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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 typeⅠ,Col1α1)、scleraxis(Scx)和tenomodulin(Tnmd)m RNA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m RNA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。
Objective To investigate the effects of cryopreservation on survival, cell viability, early apoptosis, migration and tendon related gene expression in tendon-derived stem cells (TDSCs) of patellar tendon in rats. Methods Bilateral patellar tendons of 12 male 4-month-old male Sprague-Dawley rats were randomly divided into 6 groups. Twelve patellar tendon tissues (experimental group) of 6 rats were cryopreserved. The remaining patellar tendon tissue (control group) Control. The nucleated cells were obtained by digestion of 0.3% type Ⅰ collagenase in two groups of patellar tendon tissues. The viability of nucleated cells was detected by trypan blue staining and the ability of colony formation of nucleated cells was detected by crystal violet staining. Two groups of patellar tendon tissue were separated TDSCs were cultured and passaged to the third passage. The proliferation of cells was detected by Alamar Blue assay. The rate of cell apoptosis was detected by Annexin V-FITC / PI double staining. The cell migration was detected by Transwell assay. The expression of collagen type Ⅰ (Col1α1), scleraxis (Scx) and tenomodulin (Tnmd) mRNA were detected by Western blot. Results Compared with the control group (91.00% ± 3.63%), the survival rate of primary nucleated cells in experimental group was 61.65% ± 4.76%, the difference was statistically significant (t = 12.010, P = 0.000). After cultured for 12 days, the number of colony forming colonies per 1 000 nucleated cells in the experimental group was (8.41 ± 0.33), which was significantly lower than that in the control group (15.19 ± 0.47) (t = 28.910, P = 0.000). The proportion of TUNEL positive cells in experimental group was 1.37% ± 0.09%, which was significantly lower than that in control group (1.67% ± 0.10%) (t = 5.508, P = 0.003). There was no significant difference in absorbance (A) between the two groups at each time point (P> 0.05). The early apoptotic rates of TDSCs in experimental and control groups were 1.67% ± 0.06% and 1.63% ± 0.06% respectively, with no significant difference (t = 0.707, P = 0.519). Microscopically, TDSCs adhered to the lower chamber of Transwell cells in both groups, with (445.00 ± 9.70) cells in the experimental group and (451.50 ± 12.66) in the control group, with no significant difference (t = 0.998, P = 0.342). The relative expression levels of m RNA in Col1α1, Scx and Tnmd in experimental group were 3.498 ± 0.065 and 0.062 ± 0.002, respectively (4.211 ± 0.211) × 10-5, while those in control group were 3.499 ± 0.113 and 0.062 ± 0.001 respectively (4.341 ± 0.274) × 10-5. There was no significant difference between the two groups (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 rat tendon tissue after cryopreservation is low, but there is still a large amount of active TDSCs in tendon tissue, and there is no obvious abnormality in cell proliferation, early apoptosis rate, in vitro migration and tendon differentiation .