体外培养人成骨细胞I型胶原合成与表达的增龄性改变

Influence of donor age on COL-1 expression of human osteoblastic cells cultured in vitro

目的探讨体外培养不同年龄组人成骨细胞I型胶原(COL-1)表达的增龄性改变. 方法选择＜5岁、30～40岁、50～60岁及70～79岁4个年龄组骨折患者的松质骨,用1:1 DMEM-Ham F-12培养液进行培养.第2代细胞培养至7 d和13 d,分别定量留取培养48 h的条件培养液,第2代细胞一部分经诱导培养至14 d进行电镜观察,另一部分培养至14 d抽提细胞内总RNA,用逆转录聚合酶链反应方法半定量检测COL-1基因表达,并进行各年龄组比较. 结果成骨细胞经体外诱导培养后透射电镜下观察,细胞外基质中可见大量原胶原;细胞培养至7 d和13 d I型前胶原羧基端前肽(PICP)检测值均随年龄增长逐渐降低,与年龄之间呈显著负相关(男性7 d: r=-0.503, P＜0.05, 13 d: r=-0.662, P＜0.01; 女性7 d: r=-0.658, P＜0.01, 13 d: r=-0.770, P＜0.01);各年龄组成骨细胞均检测到COL-1基因mRNA表达,表达量＜5岁组较低,30～40岁组最高,之后随年龄增长逐渐降低,其表达量和年龄呈显著正相关(男性r=0.331,女性r= 0.327,均为P＜0.05),性别间差异无统计学意义. 结论体外培养人成骨细胞可合成原胶原,PICP分泌随增龄而降低,不同年龄组人成骨细胞均表达COL-1 mRNA,30～40岁后表达量随年龄增长而降低.

Objective To investigate the differences in function and collagen type Ⅰ （COL-l） expression of cultured human ostoblastic cells between different age donors in vitro. Methods Human osteoblasts from different age donors （〈5 years, 30～ 40 years, 50 ～60 years, and ≥ 70 years） were isolated and cultured with the tissue-fragment- migrating-growth method. The osteoblasts of first passage were cultured to confluence, then trypsinized and numbered. The osteoblasts of second passage in 25 cm^2 flasks were cultured for 14 days; then observed by electromicroscope. The conditioned medium of 48 hours culture in 7th and 13th day were stored at --80℃ and detected for carboxyl terminal propeptide of type-Ⅰ precollagen （PICP） with radioimmunoassay. Total RNA was extracted from each sample when the osteoblasts of different age donors in second passage were cultured for 14 days. The mRNA expressions of COL-1 from each sample were measured with semiquantity reverse transcription-polymerase chain reaction （RT-PCR） and compared among different groups. Results Much tropocollagen could be found in the extracellular matrix by transmission electron microscope when the osteoblasts were cultured with ascorbate and 13-glycerophosphate. PICP was shown to decrease with donor aging when the osteoblasts were cultured for 7 or 13 days （P〈0. 01 or 0. 05 respectively）. Expression of COL-1 mRNA could be detected in human osteoblasts of different age donors cultured in vitro. The COL-1 mRNA expression level of the osteoblasts was shown to increase from 〈5 year-old donors to 30～40 year-old donors, then decreased with donor aging. The expressions were correlated with donor aging （P〈0. 05）. There was no significant difference between sexes. Conclusions Human osteoblasts cultured in vitro can express precollagen. PICP secretion was decreased with donor aging. All the human osteoblasts from different age donors cultured in vitro might express COL-1 mRNA. The quantity of COL-1 mRNA expression in the osteoblasts is decreased with donor aging.