CN113509482B - Dna四面体在制备预防和治疗舍格伦综合征的药物中的用途 - Google Patents
Dna四面体在制备预防和治疗舍格伦综合征的药物中的用途 Download PDFInfo
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Abstract
本发明公开了DNA四面体在制备预防和治疗舍格伦综合征的药物中的用途,属于核酸药物领域。实验证明,DNA四面体可特异性地上调调节性B细胞,抑制自身反应B细胞,减少生发中心形成,阻碍舍格伦综合征病发生发展。另一方面,DNA四面体可以重建唾液腺唾液分泌功能,以及维持下颌下腺正常状态,并维持下颌下腺细胞增殖,减少下颌下腺细胞凋亡。DNA四面体预防和治疗舍格伦综合征的效果十分优异,应用前景良好。
Description
技术领域
本发明属于核酸药物领域。
背景技术
舍格伦综合征(syndrome,SS)是一种系统性自身免疫性疾病,主要累及唾液腺、泪腺、下颌下腺等外分泌腺,局部组织出现淋巴细胞浸润,影响腺体的正常功能,并可同时出现系统性损害,临床特征包括口干、眼干、下颌下腺病变、调节性B细胞数量减少、自身反应B细胞异常增加等。
目前没有理想的治疗和预防舍格伦综合征的方法。针对唾液减少,通常使用高氟牙膏刷牙、洗必泰漱口、唾液替代品、咀嚼无糖口香糖去缓解口干症状;或者使用系统性刺激药物,比如毒蕈碱激动剂(毛果芸香碱),通过选择性直接作用于M胆碱受体,刺激唾液腺分泌唾液。但前述方法只能缓解轻微的症状,不能有效治疗。而效果更强的免疫调节药物,比如皮质类固醇、羟氯嗪、甲氨蝶呤、硫唑嘌呤、环孢素A、环磷酰胺,会引起免疫系统整体的抑制,导致使用者容易出现副作用,且降低对疾病的抵抗力。
DNA四面体,又称四面体框架核酸(tFNAs),是由DNA单链(通常为4条)通过碱基互补配对,从二维结构上看,原先的DNA单链变为螺旋双链,从三维结构上看,形成四面体结构。DNA四面体比单链DNA或普通线性双链DNA更稳定,因此可用于合成生物体内检测探针,或者充当某些核酸药物的载体。
目前尚无DNA四面体用于治疗舍格伦综合征的报道。
发明内容
本发明要解决的问题是:提供DNA四面体在制备预防和治疗舍格伦综合征的药物中的用途。
本发明的技术方案如下:
DNA四面体在制备预防和治疗舍格伦综合征的药物中的用途。
进一步地,所述药物是促进恢复唾液腺唾液分泌功能的药物。
进一步地,所述药物是保护下颌下腺的药物;
和/或,所述药物是抑制B细胞活化的药物;
其抑制B细胞活化是通过上调调节性B细胞,抑制自身反应B细胞,减少生发中心形成实现的。
进一步地,所述DNA四面体是由序列如SEQ ID NO.1~4所述DNA单链或者与其同源性大于95%的DNA单链,通过1∶1∶1∶1的摩尔比互补配对形成的DNA四面体。
本发明中“同源性”指序列比对的相似度。
进一步地,所述药物中每单位制剂中含DNA四面体7.9μmol。
一种预防和治疗舍格伦综合征的药物,所述药物以DNA四面体为活性成分,加上药学上可接受的辅料制备而成。
进一步地,所述药物是重建唾液腺唾液分泌功能的药物。
进一步地,所述药物是保护下颌下腺的药物;
和/或,所述药物可调节B细胞的药物;
所述调节B细胞为上调调节性B细胞,抑制自身反应B细胞。
进一步地,所述DNA四面体是由序列如SEQ ID NO.1~4所述DNA单链或者与其同源性大于95%的DNA单链,通过1∶1∶1∶1的摩尔比互补配对形成的DNA四面体。
进一步地,所述药物中每单位制剂中含DNA四面体7.9μmol。
实验表明,对舍格伦综合征小鼠施用DNA四面体后,能促进恢复唾液腺唾液分泌功能,保护下颌下腺,调节B细胞(上调调节性B细胞,抑制自身反应B细胞),对舍格伦综合征起到较好的治疗效果,尤其是注射250nM浓度,隔1天注射1次,连续注射4周后,舍格伦综合征小鼠得以恢复到接近正常状态。可见,可将DNA四面体用于制备治疗舍格伦综合征的药物。
对小鼠来说,药物最佳规格是每单位制剂含DNA四面体25nmol。按照70kg成人相对于小鼠的体表面积系数,可推知人用单位制剂中DNA四面体含量为:25nmol×387.9(70kg成人相对于小鼠的体表面积系数)=7.9μmol。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1:DNA四面体的合成原理示意图和鉴定。a,合成原理示意图;b,毛细电泳结果;c,PAGE胶结果;d,透射电镜结果。
图2:唾液流和H&E染色检测结果。a,实验流程图;b~c,H&E染色检测结果;d,唾液流检测结果。
图3:下颌下腺中结构蛋白的免疫荧光检测结果。a~e分别为对CK5、c-kit、α-SMA、AQP4、AQP5的检测。
图4:下颌下腺中浸润的B细胞的免疫荧光和流式细胞术检测结果。a,对CD19的免疫荧光;b,对BAFF的免疫荧光;c,CD19和BAFF的免疫荧光信号数值化统计图;d~e,流式细胞术检测结果。
具体实施方式
实施例1 DNA四面体(tFNAs)的合成与鉴定
1.合成方法
将四条单链DNA(S1、S2、S3、S4)溶解于TM Buffer(10mM Tris-HCl,50mM MgCl2,pH=8.0)中,使得四条单链DNA的终浓度为1000nM,充分混匀后迅速加热至95℃保持10分钟,之后迅速降温至4℃并维持20分钟以上,即可得到tFNAs。
四条单链的序列(5′→3′)如下:
S1:
ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA(SEQID NO.1)
S2:
ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG(SEQID NO.2)
S3:
ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC(SEQID NO.3)
S4:
ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG(SEQID NO.4)
2.鉴定
毛细电泳结果可见tFNAs大小约为164bp(图1b);由PAGE胶结果可见tFNAs大小约为200bp(图1c);透射电镜可见散在的点状物体,并能观察到部分点状物体呈现四面体形态(图1d)。
通过前述鉴定结果,可以认为tFNAs被成功合成。
以下将以实验例的形式对本发明进一步说明,实验例中所用的tFNAs由实施例1的方法制备得到。
实验例1 tFNAs对舍格伦综合征小鼠的治疗作用
1.模型小鼠
雌性NOD/ShiLtJ小鼠是舍格伦综合征常见模型(詹天乐等,基于NOD/Ltj模型鼠探讨维生素C对舍格伦综合症相关病理因子的作用调控,2019全国口腔生物医学学术年会;石欢等,Toll样受体9依赖的p38MAPK信号通路在原发性舍格伦综合征中的作用机制,口腔颌面外科杂志2015年4月第25卷第2期)。本实验例将11周龄的雌性NOD/ShiLtJ小鼠,适应性喂养一周。将12周龄的雌性NOD/ShiLtJ小鼠,作为舍格伦综合征小鼠。
2.分组处理
实验组注射250nM或500nM的tFNAs,体积为100μL,模型组注射等体积生理盐水,隔一天注射一次,注射四周。
3.检测
在整个实验给药和观察过程中,监控小鼠唾液流率。
注射药物后5周、10周、15周处死相应小鼠,收取下颌下腺进行检测。
(1)流式细胞术。收集下颌下腺进行流式细胞术检测,探究CD4+T和B细胞亚群组成。
(2)染色。收集下颌下腺,进行H&E染色,观察淋巴细胞浸润情况;通过免疫荧光染色观察与唾液分泌功能和下颌下腺正常结构有关的特殊蛋白。
4.结果
图2a为实验流程图。图2b-c表明tFNAs治疗之后,舍格伦综合征小鼠下颌下腺炎症细胞浸润团块和浸润面积明显减少。图2d表明,tFNAs治疗之后,舍格伦综合征小鼠的唾液分泌功能得到恢复,口干症缓解。其中,250nM tFNAs治疗后,舍格伦综合征小鼠的唾液分泌功能基本恢复到正常小鼠水平。这表明,tFNAs治疗恢复了舍格伦综合征小鼠的唾液分泌功能,减少了唾液腺中炎症细胞浸润。
下颌下腺中对于下颌下腺正常结构至关重要的蛋白,比如CK5,c-Kit和α-SMA在tFNAs治疗之后都恢复到了正常水平(图3a-c)。同样,AQP-4和AQP-5是唾液分泌的重要蛋白,在tFNAs治疗后也恢复到了正常水平(图3b,d)。
下颌下腺中浸润的B细胞也通过免疫荧光和流式细胞术进行了探究。tFNAs治疗后,B细胞和B细胞分泌的重要细胞因子BAFF都有所降低(图4a-c)。抑制疾病发展的CD5+CD1D+CD19+B220+B细胞(Breg)和IL-10+CD5+CD1D+CD19+B220+B细胞(IL-10+Breg)在tFNAs治疗后占比都有所增加。促进疾病发展的CD19+CD138+B细胞和CD19+CD95+GL-7+B细胞,同时,这些细胞也是生发中心(germinal centers)形成的重要细胞。在tFNAs治疗后,占比都有所下降。同样,250nM tFNAs治疗的后的下颌下腺浸润的B细胞亚群都恢复到了正常小鼠水平(图4d,e)。
舍格伦综合征是B细胞过度活化,产生大量自身抗体所引起的自身免疫性疾病。上述结果表明,tFNAs可特异性地上调调节性B细胞,抑制自身反应B细胞,减少生发中心形成,进而抑制B细胞过度活化,减少自身抗体的生成,从而治疗舍格伦综合征。
综上,tFNA可重建唾液腺唾液分泌功能,保护下颌下腺,调节B细胞(上调调节性B细胞,抑制自身反应B细胞),对舍格伦综合征起到较好的治疗效果,在特定用量下舍格伦综合征小鼠得以恢复到接近正常状态。因此,将tFNA用于制备治疗舍格伦综合征的药物具有良好的应用前景。
SEQUENCE LISTING
<110> 四川大学
<120> DNA四面体在制备预防和治疗舍格伦综合征的药物中的用途
<130> GYKH1118-2021P0112784CC
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Claims (2)
1.DNA四面体在制备治疗舍格伦综合征的药物中的用途;
所述DNA四面体是由序列如SEQ IDNO.1~4所述DNA单链,通过1∶1∶1∶1的摩尔比互补配对形成的DNA四面体;
所述DNA四面体的制备方法为:将四条单链DNA溶解于TM Buffer中,使得四条单链DNA的终浓度为1000nM,充分混匀后迅速加热至95℃保持10分钟,之后迅速降温至4℃并维持20分钟以上,即得;
所述TM Buffer为10mM Tris-HCl,50mM MgCl 2,pH=8.0。
2.如权利要求1所述的用途,其特征在于:所述药物中每单位制剂中含DNA四面体7.9μmol。
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