本文選題：腎精虧虛 + 疾病模型��； 參考：《湖北中醫(yī)學院》2009年博士論文

【摘要】： 中醫(yī)理論認為腎主藏精,為先天之本,腎精主生長、發(fā)育及生殖;腎主骨,腎所藏之精能生髓,髓能養(yǎng)骨,據(jù)此我們提出了腎主骨生長發(fā)育的觀點。并分別從理論探討和實驗研究兩方面進行了研究。 目的 本實驗采用“恐傷孕鼠”的方法復制腎精虧虛模型,研究孕鼠腎虛對其仔鼠早期長骨發(fā)育的影響及機制。 方法 ①本實驗采用3月齡SD(sprague-Dawley)大鼠,雌性30只,雄性15只,將其按2:1的比例合籠配對。受孕后的雌鼠用隨機分為正常組、模型組和補腎組,每組各10只。自懷孕第一天起,模型組和補腎組每日上、下午用貓恐嚇孕鼠各2小時直至其分娩,在仔鼠出生后20天進行指標檢測。自懷孕第一天起,補腎組每日予中藥補腎填精方煎劑2ml/100g體重灌胃,正常組及模型組每日予生理鹽水2ml/100g體重灌胃,直至孕鼠臨產(chǎn)。 ②記錄孕鼠產(chǎn)子數(shù),評估其生育力。記錄仔鼠開眼、出牙、張耳、被毛生長的時間;測量仔鼠出生至20天內(nèi)每隔4天的體重及身長,計算各時段增長值,各組間比較判斷仔鼠的生長發(fā)育狀況。 ③仔鼠20日齡時每窩隨機抽取1-2只,斷頭處死。從髖關節(jié)處切下右下肢,仔細清除所有肌肉和相連組織,去除腓骨,剩下脛骨。取右脛骨近端1/3,放入10%中性甲醛中固定1周,脫鈣,常規(guī)石蠟包埋,5um厚連續(xù)縱向切片;20日齡時,每組隨機抽取一只,斷頭處死,迅速分離右側(cè)脛骨,切取1mm~3骨組織置入戊二醛固定液固定。分別在光學顯微鏡和電子顯微鏡下觀察各組仔鼠出生后20天脛骨干骺端生長板的組織形態(tài)學改變。 ④仔鼠20日齡時每組隨機抽取5個右脛骨近端標本,進行切片檢測。原位雜交法測定仔鼠出生后20天脛骨干骺端TGF-βmRNA表達。 ⑤仔鼠出生后20天,每窩隨機抽取1-2只,每組共抽取15只,斷頭取血,酶聯(lián)免疫吸附試驗法檢測血清IGF-1、BALP含量。 ⑥仔鼠出生后20天,每窩隨機抽取3只或6只斷頭取血。化學發(fā)光法檢測各組血清游離三碘甲腺原氨酸(FT_3)、游離四碘甲腺原氨酸(FT_4)及促甲狀腺激素(TSH)的含量。⑦每組抽取30張右脛骨近端標本切片免疫組化SABC法觀察仔鼠出生20天生長板軟骨細胞Bcl-2/bax的表達。 結(jié)果 ①孕鼠產(chǎn)子數(shù)、仔鼠的生長發(fā)育狀況:模型組孕鼠的平均每胎生產(chǎn)數(shù)低于正常組和補腎組(P0.01)。從出生到出生后第20天內(nèi),模型組仔鼠平均體重顯著低于正常組和補腎組;從出生后第8天起,模型組仔鼠平均身長顯著短于正常組和補腎組;模型組各時段平均體重、身長增長情況明顯落后;模型組仔鼠出牙及被毛生長的時間略晚于補腎組和正常組。 ②仔鼠生后20天生長板形態(tài)學改變:模型組仔鼠生長板結(jié)構(gòu)紊亂,增殖區(qū)軟骨細胞發(fā)育不良,成骨作用減弱,骨量明顯減少。電鏡下模型組仔鼠生長板增殖區(qū)內(nèi)多個視野下軟骨細胞細胞器結(jié)構(gòu)異常,部分細胞核固縮。正常組與補腎組仔鼠生長板形態(tài)學則無明顯改變。 ③仔鼠生后20天時TGF-β在干骺端的表達:陽性信號的細胞呈深藍色,定位于細胞漿;陽性表達強度采用平均光度進行定量分析。模型組平均光度較正常組和補腎組下降,正常組和補腎組相比無差異。 ④仔鼠出生20天斷頭取血,血清IGF-1、BALP含量:模型組顯著降低(P0.05),正常組和補腎組相比無差異。 ⑤仔鼠生后20天斷頭取血,血清FT_3、FT_4,TSH的含量:模型組仔鼠血清FT_4的含量低于正常組(P0.05),血清FT_3,TSH的含量與正常組相比無差異。補腎組仔鼠血清FT_3,FT_4、TSH的含量與正常組相比無統(tǒng)計學差異。 ⑥仔鼠生后20天免疫組化陽性反應為棕黃色顆粒,Bcl-2表達位于細胞漿;bax表達位于細胞漿和細胞核。Bcl-2主要在靜止區(qū)、增殖區(qū)表達,bax主要在肥大區(qū)表達,各組陽性細胞的表達率情況比較:模型組仔鼠Bcl-2表達較正常組和補腎組顯著下降(p0.05);模型組仔鼠bax表達較正常組和補腎組表達上調(diào)(p0.05)。 結(jié)論 ①模型組孕鼠的每胎生產(chǎn)數(shù)低于正常組和補腎組;仔鼠從出生到出生后第20天內(nèi),平均體重、平均身長、各時段平均體重、身長增長情況、出牙、被毛生長時間較補腎組和正常組明顯落后,結(jié)合恐傷腎的病因病機,說明模型組孕鼠表現(xiàn)出生殖功能減退,存在腎精虧虛;同時,其仔鼠也存在先天腎虛,故其機體發(fā)育遲緩。 ②模型組仔鼠脛骨生長板組織學改變、增殖區(qū)軟骨細胞超微結(jié)構(gòu)變化,提示其軟骨細胞增殖未達到正常水平,生長板生長速度降低,軟骨內(nèi)成骨緩慢;成骨區(qū)成骨量明顯減少。 ③模型組仔鼠脛骨生長板TGF-β在增殖層和肥大層受到抑制,表達減少,導致軟骨內(nèi)成骨形成緩慢,影響長骨生長。 ④腎虛致骨發(fā)育障礙的分子機制可能為:模型組仔鼠存在先天腎虛、長骨發(fā)育障礙和血清TH水平下降,IGF-1含量減少。結(jié)合現(xiàn)代研究認為存在下丘腦-垂體-甲狀腺軸的功能紊亂,腎虛與IGF-1的含量密切相關,推測TH降低影響IGF-1的表達從而影響生長板的生長障礙;模型組仔鼠存在先天腎虛、長骨發(fā)育障礙、生長板TGF-β表達下降,同時血清IGF-1含量減少,伴隨著Bcl-2基因表達的下調(diào)。結(jié)合現(xiàn)代腎虛對IGF-1、TGF-β表達的影響,Bcl-2基因的表達與腎虛的密切關系,推測IGF-1、TGF-β的協(xié)同作用及調(diào)控Bcl-2/bax的表達為腎虛致骨發(fā)育障礙的另一分子調(diào)控機制。 ⑤本課題首次提出了“腎主骨發(fā)育”的觀點,并從腎對骨的結(jié)構(gòu)和功能影響的角度進行了深入的理論探討。實驗方面采用“恐傷孕鼠”的方法成功復制出腎精虧虛模型,發(fā)現(xiàn)模型組仔鼠具有先天腎虛的表現(xiàn),其長骨發(fā)育存在明顯的障礙,中藥補腎填精方可逆轉(zhuǎn)上述異常,有效阻斷腎虛對骨的正常發(fā)育的影響。
[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.
【學位授予單位】：湖北中醫(yī)學院
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R-332;R228

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