【作者】 桑红灵；

【导师】 周安方；

【作者基本信息】 湖北中医学院 ， 中医基础理论， 2009， 博士

【摘要】 中医理论认为肾主藏精,为先天之本,肾精主生长、发育及生殖;肾主骨,肾所藏之精能生髓,髓能养骨,据此我们提出了肾主骨生长发育的观点。并分别从理论探讨和实验研究两方面进行了研究。目的本实验采用“恐伤孕鼠”的方法复制肾精亏虚模型,研究孕鼠肾虚对其仔鼠早期长骨发育的影响及机制。方法①本实验采用3月龄SD（sprague-Dawley）大鼠,雌性30只,雄性15只,将其按2:1的比例合笼配对。受孕后的雌鼠用随机分为正常组、模型组和补肾组,每组各10只。自怀孕第一天起,模型组和补肾组每日上、下午用猫恐吓孕鼠各2小时直至其分娩,在仔鼠出生后20天进行指标检测。自怀孕第一天起,补肾组每日予中药补肾填精方煎剂2ml/100g体重灌胃,正常组及模型组每日予生理盐水2ml/100g体重灌胃,直至孕鼠临产。②记录孕鼠产子数,评估其生育力。记录仔鼠开眼、出牙、张耳、被毛生长的时间;测量仔鼠出生至20天内每隔4天的体重及身长,计算各时段增长值,各组间比较判断仔鼠的生长发育状况。③仔鼠20日龄时每窝随机抽取1-2只,断头处死。从髋关节处切下右下肢,仔细清除所有肌肉和相连组织,去除腓骨,剩下胫骨。取右胫骨近端1/3,放入10%中性甲醛中固定1周,脱钙,常规石蜡包埋,5um厚连续纵向切片;20日龄时,每组随机抽取一只,断头处死,迅速分离右侧胫骨,切取1mm³骨组织置入戊二醛固定液固定。分别在光学显微镜和电子显微镜下观察各组仔鼠出生后20天胫骨干骺端生长板的组织形态学改变。④仔鼠20日龄时每组随机抽取5个右胫骨近端标本,进行切片检测。原位杂交法测定仔鼠出生后20天胫骨干骺端TGF-βmRNA表达。⑤仔鼠出生后20天,每窝随机抽取1-2只,每组共抽取15只,断头取血,酶联免疫吸附试验法检测血清IGF-1、BALP含量。⑥仔鼠出生后20天,每窝随机抽取3只或6只断头取血。化学发光法检测各组血清游离三碘甲腺原氨酸（FT₃）、游离四碘甲腺原氨酸（FT₄）及促甲状腺激素（TSH）的含量。⑦每组抽取30张右胫骨近端标本切片免疫组化SABC法观察仔鼠出生20天生长板软骨细胞Bcl-2/bax的表达。结果①孕鼠产子数、仔鼠的生长发育状况:模型组孕鼠的平均每胎生产数低于正常组和补肾组（P<0.01）。从出生到出生后第20天内,模型组仔鼠平均体重显著低于正常组和补肾组;从出生后第8天起,模型组仔鼠平均身长显著短于正常组和补肾组;模型组各时段平均体重、身长增长情况明显落后;模型组仔鼠出牙及被毛生长的时间略晚于补肾组和正常组。②仔鼠生后20天生长板形态学改变:模型组仔鼠生长板结构紊乱,增殖区软骨细胞发育不良,成骨作用减弱,骨量明显减少。电镜下模型组仔鼠生长板增殖区内多个视野下软骨细胞细胞器结构异常,部分细胞核固缩。正常组与补肾组仔鼠生长板形态学则无明显改变。③仔鼠生后20天时TGF-β在干骺端的表达:阳性信号的细胞呈深蓝色,定位于细胞浆;阳性表达强度采用平均光度进行定量分析。模型组平均光度较正常组和补肾组下降,正常组和补肾组相比无差异。④仔鼠出生20天断头取血,血清IGF-1、BALP含量:模型组显著降低（P<0.05）,正常组和补肾组相比无差异。⑤仔鼠生后20天断头取血,血清FT₃、FT₄,TSH的含量:模型组仔鼠血清FT₄的含量低于正常组（P<0.05）,血清FT₃,TSH的含量与正常组相比无差异。补肾组仔鼠血清FT₃,FT₄、TSH的含量与正常组相比无统计学差异。⑥仔鼠生后20天免疫组化阳性反应为棕黄色颗粒,Bcl-2表达位于细胞浆;bax表达位于细胞浆和细胞核。Bcl-2主要在静止区、增殖区表达,bax主要在肥大区表达,各组阳性细胞的表达率情况比较:模型组仔鼠Bcl-2表达较正常组和补肾组显著下降（p<0.05）;模型组仔鼠bax表达较正常组和补肾组表达上调（p<0.05）。结论①模型组孕鼠的每胎生产数低于正常组和补肾组;仔鼠从出生到出生后第20天内,平均体重、平均身长、各时段平均体重、身长增长情况、出牙、被毛生长时间较补肾组和正常组明显落后,结合恐伤肾的病因病机,说明模型组孕鼠表现出生殖功能减退,存在肾精亏虚;同时,其仔鼠也存在先天肾虚,故其机体发育迟缓。②模型组仔鼠胫骨生长板组织学改变、增殖区软骨细胞超微结构变化,提示其软骨细胞增殖未达到正常水平,生长板生长速度降低,软骨内成骨缓慢;成骨区成骨量明显减少。③模型组仔鼠胫骨生长板TGF-β在增殖层和肥大层受到抑制,表达减少,导致软骨内成骨形成缓慢,影响长骨生长。④肾虚致骨发育障碍的分子机制可能为:模型组仔鼠存在先天肾虚、长骨发育障碍和血清TH水平下降,IGF-1含量减少。结合现代研究认为存在下丘脑-垂体-甲状腺轴的功能紊乱,肾虚与IGF-1的含量密切相关,推测TH降低影响IGF-1的表达从而影响生长板的生长障碍;模型组仔鼠存在先天肾虚、长骨发育障碍、生长板TGF-β表达下降,同时血清IGF-1含量减少,伴随着Bcl-2基因表达的下调。结合现代肾虚对IGF-1、TGF-β表达的影响,Bcl-2基因的表达与肾虚的密切关系,推测IGF-1、TGF-β的协同作用及调控Bcl-2/bax的表达为肾虚致骨发育障碍的另一分子调控机制。⑤本课题首次提出了“肾主骨发育”的观点,并从肾对骨的结构和功能影响的角度进行了深入的理论探讨。实验方面采用“恐伤孕鼠”的方法成功复制出肾精亏虚模型,发现模型组仔鼠具有先天肾虚的表现,其长骨发育存在明显的障碍,中药补肾填精方可逆转上述异常,有效阻断肾虚对骨的正常发育的影响。更多还原

【Abstract】 According to Chinese medicine theory,the main function of kidney is the possession of Shenjing,which is the essence of birth and also the main essence associated with growth,development and reproduction of our bodies.Kidney is in charge of bone,while Shenjing can made Sui,which can provide nutrition to bone.Therefore,we have put forward the main opinion of kidney controlling bone growth and development,and separately from the theoretical and experimental aspects to study.ObjectiveThe study was designed to investigate the mechanism that" horror hurt pregnant rat "had effect on the long bone development.Methods①3-month-old SD（Sprague-Dawley） female rats 30,male 15 to be combined according to the proportion of 2:1 matching cage.Female after conception using a random access method is divided into normal group,model group and bu-shen group,10 in each group.During gestational period,the rats of the model and bushen group were frighented by cat four hours（2 hours Am,2 hours Pm） a day until the birth.The results were examined at postnatal day 20.Bushen group model since the first day,a day to the Chinese side Bushen Tianjing 2ml-/100g weight decoction orally,the normal group and model group also based on body weight given normal saline orally.Time model for gestational.②Record the number of pregnant rats given birth and assess their fertility.Record the eye,the teeth,the ear,the hair growth time;measurement of rats from birth up to 20 days once every four days in length and weight to calculate the progress in the growth period,compared among the three groups of rats to determine the growth and development of the situation.③20- day-old rats were randomly selected when the litter is only 1-2,broken to death.From the hip,cutting the right lower limb,carefully remove all the muscles and connected organization,remove the fibula,and the remaining tibia. Put right proximal tibia from 1/3 removal of soft tissue into 10%neutral formaldehyde fixed one week,decalcified,paraffin-embedded conventional,5 urn thick con- tinuous vertical sections;20-day-old rats were randomly selected, decapitated executed quickly separated from the right tibia,1 mm³ cut into the bone tissue fixative glutaraldehyde fixed.Separately in the optical microscope and electron microscope observation of the growth plate of rats in each group metaphyseal tibia morphological changes of the organization.④20-day-old rats were randomly selected at each of five specimens of the right proximal tibia,the detection section.Determination of in situhybridizal pups born tibial metaphysis expression of TGF-PmRNA.The average optical density were determined by photo analysis technique.⑤Rats 20 days after birth,1-2 litter were randomly selected,each group collected a total of 15,collectting blood,enzyme-linked immunosorbent assay serum IGF-1,BALP content.⑥Rats 20 days after birth,litter three or six randomly selected,collectting blood. Chemilum inescence detection of three groups of serum FT₃?FT₄ and T-SH levels.⑦Each taking 30 right proximal tibia biopsy specimens by immunohistoche-mical SABC,growth plate chondrocytes were observed the expression of Bcl -2/bax.The positive ratio were determined by photo-analysis technique.All the results were showed by mean value±SEM.Results①The number of neonatal rats and their conditions of growth and development :the model group of pregnant rats produce lower than normal or bushen group （P<0.01）.From postnal day 1 to day 20,the average weight of the model g -roup rats have significantly reduced campared with normal group and bushen group;from the 8th day after birth,the model group rats average length compared with the normal group and bushen group have great decrease;In model group,the average weight and length of the major time were obviously lagging behind.At the same time,their teeth and hair growing out of time slightly later than the bushen group and normal group.②Rats 20 days after birth,the morphological changes of growth plate:In the model group,the structure of rat growth plate disorders,proliferative zone chondrocytes are stunted and weakened the bone formation,bone volume decreased significantly.Form electron microscope,in model group rats,a number of vision in growth plate cartilage cells have abnormal structure,some nuclear pyknosis.Normal and bushen rats showed no significant change in morpholog -y.③Rats 20 days after birth the TGF-βexpression in the metaphysis:positive signals in cells,dark blue,located in cytoplasm;positive expression intensity of the use of quantitative analysis of optical density.The average brightness of m odel group has a decline while the normal group has no difference compared with bushen group.④Rats of 20 days blood serum IGF-1,BALP concentration:a significant reduction in the model group（P<0.05）,the normal group has no difference compared with bushen group.⑤Rats of 20 days after birth,blood serum FT₃,FT₄,TSH levels:Compared with the other two groups,the model group rats FT₄ serum levels lower than normal group（P<0.05）,while serum FT3,TSH levels has no difficence.Bushen rats serum FT3,FT4,TSH levels compared with normal group have no statisstical difference.⑥Immunohistochemical positive reaction for the brown-yellow granules,Bcl-2 expression is located in cytoplasm;bax expression located in cytoplasm and nucleus.Bcl-2 mainly in the static and the proliferation areas;bax expression mainly in the hypertrophic zone;the rate of positive cells compared each other: the model group rats Bcl-2 expression than normal and bushen group has decreased significantly（p<0.05）while bax expression has increased（p<0.05）.Conclusion①Model group of pregnant rats have lower fertility than normal and bushen group;rats from birth to the first 20 days,the average weight,average length, average weight and length growth of each period,the teeth,the hair growth time are significantly behind.Combined with fear of injury etiology and pathogenesis of the kidney,the model group of pregnant rats showed decreased reproductive function,at the same time its existence essence deficiency of the kidney resulting to their offspring body growth retardation.②Model group rats tibial growth plate histology changes and the ultrastructture of proliferative zone delicate that chondrocytes of cartilage cell proliferation prompting change does not meet the normal level,together with reducing the growth rate of growth plate cartilage into bone.③The expression of TGF-βin model rats tibial growth plate（Prolierative and Hypertrophic zone）was reduced,which causing the formation from cartilage into bone slow and affecting the growth of long bones.④Bone developmental disorder caused by deficiency of the kidney of the molecular mechanisms were likely to be:the existence of the model group rats congenital deficiency of the kidney,long bones stunted growth,decreased serum levels of thyroid ho- rmones and IGF-1.Kidney deficiency of modern research studies suggest that the existence of hypothalamic-pituitary -thyroid axis dysfunction,kidney deficiency and IGF-1 is closely related,consider the impact of thyroid hormone to IGF-1 expression causing the development of the growth plate;model group congenital deficiency of the kidney,stunting of long bone,growth plate decreased the expression of TGF-β,while serum IGF 1 content,accompanied by Bcl-2 gene expression declines.There are close relationship between kidney deficiency and the expression of IGF-1,TGF-P with Bcl-2.We can conclud that IGF-1,TGF-P have synergies and their adjustment on expression of Bc-1-2/bax is another molecular regulatory mechanism.⑤The topic of "kidney is bone growth decision " was raised for the first time, and from the angles of the impacts of kidney to the bone structure and function,we made an in-depth study.Experiments using "horror hurt pregnant rats" approach successfully replicated the essence deficiency model and found that the model group rats with the performance of a congenital deficiency of the kidney,existing an obvious obstacle of the long bone development,whiile Chinese medicine can reversed the abovementioned abnormal and effectively block the impacts of kidney deficiency on bone development.更多还原