CN114480553B - Sod3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选或疫苗评价中的应用 - Google Patents
Sod3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选或疫苗评价中的应用 Download PDFInfo
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Abstract
本发明涉及一种SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选或疫苗评价中的应用;本发明的意义在于:双氢青蒿素可调控脾脏免疫细胞的异质性。SOD3‑/‑小鼠用等量的双氢青蒿素进行灌胃处理后未发现脾脏增大,脾指数也未升高。流式细胞分析发现SOD3‑/‑小鼠脾脏免疫细胞亚群中CD11b+Gr‑1+中性粒细胞和B220+B细胞、CD3+CD4+T细胞的比例也未发生显著变化。因此,SOD3蛋白缺失在逆转双氢青蒿素诱导的脾增大及免疫细胞异质性方面有一定的作用,验证SOD3是双氢青蒿素作用的靶点之一。
Description
技术领域
本发明涉及医药技术领域,主要涉及到SOD3蛋白缺失在缓解双氢青蒿素诱导的脾增大及免疫细胞异质性方面的应用。
背景技术
双氢青蒿素(DHA),作为青蒿素的一种衍生物谢物,比青蒿素本身表现出更强的抗疟疾作用,同时也被公认为是一种免疫调节剂。研究表明,高浓度的双氢青蒿素可以抑制体液免疫反应,此外,双氢青蒿素还可以抑制氧化应激以及活性氧(ROS)积累以改善炎症。而作为主要的抗氧化酶,超氧化物歧化酶(SOD) 在清除ROS中起着至关重要的作用。大多数哺乳动物细胞含有三种形式的 SOD,即SOD1、SOD2和SOD3,它们在细胞中有不同的定位:SOD1主要存在于细胞质和线粒体内膜间隙;SOD2主要分布在线粒体基质和内膜;SOD3在信号肽的引导下定位与细胞外间隙。在细胞外环境中,SOD3催化超氧阴离子的歧化,阻止O2-的形成,所以,SOD3在双氢青蒿素调控的免疫反应中是否发挥重要作用,是本领域技术人员一直渴望解决的技术难题。
发明内容
本发明针对上述现有技术中存在的问题,目的在于提供SOD3蛋白缺失在缓解双氢青蒿素诱导的脾增大及免疫细胞异质性方面的应用。
本发明的技术方案如下:
SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选或疫苗评价中的应用。
所述的双氢青蒿素在调控脾脏增大及免疫细胞异质性的检测方法,包括如下步骤:
步骤1,将0.1g羧甲基纤维素钠(CMC-Na)加入到20mL加热至100℃的蒸馏水中,在磁力搅拌器中搅拌3-5h,当温度恢复至室温时,加入0.2g双氢青蒿素(DHA),4℃避光连续搅拌10-12h,得到双氢青蒿素溶液;
步骤2,给小鼠连续灌胃100mg/kg/d双氢青蒿素溶液26天后,取小鼠脾脏;
步骤3,将小鼠脾脏置于70μm的细胞筛上,细胞筛下面放置一个50mL的离心管,用注射器尾部带棱面研磨,同时加入预冷PBS冲洗,收集细胞悬液于 15mL的离心管中;
步骤4,上述步骤3中的细胞悬液1500rpm/min离心5min,吸去上清,留沉淀,用红细胞裂解液裂解红细胞,混匀后冰上静止3min;
步骤5,上述步骤4中裂解红细胞后的细胞悬液1500rpm/min离心5min,吸去上清,留沉淀,加入预冷5mL PBS重悬,洗涤3遍,从而制备双氢青蒿素诱导的免疫细胞亚群悬浊液;
步骤6,使用荧光显微镜及血球计数板计算小鼠脾脏免疫细胞数量;
步骤7,使用流式细胞术及单细胞转录组测序方法测定双氢青蒿素诱导的免疫细胞亚群的异质性。
所述的小鼠选体重18-20g的健康雌性BALB/c小鼠,所有小鼠均饲养于无特定微生物的小动物房中,保持黑暗12小时和光照12小时。
所述的步骤4中,红细胞裂解液为155mM NH 4Cl,10mM KHCO3,0.11mM EDTA.Na2,pH 7.2。
所述的步骤7中,使用流式细胞术测定双氢青蒿素溶液诱导的免疫细胞亚群的方法,包括下述步骤:
(1)将所获得的脾脏单细胞悬浮在含有0.04%BSA的PBS中;
(2)制备不同组合的流式抗体组合,流式抗体组合如下:
①第一组抗体,7AAD、抗CD45-pacific blue抗体、抗CD3-PE抗体、抗 CD4-APC/FireTM750抗体、抗CD8a-FITC抗体、抗CD19-BV510抗体、抗 CD45R/B220-APC抗体;
②第二组抗体,7AAD、抗CD45-pacific blue抗体、抗CD11b-APC抗体、抗 Ly-6G/Ly-6C(Gr-1)-FITC抗体;
(3)将待测样本分别加入到流式管A和B中,使呈单个细胞悬液状态,并保证细胞量1×10 6/管-1×10 7/管;所述待测样本为脾脏;
(4)向步骤(3)处理得到的流式管A中加入本发明所述的试剂组合物中的第一组抗体,向步骤(3)处理得到的流式管B中加入本发明所述的试剂组合物中的第二组抗体,各流式管4度条件下避光孵育30分钟;
(5)孵育结束后,向步骤(4)每管加入700μL PBS,混匀,1500rpm/min 离心5min,洗涤2次,吸去上清,留沉淀;
(6)向步骤(5)去上清后的流式管中,分别加入PBS缓冲液洗涤,离心后去上清,用PBS缓冲液重悬细胞,即得到流式细胞上机样品。
所述的步骤7中,使用单细胞方法测定双氢青蒿素诱导的免疫细胞亚群的方法,包括细胞捕获和cDNA合成,具体步骤如下:
(1)将所获得的脾脏单细胞悬浮在含有0.04%BSA的PBS中;
(2)在芯片的每个通道加入约6,000个细胞,预计回收的目标细胞约为3,000 个细胞;
(3)捕获的细胞被裂解,释放的RNA在单个GEM中通过逆转录进行条形码标记;
(4)逆转录在S1000TM Touch Thermal Cycler(Bio Rad)上在53℃下进行45分钟,然后在85℃,5分钟,在结束后保持在4℃;
生成的cDNA在安捷伦4200分光光度计中确定质量。
本发明的优点效果如下:
使用荧光显微镜及血球计数板计算小鼠脾脏免疫细胞数量,发现双氢青蒿素灌胃可显著提高小鼠脾脏免疫细胞数;使用流式细胞术及单细胞转录组测序技术解析双氢青蒿素诱导的脾脏免疫细胞亚群悬浊液成分,为研究青蒿素对机体的免疫调节作用奠定了基础。
本发明的意义在于:双氢青蒿素可诱导小鼠脾脏增大,脾脏免疫细胞亚群中CD11b+Gr-1+中性粒细胞和B220+B细胞的比例显着增加,CD3+CD4+和 CD3+CD8+T细胞的比例显着降低,绝对计数显示双高清青蒿素处理的小鼠脾脏中CD11b+Gr-1+中性粒细胞、B220+B细胞、CD3+CD4+和CD3+CD8+T细胞的绝对计数均显著升高。因此,双氢青蒿素可调控脾脏免疫细胞的异质性。 SOD3-/-小鼠用等量的双氢青蒿素进行灌胃处理后未发现脾脏增大,脾指数也未升高。流式细胞分析发现SOD3-/-小鼠脾脏免疫细胞亚群中CD11b+Gr-1+中性粒细胞和B220+B细胞、CD3+CD4+T细胞的比例也未发生显著变化。因此,SOD3 蛋白缺失在逆转双氢青蒿素诱导的脾增大及免疫细胞异质性方面有一定的作用,验证SOD3是双氢青蒿素作用的靶点之一。
附图说明
图1A为脾脏形态学观察及脾脏指数图(脾脏指数=脾脏重量(mg)×10/ 体重(g)×100%)。
图1B为脾脏单细胞悬液镜检图及脾脏细胞血球计数板计数结果。
图2A-2D为脾脏CD11b+Gr-1+中性粒细胞、B220+B细胞、CD3+CD4+和 CD3+CD8+T细胞的比例示意图。
图2E-2H为脾脏CD11b+Gr-1+中性粒细胞、B220+B细胞、CD3+CD4+和 CD3+CD8+T细胞的数量示意图。
图3A为脾脏单细胞转录组测序流程图。
图3B为各个免疫细胞亚群比例变化示意图。
图3CDHA为脾脏免疫细胞各亚群的单细胞图。
图4A为野生型及SOD3-/-小鼠的双氢青蒿素灌胃实验流程图。
图4B为双氢青蒿素灌胃后野生型及SOD3-/-小鼠脾脏形态学观察图。
图4C为双氢青蒿素灌胃后野生型及SOD3-/-小鼠脾脏指数结果图。
图4D为双氢青蒿素灌胃后野生型及SOD3-/-小鼠脾脏HE染色图。
图4E为双氢青蒿素灌胃后野生型及SOD3-/-小鼠脾脏CD11b+Gr-1+中性粒细胞、B220+B细胞、CD3+CD4+和CD3+CD8+T细胞的比例示意图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。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。
实施例1、使用双氢青蒿素溶液制备药物处理的小鼠
制备100毫克/千克/天的双氢青蒿素溶液,步骤如下:
将0.1g羧甲基纤维素钠加入到20mL加热至100℃的蒸馏水中,在磁力搅拌器中搅拌3-5h,当温度恢复至室温时,加入0.2g双氢青蒿素(DHA),4℃避光连续搅拌10-12h,得到双氢青蒿素溶液。
使用双氢青蒿素溶液灌胃健康BALB/c小鼠,步骤如下:
(1)从辽宁长生生物有限公司购买体重18至20g的健康雌性BALB/c小鼠,所有小鼠均饲养于无特定微生物的小动物房中,保持黑暗12小时和光照12小时;
(2)待小鼠适应了小动物房饲养环境(约1周时间)后,使用0.2G灌胃针吸取双氢青蒿素-羧甲基纤维素钠溶液0.2mL灌胃至小鼠;
(3)分别连续灌胃小鼠26天;
(4)在灌胃结束后,称量小鼠体重并进行记录;
(5)使用麻醉剂将小鼠麻醉后,处死小鼠。取出脾脏后照相并称重,之后使用0.7微米的细胞筛将脾脏组织研磨成单细胞。
实施例2、双氢青蒿素溶液诱导的脾脏免疫细胞亚群异质性的鉴定
方式1使用流式细胞术测定双氢青蒿素溶液诱导的免疫细胞亚群
(1)将上述所获得的脾脏单细胞悬浮在含有0.04%BSA的PBS中;
(2)制备不同组合的流式抗体组合,流式抗体组合如下:
①第一组抗体,7AAD、抗CD45-pacific blue抗体、抗CD3-PE抗体、抗CD4-APC/FireTM750抗体、抗CD8a-FITC抗体、抗CD19-BV510抗体、抗 CD45R/B220-APC抗体;
②第二组抗体,7AAD、抗CD45-pacific blue抗体、抗CD11b-APC抗体、抗 Ly-6G/Ly-6C(Gr-1)-FITC抗体;
(3)将待测样本分别加入到流式管A和B中,使呈单个细胞悬液状态,并保证细胞量1×10 6/管-1×10 7/管;所述待测样本为脾脏;
(4)向步骤(3)处理得到的流式管A中加入本发明所述的试剂组合物中的第一组抗体,向步骤(3)处理得到的流式管B中加入本发明所述的试剂组合物中的第二组抗体,各流式管4度条件下避光孵育30分钟。抗体所用用量为商家推荐,按照制造商说明书进行;
(5)孵育结束后,向步骤(4)每管加入700μL PBS,混匀,1500rpm/min 离心5min,洗涤2次,吸去上清,留沉淀。
(6)向步骤(5)去上清后的流式管中,分别加入PBS缓冲液洗涤,离心后去上清,用PBS缓冲液重悬细胞,即得到流式细胞上机样品。
方式2使用单细胞方法测定双氢青蒿素诱导的免疫细胞亚群
细胞捕获和cDNA合成,步骤如下:
(1)将上述所获得的脾脏单细胞悬浮在含有0.04%BSA的PBS中;
(2)在芯片的每个通道加入约6,000个细胞,预计回收的目标细胞约为3,000 个细胞;
(3)捕获的细胞被裂解,释放的RNA在单个GEM中通过逆转录进行条形码标记;
(4)逆转录在S1000TM Touch Thermal Cycler(Bio Rad)上在53℃下进行45分钟,然后在85℃,5分钟,在结束后保持在4℃;
生成的cDNA在安捷伦4200分光光度计中确定质量。
单细胞文库的准备,步骤如下:
(1)单细胞文库使用Single Cell 3’Library andGel Bead Kit V3进行构建;
(2)构建好的文库使用Illumina Novaseq6000测序仪进行测序。
数据处理,步骤如下:
(1)上述获得原始测序文件使用Cell Ranger软件进行下游数据分析,CellRanger被用来进行数据对齐、过滤、条码计数,UMI计数。使用Cell Ranger计数模块生成特征条码矩阵并确定聚类。使用PCA和前十原则进行降维分别通过 K-means算法和基于图的算法使用组件生成集群;
(2)另一种聚类方法是Seurat 3.0。基因数小于200的细胞,或基因数排在前1%,或线粒体基因比例超过25%被视为异常并过滤掉。PCA降维,TSNE 实现可视化,UMAP。
实施例3、SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性中的作用
(1)将实验分4组:①野生型C57小鼠灌胃羧甲基纤维素钠溶剂组;②野生型C57小鼠灌胃双氢青蒿素溶液组;③SOD3-/-小鼠灌胃羧甲基纤维素钠溶剂组;④SOD3-/-小鼠灌胃双氢青蒿素溶液组。每组10只,各组小鼠灌胃实施例1 所获得100mg/千克/天的双氢青蒿素溶液或等体积0.5%羧甲基纤维素钠溶剂,处理26天。
(2)在灌胃结束后,称量小鼠体重并进行记录;
(3)使用麻醉剂将小鼠麻醉后,处死小鼠。取出脾脏后照相并称重,之后使用0.7微米的细胞筛将脾脏组织研磨成单细胞。
(4)使用实施例2的方式1中的方法检测双氢青蒿素诱导的个组小鼠的免疫细胞亚群的变化。
结果
在这项发明中,我们发现双氢青蒿素处理组脾脏大小及脾脏指数显著高于溶剂对照处理组(图1A,P<0.001)。利用荧光显微镜观察结果显示:双氢青蒿素处理组脾脏细胞数(包括有核细胞和无核细胞)显著高于溶剂对照处理组(图 1B,P<0.01)。流式细胞术分析显示CD11b+Gr-1+中性粒细胞和B220+B细胞在双氢青蒿素处理组的比例显著增加(图2A和2B,双氢青蒿素组vs.羧甲基纤维素钠溶剂对照组:CD11b+Gr-1+中性粒细胞,3.73±1.55vs.1.94±0.47,P<0.05; B220+B细胞,26.23±4.10vs.19.95±5.47,P<0.05),而与羧甲基纤维素钠溶剂对照组小鼠相比,双氢青蒿素灌胃组小鼠中CD3+CD4+和CD3+CD8+T细胞的比例显着降低(图2C和2D,双氢青蒿素组vs.羧甲基纤维素钠溶剂对照组: CD3+CD4+T细胞,35.18±1.64vs.42.00±4.24,P<0.01;CD3+CD8+T细胞, 15.90±1.54vs.18.88±1.21,P<0.01)。绝对计数结果显示,双氢青蒿素组处理小鼠脾脏中CD11b+Gr-1+中性粒细胞、B220+B细胞、CD3+CD4+和 CD3+CD8+T细胞数量均显著高于羧甲基纤维素钠溶剂组处理小鼠脾脏中的细胞数(图2E-H)。单细胞测序结果与流式细胞术得到的结果一致(图2B和2C)。然而,SOD3-/-小鼠用等量的双氢青蒿素进行灌胃处理后未发现脾脏增大,脾指数也未升高(图4B-D)。流式细胞分析发现SOD3-/-小鼠脾脏免疫细胞亚群中 CD11b+Gr-1+中性粒细胞和B220+B细胞、CD3+CD4+T细胞的比例也未发生显著变化(图4E)。这些结果表明双氢青蒿素可以调控脾脏增大及免疫细胞的异质性,SOD3蛋白缺失在缓解双氢青蒿素诱导的脾增大及免疫细胞异质性方面发挥了一定的作用。
实验材料
Claims (6)
1.SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选中的应用。
2.根据权利要求1所述的SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选中的应用,其特征在于所述的双氢青蒿素在调控脾脏增大及免疫细胞异质性的检测方法,包括如下步骤:
步骤1,将0.1 g羧甲基纤维素钠加入到20 mL加热至100℃的蒸馏水中,在磁力搅拌器中搅拌3-5 h,当温度恢复至室温时,加入0.2 g 双氢青蒿素,4℃避光连续搅拌10-12h,得到双氢青蒿素溶液;
步骤2,给小鼠连续灌胃100mg/kg/d双氢青蒿素溶液26天后,取小鼠脾脏;
步骤3,将小鼠脾脏置于70 μm的细胞筛上,细胞筛下面放置一个50 mL的离心管,用注射器尾部带棱面研磨,同时加入预冷PBS冲洗,收集细胞悬液于15 mL的离心管中;
步骤4,上述步骤3中的细胞悬液1500 rpm/min离心5 min,吸去上清,留沉淀,用红细胞裂解液裂解红细胞,混匀后冰上静止3 min;
步骤5,上述步骤4中裂解红细胞后的细胞悬液1500 rpm/min离心5 min,吸去上清,留沉淀,加入预冷5 mL PBS重悬,洗涤3遍,从而制备双氢青蒿素诱导的免疫细胞亚群悬浊液;
步骤6,使用荧光显微镜及血球计数板计算小鼠脾脏免疫细胞数量;
步骤7,使用流式细胞术及单细胞转录组测序方法测定双氢青蒿素诱导的免疫细胞亚群的异质性。
3.根据权利要求2所述的SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选中的应用,其特征在于所述的小鼠选体重18-20g的健康雌性BALB/c小鼠,所有小鼠均饲养于无特定微生物的小动物房中,保持黑暗12小时和光照12小时。
4.根据权利要求2所述的SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选中的应用,其特征在于所述的步骤4中,红细胞裂解液为155 mM NH 4Cl,10 mMKHCO3,0.11 mM EDTA.Na2,pH 7.2。
5.根据权利要求2所述的SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选中的应用,其特征在于所述的步骤7中,使用流式细胞术测定双氢青蒿素溶液诱导的免疫细胞亚群的方法,包括下述步骤:
(1)将所获得的脾脏单细胞悬浮在含有 0.04% BSA 的 PBS 中;
(2)制备不同组合的流式抗体组合,流式抗体组合如下:
①第一组抗体,7AAD、抗CD45-pacific blue抗体、抗CD3-PE抗体、抗CD4-APC/Fire™750抗体、抗CD8a -FITC抗体、抗CD19-BV510抗体、抗 CD45R/B220-APC抗体;
②第二组抗体,7AAD、抗CD45-pacific blue抗体、抗CD11b-APC抗体、抗 Ly-6G/Ly-6C-FITC抗体;
(3)将待测样本分别加入到流式管A和B中,使呈单个细胞悬液状态,并保证细胞量1×10 6/管-1×10 7/管;所述待测样本为脾脏;
(4)向步骤(3)处理得到的流式管A中加入步骤(2)中所述的第一组抗体,向步骤(3)处理得到的流式管B中加入步骤(2)中所述的第二组抗体,各流式管4度条件下避光孵育30分钟;
(5)孵育结束后,向步骤(4)每管加入700 μL PBS,混匀,1500 rpm/min离心5 min,洗涤2次,吸去上清,留沉淀;
(6)向步骤(5)去上清后的流式管中,分别加入PBS缓冲液洗涤,离心后去上清,用PBS缓冲液重悬细胞,即得到流式细胞上机样品。
6.根据权利要求2所述的SOD3在调控双氢青蒿素诱导的脾脏增大及免疫细胞异质性药物筛选中的应用,其特征在于所述的步骤7中,使用单细胞方法测定双氢青蒿素诱导的免疫细胞亚群的方法,包括细胞捕获和 cDNA 合成,具体步骤如下:
(1)将所获得的脾脏单细胞悬浮在含有 0.04% BSA 的 PBS 中;
(2)在芯片的每个通道加入约6,000个细胞,预计回收的目标细胞为3,000个细胞;
(3)捕获的细胞被裂解,释放的 RNA 在单个 GEM 中通过逆转录进行条形码标记;
(4)逆转录在 S1000TM Touch Thermal Cycler 上在 53°C 下进行 45 分钟,然后在85°C,5 分钟,在结束后保持在 4°C;
生成的cDNA在安捷伦4200分光光度计中确定质量。
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CN111096965A (zh) * | 2018-10-29 | 2020-05-05 | 百药智达(北京)纳米生物技术有限公司 | 含双氢青蒿素的药物、其制备方法、药物组合物及其应用 |
WO2022000492A1 (zh) * | 2020-07-03 | 2022-01-06 | 中山大学附属第一医院 | 一种青蒿素或衍生物青蒿琥酯、双氢青蒿素的用途 |
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CN111096965A (zh) * | 2018-10-29 | 2020-05-05 | 百药智达(北京)纳米生物技术有限公司 | 含双氢青蒿素的药物、其制备方法、药物组合物及其应用 |
WO2022000492A1 (zh) * | 2020-07-03 | 2022-01-06 | 中山大学附属第一医院 | 一种青蒿素或衍生物青蒿琥酯、双氢青蒿素的用途 |
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双氢青蒿素对低氧下人肺动脉内皮细胞增殖和分泌功能影响;余华;王良兴;刘静静;董一枝;夏晓茹;管华琴;;中国现代医生(第04期);全文 * |
双氢青蒿素对卡氏肺孢子虫肺炎大鼠脾细胞凋亡的影响;李文桂, 陈雅棠, 刘成伟;中国人兽共患病杂志(第02期);全文 * |
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