人退变椎间盘内3种干细胞的生物学特性比较

The comparison of biological characteristics of three kinds of stem cells in human degenerative in- tervertebral disc

目的比较来源于同一人退变椎间盘内髓核干细胞（NPSCs）、纤维环干细胞（AFSCs）及软骨终板干细胞（CESCs）的生物学特性。方法收集7例经x线、核磁共振成像（MRI）确诊为椎间盘突出症患者，将取出的椎间盘仔细分离并进行酶消化、滤网过滤后获得NPSCs、AFSCs及CESCs。培养至第3代时，采用细胞计数试剂盒（CCK-8）检测3种干细胞的生长活性，并进行成脂、成骨及成软骨诱导分化。诱导2td后分别应用油红0染色检测细胞成脂能力，茜素红染色检测细胞成骨能力，甲苯胺蓝染色检测细胞成软骨能力。提取第3代细胞总RNA，分别行反转录．聚合酶链反应（RT-PCR）检测成脂、成骨及成软骨相关基因的表达情况。结果来自于同一患者的NPSCs、AFSCs与CESCs形态上相似，差异无统计学意义（P〉0．05）。生长曲线显示CESCs[第1、3、5、7、9、11、13、15天吸光度（A）值分别为0．35±0．01、0．52±0．02、0．64±0．08、0．80±0．19、0．79±0．20、1．06±0．11、1．37±0．09、1．31±0．41]与AFSCs（第1、3、5、7、9、11、13、15天A值分别为0．38±0．01、0．50±0．03、0．72±0．06、0．82±0．13、0．87±0．08、0．87±0．09、1．20±0．05、1．43±0．05）增殖能力要比NPSCs（第1、3、5、7、9、11、13、15天A值分别为0．32±0．02、0．42±0．04、0．59±0．01、0．64±0．17、0．65±0．19、0．68±0．21、0．67±0．12、0．59±0．12）活跃。油红0染色、茜素红染色及甲苯胺蓝染色分别证实3种干细胞均可向脂肪、骨及软骨三系诱导分化。RT—PCR检测结果显示AFSCs在成脂[过氧化物酶体增殖物激活受体1（PPA脚）：6．85±0．32，脂蛋白脂肪酶（LPL）：13．72±0．37]、及成软骨[Ⅱ型胶原（COL-2）：10．45±1．10，性别决定因子相关基因-9（SOX-9）：14．39±1．22]基因表达方面比NPSCs（PPARγ：6．63±0．50，LPL：8．41±0．42，COL-2：3．53±0．62，SOX-9：7．66±0．68）与CESCs（PPARγ：7．21±0．21，LPL：10．62±0．37；COL-2：8．26±0．49，SOX-9：13．70±1．10）略强；而成骨能力方面，CESCs最强[Runt相关转录因子2（RUNX-2）：7．30±1．1，COL-2：8．22±0．84]，AFSCs（RUNX-2：6．20±O．92，COL-2：4．94±0．90）与NPSCs相当（RUNX-2：2．61±0．06，COL-2：7．05±0．82）；NPSCs的成脂基因尤其LPL基因表达较低。结论NPSCs、AFSCs及CESCs在体外培养中细胞形态相似，但生物学特性方面存在差异。体外诱导分化及RT-PCR检测结果均提示AFSCs比其他两种干细胞在椎间盘组织工程领域中更加具有优越性。

Objective To compare the biological characteristics of nucleus pulposus - derived stem cells （NPSCs）, annulus fibrosns -derived stem cells （AFSCs） and cartilage endplate -derived stem cells （CESCs） from the same patient who had degenerative disc. Methods The human discs isolated from 7 patients with intervertebral disc herniation were separated carefully and digested and filtered before getting the original NPSCs, AFSCs and CESCs. When cultivated to third passage, the cell activity of stem cells was analyzed by using cell counting kit - 8 （ CCK - 8 ） ; multilineage （ adipogenic, osteogenic and chondro- geuic） differentiation potential was analyzed to define different stem ceils. The total RNA of third -passage -cells was extracted, and then reverse transcriptase -polymerase chain reaction （RT- PCR） was used to detect the relative expression of adipogenic, osteogenic and chondrogenic differentiation - related genes. Results Cell morphology of NPSCs, AFSCs and CESCs from the same patient showed no signifi- cant difference. The growth curve showed that proliferative ability of CESCs （A value at day 1,3, 5, 7, 9, 11, 13, 15 was 0.35 ±0.01, 0.52 ±0.02, 0.64 ±0.08, 0.80 ±0.19, 0.79 ±0.2, 1.06±0.11, 1.37 ±0. 09, 1.31 ±0. 41 ） and AFSCs （0. 38 ±0. 01, 0. 50 ±0.03, 0. 72 ±0. 06, 0. 82 ±0. 13, 0. 87 ± 0. 08, 0. 87 ± 0. 09, 1.20 ± 0. 05, 1.43 ± 0. 05 ） was more active than that of NPSCs （0. 32 ± 0. 02, 0.42±0.04, 0.59±0.01, 0.64±0. 17, 0.65±0. 19, 0.68±0.21, 0.67±0. 12, 0.59±0. 12）. Mul- tilineage differentiation results showed adipocyte -like red by oil red O, osteocyte -like cells were stained red by Alizarin red, and chondrocyte - like cells blue by toluidine blue. The RT - PCR results showed that AFSCs was better than NPSCs and CESCs in the aspect of the expression of adipogenic [ the relative expres- sion of peroxisome proliferator - activated receptor gamma （ PPAR3, ） gene was 6. 85 ± 0. 32 vs. 6. 63 ± 0. 50 and 7.21 ± 0. 21 ; the relative expression of lipoprotein lipase （LPL） gene was 13.72 ± 0. 37 vs. 8.41 ± 0. 42 and 10. 62 ± 0. 37 ] and chondrogenic [ the relative expression of type 2 collagen （ COL-2 ） gene was 10.45 ± 1.10 vs. 3.53 ±0. 62 and 8. 26 ±0. 49 ; the relative expression of sex determining region Y - box 9（ SOX -9） gene was 14. 39 ± 1.22 vs. 7.66 ±0. 68 and 13.70 ± 1.10] differentiation - related genes, whereas the expression of osteogenic differentiation - related genes was best in CESCs [ the relative expression of runt related transcription factor - 2 （ RUNX - 2） gene was 7. 30 ± 1.1 vs. 2. 61 ± 0. 06 and 6. 20 ± 0. 92 ; the relative expression of COL-2 gene was 8.22 ± 0. 84 vs. 7. 05 ± 0. 82 and 4. 94 ± 0. 90 ] and the expression of adipogentic differentiation - related genes, particularly LPL gene was worse in NPSCs. Conclusion The cell morphology was similar among NPSCs, AFSCs and CESCs in vitro, but bio- logical characteristics of those were different. Induced differentiation and RT - PCR results both showed that AFSCs had more advantages than NPSCs and CESCs in the field of intervertebral disc tissue engineer- ing.