CN114934052B - 一种长链非编码rna aabr07017227的应用 - Google Patents
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Abstract
本发明提供了一种长链非编码RNA AABR07017227的应用,长链非编码RNA AABR07017227的核苷酸序列如SEQ ID NO:1所示,用于制备抑制UC‑MSCs应激性凋亡的药物。本发明的长链非编码RNAAABR07017227可用于制备抑制UC‑MSCs应激性凋亡的药物,具有广阔的临床应用前景。
Description
技术领域
本发明属于长链非编码RNA技术领域,具体涉及一种长链非编码RNAAABR07017227的应用。
背景技术
脐带血间充质干细胞(umbilical cord blood mesenchymal stem cells,UC-MSCs)具有强大的再生和多向分化能力,已用于多种疾病的移植治疗。但是,移植的UC-MSCs在病灶区复杂微环境中存在应激性凋亡现象,导致其移植疗效受限,如何抑制UC-MSCs应激性凋亡是提高其移植疗效的关键。目前尚无抑制UC-MSCs应激性凋亡的药物。
长链非编码RNA(long non-coding RNA,LncRNA)是一类转录本长度在200nt-100kb之间的非编码RNA分子,它们能以序列特异性方式调节凋亡相关基因表达,进而干预细胞凋亡等多种生命活动。LncRNA在抑制UC-MSCs应激性凋亡中的应用,仍处于研发阶段,需要不断的探索和研究。
发明内容
本发明所要解决的技术问题在于针对上述现有技术的不足,提供一种长链非编码RNA AABR07017227,该长链非编码RNA AABR07017227可用于制备抑制UC-MSCs应激性凋亡的药物,具有广阔的临床应用前景。
为解决上述技术问题,本发明采用的技术方案是:一种长链非编码RNAAABR07017227的应用,所述长链非编码RNA AABR07017227用于制备抑制UC-MSCs应激性凋亡的药物;所述长链非编码RNA AABR07017227的核苷酸序列如SEQ ID NO:1所示;所述UC-MSCs为脐带血间充质干细胞;
所述长链非编码RNA AABR07017227抑制UC-MSCs应激性凋亡,促进UC-MSCs在氧化应激环境中的存活,从而提高UC-MSCs的移植疗效。
优选地,所述长链非编码RNA AABR07017227包含长链非编码RNA AABR07017227全部序列的质粒、长链非编码RNA AABR07017227部分序列的质粒、长链非编码RNAAABR07017227全部序列的慢病毒表达载体或者长链非编码RNA AABR07017227部分序列的慢病毒表达载体。
本发明与现有技术相比具有以下优点:
本发明的长链非编码RNA AABR07017227在UC-MSCs应激性凋亡中起保护作用,长链非编码RNA AABR07017227通过抑制UC-MSCs应激性凋亡,促进UC-MSCs在氧化应激微环境中存活,从而提高UC-MSCs的移植疗效。因此,长链非编码RNA AABR07017227可用于制备抑制UC-MSCs应激性凋亡的药物,具有广阔的临床应用前景。
下面结合附图和实施例对本发明作进一步详细说明。
附图说明
图1为本发明实施例1的敲除长链非编码RNA AABR07017227后,Annexin V-FITC/PI双染色法检测UC-MSCs应激性凋亡图。
图2为本发明实施例1的敲除长链非编码RNA AABR07017227后,TUNEL染色法检测UC-MSCs应激性凋亡图。
图3为本发明实施例1的过表达长链非编码RNA AABR07017227后,Annexin V-FITC/PI双染色法检测UC-MSCs应激性凋亡图。
图4为本发明实施例1的过表达长链非编码RNA AABR07017227后,TUNEL染色法检测UC-MSCs应激性凋亡图。
图5为本发明实施例2的UC-MSCs移植术后48小时,小动物活体成像观察移植UC-MSCs在骨坏死区氧化应激微环境中的存活图。
图6为本发明实施例2的UC-MSCs移植术后48小时,TUNEL染色检测移植UC-MSCs在骨坏死区氧化应激微环境中的凋亡图。
图7为本发明实施例2的UC-MSCs移植术后12周,micro-CT观察骨坏死区的修复情况图。
图8为本发明实施例2的UC-MSCs移植术后12周,HE染色检测骨坏死区的新骨形成面积图。
图9为本发明实施例2的UC-MSCs移植术后12周,Masson染色检测骨坏死区的新骨形成图。
具体实施方式
实施例1(体外实验)
本实施例为长链非编码RNA AABR07017227的体外应用(体外实验),所述长链非编码RNA AABR07017227用于制备抑制UC-MSCs应激性凋亡的药物;所述长链非编码RNAAABR07017227的核苷酸序列如SEQ ID NO:1所示;所述UC-MSCs为脐带血间充质干细胞;
体外实验:
试验A:
分离培养原代UC-MSCs,采用CRISPR/Cas9技术敲除UC-MSCs的AABR07017227,然后采用H2O2(浓度600μM)模拟氧化应激处理UC-MSCs24小时,实验分为3组:未敲除长链非编码RNA AABR07017227(0μM H2O2)、未敲除长链非编码RNA AABR07017227(600μM H2O2)、敲除长链非编码RNA AABR07017227(600μM H2O2),最后采用TUNEL染色法和Annexin V-FITC/PI双染色法检测各组UC-MSCs凋亡情况,试验表明,与未敲除长链非编码RNA AABR07017227(600μM H2O2)组相比,敲除长链非编码RNA AABR07017227的UC-MSCs遭受氧化应激后,细胞凋亡率显著增加(TUNEL和Annexin V-FITC/PI阳性细胞数量增加)。
如图1所示,Annexin V-FITC/PI双染色法检测各组UC-MSCs凋亡情况,图中A为未敲除长链非编码RNA AABR07017227(0μM H2O2),B未敲除长链非编码RNA AABR07017227(600μM H2O2),C为敲除长链非编码RNA AABR07017227(600μM H2O2)。如图2所示,TUNEL染色法检测各组UC-MSCs凋亡情况,图中A为未敲除长链非编码RNA AABR07017227(0μM H2O2),B为未敲除长链非编码RNA AABR07017227(600μM H2O2),C为敲除长链非编码RNA AABR07017227(600μM H2O2)。由图可知,正常培养的UC-MSCs凋亡率约为(4.17±1.82)%,UC-MSCs氧化应激诱导后细胞凋亡率约为(45.92±1.46)%,当敲除UC-MSCs的长链非编码RNAAABR07017227后进行氧化应激处理,细胞凋亡率进一步升高至(69.65±2.04)%,差异显著,有统计学意义(P<0.001)。本试验说明,敲除长链非编码RNA AABR07017227,可增加UC-MSCs应激性凋亡。
试验B:
分离培养原代UC-MSCs,采用长链非编码RNA AABR07017227过表达慢病毒转染UC-MSCs,然后采用H2O2(浓度600μM)模拟氧化应激处理UC-MSCs 24小时,实验分为3组:未过表达长链非编码RNA AABR07017227(0μM H2O2)、未过表达长链非编码RNA AABR07017227(600μM H2O2)、过表达长链非编码RNA AABR07017227(600μM H2O2),最后采用TUNEL染色法和Annexin V-FITC/PI双染色法检测各组UC-MSCs凋亡情况,试验表明,与未过表达AABR07017227(600μM H2O2)组相比,过表达长链非编码RNA AABR07017227的UC-MSCs遭受氧化应激后,细胞凋亡率显著下降(TUNEL和Annexin V-FITC/PI阳性细胞数量减少)。
如图3所示,Annexin V-FITC/PI双染色法检测各组UC-MSCs凋亡情况,图中A为未过表达长链非编码RNA AABR07017227(0μM H2O2),B为未过表达长链非编码RNAAABR07017227(600μM H2O2),C为过表达长链非编码RNA AABR07017227(600μM H2O2)。如图4所示,TUNEL染色法检测各组UC-MSCs凋亡情况,图中A为未过表达长链非编码RNAAABR07017227(0μM H2O2),B为未过表达长链非编码RNA AABR07017227(600μM H2O2),C为过表达长链非编码RNA AABR07017227(600μM H2O2)。由图可知,正常培养的UC-MSCs凋亡率约为(4.32±1.72)%,UC-MSCs氧化应激诱导后细胞凋亡率约为(47.07±1.58)%,当UC-MSCs过表达长链非编码RNA AABR07017227后进行氧化应激处理,细胞凋亡率降至(19.22±1.74)%,差异显著,有统计学意义(P<0.001)。本试验说明,长链非编码RNAAABR07017227可抑制UC-MSCs应激性凋亡。
本实施例的长链非编码RNA AABR07017227可抑制UC-MSCs应激性凋亡,促进UC-MSCs在氧化应激环境中存活。可以将长链非编码RNA AABR07017227制备成药物,用于抑制UC-MSCs应激性凋亡。
实施例2(体内实验)
本实施例为长链非编码RNA AABR07017227的体内应用(体内实验),所述长链非编码RNA AABR07017227用于抑制UC-MSCs应激性凋亡,进而促进移植UC-MSCs在骨坏死区氧化应激微环境中的存活,从而显著提高UC-MSCs对骨坏死的移植疗效;本实施例的长链非编码RNA AABR07017227可用于制备用于抑制UC-MSCs应激性凋亡的药物;
所述长链非编码RNA AABR07017227的核苷酸序列如SEQ ID NO:1所示。
所述长链非编码RNA AABR07017227包含长链非编码RNA AABR07017227全部序列的质粒、长链非编码RNA AABR07017227部分序列的质粒、长链非编码RNA AABR07017227全部序列的慢病毒表达载体或者长链非编码RNA AABR07017227部分序列的慢病毒表达载体。
试验A:
首先分离培养原代UC-MSCs,在体外采用CRISPR/Cas9技术敲除UC-MSCs的长链非编码RNA AABR07017227,或者采用长链非编码RNA AABR07017227过表达慢病毒转染UC-MSCs以过表达AABR07017227,并采用DiR荧光标记所有的UC-MSCs。然后建立SD大鼠股骨头坏死模型。最后将敲除或过表达长链非编码RNA AABR07017227的UC-MSCs植入骨坏死区,实验分为3组:植入UC-MSCs、植入敲除长链非编码RNA AABR07017227的UC-MSCs、植入过表达长链非编码RNA AABR07017227的UC-MSCs。术后48小时,通过小动物活体成像技术观察移植的UC-MSCs在股骨头坏死区的存活情况,采用TUNEL染色检测各组移植的UC-MSCs在股骨头坏死区的凋亡情况,试验表明,单纯植入UC-MSCs和植入敲除AABR07017227的UC-MSCs在骨坏死区氧化应激微环境中大量凋亡,而过表达长链非编码RNA AABR07017227的UC-MSCs在骨坏死区氧化应激微环境中凋亡率显著减少(TUNEL阳性细胞数量减少,移植区DiR荧光强度增加)。术后12周,取出股骨头组织,通过micro-CT检查、HE染色和Masson染色评估各组股骨头坏死区的修复情况,试验表明,未植入UC-MSCs、植入UC-MSCs和植入敲除AABR07017227的UC-MSCs组,坏死区无明显修复,坏死区内未见新生骨小梁和成熟的骨组织,而植入过表达长链非编码RNA AABR07017227的UC-MSCs组坏死区已完全修复,骨小梁连续、重建完整,骨组织趋于成熟。
如图5所示,术后48小时,采用小动物活体成像技术观察各组移植的UC-MSCs在骨坏死区的存活情况,图中A为植入UC-MSCs,B植入过表达长链非编码RNA AABR07017227的UC-MSCs,C植入敲除长链非编码RNA AABR07017227的UC-MSCs。如图6所示,术后48小时,TUNEL染色法检测各组UC-MSCs凋亡情况,图中A为植入UC-MSCs,B植入过表达长链非编码RNA AABR07017227的UC-MSCs,C植入敲除长链非编码RNA AABR07017227的UC-MSCs。由图可知,将UC-MSCs植入股骨头坏死区氧化应激微环境中,大量细胞出现凋亡,而过表达长链非编码RNA AABR07017227的UC-MSCs植入股骨头坏死区氧化应激微环境中,凋亡率减少,敲除长链非编码RNA AABR07017227的UC-MSCs植入股骨头坏死区氧化应激微环境中,凋亡率增加(TUNEL阳性细胞数量增加,移植区DiR荧光强度下降)
如图7所示,术后12周,取出股骨头组织,通过micro-CT检测各组股骨头坏死区的修复情况,图中A为植入UC-MSCs,B植入过表达长链非编码RNA AABR07017227的UC-MSCs,C植入敲除AABR07017227的UC-MSCs。如图8所示,术后12周,取出股骨头组织,采用HE染色评估各组股骨头坏死区的修复情况,图中A为植入UC-MSCs,B植入过表达长链非编码RNAAABR07017227的UC-MSCs,C植入敲除AABR07017227的UC-MSCs。如图9所示,术后12周,取出股骨头组织,采用Masson染色评估各组股骨头坏死区的修复情况,图中A为植入UC-MSCs,B植入过表达长链非编码RNA AABR07017227的UC-MSCs,C植入敲除长链非编码RNAAABR07017227的UC-MSCs。由图可知,植入UC-MSCs和植入敲除AABR07017227的UC-MSCs组,坏死区无明显修复,坏死区内未见新生骨小梁和成熟的骨组织,而植入过表达长链非编码RNA AABR07017227的UC-MSCs组坏死区已完全修复,骨小梁连续、重建完整,骨组织趋于成熟。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何限制。凡是根据发明技术实质对以上实施例所作的任何简单修改、变更以及等效变化,均仍属于本发明技术方案的保护范围内。
序列表
<110> 贵州医科大学附属医院
<120> 一种长链非编码RNA AABR07017227的应用
<130> 2022.5.25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 928
<212> DNA
<213> 人工合成(Artificial synthesis)
<400> 1
ctgggaaatg ccgtagccta ctttctaact acctctaacc acactgacct gtttgtggag 60
tgcctttccc tgaaactcac tcttgagcca gggaataccc atttgtccat tctccttcca 120
gagctaaaga gttcttacta cttcatttca actcagttgc tgaggatagg gagagttggt 180
taaggtcttg atatgggtag aagagctttg ttaacaccca aacatctaaa gaccttggat 240
gagggagatt cttcttcctg aaatggataa acccaggctc cattcatgtg acatacagtc 300
caattcccac agtttttgca gtttcccgga ggccaatatc tttttcctac acctttaaat 360
gtatgcaggt tggagtatat cgtttcactg agtaagcctt gagaggaggc atagtttcaa 420
tatattcacc catgttgtgg agacatcgag atgcagctcc caggcaggat caatcatgcc 480
tcagatgttc ctggtatcgt gggaataacg atcttgtggt atcttgcaaa gtggaaattg 540
aggatgggag acaattctgc ttacctggga gatggatcca aacatggacc gcatcctacc 600
ctcccaggca tccaaacaaa gcagctctat tttctcctga tggaagtcct tcagagatga 660
agaggtgtcc tctgttactg aggccatggc ttctctaaag gttcataaac ggcaatggag 720
aaggatacta cagtgacaac cccattactt acgtggggtc agggtgcgtg gagaggagaa 780
tgaacacttt agtggatcct tgctgatgga atggggagat ggtggcattg agataatcga 840
ctatcctctg tggaagatcc tactgcattt caaactcatt tgtaaaagac tgccgttaca 900
ttaaaagaag ttgtgcttgc taatggaa 928
Claims (2)
1.一种长链非编码RNA AABR07017227的应用,其特征在于,所述长链非编码RNAAABR07017227用于制备抑制UC-MSCs氧化应激性凋亡的药物;所述长链非编码RNAAABR07017227的核苷酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的一种长链非编码RNA AABR07017227的应用,其特征在于,所述药物包含长链非编码RNA AABR07017227全部序列的质粒或者长链非编码RNAAABR07017227全部序列的慢病毒表达载体。
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