兔成骨细胞和脂肪细胞横向分化的研究

Reasearch of transdifferentiation between osteoblasts and adipocytes in rabbits

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0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，PPARγ／mRNA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。

Objective To investigate the possibility of transdifferentiation between osteoblasts and adipocytes in rabbits. Methods Adipocytes were isolated from the subcutaneous adipose tissue in the ab- domen 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 culntured in osteogenic me- dium for 3 weeks, and then calcium nodules of the cells were stained by the alizarin red and by immunocy- tochemical 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 osteoblasts were culutured in adipogenic medium for 3 weeks. Oil Red 0 staining was used to visual- ize 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 ma- ture 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-spidadle-shaped and positive for Oil Red O staining after culture with adipogenic medium for 3 weeks. The expression of PPAR＇,/mRNA in experimental group was positive, that in control group was neg- ative （ P 〈 0. 05 ）. Conclusion Mature adipocytes can turn into DA by in vitroculture. Adipocytes and os- teoblasts can differentiate into each other under some particular conditions.