CN114591955B - 一种促进内皮祖细胞向间质细胞转化的环状rna及其应用 - Google Patents
一种促进内皮祖细胞向间质细胞转化的环状rna及其应用 Download PDFInfo
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Abstract
本发明涉及内皮祖细胞向间质细胞转化技术领域,特别涉及一种促进内皮祖细胞向间质细胞转化的环状RNA及其应用。所述环状RNA为Circ‑1199,其序列如SEQ ID NO.1所示。所述环状RNA用于体内及体外促进内皮祖细胞向间质细胞转化,所述体外促进内皮祖细胞向间质细胞转化的实验步骤包括:(1)利用慢病毒过表达技术促使内皮祖细胞内过表达Circ‑1199;(2)利用EdU细胞增殖实验检测内皮祖细胞增殖能力的变化;(3)利用Matrigel基质胶检测内皮祖细胞成血管能力的改变;(4)利用定量RT‑PCR和蛋白免疫印迹检测其对内皮细胞标记分子CD31和VE‑Cadherin,以及间质细胞标记分子α‑SMA和SM22α基因及蛋白表达的影响。本发明的环状RNA‑‑Circ‑1199无论体内和体外都可促进内皮祖细胞发生内皮间质转化,该发现为心血管疾病的研究提供一个新的方向。
Description
技术领域
本发明涉及内皮祖细胞向间质细胞转化技术领域,特别涉及一种促进内皮祖细胞向间质细胞转化的环状RNA及其应用。
背景技术
最新研究发现,内皮祖细胞(endothelial progenitor cells,EPCs)在转化生长因子β1(TGF-β1)等刺激下,可发生内皮间质转化(EndoMT),进而造成动脉粥样斑块、动脉纤维化等病理性血管重塑。内皮祖细胞的内皮间质转化可能是病理性血管重塑的一种常见机制。我们前期研究证实:存在于主动脉弓、颈动脉分支等动脉弯曲、分叉部位的振荡剪切应力(OSS)亦可以引起内皮祖细胞发生内皮间质转化,但其内在机制尚不清晰。
因此,目前亟需证实何种物质是诱导内皮祖细胞发生内皮间质转化的关键分子,进而为病理性血管重塑等心血管疾病的研究提供一个新的方向。
发明内容
针对现有技术存在的不足,本发明要解决的技术问题是探究并验证环状RNA--Circ-1199为促进内皮祖细胞发生内皮间质转化的主要物质,为心血管疾病的研究提供一个新的方向。
本发明为实现上述目的采用的技术方案是:一种促进内皮祖细胞向间质细胞转化的环状RNA,所述环状RNA为Circ-1199,其序列如SEQ ID NO.1所示。
一种环状RNA的应用,所述环状RNA具有上述的序列,所述环状RNA用于促进内皮祖细胞向间质细胞转化。
进一步的,所述环状RNA用于体内和/或体外促进内皮祖细胞向间质细胞转化。
进一步的,所述体外促进内皮祖细胞向间质细胞转化的实验步骤包括:
(1)利用慢病毒过表达技术促使内皮祖细胞内过表达Circ-1199;
(2)利用EdU细胞增殖实验检测内皮祖细胞增殖能力的变化;
(3)利用Matrigel基质胶检测内皮祖细胞成血管能力的改变;
(4)利用定量RT-PCR和蛋白免疫印迹(Western blotting,WB)检测其对内皮细胞标记分子CD31和VE-Cadherin,以及间质细胞标记分子α-SMA和SM22α基因及蛋白表达的影响。
进一步的,
所述步骤(2)中的EdU细胞增殖实验为:
将1×105个细胞接种在含有10%FBS的EGM2培养基中;
24小时后,将1:1000稀释的EdU标记试剂的EGM2添加到细胞中;
2小时后,将细胞用4%多聚甲醛固定,在含3%牛血清白蛋白(BSA)的PBS中封闭30mins,然后在室温下在0.5%X-100PBS中孵育20mins;
使用Click-iT EdU Alexa Fluor 555检测细胞增殖情况,并用DAPI复染细胞核;
使用ImageJ-pro plus软件分析图像。
进一步的,
所述步骤(3)中的Matrigel基质胶检测实验为成血管实验:
将内皮祖细胞接种到Matrigel包被的96孔板上,每孔2×104个细胞,并在EGM2培养基中孵育,置于37℃的培养箱中;
在4,6,8,10,12小时后通过倒置显微镜观察血管样结构的形成情况;计数每个图像的成管长度,分析成血管情况。
进一步的,
所述步骤(4)中的蛋白免疫印迹实验:
使用RIPA裂解液提取各组细胞的总蛋白,BCA蛋白质测定试剂盒测定蛋白浓度;
将10μg的蛋白质进行SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳,然后转移到PVDF膜上;
用5%脱脂牛奶封闭后,将膜与针对α-SMA(1:400),SM22α(1:2000),CD31(1:500),VE-cadherin(1:500),β-actin(1:2000)的抗体孵育;
孵育辣根过氧化物酶偶联的二抗,最后使用ECL发光试剂盒检测蛋白信号;
所述步骤(4)中的RT-PCR实验:
使用Trizol试剂从内皮祖细胞中分离提取各组细胞的总RNA;
使用Prime Script RT试剂进行RNA逆转录,SYBR Premix Ex TaqTM试剂进行PCR扩增;
使用U6作为对照,通过SYBR Green测定法分析Circ-1199;使用GAPDH作为内参,通过SYBR Green测定法分析α-SMA,SM22α,CD31,VE-cadherin;
在ABI Step One-Plus检测系统中进行定量RT-PCR,用2-ΔΔCt方法确定相应基因的表达情况,每个测定重复三次。
进一步的,所述体内促进内皮祖细胞向间质细胞转化的实验步骤包括:
(1)将慢病毒过表达Circ-1199转染的内皮祖细胞及其对照内皮祖细胞,与Matrigel混匀后接种于裸鼠(BALB/c)背部皮下;
(2)两周后,取下接种后的基质胶块,通过HE染色分析体内成血管情况以及细胞免疫荧光观察内皮细胞标记分子vWF和CD31,以及间质细胞标记分子α-SMA和SM22α的表达情况;
(3)使用ImageJ-pro plus软件分析免疫荧光染色图像。
本发明探索到一种可促进内皮祖细胞发生内皮间质转化的环状RNA--Circ-1199;该环状RNA--Circ-1199无论体内和体外都可促进内皮祖细胞发生内皮间质转化,该发现为心血管疾病的研究提供一个新的方向。
附图说明
图1为本发明实施例体外过表达Circ-1199前后内皮祖细胞的成血管能力结果图;
图2为本发明实施例体外过表达Circ-1199前后内皮祖细胞的增殖能力结果图;
图3为本发明实施例体外过表达Circ-1199前后内皮细胞标记分子CD31和VE-Cadherin,以及间质细胞标记分子α-SMA和SM22α基因的表达量变化图;
图4为本发明实施例体外过表达Circ-1199前后内皮细胞标记分子CD31和VE-Cadherin,以及α-SMA和SM22α蛋白的表达量变化图;
图5为本发明实施例体内过表达Circ-1199前后内皮祖细胞的成血管能力结果图;
图6为本发明实施例体内过表达Circ-1199前后内皮细胞标记分子vWF和CD31,以及间质细胞标记分子α-SMA和SM22α蛋白的表达量变化图;
图7为本发明实施例高通量基因测序技术检测内皮祖细胞加载OSS前后差异表达显著的环状RNA;
图8为本发明实施例定量RT-PCR和RNA FISH技术手段验证Circ-1199在内皮祖细胞加载OSS前后的差异表达情况。
具体实施方式
下面结合附图与具体实施方式对本发明作进一步详细描述:
实施例1:
一种促进内皮祖细胞向间质细胞转化的环状RNA,所述环状RNA为Circ-1199,其序列如SEQ ID NO.1所示。
一种环状RNA的应用,所述环状RNA为Circ-1199,具有SEQ ID NO.1所述的序列,所述环状RNA用于促进内皮祖细胞向间质细胞转化。
所述环状RNA用于体外促进内皮祖细胞向间质细胞转化,实验步骤包括:
(1)利用慢病毒过表达技术促使内皮祖细胞内过表达Circ-1199;
(2)利用EdU细胞增殖实验检测内皮祖细胞增殖能力的变化;
(3)利用Matrigel基质胶检测内皮祖细胞成血管能力的改变;
(4)利用定量RT-PCR和蛋白免疫印迹检测其对内皮细胞标记分子CD31和VE-Cadherin,以及间质细胞标记分子α-SMA和SM22α基因及蛋白表达的影响。
所述步骤(2)中的EdU细胞增殖实验为:
将1×105个细胞接种在含有10%FBS的EGM2培养基中;
24小时后,将1:1000稀释的EdU标记试剂的EGM2添加到细胞中;
2小时后,将细胞用4%多聚甲醛固定,在含3%牛血清白蛋白的PBS中封闭30mins,然后在室温下在0.5%X-100PBS中孵育20mins;
使用Click-iT EdU Alexa Fluor 555检测细胞增殖情况,并用DAPI复染细胞核;
使用ImageJ-pro plus软件分析图像。
所述步骤(3)中的Matrigel基质胶检测实验为成血管实验:
将内皮祖细胞接种到Matrigel包被的96孔板上,每孔2×104个细胞,并在EGM2培养基中孵育,置于37℃的培养箱中;
在4,6,8,10,12小时后通过倒置显微镜观察血管样结构的形成情况;计数每个图像的成管长度,分析成血管情况。
所述步骤(4)中的蛋白免疫印迹实验:
使用RIPA裂解液提取各组细胞的总蛋白,BCA蛋白质测定试剂盒测定蛋白浓度;
将10μg的蛋白质进行SDS-聚丙烯酰胺凝胶电泳,然后转移到PVDF膜上;
用5%脱脂牛奶封闭后,将膜与针对α-SMA(1:400),SM22α(1:2000),CD31(1:500),VE-cadherin(1:500),β-actin(1:2000)的抗体孵育;
孵育辣根过氧化物酶偶联的二抗,最后使用ECL发光试剂盒检测蛋白信号;
所述步骤(4)中的RT-PCR实验:
使用Trizol试剂从内皮祖细胞中分离提取各组细胞的总RNA;
使用Prime Script RT试剂进行RNA逆转录,SYBR Premix Ex TaqTM试剂进行PCR扩增;
使用U6作为对照,通过SYBR Green测定法分析Circ-1199;使用GAPDH作为内参,通过SYBR Green测定法分析α-SMA,SM22α,CD31,VE-cadherin;
在ABI Step One-Plus检测系统中进行定量RT-PCR,用2-ΔΔCt方法确定相应基因的表达情况,每个测定重复三次。
图1为:体外过表达Circ-1199前后内皮祖细胞的成血管能力结果图,结果可知过表达后可抑制内皮祖细胞的成血管能力。
图2为:体外过表达Circ-1199前后内皮祖细胞的增殖能力结果图,结果可知过表达后可促进内皮祖细胞的增殖能力。
图3为:体外过表达Circ-1199前后内皮细胞标记分子CD31和VE-Cadherin,以及间质细胞标记分子α-SMA和SM22α基因的表达量变化图,结果可知CD31和VE-Cadherin基因表达量降低,而α-SMA和SM22α基因表达量升高。
图4为:体外过表达Circ-1199前后内皮细胞标记分子CD31和VE-Cadherin,以及α-SMA和SM22α蛋白的表达量变化图,结果可知CD31和VE-Cadherin蛋白表达量降低,而α-SMA和SM22α蛋白表达量升高。
实施例2:
一种环状RNA的应用,所述环状RNA为Circ-1199,具有SEQ ID NO.1所述的序列,所述环状RNA用于促进内皮祖细胞向间质细胞转化。
所述环状RNA用于体内促进内皮祖细胞向间质细胞转化,实验步骤包括:
(1)将慢病毒过表达Circ-1199转染的内皮祖细胞及其对照内皮祖细胞,与Matrigel混匀后接种于裸鼠(BALB/c)背部皮下;
(2)两周后,取下接种后的基质胶块,通过HE染色分析体内成血管情况以及细胞免疫荧光观察内皮细胞标记分子vWF和CD31,以及间质细胞标记分子α-SMA和SM22α的表达情况;
(3)使用ImageJ-pro plus软件分析免疫荧光染色图像。
图5为:体内过表达Circ-1199前后内皮祖细胞的成血管能力结果图,结果可知过表达后可抑制内皮祖细胞的成血管能力。
图6为:体内过表达Circ-1199前后内皮细胞标记分子CD31和vWF,以及间质细胞标记分子α-SMA和SM22α蛋白的表达量变化图,结果可知过表达后内皮细胞标记分子vWF和CD31明显降低,而间质细胞标记分子α-SMA和SM22α的蛋白表达升高,可知Circ-1199促进内皮祖细胞发生内皮间质转化。
实施例3:
一种环状RNA的应用,所述环状RNA为Circ-1199,具有SEQ ID NO.1所述的序列,所述环状RNA用于促进内皮祖细胞向间质细胞转化。
所述环状RNA用于体外促进内皮祖细胞向间质细胞转化,实验步骤包括:
(1)内皮祖细胞的分离、培养
收集孕妇脐带血50ml,常规分离培养内皮祖细胞。
(2)振荡剪切应力的加载
将内皮祖细胞接种在涂有纤维连接蛋白(Fn)的载玻片上,培养至70%-80%的密度,使用Flexcell flow STR-4000平行板流室系统,以±3.5dynes/cm2,1Hz的频率加载OSS作用于内皮祖细胞;运用高通量基因测序技术检测内皮祖细胞加载OSS前后差异表达显著的环状RNA,筛选出差异表达最显著的Circ-1199;进一步运用定量RT-PCR和RNA FISH等技术手段验证Circ-1199在内皮祖细胞加载OSS前后的差异表达情况;
(3)利用EdU细胞增殖实验检测内皮祖细胞增殖能力的变化;
(4)利用Matrigel基质胶检测内皮祖细胞成血管能力的改变;
(5)利用定量RT-PCR和蛋白免疫印迹检测其对内皮细胞标记分子CD31和VE-Cadherin,以及间质细胞标记分子α-SMA和SM22α基因及蛋白表达的影响。
图7为:高通量基因测序技术检测内皮祖细胞加载OSS前后差异表达显著的环状RNA,其中Circ-1199最为显著;
图8为:定量RT-PCR和RNA FISH技术手段验证Circ-1199在内皮祖细胞加载OSS前后的差异表达情况。
上述实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
上述实施例的统计学分析:所有实验至少3次独立重复,结果采用平均值±标准差(x±s)表示,使用T检验分析。所有统计分析均采用P<0.05作为具有显著统计学差异的检验标准,分析软件为GraphPad Prism 8。
在本发明的制备方法中,各种材料的加成顺序和具体反应步骤可由本领域技术人员进行调整,不仅适用于实验室的小规模制备,也适用于化工厂的工业化大规模生产。在工业批量生产中,具体反应参数可由本领域技术人员通过实验确定。
若无特殊说明,上述实施例中所用到的试剂、材料等,均可从商业途径获得或由商业途径所获原料合成。
上述实施例只是为了说明本发明的技术构思及特点,其目的是在于让本领域内的普通技术人员能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡是根据本发明内容的实质所做出的等效的变化或修饰,都应涵盖在本发明的保护范围内。
序列表
<110> 潍坊医学院
<120> 一种促进内皮祖细胞向间质细胞转化的环状RNA及其应用
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Claims (6)
1.一种环状RNA的应用,环状RNA为Circ-1199,其序列如SEQ ID NO.1所示,其特征在于:所述环状RNA用于体外促进内皮祖细胞向间质细胞转化。
2.根据权利要求1所述的应用,其特征是,所述体外促进内皮祖细胞向间质细胞转化的实验步骤包括:
(1)利用慢病毒过表达技术促使内皮祖细胞内过表达所述Circ-1199;
(2)利用EdU细胞增殖实验检测内皮祖细胞增殖能力的变化;
(3)利用Matrigel基质胶检测内皮祖细胞成血管能力的改变;
(4)利用定量RT-PCR和蛋白免疫印迹检测其对内皮细胞标记分子CD31和VE-Cadherin,以及间质细胞标记分子α-SMA和SM22α基因及蛋白表达的影响。
3.根据权利要求2所述的应用,其特征是:
所述步骤(2)中的EdU细胞增殖实验为:
将1×105个细胞接种在含有10%FBS的EGM2培养基中;
24小时后,将1:1000稀释的EdU标记试剂的EGM2添加到细胞中;
2小时后,将细胞用4%多聚甲醛固定,在含3%牛血清白蛋白的PBS中封闭30mins,然后在室温下在0.5%Triton®X-100 PBS中孵育20 mins;
使用Click-iT EdU Alexa Fluor 555检测细胞增殖情况,并用DAPI复染细胞核;
使用ImageJ-pro plus软件分析图像。
4.根据权利要求2所述的应用,其特征是:
所述步骤(3)中的Matrigel基质胶检测实验为成血管实验:
将内皮祖细胞接种到Matrigel包被的96孔板上,每孔2×104个细胞,并在EGM2培养基中孵育,置于37℃的培养箱中;
在4,6,8,10,12小时后通过倒置显微镜观察血管样结构的形成情况;计数每个图像的成管长度,分析成血管情况。
5.根据权利要求2所述的应用,其特征是:
所述步骤(4)中的蛋白免疫印迹实验:
使用RIPA裂解液提取各组细胞的总蛋白,BCA蛋白质测定试剂盒测定的蛋白浓度;
将10μg的蛋白质进行SDS-聚丙烯酰胺凝胶电泳,然后转移到PVDF膜上;
用5%脱脂牛奶封闭后,将膜与针对α-SMA,SM22α,CD31,VE-cadherin,β-actin的抗体孵育;
孵育辣根过氧化物酶偶联的二抗,最后使用ECL发光试剂盒检测蛋白信号。
6.根据权利要求2所述的应用,其特征是:
所述步骤(4)中的RT-PCR实验:
使用Trizol试剂从内皮祖细胞中分离提取各组细胞的总RNA;
使用Prime Script RT试剂进行RNA逆转录,SYBR Premix Ex Taq™试剂进行PCR扩增;
使用U6作为对照,通过SYBR Green测定法分析所述Circ-1199;使用GAPDH作为内参,通过SYBR Green测定法分析α-SMA,SM22α,CD31,VE-cadherin;
在ABI Step One-Plus检测系统中进行定量RT-PCR,用2-ΔΔCt方法确定相应基因的表达情况,每个测定重复三次。
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