CN110101863A - 抑制hipk1基因表达的应用 - Google Patents
抑制hipk1基因表达的应用 Download PDFInfo
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- CN110101863A CN110101863A CN201910268283.0A CN201910268283A CN110101863A CN 110101863 A CN110101863 A CN 110101863A CN 201910268283 A CN201910268283 A CN 201910268283A CN 110101863 A CN110101863 A CN 110101863A
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Abstract
本发明涉及一种抑制HIPK1基因表达在制备治疗病理性心肌肥厚和心力衰竭中的应用。本发明通过蛋白免疫印迹法发现在细胞水平的苯肾上腺素诱导的心肌细胞病理性肥大和动物水平的主动脉弓缩窄术诱导的心力衰竭模型中HIPK1表达明显升高，而抑制HIPK1的表达有抑制病理性心肌肥厚、治疗心力衰竭的作用。据此，可将HIPK1应用于病理性心肌肥厚和心力衰竭的诊断或开发抑制病理性心肌肥厚和心力衰竭的药物。
Description
技术领域
本发明涉及一种抑制HIPK1基因表达在制备治疗病理性心肌肥厚和心力衰竭药物中的应用。
背景技术
高血压、冠心病、心肌病等可诱发病理性心肌肥厚，主要表现为心脏增大伴心肌细胞丢失和纤维化增加，最终导致心室重塑、心力衰竭、甚至死亡。心力衰竭是众多心血管疾病发展的终末阶段，心肌缺血、血流动力学负荷过重、炎症等任何原因引起的心肌损伤，均可造成心肌结构和功能的变化，进而导致心室泵血和（或）充盈功能低下。目前临床上使用的β受体阻滞剂、血管紧张素转换酶抑制剂、血管紧张素受体拮抗剂及醛固酮受体拮抗剂等，可以有效改善心力衰竭患者的症状和生活质量，延缓心力衰竭的发展。但是，心力衰竭患者的患病率和死亡率仍在持续上升，且五年生存率低，心肌肥厚和心力衰竭的发生机制和治疗策略一直是心血管研究领域的重要课题。
近年来，心力衰竭的治疗已从利尿、强心、扩血管等短期血流动力学/药理学措施，转而以神经内分泌调控为主的长期、修复性的策略，目的是改变衰竭心脏的生物学性质。心力衰竭的外科治疗也出现了许多新技术，例如：冠状动脉搭桥、瓣膜修复、置换等手术，心脏功能衰减已经不是外科手术的禁区。然而，心力衰竭患者的五年生存率仍然较低，三分之一的患者在诊断一年内死亡。目前对于心力衰竭的治疗，临床上没有特效药物，大部分药物在缓解心力衰竭症状的同时具有不可避免的毒副作用。综上，从分子水平寻找到诊断病理性心肌肥厚和心力衰竭的指标与干预靶点具有重要的临床意义。
非编码RNA在基因表达中的调控作用越来越受到重视，具备多样化的功能，参与调控了几乎所有的基因表达。微小RNA是一大类长度约22个碱基的非编码RNA，主要通过作用于靶基因的3’端非翻译区（3’UTR），降低靶基因mRNA的稳定性或抑制其翻译，在转录后水平负调控靶基因的表达。微小RNA在心脏生理和心血管病中的作用已受到广泛关注。本团队前期研究发现微小RNA-222在运动诱导的生理性心肌肥厚中显著上调，进一步的功能学实验证实微小RNA-222具备抵抗病理性心肌肥厚和心力衰竭的作用，主要通过调控下游的HIPK1、HIPK2、P27和HMBOX1分子，相关研究工作已发表于Cell Metabolism杂志。此研究提示介导运动诱导的生理性心肌肥厚的分子将有望成为阻断病理性心肌肥厚和心力衰竭的潜在干预靶点。在该研究中发现HIPK1是微小RNA-222的一个靶基因，在心肌特异性过表达微小RNA-222的小鼠心脏中表达下调。目前尚无其他关于同源结构域相互作用蛋白激酶1（HIPK1）对病理性心肌肥厚和心力衰竭诊断与治疗的发明。
发明内容
本发明的目的之一在于提供一种抑制HIPK1基因表达在制备治疗病理性心肌肥厚药物中的应用
本发明的目的之二在于提供一种抑制HIPK1基因表达在制备治疗心力衰竭药物中的应用。
所述的心力衰竭为：急性心力衰竭、慢性心力衰竭。
所述的病理性心肌肥厚为：高血压、冠心病诱发的病理性心肌肥厚。
该发明中HIPK1是丝氨酸/苏氨酸蛋白激酶家族中高度保守的一员。HIPK1是一种重要的转录调控因子，可以在细胞核内通过磷酸化同源结构域转录因子的羟基团，抑制RNA聚合酶II相关的转录因子，从而抑制信使RNA（mRNA）和小RNA前体的转录。在氧化应激、DNA损伤、缺氧、感染、代谢异常等不同条件的刺激下，HIPK1参与调控细胞的生长、分化、增殖、凋亡等生物学行为。该发明中用于抑制HIPK1基因表达是一段保守的核苷酸序列，其特征在于该所述的sh-HIPK1核苷酸序列为：
Forward：ccggtcgtaccacatcttcttataactcgagttataagaagatgtggtacgatttttg；
Reverse：aattcaaaaatcgtaccacatcttcttataactcgagttataagaagatgtggtacga。
制备具有治疗病理性心肌肥厚和心力衰竭靶向药物，该药物的主要成分为HIPK1表达的抑制剂，其来源包括天然的或是人工合成的，或者使用可以过表达该药物的载体转染细胞获得；所述抑制剂能够抑制细胞和组织中HIPK1的表达、或能够破坏细胞和组织中HIPK1的稳定性、或能够降低细胞和组织中HIPK1的活性、或能够降低细胞或者组织中HIPK1的有效作用时间；抑制剂可以选自：蛋白、小分子化合物、寡核苷酸表达载体；药物载体包括但不限于稀释剂、缓冲剂、混悬剂、乳剂、颗粒剂、包囊剂、赋形剂、吸收促进剂、表面活性剂或吸附载体，最终药物疗效为能抑制细胞或组织内的HIPK1的表达量。
本团队前期研究发现HIPK1在细胞和动物的病理性心肌肥厚的过程中表达异常升高，进一步的功能学实验表明抑制HIPK1具备抵抗病理性心肌肥厚和心力衰竭的作用。在2016年，Z·尤等关于胰腺癌相关材料和方法，参见中国专利 CN 105637367 A发现HIPK1可以作为诊断胰腺肿瘤并预测治疗后对象中胰腺肿瘤复发的可能性的一种方法。2016年，K·W·武赫尔芬尼希等，参见中国专利《用于降低通过肿瘤细胞的免疫抑制的方法和组合物》，公布号为CN105431524A。发现具有肿瘤特异性的免疫应答细胞，其包含编码能够沉默抑制T细胞功能的基因的shHIPK1的载体，shHIPK1是免疫应答细胞和药学可接受的载体的组合物之一。此次基于HIPK1这个创新性的发现，拟开发用于病理性心肌肥厚和心力衰竭的HIPK1相关的诊断工具和靶向药物。
本发明通过蛋白免疫印迹法发现在细胞水平的苯肾上腺素诱导的心肌细胞病理性肥大和动物水平的主动脉弓缩窄术诱导的心力衰竭模型中HIPK1表达明显升高，而抑制HIPK1的表达有抑制病理性心肌肥厚、治疗心力衰竭的作用。据此，可将HIPK1应用于病理性心肌肥厚和心力衰竭的诊断或开发抑制病理性心肌肥厚和心力衰竭的药物。
附图说明
图1 为HIPK1在病理性心肌肥厚的模型中表达上调。（A）蛋白免疫印迹法检测HIPK1在小鼠主动脉弓缩窄（TAC）手术后2周心脏组织中的表达升高（n=3）。（B）蛋白免疫印迹法检测HIPK1在苯肾上腺素（PE，50 μM）诱导的心肌细胞病理性肥大模型中的表达升高（n=3）。*，P<0.05。
图2 为干扰HIPK1抑制苯肾上腺素诱导的心肌细胞肥大。（A）在新生大鼠心肌细胞添加HIPK1的干扰慢病毒，蛋白免疫印迹法验证HIPK1的表达下调（n=3）。（B）采用苯肾上腺素（PE，50 μM）处理新生大鼠心肌细胞48小时，同时添加HIPK1干扰慢病毒，运用α-actinin免疫荧光染色检测心肌细胞的大小（n=4），证实抑制HIPK1可以降低PE诱导的心肌细胞病理性肥大。标尺100 μm。（C）荧光定量PCR检测ANP、BNP和β-MHC的表达（n=6），证实抑制HIPK1可以降低PE诱导的ANP、BNP、β-MHC的升高。*，P<0.05；**，P<0.01。
图3 为过表达HIPK1对苯肾上腺素诱导的心肌细胞肥大没有影响。（A）采用苯肾上腺素（PE，50 μM）处理新生大鼠心肌细胞48小时，同时添加HIPK1过表达慢病毒，运用α-actinin免疫荧光染色检测心肌细胞的大小（n=4），证实过表达HIPK1不能进一步增加PE诱导的心肌细胞病理性肥大。标尺=100 μm。（B）荧光定量PCR检测ANP、BNP和β-MHC的表达（n=6），证实过表达HIPK1促进PE诱导的ANP、BNP、β-MHC的升高。**，P<0.01。
图4 为敲除HIPK1改善主动脉弓缩窄术后的小鼠心功能。（A）选取成年雄性HIPK1敲除小鼠（HIPK1 KO）及其同窝野生对照（WT），采用主动脉弓缩窄术（TAC）6周诱导病理性心肌肥厚的心脏大体观示意图。（B）TAC术后6周运用小动物心脏超声检测左室射血分数（EF，%）和左室短轴缩短率（FS，%）（n=7-10），HIPK1敲除显著改善TAC术后的心脏收缩功能。（C）荧光定量PCR检测CTGF、Col1a1、Col3a1的表达（n=6），HIPK1敲除可以降低TAC术后诱导的CTGF、Col1a1、Col3a1的异常升高。*，P<0.05；**，P<0.01。
具体实施方式
参见图1-图4，以下结合具体实施例，来进一步说明本发明的技术方案。应理解，以下实施例只用于说明本发明而不以任何形式限制本发明。
1．新生大鼠心肌细胞的分离和培养：选取出生3天内的SD大鼠，在超净台中取心脏，挤血洗净后，用镊子将组织转移到灭菌干净的安瓿瓶，剪成大小均一的组织块。用胰酶胶原酶消化液反复消化7-8次，直到组织块完全消化。用孔径为100 μm一次性滤网过滤细胞悬液到10 cm细胞培养皿，用“8”字摇法摇匀，放入细胞培养箱差速贴壁30–60 min。将未贴壁细胞吸取到50 ml离心管中，离心（1000 rpm，5 min），弃上清，用温育1×ADS重悬细胞，运用Percoll法获得高纯度的心肌细胞，即为原代大鼠心肌细胞。分离的新生大鼠心肌细胞采用心肌细胞培养基（DMEM高糖培养基+10%马血清+5%胎牛血清+1%青链霉素）培养。
2．心肌细胞的病理性肥大模型：将新生大鼠心肌细胞（20万/ml）铺入明胶包被的细胞培养皿，以无血清培养基饥饿过夜，次日在避光条件下，采用苯肾上腺素（PE，50 μM）处理心肌细胞48 hr，诱导心肌细胞发生病理性肥大。
3．细胞和组织RNA提取及ANP、BNP、β-MHC、CTGF、Col1a1、Col3a1的检测：利用RNeasy Mini Kit （Qiagen）提取细胞和组织中的总RNA。利用SYBR法检测ANP、BNP、β-MHC、CTGF、Col1a1、Col3a1的相对表达量。以18S作为内参引物，采用2-ΔΔCt法进行计算。
4．α-actinin免疫荧光染色：细胞处理48 hr后，用4%多聚甲醛固定细胞，室温孵育20 min。PBS清洗后，用0.5% Triton破膜，室温孵育30 min。PBS清洗后，用3%–5%牛血清白蛋白（BSA）室温封闭60 min。随后，用3%–5% BSA按照1:200比例稀释一抗α-actinin（Sigma），4°C孵育过夜。次日，PBS清洗后，用3%–5% BSA按照1:200比例稀释对应的二抗，室温避光孵育60 min。PBS清洗后，按照1:2000比例稀释DAPI染细胞核，室温孵育20 min。PBS清洗后，低温避光保存。在倒置荧光显微镜下观察和拍照，采用Image J软件分析α-actinin阳性心肌细胞的大小。
5. 主动脉弓缩窄术：用1%戊巴比妥钠腹腔注射麻醉小鼠。待小鼠充分麻醉后将其取仰卧位固定，备皮。显微镜下在颈部插入气管插管。于小鼠左胸口剪开一0.8 cm长小口，分离开口处肌肉，显微镜下用小剪刀将胸骨开至第二肋骨，分离胸腺，拨开脂肪组织找到主动脉弓，将一枚27 G规格的注射器针头并排平行放置与主动脉弓旁，显微镊将线打结结扎主动脉弓和针头，再次打结固定，之后迅速抽出针头。逐层缝合肋骨、肌肉和皮肤并对伤口进行碘酒消毒。
6. 蛋白免疫印迹法：运用RIPA裂解液添加1% PMSF裂解细胞和组织蛋白，运用BCAProtein Assay Kit测定总蛋白浓度。将蛋白上清液和5×Loading buffer（4:1）混匀后，用100°C水浴5 min，待温度冷却后放置–20℃储存。配制所需浓度的SDS凝胶，将等量总蛋白上样，经凝胶电泳后转移至PVDF膜。用5%脱脂奶粉封闭2 hr。用5%脱脂奶粉按1:1000比例配置一抗HIPK1，于4°C摇床孵育一抗过夜。次日，用TBST清洗（每次10 min，洗3次），用5%脱脂奶粉按1:10000比例配置相应二抗，室温孵育2 hr。用TBST清洗，最后使用ECL发光试剂盒在显色仪中曝光显影，蛋白条带使用Image J软件进行灰度值测量，用GAPDH作为内参。
Claims (4)
1.一种抑制HIPK1基因表达在制备治疗病理性心肌肥厚药物中的应用。
2.根据权利要求1所述的应用，特征在于所述的病理性心肌肥厚为：高血压、冠心病诱发的病理性心肌肥厚。
3.一种抑制HIPK1基因表达在制备治疗心力衰竭药物中的应用。
4.根据权利要求3所述的应用，特征在于所述的心力衰竭为：急性心力衰竭、慢性心力衰竭。
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