目的：生酮饮食(Ketogenic diet，KD)是由极低碳水化合物、高脂以及适量蛋白质组成的特殊高脂饮食，主要在难治性小儿癫痫上有较好的临床疗效。KD通过严格限制每日摄取的总热量和蛋白质的摄入，从而迫使机体将显著升高的酮体(β-羟丁酸、乙酰乙酸、丙酮)作为主要供能物质。KD被用在难治性癫痫已有近百年的历史，其对于神经损伤、心血管疾病甚至肿瘤等方面的疗效也被越来越多的实验研究所证实。有研究称KD治疗小儿难治性癫痫会引起患儿骨密度下降及生长发育受限，但这一结论尚不明确，且缺乏对不同部位的骨结构影响的全面评估和对比。本实验通过喂养定制KD建立相应的大鼠模型，探究KD对于长骨和椎骨微观结构和力学性能的影响，同时研究KD对于骨代谢的影响以及对骨髓间充质干细胞(BMSC)分化的影响。 方法：28只6周龄雄性SD大鼠适应性喂养2周后，随机等分为标准饮食组(Contol组)和生酮饮食组(KD组)。生酮饲料是脂肪比碳水化合物加蛋白质为3:1的纯化生酮饲料，Control组则喂食标准大鼠饲料。开始喂养后每周监测体重，每两周穿刺尾静脉监测酮体与血糖。大鼠差别喂养12周后处死，每组随机选择7只，取右侧下肢胫骨和股骨骨髓行BMSC原代培养。剩余大鼠取血清，测量钙、磷和维生素D的含量，以及血清和骨组织中成骨和破骨指标的表达。取材大鼠的右侧肱骨、右侧胫骨和腰椎，使用显微CT分别扫描肱骨远端、胫骨平台下以及腰4椎体中部180层。建立扫描部位的3D模型，使用微结构有限元模型(FE)软件模拟压缩试验，得到相应部位骨的压缩刚度和最大破坏载荷。 结果：(1)12周的喂养下两组间体重差异有显著性，血清钙、磷和维生素D浓度的差异均无显著性；(2)KD组的血酮水平为0.352 mmol／L，显著高于对照组的1.63 mmol／L；(3)全身骨密度测量显示KD组大鼠的全身骨密度较对照组显著性降低(0.162±0.008 g／㎜² vs 0.153±0.007 g／㎜²)；(4)显微CT结果显示KD组肱骨和胫骨的皮质骨厚度和松质骨骨体积分数(BV／TV)、骨密度(BMD)、骨小梁数目(Tb.N)、骨小梁厚度(Tb.Th)等均有显著低于对照组，而KD组骨小梁间隔(Tb.Sp)显著增大；(5)KD组椎体的皮质骨和松质骨微观结构参数与对照组相比均没有显著性差异；(6)长骨和椎骨皮质骨的变化比率相似，胫骨松质骨各参数变化比率则明显大于肱骨和椎骨；(7)对照组肱骨和胫骨压缩刚度和最大破坏载荷均高于KD组，且差异有显著性；(8)两组间椎体压缩刚度和最大破坏载荷的差异没有显著性；(9)KD组血清ALP表达较对照组显著减少(1.88± 0.04 OD／5min vs.2.19±0.13 OD／5min)，而KD组TRAP的表达显著高于对照组(0.67±0.07 OD／10min vs.0.37±0.01 OD／10min)(P<0.05)；(10)对照组和KD组OCN表达之间的差异没有显著性。对照组Col I高于KD组(p<0.05)。 KD组的TRAP水平显著高于对照组(P<0.05)；(11)在7天的成骨诱导后，KD组的ALP活性较对照组降低(31.0±3.0 OD／min／㎎ protein vs 44.5±4.5 OD／min／㎎ protein)。经过14天的成骨诱导后，对照组的钙结节数量较KD组更高。 结论：(1)KD同时引起长骨松质骨和皮质骨的骨丢失，但松质骨骨丢失更为明显；(2)KD引起胫骨松质骨骨丢失程度大于肱骨；(3)KD不导致椎体骨丢失；(4)KD导致长骨力学性能下降，但不影响椎体力学性；(5)KD对于大鼠的钙、磷、生素D吸收没有影响；(6)KD喂养对体重有显著性影响，但升高体脂率；(7)KD可能是通过抑制成骨和促进破骨双重作用引起骨丢失；(8)KD导致大鼠BMSC成骨分化能力下降。 关键词：生酮饮食 骨丢失 微观结构 力学性能 骨代谢

 Objective Ketogenic diet ( KD ) is a special high fat diet characters of very low-carbohydrate, high fat and adequate protein which mainly used for pediatric intractable epilepsy. Previous research has found that reduction has occurred in part of children whose' treated with ketogenic diet, but the effect of ketogenic on bone is not clear yet, and also not has a contrast between axial bones and limb bones. The daily intake of total calories and protein was restricted in KD, forcing the body to peripheral obvious increase of ketone body (β-hydroxybutyric, acetoacetate, acetone) as the main energy material. KD was used in refractory epilepsy for more than hundred years in the past, even the effect of ketone body on the nerve injury prevention of cardiovascular disease and even cancer treatment has been confirmed. The objective of the experiment is to establish a ketogenic rat model, verify the affection of ketogenic diet on microstructure and biomechanical of limb bone and axial bone. Ketogenic diet (KD) is a special high-fat diet consisting of extremely low carbohydrate, high fat and a moderate amount of protein. Meanwhile, the effects of KD on bone metabolism and bone mesenchymal stem cells osteogenic differentiation in short-term KD were studied. Methods In order to investigate the effects of KD on the microstructure, compressive stiffness and strength of tibia, humerus and L4 body in rats, fourteen male Sprague-Dowley rats were fed with ketogenic (fat to carbohydrate and protein=3:1) and standard fodder 12 weeks respectively. Body weight, blood glucose and blood ketone were monitored fortnightly. Then the proximal humerus, tibial plateau and middle-one-third L4 body were scanned by micro-CT, and the total bone mass density (TBMD) was calculated by DEXA. The compressive stiffness and strength of scanned skeleton sites were calculated with micro-finite-element models. Result The KD led to ketone level higher, and glucose level and TBMD lower, but serum calcium or phosphate concentration unchanged. It also resulted significant changes in BV/TV, BMD, Tb.N, Tb. sp of cancellous bone and Ct. Th, Tarea and Barea of cortical bone in the tibia and humerus, and changes lesser in the vertebral body, but unchanged in the trabecular thickness. The stiffness and strength decreased significantly after KD in tibia and humerus, and non-significantly in the vertebral body. BMD and Barea were correlated significantly to the stiffness and strength of corresponding bones respectively. The serum ALP express in KD group is significant lower than Control group(Control group: 2.19±0.13 OD / 5 min and KD group: 1.88±0.04 OD / 5 min). On contrast, control group significantly reduced the TRAP express in serum (0.37 0.01 OD /10 min vs 0.67 0.07 OD /10 min) (P < 0.05); (10) The express of OCN in bone tissue is no difference between two groups and Col express in the control group is higher than KD group (p<0.05). On the contray, TRAP level of KD group is higher than control group in bone tissue(p<0.05). The ALP activity of cells in KD group is lower than control group (31.0±3.0 OD/min/㎎ protein vs 44.5±4.5 OD/min/㎎ protein) after 7 days of osteoinductive culture of BMSC. After 14 days of osteoinductive culture of BMSC, control group has more calcium nodules than KD group. Conclusion (1)The KD causes bone loss in both cancellous and cortical bone of tibia and humerus. (2)The bone loss of cancellous bone in tibia is more serious than humerus. (3)The KD results bone loss of vertebral body, but the change was not significantly. (4)TheKD leads decline of biomechanical properties in long bones, but not affect axial bone. (5) The KD had no effect on the uptake of calcium and phosphorus in rats. (6)The KD caused siginificant weight decline after 12 weeks feeding, but the increase the body fat rate in rats. (7) The KD may inhibits osteogenesis and promotes osteoclast in rats. (8) The KD may inhabits BMSC osteogenic differentiation in rats. Key words Ketogenic diet; Bone loss; Microstructure; Biomechanical property; Bone metabolism