CN115851972B - 一种绵羊毛囊发育标志物miR-23b及其应用 - Google Patents
一种绵羊毛囊发育标志物miR-23b及其应用 Download PDFInfo
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Abstract
本发明提供了一种绵羊毛囊发育标志物miR‑23b及其应用,属于生物学技术领域。本发明收集苏博美利奴羊E65、E85、E105和E135胚胎日的胚胎皮肤组织和D7和D30出生后的羔羊的皮肤组织提取RNA进行测序筛选。本发明发现随着胚胎毛囊的发育miR‑23b的表达量逐渐升高,同时,本发明发现通过调节miR‑23b的表达可以调节绵羊真皮成纤维细胞的增殖和细胞中毛囊发育相关基因的表达。
Description
技术领域
本发明属于生物学技术领域,尤其涉及一种绵羊毛囊发育标志物miR-23b及其应用。
背景技术
苏博美利奴是中国自主培育的超细羊毛品种,其平均羊毛纤维直径在17-19 µm,超过了80 Nm 的标准纺织品支数,该品种对细毛羊产业影响深远。羊毛的生长发育受毛囊控制,毛囊是附着在皮肤上的微小器官,具有复杂的形态、复杂的结构和周期性的生长模式。因此,研究毛囊的发育的标志物以及毛囊发育的机制将有助于羊毛产业的发展。
MicroRNA(miRNA)是一类长度约为19-25nt的内源性非编码RNA,广泛参与基因转录后调控活动,具有高度序列保守性、表达时序性和组织特异性。研究表明miRNA参与各种各样的调节途径,包括发育、病毒防御、造血过程、脂肪代谢等,具有重要的基因表达调控作用。目前,关于MicroRNA在毛囊发育中的功能尚不完全清楚,因此本发明对MicroRNA在毛囊发育中的表达情况和功能进行研究。
发明内容
本发明的目的在于提供一种和绵羊毛囊发育相关的标志物miR-23b并且探究miR-23b在毛囊发育中的功能。
为实现上述目的,本发明提供了如下技术方案:
本发明提供了检测miR-23b表达量的引物在制备检测苏博美利奴羊胚胎毛囊发育检测试剂盒中的应用,所述引物的序列如SEQ ID NO.1所示;
所述miR-23b在毛囊发育初期表达量低,所述miR-23b在毛囊发育后期表达量高。
本发明提供了miR-23b抑制剂在制备毛囊发育促进剂中的应用,所述miR-23b抑制剂为miR-23b inhibitor,所述miR-23b inhibitor的序列如SEQ ID NO.6;
所述miR-23b抑制剂促进绵羊真皮成纤维细胞的增殖,毛囊发育相关基因NOTCH1、WNT10A和TGFβ2的表达,加快细胞周期,降低细胞凋亡,促进细胞迁移。
本发明提供了miR-23b抑制剂在制备绵羊真皮成纤维细胞增殖促进剂中的应用,所述miR-23b抑制剂为miR-23b inhibitor,所述miR-23b inhibitor的序列如SEQ ID NO.6所示。
本发明提供了 miR-23b抑制剂在制备绵羊真皮成纤维细胞中毛囊发育相关基因表达促进剂中的应用,所述miR-23b抑制剂为miR-23b inhibitor,所述miR-23b inhibitor的序列如SEQ ID NO.6;
所述毛囊发育相关基因为:NOTCH1、WNT10A和TGFβ2;
所述基因表达促进剂为mRNA表达促进剂和蛋白表达促进剂。
本发明提供了miR-23b抑制剂在制备绵羊真皮成纤维细胞的细胞凋亡抑制剂中的应用,所述miR-23b抑制剂为miR-23b inhibitor,所述miR-23b inhibitor的序列如SEQ IDNO.6。
本发明提供了miR-23b抑制剂在制备绵羊真皮成纤维细胞的细胞迁移促进剂中的应用,所述miR-23b抑制剂为miR-23b inhibitor,所述miR-23b inhibitor的序列如SEQ IDNO.6。
本发明提供了miR-23b促进剂在制备毛囊发育抑制剂中的应用,所述miR-23b促进剂为miR-23b mimic,所述miR-23b mimic的序列如SEQ ID NO.4和SEQ ID NO.5所示;
所述miR-23b促进剂抑制绵羊真皮成纤维细胞的增殖,毛囊发育相关基因NOTCH1、WNT10A和TGFβ2的表达,减慢细胞周期,促进细胞凋亡,抑制细胞迁移。
本发明提供了 miR-23b促进剂在制备绵羊真皮成纤维细胞增殖抑制剂中的应用,所述miR-23b促进剂为miR-23b mimic,所述miR-23b mimic的序列如SEQ ID NO.4和SEQ IDNO.5所示。
本发明提供了miR-23b促进剂在制备绵羊真皮成纤维细胞中毛囊发育相关基因表达抑制剂中的应用,所述miR-23b促进剂为miR-23b mimic,所述miR-23b mimic的序列如SEQ ID NO.4和SEQ ID NO.5所示;
所述毛囊发育相关基因为:NOTCH1、WNT10A和TGFβ2;
所述基因表达抑制剂为mRNA表达抑制剂和蛋白表达抑制剂。
本发明提供了miR-23b促进剂在制备绵羊真皮成纤维细胞中细胞凋亡促进剂中的应用,所述miR-23b促进剂为miR-23b mimic,所述miR-23b mimic的序列如SEQ ID NO.4和SEQ ID NO.5所示。
本发明提供了miR-23b促进剂在制备绵羊真皮成纤维细胞中细胞迁移抑制剂中的应用,所述miR-23b促进剂为miR-23b mimic,所述miR-23b mimic的序列如SEQ ID NO.4和SEQ ID NO.5所示。
本发明的有益效果是:
1.本发明通过测序和荧光定量PCR确定了miR-23b会随着的胎儿毛囊发育而表达量逐渐升高,因此可以根据miR-23b的引物来制备试剂盒,提供检测miR-23b的表达情况来确定毛囊的发育情况。
2.本发明通过实验发现,过表达miR-23b抑制绵羊真皮成纤维细胞的增殖,减慢细胞的细胞周期,促进细胞凋亡,抑制细胞迁移;
而抑制miR-23b促进绵羊真皮成纤维细胞的增殖,加快细胞的细胞周期,抑制细胞凋亡,促进细胞迁移。
3.本发明通过实验发现,过表达miR-23b会抑制绵羊真皮成纤维细胞中毛囊发育相关的基因NOTCH1,WNT10A和TGFβ2的基因和蛋白表达,而抑制miR-23bmiR-23b则会促进绵羊真皮成纤维细胞中毛囊发育相关的基因NOTCH1,WNT10A和TGFβ2的基因和蛋白表达。
附图说明
图1为测序筛选结果;
a为差异表达miRNA的热图,b为miRNA维恩图,c为组间的DE-miRNA,d为DE-miRNA的维恩图(up 表示显着上调的基因; down 表示显着的下调基因);
图2为miR-23b的基因测序和荧光定量PCR的结果;
图3为过表达和抑制miR-23b后miR-23b的表达情况;
图4为转染miR-23b模拟物及抑制物后CCK-8检测的SDF的增殖水平;
a为过表达miR-23b后SDF的增殖情况,b为抑制内源性miR-23b后SDF细胞的增殖情况;*表示差异显著(P<0.05),**表示差异极显著(P<0.01);
图5为miR-23b对NOTCH1在SDF中mRNA及蛋白水平表达的影响;
a为过表达和抑制内源性miR-23b后NOTCH1的mRNA表达情况;b为miR-23b调控NOTCH1的蛋白表达图;*表示差异显著(P<0.05),**表示差异极显著(P<0.01);
图6为miR-23b对WNT10A在SDF中mRNA及蛋白水平表达的影响;
a为过表达和抑制内源性miR-23b后WNT10A的mRNA表达情况;b为miR-23b调控WNT10A的蛋白表达图;*表示差异显著(P<0.05),**表示差异极显著(P<0.01);
图7为miR-23b对TGFβ2在SDF中mRNA及蛋白水平表达的影响;
a为过表达和抑制内源性miR-23b后TGFβ2的mRNA表达情况;b为miR-23b调控TGFβ2的蛋白表达图;*表示差异显著(P<0.05),**表示差异极显著(P<0.01)。
图8为miR-23b对绵羊真皮成纤维细胞的细胞凋亡的影响;
a为流式结果;b为统计结果;*表示差异显著(P<0.05),**表示差异极显著(P<0.01);
图9为miR-23b对绵羊真皮成纤维细胞的细胞周期的影响;
a为流式结果;b为统计结果;*表示差异显著(P<0.05),**表示差异极显著(P<0.01);
图10为miR-23b对绵羊真皮成纤维细胞的细胞周期蛋白的影响;
a为Western Blot蛋白检测结果;b为统计结果;*表示差异显著(P<0.05),**表示差异极显著(P<0.01);
图11为miR-23b对绵羊真皮成纤维细胞的细胞迁移的影响;
a为显微镜检测结果;b为统计结果;*表示差异显著(P<0.05),**表示差异极显著(P<0.01)。
具体实施方式
所举实施例是为了更好地对本发明进行说明,但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
实施例1
实验动物和样品制备
(1)所选用的受试个体选自新疆科创繁育中心羊群,使用来自苏博美利奴公羊(3岁;MFD,19.0±0.4 μm)的新鲜精子对18只健康的苏博美利奴母羊(2-3 岁;平均纤维直径(MFD),18.1±0.5 μm)进行人工授精;
(2)受精日被指定为胚胎第0天(E0),之后,我们在四个不同的胚胎日(即 E65、E85、E105 和 E135)从12只怀孕的母羊身上收集胚胎,并且立即收集胚胎皮肤组织;
(3)同时,我们收集D7和D30出生后羔羊的皮肤组织收集,深度约为2cm2×3 mm,上述六个发育阶段中的每一个生成三个生物学重复。
(4)将收集到的所有18个皮肤组织样本使用1×PBS冲洗,之后将样品切成小块,迅速放入液氮中,随后在-80°C下储存用于RNA提取。
实施例2
筛选差异miRNA
(1)使用miRNeasy Mini Kit试剂从18个皮肤组织中分离总RNA,使用通过Qubit®2.0荧光计和Nanodrop One 分光光度计测定RNA浓度和质量,同时,使用Agilent 2100Bioanalyzer评估总RNA样品的完整性;
(2)将RNA完整性数评分>7的样本用于测序,使用QIAseq miRNA Library Kit按照QIAseq miRNA Library Kit Guide合成双末端文库,然后用PCR纯化和富集产物以创建最终的cDNA文库;
(3)纯化的文库通过Qubit® 2.0 荧光计进行量化,并通过Agilent 2100Bioanalyzer进行验证,以确认插入大小并计算摩尔浓度;
(4)簇由cBot从稀释至10 pM的文库中生成,然后在 Illumina NovaSeq 6000(Illumina,USA)上测序,得到的结果如图1所示。
实施例3
(1)使用EdgeR软件对样本进行差异基因分析,得到p值后进行多重假设检验校正。通过控制 FDR(false discovery rate)来确定 p 值的阈值。同时,还根据FPKM值计算了差异表达倍数,即倍数变化,其中q值≤0.05且倍数变化≥2的基因被认为是差异表达的;
(2)从差异miRNA中挑选出miR-23b进行qRT-PCR验证;
(3)使用TRIzol试剂提取总RNA,根据Poly(A) Tailing Kit 的方案将Poly(A)尾添加到miRNA中;
(4)使用PrimeScriptTM RT试剂盒与gDNA Eraser和基因特异性引物或随机引物用于生成 cDNA;
(5)使用miRcute Plus miRNA qPCR Kit进行miRNA的qRT-PCR检测,
引物序列如下:
miR-23b
F:CGCGCGatcacattgccagggatt,SEQ ID NO.1;
U6
F:CTCGCTTCGGCAGCACA,SEQ ID NO.2;
R:AACGCTTCACGAATTTGCGT,SEQ ID NO.3;
反应条件为:
95℃ 15 分钟; 94℃ 20 s,60℃ 34s,进行45个循环;
(6)qRT-PCR实验一式三份进行, 2−ΔΔCt方法用于确定相对表达量,实验得到的结果如图2所示。
实施例4
设计miR-23b模拟物和抑制物并验证
(1)miR-23b mimic的序列如下:
Sense: AUCACAUUGCCAGGGAUU,SEQ ID NO.4;
Antisense: UCCCUGGCAAUGUGAUUU, SEQ ID NO.5
miR-23b inhibitor的序列如下:
Sense: AAUCCCUGGCAAUGUGAU,SEQ ID NO.6;
mimic-NC的序列如下:
Sense: UUGUACUACACAAAAGUACUG,SEQ ID NO.7;
Antisense: GUACUUUUGUGUAGUACAAUU,SEQ ID NO.8;
inhibitor-NC的序列如下:
Sense: CAGUACUUUUGUGUAGUACAA,SEQ ID NO.9;
(2)将绵羊真皮成纤维细胞接种于6孔培养板中,待绵羊真皮成纤维细胞生长汇合度达80%时,进行细胞转染,具体转染步骤参照Lipofectamine3000说明书;
(3)将稀释好的miR-23b mimic,miR-23b inhibitor和阴性对照分别与脂质体混匀,静置15-20min,将混合物滴加到6孔板,6h后换完全培养基,转染48h后收集细胞进行RNA提取,转染72h后收集细胞进行RNA提取;
(4)提取RNA后,进行逆转录和qRT-PCR检测转染miR-23b mimic和miR-23binhibitor 后,细胞中miR-23b的表达情况,得到的结果如图3所示。
实施例5
过表达和抑制后miR-23b对于绵羊真皮成纤维细胞增殖的影响
(1)将转染miR-23b mimic,miR-23b inhibitor以及miR-23b mimic-NC 36h后的绵羊真皮成纤维细胞使用0.25%胰酶消化;
(2)加入工作培养基终止消化后,离心弃去培养基,加入适量的维持培养基制备单细胞悬液;
(3)按照600µL/孔(约2×103个细胞)接种至24孔板中进行培养,每组设置4个时间点,分别为24h、48h、72h和96h;
(4)分别在培养24、48、72h和96h后对24孔板每孔给予60µL CCK-8溶液,放回培养箱中继续培养2h后,将培养孔中的液体转移至96孔板(100µL/孔)每个样品设置6个重复;
(5)然后用酶标仪测定在450nm处的吸光度,以OD值代表绵羊真皮成纤维细胞的相对增殖水平,绘制细胞增殖曲线,实验得到的结果如图4和表1所示。
表1 不同组间OD值的差异
miR-23b mimic | mimic-NC | miR-23b inhibitor | inhibitor-NC | |
24h | 0.159 | 0.161 | 0.157 | 0.161 |
48h | 0.170 | 0.172 | 0.169 | 0.159 |
72h | 0.207 | 0.246 | 0.225 | 0.231 |
96h | 0.310 | 0.421 | 0.371 | 0.349 |
实施例6
miR-23b对于毛囊发育相关蛋白NOTCH1、WNT10A和TGFβ2的影响
(1)将绵羊真皮成纤维细胞接种于6孔培养板中,待绵羊真皮成纤维细胞生长汇合度达80%时,进行细胞转染,具体转染步骤参照Lipofectamine3000说明书;
(2)将稀释好的miR-23b mimic,miR-23b inhibitor和阴性对照分别与脂质体混匀,静置15-20min,将混合物滴加到6孔板,6h后换完全培养基,转染48h后一部分,去除培养基,PBS清洗后,收集细胞进行RNA提取;
RNA提取和检测步骤参见实施例3,NOTCH1,WNT10A和TGFβ2的引物序列如下:
NOTCH1
F: TATCTGCATGCCTGGCTACG,SEQ ID NO.10;
R: GTCCACGTCATACTGGCACA,SEQ ID NO.11;
WNT10A
F: TCCTGACTTCTGCGAGCGAGAC, SEQ ID NO.12;
R: TGCGGCACTCCTCACAGACC, SEQ ID NO.13;
TGFβ2
F: AGCGGAGCGACGAGGAATACTAC, SEQ ID NO.14;
R: CACTGAGCCAGAGGGTGTTGTAAC, SEQ ID NO.15;
(3)另一部分去除培养基,使用PBS清洗细胞后,加入蛋白裂解液,使其与细胞充分接触,冰上放置,期间使用移液枪轻轻吹打细胞2-3次,每次10s;
(4)收集细胞至1.5ml离心管中,在冰上放置5min,期间剧烈振荡3-4次,每次30s;
(5)12000rpm,4℃离心5min,取上清即可进行后续的Western操作;
(6)按照下表配置SDS-PAG:
表1 5%浓缩胶和10%分离胶配置
5%浓缩胶 | For 1gel | For 2gel |
ddH2O | 1.4mL | 2.7mL |
0.5M Tris pH6.8 | 250μL | 500μL |
30% Acrylamide | 330μL | 670μL |
10% SDS | 20μL | 40μL |
10% APS | 20μL | 40μL |
TMEM | 2μL | 4μL |
10%分离胶 | For 1gel | For 2gel |
ddH2O | 1.9 mL | 4.0 mL |
1.5M Tris pH8.8 | 1.3 mL | 2.5 mL |
30% Acrylamide | 1.7 mL | 3.3 mL |
10% SDS | 50μL | 100μL |
10% APS | 50μL | 100μL |
TMEM | 2μL | 4μL |
(7)将电泳液加入到电泳槽中,将变性好的蛋白样品每孔按蛋白总量50μg按顺序加入至胶孔中,同时在每组两侧加入适量预染蛋白marker,设定恒压80V,30min,待loadingbuffer的蓝色指示带到达分离胶时加大电压为120V,至跑完全程;
(8)将凝胶自胶板中取出,按照“三明治”结构,将转膜夹组装好,夹紧放置转膜槽中,加满转膜液,于冰上转膜,转膜参数设定为:恒流200mA,120min;
(9)转膜完成后将膜取出置于封闭液中,常温摇床上封闭1h;
(10)将PVDF膜按照需要的分子量剪开,将膜完全孵育到特定的一抗中,4℃孵育过夜。
(11)自一抗中取出条带,TBST洗涤3min×3次,将条带放到相应的二抗中,摇床上缓慢摇晃常温孵育1h,孵育完成后用摇床上高速TBST洗涤10min ×3次;
(12)洗膜完成后将膜放在发光仪中,滴加ECL化学发光液,将发出的条带拍照、保存,实验得到的结果如图5-7所示。
实施例7
miR-23b对绵羊皮肤成纤维细胞的凋亡能力的影响
(1)取处于对数生长期,生长状态良好的绵羊皮肤成纤维细胞,以5×105个/孔接种于细胞培养6孔板,37℃、5%CO2培养箱中培养过夜;(在细胞孔周围孔内加入100µL无菌PBS);
(2)按照如下分组对细胞进行处理:miR-23b mimic;miR-23b inhibitor;mimic-NC;inhibitor-NC;
(3)细胞处理48h后,胰酶消化收集细胞,用PBS将细胞润洗2次,1200rpm,5min离心;
(4)按照AnnexinV-FITC/PI细胞凋亡检测试剂盒操作说明进行:①加入500μLBinding Buffer,重悬细胞;②加入5μL AnnexinV-FITC混匀后加入5μL PI,混匀;③室温避光反应5-15min(同时设阴性对照,即正常细胞不加AnnexinV-FITC和PI);
(5)流式细胞仪上机检测。
实施例8
miR-23b对绵羊皮肤成纤维细胞的细胞周期的影响
(1)取处于对数生长期,生长状态良好的绵羊真皮绵羊皮肤成纤维细胞,以5×105个/孔接种于细胞培养6孔板,37℃、5%CO2培养箱中培养过夜;(在细胞孔周围孔内加入100µL无菌PBS);
(2)按照如下分组对细胞进行处理:miR-23b mimic;miR-23b inhibitor;mimic-NC;inhibitor-NC;
(3)流式检测细胞周期:①细胞处理48h时间后,胰酶消化收集细胞,1200rpm,5min,去上清,PBS重悬润洗2次;②100µL PBS重悬细胞,缓慢加入700µL预冷的80%乙醇,使乙醇终浓度为70%;③4℃固定4h以上;④1500rpm,5min,预冷PBS润洗2次,200µL PBS重悬细胞;⑤加入10µL RNase(1mg/mL),37℃孵育30min;⑥加入10µL PI(400μg/ml),4℃避光染色30min;
(4)流式细胞仪上机检测。
实施例9
miR-23b对绵羊皮肤成纤维细胞的细胞周期蛋白的影响
(1)将绵羊真皮成纤维细胞接种于6孔培养板中,待绵羊真皮成纤维细胞生长汇合度达80%时,进行细胞转染,具体转染步骤参照Lipofectamine3000说明书;
(2)按照如下分组对细胞进行处理:miR-23b mimic;miR-23b inhibitor;mimic-NC;inhibitor-NC;
(3)转染48h后,参照上述实施例的Western Blot检测步骤检测Cyclin D1、CDKN1A、BAX和P53蛋白的表达。
实施例10
(1)取处于对数生长期,生长状态良好的绵羊皮肤成纤维细胞,以5×105个/孔接种于细胞培养6孔板,37℃、5%CO2培养箱中培养过夜;(在细胞孔周围孔内加入100µL无菌PBS);
(2)按照如下分组对细胞进行处理:miR-23b mimic;miR-23b inhibitor;mimic-NC;inhibitor-NC;
(3)细胞处理48h后,分别消化收集细胞,1000rpm,5min离心,去上清,PBS润洗一次,清洗掉残余血清;
(4)无血清DMEM培养基重悬细胞,细胞计数板计数,无血清DMEM培养基稀释细胞浓度至3×105个/ml,备用;
(5)在24孔板中预先加入700µL 10% FBS 的DMEM培养基(含双抗),并放入Transwell小室,在Transwell上室分别接入200µL各组细胞悬液,37℃,5% CO2培养箱培养24h;
(6)取出Transwell,用PBS小心清洗小室一遍,用70%冰乙醇溶液固定细胞1h;
(7)用0.5%结晶紫染液染色,室温中放置20min,PBS清洗一下,用干净的棉球将上室一侧的未迁移的细胞擦干净,显微镜下观察拍照。
实验结果
如图1所示,通过测序选出了15个DE-miRNA是与Stage A相关的候选miRNA,22个DE-miRNA是与Stage B相关的候选miRNA,从中我们选出miR-23b进行后续实验。
如图2所示,可以看出qRT-PCR 结果和 RNA-seq 结果一致。其中,E65时,RT-PCR的结果为1,miRNA-seq的结果为38.75;E85时,RT-PCR的结果为15.03,miRNA-seq的结果为83.80;E105时,RT-PCR的结果为62.97,miRNA-seq的结果为192.66;E135时,RT-PCR的结果为242.80,miRNA-seq的结果为532.68;D7时,RT-PCR的结果为360.72,miRNA-seq的结果为888.01;D30时,RT-PCR的结果为941.03,miRNA-seq的结果为1301.53;可以看出miR-23b的表达量随着毛囊发育不断升高。
如图3所示,miR-23b mimic的相对表达量为2.503 ± 0.246,miR-23b inhibitor的相对表达量为0.553 ± 0.145,可以看出转染miR-23b mimic有效的促进了miR-23b的表达量,转染miR-23b inhibitor有效的抑制了miR-23b的表达量。
如图4和表1所示,可以看出,转染了miR-23b mimic 72和96小时的细胞增殖水平均极显著降低 (P<0.01);而转染了miR-23b inhibitor 48小时的细胞增殖水平较inhibitor-NC极显著升高(P<0.01),72和96小时的细胞增殖水平显著升高(P<0.05)。实验结果表明,过表达miR-23b抑制绵羊真皮成纤维细胞的增殖,而抑制miR-23b促进绵羊真皮成纤维细胞的增殖。
如图5所示,可以看出,转染了miR-23b mimic后,绵羊真皮成纤维细胞中NOTCH1的mRNA表达和蛋白表达显著下降,而转染了miR-23b inhibitor后,绵羊真皮成纤维细胞中NOTCH1的mRNA表达和蛋白表达显著上调。
如图6所示,可以看出,转染了miR-23b mimic后,绵羊真皮成纤维细胞中WNT10A的mRNA表达和蛋白表达显著下降,而转染了miR-23b inhibitor后,绵羊真皮成纤维细胞中WNT10A的mRNA表达和蛋白表达显著上调。
如图7所示,可以看出,转染了miR-23b mimic后,绵羊真皮成纤维细胞中TGFβ2的mRNA表达和蛋白表达显著下降,而转染了miR-23b inhibitor后,绵羊真皮成纤维细胞中TGFβ2的mRNA表达和蛋白表达显著上调。
如图8所示,可以看出,与mimic-NC相比,过表达内源性miR-23b可以增加绵羊皮肤成纤维细胞的凋亡率。与inhibitor-NC相比抑制内源性miR-23b可以降低绵羊皮肤成纤维细胞的凋亡率。且经过统计分析表明,过表达miR-23b极显著(P<0.01)促进绵羊皮肤成纤维细胞的凋亡,而抑制miR-23b的表达可极显著(P<0.01)抑制绵羊皮肤成纤维细胞的凋亡。
如图9所示,可以看出,过表达miR-23b可极显著促进细胞周期的G1期和G2期,而极显著抑制细胞周期S期。而抑制miR-23b结果相反。结果表明,过表达miR-23b促进了细胞周期进程,并增加了绵羊皮肤成纤维细胞的G1/S比例。相反,抑制miR-23b导致G1/S期细胞数量减少。
如图10所示,可以看出,与mimic-NC相比过表达miR-23b能够有效抑制Cyclin D1的表达,但是显著促进CDKN1A、BAX和P53蛋白的表达。相反抑制miR-23b能够有效地促进Cyclin D1蛋白的表达,而抑制miR-23b能够显著促进Cyclin D1的表达,抑制CDKN1A、BAX和P53蛋白的表达。对条带就那些统计分析发现,过表达miR-23b能够极显著(P<0.01)抑制Cyclin D1蛋白的表达,而极显著(P<0.01)促进CDKN1A、BAX和P53蛋白的表达。抑制miR-23b结果与之相反。结合前面的增殖和凋亡结果可以得出miR-23b抑制细胞增殖而促进细胞凋亡。
如图11所示,可以看出,过表达miR-23b能够抑制细胞的迁移,而抑制miR-23b促进细胞迁移。对迁移细胞数进行统计分析发现,与mimic-NC相比,过表达miR-23b极显著(P<0.01)抑制绵羊皮肤成纤维细胞的迁移。与inhibitor-NC相比抑制miR-23b极显著(P<0.01)促进细胞的迁移。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (3)
1.检测miR-23b表达量的引物在制备检测苏博美利奴羊胚胎毛囊发育检测试剂盒中的应用,其特征在于,所述引物的序列如SEQ ID NO.1所示。
2.miR-23b抑制剂在制备绵羊毛囊发育促进剂中的应用,其特征在于,所述miR-23b抑制剂为miR-23b inhibitor,所述miR-23b inhibitor的序列如SEQ ID NO.6;
所述miR-23b抑制剂促进绵羊真皮成纤维细胞的增殖,促进毛囊发育相关基因NOTCH1、WNT10A和TGFβ2的表达,加快细胞周期,降低细胞凋亡,促进细胞迁移。
3. miR-23b促进剂在制备绵羊毛囊发育抑制剂中的应用,其特征在于,所述miR-23b促进剂为miR-23b mimic,所述miR-23b mimic的序列如SEQ ID NO.4和SEQ ID NO.5所示;
所述miR-23b促进剂抑制绵羊真皮成纤维细胞的增殖,抑制毛囊发育相关基因NOTCH1、WNT10A和TGFβ2的表达,减慢细胞周期,促进细胞凋亡,抑制细胞迁移。
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