CN114807024A - lncRNA CASC2激活剂在脐带间充质干细胞成心肌分化中的应用 - Google Patents
lncRNA CASC2激活剂在脐带间充质干细胞成心肌分化中的应用 Download PDFInfo
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Abstract
本发明公开了lncRNA CASC2激活剂在脐带间充质干细胞成心肌分化中的应用。间充质干细胞具有来源丰富、易于获得、体外增殖能力强和不受伦理问题限制等优点，越来越被青睐。脐带间充质干细胞指存在于新生儿脐带组织中的一种间充质干细胞，临床应用前景广阔，定向诱导脐带间充质干细胞向心肌细胞分化治疗心肌梗死等成为热门研究。本发明发现，提高lncRNA CASC2含量水平可以诱导人脐带间充质干细胞向心肌细胞分化，因此lncRNACASC2激活剂具有体外诱导人脐带间充质干细胞成心肌分化的作用。
Description
技术领域
本发明属于细胞领域，涉及干细胞治疗，具体涉及lncRNA CASC2激活剂在脐带间充质干细胞成心肌分化中的应用。
背景技术
近年来，尽管社会经济稳步提高，但是心血管疾病的患病率却居高不下。在临床上发现，心肌梗死伴随着心肌细胞的缺血、缺氧、凋亡坏死，梗死的组织被瘢痕组织代替，造成心肌局部功能障碍、心室重塑和扩张，最终演变成心力衰竭。目前心肌梗死的治疗手段包括药物、介入、手术等综合治疗方法，但是不能从根本上治愈，只是在一定程度上缓解病情。
间充质干细胞具有来源丰富、易于获得、体外增殖能力强和不受伦理问题限制等优点，越来越被青睐。脐带间充质干细胞指存在于新生儿脐带组织中的一种间充质干细胞，临床应用前景广阔，定向诱导脐带间充质干细胞向心肌细胞分化治疗心肌梗死等成为热门研究。
在定向诱导脐带间充质干细胞向心肌细胞分化治疗心肌梗死的研究中，一个重要的研究方向就是如果提高脐带间充质干细胞向心肌细胞的分化能力。
发明内容
为了克服现有技术的不足，本发明提供lncRNA CASC2激活剂在脐带间充质干细胞成心肌分化中的应用。
技术方案如下：
lncRNA CASC2激活剂在体外诱导人脐带间充质干细胞成心肌分化中的应用，所述lncRNA CASC2激活剂为能提高lncRNA CASC2含量水平的物质。
进一步地，所述能提高lncRNA CASC2含量水平的物质为能提高lncRNA CASC2含量水平的核苷酸片段。
技术效果如下：
本发明发现，提高lncRNA CASC2含量水平可以诱导人脐带间充质干细胞向心肌细胞分化，因此lncRNA CASC2激活剂具有体外诱导人脐带间充质干细胞成心肌分化的作用。
附图说明
图1为脐带间充质干细胞在倒置显微镜下观察、拍照结果(100×)；
图2为流式细胞术测定的表型结果；
图3为免疫细胞化学法检测结果；
图4为Western blot法检测结果。
具体实施方式
一、材料和试剂
人脐带间充质干细胞购自武汉普诺赛生命科技有限公司，冻存发货。
α-MEM培养基、胎牛血清、FITC-抗人CD34、FITC-抗人CD45、PE-抗人CD73、PE-抗人CD90和PE-抗人CD105购自美国Thermo Fisher公司。
LV-CASC2质粒和LV-Scramble质粒由苏州金唯智生物科技有限公司设计并合成，前者转染进细胞后细胞会高表达lncRNA CASC2，后者为不含有目的基因的空白质粒核苷酸片段(转染进细胞后细胞中lncRNA CASC2表达水平不变，属于转染对照)，参考文献“长链非编码RNA CASC2调节HepG2肝癌细胞的炎症因子分泌并诱导细胞凋亡和生长阻滞；免疫学杂志，2021年11月第37卷第11期”。qRT-PCR引物也由苏州金唯智生物科技有限公司设计并合成，lncRNA CASC2、β-actin引物按文献“长链非编码RNA CASC2、miR-155-5p、FOXO1在甲状腺乳头状癌中的表达及临床意义，华北理工大学硕士学位论文，2021”设计。
lncRNA CASC2上游引物：5’-GAGACCATCAGGCTCACAA-3’
lncRNA CASC2下游引物：5’-GAGGGACTCAGTAAAGACACG-3’
β-actin上游引物：5’-AAGGCCAACCGCGAGAA-3’
β-actin下游引物：5’-ATGGGGGAGGGCATACC-3’
Lipofectamine 2000试剂盒、TRIzol试剂购自美国Thermo Fisher公司。
cDNA逆转录试剂盒购自上海联迈生物工程有限公司。
SYBR-Green PCR试剂盒购自北京百奥莱博科技有限公司。
免疫细胞化学法和Western blot法用到的试剂购自上海碧云天生物技术有限公司。
二、方法
1、脐带间充质干细胞的培养和鉴定
按常规复苏方法将人脐带间充质干细胞复苏后接种于75cm2细胞培养瓶中，用含10％胎牛血清和1％双抗的α-MEM培养基于37℃，5％CO2条件下培养，待细胞融合度达80％，弃去原培养基，PBS缓冲液洗涤，胰酶消化成单细胞悬液，离心，α-MEM完全培养基重悬，继续培养传代1次。将传代细胞消化重悬，取适量细胞接种于培养皿中，吉姆萨染色，倒置显微镜下观察、拍照；另取适量细胞用流式细胞术测定细胞表型，流式抗体为FITC-抗人CD34、FITC-抗人CD45、PE-抗人CD73、PE-抗人CD90和PE-抗人CD105。
2、细胞转染
取生长状态良好的人脐带间充质干细胞，α-MEM完全培养基重悬成细胞悬液，以每孔约5×106个接种于6孔板，于37℃、5％CO2条件下培养。待细胞融合度达80％，根据Lipofectamine 2000说明书将100nmol/L的LV-CASC2质粒或LV-Scramble质粒分别转染进入人脐带间充质干细胞。转染48h后，用于后续实验。同时设置不进行任何转染的空白对照组。
3、qRT-PCR检测lncRNA CASC2表达
转染48h后，使用TRIzol试剂从各组细胞中分离总RNA，使用cDNA逆转录试剂盒合成cDNA。按照SYBR-Green PCR试剂盒说明书进行PCR，所有反应均在20μL反应体系中进行，PCR反应条件：5℃2min、95℃10min、96℃15s、60℃1min，40个循环。以β-actin为内参，利用2-ΔΔCt法分析lncRNA CASC2的相对表达量。
4、成心肌样细胞分化检测(免疫细胞化学法)
将转染48h后的人脐带间充质干细胞用α-MEM完全培养基重悬成1×105个/mL细胞悬液接种于24孔板中(接种前放置细胞爬片)，每孔500μL，于37℃，5％CO2条件下培养72h后，PBS冲洗，多聚甲醛固定，弃掉固定液并再次用PBS冲洗，Triton X-100溶液(1％)室温下通透，用PBS冲洗，内源性过氧化物酶阻断剂室温孵育阻断15min，用PBS冲洗，山羊血清工作液37℃孵育15min进行封闭，滤纸吸干，滴加cTnT一抗4℃孵育过夜，恢复至室温后PBS冲洗，用生物素标记的羊抗兔IgG室温孵育20min，PBS冲洗，再用辣根酶标记链霉卵白素工作液室温孵育15min，PBS冲洗，二氨基联苯胺显色液染色，自来水冲洗，苏木精复染，分化、返蓝、脱水、透明、封固处理后于光学显微镜下观察。
5、成心肌样细胞分化检测(Western blot法)
将转染48h后的人脐带间充质干细胞用α-MEM完全培养基重悬成1×105个/mL细胞悬液接种于24孔板中，每孔500μL，于37℃，5％CO2条件下培养72h后，收集细胞，RIPA裂解缓冲液提取蛋白质并用BCA试剂盒测定蛋白浓度。取30μg总蛋白行SDS-PAGE电泳，转膜，5％脱脂牛奶封闭，PBST洗膜，加入稀释的cTnI、cTnT、Desmin、GAPDH一抗4℃孵育过夜，次日洗膜，加入稀释的二抗溶液室温孵育1h，ECL化学发光试剂盒检测蛋白质表达水平。
6、统计分析
统计分析采用SPSS 17.0软件，结果用均数±标准差表示，两组间比较采用t检验，P<0.05为差异有显著性。
三、结果
1、脐带间充质干细胞的鉴定
倒置显微镜下观察、拍照如图1所示，细胞形态多呈长梭形，排列紧密且具有方向性；流式细胞术测定的表型如图2所示，弱表达CD34和CD45，强表达CD73、CD90和CD105。倒置显微镜观察结果和表型测定结果均符合人脐带间充质干细胞的特点。
2、qRT-PCR检测lncRNA CASC2表达
与空白对照组(不进行任何转染)相比，CASC2转染组(转染LV-CASC2)lncRNACASC2的含量明显升高，而转染对照组(转染LV-Scramble)lncRNA CASC2的含量无明显变化。结果如表1。该结果说明转染操作达到预期效果，CASC2转染组构建得到lncRNA CASC2高表达、含量水平明显升高的人脐带间充质干细胞。
表1 lncRNA CASC2相对表达量
| 空白对照组 | CASC2转染组 | 转染对照组 | |
| lncRNA CASC2/β-actin | 1.00±0.06 | 3.95±0.18 | 1.02±0.09 |
3、免疫细胞化学法检测结果
结果如图3所示，CASC2转染组cTnT呈现强阳性表达，空白对照组和转染对照组cTnT表达不明显。cTnT为心肌细胞的特异性蛋白，说明CASC2转染组出现了明显的向心肌分化。
4、Western blot法检测结果
结果如图4所示，CASC2转染组cTnI、cTnT、Desmin呈现强阳性表达，空白对照组和转染对照组cTnI、cTnT、Desmin表达不明显。cTnI、cTnT、Desmin均为心肌细胞的特异性蛋白，说明CASC2转染组出现了明显的向心肌分化。
上述结果说明，提高lncRNA CASC2含量水平可以诱导人脐带间充质干细胞向心肌细胞分化，因此lncRNA CASC2激活剂具有体外诱导人脐带间充质干细胞成心肌分化的作用。
Claims (2)
1.lncRNA CASC2激活剂在体外诱导人脐带间充质干细胞成心肌分化中的应用，所述lncRNA CASC2激活剂为能提高lncRNA CASC2含量水平的物质。
2.根据权利要求1所述的应用，其特征在于：所述能提高lncRNA CASC2含量水平的物质为能提高lncRNACASC2含量水平的核苷酸片段。
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