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目的探讨不同复温温度对低温处理人成纤维细胞形态和Ⅰ型胶原代谢的影响。方法将体外培养的人成纤维细胞制成悬液后接种于培养皿中,随机分为对照组、20℃复温组、37℃复温组。将两个复温组培养细胞渐冷冻至-10℃制成冻伤模型,然后各自于20、37℃下复温,再与对照组一起继续培养,于伤前及伤后即刻、24、48、72、96h进行观察,每组各时相点9个培养皿。采用噻唑蓝法观察细胞活力的变化,以波长570nm下的吸光度(A)值表示;倒置相差显微镜观察细胞形态变化;免疫组织化学染色法及IMAGE-J图像分析软件测定细胞内胶原含量变化,以灰度值表示;双抗夹心亲和素-生物素酶联免疫吸附测定法测定细胞外胶原含量,以A值表示。结果20℃复温组细胞活力伤后先降低后升高,各时相点明显低于伤前值(0.95±0.16,P<0.05或0.01);细胞伤后即刻脱水,胞浆丢失、核浆比例增大,伤后72、96h细胞增生活跃,核分裂相增多,部分细胞出现漂浮后再贴壁现象,细胞排列杂乱;细胞外胶原含量较伤前值(96.4±2.9)呈先升高后降低再缓慢升高的趋势,细胞内胶原含量则较伤前值(0.0479±0.0027)呈先降低后逐渐升高趋势,与对照组比较,差异均有统计学意义(P<0.05或0.01)。37℃复温组伤后细胞活力各时相点与伤前值相近(P>0.05);与对照组比较,其细胞形态、胶原代谢基本无变化(P>0.05).结论低温处理后细胞脱水是一种保护机制,适宜的复温条件能使细胞复苏;与20℃相比,37℃复温对细胞的损伤较轻。
Objective To investigate the effects of different rewarming temperatures on the morphology of human fibroblasts and the type Ⅰ collagen metabolism. Methods Human fibroblasts cultured in vitro were inoculated into petri dishes and randomly divided into control group, 20 ℃ rewarming group and 37 ℃ rewarming group. The cells in two rewarming groups were gradually frozen to -10 ° C to make frostbite models, and each was rewarmed at 20 and 37 ° C before continuing to culture with the control group. Immediately before and immediately after the injury, 24, 48, 72,96h were observed, each time point nine dishes. The changes of cell viability were observed by the method of thiazolyl blue staining and the absorbance (A) was measured at 570nm. The morphological changes of cells were observed by inverted phase contrast microscope. The changes of intracellular collagen content were measured by immunohistochemical staining and IMAGE-J image analysis software Gray scale value; double-antibody sandwich avidin-biotin enzyme-linked immunosorbent assay for determination of extracellular collagen content, expressed as A value. Results The cell viability in 20 ℃ rewarming group decreased first and then increased, and the time points at each time point were significantly lower than those before injury (0.95 ± 0.16, P <0.05 or 0.01); dehydration immediately after injury, cytosolic loss, The percentage of extracellular collagen increased first and then decreased (96.4 ± 2.9) compared with the pre-injury value (96.4 ± 2.9) Then increased slowly. The intracellular collagen content decreased first and then increased gradually compared with the pre-injury value (0.0479 ± 0.0027). Compared with the control group, the difference was statistically significant (P <0.05 or 0.01). Compared with the control group, the cell morphology and collagen metabolism were almost unchanged (P> 0.05) .Conclusion Cell dehydration after cryopreservation Is a kind of protection mechanism. Appropriate rewarming conditions can make cell recovery; compared with 20 ℃, 37 ℃ rewarming cell damage less.