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  

Effects of cryopreservation on biological characteristics of tendon-derived stem cells in rat patellar tendon

作　　者：代广春 李荥娟[3] 徐宏亮[4] 林禹丞 刘俊延[1,2] 徐林[4] 芮云峰[1,2,4] 

机构地区：[1]东南大学医学院附属中大医院骨科,南京210000 [2]东南大学医学院,南京210000 [3]东南大学医学院附属中大医院老年科,南京210000 [4]东南大学医学院附属中大医院无锡分院骨科,江苏无锡214000

出　　处：《中国修复重建外科杂志》2017年第7期845-852,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金青年基金资助项目(81201422);国家自然科学基金资助项目(81572187);中国博士后科学基金资助项目(2012M520983);中国博士后基金资助特别项目(2014T70461);江苏省自然科学基金青年基金资助项目(BK2012334);江苏省"六大人才高峰"D类资助项目(2013-WSW-054)~~

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(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=0.5Objective 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 ceils 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 （Collα1）, scleraxis （Scx）, and tenomodulin （Tnmd）] by real-time quantitative PCR. Results The survival rate of nucleated ceils 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.

关 键 词：深低温冷冻 肌腱干细胞 肌腱缺损 同种异体肌腱移植 成肌腱分化 

分 类 号：R686[医药卫生—骨科学]