CN117448440A - Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用 - Google Patents
Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体为Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,提供了对创伤后应激障碍异常恐惧记忆消退的针对性的治疗靶点。
Description
技术领域
本发明涉及生物技术领域,具体涉及Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用。
背景技术
创伤后应激障碍(PTSD)是指个体遭受异常强烈的精神刺激后,延迟出现的一种精神心理疾病。恐惧记忆消退异常是PTSD患者的一个核心症状,严重影响患者的预后和日常生活。目前,临床上缺乏有效的干预手段。既往研究认为丘脑是感觉信息的中继站,然而近年来大量研究已经证实丘脑尤其是高阶丘脑与多种高级脑功能相关。本研究发现PTSD模型小鼠丘脑背内侧核(MD)在恐惧记忆消退过程中过度激活,且光遗传学抑制该脑区后可以有效促进PTSD小鼠的恐惧记忆消退。国内外大量研究主要集中在海马、杏仁核和前额叶皮质等脑区,更多的是环路层面的研究,对于具体分子层面的研究比较少。丘脑作为当前研究的热点,其在恐惧记忆消退过程中的功能逐渐被揭示。然而,具体是什么丘脑在发挥作用,又是通过何种途径参与恐惧记忆消退过程尚不清楚。
恐惧记忆消退异常是严重困扰PTSD患者的一个核心症状,临床上亟需找到针对性的治疗靶点。但是现在鲜有找到有效的干预靶点,如果不明确其中涉及的功能环路,以及异常变化的分子,就无法精确靶向蛋白水平去干预。
发明内容
为解决上述技术问题,本发明提供了Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,
Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用。
优选的,所述Kv3具体为Kv3.2。
优选的,所述Kv3.2被磷酸化后用作分子标志物用于制备诊断创伤后应激障碍异常恐惧记忆消退的产品。
优选的,用作分子标志物时,Kv3.2磷酸化水平降低。
优选的,所述Kv3.2作为治疗靶点用于制备治疗创伤后应激障碍异常恐惧记忆消退的产品。
优选的,所述治疗靶点还包括与Kv3.2互相作用的PPP6C。
优选的,负调控PPP6C时Kv3.2磷酸化水平上调。
优选的,纳米脂质体包被siRNA后负调控PPP6C。
优选的,被纳米脂质体包被的siRNA在MD脑区发挥作用的时间为48h。
优选的,治疗创伤后应激障碍异常恐惧记忆消退的产品负调控PPP6C。
与现有技术相比,本发明的有益效果在于:
1、本发明提出Kv3.2离子通道作为分子标志物和治疗靶点,同时提出与Kv3.2相互作用的PPP6C作为分子标志物和治疗靶点,提供了对创伤后应激障碍异常恐惧记忆消退异常的针对性的治疗靶点。
2、本发明提供的LNP-siNRA策略,可以更加精准的靶向PPP6C的表达,避免药物治疗带来的副反应及耐药性。
附图说明
图1为PTSD模型构建示意图和行为测试图,A:PTSD模型构建,B:第12天进行条件恐惧的训练,第14天进行恐惧记忆消退实验.
图2为条件恐惧行为检测及Arc染色统计图,A:恐惧记忆消退实验结果,B:恐惧记忆消退期间Arc染色的典型图,C:恐惧记忆消退期间Arc表达的统计图,****P<0.0001.
图3为MD脑区离体脑片膜片钳示意图和统计图,A:MD膜片钳示意图(上)和实物图(下),B:PTSD模型组和对照组放电典型图比较,C:PTSD模型组和对照组半宽比较的典型图,D:PTSD模型组和对照组放电数比较的统计图,E:PTSD模型组和对照组主被动膜特性比较的统计图,**P<0.01,****P<0.0001;
图4为MD脑区光纤记录统计图,A:PTSD模型组和对照组MD光纤记录的典型图,B:PTSD模型组和对照组归一化峰值的统计图,C:PTSD模型组和对照组恐惧消退期间MD光纤记录的热图,D:PTSD模型组和对照组恐惧消退期间MD光纤记录的曲线图,**P<0.01;
图5为MD脑区Kv3通道电流变化,A:对照组MD脑区Kv3电流重复结果,B:PTSD模型组MD脑区Kv3电流重复结果,C:PTSD模型组和对照组MD脑区Kv3电流峰值的统计图及实验方法示意图,D:PTSD模型组和对照组MD脑区Kv3电流均值的统计图,****P<0.0001;
图6为MD脑区Kv3家族的表达差异,A:MD脑区Kcnc1RNA表达图,B:MD脑区Kcnc2RNA表达图,C:MD脑区Kcnc3RNA表达图,D:MD脑区Kcnc4RNA表达图;
图7为MD脑区Kv3.1和Kv3.2蛋白表达水平,A:PTSD模型组和对照组MD脑区Kv3.1和Kv3.2蛋白表达典型图,B:PTSD模型组和对照组MD脑区Kv3.1蛋白水平统计图,C:PTSD模型组和对照组MD脑区Kv3.2蛋白水平统计图;
图8为MD脑区Kv3.1和Kv3.2蛋白磷酸化水平,A:PTSD模型组和对照组MD脑区Kv3.1蛋白磷酸化统计图,B:PTSD模型组和对照组MD脑区Kv3.2蛋白磷酸化统计图,***P<0.001;
图9为MD脑区质谱分析;
图10为CSNK2B和PPP6C与Kv3.2的互作水平,A:PTSD模型组和对照组MD脑区CSNK2B和PPP6C蛋白表达典型图,B:PTSD模型组和对照组MD脑区CSNK2B蛋白表达统计图,C:PTSD模型组和对照组MD脑区PPP6C蛋白表达统计图,**P<0.01;
图11为设计的siPPP6C序列及体外敲除效果图,A:设计的4条siPPP6C,B:4条序列体外敲除后mRNA水平验证统计图,****P<0.0001;
图12为LNP包被siPPP6C的示意图及给药方式,A:LNP包被siPPP6C的结构示意图,B:LNP包被所用材料的分子结构,C:LNP体内作用示意图,D:LNP给药方式示意图;
图13为siRNA条件性敲低PPP6C后PPP6C的表达水平及Kv3.2的磷酸化水平,A:分别在给予MD脑区siPPP6C24h,48h,72h后PPP6C蛋白表达水平典型图,B:分别在给予siPPP6C24h,48h,72h后PPP6C蛋白表达水平统计图,C:siPPP6C组和siCon组MD脑区Kv3.2磷酸化水平典型图,D:siPPP6C组和siCon组MD脑区Kv3.2磷酸化水平统计图,**P<0.01,***P<0.001;
图14为siRNA条件性敲低PPP6C后MD脑区Kv3电流的变化,A:LNP-empty组MD脑区Kv3电流,B:LNP-siRNA组MD脑区Kv3电流,C:LNP-empty组和LNP-siRNA组MD脑区Kv3电流峰值的统计图,D:LNP-empty组和LNP-siRNA组MD脑区Kv3电流均值的统计图,****P<0.0001;
图15为siRNA条件性敲低PPP6C后MD脑区的电生理变化,A:给予MD脑区LNP-siPPP6C并进行膜片钳的示意图,B:siRNA组和对照组MD脑区神经元放电数统计图,C:siRNA组和对照组MD脑区神经元半宽典型图,D:siRNA组和对照组MD脑区神经元主被动膜特性统计图,****P<0.0001;
图16为siRNA条件性敲低PPP6C后小鼠的行为学表型,A:PTSD模型组和对照组手术及行为学检测示意图,B和C:PTSD模型组和对照组恐惧记忆消退统计图,D:PTSD模型组和对照组恐惧消退回忆的统计图,****P<0.0001;
具体实施方式
下面对本发明的具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。本发明各实施例中所述实验方法,如无特殊说明,均为常规方法。
本发明实施例中,小鼠立体定位仪购自深圳瑞沃德公司,玻璃微量注射器购自瑞士Hamilton公司,使用Leica冰冻切片机、Olympus数字切片扫描仪VS200和激光共聚焦显微镜FV 3000。所用病毒购自武汉布林凯斯生物技术有限公司。光遗传及光纤记录设备购自千奥兴科南京生物技术有限公司。
本发明实施例中,使用的材料包括:PPP6C siRNA(siPPP6C-4),LNP-siPPP6C(LNP-siRNA)。
本发明实施例中,具体的实验步骤如下:
(1)SPS&S模型构建
首先将C57小鼠放入束缚装置中,而后将其放入鼠笼束缚4h;间隔30min后,将小鼠置于盛有水的透明桶中进行强迫游泳20min,透明桶的直径20cm,高30cm,桶中水位线20cm,水温25℃;间隔30min后,使用乙醚对小鼠进行深度麻醉,直到小鼠对于捏尾巴和脚趾反应消失;间隔30min后,在带有电击装置的箱体内施加非条件足底电刺激,箱体尺寸为25cm x25cm x 25cm,电刺激条件为0.8mA,5s,对照组小鼠放入箱体中但不施加任何刺激。
判断模型是否成功的方法参考文章Xi K,Huang X,Liu T,Liu Y,Mao H,Wang M,et al.Translational relevance of behavioral,neural,andelectroencephalographic profiles in a mouse model ofpost-traumatic stressdisorder.Neurobiol Stress.2021;15:100391.doi:10.1016/j.ynstr.2021.100391。
(2)脑立体定位注射
用异氟烷麻醉小鼠并固定于脑立体定位仪上,麻醉时诱导2.5%,维持1.5%,氧气流量1L/min,剃毛使颅骨暴露,碘伏常规消毒颅顶手术区,红霉素眼膏涂抹眼表面保护动物眼球。待消毒液干燥后用眼科剪沿颅顶正中线做一长1.5~2.0cm的纵行皮肤切口,钝性分离皮下组织,充分暴露小鼠的前、后囟,调平定位仪,用微型颅骨钻打孔洞,在体视显微镜下用显微镊小心撕除孔洞区硬脑膜并暴露软脑膜,缓慢调节微量注射器,用玻璃微管注射器注射病毒溶液,定位MD:前囟前:-1.70mm,中缝左:+0.30mm,颅骨下深度:-3.40mm。颅内注射后至少留针10min后再缓慢退针,使病毒充分被组织吸收,颅内注射完毕后再次用碘伏消毒术区,缝合切口表皮,并在切口周围涂抹抗生素软膏预防感染,腹腔注射2mg/kg的美洛昔康作为镇痛药物,放在电热毯上保暖至苏醒,待病毒表达3周后进行后续实验。
(3)灌注取脑
异氟烷麻醉动物后,动物成仰卧位固定,剪开胸骨下方腹壁,提起胸廓充分暴露心脏,剪开右心耳,使用0.1%磷酸盐缓冲液(PBS)滴注,待到右心耳流出的缓冲液澄清后换含4%多聚甲醛的磷酸缓冲液滴注约15min,含4%多聚甲醛的磷酸缓冲液作为固定液。结束后取脑,放入上述相同的固定液中后固定4h,然后再将脑分别放入含10%、20%、30%蔗糖的磷酸缓冲液中梯度脱水,直至脑组织沉底。
(4)冰冻切片
将脱水后的脑组织使用OCT包埋剂在切片机中快速冷冻,固定在冰冻切片机的平台上,修平脑组织至与冠状面水平,切片厚度50μm,然后按切片顺序放置在0.1%磷酸盐缓冲溶液中保存。
(5)免疫荧光染色
使用0.1%的PBS在摇床上清洗3遍脑切片,每次10min,而后进行封闭液封闭,2小时后进行一抗染色,4°过夜,18小时后使用0.1%的PBS在摇床上清洗3遍脑切片,每次10min,而后进行二抗染色,3小时后使用0.1%的PBS在摇床上清洗3遍脑切片,每次10min。然后进行DAPI染色,裱片及封片后避光保存。
(6)离体脑片膜片钳
小鼠用异氟烷麻醉后断头取脑,剪开头部皮肤,暴露颅骨,将颅骨沿左侧剪开,向右掀开颅骨,使脑组织充分暴露,用挖脑勺迅速将小鼠脑组织取出置于冰水混合的切片液中,切片液应预先通氧,通氧的成分为95%O2+5%CO2。将取下的脑组织稍作修整,用胶水将小鼠脑固定在振荡切片机的平台上,切片机的槽内应预先放入切片液的冰水混合物,同时通入混合气,进行冠状切片,切片厚度为300μm,切好的脑片放入充分氧合的切片液中,32℃条件下持续通氧孵育30min,之后将切片液更换为人工脑脊液,继续孵育1h。孵育结束后将脑片置于显微镜平台的浴槽内,辅以U形盖网固定,人工脑脊液持续通氧灌流。在显微镜下找到MD。玻璃电极内充灌电极内液,电机内液为K-Glu,手指轻弹排空气泡,入液前给予一定正压,以防灌流液中杂质堵塞玻璃电极尖端,利用显微操作系统将玻璃电极尖端贴住神经元,释放正压的同时给予负压吸引,待形成高阻封接后,用嘴给予负压破膜,利用Axonclamp-200B/700B放大器进行记录。
(7)光纤记录
注射病毒和光纤植入2-3周后,将小鼠连接到记录设备上,确认荧光信号的存在。在每次记录之前,植入的光纤通过外部光纤连接到记录装置,中国。为了激发GCaMP6s,一个473nm的发光二极管(LED)被一个用0.4数值孔径(NA)20倍物镜聚焦的二向镜反射,并耦合到一个光学转向器(Doris lens)。光纤在转向器和植入的光纤之间引导光,光纤的光密度230μm,0.37NA。将光纤尖端处的激光功率调整为0.01~0.02mW,以减少激光漂白。荧光进行带通滤波,并使用放大器将CMOS(互补金属氧化物半导体)电流输出转换为信号,通过低通滤波器进一步滤波,40Hz截止。模拟电压信号在50Hz的频率下被数字化,并由多通道光纤测光记录系统记录。其中,记录装置品牌为千奥兴科,20倍物镜的品牌为Olympus,二向镜的品牌为MD498,Thorlabs,带通滤波的品牌为MF525-39,Thorlabs,CMOS的品牌为DCC3240M,Thorlabs)。
(8)光遗传学
无线光遗传器件集成了各种功能层,屏障膜,以及在75μm厚度的聚酰亚胺衬底上制造的活性元件,以整体平面几何形状便于传统制造技术的加工,功能层为铜金属化,屏障膜为苝和聚二甲基硅氧烷,活性元件表面贴装芯片和μILEDs。电气接口由一对金属线组成,沿着一条蛇状互连走线,允许相对于连接的圆形线圈的垂直和水平运动自由,表面安装芯片,圆形线圈直径9.8mm,铜走线:8匝,60μm宽,18μm厚,80μm间距,通过磁耦合将电力传输和控制到一个独立的射频传输环路天线,工作频率为13.56MHz。一个23pf的电容提供阻抗匹配。二极管整流接收到的射频信号,产生μ-ILEDs的电流源。在光遗传神经元活动操纵实验中,用589nm激光激活eNpHR 3.0,激光为5mW,恒定黄光。
(9)蛋白免疫印记(WesternBlotting)
①-80℃冰箱取出的蛋白样品放置冰上,融化后高速离心机4℃,12000rpm高速离心10分钟取上清,加入5×蛋白上样缓冲液制样;同时按照TGX Stain-FreeFastCastAcrylamide Kit,10%使用说明书配置凝胶(cat.#161-1083);配置1×RunningBuffer电泳液,电泳液每L含25mM Tris、192mM Glycine和0.1%SDS,混匀;
②用清水反复冲洗跑胶需要使用的电泳槽和架子,使用配置好凝胶的玻板组装好设备,需要注意凝胶的玻板和架子之间的紧密结合,其是防止电泳过程中漏液的关键步骤;电泳槽中慢慢倒入混匀好的1×Running Buffer电泳液,最后富含气泡的电泳液体不要倒入中间玻板之间的槽内;
③把加热好的蛋白样品依次加入凝胶玻板里面,完成加样后样品立刻放回冰箱继续保存;加样过程中,枪头一定要从加样孔的上方中间垂直插入,从靠近加样孔的底部位置缓慢开始加入样品,并且要随着样品的不断地加入加样孔过程,不断地将移液枪往上提,从而始终保持移液枪枪头口在刚加入的样品液面上方,这样以免最后排出样品时会产生一些气泡将加入的蛋白样品带出刚加入的加样孔;
④记录好加样的顺序,上样蛋白Marker以标记蛋白上样的顺序和蛋白的分子量,插好电源,先以恒压80V电泳使蛋白跑至分离胶,大约30分钟,再用120V恒压跑胶50分钟左右,直到loading buffer中的溴芬蓝指示液到了底部凝胶边缘,停止跑胶;
⑤用清水反复洗净所有将要使用的设备,包括:转膜使用的架子、电泳槽、绿色的取胶片、白色滤纸和黑色海绵等;白色的滤纸和黑色的海绵分别浸泡在不同的清水中待用;
⑥配制电泳转膜缓冲液,1L需要加200mL甲醇和100mL10×Tris/GlycineBuffer,然后补充水至1L,补充的水要提前预冷;裁好的PVDF膜放置于甲醇中泡5分钟左右,再转至刚配制好的转膜缓冲液里面浸泡;
⑦将待转膜的架子放置于大量清水里面,打开摆放好,黑色在下面,再依次放置一张黑色的海绵、两张白色的滤纸和浸泡好的PVDF膜,放置时使用平头镊子取;
⑧用配制的撬胶器轻轻地取出凝胶,用绿色的取胶片裁去除掉多余部分,以便凝胶方便展平,先将凝胶放置于转膜缓冲液,再转移放置上步准备的PVDF膜上;再依次在PVDF膜上放一张白色的滤纸、一张黑色的海绵,注意整个实验过程中一定不能产生气泡。注意,滤纸尽量使用新的,如果滤纸用的次数太多会产生一些碎屑,这些会影响转膜的效果,所以一定要将使用的滤纸先用清水洗净浸泡好。如果产生一些明显碎屑,需要丢掉更换新的滤纸。
⑨使用清洗过的滚棒轻轻地滚动摆放好的转膜体系,赶出可能存在转膜体系中的气泡,再把加架子慢慢合起来扣好,从清水中拿出转膜体系,待流掉多余的清水后,转到转膜缓冲液中浸泡,等待下一张凝胶的安装。再一起将两张转膜架子放到放了转膜液的转膜电泳槽里,转膜架子的白色对转膜电泳槽红色,黑色对着黑色,倒入足量的转膜缓冲液充满整个转膜电泳槽,并加入存放在-20℃预冰过的冰盒,然后插好电源,在整个电泳槽埋放在冰水混合物中,根据分子量大小来选择转膜时间。
⑩转膜结束后,转移至5%的脱脂奶粉中封闭1小时,先在TBST缓冲液中轻轻漂洗两次,再去除TBST缓冲液,根据产品说明稀释倍数加入第一抗体稀释液,4℃冰箱里的摇床上轻轻地晃动,第二天回收第一抗体稀释液,再用TBST缓冲液轻轻漂洗三次,每次10分钟,然后加同样属性的第二抗体稀释液,常温孵育2小时,再用TBST缓冲液中轻轻漂洗三次,每次15分钟。显影仪曝光显影及其对图片进行定量分析。
(10)免疫共沉淀(co-IP)
①对收集的每种处理组细胞,用新鲜配置的裂解液在冰上裂解细胞,裂解液在用前补加25×Complete inhibitor、10×PhosphoStop inhibitor和100×PMSF,每管细胞加800μL IP裂解液,添加过程中用移液枪上下反复吸裂解液充分打撒细胞,放在4℃摇床上裂解4小时;4℃条件下高速离心机12000rpm离心15分钟,离心结束后转移上清液至新EP管中做预清除;
②先取共约150μL*4磁珠,先用不含抑制剂的裂解液洗一遍,再用150μL*4裂解液重悬磁珠,再将装蛋白的12个EP管中分别加入20μL磁珠预清除处理,使用200μL的黄色枪头前需要用剪刀剪掉一小节,以免破环磁珠,,4℃旋转一个小时;同时再取90μL*4磁珠加12μL*4flag M2抗体形成磁珠-抗体复合物4℃旋转一个小时;
③取预清除后装蛋白的EP管放置于磁力架上,轻轻地吸取上清液至新的EP管中,磁珠裂解液冲洗后,再加100μL PBS重悬4℃保存,PBS中含0.5%的NaN3;
④预清除后的蛋白共12管上清蛋白液,每管取出80μL做input,剩余的720μL上清液,再各取出55μL混合在一起共660μL做IgG对照;
⑤剩余665μL的12管上清蛋白液中分别加入32μL上述处理好的磁珠-抗体复合物,IgG对照组中加入上述所有的磁珠-IgG复合物,4℃旋转2小时;
⑥将上述12个EP管和IgG对照组的EP管放置于磁力架上,轻轻吸取上清液做elution,再用1mL裂解液清洗磁珠,15分钟*3,4℃旋转,每次洗过的上清液都丢弃;PBS清洗磁珠2分钟*5,每次使用1mLPBS,4℃旋转,保留磁珠,最后一次用1mL PBS重悬后,每管都取200μL分一管,800μL分一管,放置磁力架上吸干PBS后,分装200μL磁珠的EP管吸干液体后加50μL 1*loading,分装800μL磁珠的EP管吸干液体后放置-80℃冰箱待送样检测;
⑦input和elution分别加入26.7μL,192μL4*loading;
⑧进行与Western Blotting相同的实验步骤。
实施例1
行为学试验
首先使用SPS&S方法进行PTSD小鼠模型构建,如图1A所示,包括束缚,强迫游泳,深度麻醉和无条件足底电刺激,设置对照组和PTSD模型组,对照组为未进行处理的野生型小鼠,然后进行条件性恐惧行为实验的测试(图1B)。
第1天将小鼠放在环境A中进行训练,首先让小鼠适应2分钟,而后给30秒的连续声音刺激,并在最后2秒给予0.8mA的足底电刺激,而后休息1分钟,依次进行10个循环。48小时后即第3天,将小鼠置于环境B,适应2分钟后,给予30秒的连续声音刺激,而后休息1分钟,依此进行30个循环,检测小鼠对于第1天习得的恐惧记忆的消退情况。24小时后(第4天),将小鼠置于环境B,适应2分钟后给予30秒的连续声音刺激,而后休息1分钟,依此进行5个循环,检测小鼠恐惧记忆消退水平,其中声音刺激条件为75dB,4500Hz。
环境A:25cm*25cm*25cm的正方箱体,内部有发声装置和电击装置,环境B:直径20cm,高30cm的透明桶。
结果显示,PTSD模型小鼠在第3天恐惧消退行为中保持较高的冻结水平,表示小鼠持续的恐惧,而对照组小鼠随着实验数的增加,冻结水平逐渐降低,提示PTSD小鼠恐惧消退异常,如图2A。
实施例2
电压门控钾离子通道Kv3.2影响MD脑区神经元
采用免疫荧光染色的方法发现MD脑区即时反应基因(IEG)蛋白Arc在PTSD小鼠中显著增多,如图2B和图2C,提示MD脑区活动增强。同时,采用离体脑片膜片钳技术检测了MD脑区神经元的电生理特性,发现PTSD小鼠神经元放电显著高于对照组,如图3A、图3B和图3D,神经元的半宽显著低于对照组如图3C和图3E。进一步,利用光纤记录的方法记录了MD脑区神经元在恐惧记忆消退过程中的活动变化,发现PTSD小鼠MD脑区钙活动在恐惧记忆消退过程中显著高于对照组,如图4A和图4B,且随着实验次数的增加PTSD小鼠MD脑区钙信号峰值持续保持较高水平,而对照组小鼠逐渐降低,如图4C和图4D。
既往文献报道,电压门控钾离子通道Kv3参与快速放电神经元的复极化过程。因此推测MD脑区神经元半宽降低可能是Kv3通道变化所致。利用离体脑片膜片钳技术记录了MD脑区Kv3电流,发现PTSD小鼠Kv3电流强度显著高于对照组,如图5A-D。Kv3家族包括Kv3.1、Kv3.2、Kv3.3和Kv3.4。此外,发现MD脑区仅表达Kv3.1和Kv3.2,如图6A-D。因此,接下来用蛋白质印迹技术(WB)检测了MD脑区Kv3.1和Kv3.2的蛋白水平,发现均无显著差异,如图7A-C。
考虑到磷酸化修饰可能影响蛋白的活性。紧接着利用免疫共沉淀技术(Co-IP)检测了两种分子的磷酸化水平,发现Kv3.2磷酸化水平显著降低,如图8C和图8D,而Kv3.1磷酸化水平无显著变化,如图8A和图8B,提示Kv3.2可能发挥重要作用。
实施例3
PPP6C影响Kv3.2的磷酸化,导致恐惧记忆消退异常
对MD脑区进行了质谱分析,找到了两种与Kv3.2互相作用的分子,即CSNK2B和PPP6C,如图9,发现两种分子的表达水平均显著升高,如图10A-C。CSNK2B是一种磷酸化酶,而PPP6C负责对靶向蛋白去磷酸化。考虑到Kv3.2磷酸化水平降低,推测PPP6C可能发挥更重要的作用。随后设计了4条小干扰RNA(siRNA)以条件性敲除PPP6C,如图11A。体外实验结果提示siPPP6C-4具有更好的效果,因此后续实验采用了该序列,如图11B。
考虑到siRNA体内给药的不稳定性,采用一种新的递送技术-纳米脂质体包被siRNA(LNP-siRNA),如图12A和图12B,从而提高siRNA的转染效率,避免被机体代谢,如图12C和图12D。
首先在不同时间段给药,即24小时,48小时,72小时,以确定实验的最佳时间。
WB结果提示,立体定位给予MD脑区LNP-siRNA48小时后,PPP6C的敲低效果更好,如图13A和B,且Kv3.2磷酸化水平也显著上调,如图13C和图13D,因此后续行为学及离体电生理实验均采用48小时的时间窗口。
离体电生理结果提示,给予LNP-siRNA后MD脑区Kv3电流明显降低(图14A-D,且MD神经元半宽也显著增高,如图15A-D,进一步证实了PPP6C的重要作用。
最后进行了行为学检测,如图16A,发现给予LNP-siRNA条件性敲低MD脑区PPP6C后PTSD小鼠在第3天恐惧消退过程中冻结水平显著降低,如图16B和图16C,提示小鼠恐惧记忆逐渐消退。同样地,我们在第4天恐惧回忆过程中也观察到条件性敲低MD脑区PPP6C后PTSD小鼠冻结水平显著降低,如图16D。
需要说明的是,本发明权利要求书中涉及数值范围时,应理解为每个数值范围的两个端点以及两个端点之间任何一个数值均可选用,为了防止赘述,本发明描述了优选的实施例。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用。
2.根据权利要求1所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,所述Kv3具体为Kv3.2。
3.根据权利要求2所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,所述Kv3.2被磷酸化后用作分子标志物,用于制备诊断创伤后应激障碍异常恐惧记忆消退的产品。
4.根据权利要求3所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,用作分子标志物时,Kv3.2磷酸化水平降低。
5.根据权利要求2所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,所述Kv3.2作为治疗靶点,用于制备治疗创伤后应激障碍异常恐惧记忆消退的产品。
6.根据权利要求5所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,所述治疗靶点还包括与Kv3.2互相作用的PPP6C。
7.根据权利要求6所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,负调控PPP6C时Kv3.2磷酸化水平上调。
8.根据权利要求7所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,纳米脂质体包被siRNA后负调控PPP6C。
9.根据权利要求8所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,被纳米脂质体包被的siRNA在MD脑区发挥作用的时间为48h。
10.根据权利要求6所述的Kv3在制备诊断或治疗创伤后应激障碍异常恐惧记忆消退产品中的应用,其特征在于,治疗创伤后应激障碍异常恐惧记忆消退的产品负调控PPP6C。
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