塔里木马鹿鹿茸骨化及其相关因子关系研究

发布时间：2018-06-23 17:56

本文选题：塔里木马鹿 + 鹿茸　；参考：《华中农业大学》2016年博士论文

【摘要】：鹿茸是一种名贵的中药材,具有温肾壮阳、生精益血、强筋补髓的功效。它是鹿科动物特有的组织,具有很强的再生能力,在自然生长情况下每年都要经历发生、生长、骨化、脱落等过程。塔里木马鹿是单位体重产茸量最高的鹿科亚种,在生茸期鹿茸生长迅速,鹿茸角的生长的本质就是在软骨骨架上的成骨过程,期间大量的矿物质元素尤其是钙将沉积在鹿茸角中。钙离子的沉积需要多种因素调控,关系比较密切的因素有钙敏感受体(CaSR)、甲状旁腺素(PTH)、雄激素(ADG)、降钙素(CT)、骨特异性碱性磷酸酶(BALP)、骨唾液酸糖蛋白(BSP)、骨钙素(BGP)等激素。为了明确钙离子在鹿茸中的沉积规律、以及鹿茸骨化与调控因素之间的关系,本试验通过测定生茸期鹿茸钙、血钙、骨钙、血液中ADG、PTH、CT、BALP、BGP及BSP等激素的含量和变化规律,对塔里木马鹿鹿茸间充质细胞、前成软骨细胞、软骨细胞、骨膜细胞进行体外培养,采用激光共聚焦显微技术观察细胞外钙离子向胞内流动以及测定胞内钙离子浓度,采用Real-time PCR、Western Blot等方法,分析CaSR、BSP、Runx2、GAPDH等基因表达情况,分析研究鹿茸角骨化机制。通过试验研究,取得如下成果:1、塔里木马鹿鹿茸生长状况,及鹿茸钙、血钙、骨钙变化规律。青年组塔里木马鹿鹿茸角的生长速度在鹿茸角开始生长的第84天达到最大值,血Ca~(2+)含量在鹿茸角开始生长的第70天出现下降趋势,而后升高,于鹿茸角开始生长的第84天达到最高值。中年组、老年组马鹿在鹿茸角开始生长的第70天鹿茸角生长速度达到最大值,血液中Ca~(2+)含量于鹿茸角开始生长的第57天达到最高值,在鹿茸角开始生长的第70天出现降低态势,此后Ca~(2+)含量又缓慢攀升。塔里木马鹿鹿茸生长期约有100天(从春分至夏至),年龄大的鹿只其鹿茸角先开始生长、先进入骨化阶段。鹿茸钙含量是不断增加的,鹿茸骨化过程是分阶段进行的,在生长期表现为钙在软骨内沉积,在骨化期则是软骨组织迅速钙化。马鹿主要骨骼均存在脱钙现象,鹿茸角中的钙主要来源于肋骨,其次是骨盆骨,而小腿骨与肩胛骨脱钙程度最轻。2、鹿茸骨化与血液中相关激素之间的关系。在鹿茸角的不同生长阶段,塔里木马鹿外周血中血清ADG、PTH、CT、BALP、BGP及BSP的含量存在着不断的变化,尤其是骨化阶段变化最为明显。血清ADG水平的变化最明显的是在锯茸后,在此之前都一直处于低水平状态,然后迅速升高,达到显著性差异(P0.01)。血清PTH在鹿茸角生长阶段变化一直不明显,骨化期之前一直处于低水平状态,但在进入骨化后期,各组马鹿血清PTH含量显著增高(P0.01)。各组马鹿血清BALP含量在生茸初期至快速生长期均有下降趋势,快速生长期至骨化后期,各组马鹿血清BALP含量均增加,在鹿茸收获以后,明显下降。血清BGP、BSP均是随着鹿茸角的生长而在升高,进入骨化期后开始下降,鹿茸角收获后就一直在下调,与鹿茸角的生长速度呈正相关。各组血清CT的浓度变化趋势与鹿茸角生长率相同,但滞后于鹿茸角生长速度的变化,在生茸初期、茸快速生长期均缓慢升高,到骨化后期达到最高,然后开始下降。说明血液中ADG、BALP、BGP、BSP等激素与鹿茸角快速骨化有着密切关系。血液中PTH、CT等激素与脱钙的骨骼重建有着密切关系。3、鹿茸细胞培养及与钙调节相关基因的关系。采集处于生长期鹿茸顶端部分进行细胞培养,实验结果表明:前软骨细胞(ACC)、间充质细胞(MSC)易培养,而软骨细胞(CC)生长较慢。用激光共聚焦方法检测[Ca~(2+)]i的变化,结果发现MSC的[Ca~(2+)]i高于ACC、CC,补充外源性钙时,胞外Ca~(2+)流入胞内,MSC、ACC细胞需要的时间较短,而CC细胞则需要的时间较长。说明越临近骨化期,鹿茸角中软骨细胞的数量越多,需要的钙量也越来越多。用实时荧光定量PCR扩增CaSR、BSP、Runx2等基因,前软骨细胞的BSP、CaSR、Runx2等基因mRNA表达水平明显高于间充质细胞,而软骨细胞中只有Runx2基因的mRNA表达水平高于间充质细胞、前软骨细胞。Western Blot检测得到了相同的结果。说明鹿茸的生长与MSC、ACC的分化能力有关,鹿茸的钙化与CC的数量、胞外钙的浓度有关。在鹿茸生长期内,BSP调节Ca~(2+)进入到软骨细胞形成骨小梁的过程中起到重要作用;Ca~(2+)将通过CaSR介导在软骨细胞中沉积;Runx2在软骨成熟中可以起到一定的作用。
[Abstract]:Antler is a kind of precious Chinese herbal medicine, which has the effect of warming and kidney yang, producing lean blood and reinforcing the marrow. It is a special tissue of the deer family and has a strong regenerative ability. In natural growth, it has to experience, grow, ossification and fall off every year. The growth of antler is rapid. The essence of the growth of antler horn is the process of osteogenesis on the cartilaginous skeleton. During the period, a large number of mineral elements, especially calcium, will be deposited in the antler horn. Calcium ion deposition requires a variety of factors, such as calcium sensitive receptor (CaSR), parathyroid hormone (PTH), androgen (ADG), calcitonin (CT). Bone specific alkaline phosphatase (BALP), bone sialic acid glycoprotein (BSP), osteocalcin (BGP) and other hormones. In order to determine the relationship between calcium ion deposition in deer antler, and the relationship between antler ossification and regulatory factors, this test was conducted by measuring the content and changes of the antler Calc, blood calcium, bone calcium, ADG, PTH, CT, BALP, BGP and BSP in the blood of the pilose antler. The cytoplasm cells, anterior chondrocytes, chondrocytes and periosteum cells were cultured in vitro. The intracellular calcium ion flow and intracellular calcium concentration were observed by laser confocal microscopy. The gene tables such as CaSR, BSP, Runx2, GAPDH and other genes were analyzed by Real-time PCR and Western Blot. Through the experimental study, the following results were obtained: 1, the growth of antler deer antler, calcium antler, blood calcium, bone calcium. The growth rate of the deer antler horns in the youth group reached the maximum at the eighty-fourth days of the growth of the deer antler angle, and the blood Ca~ (2+) content began to grow in the antler horn. On the seventieth day, there was a downward trend, then increased, and reached the highest value on the eighty-fourth day of the growth of the antler angle. In the middle age group, the growth rate of the antler angle of the deer antler angle reached the maximum at the seventieth day of the growth of the antler horn, and the content of Ca~ (2+) in the blood reached the highest value on the fifty-seventh day of the growth of the deer antler angle, and the seventieth day of the growth of the deer antler angle. The content of Ca~ (2+) increased slowly since then. The growth period of antler deer antler of Tarim deer was about 100 days (from Vernal Equinox to summer solstice), and the deer antler angle of the older deer began to grow first and advanced into the ossification stage. The calcium content of the deer antler was increasing, the process of the pilose antler ossification was carried out in stages, and the calcium was deposited in the cartilage during the growth period. In the period of ossification, there is a rapid calcification of the cartilage tissue. The main skeleton of the deer is decalcification. The calcium in the horn of the deer antler mainly comes from the ribs, followed by the pelvis, and the decalcification of the leg bone and the scapula is the lightest.2, and the relationship between the antler ossification and the related hormones in the blood. The content of ADG, PTH, CT, BALP, BGP and BSP in serum is constantly changing, especially in the stage of ossification. The most obvious change of serum ADG level is in low level before sawing, and then rapidly rising to achieve significant difference (P0.01). The change of serum PTH at the growth stage of antler angle has not been changed. Obviously, the period of ossification was at a low level before the period of ossification, but the content of serum PTH in each group was significantly increased (P0.01) in the late stage of ossification. The serum BALP content of the deer's serum decreased in the early stage to the rapid growth period, and the rapid growth period to the later stage of the ossification, the serum BALP content of each group increased. After the pilose antler harvest, the content of the serum was obviously increased. The serum BGP and BSP were increased with the growth of the antler horn, and began to decline after the ossification period. After the harvest of antler horn, the growth rate of the antler angle was positively correlated. The trend of serum CT concentration in each group was the same as the growth rate of antler angle, but lagged behind the growth rate of antler angle, and it was fast in the early stage of the pilose antler. The rapid growth period increased slowly and reached the highest in the late ossification, and then began to fall. It indicated that the ADG, BALP, BGP, BSP and other hormones in the blood were closely related to the rapid ossification of the antler horn. The PTH, CT and other hormones in the blood have a close relationship with the decalcified bone reconstruction. The relationship between the culture of the pilose antler cells and the genes related to calcium regulation. Cell culture at the top part of the long pilose antler was carried out. The experimental results showed that the precursor chondrocytes (ACC), the mesenchymal cells (MSC) were easily cultured, and the chondrocytes (CC) grew slowly. The changes of [Ca~ (2+)]i were detected by laser confocal method. The results showed that [Ca~ (2+)]i of MSC was higher than ACC, CC, and supplemented exogenous calcium. The time needed is shorter and the CC cell needs a longer time. It shows that the closer the ossification period, the more the number of cartilage cells in the antler angle, the more the amount of calcium needed. The CaSR, BSP, Runx2 and other genes of the BSP, CaSR, Runx2, and other genes in the anterior chondrocytes are significantly higher than those of mesenchymal cells, and the expression level of the BSP, CaSR, Runx2 and other genes in the anterior chondrocytes is obviously higher than that of the mesenchymal cells, and the soft expression level of the precursor cells is obviously higher than that of the mesenchymal cells. The mRNA expression level of only Runx2 gene in bone cells was higher than that of mesenchymal cells, and the.Western Blot of anterior chondrocytes was detected by the same result. The growth of pilose antler was related to the differentiation ability of MSC and ACC. The calcification of pilose antler was related to the quantity of CC and the concentration of extracellular calcium. In the growth period of pilose antler, BSP regulated Ca~ (2+) into the cartilage cell shape. Trabecular bone plays an important role; Ca~ (2+) is mediated through CaSR in chondrocytes; Runx2 plays a role in cartilage maturation.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S825

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