恐伤孕鼠对其仔鼠早期长骨发育的影响及其机理研究

发布时间：2018-05-28 20:30

本文选题：肾精亏虚 + 疾病模型　；参考：《湖北中医学院》2009年博士论文

【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1mm~3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(FT_3)、游离四碘甲腺原氨酸(FT_4)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(P0.01)。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(P0.05),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FT_3、FT_4,TSH的含量:模型组仔鼠血清FT_4的含量低于正常组(P0.05),血清FT_3,TSH的含量与正常组相比无差异。补肾组仔鼠血清FT_3,FT_4、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(p0.05);模型组仔鼠bax表达较正常组和补肾组表达上调(p0.05)。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。
[Abstract]:The theory of traditional Chinese medicine (TCM) holds that the kidney is main hidden essence, which is the origin of the kidney. The kidney essence is the main growth, development and reproduction. The main bone of the kidney, the essence of the kidney can produce the marrow and the marrow can raise the bone. According to this, we put forward the view of the growth and development of the kidney main bone. The two aspects of the theoretical study and experimental study are studied respectively.
objective
In this experiment, we use the method of "fear of injuring pregnant rats" to reproduce the kidney essence deficiency model, and study the effect and mechanism of kidney deficiency on the development of long bones in the early stage of offspring rats.
Method
(1) 3 month old SD (sprague-Dawley) rats, 30 females and 15 males, were paired in proportion to 2:1. The female rats after pregnancy were randomly divided into normal group, model group and kidney tonifying group, 10 in each group. From the first day of pregnancy, the model group and the kidney tonifying group were intimidated by the cat for 2 hours in the afternoon until their childbirth. In the baby, the offspring were in childbirth, in the baby. From the first day of the pregnancy, the rats were given the 2ml/100g weight of the decoction of Tonifying the kidney and filling the kidney with the decoction of 2ml/100g, and the normal group and the model group were given the 2ml/100g weight of the normal saline daily to the pregnant rats.
(2) record the birth number of pregnant rats and assess their fertility. Record the time of eyes, teeth, ears, and hair growth; measure the weight and length of the offspring every 4 days from birth to 20 days, calculate the growth of each time, and compare the growth and development of the offspring.
(3) 1-2 rats were randomly selected from each nest at 20 days of age. Cut the right lower extremities from the hip joint, carefully remove all the muscles and connected tissues, remove the fibula and the left tibia, take the proximal 1/3 of the right tibia and put it in 10% neutral formaldehyde for 1 weeks, decalcified, conventional paraffin embedding, and 5um thick continuous longitudinal section; at 20 days of age, one group randomly extracts one group at 20 days of age. The right tibia was separated quickly and the 1mm~3 bone tissue was inserted into the fixed solution of glutaraldehyde. The histomorphological changes of the growth plate of the tibial metaphysis at 20 days after birth were observed under the optical and electronic microscope respectively.
(4) at 20 days of age, 5 right tibial proximal specimens were randomly selected from each group at 20 days of age to perform slice detection. In situ hybridization was used to determine the expression of TGF- beta mRNA in the tibial diaphysis epiphysis at 20 days after birth.
20 days after birth, 1-2 rats were randomly sampled from each nest. 15 rats in each group were selected. Blood samples were taken from decapitated blood. The levels of IGF-1 and BALP were detected by ELISA.
6. 20 days after birth, 3 or 6 broken heads were taken randomly from each litter. The serum free three iodosine adenoprolysine (FT_3), free four iodotiprotic acid (FT_4) and thyroid stimulating hormone (TSH) were detected by chemiluminescence. 20 groups of 30 right tibia specimens from each group were selected to observe the growth of the offspring for 20 days. Expression of Bcl-2/bax in the chondrocytes of the plate.
Result
The average per tire production number of pregnant rats in model group was lower than that of normal group and kidney tonifying group (P0.01). From birth to twentieth days after birth, the average weight of the model group was significantly lower than that of the normal group and the kidney tonifying group. From the eighth day after birth, the average length of the model group was significantly shorter than that of the normal group and the kidney tonifying group. The average body weight and body length growth of the model group were significantly lagged behind. The growth time of the teeth and hair in the model group was slightly later than that in the kidney tonifying group and the normal group.
(2) the morphological changes of the growth plate in the 20 days after birth: the growth plate structure of the model group was disordered, the chondrocytes in the proliferating area were poorly developed, the osteogenesis was weakened, and the bone mass decreased obviously. Under the electron microscope, the structure of the chondrocyte organelles in the proliferation area of the model group of the offspring rats was abnormal, and the part of the nucleus retracted. The normal group and the kidney tonifying group were the offspring. There was no obvious change in the morphology of the growth plate.
(3) the expression of TGF- beta in the metaphysis at 20 days after birth: the positive signal cells were dark blue and located in the cytoplasm; the positive expression intensity was quantified by average photometric analysis. The average luminosity of the model group was lower than that of the normal group and the tonifying group, and there was no difference between the normal group and the kidney tonifying group.
4. After taking the blood for 20 days, the serum IGF-1 and BALP contents in the model group were significantly lower than those in the model group (P0.05), and there was no difference between the normal group and the kidney tonifying group.
The content of serum FT_3, FT_4, TSH in the sera of the model group was lower than that of the normal group (P0.05), and the content of serum FT_3 and TSH was no difference compared with that of the normal group. There was no statistical difference between the serum FT_3, FT_4 and TSH in the serum of the young rats of the kidney group.
(6) 20 days after birth, the positive reaction was brown yellow granules and Bcl-2 expression was located in the cytoplasm. The expression of Bax was located in the cytoplasm and nucleus.Bcl-2 mainly in the static region, the proliferation area was expressed, the expression of Bax was mainly in the hypertrophic region. The expression rate of the positive cells in each group was compared: the expression of Bcl-2 in the model group was significantly lower than that in the normal group and the kidney tonifying group. The expression of Bax in the model group was higher than that in the normal group and the kidney tonifying group (P0.05). (P0.05).
conclusion
The average weight, average length, average body weight, average body weight, length of body growth, tooth growth, hair growth time behind the kidneys group and the normal group were significantly lower than that of the kidney group and the normal group, which showed that the model group showed reproduction in the model group. Dysfunction of kidney, deficiency of kidney essence, and congenital deficiency of kidneys in the offspring of rats.
The histological changes of the tibial growth plate in the model group and the ultrastructural changes of the chondrocytes in the proliferating area showed that the proliferation of the chondrocytes was not reached to the normal level, the growth speed of the growth plate decreased, the osteogenesis in the cartilage was slow, and the bone formation in the osteogenic area decreased significantly.
(3) in the model group, the growth of TGF- beta in the tibial growth plate was inhibited and the expression decreased in the proliferative layer and hypertrophy layer, resulting in slow formation of osteogenesis in the cartilage and affecting the growth of long bones.
(4) the molecular mechanism of bone development disorder caused by kidney deficiency may be as follows: in the model group, there are congenital kidney deficiency, the dysplasia of long bone and the decrease of serum TH level, and the decrease of IGF-1 content. In combination with modern research, there is a dysfunction of the hypothalamus hypophysis thyroid axis, the kidney deficiency is closely related to the content of IGF-1, and the decrease of the expression of IGF-1 by TH is presumed so that the expression of the IGF-1 is reduced. The growth barrier of the growth plate was influenced by the congenital kidney deficiency, the dysplasia of the long bone, the decrease of the expression of TGF- beta in the growth plate, the decrease of the IGF-1 content of the serum, the decrease of the expression of Bcl-2 gene, the influence of the expression of IGF-1, the expression of TGF- beta and the close relationship between the expression of the Bcl-2 gene and the deficiency of the kidney, and the coordination of the coordination of IGF-1 and TGF- beta in the model group. The regulation of Bcl-2/bax expression is another molecular regulation mechanism of bone deficiency caused by kidney deficiency.
(5) the concept of "kidney main bone development" was put forward for the first time, and the effect of kidney on the structure and function of bone was discussed in depth. In the experiment, the model of kidney deficiency deficiency was successfully replicated by means of "fright gestation mice". It was found that the model group had a congenital kidney deficiency, and the development of the long bone had obvious barrier. The Chinese medicine Bushen Tianjing decoction can reverse the abnormality and effectively block the effect of kidney deficiency on the normal development of bone.
【学位授予单位】：湖北中医学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R-332;R228

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