CN116515836B - 抑制MerTK表达的shRNA、腺相关病毒及其应用 - Google Patents
抑制MerTK表达的shRNA、腺相关病毒及其应用 Download PDFInfo
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Abstract
本申请公开了一种抑制星形胶质细胞吞噬受体MerTK表达的shRNA、腺相关病毒及其在治疗学习记忆障碍中的应用。实验结果表明：MerTK‑shRNA干扰质粒能显著特异性抑制大鼠海马神经胶质细胞MerTK的表达；应激诱导海马星形胶质细胞MerTK表达升高，改变星形胶质细胞吞噬功能，导致学习记忆功能降低；MerTK‑shRNA腺相关病毒能够改善神经元突触可塑性，从而起到抵抗应激诱导的神经元损伤的作用，在治疗各种应激压力所致的学习记忆功能下降方面具有潜在应用价值。
Description
技术领域
本发明属于生物技术领域，具体涉及一种抑制星形胶质细胞吞噬受体MerTK表达的shRNA、腺相关病毒及其应用。
背景技术
随着现代社会生活节奏的加快和社会竞争的加剧，多数人都处在不同程度的应激负荷之下，长期过度应激已被确认为人类多种重大致死性疾病的重要推动因素，人类约70％的疾病的发生发展与机体应激损伤密切相关。大脑是机体感应和应对应激的调控中枢，同时也是应激易损伤的靶器官，尤其是涉及高级脑活动的认知功能在面对应激时显得尤为脆弱。流行病学调查显示，长期处于应激状态的人群其轻度认知障碍和痴呆的患病率是同年龄非应激人群的两倍以上。海马是调控学习、记忆等认知功能的重要脑区，慢性应激可导致海马的结构和功能改变，包括引起海马体积缩小、树突棘数量减少、突触密度降低、突触可塑性异常、抑制齿状回神经发生等。鉴于目前应激诱导学习记忆能力损伤的调控机制仍不清楚，临床上尚缺乏针对应激性学习记忆障碍的有效防治药物。
星形胶质细胞是大脑中含量最丰富的一类细胞，约占脑细胞的40％，在调节大脑的各种功能中起着重要的作用，其中调节突触形成、成熟和修剪功能和免疫反应功能相关的吞噬作用，与学习记忆等认知功能密切相关。在正常和健康的情况下，吞噬功能通过清除不需要的突触和神经突调节重构神经回路，介导凋亡细胞和神经碎片的清除，维持脑内稳态。在病理条件下，星形胶质细胞介导蛋白聚集物，如Aβ、Htt和α-Syn的吞噬，以清除神经退行性疾病患者大脑中积累的蛋白，并清除神经损伤中凋亡的细胞。过度激活的星形胶质细胞吞噬功能产生有害作用，吞噬和清除完整的突触和神经元，促进神经退行性变、发起和推动认知功能下降。MerTK是属于受体酪氨酸激酶家族TAM(Tyro3、Axl和MerTK)的单跨膜受体，高度表达于单核-吞噬系统细胞、睾丸细胞和上皮细胞，近年来发现亦存在于星形胶质细胞。通过结合多种配体，包括LGALS3、TUB、TULP1或GAS6，将信号从细胞外基质转导至细胞质，调节许多生理过程，包括细胞存活、迁移、分化和凋亡细胞的吞噬。MerTK在星形胶质细胞清除过多的突触、神经元、凋亡细胞和神经碎片中发挥受体和信号转导作用，其表达水平代表着星形胶质细胞吞噬功能水平。但MerTK在应激性学习记忆障碍中的作用迄今未见报道，目前也并没有靶向MerTK治疗应激性学习记忆障碍的相关技术。
近年来，腺相关病毒(AAV)作为载体已成为基因治疗的关键组成部分，鉴于其具有安全性高、免疫原性低，组织特异性亲和能力强，且可以在体内持续稳定表达等特性，使AAV载体成为中枢神经系统疾病基因治疗途径的候选载体。
发明内容
针对上述情况：
一方面，本申请提供了抑制MerTK表达的shRNA，所述shRNA由包含SEQ ID NO.1靶点序列生成。
进一步地，所述shRNA序列为SEQ ID NO.5、6所示。
另一方面，本申请提供了一种质粒，其携带上述shRNA。
另一方面，本申请提供了一种病毒载体，其携带上述shRNA或者质粒。
进一步地，所述病毒载体为腺相关病毒。
另一方面，本申请提供了上述shRNA、质粒或者病毒载体在制备治疗学习记忆功能障碍中的应用。
进一步地，所述学习记忆功能障碍为应激诱导的学习记忆功能障碍。
进一步地，所述治疗学习记忆功能障碍包括保护海马神经元。
进一步地，所述保护海马神经为改善海马突触结构蛋白PSD95、GSP43、SYP。
另一方面，本申请提供了一种治疗学习记忆功能障碍的药物，其包含上述shRNA、质粒或者病毒载体。
本发明的MerTK-shRNA干扰质粒能显著特异性抑制大鼠海马神经胶质细胞MerTK的表达；应激诱导海马星形胶质细胞MerTK表达升高，改变星形胶质细胞吞噬功能，导致学习记忆功能降低。MerTK-shRNA腺相关病毒能够改善神经元突触可塑性，从而起到抵抗应激诱导的神经元损伤的作用，在治疗各种应激压力所致的学习记忆功能下降方面具有潜在应用价值。。
附图说明
图1为腺相关病毒载体结构；
图2为MerTK-shRNA腺相关病毒载体对MerTK表达的影响(**P<0.01)；
图3为MerTK-shRNA腺相关病毒抑制大鼠海马组织MerTK表达(*P<0.05，**P<0.01)；
图4为MerTK-shRNA腺相关病毒对大鼠前额叶皮质MerTK表达无影响(*P<0.05，**P<0.01)；
图5为MerTK-shRNA腺相关病毒改善应激大鼠海马突触结构蛋白PSD95损伤(**P<0.01)；
图6为MerTK-shRNA腺相关病毒改善应激大鼠海马突触结构蛋白GSP43损伤(*P<0.05，**P<0.01)；
图7为MerTK-shRNA腺相关病毒改善应激大鼠海马突触结构蛋白SYP损伤(*P<0.05，**P<0.01)；
图8为MerTK-shRNA腺相关病毒缓解应激大鼠空间记忆能力损伤(*P<0.05，**P<0.01)；
图9为MerTK-shRNA腺相关病毒缓解应激大鼠自发活动和自主探究能力损伤(*P<0.05，**P<0.01)。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1MerTK-shRNA腺相关病毒载体构建及鉴定
针对MerTK转录本序列，设计并合成特异性的RNAi序列，退火形成双链，克隆至腺相关病毒表达载体中，获得重组质粒。
MerTK干扰序列设计
从NCBI获得MerTK转录本序列(NM_022943)，设计针对MerTK的shRNA干扰位点，靶点序列如下：MerTK-shRNA1：GGAAGAAATCAAGCCAGATCA(SEQ ID NO.1)，MerTK-shRNA2：GCGTGAATGTCACCAGAAACA(SEQ ID NO.2)，MerTK-shRNA3：GGAGCACAGTAGGGTAGATTA(SEQ IDNO.3)，无效序列shRNA-scramble:CCTAAGGTTAAGTCGCCCTCG(SEQ ID NO.4)，化学合成MerTK-shRNA oligo及阴性对照shRNA oligo，并筛选出抑制效果最好的位点。
将合成的DNA干粉溶解于退火缓冲液中，90℃水浴15min，自然冷却至室温形成双链，配对结果如下：
ACCGGCGCTGAGTACTTCGAAATGTCCTCGAGGACATTTCGAAGTACTCAGCGTTTTT(SEQ IDNO.5)；
CGCGACTCATGAAGCTTTACAGGAGCTCCTGTAAAGCTTCATGAGTCGCAAAAAATCT(SEQ IDNO.6)。
载体酶切及回收
(1)所用腺相关病毒载体为pAAV-U6-shRNA-CMV bGlobin-eGFP-3Flag(GV390)(图1)。
(2)用限制性内切酶BsmB I对载体进行切割。反应溶液混匀后37℃水浴锅中酶切过夜。
(3)向载体的酶切产物中加入10μL 6×loading buffer，进行琼脂糖凝胶电泳，将目的条带在紫外灯下切下，进行胶回收。
连接
通过T4 DNA连接酶将线性化的载体和退火双链DNA进行连接反应，按如下体系配制反应溶液，混匀后于16℃连接过夜。
转化
(1)取一只E.coli DH5感受态细胞，置于冰上自然融化。将2μL连接产物加到感受态细胞中，冰上静置30min。42℃水浴锅中热休克90sec，立即插到冰上，放置2min。加入800μL SOC培养基，37℃摇床摇菌45min。
(2)将菌液均匀涂到含氨苄青霉素的琼脂平板上。先在37℃烘箱里正置30min，后倒置培养16hr。
(3)挑取多个单克隆分别放入摇菌管，加3mL含氨苄青霉素的LB培养基中，37℃摇床摇菌12-16hr。
阳性重组子的鉴定
(1)设计并合成鉴定PCR用引物，序列如下：P1 5’CCGTTGTCAGGCAACGTG 3’(SEQ IDNO.7)，P2 5’AGCTGACAGGTGGTGGCAAT 3’(SEQ ID NO.8)
(2)以菌液为模板，进行菌液PCR鉴定实验，反应体系如下：
在PCR仪中按如下程序进行PCR反应：95℃预变性3min；95℃变性15sec，60℃退火30sec，72℃延伸30sec，共进行40个循环；最后72℃反应10min。
(3)对PCR初步鉴定出的阳性菌群进行测序，测序结果正确，与合成的序列一致。
(4)将测序正确的菌液加入10mL含氨苄抗生素的LB培养基中，37℃摇床摇菌12～16hr。提取重组质粒。
实施例2MerTK-shRNA质粒干扰效果检测
将质粒转染HEK293T细胞，收集细胞提取RNA，通过real-time PCR实验检测重组质粒对MerTK表达的影响，选择干扰效果最为显著的质粒进行腺相关病毒包装。
细胞转染
胰酶消化HEK293T细胞(DMEM+10％FBS)，制成细胞悬液，调整细胞密度为1.0×105个/ml，接种细胞于6孔板中，2000ul/孔，每孔细胞数量为2×105个，放置37℃，5％ CO2中培养过夜。配制质粒与转染试剂的混合物，逐滴加入细胞，设置3个重复，转染完后放置37℃，5％CO2培养箱，培养72h。
实时荧光定量PCR(Real-time PCR)
(1)设计并合成检测MerTK表达水平所用的real-time PCR引物，序列如下：Forward Primer 5′CCCCGTCTGTCCTAACTGTCGCTGGT 3′(SEQ ID NO.9)，Reverse Primer 5′TGCGCTGTGCTGTTGAGGATATGGACT 3′(SEQ ID NO.10)。
(2)提取细胞总RNA，使用HiScript II One Step qPCR-PCR-SYBR Green Kit进行Real-time PCR，反应溶液混匀后进行扩增，设置3个复孔。
(3)GAPDH作为内参，对检测结果进行分析，如图2所示，相对其它干扰序列，shRNA1对MerTK的干扰下调效果最为显著。
实施例3MerTK干扰重组腺相关病毒的包装与滴度检测
rAAV-shMerTK病毒包装纯化
为了提高病毒在中枢神经胶质细胞中的转导效率和靶向性，建立标记星形胶质细胞特异性启动子GFAP载体和AAV2/5杂合型包装策略，样品纯度达到90％以上。
rAAV-shMerTK病毒滴度测定
qPCR法定量腺相关病毒滴度，样品稀释30000倍后测定，病毒直接滴度为4.56×1014vg/mL，校准后滴度为9.28×1013vg/mL，样品稀释24倍后分装滴度为3.87×1012vg/mL，待大鼠海马注射备用。
实施例3rAAV-shMerTK对应激性学习记忆功能损伤的保护作用
为观察MerTK-shRNA腺相关病毒对神经元的调控作用，我们建立了稳定干扰海马星形胶质细胞MerTK表达的大鼠应激损伤模型，检测抑制MerTK表达对神经元突触可塑性的影响，并通过动物行为学实验检测大鼠学习记忆能力的改变。
通过脑立体定位注射法对大鼠海马进行MerTK-shRNA腺相关病毒(AAV-shMerTK和对照AAV-GFP)注射，注射滴度为3.87×1012vg/mL。待动物恢复5-7天后，通过给予SD大鼠慢性束缚应激，建立应激认知功能损伤动物模型。通过real-time PCR和western blot实验检测大鼠海马和前额叶皮质区MerTK表达水平，结果显示应激诱导大鼠海马和前额叶皮质组织MerTK表达增加，而MerTK-shRNA腺相关病毒可显著抑制大鼠海马组织MerTK表达，但前额叶皮质未见影响(图3-4)。
为了进一步观察抑制MerTK表达的腺相关病毒在应激致学习记忆功能损伤中对神经元的调控作用，我们检测了大鼠海马神经元突触结构标志蛋白突触后致密物(PSD95)、神经生长相关蛋白(GAP43)和突触素(SYP)水平。结果显示应激大鼠海马组织神经元突触结构标志蛋白水平均显著下降，而MerTK-shRNA腺相关病毒干预可显著缓解该损伤，改善应激大鼠海马神经元突触可塑性(图5-7)。
通过水迷宫实验和旷场实验检测应激大鼠学习记忆功能，验证MerTK-shRNA腺相关病毒对慢性应激诱导的空间学习记忆损伤的调控作用。结果显示应激大鼠水迷宫实验平台停留时间和穿台次数减少，旷场实验中央区穿越次数和停留时间减少，而MerTK-shRNA腺相关病毒干预可显著缓解应激诱导的空间学习记忆损伤的发生(图8-9)。
Claims (5)
1. 抑制MerTK表达的shRNA在制备治疗应激诱导的学习记忆功能障碍的药物中的应用，其特征在于，所述shRNA序列为SEQ ID NO.5和SEQ ID NO.6所示。
2. 质粒在制备治疗应激诱导的学习记忆功能障碍的药物中的应用，其特征在于，所述质粒上携带有序列如SEQ ID NO.5和SEQ ID NO.6所示的shRNA。
3. 病毒载体在制备治疗应激诱导的学习记忆功能障碍的药物中的应用，其特征在于，所述病毒载体上携带有序列如SEQ ID NO.5和SEQ ID NO.6所示的shRNA。
4.根据权利要求3所述的应用，其中所述病毒载体为腺相关病毒。
5.根据权利要求1-4任一项所述的用途，其中所述治疗应激诱导的学习记忆功能障碍包括保护海马神经元。
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