CAJ | 학술논문

目的探讨兔成骨细胞和脂肪细胞两者横向分化的能力和方法.方法选取3个月龄新西兰大白兔,获取、分离培养脂肪细胞,天花板贴壁法使其去分化,去分化脂肪细胞进行成骨诱导3周后行茜素红、碱性磷酸酶(ALP)和Ⅰ型胶原酶免疫组织化学染色.另选取出生7d内的乳兔,取颅骨骨块利用酶消-组织块培养法培养成骨细胞并传代培养,对其进行成脂诱导3周后行油红O染色、逆转录-聚合酶链反应(RT-PCR)检测过氧化物酶体增殖物激活受体γ(PPARγ) mRNA的表达.结果提取的成熟脂肪细胞去分化后为长梭形成纤维细胞状；成骨诱导后,Ⅰ型胶原免疫组织化学染色显示实验组细胞内表达出Ⅰ型胶原,与对照组比较差异有统计学意义(P＜0.05)；各组细胞第3天、第7天、第14天不同时段的ALP活性实验组较对照组高(P＜0.05),分别为0.165±0.007、0.253±0.005、0.345±0.007和0.067±0.004、0.076±0.006、0.082±0.003,且随着培养时间的延长实验组ALP活性逐渐增强(组内比较P＜0.05)；提取的乳兔颅骨成骨细胞呈短梭形,成脂诱导3周后油红O染色阳性,PPARγmRNA表达阳性,对照组为阴性,实验组与对照组比较差异有统计学意义(P＜0.05).结论成熟脂肪细胞可以通过体外培养实现去分化,脂肪细胞和成骨细胞在一定条件下可以相互转化.
목적 탐토토성골세포화지방세포량자횡향분화적능력화방법.방법 선취3개월령신서란대백토,획취、분리배양지방세포,천화판첩벽법사기거분화,거분화지방세포진행성골유도3주후행천소홍、감성린산매(ALP)화Ⅰ형효원매면역조직화학염색.령선취출생7d내적유토,취로골골괴이용매소-조직괴배양법배양성골세포병전대배양,대기진행성지유도3주후행유홍O염색、역전록-취합매련반응(RT-PCR)검측과양화물매체증식물격활수체γ(PPARγ) mRNA적표체.결과 제취적성숙지방세포거분화후위장사형성섬유세포상；성골유도후,Ⅰ형효원면역조직화학염색현시실험조세포내표체출Ⅰ형효원,여대조조비교차이유통계학의의(P＜0.05)；각조세포제3천、제7천、제14천불동시단적ALP활성실험조교대조조고(P＜0.05),분별위0.165±0.007、0.253±0.005、0.345±0.007화0.067±0.004、0.076±0.006、0.082±0.003,차수착배양시간적연장실험조ALP활성축점증강(조내비교P＜0.05)；제취적유토로골성골세포정단사형,성지유도3주후유홍O염색양성,PPARγmRNA표체양성,대조조위음성,실험조여대조조비교차이유통계학의의(P＜0.05).결론 성숙지방세포가이통과체외배양실현거분화,지방세포화성골세포재일정조건하가이상호전화.
Objective To investigate the possibility of transdifferentiation between osteoblasts and adipocytes in rabbits.Methods Adipocytes were isolated from the subcutaneous adipose tissue in the abdomen of 3-month-old New Zealand white rabbits.Then mature adipocytes were cultured and induced to dedifferentiated adipocytes (DA) by ceiling adherent culture method.DA were culutured in osteogenic medium for 3 weeks,and then calcium nodules of the cells were stained by the alizarin red and by immunocytochemical staining for collagen I and alkaline phosphatase (ALP).Osteoblasts were isolated from the skull of one-week-old rabbits by mechanical digestion and enzyme digestion and cultured and amplified in vitro.Then osteoblasta were culutured in adipogenic medium for 3 weeks.Oil Red O staining was used to visualize lipid-rich vacuoles.The expression of peroxisome proliferator activated receptor γ (PPARγ) mRNA was detected by using reverse transcription-polymerase chain reaction (RT-PCR).Results The extracted mature adipocytes turned into fibroblast-like cells (spindle-shaped) after dedifferentiation.After osteogenic induction,the experiment group expressed type Ⅰ collagen,and there was significant difference as compared with control group (P ＜0.05).The ALP activity in experimental group at 3rd,7th and 14th day (0.165± 0.007,0.253 ± 0.005,0.345 ± 0.007 respectively) was higher than that in control group (0.067 ±0.004,0.076 ± 0.006,0.082 ± 0.003 respectively) ( P ＜ 0.05 ).The osteoblasts obtained from the skull were shot-spindle-shaped and positive for Oil Red O staining after culture with adipogenic medium for 3 weeks.The expression of PPARγmRNA in experimental group was positive,that in control group was negative (P ＜0.05).Conclusion Mature adipocytes can turn into DA by in vitroculture.Adipocytes and osteoblasts can differentiate into each other under some particular conditions.