体外培养不同代次人成纤维细胞的衰老程度

Diverse senescence of human fibroblasts cultured in vitro of different passages

目的:探讨体外培养不同代次人成纤维细胞的衰老程度,为组织工程化皮肤提供合适的种子细胞。方法:实验于2000-09/2002-06在潍坊医学院整形外科研究所完成。取6～8岁正常男性儿童20例包皮环切术切除的健康包皮组织,将包皮二倍体成纤维细胞进行传代培养,以不同代次(P0,P5,P10,…,P65)的细胞为实验对象,通过倒置相差显微镜、透射电镜、衰老相关β-半乳糖苷酶染色一系列检测方法,对不同代次成纤维细胞的衰老程度进行观察。结果:①成纤维细胞的形态学:P45以内成纤维细胞生长迅速,两三天即呈汇合状态,细胞排列紧密,多呈放射状、编织状或漩涡状走行,有的交叉重叠生长。P46以后细胞排列不规则,体积较大,形状扁平,胞浆区域增大,胞质突起多而大,胞内出现颗粒和空泡,边界不清,形态不规则;成批的细胞出现固缩、脱落,有离壁漂起的现象。②透射电镜结果:P0～P40:成纤维细胞胞质内有丰富的粗面内质网;细胞核大,核仁明显;细胞表面有许多短小突起,可见微绒毛和胞浆皱褶。P41～P65:细胞核/浆比例减小,细胞表面突起和微绒毛减少;核膜内折,有凋亡现象。③细胞生物学行为的改变:P60增殖速度较P10明显减慢。P10最大增殖数为47.3×105,对数生长期在3～6d,细胞倍增时间为2.18d;P60最大增殖数为8.5×104,对数生长期在4.0～7.5d,群体倍增时间为3.86d。④衰老相关β-半乳糖苷酶染色结果:与P10相比,P60中阳性细胞率大为增强(阳性细胞率为4%和60%);直线相关分析结果显示,β-半乳糖苷酶染色的阳性率和细胞代龄之间呈显著正相关(r=0.92,P<0.01)。结论:各代成纤维细胞老化程度不同,46代后的细胞老化明显,不适合作为组织工程化皮肤的种子细胞。

AIM： To evaluate the aging of fibroblasts isolated from human skin cultured in vitro for different passages so as to provide optimal seeding cells for skin tissue engineering. METHODS： The experiment was finished at the Institute of Plastic Surgery of Weifang Medical College from September 2000 to June 2002. Human diploid fibroblasts were taken from the healthy foreskin for successive culture which had been resected from 20 normal boys （6 to 8 years old） by circumcision. The fibroblasts of different passages（P0, P5, P10, ……,P65） were used as the material. The aging process of fibroblasts was displayed by inverted phase contrast microscopy, transmission electron microscopy and senescence-associated β-galactosidase（SA-β-Gal） staining. RESULTS： ①Morphology of fibroblasts： Fibroblasts within 45 passages proliferated rapidly, which were syncretic within 2 to 3 days. Fibroblasts were closely arranged, radial, knitted or circinate, and some grew interlacedly and overlappingly. Fibroblasts beyond passage 46 arranged irregularly in a great volume and a flat shape. The cytoplasm was enlarged in area with lots of and big cytoplasm processus, in which many particles and vacuoles were found, and the margin was undistinguished in irregular form. Abundant fibroblasts were contracted and shedding, floating from cellular wall ② Resuhs of the transmission electron microscopy： The large nucleus of fibroblasts from passage 0 to 40 was seen apparently and surrounded by cytoplasm rich in rough endoplasmic reticula. There were many short and small processus on the surfaces of fibroblasts, also with microvilli and creased cytoplasm. From passage 41 to 65, the ratio of nuclei to endoplasm was reduced, and the processus and microvill of fibroblast surfaces were became less; The cytomembranes of fibroblasts were inwardly folded, and apoptotic bodies were seen in fibroblasts.③ Changes of the cellular biology： Fibroblasts of passage 60 proliferated more slowly than those of passage 10. The most proliferating fibroblasts of passage 10 were 47.3×10^5 cells, logarithmic growth was 3 to 6 days and the population doubling time （PDT） was 2.18 days; Those of fibroblasts of passage 60 were 8.5×10^4 cells, 4.0 to 7.5 days and 3.86 days respectively. ④Results of the expression of SA-β-Gal staining： The expression of SA-β- Gal became to be more apparent in fibroblasts of passage 60 （60% positive cells） than in fibroblasts of passage 10（4% positive cells）. The analysis of linear correlation showed that the percentage of positive fibroblasts stained by SA-β-Gal was in evidently positive correlation with passages of fibroblasts（r=0.92, P 〈 0.01）. CONCLUSION： Fibroblasts of different passages were of a diverse senescence, and those beyond passage 46 showed a remarkable senescence, which were unsuitable as seeding cells for skin tissue engineering.