三维培养下胰岛素样生长因子Ⅰ对人牙周膜细胞增殖及早期成骨向分化的影响

Effect of insulin-like growth factor on the proliferation and early stage osteogenesis of human periodontal ligament stem cells under three-dimensional culture system

目的 了解胰岛素样生长因子Ⅰ（insudin-like growth factor-Ⅰ,IGF-Ⅰ）在三维培养环境中对人牙周膜细胞（human periodontal ligament cells,hPDLC）生长、分化的影响,以期为进一步研究牙周组织的再生提供实验依据.方法 从手术拔除的8颗前磨牙或第三磨牙中获取牙周膜组织,利用有限稀释法培养获得hPDLC.采用旋转细胞培养系统（rotary cell culture system,RCCS）建立三维培养环境,根据培养液中加入IGF-Ⅰ的不同质量浓度分为对照组（0 μg/L）和5个实验组（0.1、1、10、50及100μg/L）,甲基噻唑基四唑（MTT）法检测细胞增殖情况.成骨诱导培养中加入上述不同质量浓度的IGF-Ⅰ,分光光度计法检测碱性磷酸酶（alkine phosphatase,ALP）浓度,反转录聚合酶链反应（RT-PCR）检测骨钙素及Ⅰ型胶原基因表达水平.结果 三维培养下,各实验组IGF-Ⅰ对细胞增殖的作用均显著高于对照组（P＜0.05）,其中IGF-Ⅰ 0.1μg/L组细胞增殖活性（0.219±0.021）与50μg/L组（0.287±0.011）、100 μg/L组（0.293±0.012）相比差异均有统计学意义（P＜0.05）,其余组间差异均无统计学意义（P＞0.05）.各实验组ALP活性均显著高于对照组（P＜0.05）,1μg/L组（0.304±0.020）与10 μg/L组（0.310±0.013）相比差异无统计学意义,50 μg/L组（0.347±0.011）与100μg/L组（0.344±0.010）相比差异无统计学意义,但50、100 μg/L组显著高于1、10μg/L组,且四组均显著高于0.1 μg/L组（0.212±0.011）,P＜0.05.Ⅰ型胶原及骨钙素的产物表达在mRNA水平及蛋白水平均呈现随浓度升高而升高的趋势.结论 在本实验三维培养条件下,IGF-Ⅰ对hPDLC的促增殖作用在0.1～100μg/L范围内呈浓度依赖性增强;100 μg/L IGF-Ⅰ在三维培养条件下具有明确的促hPDLC成骨向分化的作用.

Objective To investigate the effect of insulin-like growth factor- Ⅰ （ IGF- Ⅰ ） on the proliferation and osteogenesis of human periodontal ligament stem cells （ hPDLC ） under three-dimensional （3D） culture system. Methods Human periodontal cells were isolated from the ligament of surgically extracted human teeth, and through the limiting dilution assay, got mono-clone of the cell, hPDLCs were isolated from MesenPRO RS medium. Rotary cell culture system （RCCS） was enrolled to set 3D environment. Control group and experiment groups were assigned according to the concentration of IGF- Ⅰ .There were 5 level of experiment groups （0.1,1,10,50,100 μg/L）. Proliferation was tested with methyl thiazolyl tetrazolium （ MTT）, and alkine phosphatase （ALP） level was assayed by spectrophotometer to analyze the osteogenesis of hPDLCs. Gene expression of ostetocalcin（OCN）and type Ⅰ collagen （Col Ⅰ ）were assayed by reverse transcriptase polymerase chain reaction（RT-PCR）. Results In 3D culture system,the effect of IGF- Ⅰ on cell proliferation was significantly different between control group and experiment groups（ P 〈 0.05 ）, and there showed significant differences between the group of 0.1 μg/L （ 0.219 ±0.021 ） IGF- Ⅰ and the groups of 50, 100 μg/L（0.287 ±0.011,0.293 ±0.012）. However, there showed no significant differences among other groups. Significant differences of ALP activity were observed between the control group and experiment groups, and between the groups of 1, 10 μg/L（0.304 ±0.020, 0.310 ±0.013） and that of 50, 100 μg/L （0.347 ±0.011, 0.344 ±0.010） （P 〈0.05）. While no significantdifferences were detected between the group of 1 μg/L and that of 10 μg/L, nor between the group of 50 μg/L and that of 100 μg/L. Expressions of Col Ⅰ and OCN in mRNA and protein level both showed dose-dependent increase. Conclusions In 3D culture system, in the scale of 0.1-100 μg/L, the effect of IGF-Ⅰ on the proliferation of hPDLCs increased dose-dependently. 100 μg/L IGF- Ⅰ promotes osteogenesis of the cells significantly.