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醫(yī)學(xué)論文范文:重組腺病毒載體Ad-LMP-1的構(gòu)建及其轉(zhuǎn)染MSC后成骨活性
【摘要】 【目的】 快速構(gòu)建大鼠LIM礦化蛋白-1基因重組腺病毒載體,轉(zhuǎn)染MSC,探討LMP-1基因?qū)SC成骨分化和成骨活性的影響。 【方法】 從大鼠成骨細(xì)胞內(nèi)提取總RNA,并設(shè)計(jì)LMP-1特異性引物進(jìn)行RT-PCR,獲取LMP-1基因后利用Invitrogen公司的TOPO定向克隆技術(shù)創(chuàng)建入門克隆pENTR/D-LMP-1。轉(zhuǎn)化TOP10細(xì)菌后,挑取陽(yáng)性克隆搖菌提取質(zhì)粒,入門克隆再與表達(dá)載體pAD/CMV/V5-DEST進(jìn)行同源重組反應(yīng)得到病毒載體pAD/CMV/V5-LMP-1,轉(zhuǎn)化細(xì)菌后挑選陽(yáng)性克隆搖菌提取重組病毒質(zhì)粒,用PacI內(nèi)切酶線性化后用轉(zhuǎn)染293A細(xì)胞后得到重組腺病毒載體。以腺病毒為載體,將大鼠LMP-1基因體外轉(zhuǎn)染于3代大鼠MSC細(xì)胞內(nèi),檢測(cè)LMP-1基因在MSC細(xì)胞的表達(dá),分別觀察轉(zhuǎn)染后實(shí)驗(yàn)組與對(duì)照組I型膠原。堿性磷酸酶。骨鈣素表達(dá)變化以及骨鈣結(jié)節(jié)的形成情況,評(píng)價(jià)LMP-1基因的成骨能力�！窘Y(jié)果】 成功獲取大鼠LMP-1基因,入門質(zhì)粒與病毒表達(dá)質(zhì)粒經(jīng)過(guò)酶切鑒定以及測(cè)序驗(yàn)證。LMP-1基因能在MSC中高效表達(dá),轉(zhuǎn)然后MSC的I型膠原,堿性磷酸酶。骨鈣素的表達(dá)明顯增強(qiáng),骨鈣結(jié)節(jié)形成增多。 【結(jié)論】 利用Gateway 技術(shù)構(gòu)建LMP-1重組腺病毒載體相對(duì)簡(jiǎn)單,可快捷獲得的pAd-LMP-1。pAd-LMP-1轉(zhuǎn)染MSC后,LMP-1基因能促進(jìn)MSC向成骨細(xì)胞分化,增強(qiáng)其成骨作用。
【關(guān)鍵詞】 LIM-1礦化蛋白; 間充質(zhì)干細(xì)胞; 成骨活性; 基因轉(zhuǎn)染
中圖分類號(hào): R329.2+8 文獻(xiàn)標(biāo)志碼: A 文章編號(hào): 1672-3554(2010)02-0199-07
Osteogenic Activity of MSC Infected by Recombinant Adenovirus Vector Ad-LMP-1
CHENG Zhi-an1, LIU Dong-bin3, WU Yan-feng2, HUANG Lin2, SHEN Hui-yong2, LIU Shang-li2
1. Department of Orthopaedices, The Second Affiliated Hospital of Guangzhou Chinese Medicine University Guangdong Provincial Hospital of Traditional Chinese Medicine), Guangzhou 510120, China;2. Department of Orthopaedics, The Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, China;3. Department of Orthopadics, The People’s Hospital of Nanhai District, Foshan 528200, China
Abstract: 【Objective】 This study was designed to construct a recombinant adenovirus vector contains LMP-1 gene,and investigate the osteoinductive activity of MSC which were transfected recombinated adenoviral vector carrying LMP-1 gene. 【Methods】 Total RNA was extracted from rat osteoblast and the LMP-1 gene was acquired by RT-PCR, the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology, then the entry clone and the expression vector were used to create the expression clone through the LR recombination reaction. The adenovirus expression clone was linearized by PacI and transfected to the 293A cell line to harvest a high titer. Ad-LMP-1 was infected into the 3rd passage MSC, the expression of LMP-1 was detected by Western blot. The osteogenic activity of MSC was evaluated by the expression of collagen I, ALP, osteocalcin and the formation of bone nodule. 【Result】 The LMP-1 gene was successfully acquired and confirmed, the entry clone and the expression clone were both verified by enzymes digestion, and the expression clone was further confirmed by sequenced. The expression of LMP-1 was detected successfully in MSC. The increasing expression of collagen I, osteocalcin, ALP and bone nodule were observed by comparing to the control group. 【Conclusion】 Gateway technology not only LIM make construction of the pAd-LMP-1 recombination adenovirus vector simple and fast, but also get a high transfection efficacy in MSC. LMP-1 gene can induce the osteoblast differention of MSCs, and improve its osteogenic activity. The adenovirus vector is reliable to be used in further gene therapy research.
Key words: LIM mineralization protein-1; mesenchymal stem cells; osteogenic activity; gene transfect
[J SUN Yat-sen Univ(Med Sci),2010,31(2):199-206]
礦化蛋白-1(LIM mineralization protein-1, LMP-1)是直接參與成骨分化的細(xì)胞內(nèi)因子,它的成骨作用可能表現(xiàn)在參與合成和分泌其他的成骨誘導(dǎo)蛋白如骨形態(tài)發(fā)生蛋白(bone morphogenetic proteins,BMP)以及轉(zhuǎn)化生長(zhǎng)因子子β1(transforming growth factor β1,TGF-β1),同時(shí)招募附近的細(xì)胞分化直接參與骨膜或軟骨內(nèi)成骨。在動(dòng)物或體外實(shí)驗(yàn)中,小劑量的含LMP-1基因的腺病毒或質(zhì)粒載體轉(zhuǎn)染骨髓基質(zhì)細(xì)胞,可持續(xù)穩(wěn)定地誘導(dǎo)骨形成[1]。LMP-1的高效骨誘導(dǎo)特性為治療骨質(zhì)疏松性骨折提供了新的選擇。本研究用Gateway技術(shù)構(gòu)建LMP-1重組腺病毒載體,以腺病毒為載體,將LMP-1基因?qū)腴g充質(zhì)干細(xì)胞(mesenchymal stem cells,MSC),通過(guò)比較實(shí)驗(yàn)組與對(duì)照組中MSC成骨活性的差異,來(lái)進(jìn)一步驗(yàn)證LMP-1的骨誘導(dǎo)作用,為后續(xù)的LMP-1基因治療骨質(zhì)疏松及其骨折研究奠定基礎(chǔ)。
