CN117442733B - Irf7的表达抑制剂在制备防治1,2-二氯乙烷引起的生殖毒性的药物中的应用 - Google Patents
Irf7的表达抑制剂在制备防治1,2-二氯乙烷引起的生殖毒性的药物中的应用 Download PDFInfo
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Abstract
本发明公开了IRF7的表达抑制剂在制备防治1,2‑二氯乙烷引起的生殖毒性的药物中的应用。本发明研究发现,1,2‑DCE暴露可引起小鼠精母细胞焦亡,引起小鼠睾丸上皮细胞萎缩脱落伴随焦亡的发生;进一步研究显示,1,2‑DCE是通过上调干扰素调节因子7(IRF7)的表达,激活RIG‑I‑like通路,介导精母细胞焦亡,可以IRF7的RIG‑I‑like通路作为靶点,治疗1,2‑DCE引起的生殖毒性。通过褪黑素处理可抑制IRF7,改善1,2‑DCE引起的精母细胞焦亡及病理损伤,减少精子畸形。本发明为治疗由1,2‑DCE职业中毒引起的生殖毒性提供了一种全新的治疗思路和策略。
Description
技术领域
本发明涉及生物医药技术领域,更具体地,涉及以干扰素调节因子7(IRF7)作为治疗靶点,具体为IRF7的表达抑制剂用于制备防治1,2-二氯乙烷引起的生殖毒性的药物中的应用。
背景技术
1,2-二氯乙烷(1,2-dichloroethane,1,2-DCE)是卤代烃类化合物,CAS:107-06-2,易挥发,作为化学合成原料、粘合剂、工业溶剂被广泛应用于五金、皮具、电子等多个行业。1,2-DCE属于高毒类物质,可经口、吸入进入人体引起急性、亚急性中毒或慢性中毒,主要损伤中枢神经系统和生殖系统,可累及肝、肾、循环系统等。目前有关1,2-DCE中毒的报告病例以急性、亚急性多见,但对其引起的男性睾丸的生殖影响的研究有限。且1,2-DCE破坏男性睾丸生殖毒性分子的潜在机制仍然很大程度上未知,更重要的是目前尚无任何治疗方法。
外源性化学物质引起的男性生殖损伤的很大一部分以睾丸组织细胞死亡为特征,包括凋亡、坏死和铁下垂。焦亡是一种程序性溶解性细胞死亡,其特征是快速的质膜破坏、DNA损伤和细胞内促炎物质的释放。最近的研究表明,外源性化学物质对男性睾丸组织的损伤在小鼠睾丸和GC-2 spd细胞中都表现出焦亡的特征。鉴于此,有必要开发一种治疗由1,2-DCE引起生殖毒性(精母细胞焦亡)的有效治疗手段。
褪黑激素是由松果体合成和分泌的一种激素,也被发现在外周生殖道中产生。有证据表明,褪黑激素可以穿过血睾丸屏障,存在于睾丸中。但褪黑激素是否可以治疗1,2-DCE职业中毒导致的男性生殖毒性损伤目前尚不明确。干扰素调节因子7(IRF7)是IRF家族成员之一,与IRF3是二聚体,位于模式识别受体(PRRs)介导的信号通路下游,对Ⅰ型干扰素(IFN-Ⅰ)的产生至关重要。IRF7激活可能抑制细菌感染以及部分呼吸道病毒诱导的炎症,但却促进过敏性哮喘的发生。IRF7可能抑制某些肿瘤生长和转移,尤其是骨转移,但可能由于影响肿瘤微环境而促进胶质母细胞瘤和非小细胞肺癌的生长。目前关于IRF7在1,2-DCE引起的男性生殖毒性损伤中的作用也尚不明确。
发明内容
本发明的目的在于克服现有技术中存在的上述缺陷和不足,提供IRF7的表达抑制剂在制备预防和/或治疗1,2-二氯乙烷引起的雄性生殖毒性的药物中的应用。
本发明的第二个目的在于提供一种预防和/或治疗1,2-二氯乙烷引起的雄性生殖毒性的药物。
本发明的上述目的是通过以下技术方案给予实现的:
本发明以小鼠精子发生途径细胞系GC-2 spd(ts)细胞和NIH Swiss系雄性小鼠作为实验对象,通过实验发现,1,2-DCE暴露可引起小鼠精母细胞焦亡,引起小鼠睾丸上皮细胞萎缩脱落伴随焦亡的发生;进一步研究发现,1,2-DCE是通过上调干扰素调节因子7(IRF7)的表达,激活RIG-I-like通路,介导精母细胞焦亡。IRF7过表达导致GC-2 spd细胞培养基上清液中乳酸脱氢酶(LDH)泄漏增加,伴随着焦亡蛋白的激活;而通过沉默IRF7可降低GC-2 spd细胞培养基上清液中乳酸脱氢酶的泄漏,并抑制焦亡蛋白表达,且沉默的IRF7可逆转由1,2-DCE处理引起的乳酸脱氢酶泄漏增加以及焦亡蛋白的激活,并减少焦亡细胞比例,表明可以IRF7的RIG-I-like通路作为靶点,采用IRF7表达抑制剂预防或治疗1,2-DCE引起的生殖毒性。进一步,通过褪黑素处理,可以逆转1,2-DCE上调IRF7介导的RIG-I-like通路激活、体内生殖细胞焦亡,可有效减轻1,2-DCE诱导的小鼠睾丸部分生殖细胞上皮的萎缩,减少1,2-DCE暴露引起的精子畸形,表明褪黑素可靶向治疗由1,2-DCE引起的雄性精母细胞焦亡,可使用褪黑素抑制细胞组织的IRF7表达,制备预防或治疗由1,2-DCE引起的生殖毒性的药物。
因此,本发明提供关于IRF7的表达抑制剂的以下新用途:
IRF7的表达抑制剂在制备预防和/或治疗1,2-二氯乙烷引起的雄性生殖毒性的药物中的应用;所述IRF7的表达抑制剂为靶向沉默IRF7或下调IRF7表达量的试剂;所述试剂为褪黑素或IRF7的siRNA;所述siRNA序列为Si-Irf7-1:正向:GCACUUUCUUCCGAGAACUTT,反向:AGUUCUCGGAAGAAAGUGCTT;Si-Irf7-2:正向:CUUGCGCCAAGACAAUUCATT,反向:UGAAUUGUCUUGGCGCAAGTT。
具体地,所述预防为在1,2-二氯乙烷暴露前,通过预先采用IRF7的表达抑制剂进行处理,从而降低1,2-二氯乙烷暴露引起的雄性生殖毒性风险。所述治疗为在1,2-二氯乙烷暴露引起的雄性生殖毒性之后再采用IRF7的表达抑制剂进行处理以缓解雄性生殖毒性。
进一步地,所述雄性生殖毒性包括精母细胞焦亡、睾丸上皮细胞萎缩脱落、精子畸形。
具体地,所述IRF7的表达抑制剂为通过改善精母细胞焦亡,减轻睾丸生殖细胞上皮的萎缩,减少精子畸形从而实现预防和/或治疗1,2-二氯乙烷引起的雄性生殖毒性。
本发明还提供一种预防和/或治疗1,2-二氯乙烷引起的雄性生殖毒性的药物,包括IRF7的表达抑制剂;所述IRF7的表达抑制剂为靶向沉默IRF7或下调IRF7表达量的试剂;所述试剂为IRF7的siRNA;所述siRNA序列为Si-Irf7-1:正向:GCACUUUCUUCCGAGAACUTT,反向:AGUUCUCGGAAGAAAGUGCTT;Si-Irf7-2:正向:CUUGCGCCAAGACAAUUCATT,反向:UGAAUUGUCUUGGCGCAAGTT。
与现有技术相比,本发明具有以下有益效果:
本发明提供了IRF7抑制剂在制备防治1,2-二氯乙烷引起的生殖毒性的药物中的应用。本发明研究发现,1,2-DCE暴露可引起小鼠精母细胞焦亡,引起小鼠睾丸上皮细胞萎缩脱落伴随焦亡的发生;进一步研究显示,1,2-DCE是通过上调IRF7的表达,激活RIG-I-like通路,介导精母细胞焦亡,可以IRF7的RIG-I-like通路作为靶点,采用IRF7表达抑制剂治疗1,2-DCE引起的生殖毒性。通过褪黑素处理可抑制IRF7,可改善1,2-DCE引起的精母细胞焦亡及病理损伤,减少精子畸形。本发明为治疗由1,2-DCE职业中毒引起的生殖毒性提供了一种全新的治疗思路和策略。
附图说明
图1为1,2-DCE处理GC-2 spd细胞后对精母细胞的影响。其中图1中A为不同浓度的1,2-DCE处理GC-2 spd细胞后对细胞活性的影响图;B为采用不同的细胞死亡抑制剂预处理GC-2 spd细胞后,再使用30 mM的1,2-DCE处理GC-2 spd细胞后对细胞活性的影响图;C为不同浓度的1,2-DCE处理GC-2 spd细胞后对细胞内焦亡相关蛋白表达的WB图;D为不同浓度的1,2-DCE处理GC-2 spd细胞后对细胞内焦亡相关蛋白水平的影响图;E为不同浓度的1,2-DCE处理GC-2 spd细胞后电镜观察结果;F为不同浓度的1,2-DCE处理GC-2 spd细胞后LDH释放实验结果;G为不同浓度的1,2-DCE处理GC-2 spd细胞后焦亡细胞的流式细胞术结果;H为G图不同浓度的1,2-DCE处理GC-2 spd细胞后焦亡细胞的流式细胞术结果焦亡细胞比例定量结果。
图2为1,2-DCE处理NIH Swiss小鼠后对其睾丸组织的影响。其中图2中A为不同浓度1,2-DCE处理NIH Swiss小鼠后其睾丸组织免疫组化结果;B为不同浓度1,2-DCE处理NIHSwiss小鼠后其睾丸组织细胞蛋白水平检测结果。
图3为1,2-DCE暴露的作用机理研究结果。其中图3中A为经1,2-DCE染毒处理24小时(0,30 mM)的GC-2 spd细胞GSEA研究结果;B为不同浓度1,2-DCE处理后GC-2 spd细胞中IRF7蛋白表达情况;C为不同浓度1,2-DCE处理NIH Swiss小鼠后其睾丸组织中IRF7蛋白表达情况。
图4为IRF7过表达和沉默IRF7对于GC-2 spd细胞的影响结果。其中图4中A为IRF7过表达对GC-2 spd细胞乳酸脱氢酶(LDH)的影响;B为IRF7过表达对GC-2 spd细胞相关蛋白相对表达量的影响;C为IRF7过表达对GC-2 spd细胞相关蛋白表达的WB图;D为沉默IRF7对GC-2 spd细胞乳酸脱氢酶(LDH)的影响;E为沉默IRF7对相关蛋白相对表达量的影响;F为沉默IRF7对GC-2 spd细胞相关蛋白表达的WB图;G为沉默IRF7后经1,2-DCE暴露处理对GC-2spd细胞乳酸脱氢酶(LDH)的影响;H为沉默IRF7及1,2-DCE暴露处理对相关蛋白相对表达量的影响;I为沉默IRF7及1,2-DCE暴露处理对GC-2 spd细胞相关蛋白表达的WB图;J为沉默IRF7及1,2-DCE暴露后GC-2 spd细胞中焦亡细胞的流式细胞术结果;K为沉默IRF7及1,2-DCE暴露后GC-2 spd细胞中焦亡细胞的流式细胞术结果焦亡细胞比例定量结果。
图5为体外、体内实验中褪黑素处理对于小鼠及小鼠精母细胞的影响。其中图5中A为体外实验中褪黑素处理对于小鼠精母细胞中IRF7及焦亡相关标记物的蛋白水平的影响结果;B为体外实验中褪黑素处理对于小鼠精母细胞中IRF7及焦亡相关标记物的蛋白的WB图;C为体外实验中褪黑素处理对于小鼠精母细胞乳酸脱氢酶(LDH)的影响;D为体内实验中褪黑素处理后小鼠睾丸组织免疫组化结果;E为体内实验中褪黑素处理后对于小鼠精子异常率的影响;F为体内实验中褪黑素处理后对IRF7及焦亡相关标记物的蛋白水平的影响结果;G为体内实验中褪黑素处理后对IRF7及焦亡相关标记物的蛋白的WB图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
本发明包括体外实验和体内实验两部分,所采用的细胞株为:GC-2 spd(ts)小鼠精母细胞系,实验动物为:NIH Swiss系SPF级雄性小鼠。
实施例1 1,2-DCE暴露可引起精母细胞焦亡
S1.取对数生长期,状态良好的GC-2 spd细胞制成细胞悬液,按8000每孔的数量接种于96孔板中,于5%、CO2细胞培养箱中37 ℃培养12 h以上,待细胞贴壁后,用1,2-DCE(0、5、10、20、30、40、50、60、70、80 mM)在DMEM培养基中处理24 h,加入CCK-8溶液10µL /孔,在37℃培养箱中继续孵育4小时,酶标仪测定450 nm处吸光度。确定体外研究的1,2-DCE的最佳浓度(0、7.5、15、30 mM)。
S2.取对数生长期,状态良好的GC-2 spd细胞制成细胞悬液,按4×105每孔的数量接种于6孔板中,于5% CO2细胞培养箱中37 ℃培养12 h以上,待细胞贴壁后,进行1,2-DCE(0、7.5、15、30 mM)处理,进行CCK8实验检测细胞毒性,步骤同S1。
S3.在1,2-DCE染毒前用不同的细胞死亡抑制剂的有效浓度(VX-765、双唑仑、Z-VAD-FMK、peptide V5、3-甲基腺嘌呤、氯喹、necrosµLfonamide、坏死抑素-1、铁死亡抑制剂-1、去铁胺)预处理细胞,CCK8实验检测细胞毒性,步骤同S1。
S4.LDH释放实验:1,2-DCE(0、7.5、15、30 mM)处理24 h后收集细胞培养上清,根据LDH试剂盒说明书(Nanjing Jiancheng Bioengineering Institute, Nanjing, China)取新的96孔板每孔加入20 μL上清液、25 μL基质缓冲液及5 μL辅酶Ⅰ,混匀后37℃温浴15min,再加入25 μL 2,4-二硝基苯肼,37℃温浴15min,加入0.4 mol/L NaOH溶液250μL,混匀后室温放置5min,使用Bio-Rad酶标仪在450 nm处测量吸光度。
S5.电镜观察:弃培养基,用PBS轻轻漂洗后,弃PBS加电镜固定液(2.5%戊二醛固定液),黑暗处室温放置2 h,用0.1% PBS清洗三次,乙醇梯度脱水,用CO2临界点干燥仪干燥后,将样品用双面导电胶带粘在样品台上,用日立303 MC1000离子溅射器(Hitachi,Tokyo, Japan)喷涂镀金,在日立RegµLus 8100扫描电镜在15kV下进行观察并拍摄图像。
S6.流式细胞术:细胞(5×105个/mL)在295 µL PBS和5 µL FLICA 660-YVAD-FMK溶液中重悬,在黑暗中培养60 min,反应结束后,用1 L洗涤缓冲液洗涤细胞2次,200 g离心5 min,弃上清收集细胞;每管加入300 µL洗涤缓冲液和5µL PI重悬细胞,轻轻混合,4℃下避光反应5 min,AccuriC6流式细胞仪(BD Biosciences, San Jose, CA, USA)分析,FL3通道测定PI,FL4通道测定FLICA 660,FLICA 660-YVAD-FMK和364 PI双阳性定义为焦亡细胞。
S7.细胞蛋白水平检测:弃除旧培养基,加入预冷的PBS缓冲液冲洗三遍后,弃除。按照100:1的比例配制适量的裂解液(RIPA裂解液+磷酸酶/蛋白酶抑制剂),充分混匀后每皿加入100 µL裂解液。使用干净细胞刮匙将裂解液均匀铺在细胞表面,冰上裂解30 min,期间经常摇晃培养皿。用细胞刮匙将细胞刮于培养皿一侧后,用移液器将细胞裂解液转移至无酶1.5 mL EP管内。13000 r/min,4 ℃离心30 min,所得上清液为总蛋白溶液,将其转移至新的1.5 mL EP管中,并记录体积。
①按照1:4配制适量考马斯亮蓝应用液(考马斯亮蓝染料+去离子水)。BSA蛋白标准品浓度为0.5 µg/µL,所需体积依次为100 µL、80 µL、60 µL、40 µL、20 µL、0,加入去离子水补充至终体积为100 µL,混匀后取10µL分别加入96孔板中,每个浓度设置三个重复孔。取待测蛋白样品2 µL,加入去离子水58 µL,充分混匀。取10 µL加入96孔板中,每个蛋白样品设置三个重复孔。加入200 µL考马斯亮蓝应用液,贴膜后置于37 ℃培养箱孵育15 min。将96孔板放入酶标仪,测得各浓度蛋白标准品及待测蛋白样品在595 nm波长下的OD值。以蛋白标品的浓度为横坐标,测得的OD值为纵坐标,绘制标准曲线。根据标准曲线和待测蛋白样品的OD值计算样品总蛋白浓度。
②蛋白变性:根据所测得的蛋白样品浓度,分别加入适量的RIPA裂解液以调整蛋白样品浓度为2.5 µg/µL,并按4体积的蛋白加入1体积的5× Loading buffer。涡旋混匀后瞬离,在金属浴中100 ℃变性10 min,-80 ℃冰箱保存。
③电泳:清洁烘干玻璃板后,将长板置于外侧,短板置于内侧,确保底面平齐,用楔子固定并封闭玻璃板底部,检查两面夹紧后开始灌胶。配制含有8%丙烯酰胺的分离胶,充分混匀后,灌入玻璃板至指定位置,使用异丙醇液封以促进分离胶聚合。室温静置40 min,使分离胶充分凝固。弃除异丙醇并用去离子水清洗后,将混合均匀的4%丙烯酰胺的浓缩胶灌入玻璃板,去除边缘气泡后小心插入梳子,用夹子固定,室温静置40 min。待浓缩胶充分凝固。组装好电泳设备,倒入电泳缓冲液,垂直向上拔出梳子,每孔蛋白样品上样10 µL,蛋白Marker上样5 µL。连接电源后,设置电压为60 V,30 min后更改电压为95 V,继续电泳1.5h,待蓝紫色条带电泳出底端可终止电泳。
④转膜:将配制的电转缓冲液置于4 ℃冰箱预冷。将待用的海绵、滤纸浸泡于电转缓冲液中备用。裁剪一张适当大小的PVDF膜浸泡于甲醇1 min,使其活化。电泳完毕后,切好胶置于电转液中浸泡5 min。按从正极到负极依次是海绵、3张滤纸、PVDF膜、凝胶、3张滤纸、海绵,制备“三明治胶”,应保持各层湿润且无气泡。将对应的正负极插入转膜槽中,加入足量的电转缓冲液,连接电源,设置电压为100 V,转膜时间约为1.5 h。
⑤免疫反应:将转膜完毕的PVDF膜做好标记后,浸泡在5%脱脂奶粉中,在摇床室温封闭2 h。一抗孵育:用TBST洗涤PVDF膜三次,每次10 min。将一抗用TBST溶液稀释至1:500,内参β-ACTIN稀释至1:1000。将PVDF膜置于抗体液中孵育1 h后,转入4 ℃冰箱过夜。二抗孵育:用TBST在摇床上洗涤PVDF膜三次,每次10 min。用TBST将二抗稀释至1: 5000。将PVDF膜置于抗体液中室温孵育2 h。用TBST洗涤三次,每次10 min。
⑥化学发光显影:在避光条件下,吸取ECL发光液A液和B液各1 mL并混合均匀,将PVDF膜浸泡在发光混合液中,反应1~2 min,然后将PVDF膜移至荧光图像分析系统的平板上,再放入荧光图像分析系统显影区。待出现清晰的蛋白条带后可终止显影,保存条带图像。
⑦凝胶成像分析:用ImageJ软件对目的条带和内参条带进行定量分析,蛋白表达水平为目的条带定量结果/内参条带定量结果。
结果如图1所示,1,2-DCE可引起生殖细胞的焦亡;1,2-DCE引起GC-2 spd细胞细胞活性下降(图1中的A),而焦亡抑制剂可挽救染毒引起的细胞活性下降(图1中的B),1,2-DCE染毒以剂量依赖性方式上调细胞内焦亡相关蛋白水平(图1中的C、图1中的D),伴随膜孔形成(图1中的E、图1中的F)。流式细胞术显示焦亡细胞比例呈剂量依赖增加(图1中的G、图1中的H)。
实施例2 1,2-DCE暴露可引起小鼠睾丸上皮细胞萎缩脱落,伴随焦亡的发生
S1.40只NIH Swiss小鼠随机分为4组,每组10只,使用HOPE-MED 8050F全身动态吸入系统暴露于1,2-DCE(0、100、350、700 mg/m3),6 h/d,连续28天,最后一次暴露后24 h内注射3%戊巴比妥麻醉并处死小鼠,收集睾丸和附睾,一侧液氮冷冻并在-80°C下保存,一侧保存于多聚甲醛固定液中。
S2.将固定后的睾丸组织从多聚甲醛固定液中取出,在用手术刀将组织修平整后,和对应的标签至于脱水盒内。将脱水盒放进脱水机内依次梯度酒精进行脱水。将浸好蜡的组织于包埋机内进行包埋。先将融化的蜡放入包埋框,待蜡凝固之前将组织从脱水盒内取出按照包埋面的要求放入包埋框并贴上对应的标签,-20℃冷却。蜡凝固后将蜡块从包埋框中取出并修整蜡块。采用石蜡切片机切片,厚4 μm,用于苏木素-伊红(H&E)染色。切片漂浮于摊片机40℃温水上将组织展平,载玻片将组织捞起,60℃烘箱内烤片。水烤干、蜡烤化后取出常温保存备用。石蜡切片进行脱蜡至水并采用H&E染色,采用中性树胶进行封片。
S3.组织蛋白水平检测:睾丸组织解冻后剪取适量组织并称重,转移至无酶1.5 mLEP管内研磨匀浆,每管加入100 µL裂解液,冰上裂解30 min,13000 r/min,4 ℃离心30min,取上清液转移至新的1.5 mL EP管中,并记录体积。其余步骤同实施例1步骤S7。
结果如图2所示,1,2-DCE可引起小鼠睾丸精管生殖细胞出现了广泛的空泡变性,生精细胞脱落到睾丸腔内(图2中的A),睾丸组织细胞发生焦亡(图2中的B)。
实施例3 1,2-DCE上调IRF7的表达,激活RIG-I-like通路
S1.提取经1,2-DCE染毒处理24小时(0,30 mM)的GC-2 spd细胞总RNA,并通过RNA清洁XP 工具和RNase-Free DNase Set进一步纯化总RNA。使用NovaSeq6000测序器进行测序,模式为PE150,使用M值方法的剪切均值将不同样本的表达水平规范化,并将样本的标准化表达式级别转换为FPKM(fragments per kilobase of transcript per millionmapped fragments)。将mRNA水平标准化,使用R语言软件的“clusterProfiler” 包(v.3.14.0),通过京都基因和基因组(Kyoto Encyclopedia of Genes and Genomes, KEGG)的数据库进行了GSEA研究,其意义路径设置在FDR≤0.1和P≤0.05。
S2.细胞和组织IRF7水平验证:步骤同实施例1步骤S7和实施例2步骤S3。
结果如图3所示,1,2-DCE暴露激活RIG-I-like的通路,与细胞焦亡的发生密切相关(图3中的A);通路的激活主要通过上调IRF7引起的(图3中的B、图3中的C)。
实施例4 1,2-DCE通过上调IRF7的表达,介导精母细胞焦亡
S1.细胞培养:选取呈对数生长期的GC-2 spd细胞株进行实验。
S2.质粒转染:稀释过表达Irf7质粒的DNA(购自擎科生物):加入适量无血清培养基稀释1.6 μg质粒DNA至终体积为100 μL。稀释LipoFit 3.0转染试剂:取3.2 μL转染试剂加入100 μL无血清培养基混匀,静置5 min。将稀释的LipoFit 3.0转染试剂和稀释的质粒DNA以1:1的比例混匀,室温孵育20 min。将上述混合液加入12孔板中,缓慢摇晃使其混匀。在细胞培养箱内培养24 h。检测LDH和蛋白水平,处理方式同实施例1步骤S4,S7。
S3.RNA转染:起始浓度为20 µM siRNA(Si-Irf7-1:正向:GCACUUUCUUCCGAGAACUTT,反向:AGUUCUCGGAAGAAAGUGCTT;Si-Irf7-2:正向:CUUGCGCCAAGACAAUUCATT,反向:UGAAUUGUCUUGGCGCAAGTT。)以及scramble siRNA,溶解后吸取对应体积RNA原液和无血清培养基在1.5 mLEP管中预混,在每管中加入对1体积转染试剂之后的体系即为转染工作液,如表1,室温孵育10分钟,将上述转染工作液按表1的体积加入以经更换1 mL新DMEM完全培养基的12孔板中,缓慢摇晃使其混匀。在细胞培养箱内培养24小时,以备下一步实验。检测LDH和蛋白水平,处理方式同实施例1步骤S4,S7。
表1 转染体系
RNA | 20 µM RNA原液的体积(µL/mL) | 转染试剂 | 转染试剂体积(µL/mL) | 转染工作液总体积(µL/孔) | 体系终浓度(nmol/mL) |
Si-Irf7-1 | 10 | Tesfect | 1.5 | 25 | 200 |
Si-Irf7-2 | 10 | Tesfect | 1.5 | 25 | 200 |
Scramble | 10 | Tesfect | 1.5 | 25 | 200 |
S4.挽救实验:细胞经S3步骤处理后,更换为30 μM浓度的1,2-DCE处理24 h,检测指标检测LDH、蛋白水平和焦亡细胞,处理方式同实施例1 S4,S6,S7。
结果如图4所示,Irf7过表达导致GC-2 spd细胞培养基上清液中乳酸脱氢酶泄漏增加(图4中的A),并伴随着焦亡蛋白的激活(图4中的B、图4中的C)。而沉默Irf7可降低GC-2spd细胞培养基上清液中乳酸脱氢酶的泄漏(图4中的D),并抑制焦亡蛋白(图4中的E、图4中的F)。且沉默的Irf7可逆转由1,2-DCE处理引起的乳酸脱氢酶(LDH)泄漏增加(图4中的G)以及焦亡蛋白的激活(图4中的H、图4中的I),并减少焦亡细胞比例(图4中的J,图4中的K)。
实施例5 褪黑素可通过抑制IRF7,缓解由1,2-DCE引起的精母细胞焦亡
S1.将40只雄性小鼠随机分为4组,每组包括10只小鼠:阴性对照、褪黑素、1,2-DCE和1,2-DCE+褪黑素。阴性对照组和褪黑素组每天吸入清洁空气,1,2-DCE和1,2-DCE+褪黑素组动态吸入1,2-DCE(700 mg/m3)持续28天,每天6小时;1,2-DCE+褪黑素和褪黑素组,小鼠在每次动态吸入前1小时单次接受经口剂量20 mg/kg的褪黑素治疗,所有治疗均在每天早上8点到8:30之间进行,阴性对照组和1,2-DCE暴露组口服纯净水,而后再进行700 mg/m3的1,2-DCE动式吸入暴露。
S2.染毒和治疗结束后麻醉处死小鼠,收集睾丸和附睾,进行精子参数分析、病理镜检和分子检测。
S3.6孔板内细胞在1,2-DCE染毒前使用褪黑素(125 μM)预处理2 h,染毒24 h后弃培养基,收集细胞,进行LDH释放检测和Western blot,方法同实施例1 S4,S7。
结果如图5所示,在体外试验中,褪黑素可逆转1,2-DCE诱导的细胞中IRF7上调(图5中的A),下调焦亡相关标记物的蛋白水平(图5中的B),伴随着LDH释放减少(图5中的C)。而在体内试验中,褪黑素能有效地减轻1,2-DCE诱导的小鼠睾丸切片中生发上皮的萎缩(图5中的D)以及减少精子异常率(图5中的E)。褪黑素的保护作用主要通过抑制IRF7介导的焦亡途径发挥(图5中的F、图5中的G)。
本实施例在染毒前给予褪黑素治疗可通过抑制IRF7介导的RIG-I-like通路,逆转1,2-DCE引起的生殖细胞焦亡,减少LDH释放。在动物水平上,证实了褪黑素通过抑制IRF7逆转1,2-DCE引起的生殖细胞焦亡,可以有效的缓解1,2-DCE引起的睾丸上皮细胞萎缩和精子畸形。
本发明上述结果表明,1,2-DCE是通过上调IRF7的表达,激活RIG-I-like通路,介导精母细胞焦亡,可以IRF7的RIG-I-like通路作为靶点,采用IRF7表达抑制剂预防或治疗1,2-DCE引起的生殖毒性。通过褪黑素治疗,可以逆转体内外生殖细胞焦亡导致的生殖损伤。这为治疗由1,2-DCE引起的生殖毒性提供了一种全新的思路和策略。
Claims (1)
1. IRF7的表达抑制剂在制备预防和/或治疗1,2-二氯乙烷引起的雄性生殖毒性的药物中的应用;所述IRF7的表达抑制剂为靶向沉默IRF7或下调IRF7表达量的试剂;所述试剂为IRF7的siRNA;所述siRNA序列为Si-Irf7-1:正向:GCACUUUCUUCCGAGAACUTT,反向:AGUUCUCGGAAGAAAGUGCTT;Si-Irf7-2:正向:CUUGCGCCAAGACAAUUCATT,反向:UGAAUUGUCUUGGCGCAAGTT;所述雄性生殖毒性为精母细胞焦亡。
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