【摘要】：目的探討深低溫冷凍處理對大鼠髕腱組織中肌腱干細胞(tendon-derived stem cells,TDSCs)的成活、細胞活力、早期凋亡、遷移能力及肌腱相關(guān)基因表達的影響。方法取12只4月齡雄性SD大鼠雙側(cè)髕腱組織,其中6只大鼠12條髕腱組織(實驗組)進行深低溫冷凍處理;剩余髕腱組織(對照組)不作處理,作為正常對照。取兩組髕腱組織用0.3%Ⅰ型膠原酶消化獲得有核細胞,分別用錐蟲藍染色檢測有核細胞成活率、結(jié)晶紫染色檢測有核細胞克隆形成能力;取兩組髕腱組織分離培養(yǎng)TDSCs,并傳至第3代,采用Alamar Blue法檢測細胞體外增殖能力;Annexin V-FITC/PI雙染法檢測細胞早期凋亡率;Transwell法檢測細胞遷移能力;實時熒光定量PCR檢測細胞肌腱相關(guān)基因Ⅰ型膠原(collagen typeⅠ,Col1α1)、scleraxis(Scx)和tenomodulin(Tnmd)m RNA表達。結(jié)果與對照組(91.00%±3.63%)比較,實驗組原代有核細胞成活率為61.65%±4.76%,差異有統(tǒng)計學(xué)意義(t=12.010,P=0.000)。兩組有核細胞接種后,均呈克隆樣生長;培養(yǎng)12 d,實驗組每1 000個有核細胞克隆集落形成數(shù)為(8.41±0.33)個,較對照組(15.19±0.47)個顯著減少(t=28.910,P=0.000)。實驗組TDSCs占活性有核細胞比例為1.37%±0.09%,較對照組1.67%±0.10%顯著減少(t=5.508,P=0.003)。第3代TDSCs培養(yǎng)14 d內(nèi)兩組細胞生長趨勢一致;兩組各時間點吸光度(A)值比較,差異均無統(tǒng)計學(xué)意義(P0.05)。實驗組及對照組TDSCs早期凋亡率分別為1.67%±0.06%、1.63%±0.06%,比較差異無統(tǒng)計學(xué)意義(t=0.707,P=0.519)。鏡下見,兩組均有TDSCs黏附于Transwell小室下室,實驗組細胞數(shù)為(445.00±9.70)個,對照組為(451.50±12.66)個,比較差異無統(tǒng)計學(xué)意義(t=0.998,P=0.342)。實驗組Col1α1、Scx、Tnmd的m RNA相對表達量分別為3.498±0.065、0.062±0.002、(4.211±0.211)×10 5,對照組分別為3.499±0.113、0.062±0.001、(4.341±0.274)×10 5,比較差異均無統(tǒng)計學(xué)意義(t=0.013,P=0.991;t=0.042,P=0.969;t=0.653,P=0.549)。結(jié)論大鼠肌腱組織經(jīng)深低溫冷凍后其有核細胞成活率降低,但肌腱組織仍保留大量有活性TDSCs,且細胞增殖能力、早期凋亡率、體外遷移能力及成肌腱分化能力未見明顯異常。
[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，

本文編號：2240075