天堂国产午夜亚洲专区-少妇人妻综合久久蜜臀-国产成人户外露出视频在线-国产91传媒一区二区三区

Col X基因表達下調(diào)對鹿茸干細胞向軟骨細胞分化的影響

發(fā)布時間:2018-06-29 10:24

  本文選題:X型膠原蛋白(Col + X); 參考:《農(nóng)業(yè)生物技術學報》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(RNA interface,RNAi)下調(diào)鹿茸干細胞中Col X的表達并研究離體情況下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)并進行原代培養(yǎng),同時利用間充質(zhì)干細胞表面標記物(Stro-1)對AP細胞進行定性分析。其次,針對東北梅花鹿(Cervus nippon)Col X mRNA,設計了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細胞包裝慢病毒、收集濃縮病毒后利用懸液法感染AP細胞,同時利用微粒培養(yǎng)對其進行成軟骨誘導。成軟骨誘導培養(yǎng)21 d后,利用qRT-PCR和Western blot技術驗證Col X的表達變化,最后通過組織切片結(jié)合HE染色和阿辛藍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(P0.05)。阿辛藍染色發(fā)現(xiàn),與陰性對照相比Col X下調(diào)后微粒體基質(zhì)中阿辛藍著色顯著減少,說明基質(zhì)中酸性粘多糖比例下降。同時,HE染色表明在粘多糖下降的同時伴隨著部分軟骨細胞的死亡表現(xiàn)為基質(zhì)中無細胞陷窩空泡的增加。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,本研究為進一步研究Col X在哺乳動物軟骨內(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)科學院特產(chǎn)研究所/特種經(jīng)濟動物分子生物學國家重點實驗室/吉林省鹿茸工程研究中心;吉林農(nóng)業(yè)大學中藥材學院;
【基金】:吉林省自然科學基金(No.20170101032JC) 中國農(nóng)業(yè)科學院科技創(chuàng)新工程項目(No.CAAS-ASTIP-2014-ISAPS-04) 吉林省科技發(fā)展計劃項目(No.20140204010YY)
【分類號】:S825


本文編號:2081810

資料下載
論文發(fā)表

本文鏈接:http://sikaile.net/yixuelunwen/dongwuyixue/2081810.html


Copyright(c)文論論文網(wǎng)All Rights Reserved | 網(wǎng)站地圖 |

版權申明:資料由用戶1e572***提供,本站僅收錄摘要或目錄,作者需要刪除請E-mail郵箱bigeng88@qq.com
精品国产av一区二区三区不卡蜜 | 观看日韩精品在线视频| 草草夜色精品国产噜噜竹菊| 欧美一本在线免费观看| 国产传媒一区二区三区| 国产精品一区二区视频大全| 激情五月激情婷婷丁香| 老熟妇乱视频一区二区| 日韩中文字幕视频在线高清版| 欧美精品亚洲精品日韩精品| 亚洲欧美日韩综合在线成成| 国产又爽又猛又粗又色对黄| 日本高清中文精品在线不卡| 日韩视频在线观看成人| 欧美日韩国产免费看黄片| 亚洲国产精品久久综合网| 欧美自拍偷自拍亚洲精品| 久久碰国产一区二区三区| 国产精品亚洲精品亚洲| 日韩黄色大片免费在线| 91久久精品国产成人| 国产人妻精品区一区二区三区| 日本高清不卡一二三区| 日本少妇中文字幕不卡视频| 在线观看国产午夜福利| 国产成人在线一区二区三区| 免费在线成人午夜视频| 欧美熟妇喷浆一区二区| 日本欧美一区二区三区高清| 婷婷激情四射在线观看视频| 国产无摭挡又爽又色又刺激| 中文字幕亚洲精品乱码加勒比| 色婷婷国产熟妇人妻露脸| 男女午夜视频在线观看免费| 黄色国产自拍在线观看| 色婷婷视频在线精品免费观看| 国产精品亚洲二区三区| 欧美在线视频一区观看| 亚洲伦理中文字幕在线观看| 国内自拍偷拍福利视频| 日韩精品一区二区三区四区|