Objective: 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.
Methods: 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.
Results: 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 ( t=12.010, P=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] ( t=28.910, P=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%) ( t=5.508, P=0.003). The growth trend of TDSCs (P3) in the 2 groups was consistent within 14 days. There was no significant difference in absorbance ( A) value between 2 groups at each time point ( P>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 ( t=0.707, P=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 ( t=0.998, P=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 -5 in the experimental group and were 3.499±0.113, 0.062±0.001, and (4.341±0.274)×10 -5 in the con-trol group, showing no significant difference ( t=0.013, P=0.991; t=0.042, P=0.969; t=0.653, P=0.549).
Conclusion: 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 in vitro, and cell tenogenic differentiation ability are remained.
目的: 探讨深低温冷冻处理对大鼠髌腱组织中肌腱干细胞（tendon-derived stem cells，TDSCs）的成活、细胞活力、早期凋亡、迁移能力及肌腱相关基因表达的影响。.
方法: 取 12 只 4 月龄雄性 SD 大鼠双侧髌腱组织，其中 6 只大鼠 12 条髌腱组织（实验组）进行深低温冷冻处理；剩余髌腱组织（对照组）不作处理，作为正常对照。取两组髌腱组织用 0.3% Ⅰ 型胶原酶消化获得有核细胞，分别用锥虫蓝染色检测有核细胞成活率、结晶紫染色检测有核细胞克隆形成能力；取两组髌腱组织分离培养 TDSCs，并传至第 3 代，采用 Alamar Blue 法检测细胞体外增殖能力；Annexin V-FITC/PI 双染法检测细胞早期凋亡率；Transwell 法检测细胞迁移能力；实时荧光定量 PCR 检测细胞肌腱相关基因 Ⅰ 型胶原（collagen type Ⅰ，Col1α1）、scleraxis（Scx）和 tenomodulin（Tnmd） mRNA 表达。.
结果: 与对照组（91.00%±3.63%）比较，实验组原代有核细胞成活率为 61.65%±4.76%，差异有统计学意义（ t=12.010， P=0.000）。两组有核细胞接种后，均呈克隆样生长；培养 12 d，实验组每 1 000 个有核细胞克隆集落形成数为（8.41±0.33）个，较对照组（15.19±0.47）个显著减少（ t=28.910， P=0.000）。实验组 TDSCs 占活性有核细胞比例为 1.37%±0.09%，较对照组 1.67%±0.10% 显著减少（ t=5.508， P=0.003）。第 3 代 TDSCs 培养 14 d 内两组细胞生长趋势一致；两组各时间点吸光度（ A）值比较，差异均无统计学意义（ P>0.05）。实验组及对照组 TDSCs 早期凋亡率分别为 1.67%±0.06%、1.63%±0.06%，比较差异无统计学意义（ t=0.707， P=0.519）。镜下见，两组均有 TDSCs 黏附于 Transwell 小室下室，实验组细胞数为（445.00±9.70）个，对照组为（451.50±12.66）个，比较差异无统计学意义（ t=0.998， P=0.342）。实验组 Col1α1、Scx、Tnmd 的 mRNA 相对表达量分别为 3.498±0.065、0.062±0.002、（4.211±0.211）×10 –5，对照组分别为 3.499±0.113、0.062±0.001、（4.341±0.274）×10 –5，比较差异均无统计学意义（ t=0.013， P=0.991； t=0.042， P=0.969； t=0.653， P=0.549）。.
结论: 大鼠肌腱组织经深低温冷冻后其有核细胞成活率降低，但肌腱组织仍保留大量有活性 TDSCs，且细胞增殖能力、早期凋亡率、体外迁移能力及成肌腱分化能力未见明显异常。.
Keywords: Cryopreservation; allogeneic tendon transplantation; tendon defect; tendon-derived stem cells; tenogenic differentiation.