CN113156116A - 一种非诊断目的的脑组织外泌体水平的检测方法及其应用以及脑组织外泌体的应用 - Google Patents
一种非诊断目的的脑组织外泌体水平的检测方法及其应用以及脑组织外泌体的应用 Download PDFInfo
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Abstract
本发明公开了一种非诊断目的的脑组织外泌体水平的检测方法及其应用以及脑组织外泌体的应用，所述检测方法包括：通过免疫荧光技术结合超高分辨显微技术检测脑组织外泌体水平；本发明通过免疫荧光结合超高分辨显微技术对脑组织外泌体水平进行检测，能够实现可视化监测，对脑组织外泌体与中枢神经系统疾病相关研究具有很大的实际意义，此外，本发明通过构建Rab27a敲除小鼠，获得脑组织外泌体释放水平显著减少的小鼠模型，该小鼠模型只影响脑组织外泌体的释放水平，而不影响相同数量外泌体对脑缺血损伤调控的功能，能够可靠的用于评价脑组织外泌体释放调控对脑缺血损伤，为脑组织及中枢神经系统疾病外泌体相关研究提供新的手段。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种非诊断目的的脑组织外泌体水平的检测方法及其应用以及脑组织外泌体的应用。
背景技术
外泌体(exosomes，EXs)是由细胞活化或凋亡产生的一种直径30-150nm的细胞膜微小囊泡，其通过传递来源细胞的蛋白，脂质体及核酸调节受体细胞功能，是一条新的重要的细胞间信息交流途径。
近年来，科研工作者们聚焦外泌体释放调控的研究。GW4689(鞘磷脂酶抑制剂)是已知能够通过抑制鞘磷脂酶从而抑制外泌体释放的化学试剂，是目前普遍被应用于体外培养细胞的外泌体释放抑制剂，然而其作为一种化学试剂其在体内尤其是中枢神经系统中的应用受限。同时，由于外泌体结构微小，不利于组织水平可视化监测。
现有技术中研究了Rab家族GTP酶通过调节囊泡转运、核小体内吞及囊泡与细胞膜融合广泛参与调控外泌体(EXs)的释放，其家族成员Rab27a能通过介导Hela细胞中多囊泡小体与细胞膜融合调控外泌体释放，Rab27a敲除能抑制小鼠循环外泌体水平。然而未见报道脑组织外泌体水平检测方法以及如何可靠的评价脑组织外泌体释放调控对脑缺血损伤。
发明内容
本发明的目的在于提供一种非诊断目的的脑组织外泌体水平的检测方法及其应用以及脑组织外泌体的应用，该检测方法通过免疫荧光结合超高分辨显微技术对脑组织外泌体水平进行可视化监测，能够为脑组织及中枢神经系统疾病外泌体相关研究提供新的手段。
为了实现本发明的上述目的，特采用以下技术方案：
本发明第一方面提供一种非诊断目的的脑组织外泌体水平的检测方法，所述检测方法包括：
通过免疫荧光技术结合超高分辨显微技术检测脑组织外泌体水平。
优选地，所述通过免疫荧光技术结合超高分辨显微技术检测脑组织外泌体水平包括如下步骤：
(a)分离全脑组织并冷冻，再将脑组织冠状切成脑片；
(b)采用外泌体特异性标记抗体一抗孵育脑片，再采用星形胶质细胞特异性标记抗体一抗孵育脑片，然后，分别采用第一荧光染料标记的第一酶标记抗体和第二荧光染料标记的第二酶标记抗体孵育脑片，随后，采用第三荧光染料标记细胞核；
(c)滴加抗荧光猝灭剂，采用超高分辨显微镜拍照、分析，得到外泌体数量。
本发明第二方面提供一种脑组织外泌体释放调控对脑缺血损伤的评估方法，包括如下步骤：
S1，构建Rab27a基因敲除小鼠模型；
S2，分别构建Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型；
S3，采用上述检测方法分别检测Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型小鼠的脑组织外泌体水平；
S4，将野生型小鼠脑组织外泌体和不同浓度的Rab27a基因敲除小鼠模型脑组织外泌体分别静脉注射至Rab27a基因敲除小鼠模型的缺血性脑卒中模型小鼠中，并监测注射不同外泌体后的小鼠脑梗死体积、神经功能缺损评分和脑血流；
S5，根据监测结果评估外泌体释放调控与脑缺血损伤的相关性。
本发明上述评估方法，通过构建Rab27a基因敲除小鼠模型能够稳定降低脑组织外泌体释放水平，并且不影响相同数量外泌体对脑缺血损伤调功的功能，进而能够可靠的用于评价脑组织外泌体释放调控对脑缺血损伤。
优选地，所述Rab27a基因敲除小鼠模型的构建方法包括如下步骤：
S1，设计小鼠Rab27a基因第2外显子为敲除位点；
S2，构建TALEN载体；
S3，体外转录TALEN获得mRNA并注射到野生型小鼠受精卵中，培育获得Rab27a基因敲除小鼠；
S4，对Rab27a基因敲除小鼠进行鉴定，得到Rab27a基因敲除小鼠模型。
优选地，所述对Rab27a基因敲除小鼠进行鉴定包括：
通过PCR结合DNA序列分析对对Rab27a基因敲除小鼠进行鉴定。
优选地，所述监测注射不同外泌体后的小鼠脑梗死体积、神经功能缺损评分和脑血流具体包括：
通过TTC染色、5点评分法和多普勒血流仪分别监测小鼠脑梗死体积、神经功能缺损评分及脑血流。
本发明第三方面提供一种Rab27a基因敲除小鼠模型的脑组织外泌体在制备治疗脑组织疾病和/或中枢申请系统疾病药物中的应用。
本发明第四方面提供一种用于治疗脑组织疾病和/或中枢申请系统疾病的注射制剂，所述注射制剂含有Rab27a基因敲除小鼠模型脑的组织外泌体，并且浓度为50μg/ml。
与现有技术相比，本发明的有益效果至少包括：
本发明通过免疫荧光结合超高分辨显微技术对脑组织外泌体水平进行检测，能够实现可视化监测，对脑组织外泌体与中枢神经系统疾病相关研究具有很大的实际意义。此外，本发明通过构建Rab27a敲除小鼠，获得脑组织外泌体释放水平显著减少的小鼠模型，该小鼠模型只影响脑组织外泌体的释放水平，而不影响相同数量外泌体对脑缺血损伤调控的功能，能够可靠的用于评价脑组织外泌体释放调控对脑缺血损伤，为脑组织及中枢神经系统疾病外泌体相关研究提供新的手段。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。在所有附图中，类似的元件或部分一般由类似的附图标记标识。附图中，各元件或部分并不一定按照实际的比例绘制。
图1为蛋白免疫印迹检测的野生型小鼠和Rab27a基因敲除小鼠脑组织中Rab27a表达水平；
图2为纳米颗粒分析野生型小鼠和Rab27a基因敲除小鼠脑组织外泌体水平；
图3为电子透射显微镜分析野生型小鼠和Rab27a基因敲除小鼠脑组织外泌体水平；
图4为蛋白免疫印迹鉴定分析野生型小鼠和Rab27a基因敲除小鼠脑组织外泌体水平；
图5为超高分辨显微镜检测Rab27a基因敲除小鼠模型和野生型小鼠脑组织外泌体水平；
图6为野生型小鼠和Rab27a基因敲除小鼠脑梗死面积；
图7为野生型小鼠和Rab27a基因敲除小鼠神经功能缺损评分；
图8为野生型小鼠和Rab27a基因敲除小鼠脑血流；
图9为输注EXWT、不同浓度的EXRab27aKO以及空白组Rab27a基因敲除小鼠的脑梗死面积；
图10为输注EXWT、不同浓度的EXRab27aKO以及空白组Rab27a基因敲除小鼠的神经功能缺损评分；
图11为输注EXWT、不同浓度的EXRab27aKO以及空白组Rab27a基因敲除小鼠的脑微血管密度；
图12为输注EXWT、不同浓度的EXRab27aKO以及空白组Rab27a基因敲除小鼠的脑血流。
图中：*p＜0.05。
具体实施方式
下面将结合实施例对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案，因此只作为示例，而不能以此来限制本发明的保护范围。
需要注意的是，除非另有说明，本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
本发明中采用的原料及定义如下：
C57BL/6小鼠是一种常见的近交品系实验鼠，在遗传学试验中广泛用作转基因鼠以模拟人类的基因缺陷类疾病。
基因敲除(knockout，简称ko)是用含有一定已知序列的DNA片段与受体细胞基因组中序列相同或相近的基因发生同源重组，整合至受体细胞基因组中并得到表达的一种外源DNA导入技术。
小鼠Rab27a基因(基因库编号：NM_023635.6；数据库：ENSMUSG00000032202)位于小鼠9号染色体上。利用显微注射技术和转录激活样效应因子核酸酶(transcriptionactivator-like effector nuclease，TALEN)技术构建Rab27a敲除小鼠并对其进行表型分析。
实施例1
本实施例为一种Rab27a敲除C57BL/6小鼠模型的构建方法，该构建方法包括如下步骤：
S1，设计C57BL/6小鼠Rab27a基因第2外显子为敲除位点；
S2，构建TALEN载体；
S3，体外转录TALEN获得mRNA并注射到野生型小鼠受精卵中，培育获得Rab27a基因敲除小鼠；
S4，采用聚合酶链式反应(PCR)结合DNA序列分析鉴定Rab27a基因敲除小，得到Rab27a基因敲除小鼠模型。
本发明构建得到的Rab27a基因敲除小鼠模型，能够稳定降低脑组织外泌体释放水平，并且不影响相同数量外泌体对脑缺血损伤调功的功能，进而能够可靠的用于评价脑组织外泌体释放调控对脑缺血损伤。
实施例2
本实施例为脑组织外泌体水平的检测方法，检测方法包括：
(a)分离全脑组织并冷冻，再将脑组织冠状切成脑片；
(b)采用外泌体特异性标记抗体一抗孵育脑片，再采用星形胶质细胞特异性标记抗体一抗孵育脑片，然后，分别采用第一荧光染料标记的第一酶标记抗体和第二荧光染料标记的第二酶标记抗体孵育脑片，随后，采用第三荧光染料标记细胞核；
(c)滴加抗荧光猝灭剂，采用超高分辨显微镜拍照、分析，得到外泌体数量。
实施例3
本实施例为一种脑组织外泌体释放调控对脑缺血损伤的评估方法，该评估方法包括如下步骤：
S1，采用大脑中动脉栓塞法分别构建实施例1中的Rab27a基因敲除小鼠模型和C57BL/6野生型小鼠的缺血性脑卒中模型；
S2，分别检测Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型小鼠的脑组织外泌体水平；其中，检测方法如下：
(a)分离全脑组织并冷冻，再将脑组织冠状切成20μm厚脑片；
(b)采用兔抗CD63(外泌体特异性标记抗体，1:100,购于abcam公司)一抗于4℃孵育脑片过夜，山羊抗GFAP(星形胶质细胞特异性标记抗体，1:100,购于abcam公司)一抗于4℃孵育脑片过夜，再分别采用Alexia fluor647染料标记的驴抗兔二抗(1:500,购于abcam公司)和Alexia fluor568染料标记的驴抗山二抗室温条件孵育1h，采用4',6-二脒基-2-苯基吲哚DAPI(1:1000,购于abcam公司)染料标记细胞核；
(c)PBS(磷酸缓冲盐溶液)清洗三次后,滴加抗荧光猝灭剂封片，采用尼康超高分辨纤维镜于100×硅油物镜下观察脑组织中外泌体水平并拍照，Image J软件分析外泌体数量；
S3，将野生型小鼠脑组织外泌体(浓度为50μg/ml)和不同浓度的Rab27a基因敲除小鼠模型脑组织外泌体(低浓度为20μg/ml，高浓度为50μg/ml)分别静脉注射至Rab27a基因敲除小鼠模型的缺血性脑卒中模型小鼠中，并通过TTC染色、5点评分法和多普勒血流仪分别监测小鼠脑梗死体积、神经功能缺损评分及脑血流；
S4，根据监测结果评估外泌体释放调控与脑缺血损伤的相关性。
实验例
1、采用蛋白免疫印迹检测实施例3中Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型小鼠脑组织中Rab27a蛋白表达；
监测结果如图1所示；
由图1可知：Rab27a基因敲除小鼠模型脑组织中Rab27a表达缺失。
2、采用纳米颗粒示踪技术及投射电子显微镜检测Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型小鼠的脑组织外泌体的粒径大小以及免疫印迹鉴定野生型小鼠和Rab27a基因敲除小鼠特异性蛋白CD63和TSG101的表达水平；
检测结果如图2～4所示；
由图2～4可知：纳米颗粒示踪技术及透射电子显微镜检测发现两组小鼠脑组织外泌体粒径为100nm左右，并表达外泌体特异性蛋白CD63和TSG101。
3、实施例2中Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型小鼠的脑组织外泌体水平检测结果如图5所示；
由图5可知：与野生型组小鼠相比，Rab27a敲除组小鼠脑组织中外泌体水平减少达60％，而且与上述纳米颗粒分析仪检测结果相符。
4、通过TTC染色、5点评分法和多普勒血流仪分别监测Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型小鼠脑梗死体积、神经功能缺损评分及脑血流的监测结果如图6～8所示；
由图6～8可知：
相对于野生型小鼠，Rab27a敲除小鼠梗死体积、神经功能缺损评分显著增加，脑血流显著减少。
5、通过TTC染色、5点评分法和多普勒血流仪分别监测尾静脉输注野生型小鼠脑组织外泌体(EXWT，浓度为50μg/ml)和不同浓度的Rab27a基因敲除小鼠模型脑组织外泌体(L-EXRab27a KO，低浓度为20μg/ml，H-EXRab27a KO高浓度为50μg/ml)Rab27a基因敲除小鼠模型的缺血性脑卒中模型小鼠以及未输注外泌体(Vehicle)小鼠脑梗死体积、神经功能缺损评分及脑血流进行检测；
检测结果如图9～12所示，图11中标尺为40μm；
由图9～12可知：
在Rab27a敲除的小鼠中，输注EXWT显著减少小鼠梗死体积、神经功能缺损评分，脑微血管密度及脑血流；与输注EXWT相比，输注低浓度Rab27a敲除鼠脑组织外泌体(L-EXRab27a KO)对Rab27a敲除的小鼠的上述作用效果显著减少，而输注高浓度Rab27a敲除鼠脑组织(H-EXRab27a KO)具有与输注EXWT相同的缺血损伤保护作用。
实验结果表明，采用Rab27a敲除构建的抑制脑组织外泌体释放的模型只影响脑组织外泌体的释放水平，而不影响相同数量外泌体对脑缺血损伤调控的功能。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围，其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (8)
1.一种非诊断目的的脑组织外泌体水平的检测方法，其特征在于，所述检测方法包括：
通过免疫荧光技术结合超高分辨显微技术检测脑组织外泌体水平。
2.根据权利要求1所述的检测方法，其特征在于，所述通过免疫荧光技术结合超高分辨显微技术检测脑组织外泌体水平包括如下步骤：
(a)分离全脑组织并冷冻，再将脑组织冠状切成脑片；
(b)采用外泌体特异性标记抗体一抗孵育脑片，再采用星形胶质细胞特异性标记抗体一抗孵育脑片，然后，分别采用第一荧光染料标记的第一酶标记抗体和第二荧光染料标记的第二酶标记抗体孵育脑片，随后，采用第三荧光染料标记细胞核；
(c)滴加抗荧光猝灭剂，采用超高分辨显微镜拍照、分析，得到外泌体数量。
3.一种脑组织外泌体释放调控对脑缺血损伤的评估方法，其特征在于，包括如下步骤：
S1，构建Rab27a基因敲除小鼠模型；
S2，分别构建Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型；
S3，采用权利要求1或2中所述检测方法分别检测Rab27a基因敲除小鼠模型和野生型小鼠的缺血性脑卒中模型小鼠的脑组织外泌体水平；
S4，将野生型小鼠脑组织外泌体和不同浓度的Rab27a基因敲除小鼠模型脑组织外泌体分别静脉注射至Rab27a基因敲除小鼠模型的缺血性脑卒中模型小鼠中，并监测注射不同外泌体后的小鼠脑梗死体积、神经功能缺损评分和脑血流；
S5，根据监测结果评估外泌体释放调控与脑缺血损伤的相关性。
4.根据权利要求3所述的评估方法，其特征在于，所述Rab27a基因敲除小鼠模型的构建方法包括如下步骤：
S1，设计小鼠Rab27a基因第2外显子为敲除位点；
S2，构建TALEN载体；
S3，体外转录TALEN获得mRNA并注射到野生型小鼠受精卵中，培育获得Rab27a基因敲除小鼠；
S4，对Rab27a基因敲除小鼠进行鉴定，得到Rab27a基因敲除小鼠模型。
5.根据权利要求4所述的评估方法，其特征在于，所述对Rab27a基因敲除小鼠进行鉴定包括：
通过PCR结合DNA序列分析对对Rab27a基因敲除小鼠进行鉴定。
6.根据权利要求3所述的评估方法，其特征在于，所述监测注射不同外泌体后的小鼠脑梗死体积、神经功能缺损评分和脑血流具体包括：
通过TTC染色、5点评分法和多普勒血流仪分别监测小鼠脑梗死体积、神经功能缺损评分及脑血流。
7.Rab27a基因敲除小鼠模型的脑组织外泌体在制备治疗脑组织疾病和/或中枢申请系统疾病药物中的应用。
8.一种用于治疗脑组织疾病和/或中枢申请系统疾病的注射制剂，其特征在于，所述注射制剂含有Rab27a基因敲除小鼠模型脑的组织外泌体，并且浓度为50μg/ml。
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