。2接种圆形BMSC培养6 h后贴壁,分化成圆形、类圆形或多角形;72 h后有少量短梭形、星形细胞分散、贴壁生长,部分细胞伸出一支或多支伪足,类似于成纤维细胞;约6 d后BMSCs细胞集落呈放射状排列,伸出长短及粗细均不规则,伴形态各异的伪足、细胞胞核大、核仁清晰;约10 d细胞能融合80%~90%培养面;传代后24 h内可见细胞已完全贴壁,3 d可见以梭形为主的细胞铺满80%~90%培养面;经过1~2次传代后,细胞呈放射状或平行排列,形态趋于一致。3传代2 d后,细胞缓慢生长,处于生长停滞期;3 d起细胞转入对数生长期,4~5 d至高峰后转入平台期,细胞倍增时间约39.5 h。4传代BMSc存在CD44抗原表达,阳性率98.3%,而CD34呈阴性表达。结论全骨髓贴壁筛选法是一种获得高纯度BMSCs简单、有效的方法;在适宜的培养条件下(SD大鼠育龄为2~3 w,接种密度80%~90%培养面,培养基为DMEM/F12,合适血清体积分数为10%,72 h首次全换液)BMSCs在体外生长状态良好,增殖速度快,DAPI可作为标记BMSCs的一种有效手段,也可作为细胞免疫组化的基础染色。
[Abstract]:Objective to investigate the optimal conditions for isolation and culture of bone marrow mesenchymal stem cells (BMSCs) from SD rats. Methods Sprague-Dawley rats (SD rats of different weeks) were selected and cultured in different culture medium (L-DMEMU DMEM / F12), serum volume fraction (7.5%), and the first time of fluid exchange (487296 h), method (total exchange, half exchange) by using the method of whole bone marrow adherent screening (L-DMEM-1 / F12), the serum volume fraction (7.5%) and the first time of fluid exchange (487296 h),) were used to culture the SD rats of different weeks (2 weeks, 3 weeks and 4 weeks old), and then cultured in different culture medium (L-DMEMU DMEM / F12). The changes of cell morphology and number were observed under inverted microscope every day. The cells were digested and counted at the time of cell fusion 80 ~ 90. The time to reach the requirement of passage was recorded. The BMSCs growth curve was drawn from the third generation of cells. The expression rate of CD34 and CD44 was detected by immunofluorescence. Results (1) when SD rats reached the requirement of passage at the 2nd week, the number of BMSCs was the most, the time required was the shortest, the content of BMSCs decreased obviously at the 8th week, and it was difficult to form colony growth. The cells in the medium of cell fusion were less than 50% DMEM / F12 and adhered to the wall early. The number of cells in the active L-DMEM medium was slow, and the difference between the two groups was significant (P0.05). The number of cells in the 72 h and 48 h groups was more than that in the control group (P0.05), and the number of the total cells in the 24 h group was less than that in the 24 h group (P0.05), and the number of the mixed cells in the 24 h group was more than that in the control group. The cells were distributed completely, and the increment rate of the two cells was slower than that of the former (P0.05); the higher the volume fraction, the faster the cell growth and differentiation, the shorter the time. However, there was no significant difference between the three groups (P0.05). The cells with high volume fraction in the subculture were easy to be aged by inoculating circular BMSC for 6 h and then differentiated into round cells. After 72 h, there were a few short fusiform cells in round or polygonal cells, and the astrocytes were dispersed. After 6 days, the colony of BMSCs cells were arranged radially, the length and thickness of the cells were irregular, with different pseudopodia, the nucleus of the cells was large, and the nucleoli were clear. After about 10 days, 90% of the cells were fused to 80% of the surface of culture, and within 24 h after passage, the cells were completely attached to the wall. The fusiform cells were covered with 80% or 90% of the surface of culture within 3 days. After 1 or 2 passages, the cells were arranged radially or in parallel. The morphology tended to converge for 2 days, the cells grew slowly, and the cells were transferred to the logarithmic growth period from 4 days to the peak after 3 days of growth stasis. The cell doubling time was about 39.5 h. 4. There was CD44 antigen expression in BMSc. The positive rate was 98.3%, while the expression of CD34 was negative. Conclusion the method of whole bone marrow adherent screening is a simple and effective method for obtaining high purity BMSCs. The culture medium was DMEM / F12, and the appropriate serum volume fraction was 1072 h.) BMSCs grew well in vitro. DAPI could be used as an effective method to label BMSCs, and it could also be used as the basic staining of cell immunohistochemistry.
【作者单位】：杭州市红十字会医院;大连大学附属中山医院;
【基金】：杭州市医疗卫生及重点专科专病科科研攻关专项(20140733Q26)
【分类号】：R329.2

【参考文献】