Dai Guangchun, Li Yingjuan, Xu Hongliang, Lin Yucheng, Liu Junyan, Xu Lin, Rui Yunfeng
Department of Orthpaedics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing Jiangsu, 210000, P.R.China;School of Medicine, Southeast University, Nanjing Jiangsu, 210000, P.R.China.
Department of Geriatrics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing Jiangsu, 210000, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2017 Jul 15;31(7):845-852. doi: 10.7507/1002-1892.201703033.
To explore the effects of cryopreservation on the cell survival rate, cell viability, early apoptosis, migration ability, and tendon-related marker expression of tendon-derived stem cells (TDSCs) in rat patellar tendons.
The patellar tendon tissues were harvested from 12 4-month-old male Sprague Dawley rats; 12 patellar tendon tissues from 6 rats were cryopreserved (the experimental group), and the other 12 patellar tendon tissues were not treated (the control group). The patellar tendons were digested with 0.3% type I collagenase to obtain nucleated cells. The survival rate of nucleated cells was detected by trypan blue exclusion assay, and colony-forming ability by crystal violet staining. TDSCs were isolated and cultured to passage 3 (P3). The cell viability of TDSCs was detected by Alamar Blue method, the early apoptosis by Annexin V-FITC/PI assay, the cell migration ability by Transwell method, and the mRNA expressions of tendon-related markers [collagen type I (Col1α1), scleraxis (Scx), and tenomodulin (Tnmd)] by real-time quantitative PCR.
The survival rate of nucleated cells was 91.00%±3.63% in the control group, and was 61.65%±4.76% in the experimental group, showing significant difference ( =12.010, =0.000). The formation of the primary nucleated cell clones was observed in 2 groups. At 12 days, the number of colonies forming of the experimental group [(8.41±0.33)/1 000 nucleated cells] was significantly lower than that of the control group [(15.19±0.47)/1 000 nucleated cells] ( =28.910, =0.000). The percentage of TDSCs in the active nucleated cells in the experimental group (1.37%±0.09%) was significantly lower than that in the control group (1.67%±0.10%) ( =5.508, =0.003). The growth trend of TDSCs (P3) in the 2 groups was consistent within 14 days. There was no significant difference in absorbance ( ) value between 2 groups at each time point ( >0.05). The early apoptotic rate of TDSCs was 1.67%±0.06% in the experimental group and was 1.63%±0.06% in the control group, showing no significant difference ( =0.707, =0.519). Under microscope, TDSCs adhered to the lower chamber of the Transwell chamber; the number of cells was 445.00±9.70 in the experimental group and was 451.50±12.66 in the control group, showing no significant difference ( =0.998, =0.342). The relative mRNA expressions of Col1α1, Scx, and Tnmd were 3.498±0.065, 0.062±0.002, and (4.211±0.211)×10 in the experimental group and were 3.499±0.113, 0.062±0.001, and (4.341±0.274)×10 in the con-trol group, showing no significant difference ( =0.013, =0.991; =0.042, =0.969; =0.653, =0.549).
The survival rate of nucleated cells in cryopreserved rat tendon tissues is lower, but a large number of active TDSCs, and its cell viability, early apoptosis rate, migration ability , and cell tenogenic differentiation ability are remained.
探讨冷冻保存对大鼠髌腱中肌腱源性干细胞（TDSCs）的细胞存活率、细胞活力、早期凋亡、迁移能力及肌腱相关标志物表达的影响。
从12只4月龄雄性Sprague Dawley大鼠获取髌腱组织；将6只大鼠的12条髌腱组织进行冷冻保存（实验组），另12条髌腱组织不做处理（对照组）。用0.3%Ⅰ型胶原酶消化髌腱以获得有核细胞。通过台盼蓝排斥试验检测有核细胞的存活率，用结晶紫染色检测集落形成能力。分离并培养TDSCs至第3代（P3）。采用Alamar Blue法检测TDSCs的细胞活力，用Annexin V-FITC/PI法检测早期凋亡，用Transwell法检测细胞迁移能力，用实时定量PCR检测肌腱相关标志物[Ⅰ型胶原（Col1α1）、硬骨素（Scx）和肌腱调节蛋白（Tnmd）]的mRNA表达。
对照组有核细胞存活率为91.00%±3.63%，实验组为61.65%±4.76%，差异有统计学意义（ =12.010， =0.000）。两组均观察到原代有核细胞克隆形成。第12天时，实验组集落形成数[（8.41±0.33）/1 000个有核细胞]明显低于对照组[（15.19±0.47）/1 000个有核细胞]（ =28.910， =0.000）。实验组活性有核细胞中TDSCs的百分比（1.37%±0.09%）明显低于对照组（1.67%±0.10%）（ =5.508， =0.003）。两组TDSCs（P3）在14天内生长趋势一致。各时间点两组吸光度（ ）值差异无统计学意义（ >0.05）。实验组TDSCs早期凋亡率为1.67%±0.06%，对照组为1.63%±0.06%，差异无统计学意义（ =0.707， =0.519）。显微镜下，TDSCs黏附于Transwell小室下层；实验组细胞数为445.00±9.70，对照组为451.50±12.66，差异无统计学意义（ =0.998， =0.342）。实验组Col1α1、Scx和Tnmd的相对mRNA表达分别为3.498±0.065、0.062±0.002和（4.211±0.211）×10 ，对照组分别为3.499±0.113、0.062±0.001和（4.341±0.274）×10 ，差异无统计学意义（ =0.013， =0.991； =0.042， =0.969； =0.653， =0.549）。
冷冻保存的大鼠肌腱组织中有核细胞存活率较低，但仍存在大量活性TDSCs，且其细胞活力、早期凋亡率、迁移能力及细胞成腱分化能力均得以保留。
