本文選題：塔里木馬鹿 + 鹿茸　； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：鹿茸是一種名貴的中藥材,具有溫腎壯陽、生精益血、強(qiáng)筋補(bǔ)髓的功效。它是鹿科動(dòng)物特有的組織,具有很強(qiáng)的再生能力,在自然生長情況下每年都要經(jīng)歷發(fā)生、生長、骨化、脫落等過程。塔里木馬鹿是單位體重產(chǎn)茸量最高的鹿科亞種,在生茸期鹿茸生長迅速,鹿茸角的生長的本質(zhì)就是在軟骨骨架上的成骨過程,期間大量的礦物質(zhì)元素尤其是鈣將沉積在鹿茸角中。鈣離子的沉積需要多種因素調(diào)控,關(guān)系比較密切的因素有鈣敏感受體(CaSR)、甲狀旁腺素(PTH)、雄激素(ADG)、降鈣素(CT)、骨特異性堿性磷酸酶(BALP)、骨唾液酸糖蛋白(BSP)、骨鈣素(BGP)等激素。為了明確鈣離子在鹿茸中的沉積規(guī)律、以及鹿茸骨化與調(diào)控因素之間的關(guān)系,本試驗(yàn)通過測定生茸期鹿茸鈣、血鈣、骨鈣、血液中ADG、PTH、CT、BALP、BGP及BSP等激素的含量和變化規(guī)律,對(duì)塔里木馬鹿鹿茸間充質(zhì)細(xì)胞、前成軟骨細(xì)胞、軟骨細(xì)胞、骨膜細(xì)胞進(jìn)行體外培養(yǎng),采用激光共聚焦顯微技術(shù)觀察細(xì)胞外鈣離子向胞內(nèi)流動(dòng)以及測定胞內(nèi)鈣離子濃度,采用Real-time PCR、Western Blot等方法,分析CaSR、BSP、Runx2、GAPDH等基因表達(dá)情況,分析研究鹿茸角骨化機(jī)制。通過試驗(yàn)研究,取得如下成果:1、塔里木馬鹿鹿茸生長狀況,及鹿茸鈣、血鈣、骨鈣變化規(guī)律。青年組塔里木馬鹿鹿茸角的生長速度在鹿茸角開始生長的第84天達(dá)到最大值,血Ca~(2+)含量在鹿茸角開始生長的第70天出現(xiàn)下降趨勢,而后升高,于鹿茸角開始生長的第84天達(dá)到最高值。中年組、老年組馬鹿在鹿茸角開始生長的第70天鹿茸角生長速度達(dá)到最大值,血液中Ca~(2+)含量于鹿茸角開始生長的第57天達(dá)到最高值,在鹿茸角開始生長的第70天出現(xiàn)降低態(tài)勢,此后Ca~(2+)含量又緩慢攀升。塔里木馬鹿鹿茸生長期約有100天(從春分至夏至),年齡大的鹿只其鹿茸角先開始生長、先進(jìn)入骨化階段。鹿茸鈣含量是不斷增加的,鹿茸骨化過程是分階段進(jìn)行的,在生長期表現(xiàn)為鈣在軟骨內(nèi)沉積,在骨化期則是軟骨組織迅速鈣化。馬鹿主要骨骼均存在脫鈣現(xiàn)象,鹿茸角中的鈣主要來源于肋骨,其次是骨盆骨,而小腿骨與肩胛骨脫鈣程度最輕。2、鹿茸骨化與血液中相關(guān)激素之間的關(guān)系。在鹿茸角的不同生長階段,塔里木馬鹿外周血中血清ADG、PTH、CT、BALP、BGP及BSP的含量存在著不斷的變化,尤其是骨化階段變化最為明顯。血清ADG水平的變化最明顯的是在鋸茸后,在此之前都一直處于低水平狀態(tài),然后迅速升高,達(dá)到顯著性差異(P0.01)。血清PTH在鹿茸角生長階段變化一直不明顯,骨化期之前一直處于低水平狀態(tài),但在進(jìn)入骨化后期,各組馬鹿血清PTH含量顯著增高(P0.01)。各組馬鹿血清BALP含量在生茸初期至快速生長期均有下降趨勢,快速生長期至骨化后期,各組馬鹿血清BALP含量均增加,在鹿茸收獲以后,明顯下降。血清BGP、BSP均是隨著鹿茸角的生長而在升高,進(jìn)入骨化期后開始下降,鹿茸角收獲后就一直在下調(diào),與鹿茸角的生長速度呈正相關(guān)。各組血清CT的濃度變化趨勢與鹿茸角生長率相同,但滯后于鹿茸角生長速度的變化,在生茸初期、茸快速生長期均緩慢升高,到骨化后期達(dá)到最高,然后開始下降。說明血液中ADG、BALP、BGP、BSP等激素與鹿茸角快速骨化有著密切關(guān)系。血液中PTH、CT等激素與脫鈣的骨骼重建有著密切關(guān)系。3、鹿茸細(xì)胞培養(yǎng)及與鈣調(diào)節(jié)相關(guān)基因的關(guān)系。采集處于生長期鹿茸頂端部分進(jìn)行細(xì)胞培養(yǎng),實(shí)驗(yàn)結(jié)果表明:前軟骨細(xì)胞(ACC)、間充質(zhì)細(xì)胞(MSC)易培養(yǎng),而軟骨細(xì)胞(CC)生長較慢。用激光共聚焦方法檢測[Ca~(2+)]i的變化,結(jié)果發(fā)現(xiàn)MSC的[Ca~(2+)]i高于ACC、CC,補(bǔ)充外源性鈣時(shí),胞外Ca~(2+)流入胞內(nèi),MSC、ACC細(xì)胞需要的時(shí)間較短,而CC細(xì)胞則需要的時(shí)間較長。說明越臨近骨化期,鹿茸角中軟骨細(xì)胞的數(shù)量越多,需要的鈣量也越來越多。用實(shí)時(shí)熒光定量PCR擴(kuò)增CaSR、BSP、Runx2等基因,前軟骨細(xì)胞的BSP、CaSR、Runx2等基因mRNA表達(dá)水平明顯高于間充質(zhì)細(xì)胞,而軟骨細(xì)胞中只有Runx2基因的mRNA表達(dá)水平高于間充質(zhì)細(xì)胞、前軟骨細(xì)胞。Western Blot檢測得到了相同的結(jié)果。說明鹿茸的生長與MSC、ACC的分化能力有關(guān),鹿茸的鈣化與CC的數(shù)量、胞外鈣的濃度有關(guān)。在鹿茸生長期內(nèi),BSP調(diào)節(jié)Ca~(2+)進(jìn)入到軟骨細(xì)胞形成骨小梁的過程中起到重要作用;Ca~(2+)將通過CaSR介導(dǎo)在軟骨細(xì)胞中沉積;Runx2在軟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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S825

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相關(guān)期刊論文 前10條

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本文編號(hào)：2057934