深低温冷冻对大鼠髌腱组织内肌腱干细胞生物学特性影响的研究
发布时间：2018-09-12 19:59
【摘要】：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(P0.05)。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。
[Abstract]:Objective to investigate the effects of cryopreservation on survival, cell viability, early apoptosis, migration ability and tendon related gene expression of tendon stem cells (tendon-derived stem cells,TDSCs) in rat patellar tendon. Methods 12 4-month-old male SD rats with bilateral patellar tendons were collected, 6 of them were treated with cryopreservation, while the remaining patellar tendons (control group) were not treated as normal control. Nucleated cells were obtained from patellar tendon tissue digested with 0.3% collagenase. Survival rate of nucleated cells was detected by trypanosoma blue staining and colony forming ability of nucleated cells was detected by crystal violet staining. TDSCs, was isolated from two groups of patellar tendon tissues and transferred to the third passage. The proliferation ability of the cells in vitro was detected by Alamar Blue method. The rate of early apoptosis was detected by Annexin V-FITC/PI double staining and the ability of cell migration was detected by Transwell method. Real-time quantitative PCR was used to detect the expression of type 鈪，
本文编号：2240075
[Abstract]:Objective to investigate the effects of cryopreservation on survival, cell viability, early apoptosis, migration ability and tendon related gene expression of tendon stem cells (tendon-derived stem cells,TDSCs) in rat patellar tendon. Methods 12 4-month-old male SD rats with bilateral patellar tendons were collected, 6 of them were treated with cryopreservation, while the remaining patellar tendons (control group) were not treated as normal control. Nucleated cells were obtained from patellar tendon tissue digested with 0.3% collagenase. Survival rate of nucleated cells was detected by trypanosoma blue staining and colony forming ability of nucleated cells was detected by crystal violet staining. TDSCs, was isolated from two groups of patellar tendon tissues and transferred to the third passage. The proliferation ability of the cells in vitro was detected by Alamar Blue method. The rate of early apoptosis was detected by Annexin V-FITC/PI double staining and the ability of cell migration was detected by Transwell method. Real-time quantitative PCR was used to detect the expression of type 鈪，
本文编号：2240075
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