CN117126887A - Plpp3基因在调控人卵巢颗粒细胞自噬中的应用 - Google Patents
Plpp3基因在调控人卵巢颗粒细胞自噬中的应用 Download PDFInfo
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Abstract
本发明公开了PLPP3基因在调控人卵巢颗粒细胞自噬中的应用,属于细胞工程和基因工程技术领域。本发明首次发现,超表达PLPP3基因,能够显著提高颗粒细胞自噬和凋亡水平;而干扰PLPP3基因的表达,则显著降低颗粒细胞自噬、凋亡水平和细胞活性。同时发现,在中年人卵巢组织中,PLPP3蛋白水平低;而在老年人卵巢组织中,PLPP3蛋白水平高,PLPP3蛋白水平与人卵巢衰老程度呈正相关,可以作为标志物诊断/评估人卵巢衰老水平。本发明对研究自噬调控在卵巢卵泡发育、繁殖能力及卵巢相关疾病中的影响机制具有很好的应用价值,在卵巢功能保护和卵巢相关疾病治疗中具有重要临床价值。
Description
技术领域
本发明属于细胞工程和基因工程技术领域,具体涉及PLPP3基因在调控人卵巢颗粒细胞自噬中的应用。
背景技术
现有研究表明,颗粒细胞凋亡,又名程序性细胞死亡,通常被认为是卵泡闭锁的主要原因,常导致女性卵巢卵泡质量下降和卵巢早衰。自噬是一种溶酶体降解机制,其对细胞生存、分化、发育和稳态至关重要。适度的细胞自噬可维持原始卵泡的数量和生殖细胞的存活。但是,细胞过度自噬将导致细胞死亡,又被称为自噬细胞死亡(或II型细胞死亡)。研究表明,自噬能促进细胞凋亡,并参与调控原始卵泡发育和卵泡闭锁,且自噬也被发现参与女性多囊卵巢综合征的调控。
磷脂磷酸酶3(PLPP3)属于磷脂酸磷酸酶家族,是一种跨膜酶,包含6个跨膜结构域。磷脂磷酸酶作为一种糖蛋白定位于细胞质膜上,可水解溶血磷脂酸和短链磷脂酸,把磷脂酸转化为二酰甘油,在甘油脂类的新生合成及受体激活的信号通路中发挥作用。此外,有报道称PLPP3蛋白还可水解磷酸神经酰胺和磷酸醇。通过生成的这些产物,PLPP3基因参与细胞黏附、细胞间互作、血管形成及其相关疾病炎症反应和细胞侵袭等。尽管PLPP3基因具有以上重要的生物学功能,但其在卵巢颗粒细胞和卵泡发育中的作用尚不清楚。
发明内容
为了克服现有技术的缺点与不足,本发明的目的在于提供PLPP3基因在调控人卵巢颗粒细胞自噬中的应用。
本发明构建了PLPP3基因的超表达质粒pcDNA3.1-PLPP3(OE-PLPP3),并合成了其干扰序列si-PLPP3(东泽公司合成)。使用自噬双标腺病毒(mRFP-GFP-LC3)来检测颗粒细胞自噬水平;通过qRT-PCR和Western blot(WB)实验检测PLPP3基因对自噬信号通路标志基因mRNA和蛋白水平的影响;通过RNA免疫沉淀(RIP)和共免疫沉淀(Co-IP)方法检测PLPP3基因与自噬信号通路标志基因在mRNA和蛋白水平的互作情况;通过RNA和蛋白质核质分离实验,检测PLPP3基因和自噬信号通路标志基因的表达定位及互作;通过构建GFP-PLPP3融合蛋白的截断片段,分别为Full(含PLPP3蛋白全长,1aa-311aa)、TransR-acidPPc(1aa-275aa)和TransR(1aa-125aa),使用Co-IP实验检测自噬标志蛋白与PLPP3蛋白互作的特异性位点;通过组织免疫荧光实验在组织水平检测PLPP3蛋白与自噬标志蛋白的定位及互作;通过Annexin V/PI实验方法探究PLPP3对人卵巢颗粒细胞凋亡的影响;通过EdU和CCK8方法检测PLPP3对人卵巢颗粒细胞增殖和细胞活性的影响。
本发明的目的通过下述技术方案实现:
PLPP3基因在调控人卵巢颗粒细胞自噬中的应用,所述的应用为如下应用中的任意一种或多种:
I.超表达外源性PLPP3基因在体外环境下促进卵巢颗粒细胞自噬的应用;
II.抑制PLPP3基因表达在体外环境下抑制卵巢颗粒细胞自噬的应用。
进一步地,超表达外源性PLPP3基因,卵巢颗粒细胞中自噬小体和自噬溶酶体数量显著增加,自噬标志基因p62和mTOR的mRNA和蛋白水平显著降低,自噬标志基因LC3B和ATG7的mRNA和蛋白水平显著提高;抑制PLPP3基因表达,卵巢颗粒细胞中自噬小体和自噬溶酶体数量显著减少,自噬标志基因p62和mTOR的mRNA和蛋白水平显著提高,自噬标志基因LC3B和ATG7的mRNA和蛋白水平显著降低。
进一步地,PLPP3蛋白通过与p62蛋白相互作用调控巢颗粒细胞自噬。
PLPP3基因在调控人卵巢颗粒细胞p62基因转录和/或翻译活性中的应用,所述的应用为如下应用中的任意一种或多种:
III.超表达外源性PLPP3基因在体外环境下抑制p62基因转录和/或翻译活性的应用;
IV.抑制PLPP3基因表达在体外环境下提高p62基因转录和/或翻译活性的应用。
PLPP3基因在调控人卵巢颗粒细胞活性中的应用,所述的应用为如下应用中的任意一种或多种:
V.超表达外源性PLPP3基因在体外环境下抑制卵巢颗粒细胞活性的应用;
VI.抑制PLPP3基因表达在体外环境下提高卵巢颗粒细胞活性的应用。
进一步地,所述的超表达外源性PLPP3基因通过基因超表达技术实现。
更进一步地,所述的基因超表达技术采用的基因超表达质粒通过如下方式制备得到:
(1)提取卵巢颗粒细胞的cDNA,以cDNA为模板进行PCR扩增,得到目的片段;
(2)将目的片段连接到用限制性内切酶XbaI和kpnl酶切后的pcDNA3.1载体上,得到重组质粒。
更进一步地,步骤(1)中进行PCR扩增所用引物如下所示:
Forward:5′-GGGGTACCCCATGCTGATGGTCCTCCTTGTATC-3′;
Reverse:5′-GCTCTAGAGCCTACACCATGTTGTGGTGATTGTT-3′。
进一步地,所述的抑制PLPP3基因表达通过RNA干扰技术实现。
更进一步地,所述的RNA干扰技术采用的小干扰片段序列如下:
si-PLPP3:5′-CTGATGGTCCTCCTTGTAT-3′。
抑制PLPP3基因表达的试剂在制备抑制人卵巢颗粒细胞自噬的试剂、促进卵巢颗粒细胞p62基因转录和/或翻译活性和/或促进人卵巢颗粒细胞活性的试剂中的应用。
进一步地,所述的抑制PLPP3基因表达的试剂为小干扰片段,序列如下:
si-PLPP3:5′-CTGATGGTCCTCCTTGTAT-3′。
PLPP3蛋白定量试剂在制备人卵巢衰老诊断/评估试剂盒中的应用。
进一步地,所述的PLPP3蛋白水平与人卵巢衰老程度呈正相关。
进一步地,所述的诊断/评估试剂盒中包含PLPP3蛋白特异性抗体、PLPP3蛋白免疫印记实验试剂中的至少一种。
本发明以蛋白之间互作为切入点,采用细胞生物学方法研究PLPP3基因对颗粒细胞自噬水平的影响。具体的,本发明以PLPP3对颗粒细胞中的自噬小体和自噬溶酶体数量变化及自噬信号通路标志基因的表达调控为研究对象,来分析PLPP3对颗粒细胞自噬水平的影响。验证超表达PLPP3提高了颗粒细胞中的自噬小体和自噬溶酶体数量,降低了自噬标志基因p62和mTOR的mRNA及蛋白水平,提高了LC3B和ATG7的mRNA和蛋白水平。通过RIP和Co-IP实验发现,PLPP3蛋白仅跟p62 mRNA及p62蛋白均有互作;RNA和蛋白质核质分离实验表明,PLPP3和p62基因在细胞质和细胞核中均有定位及互作,且PLPP3负调控p62基因的表达。进一步,通过GFP-PLPP3融合蛋白的Co-IP实验,确定了p62蛋白跟PLPP3蛋白的跨膜结构域(TransR)互作。同时,在人的卵巢组织上,通过Co-IP及免疫荧光实验再次验证了PLPP3蛋白和p62及LC3B蛋白的互作关系。最后,在人的颗粒细胞层面,发现超表达PLPP3促进颗粒细胞凋亡,抑制细胞增殖,降低细胞活力。本发明通过探究PLPP3通过与自噬标志基因p62互作调控颗粒细胞自噬水平,进而提高了颗粒细胞凋亡水平,该发明对研究PLPP3基因参与卵泡发育调控及卵巢衰老分子机制研究具有具有很好的应用价值。
本发明的验证结果如下:
体外环境下,超表达PLPP3基因,显著增加了自噬小体和自噬溶酶体数量;而干扰PLPP3基因的表达,则显著减少了自噬小体和自噬溶酶体数量。
体外环境下,超表达PLPP3,能够显著降低自噬标志基因p62和mTOR的mRNA和蛋白水平,显著提高自噬标志基因LC3B和ATG7的mRNA和蛋白水平,同时能够显著提高自噬标志基因ATG5、BECN1和ATG2B的mRNA水平;而干扰PLPP3的表达,能够显著提高自噬标志基因p62和mTOR的mRNA和蛋白水平,显著降低自噬标志基因LC3B和ATG7的mRNA和蛋白水平,同时能够显著降低自噬标志基因ULK1和ATG2B的mRNA水平。
体外环境下,anti-PLPP3抗体能显著富集p62 mRNA和p62蛋白,而anti-p62抗体能够显著富集PLPP3蛋白。
体外环境下,PLPP3在细胞核和细胞质中能够跟p62 mRNA和p62蛋白互作,并能够负调控p62基因的表达。
体外环境下,p62蛋白能够跟PLPP3的跨膜区域(TransR)结合。
体外环境下,在人的卵巢组织中,anti-PLPP3抗体明显富集p62蛋白。PLPP3、p62及LC3B蛋白能够在人的卵巢组织中互作,PLPP3跟p62蛋白水平负相关,但是跟LC3B蛋白水平正相关。在中年人(约40岁)卵巢组织中,PLPP3和LC3B蛋白水平低,但p62蛋白表达量高;而在老年人(约60岁)卵巢组织中,PLPP3和LC3B蛋白水平高,但p62蛋白水平低。
体外环境下,超表达PLPP3,能够显著提高颗粒细胞凋亡率,而干扰PLPP3基因的表达能显著降低颗粒细胞凋亡率。
体外环境下,超表达PLPP3能够显著提高颗粒细胞中细胞凋亡信号通路标志基因CASP3、CASP8、CASP9、BID、p53、PLCY1、BAX和BIM的mRNA水平影响,降低MCL1和CREB1的mRNA水平;而干扰PLPP3的表达能够显著降低细胞凋亡信号通路标志基因CASP3、CASP8、CASP9、BID、p53、PLCY1、BAX和BIM的mRNA水平影响,提高MCL1和CREB1的mRNA水平;超表达PLPP3能够显著提高促凋亡标志蛋白BIM、BAX和CASP9的表达,降低抗凋亡标志蛋白MCL1的表达,而si-PLPP3能够显著降低促凋亡标志蛋白BIM、BAX和CASP9的表达,提高抗凋亡标志蛋白MCL1的的表达。
体外环境下,超表达PLPP3基因,能够显著降低人卵巢颗粒细胞增殖率;而干扰PLPP3基因的表达,则显著提高颗粒细胞增殖率。
体外环境下,超表达PLPP3,能够显著降低人卵巢颗粒细胞活力,而干扰PLPP3基因的表达,则显著提高颗粒细胞活力。
体外环境下,超表达PLPP3,能够显著降低颗粒细胞中增殖信号通路标志基因CDK4,SP1,PCNA,IKBA和p65的mRNA水平,且显著降低CDK4,PCNA和p65基因的蛋白水平;而干扰PLPP3基因的表达,则显著提高了增殖信号通路标志基因CDK4,SP1,PCNA,IKBA和p65的mRNA水平,且显著提高CDK4,PCNA和p65基因的蛋白水平。
本发明相对于现有技术具有如下的优点及效果:
本发明技术方案设计周详,结果可靠。为证实PLPP3调控细胞自噬水平对人卵巢颗粒细胞功能的影响,本发明从多层次、多角度验证。首先从自噬细胞表型、自噬信号通路标志基因的mRNA及蛋白水平调控进行验证;接着又用RIP、Co-IP及构建GFP-PLPP3融合蛋白来验证蛋白之间的互作关系,检测蛋白互作的特异区域;最后在组织水平利用Co-IP和免疫荧光实验进一步验证蛋白在组织中的定位及互作,并证明了超表达PLPP3提高了颗粒细胞凋亡水平,降低了颗粒细胞增殖率和颗粒细胞活力。
本发明首次发现,超表达PLPP3基因,能够显著提高颗粒细胞自噬水平和凋亡水平;而干扰PLPP3基因的表达,则显著降低颗粒细胞自噬水平和凋亡水平。同时发现,在中年人(约40岁)卵巢组织中,PLPP3蛋白水平低;而在老年人(约60岁)卵巢组织中,PLPP3蛋白水平高,PLPP3蛋白水平与人卵巢衰老程度呈正相关,可以为标志物诊断/评估人卵巢衰老水平提供参考。
本发明对研究自噬调控在卵巢卵泡发育、繁殖能力及卵巢相关疾病中的影响机制具有很好的应用价值,在卵巢功能保护和卵巢相关疾病治疗中具有重要参考价值。
附图说明
图1是PLPP3对颗粒细胞自噬水平的影响情况研究结果图;其中,a、b是PLPP3影响颗粒细胞自噬小体和自噬溶酶体数量变化的荧光图(a)和自噬小体及自噬溶酶体数量变化统计情况(b);c是OE-PLPP3和si-PLPP3对自噬信号通路标志基因mTOR、p62、LC3B、ATG5、ATG7、BECN1、ATG2B和ULK1的mRNA水平影响;d是OE-PLPP3及si-PLPP3对自噬信号通路标志基因mTOR、p62、LC3B和ATG7的蛋白水平影响。
图2是PLPP3蛋白跟p62基因在mRNA和蛋白水平互作情况研究结果图;其中,a是RIP实验中,自噬信号通路标志基因在anti-PLPP3抗体上的mRNA富集情况;b是在Co-IP实验中,自噬信号通路标志基因在anti-PLPP3抗体上的蛋白富集情况;c是在Co-IP实验中,PLPP3蛋白在anti-p62抗体上的蛋白富集情况;d、e是RNA和蛋白质核质分离实验中,PLPP3跟p62基因在细胞质和细胞核中的定位,PLPP3对p62 mRNA水平(d)及p62蛋白水平(e)的影响情况。
图3是p62蛋白跟PLPP3蛋白互作的特异性位点情况研究结果图;其中,a是PLPP3的蛋白结构域及GFP-PLPP3融合蛋白截断处理示意情况;b是Co-IP实验中,p62蛋白跟PLPP3蛋白特异位点互作情况。
图4是PLPP3跟p62及LC3B蛋白在人的卵巢组织中互作情况研究结果图;其中,a是人的卵巢颗粒细胞中p62基因在anti-PLPP3抗体上的蛋白富集情况;b,c是组织免疫荧光实验中,PLPP3、p62和LC3B蛋白在中年人(Middle age)卵巢组织和老年人(Old age)的卵巢组织中共定位及互作情况。
图5是PLPP3对颗粒细胞凋亡水平的影响情况研究结果图;其中,a、b是OE-PLPP3(a)和si-PLPP3(b)对颗粒细胞凋亡率的影响;c是OE-PLPP3和si-PLPP3对细胞凋亡信号通路标志基因CASP3、CASP8、CASP9、BID、p53、PLCY1、BAX,BIM,MCL1和CREB1的mRNA水平影响;d是OE-PLPP3和si-PLPP3对凋亡信号通路标志基因BIM、BAX和CASP9的蛋白水平影响;
图6是PLPP3对颗粒细胞增殖率和细胞活力的影响情况研究结果图;其中,a、b是EdU实验中,OE-PLPP3和si-PLPP3对颗粒细胞增殖率影响荧光图(a)和细胞增殖率统计图(b);c、d是OE-PLPP3(c)和si-PLPP3(d)对颗粒细胞活力的影响情况;e是OE-PLPP3和si-PLPP3对增殖信号通路标志基因CDK4,SP1,PCNA,IKBA和p65的mRNA水平影响情况;f是OE-PLPP3及si-PLPP3对增殖信号通路标志基因CDK4,PCNA和p65的蛋白水平影响情况。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下列实施例中未注明具体条件的实验方法,通常按照常规条件。
下列实施例中应用统计学方法,分析各实施例中3次独立实验的结果,分别计算“平均值±标准差”,使用单因素方差分析进行差异显著性分析(图中“*”表示P<0.05,“**”表示P<0.01)。
实施例1:人卵巢癌颗粒细胞COV434细胞系的培养
本发明使用COV434细胞系(ATCC),采用完全培养基(含10%小牛血清和1%双抗),置于37℃,5%CO2培养箱进行培养,待细胞融合度达到50%-70%进行转染或者药物处理,24h后可进行后续实验。
实施例2:构建PLPP3的超表达载体和干扰片段
(1)利用NCBI网站设计引物,以抽提的人颗粒细胞的cDNA为模板扩增PLPP3的CDS区(Gene ID:8613);扩增的片段经纯化回收、连接pMD18T载体(购自Takara公司)、转化、筛选、测序鉴定正确后抽提普通质粒。
(2)在PLPP3上下游引物分别加上Xba I和Kpn I酶切位点序列。以PLPP3的CDS区重组pMD18T普通质粒为模版,进行PCR扩增;片段经纯化回收、双酶切、连接pcDNA3.1载体、转化、筛选、测序鉴定正确后抽提无内毒素质粒(Magen公司,美国),命名为pcDNA3.1-PLPP3(OE-PLPP3)。
本发明所用到的PLPP3基因CDS区引物:
Forward:5′-GGGGTACCCCATGCTGATGGTCCTCCTTGTATC-3′;
Reverse:5′-GCTCTAGAGCCTACACCATGTTGTGGTGATTGTT-3′。
(3)设计并委托东泽公司合成PLPP3基因的的小干扰片段序列:
si-PLPP3:5′-CTGATGGTCCTCCTTGTAT-3′。
(4)观察细胞状态,当细胞融合度达到50%-70%左右时,分别将1000ng/mL浓度的PLPP3超表达载体(OE-PLPP3)和超表达对照(OE-NC)转染至细胞及100nmol/L浓度的PLPP3干扰片段(si-PLPP3)和干扰对照(si-NC)转染至细胞,24h后开展其他后续实验。
实施例3:自噬水平检测
委托汉恒生物合成自噬双标腺病毒(HBAD-mRFP-GFP-LC3)。在细胞汇合度在50%时,PBS洗2次细胞,添加1/2新的完全培养基,用30MOI自噬双标腺病毒侵染细胞,4h后吸掉原来的培养基后再添加新的培养基,同时用PLPP3的超表达载体和干扰片段转染细胞,培养箱孵育24h,PBS洗2次。使用共聚焦荧光显微镜拍照,计算细胞自噬小体和自噬溶酶体数量。mRFP标记自噬小体为红色,GFP标记自噬小体为绿色,自噬小体和溶酶体融合后,会形成自噬溶酶体,GFP淬灭。mRFP点的积累代表自噬溶酶体,mRFP和GEP点的共定位表明自噬。
实施例4:qRT-PCR
本发明中基因的qRT-PCR检测采用YEASEN公司的SYBR Green qPCRMaster Mix(2X)试剂盒。实验采用比较Ct值法检测样品基因的含量,具体计算公式如下:
基因相对表达量=2-{〈﹙实验组目的基因Ct值﹚-﹙实验组内参基因Ct值﹚〉-〈﹙对照组目的基因Ct值﹚-﹙对照组内参基因Ct值﹚〉}
检测基因用GAPDH做内参,本发明所用到的qRT-PCR引物为:
qPLPP3 Forward:5′-GGAGGAGCAGGAGGAGCCG-3′;
Reverse:5′-AGTCCAAGGGGAAGAGAGC-3′;
qP62 Forward:5′-TGTGTAGCGTCTGCGAGGGAAA-3′;
Reverse:5′-AGTGTCCGTGTTTCACCTTCCG-3′;
qLC3B Forward:5′-GAGAAGCAGCTTCCTGTTCTGG-3′;
Reverse:5′-GTGTCCGTTCACCAACAGGAAG-3′;
qATG5 Forward:5′-GCAGATGGACAGTTGCACACAC-3′;
Reverse:5′-GAGGTGTTTCCAACATTGGCTCA-3′;
qATG7 Forward:5′-CGTTGCCCACAGCATCATCTTC-3′;
Reverse:5′-CACTGAGGTTCACCATCCTTGG-3′;
qBECN1 Forward:5′-CTGGACACTCAGCTCAACGTCA-3′;
Reverse:5′-CTCTAGTGCCAGCTCCTTTAGC-3′;
qULK1 Forward:5′-GCAAGGACTCTTCCTGTGACAC-3′;
Reverse:5′-CCACTGCACATCAGGCTGTCTG-3′;
qmTOR Forward:5′-AGCATCGGATGCTTAGGAGTGG-3′;
Reverse:5′-CAGCCAGTCATCTTTGGAGACC-3′;
qATG2B Forward:5′-CTTCAGATGGAGTTGGAGGAGAC-3′;
Reverse:5′-AGTGGCTCCTTTCAGTCCTACG-3′;
qGAPDH Forward:5′-TCACCAGGGCTGCTTTTAACT-3′;
Reverse:5′-CTTGACTGTGCCGTGGAACT-3′;
细胞的总RNA提取参照上海飞捷公司RNA fast 200―总RNA极速抽提试剂盒操作说明书,具体步骤如下:
(1)往用胰酶消化下来的颗粒细胞中,加入500μl RA2液,充分颠倒混匀1min。
(2)将样本裂解物吸入或倒入内套管,离心1min。
(3)弃去外套管中液体,内套管中加入500μl洗液,离心1min。再重复此过程洗一次。
(4)取出内套管,弃去外套管中液体,仍然套回内套管,不加洗液,离心1min。
(5)将内套管移入新的eppendorf管中,在膜中央加入洗脱液(或pH>7.0的DEPC处理水)25-50μl,室温静置1min,离心1min,获得总RNA。
使用TaKaRa公司的PrimeScriptTM RT Master Mix(Perfect Real Time)cDNA反转录试剂盒反转录总RNA。
实施例5:Western Blot
(1)细胞总蛋白抽提
①配置蛋白裂解液:RIPA蛋白裂解液(白鲨公司)中加入1%的蛋白酶体抑制剂,上下颠倒混匀。
②每孔(六孔板)加入120μl蛋白裂解液,置于冰上裂解10-15min,中间拿出来轻轻晃动,促进细胞脱落。
③收集裂解的血细胞,12,000g,4℃离心10min,小心吸取上清液,去除下层沉淀。
(2)SDS-PAGE:
①蛋白定量(碧云天公司BCA蛋白定量试剂盒):使用多功能酶标仪测定A562波长的OD值,绘制标准曲线,通过回归方程和各个蛋白标准的平均OD值,计算各蛋白样品浓度;
②样品制备:混合20μg总蛋白与5×上样缓冲液按5:1,煮沸10min;
③制胶点样:每孔加入20ug蛋白,跑胶电压120~150V,约40min(当溴芬蓝跑至离胶底端1~2cm左右,停止电泳;或根据具体情况调整跑胶电压与时间);
④根据蛋白Marker的分子量标准切下含有目的蛋白的胶条,选择转膜电流300mA,20min;
⑤转膜结束后,用TBST轻轻冲洗膜5min,用5%的脱脂奶粉室温封闭2h;
⑥用TBST轻轻冲洗膜2次,每次5min,用TBST按照抗体说明书比例稀释一抗PLPP3(购自北京博奥森公司),4℃孵育过夜;
⑦用TBST轻轻冲洗膜3次,每次10min,1:10000稀释二抗Goat anti-rabbit IgG(购自SAB公司),室温孵育2h;
⑧用TBST轻轻冲洗膜3次,每次10min;
⑨用极超敏ECL化学发光试剂盒(碧云天公司,中国)显色,使用天能成胶系统(天能公司,中国)观察蛋白条带并进行拍照,采用Image J软件分析胶片中的蛋白条带。
实施例6:RIP实验
使用EZ-Magna RIP试剂盒(Millipore,美国)根据制造商的说明进行RIP检测。用胰酶从6孔板里消化并收集颗粒细胞,再用试剂盒里的裂解液进行裂解后,分为IP,IgG和Input三组,IP组用anti-PLPP3抗体在4℃免疫沉淀过夜,以同源IgG作为对照。洗涤、洗脱后,采用RT-qPCR检测富集的RNA样本。qRT-PCR的特异性引物详见实施例4。
实施例7:Co-IP实验
使用EZ-Magna Co-IP试剂盒(Millipore,美国)进行Co-IP实验。收集的细胞用PLPP3(博奥森,中国)、p62(Affinity,美国)、LC3B(Affinity,美国)、ATG7(Affinity,美国)、ATG5(Affinity,美国)和BECN1抗体(Affinity,美国)在4℃下免疫共沉淀过夜,进行阴性验证,以IgG为对照。用WB法检测富集的蛋白。
实施例8:RNA核质分离实验
使用细胞质和细胞核RNA提取试剂盒(Norgen biotek,加拿大)来提取和纯化细胞质和细胞核RNA。具体操作步骤如下:
①将消化下来的细胞转移至无RNase的试管,离心10min,收集细胞沉淀。往沉淀中加入Lysis Buffer J,涡旋以致充分裂解细胞。
②将裂解液转移至新无RNase的微量离心管中,台式离心机最大转速离心10min,将含有细胞质RNA的上清液转移至另一个无RNase的试管。沉淀部分即为细胞核RNA组分。
③提取并纯化RNA。向含有细胞质RNA的上清液中和含有细胞核RNA的沉淀中分别加入Buffer,涡旋混合10s,再加入96%-100%的乙醇,涡旋混匀10s。离心1min后用WashSolution A洗涤三次,用Elution Buffer E洗脱纯化RNA。
④纯化的RNA反转录成cDNA后用qRT-PCR检测目标基因的表达量。
实施例9:蛋白质核质分离实验
使用NE-PER Nuclear and Cytoplasmic Extraction Reagents(美国,赛默飞)进行细胞质和细胞核蛋白质的提取和纯化。具体操作步骤如下:
①向收集的细胞沉淀中加入冰冷的细胞质萃取试剂I(CER I),涡旋15s,使细胞颗粒完全悬浮,冰上孵育10min。
②加入冰冷的细胞质萃取试剂IICER II,涡旋5s,冰上孵育1min。再次涡旋,离心后收集上清,即为细胞质蛋白,将其转移至新的离心管,保存待用。下层细胞沉淀即为细胞核。
③在细胞核沉淀中加入冰冷的细胞核萃取试剂NER,每10min涡旋15s,共涡旋4次,持续40min。
④离心10min,转移上清液,即核蛋白质,至一个干净的预冷管中,保存备用。
⑤将提取的细胞质、细胞质蛋白质加入SDS经变性后用于后续的WB实验,检测目标蛋白的表达量。
实施例10:组织免疫荧光
具体操作步骤如下:
①将人卵巢(来自中山大学附属第一医院-妇科生殖医学中心,已获得医学伦理委员会批准)石蜡切片分别置于脱蜡溶液和无水乙醇中10-15min。
②将洗涤后的卵巢切片放置在充满柠檬酸抗原修复溶液的修复盒中,并在微波炉中进行抗原修复。
③自然冷却后,在脱色摇台上用PBS清洗载玻片三次,每次5min。使用组织化学笔在组织周围画圆圈以防止液体流失。
④用3%-5%BSA密封载玻片30min后,与第一抗体在4℃孵育过夜,第二抗体在37℃孵育3h。
⑤加入DAPI染料溶液,避光环境下室温孵育5min,定位靶蛋白。随后用树脂密封剂密封切片,并在荧光显微镜下观察和收集切片图像。
实施例11:颗粒细胞凋亡检测
本发明使用Annexin V-FITC技术检测细胞凋亡,参照参照BioVision公司AnnexinV-FITC Apoptosis Detection Kit试剂盒说明书,具体操作步骤如下:
(1)取细胞接种于6孔板,培养细胞至融合度为50%~70%时,使用lip3000试剂(赛默飞,美国)转染PLPP3的超表达质粒和干扰片段,继续培养24h,用PBS溶液清洗细胞;
(2)使用不含EDTA的胰酶消化细胞,用2mL PBS溶液清洗细胞;
(3)取0.5mL细胞悬液(约5×105个细胞),加入500μL 1×Binding Buffer;
(4)加入5μL Annexin V-FITC及室温5μL Propidium Iodide,室温避光孵育5min;
(5)立即用流式细胞仪检测分析(每组3个重复)。
实施例12:细胞增殖检测
本发明使用EdU试剂盒(锐博,中国)检测颗粒细胞增殖,参照锐博公司Cell-LightTMEdU Apollo 567In vitro Kit检测试剂盒说明书,具体操作步骤如下:
(1)取细胞接种于48孔板,培养细胞至融合度为50%~70%时,使用lip3000试剂(赛默飞,美国)转染PLPP3的超表达质粒和干扰片段,继续培养24h;
(2)制备50μMEdU培养基,即为用细胞培养基稀释按照1000:1稀释EdU溶液,每孔加入200μL 50μMEdU培养基孵育2h,PBS清洗细胞,每次3~5min;
(3)细胞固定化:向每孔加入200μL细胞固定液(用PBS稀释至80%的丙酮),室温孵育15~30min,PBS清洗细胞,每次3~5min;
(4)细胞透化:每孔加入200μL渗透剂(0.5%TritonX-100的PBS)透化细胞,透化孵育10min,PBS清洗细胞;
(5)EdU检测:每孔加入200μL的染色反应液(避光配置),避光在细胞培养中孵育30min,PBS清洗细胞;
(6)细胞再次透化:每孔加入200μL渗透剂(0.5%TritonX-100的PBS)透化细胞,透化孵育10min,PBS清洗细胞;
(7)DNA染色:每孔加入200μL DAPI反应液,避光室温孵育30min;
(8)荧光显微镜镜检(每组三个重复)。
实施例13:颗粒细胞活性检测
本发明使用CCK-8检测试剂盒(碧云天,中国)检测细胞活性,具体操作步骤如下:
(1)用96孔板接种细胞,当细胞融合度为50%-70%时,使用lip3000试剂(赛默飞,美国)转染PLPP3的超表达质粒和干扰片段,继续培养24h;
(2)每孔加入10μLCCK-8溶液(培养基:CCK8溶液=10:1),放置细胞培养箱孵育1h,用酶标仪A450测定OD值。
结果:
1、PLPP3显著提高了颗粒细胞中的自噬水平
使用自噬双标腺病毒(mRFP-GFP-LC3)(汉恒生物,中国)检测PLPP3对颗粒细胞自噬小体和自噬溶酶体数量变化的影响;通过qRT-PCR和WB实验检测PLPP3对自噬信号通路标志基因的mRNA和蛋白水平的影响。
自噬双标腺病毒检测结果显示,OE-PLPP3显著提高了颗粒细胞中自噬小体和自噬溶酶体数量(图1中的a、b),而si-PLPP3则显著降低了颗粒细胞中自噬小体和自噬溶酶体数量(图1中的a、b)。
qRT-PCR结果显示,OE-PLPP3显著降低了自噬信号通路标志基因mTOR和p62的mRNA水平,显著提高了LC3B、ATG5、ATG7、BECN1和ATG2B和的mRNA水平(图1中的c),但对ULK1基因影响不显著;而si-PLPP3显著提高了自噬信号通路关键标志基因mTOR和p62的mRNA水平,显著降低了自噬信号通路标志基因LC3B、ATG7、ATG2B和ULK1的mRNA水平,但对ATG5和BECN1基因影响不显著(图1中的c)。
WB结果显示,OE-PLPP3显著降低了自噬标志基因mTOR和p62的蛋白水平(图1中的d),显著提高了LC3B和ATG7的蛋白水平;而si-PLPP3显著提高了自噬标志基因mTOR和p62的蛋白水平,显著降低了LC3B和ATG7的蛋白水平(图1中的d)。
2、PLPP3跟p62基因在mRNA和蛋白水平互作
通过RIP、Co-IP和核质分离实验检测PLPP3和自噬信号通路标志基因在mRNA和蛋白水平的互作情况。
RIP和Co-IP结果显示,跟自噬信号通路其他基因相比,anti-PLPP3抗体明显富集大量的p62 mRNA(图2中的a)和p62蛋白(图2中的b),且anti-p62抗体明显富集大量的PLPP3蛋白(图2中的c);
RNA和蛋白质核质分离实验结果显示,颗粒细胞中,PLPP3跟p62 mRNA和p62蛋白在细胞质和细胞核中均有互作,且OE-PLPP3抑制了p62基因的转录(图2中的d)和翻译(图2中的e)。
3、p62蛋白结合在PLPP3蛋白的跨膜区域(TransR)
使用GFP-PLPP3融合蛋白截断处理(图3中的a)并开展Co-IP实验,检测p62蛋白和PLPP3蛋白结合的特异区域。
Co-IP实验显示,p62蛋白与PLPP3的跨膜区(TransR)(1-125aa)互作(图3中的b)。
4、PLPP3跟p62蛋白在人的卵巢中互作
使用Co-IP和组织免疫荧光技术,检测PLPP3蛋白和p62蛋白在中年人和老年人卵巢的定位及互作情况。
Co-IP实验显示,人的卵巢颗粒细胞中,anti-PLPP3抗体明显富集p62蛋白(图4中的a);组织免疫荧光实验显示,PLPP3蛋白和p62蛋白在人的卵巢组织中共定位,且在中年人(约40岁)卵巢组织中,PLPP3和LC3B蛋白水平低,但p62蛋白水平高(图4中的b);而在老年人(约60岁)卵巢组织中,PLPP3和LC3B蛋白水平高,但p62蛋白水平低(图4中的c)。
5、PLPP3基因显著提高了卵巢颗粒细胞凋亡水平
通过Annexin V/PI检测PLPP3对颗粒细胞凋亡率的影响情况;通过qRT-PCR和Western Blot实验检测PLPP3对细胞凋亡信号通路标志基因的mRNA和蛋白水平的影响情况。
Annexin V/PI实验结果显示,OE-PLPP3显著提高了颗粒细胞凋亡率(图5中的a),而si-PLPP3显著降低了颗粒细胞凋亡率(图5中的b)。
qRT-PCR结果显示,OE-PLPP3显著提高了细胞凋亡信号通路标志基因CASP3、CASP8、CASP9、BID、p53、PLCY1、BAX和BIM的mRNA水平,显著降低了MCL1和CREB1的mRNA水平(图5中的c),而si-PLPP3则显著降低了细胞凋亡信号通路标志基因CASP3、CASP8、CASP9、BID、p53、PLCY1和BIM的mRNA水平影响,显著提高了MCL1的mRNA水平,但对BAX和CREB1影响不显著(图5中的c);
WB结果显示,OE-PLPP3显著提高了促凋亡标志蛋白BIM、BAX和CASP9的蛋白水平,降低了抗凋亡标志蛋白MCL1的蛋白水平(图5中的d),而si-PLPP3则显著降低了促凋亡标志蛋白BIM、BAX和CASP9的蛋白水平,提高了抗凋亡关键蛋白MCL1的蛋白水平(图5中的d)。
6、PLPP3基因显著降低了颗粒细胞增殖率和细胞活力
使用EdU(5-Ethynyl-2’-deoxyuridine)试剂盒(锐博,中国)和CCK8试剂盒(碧云天,中国)分别检测PLPP3对颗粒细胞增殖率和细胞活力的影响;通过qRT-PCR和WesternBlot实验检测PLPP3对细胞增殖信号通路标志基因的mRNA和蛋白水平的影响。
EdU实验结果显示,OE-PLPP3显著降低了颗粒细胞增殖率(图6中的a、b),而si-PLPP3则显著提高了颗粒细胞增殖率(图6中的a、b)。
CCK8实验结果显示,OE-PLPP3显著降低了颗粒细胞活力(图6中的c),而si-PLPP3则显著提高了颗粒细胞活力(图6中的d)。
qRT-PCR结果显示,OE-PLPP3显著降低了细胞增殖信号通路标志基因CDK4、SP1、PCNA、IKBA和p65的mRNA水平;而si-PLPP3则显著提高了细胞增殖信号通路标志基因SP1、PCNA、IKBA和p65的mRNA水平,但是对CDK4的影响不显著(图5中的e);
WB结果显示,OE-PLPP3显著降低了细胞增殖信号通路标志基因CDK4和PCNA的蛋白水平,但对p65的蛋白水平影响不显著;而si-PLPP3则显著提高了细胞增殖信号通路标志基因PCNA、CDK4和p65的蛋白水平(图5中的f)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.PLPP3基因在调控人卵巢颗粒细胞自噬中的应用,其特征在于:所述的应用为如下应用中的任意一种或多种:
I.超表达外源性PLPP3基因在体外环境下促进卵巢颗粒细胞自噬的应用;
II.抑制PLPP3基因表达在体外环境下抑制卵巢颗粒细胞自噬的应用。
2.根据权利要求1所述的PLPP3基因在调控人卵巢颗粒细胞自噬中的应用,其特征在于:
超表达外源性PLPP3基因,卵巢颗粒细胞中自噬小体和自噬溶酶体数量显著增加,自噬标志基因p62和mTOR的mRNA和蛋白水平显著降低,自噬标志基因LC3B和ATG7的mRNA和蛋白水平显著提高;抑制PLPP3基因表达,卵巢颗粒细胞中自噬小体和自噬溶酶体数量显著减少,自噬标志基因p62和mTOR的mRNA和蛋白水平显著提高,自噬标志基因LC3B和ATG7的mRNA和蛋白水平显著降低。
3.根据权利要求1或2所述的PLPP3基因在调控人卵巢颗粒颗粒细胞自噬中的应用,其特征在于:
所述的PLPP3基因的蛋白通过与p62基因互作调控卵巢颗粒细胞自噬。
4.PLPP3基因在调控人卵巢颗粒细胞p62基因转录和/或翻译活性中的应用,其特征在于:所述的应用为如下应用中的任意一种或多种:
III.超表达外源性PLPP3基因在体外环境下抑制p62基因转录和/或翻译活性的应用;
IV.抑制PLPP3基因表达在体外环境下促进p62基因转录和/或翻译活性的应用。
5.PLPP3基因在调控人卵巢颗粒细胞活性中的应用,其特征在于:所述的应用为如下应用中的任意一种或多种:
V.超表达外源性PLPP3基因在体外环境下抑制卵巢颗粒细胞活性的应用;
VI.抑制PLPP3基因表达在体外环境下提高卵巢颗粒细胞活性的应用。
6.根据权利要求1-5任一项所述的应用,其特征在于:
所述的超表达外源性PLPP3基因通过基因超表达技术实现;
所述的抑制PLPP3基因表达通过RNA干扰技术实现。
7.根据权利要求6所述的应用,其特征在于:
所述的基因超表达技术采用的基因超表达质粒通过如下方式制备得到:
(1)提取卵巢颗粒细胞的cDNA,以cDNA为模板进行PCR扩增,得到目的片段;
(2)将目的片段连接到用限制性内切酶XbaI和kpnl酶切后的pcDNA3.1载体上,得到重组质粒;
步骤(1)中进行PCR扩增所用引物如下所示:
Forward:5′-GGGGTACCCCATGCTGATGGTCCTCCTTGTATC-3′;
Reverse:5′-GCTCTAGAGCCTACACCATGTTGTGGTGATTGTT-3′;
所述的RNA干扰技术采用的小干扰片段序列如下:
si-PLPP3:5′-CTGATGGTCCTCCTTGTAT-3′。
8.抑制PLPP3基因表达的试剂在制备抑制人卵巢颗粒细胞自噬的试剂、促进卵巢颗粒颗粒细胞p62基因转录和/或翻译活性和/或促进人卵巢颗粒细胞活性的试剂中的应用。
9.PLPP3蛋白定量试剂在制备人卵巢衰老诊断/评估试剂盒中的应用。
10.根据权利要求9所述的PLPP3蛋白定量试剂在制备人卵巢衰老诊断/评估试剂盒中的应用,其特征在于:
所述的PLPP3蛋白水平与人卵巢衰老程度呈正相关;
所述的诊断/评估试剂盒中包含PLPP3蛋白特异性抗体、PLPP3蛋白免疫印记实验试剂中的至少一种。
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