CN112825816A - 一种新的小胶质细胞激活方法 - Google Patents
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Abstract
本发明提供一种Cx3cr1CreER‑Ai27基因型双转鼠以及一种新的小胶质细胞激活方法，通过Cx3cr1CreER小鼠与Ai27基因小鼠进行杂交获得Cx3cr1CreER‑Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光(450‑490nm)刺激，即可特异性激活光纤下方区域小胶质细胞。本发明的小胶质细胞激活方法可以特异性激活小胶质细胞，准确地区分小胶质细胞与其他细胞，在研究小胶质细胞的发育、行为和功能等方面具有重要意义，本发明操作简便，研究成本和风险降低，具有广阔的应用前景。
Description
技术领域
本发明涉及生物领域，具体涉及一种Cx3cr1CreER-Ai27基因型双转鼠、一种新的小胶质细胞的激活方法及其应用。
背景技术
小胶质细胞(Microglia)是中枢神经系统(CNS)最小的一种神经胶质细胞，分布于整个中枢神经系统，约占胶质细胞总数的5％-10％。作为常驻中枢神经系统的免疫效应细胞，小胶质细胞隶属于单核吞噬细胞族，被广泛地认为是中枢神经系统的主要免疫效应器，小胶质细胞的形态具有高度可塑性，与其生物学功能状态密切相关。正常脑组织中，小胶质细胞呈高度分枝状，具有三级和四级分枝结构，且细胞间的分枝很少发生重叠。分枝状的小胶质细胞通常被称为“静息小胶质细胞”。正常情况下，高度分枝状静息状态的小胶质细胞为大脑提供了一个高度动态和高效的监测系统。当脑内发生炎症、感染、创伤或其他神经系统疾病时，小胶质细胞迅速被激活并获得吞噬功能。小胶质细胞及其介导的神经炎症在中枢神经系统的损伤及疾病的转归过程中起着非常重要的作用，参与例如HIV脑病，阿尔兹海默病，多发性硬化等人神经系统紊乱疾病。
Cre是一种来源于P1噬菌体的位点特异性络氨酸重组酶，属于λ整合酶超家族，由一个五价物[Arg-Lys-(His/Lys)-Arg-(His/Trp)]协助催化DNA重组过程中DNA链的断裂和重连，cre插入内含子后形成icre。loxP位点是一段总长度为34bp的反向重复DNA序列，Cre重组酶介导两个loxP位点之间的重组，而且，两个loxP位点的方向和位置可以决定重组后的三种结果：删除、翻转或整合。由于Cre-loxP系统不需要任何辅助因子，经逐步改造，被广泛应用于真核细胞，目前已经在细胞发育追踪、基因敲除、基因条件表达等领域发挥了显著作用。
趋化因子Fractalkine具有CX3C趋化因子域，并根据N-末端的半胱氨酸的间距命名，此构成CX3C家族，不同于其他已知的趋化因子，CX3C模块有两种异构体存在。Fractalkine(CX3CL1)是CX3CR1的特异性配体，它是一种跨膜糖蛋白，其典型的功能是与CX3CR1高亲和性的相互作用，从而介导白细胞在流动状态下的阻滞，该可溶性的趋化因子可以被蛋白水解酶作用而释放。CX3CR1/CX3CX1信号在不同组织发挥不同的作用。在循环系统中，CX3CR1基因可在单核细胞、树突状细胞(DC)、T细胞亚型和自然杀伤细胞(NK)中表达。在体外，CX3CL1可促进神经元的存活，抑制小胶质细胞的调亡，但在完整的中枢神经中，CX3CR1/CX3CL1信号的功能还是未知的。CX3CR1可在外周单核细胞、NK细胞、DC细胞、小胶质细胞等中表达，而在中枢神经系统中，CX3CR1基因只在小胶质细胞中表达当FKN和CX3CR1分别在神经元和小胶质细胞中表达时，该受体-配体对神经元-胶质间的相互联系至关重要。通过细胞之间的交流及因子的分泌，可维持小胶质细胞在稳定条件下的静息状态，其中一种方式即为神经元通过CX3CR1/CX3CL1信号通路抑制小胶质细胞的活化。使用CX3CR1启动子驱动CreER的表达，通过大量CreER的表达，可有效进行Cre介导的重组。然而，CX3CR1基因不仅在小胶质细胞中表达，也在外周髓系细胞中表达。因而，当仅仅使用CX3CR1敲除小鼠时，并不能准确指明小胶质细胞的功能。
Ai27小鼠暴露于Cre重组酶后表达改良的hChR2/tdTomato融合蛋白。该小鼠可用于在蓝光(450-490nm)照射下快速激活体内可兴奋细胞的光遗传学研究。
目前，区分或标记小胶质细胞的主要方法包括形态学观察、特征性蛋白免疫组织化学染色和单启动子荧光蛋白标记，然而这些方法特异性较差，无法准确地区分小胶质细胞与中枢神经系统的其他细胞，且标记过程中需要处死小鼠，无法在活体状态下进行特异性标记。
因而建立有效的离体和在体激活小胶质细胞的方法以及小鼠模型，是现有技术中亟需解决的技术难题。但目前未见类似报道或产品。
基于此，本发明提供了一种新的小胶质细胞激活方法，所述方法通过Cx3cr1CreERknock-in/knock-out小鼠在大脑小胶质细胞中表达一种Cre-ERT2融合蛋白和EYFP蛋白，而后与Ai27(RCL-hChR2(H134R)/tdT)-D基因小鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光(450-490nm)刺激，即可特异性激活光纤下方区域小胶质细胞。本发明的小胶质细胞激活方法可以特异性激活小胶质细胞，准确地区分小胶质细胞与其他细胞，在研究小胶质细胞的发育、行为和功能等方面具有重要意义，，在生物医药研究领域，有广阔的应用前景。
发明内容
为了解决上述现有技术中存在的问题，本发明的一个目的在于提供一种新的小胶质细胞的激活方法，其特征在于，通过Cx3cr1CreER转基因鼠与Ai27转基因鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光刺激，即可特异性激活光纤下方区域小胶质细胞。
优选地，所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。所述蓝光的波长为450-490nm，所述腹腔注射Tamoxifen的浓度为15-25mg/mL，优选地，所述腹腔注射Tamoxifen的浓度为20mg/mL。
本发明的另一个目的在于提供一种上述小胶质细胞的激活方法获得的Cx3cr1CreER-Ai27基因型双转鼠。所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。
本发明的第三个目的在于上述小胶质细胞的激活方法在小胶质细胞的发育、行为或功能研究中的应用。
本发明的第四个目的在于上述双转鼠在小胶质细胞的发育、行为或功能研究中的应用。
优选地，所述应用可在正常、疾病或损伤条件下进行。
与现有技术相比，本发明具有以下有益效果：
(1)本发明提供的设计一种新型的激活小胶质细胞方法，该方法可在活体特异性标记激活小鼠小胶质细胞，且仅小胶质细胞表达绿色荧光蛋白，准确的区分小胶质细胞与其他神经细胞，实现了活体状态下对小胶质细胞的特异性标记、检测和追踪，可用于实时观测小胶质细胞发育、行为或功能，目前还没有类似的方法或转基因鼠报道。
(2)目前激活小胶质细胞方法的特异性不够强，操作复杂，成本高，本发明的激活小胶质细胞方法操作简便，成本和风险大大降低。
(3)目前小胶质细胞的研究还可以采用特异性的标记物进行离体染色研究，采用本发明的方法可以实现在体和离体对小胶质细胞的特异性标记和激活，在研究小胶质细胞的发育、行为和功能等方面具有重要意义，具有广阔的应用前景。
附图说明
图1本发明转基因杂交示意图。其中Cx3cr1-CreER-yfp代表Cx3cr1-CreER转基因小鼠，ChR2-tdTomato代表Ai27转基因小鼠。
图2是Cx3cr1-CreER转基因小鼠与Cx3cr1-CreER-Ai27转基因小鼠特异性标记和激活荧光对比图。
图3是激活的小胶质细胞与未激活小胶质细胞荧光对比图。不同行代表不同的放大倍数。A和B为激活的小胶质细胞，C和D为未激活小胶质细胞。
具体实施方式
以下通过具体实施例对本发明作进一步详细说明，以使本领域技术人员能够更好地理解本发明并予以实施，但实施例并不作为本发明的限定。
以下实施例中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。其中，
实施例1 Cx3cr1CreER-Ai27基因型双转鼠的构建
Cx3cr1CreER转基因小鼠与Ai27转基因小鼠均构自美国杰克森实验室。鼠龄为2-3月龄，体重20-30g，雌雄不限。实验小鼠水和食物供给充足，饲养于12h黑暗，12h光照的环境中，室温控制在20-25℃之间。挑选生长正常的Cx3cr1CreER小鼠与Ai27基因小鼠进行杂交，杂交后剪取剪取小鼠尾尖端4mm左右，用电烙铁为小鼠烫伤止血，将鼠尾置于1.5mlEppendorf管中，加入500μL鼠尾消化缓冲液和4μL蛋白酶K,60℃水浴锅消化12h。将消化好的组织摇匀后，置于离心机中10000rpm离心6min，弃沉淀。再加入500μL的酚氯仿抽提液，颠倒摇匀后，12000rpm离心10min，吸取上层清液200μL。再加入400μL无水乙醇，轻柔颠倒混匀，DNA呈白色絮状沉淀析出，12000rpm离心5min，弃上清，加入400μL的75％乙醇，12000rpm离心5min，弃上清，待乙醇干透后，加入50μL的ddH2O溶解DNA。置于4℃进行保存。根据美国杰克森实验室提供的转基因的小鼠的目的基因序列信息设计引物，使用百泰克生物技术公司的PCR反应试剂盒对转基因小鼠进行扩增后测序进行验证，测序工作委托华大基因公司进行。经过测序检测，Cx3cr1CreER-Ai27基因型双转鼠的序列与预期一致，杂交小鼠构建成功。
实施例2小胶质细胞激活验证
参见实施例1的构建方法，通过Cx3cr1CreER转基因小鼠与Ai27转基因小鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转小鼠，为了诱导Cre重组酶，将实施例1构建的Cx3cr1CreER-Ai27基因型双转小鼠用溶于200μl玉米油(Sigma)的4mg Tamoxifen(购自Sigma)进行刺激，在两个时间点分别隔48小时皮下注射或在腹腔注射Tamoxifen，Cx3cr1CreER转基因小鼠作为对照组，采用同样的处理方式。
实验处理结束后，对小鼠腹腔注射氨基甲酸乙酷溶液，进行麻醉后，将其固定于解剖盘上，剪开胸腔皮肤层和肌肉层后，暴露心脏，剪开右心耳，将灌流针插入左心室，注射0.15M PBS 15mL，然后再注射4％PFA溶液15mL，从颈部剪断后，用镊子从颅骨中剥离出大脑组织、小脑、以及部分脑干，置于20mL 4％PFA溶液中，于4℃固定2天。将脑组织从固定液中取出，以冠状切方向修平小脑，并将修整好的组织粘于振动切片机上，调整刀片位置，切片厚度30μm。采用常规的免疫组化染色后，使用蓝光(450-490nm)刺激，即可特异性激活小胶质细胞。由图2可知，Cx3cr1CreER-Ai27基因型双转小鼠与Cx3cr1CreER转基因小鼠相比，更能特异性地区分小胶质细胞与其他细胞，荧光强度更强，小胶质细胞清晰可见。
另外，Tamoxifen实验处理结束后还可以直接在体于目标脑区植入光纤，使用蓝光(450-490nm)刺激，即可特异性激活光纤下方区域小胶质细胞。由图3可知，蓝光照射后，Cx3cr1CreER-Ai27基因型双转小鼠激活的小胶质细胞清晰可见，能特异性地区分小胶质细胞与其他细胞，而未激活的小胶质细胞未能检测到清晰可见的荧光。实现了活体状态下对小胶质细胞的特异性标记、检测和追踪，可用于实时观测小胶质细胞发育、行为或功能。
由上可见，本发明已成功构建Cx3cr1CreER-Ai27基因型双转小鼠。还提供了一种简单高效的新的小胶质细胞激活方法，可以特异性激活小胶质细胞，准确地区分小胶质细胞与其他细胞。将本发明的转基因小鼠进行蓝光刺激，只有小胶质细胞表达绿色荧光蛋白，实现了活体状态下对小胶质细胞的特异性标记、检测和追踪，可用于实时观测小胶质细胞发育、行为或功能。另外，本发明的转基因小鼠还可以通过离体免疫组化染色的方法准确地区分小胶质细胞与其他细胞。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围。
Claims (9)
1.一种新的小胶质细胞的激活方法，其特征在于，通过Cx3cr1CreER转基因鼠与Ai27转基因鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光刺激，即可特异性激活光纤下方区域小胶质细胞。
2.根据权利要求1所述的小胶质细胞的激活方法，所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。
3.根据权利要求1或2所述的小胶质细胞的激活方法，所述蓝光的波长为450-490nm。
4.根据权利要求1-3任一项所述的小胶质细胞的激活方法，所述腹腔注射Tamoxifen的浓度为15-25mg/mL，优选地，所述腹腔注射Tamoxifen的浓度为20mg/mL。
5.权利要求1-4任一项所述小胶质细胞的激活方法获得的Cx3cr1CreER-Ai27基因型双转鼠。
6.根据权利要求5所述的Cx3cr1CreER-Ai27基因型双转鼠，所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。
7.权利要求1-4任一项所述小胶质细胞的激活方法在小胶质细胞的发育、行为或功能研究中的应用。
8.权利要求5或6所述的双转鼠在小胶质细胞的发育、行为或功能研究中的应用。
9.根据权利要求7或8所述的应用，所述应用可在正常、疾病或损伤条件下进行。
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