本文選題：X型膠原蛋白(Col + X)��； 參考：《農(nóng)業(yè)生物技術(shù)學(xué)報(bào)》2017年05期

【摘要】：X型膠原蛋白(collagen type X,Col X)是影響軟骨內(nèi)成骨的關(guān)鍵因子之一。為研究Col X在鹿茸軟骨內(nèi)成骨中的作用,本研究采用RNA技術(shù)(RNA interface,RNAi)下調(diào)鹿茸干細(xì)胞中Col X的表達(dá)并研究離體情況下Col X下調(diào)后鹿茸軟骨內(nèi)成骨的變化。首先,本研究采集了生茸區(qū)骨膜組織(antlerogenic periosteum,AP)并進(jìn)行原代培養(yǎng),同時(shí)利用間充質(zhì)干細(xì)胞表面標(biāo)記物(Stro-1)對AP細(xì)胞進(jìn)行定性分析。其次,針對東北梅花鹿(Cervus nippon)Col X mRNA,設(shè)計(jì)了4對特異性的sh RNA(S1~S4),并通過酶切連接法將其構(gòu)建至慢病毒載體pLVTHM中。利用293T細(xì)胞包裝慢病毒、收集濃縮病毒后利用懸液法感染AP細(xì)胞,同時(shí)利用微粒培養(yǎng)對其進(jìn)行成軟骨誘導(dǎo)。成軟骨誘導(dǎo)培養(yǎng)21 d后,利用qRT-PCR和Western blot技術(shù)驗(yàn)證Col X的表達(dá)變化,最后通過組織切片結(jié)合HE染色和阿辛藍(lán)染色鑒定成軟骨過程變化。結(jié)果表明,本研究成功構(gòu)建了包含sh RNA(S1~S4)的pLVTHM載體,慢病毒包裝成功且病毒滴度達(dá)到1×108TU/m L。qRT-PCR和Western blot結(jié)果表明,4組sh RNA均能顯著下調(diào)微粒體中Col X的表達(dá)(P0.05)。阿辛藍(lán)染色發(fā)現(xiàn),與陰性對照相比Col X下調(diào)后微粒體基質(zhì)中阿辛藍(lán)著色顯著減少,說明基質(zhì)中酸性粘多糖比例下降。同時(shí),HE染色表明在粘多糖下降的同時(shí)伴隨著部分軟骨細(xì)胞的死亡表現(xiàn)為基質(zhì)中無細(xì)胞陷窩空泡的增加。因此,綜合判斷Col X下調(diào)顯著抑制或減緩微粒體成軟骨過程,本研究為進(jìn)一步研究Col X在哺乳動(dòng)物軟骨內(nèi)成骨過程的作用提供理論依據(jù)。
[Abstract]:Type X collagen (collagen type XCol X) is one of the key factors affecting endochondral osteogenesis. In order to study the role of Col X in the osteogenesis of antler cartilage, RNA interface RNAi (RNAi) was used to down-regulate the expression of Col X in pilose antler stem cells and to study the changes of osteogenesis in antler cartilage after down-regulation of Col X in vitro. Firstly, antlerogenic peristeal AP was collected and cultured in primary culture, and the AP cells were qualitatively analyzed using Stro-1 as a surface marker of mesenchymal stem cells. Secondly, four pairs of specific sh RNAs (S1nS4) were designed for Cervus nippon Col X mRNAs and constructed into lentivirus vector pLVTHM by restriction endonuclease ligation. The lentivirus was packaged with 293T cells, then concentrated virus was collected and infected with AP cells by suspension method. Meanwhile, cartilage formation was induced by microparticle culture. After 21 days of chondrogenic induction and culture, the expression of ColX was confirmed by qRT-PCR and Western blot. Finally, the changes of cartilage formation were identified by tissue sections combined with HE staining and acinyl blue staining. The results showed that pLVTHM vector containing sh RNA (S1hS4) was successfully constructed. The results of lentivirus packaging and viral titer up to 1 脳 108TU / m L.qRT-PCR and Western blot showed that the expression of Col X in microsomes was significantly down-regulated in the four groups (P0.05). The results of acinyl blue staining showed that the percentage of acid mucopolysaccharide in microsomal matrix decreased significantly after down-regulation of Col X compared with negative control. At the same time, HE staining showed that the decrease of mucopolysaccharide was accompanied by the death of some chondrocytes. Therefore, the down-regulation of Col X could significantly inhibit or slow down the process of microsomal chondrogenesis. This study provides a theoretical basis for further study of the role of Col X in the process of endochondral osteogenesis in mammals.
【作者單位】： 中國農(nóng)業(yè)科學(xué)院特產(chǎn)研究所/特種經(jīng)濟(jì)動(dòng)物分子生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室/吉林省鹿茸工程研究中心;吉林農(nóng)業(yè)大學(xué)中藥材學(xué)院;
【基金】：吉林省自然科學(xué)基金(No.20170101032JC) 中國農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程項(xiàng)目(No.CAAS-ASTIP-2014-ISAPS-04) 吉林省科技發(fā)展計(jì)劃項(xiàng)目(No.20140204010YY)
【分類號(hào)】：S825
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本文編號(hào)：2081810