【摘要】：鹿茸是梅花鹿第二顯著性征,具有周期性再生的特性。在它生長(zhǎng)最旺盛時(shí)期,其生長(zhǎng)速度可達(dá)到2cm/d。已有的研究表明:鹿茸的再生是一個(gè)基于鹿茸干細(xì)胞代謝且受多種因子調(diào)控的復(fù)雜過(guò)程,此外有大量研究報(bào)道IGF1R基因參與多種細(xì)胞的增殖與代謝。然而,IGF1R基因在鹿茸軟骨細(xì)胞的研究鮮有報(bào)道,因此本試驗(yàn)旨在從鹿茸軟骨細(xì)胞生長(zhǎng)、增殖、周期和凋亡的角度探究IGF1R的功能,為進(jìn)一步深入研究鹿茸生長(zhǎng)發(fā)育分子機(jī)理提供新的觀點(diǎn)。本試驗(yàn)采用RNAi技術(shù)干擾鹿茸軟骨細(xì)胞IGF1R m RNA的表達(dá),從不同角度分析其對(duì)軟骨細(xì)胞的影響。本研究以RNAi-Ready p SIREN-Retro QZs Green plasmid為載體,以IGF1R作為候選基因研究其對(duì)鹿茸軟骨細(xì)胞生長(zhǎng)的調(diào)控機(jī)制,成功構(gòu)建了2個(gè)RNAi重組質(zhì)粒,分別命名為psh RNA-1和psh RNA-2,用于轉(zhuǎn)染軟骨細(xì)胞。利用Real-Time PCR和Western blot技術(shù),對(duì)轉(zhuǎn)染48h后的RNAi效果進(jìn)行分析。研究IGF1R對(duì)鹿茸軟骨細(xì)胞增殖、凋亡及自身m RNA和蛋白表達(dá)的調(diào)控,進(jìn)一步探討鹿茸生長(zhǎng)調(diào)節(jié)機(jī)制。研究?jī)?nèi)容和結(jié)果如下:(1)轉(zhuǎn)染軟骨細(xì)胞48h,相比陰性對(duì)照組,軟骨細(xì)胞經(jīng)psh RNA-1和psh RNA-2處理,IGF1R基因m RNA表達(dá)量分別為51%和75%;蛋白質(zhì)的表達(dá)量也有明顯下降。表明干擾細(xì)胞效果明顯。(2)采用CCK-8方法檢測(cè)轉(zhuǎn)染24h、48h和72h后細(xì)胞增殖情況。相比于陰性對(duì)照組,試驗(yàn)組的細(xì)胞增殖呈下降趨勢(shì),尤其是psh RNA-1質(zhì)粒干擾效果明顯((1.09±0.10 vs 1.16±0.09(P0.01),1.04±0.02 vs 1.10±0.06(P0.05),0.79±0.06 vs1.01±0.07(P0.01))。(3)轉(zhuǎn)染軟骨細(xì)胞48h,相比于陰性對(duì)照組psh RNA-negative,試驗(yàn)組細(xì)胞更多的處于G1期(79.47±0.11vs 82.27±0.35(P0.01)),而S期細(xì)胞數(shù)量顯著下降(16.56±0.10 vs 14.82±0.014(P0.05))。(4)轉(zhuǎn)染軟骨細(xì)胞48h后,試驗(yàn)組psh RNA-1和陰性對(duì)照組psh RNA-negative的早期凋亡水平分別為(22.12±2.72%vs 8.56±0.16%,P0.01);正�；罴�(xì)胞分別為(62.85±5.97 vs 77.39±4.46,P0.05)。相關(guān)凋亡基因p53(P0.01)、bcl-2(P0.05)和bax的表達(dá)水平顯著下降(P0.01)。表明轉(zhuǎn)染psh RNA-1可促進(jìn)軟骨細(xì)胞的凋亡。結(jié)果表明,IGFR對(duì)鹿茸的生長(zhǎng)可能具有一定的調(diào)控作用,特別是對(duì)鹿茸軟骨細(xì)胞的增殖和凋亡具有顯著的調(diào)控作用,此外IGFR還可能通過(guò)調(diào)控軟骨細(xì)胞的細(xì)胞周期調(diào)節(jié)其生長(zhǎng)發(fā)育。
[Abstract]:Velvet antler is the second significant sexual characteristic of sika deer and has the characteristic of periodic regeneration. At its peak, it can grow at a rate of 2 cm / d. Previous studies have shown that the regeneration of velvet antler is a complex process based on the metabolism of pilose antler stem cells and regulated by many factors. In addition, a large number of studies have reported that IGF1R gene is involved in the proliferation and metabolism of many kinds of cells. However, the study of IGF1R gene in antler chondrocytes is rarely reported. Therefore, the purpose of this study was to investigate the function of IGF1R from the perspectives of growth, proliferation, cycle and apoptosis of antler chondrocytes. It provides a new viewpoint for further studying the molecular mechanism of velvet antler growth and development. In this experiment, RNAi technique was used to interfere the expression of IGF1R m RNA in velvet antler chondrocytes, and the effects of IGF1R m RNA on chondrocytes were analyzed from different angles. In this study, RNAi-Ready p SIREN-Retro QZs Green plasmid was used as vector and IGF1R as candidate gene to study its regulatory mechanism on the growth of velvet antler chondrocytes. Two RNAi recombinant plasmids named psh RNA-1 and psh RNA-2, were successfully constructed. It is used to transfect chondrocytes. Real-Time PCR and Western blot techniques were used to analyze the effect of RNAi transfection for 48 h. To study the regulation of IGF1R on the proliferation, apoptosis and the expression of m RNA and protein of antler chondrocytes, and to further explore the mechanism of antler growth regulation. The results were as follows: (1) the expression of m RNA of IGF1R gene in chondrocytes treated with psh RNA-1 and psh RNA-2 was 51% and 75%, respectively. The results showed that the effect of interfering cells was obvious. (2) CCK-8 method was used to detect the proliferation of cells after transfection for 48 h and 72 h. Compared with the negative control group, the cell proliferation of the test group showed a downward trend, especially the effect of psh RNA-1 plasmid interference was obvious (1.09 鹵0.10 vs 1.16 鹵0.09 (P0.01), 1.04 鹵0.02 vs 1.10 鹵0.06 (P0.05). Chondrocytes were transfected with 0.79 鹵0.06 vs1.01 鹵0.07 (P0.01). (3) for 48h. Compared with the negative control group, the cells were in G1 phase (79.47 鹵0.11vs 82.27 鹵0.35, P0.01),). The number of S phase cells decreased significantly (16.56 鹵0.10 vs 14.82 鹵0.014 (P0.05). (4) 48 h after transfection of chondrocytes. The early apoptotic level of psh RNA-1 in the experimental group and psh RNA-negative in the negative control group was (22.12 鹵2.72%vs 8.56 鹵0.16); The normal living cells were (62.85 鹵5.97 vs, 77.39 鹵4.46, P 0.05). The expression of p53 (P0.01), bcl-2 (P0.05) and bax decreased significantly (P0.01). The results showed that psh RNA-1 transfection could promote the apoptosis of chondrocytes. The results showed that IGFR could regulate the growth of velvet antler, especially the proliferation and apoptosis of antler chondrocytes. In addition, IGFR might regulate the growth and development of chondrocytes by regulating the cell cycle of chondrocytes.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S825

【引證文獻(xiàn)】

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1 王欣;李光玉;李春義;;IGF1與睪酮激素對(duì)鹿角柄發(fā)生影響的研究[A];2011中國(guó)鹿業(yè)進(jìn)展[C];2011年

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本文編號(hào)：2403458