CN111534490A - 羟氯喹治疗玫瑰痤疮的模型构建方法 - Google Patents
羟氯喹治疗玫瑰痤疮的模型构建方法 Download PDFInfo
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Abstract
本发明公开了动物和细胞模型构建领域的羟氯喹(Hydroxychloroquine,HCQ)治疗玫瑰痤疮的模型构建方法,以实现通过动物和细胞模型的构建来阐述HCQ对玫瑰痤疮治疗效果的潜在分子机制。为了实现上述目的,本技术方案的实验方法如下,选用7周大小的BALB/C小鼠,然后用640μm浓度的LL37皮内注射可以诱导玫瑰痤疮样皮损并观测羟氯喹对玫瑰痤疮样皮损的改善程度;在体外实验中,构建玫瑰痤疮相关肥大细胞模型,检测羟氯喹对肥大细胞激活的影响。相对于其他实验方式的技术,本技术方案中利用对比实验对同种类小鼠进行诱变和治疗实验,降低了变量条件,提高了实验的准确性。
Description
技术领域
本发明属于动物和细胞模型构建领域,具体是羟氯喹治疗玫瑰痤疮的模型构建方法。
背景技术
玫瑰痤疮是一种常见的慢性面部炎症性疾病,通常分为4种主要亚型:红斑血管扩张性玫瑰痤疮(ETR),丘疹脓疱性玫瑰痤疮(PPR),玫瑰痤疮(PHR)和眼部(OR)玫瑰痤疮。玫瑰痤疮的临床特征包括面部红斑,丘疹,脓疱,毛细血管扩张和反复潮红,尽管它不是一种致命的疾病,但对玫瑰痤疮患者的生活质量有不利影响。此外,最近的研究表明,玫瑰痤疮与痴呆和癌症发病风险的增加有关。这些发现表明玫瑰痤疮可能是全身性疾病的反应,值得关注。
美国国家玫瑰痤疮协会的诊疗指南认为,玫瑰痤疮应按照不同亚型给予相应的治疗。本技术主要针对红斑毛细血管扩张型。目前针对红斑毛细血管扩张型的药物包括局部治疗、系统治疗、以及激光治疗。目前中外各国治疗指南中出现的外用药物包括肾上腺素受体激动剂、壬二酸等。α肾上腺素受体激动剂,可以通过促进血管平滑肌收缩,从而收缩血管,因此仅对有平滑肌层的血管有作用,而对于缺乏平滑肌层的细小血管及毛细血管无收缩作用。
大多数其他口服方案的研究已经表明,口服四环素或米诺环素具有高或不明确的偏倚风险。因此,需要新的、有效的、良好的耐受性和方便的口服药来减轻玫瑰痤疮的症状和体征,并为玫瑰痤疮患者提供更广泛的治疗,特别是对那些有多西环素耐受不良的患者。羟氯喹(HCQ)于1955年被FDA批准用于狼疮治疗,目前常用于治疗系统性自身免疫性疾病患者,并被认为在妊娠期间是安全的。
LL37(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE)是人抗菌肽CAMP的水解产物,在基础研究中可以作为玫瑰痤疮样模型的建立。
近年来,虽然HCQ在中国成功应用于玫瑰痤疮的治疗,但玫瑰痤疮的治疗缺乏高质量的数据。综上所述,现有技术存在的问题是:HCQ被用作玫瑰痤疮的治疗选择,但其治疗效果的潜在分子机制尚不清楚。
发明内容
为了解决上述问题,本发明的目的是通过动物和细胞模型的构建以阐述HCQ对玫瑰痤疮治疗效果的潜在分子机制,为玫瑰痤疮提供一种新的治疗方案。
为了实现上述目的,本发明的技术方案如下:羟氯喹治疗玫瑰痤疮的模型构建方法,包括以下步骤,
S1、选用7周大小的BALB/C实验鼠;
S2、随后将小鼠分为四组,分别为A组阴性对照组、B组羟氯喹处理组、C组玫瑰痤疮模型组和D组LL37加羟氯喹治疗组;
S3、对每组的造模时间均为一周,其中A组给予100μlPBS灌胃7天,B组给予100μlHCQ溶液灌胃连续7天,最后两天A、B组同时进行PBS皮内注射;C组给予100μlPBS灌胃7天,D组给予100μlHCQ溶液灌胃7天,最后两天C、D组同时予以LL37皮内注射;
S4、造模完成24小时之后,收取小鼠背部皮肤,将剪下的背部皮肤分别进行-80℃、液氮冷冻处理以及组织固定液中保存;
S5、对冷冻处理完成后的组织进行样本化处理,其中-80℃冷冻的组织进行免疫荧光和免疫组化检测,液氮冷冻的组织用来提取RNA,组织固定液中的用以HE或甲苯胺蓝染色;
S6、最后用新生鼠的肥大细胞构建体外的玫瑰痤疮细胞模型,并在体外进行验证,对比治疗效果。
采用上述方案后实现了以下有益效果:
进一步,所述S1中采用的小鼠为BALB/C小鼠,S3中任意组注射的LL37每次注射50μL,每日间隔12小时一次,连续注射两天。
进一步,在步骤S4中,剪下背部造模处皮肤并分为四块。一块放入于冻存管中置于液氮冷冻以备RNA提取;一块铺平贴于锡箔纸放置于-80℃冰箱冷冻,以备冰冻切片使用;两块铺平面贴于滤纸,放置于装有通用性组织固定液的1.5mlEP管中用以HE或甲苯胺蓝染色。
进一步,所述S6中肥大细胞培养和建模方法如下;
选取刚出生2-3天的胎鼠,酒精擦拭身体,减去头部和四肢取皮,取胎鼠皮肤经含有5X、2X、1X双抗的PBS溶液中清洗过后,在生物安全柜中用剪刀将其剪碎;
之后,取适量细胞分离液悬浮剪碎的皮肤组织,置于15ml离心管中,然后放置于37℃震荡箱中160转/分,震荡消化60分钟,待消化完毕后,用等量的含血清的培养基终止消化,然后经过8mm的分子筛滤过细胞;
含有细胞的浑浊液经800转/分离心10分钟后,弃上清,然后加入适量红细胞裂解液轻轻吹打混匀,静置5分钟裂解红细胞,之后再用800转/分离心5分钟,弃上清,用培养基重悬细胞后放入悬浮细胞培养皿中进行培养。
进一步,培养基选用肥大细胞完全培养基,培养基的成分含有500μl三抗,其中三抗包括100μg/ml青霉素、100μg/ml链霉素和0.25mg/ml两性霉素B,500μl非必需氨基酸100mmol/L,500μl丙酮酸钠100mmol/L,500mol/Lβ-巯基乙醇,10mg/mL重组鼠IL-3,10mg/mL重组鼠SCF和10%的胎牛血清;培养环境和时间为37℃、5%CO2,连续培养14天。进一步,随后对肥大细胞进行纯化,将原代的肥大细胞培养14天后,取培养皿中悬浮细胞,800转/分、5分钟离心重悬后加入到40%的percoll分离液(经过聚乙烯吡咯烷酮(polyvinylpyrolidone,PVP)处理的硅胶颗粒混悬液)(60%培养基加40%等渗percoll分离液),1600转/分、10分钟离心,弃上清并取最下层的细胞,即为原代皮肤肥大细胞,可用甲苯胺蓝染色鉴定细胞成熟度。
进一步,皮肤肥大细胞建模将2×106的肥大细胞置于30mm细胞培养皿中,模型组给予4μm的LL37刺激6小时;干预组给予10μm的HCQ或300nm的TRAM-34提前处理肥大细胞1小时,然后给予4μm的LL37刺激6小时;同时也需设置药物对照组,给予10μm的HCQ或300nm的TRAM-34,而空白对照组不给予任何处理,收集细胞RNA,置于-80℃冰箱中保存。
附图说明
图1为本发明的实施流程图;
图2为本发明不同小鼠模型分组中红斑的平均严重程度和面积,肥大细胞的浸润以及炎症因子的水平差异;
图3为LL37明显诱导皮肤肥大细胞Tpsab1、CMA1、MMP9、IL1β、IL17A、TNFα、CCL2、CXCL2炎症因子的表达,羟氯喹干预后,除了CMA1其它的炎症因子表达均可被一定程度地抑制;
图4为不同小鼠模型中每孔细胞内钙离子流的比较的时间曲线图;
具体实施方式
下面通过具体实施方式进一步详细说明:
材料与方法
1.主要实验溶液的配制
1.10XPBS溶液(PH=7.4):NaCl80g,Na2HPO414.4g,KH2PO42.4g,KCl2g,用双蒸水定容至1000ml,震荡搅拌均匀,充分溶解后用PH仪调节PH值到7.4,放置于高压灭菌锅灭菌后室温保存。
2.1XPBS溶液:取50ml10XPBS,加入450ml双蒸水,高压灭菌后放置于4℃冰箱保存。
3.细胞分离液:该细胞分离液用于消化和分离胎鼠皮肤组织,即配即用。取适量RPMI1640培养液(根据胎鼠皮肤组织确定具体用量),加入10%的胶原酶4和5%的分离酶2,振荡混匀后用0.22μm的滤头过滤除菌。
4.肥大细胞完全培养基:在43mlRPMI1640培基中加入500μl三抗(100μg/ml青霉素+100μg/ml链霉素+0.25mg/ml两性霉素B),500μl非必需氨基酸(100mmol/L),500μl丙酮酸钠(100mmol/L),500mol/Lβ-巯基乙醇,10mg/mL重组鼠IL-3,10mg/mL重组鼠SCF,最后加入5ml胎牛血清,混合均匀即为完全培基。
5.LL37溶液:LL37(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE)是人抗菌肽CAMP的水解产物,是由37个氨基酸构成的多肽。本实验LL37来源于上海生工生物工程有限公司,每支包装25mg,经过质谱鉴定纯度大于95%。取一支LL37粉(25mg),在生物安全柜中加入8.69ml1XPBS,震荡均匀充分溶解,浓度即为640μM的LL37溶液。分装到200μl无菌PCR管中,-20℃冰箱保存。
6.细胞用羟氯喹溶液:HCQ(每支10mg)溶于23ml1XPBS溶液中制成1mM/L的存储液放置于-20℃冰箱中,实验时每1ml细胞培养液加入10μl储存液,既为HCQ10μM/L处理浓度。
7.细胞用TRAM-34:KCa3.1通道抑制剂TRAM-34(5mg)溶于14.5mlDMSO中制成1mM/L的储存液,实验时用DMSO稀释为300nM的浓度工作液,当涉及TRAM-34的细胞实验时,其它对照皿也应加入相应的DMSO。
8.小鼠用羟氯喹溶液:羟氯喹100mg溶于10ml1XPBS溶液制成10mg/mlHCQ灌胃用溶液,放置于4℃冰箱。
2.实验方法
2.1小鼠造模
2.1.1实验分组
请参考图1,BALB/c(7-8周)雌性小鼠随机分为四组:空白对照组(PBS+PBS)、HCQ对照组(HCQ+PBS)、玫瑰痤疮模型组(PBS+LL37)、HCQ治疗组(HCQ+LL37)。
2.1.2造模
LL37(640μM)皮内注射,每次注射50μL,每日间隔12小时一次,连续注射两天即可构建玫瑰痤疮样皮炎模型。
HCQ按照40mg/kg的计量进行灌胃,7-8周小鼠体重约为25g,则每次需要灌胃1mg,配置的HCQ溶液浓度为10mg/ml,则每次灌胃100μl。
请参考图2,在LL37皮内注射造模前一天,用专用剃毛器给小鼠背部皮肤剃毛,面积约为2×3cm2。玫瑰痤疮模型组小鼠每日给予100μlPBS灌胃,连续7天,第6、7天同时予以LL37皮内注射进行玫瑰痤疮造模;HCQ治疗组给予100μlHCQ溶液灌胃,连续7天,第6、7天同时进行LL37皮内注射;空白对照组给予100μlPBS灌胃7天,最后两天同时进行PBS皮内注射;HCQ对照组给予100μlHCQ溶液灌胃7天,最后两天予以PBS皮内注射。
2.1.3样本
请参考图3,在最后一次注射LL37之后24小时,用体视镜对各组小鼠背部皮肤进行拍照。拍照后将小鼠颈椎脱臼处死,剪下背部造模处皮肤并分为4块。一块放入于冻存管中置于液氮冷冻以备RNA提取;一块铺平贴于锡箔纸放置于-80℃冰箱冷冻,以备冰冻切片使用;一块铺平面贴于滤纸,放置于装有通用性组织固定液的1.5mlEP管中用以HE或甲苯胺蓝染色。
2.2肥大细胞培养及建模
2.2.1皮肤肥大细胞的提取及培养
选取刚出生2-3天的胎鼠,酒精擦拭身体,减去头部和四肢取皮,取胎鼠皮肤经含有5X、2X、1X双抗的PBS溶液中清洗过后,在生物安全柜中用剪刀将其剪碎,这一过程约为30分钟。之后,取适量细胞分离液悬浮剪碎的皮肤组织,置于15ml离心管中,然后放置于37℃震荡箱中160转/分,震荡消化60分钟。
待消化完毕后,用等量的含血清的培养基终止消化,然后经过8mm的分子筛滤过细胞。含有细胞的浑浊液经800转/分离心10分钟后,弃上清,然后加入适量红细胞裂解液轻轻吹打混匀,静置5分钟裂解红细胞。之后再用800转/分离心5分钟,弃上清,用培养基重悬细胞后放入悬浮细胞培养皿中进行培养。培养基为肥大细胞完全培养基,含有500μl三抗(100μg/ml青霉素+100μg/ml链霉素+0.25mg/ml两性霉素B),500μl非必需氨基酸(100mmol/L),500μl丙酮酸钠(100mmol/L),500mol/Lβ-巯基乙醇,10mg/mL重组鼠IL-3,10mg/mL重组鼠SCF和10%的胎牛血清;培养环境和时间为37℃、5%CO2,连续培养14天。
2.2.2肥大细胞的纯化
原代细胞培养14天后,取培养皿中悬浮细胞,800转/分、5分钟离心重悬后加入到40%的percoll分离液中(60%培养基加40%等渗percoll分离液),1600转/分、10分钟离心,弃上清并取最下层的细胞,即为原代皮肤肥大细胞,可用甲苯胺蓝染色鉴定细胞成熟度。
2.2.3皮肤肥大细胞建模
将2×106的肥大细胞置于30mm细胞培养皿中,模型组给予LL37(4μM)刺激6小时;干预组给予HCQ(10μM)或TRAM-34(300nM)提前处理肥大细胞1小时,然后给予LL37(4μM)刺激6小时;同时也需设置药物对照组,给予HCQ(10μM)或TRAM-34(300nM),而空白对照组不给予任何处理。收集细胞RNA,置于-80℃冰箱中保存。
(1)本实验中的基因PCR引物见表2-3
表2-3引物序列
2.3免疫组织化学实验
2.3.1HE染色
(1)脱蜡:将小鼠组织切片依次放入松节油(Ⅰ)和松节油(Ⅱ)中,时间均为5分钟,之后从松节油(Ⅱ)中取出切片依次置入100%、95%、80%、70%的无水乙醇、蒸馏水中,在每一浓度梯度中切片停留的时间约为1分钟。
(2)脱蜡完毕后将切片放入苏木素中染核5分钟,然后用自来水洗去切片上残余的染液。
(3)将切片置入1%盐酸酒精脱色5秒,再用清水反复清洗反蓝,摇床上进行。
(4)最后将切片放入伊红染液,染色3分钟。
(5)透明:将切片依次放入70%、80%、95%、100%的无水乙醇,在每一浓度梯度中切片停留的时间约为1分钟;然后再将切片依次放入松节油(Ⅰ)和松节油(Ⅱ)中,时间均为5分钟,
(6)封片:小心擦去样本周围多余的松节油,然后利用100mLTIP头将中性树脂滴加在样本周围,应注意避免产生气泡。之后,用盖玻片封片、晾干。
(7)切片可使用普通光学显微镜进行观察。
2.3.2甲苯胺蓝染色
染液的配制:将1g甲苯胺蓝溶于200mL蒸馏水中配成染色液,将1mL冰醋酸溶于199mL蒸馏水中配成分色液。
(1)脱蜡:将小鼠组织切片依次放入松节油(Ⅰ)和松节油(Ⅱ)中,时间均为5分钟,之后从松节油(Ⅱ)中取出切片依次置入100%、95%、80%、70%的无水乙醇、蒸馏水中,在每一浓度梯度中切片停留的时间约为1分钟。
(2)染色:脱蜡完毕后将切片放入染色液中30分钟,然后用自来水洗去切片上残余的染液。
(3)分色:将切片放入分色液中,直到胞核和颗粒显示清晰(在显微镜下观察控制),然后清水洗涤。
(4)透明:将切片依次放入70%、80%、95%、100%的无水乙醇,在每一浓度梯度中切片停留的时间约为1分钟;然后再将切片依次放入松节油(Ⅰ)和松节油(Ⅱ)中,时间均为5分钟,
(5)封片:小心擦去样本周围多余的松节油,然后利用100mLTIP头将中性树脂滴加在样本周围,应注意避免产生气泡。之后,用盖玻片封片、晾干。
(6)切片可使用普通光学显微镜进行观察。
2.4免疫荧光实验
(1)固定:从-80℃冰箱中取出已经切好的小鼠皮肤组织冰冻切片,用4%的多聚甲醛在室温下固定15分钟。
(2)在摇床上用PBS洗三次,每次15分钟。
(3)封闭与破膜:用纸巾小心擦干玻片上的水分,然后使用免疫组化笔在皮肤组织周围画圈包绕组织,在组织上滴加封闭液(5%驴血清+0.2-0.5%的TritonX100),室温下封闭1小时。
(4)孵育一抗:甩掉玻片上的封闭液,在圈内加入1%驴血清稀释的一抗,然后将湿盒放置于4℃冰箱过夜。
(5)次日从冰箱拿出湿盒后,甩掉切片上的抗体,之后将切片置入PBS中并在摇床上清洗三次,每次15分钟。
注意:以下步骤均需避光
(6)孵育二抗:清洗玻片后,小心擦干水分,然后向组织上滴加入5%驴血清稀释的二抗(1:500),室温下孵育1小时。然后再将玻片放入PBS中洗三次,每次15分钟。
(7)染核:用PBS1:2000稀释DAPI,然后将染液滴加到组织上,染色5分钟。之后用PBS在摇床上清洗,每次5分钟,三次。
(8)封片:擦干玻片上多余水分,在组织上滴加防荧光猝灭封片剂封片,滴加过程应轻柔注意避免产生气泡,然后用盖玻片封片,置于4℃冰箱保存。待荧光显微镜荧光观察。
2.5肥大细胞脱颗粒实验
本研究中我们通过检测肥大细胞向培养基中释放的氨基己糖苷酶数量来评估细胞脱颗粒的能力。将1×105的肥大细胞种于24孔板中,用台氏液培养。在肥大细胞中加入10μMHCQ或300nMTRAM-34在37℃、5%CO2细胞培养箱中预处理1小时,然后再向肥大细胞中加入4μM LL37继续在细胞培养箱中刺激5小时。
将细胞培养皿置于冰盒上10分钟终止反应,后将培养皿中的细胞培养液转移至1.5mLEP管中,800转/分、离心5分钟,取50μL上清液置于96孔板中。总的氨基己糖苷酶是通过在24孔板中,加入1%的TritonX100裂解细胞10分钟,然后吸取培养液后转移至1.5mLEP管中,800转/分、离心5分钟,取50μL上清液置于96孔板中。上清液加入完毕后,再向96孔板中加入50μL2mM的硝基苯基-2-乙酰氨基-2-脱氧-b-D-葡萄糖苷,后将96孔板置于37℃温箱中反应3小时。最后在每孔中添加200μL0.4M甘氨酸(PH值10.7)终止反应,用多功能酶标仪读检测每孔405nm处测量吸光度。
2.6肥大细胞趋化实验
8mm孔径的趋化小室被用来进行细胞趋化实验。在24孔板中放入500μL含10%血清的完全培养基,根据实验需要,设置对照组、单用药物组、LL37组、LL37加药物组。在LL37组和LL37加药物组中,24孔板加入4μM/L的LL37。将趋化小室置入24孔板中,并种入肥大细胞(2×103),小室中的培养基为200μL且不含血清。
根据实验需要,向相应的设置组加入HCQ(10μM)或TRAM-34(300nM),将24孔板置于37℃、5%CO2细胞培养箱中培育。24小时后取出24孔板,用棉签小心拭去小室上层膜上的细胞,并用PBS清洗后加入4%多聚甲醛固定15分钟。再次清洗小室后,用0.5%的甲苯胺蓝染色30分钟,后用清水洗净小室,晾干后于显微镜下观测粘附于小室下层膜上的细胞并拍照。
2.7肥大细胞钙离子内流检测
请参考图4,Fura-2是细胞生物学常用的一种钙荧光探针,能特异性地结合钙离子(结合比例为1:1),同时可发出荧光。它结合钙离子后的最大激发波长从结合前的380nm向340nm偏移,其发射荧光强度与结合Ca2+的浓度存在定量关系。一般用340nm和380nm波长激发Fura-2,用两种激发所得到的荧光强度比率来计算细胞内的钙离子浓度。
在本实验中用PBS将肥大细胞清洗三次后,将1×106的肥大细胞重悬于不含钙的台氏液中,然后给予2μM/L的Fura-2AM钙离子探针,细胞培养箱中孵育20分钟,之后再次用PBS清洗肥大细胞三次,去除多余的钙离子探针。将已装载钙离子探针的肥大细胞重悬于台氏液中,根据实验需要设置对照组、单用药物组、LL37组、LL37加药物组。细胞的干预处理浓度为HCQ10μM/L或TRAM-34300nM/L。
然后将细胞置于细胞培养相中箱孵育约30分钟,一方面确保AM体在细胞内完全去酯化,一方面让药物进入细胞。之后将细胞转移至黑底不透光的96孔板中,置于多功能荧光酶标仪中记录每孔以340nm和380nm为激发波、540nm为吸收波的荧光读数,持续记录30秒。然后停止记录,并立即向相应细胞孔中加入4μM/L的LL37,并以10秒钟的间隔记录340nm和380nm为激发波、540nm为吸收波的荧光读数。每孔细胞内钙离子流的比较是通过340nm荧光读数值除以380nm荧光读数值而得到,并制成时间曲线图。
2.8统计学方法
实验中的数据都用均数±标准误(SEM)的形式表示,使用GraphPadPrism5软件作图并用其中的t检验检测两组数据的差异,取双侧检测标准、并以*P<0.05,**P<0.01和***p<0.001被认为具有显著性差异。
结果
3.1羟氯喹减轻LL37诱导的小鼠玫瑰痤疮样皮肤炎症
我们构建了一个玫瑰痤疮样皮肤炎症的小鼠模型,用于研究羟氯喹治疗玫瑰痤疮的药理机制。如图1所显示的那样,LL37皮内注射可以诱导小鼠背部皮肤呈现玫瑰痤疮样的红斑,而羟氯喹可以明显抑制皮肤炎症性红斑的表型。
红斑的平均严重程度和面积分别降低了约57%和35%。在免疫组化分析中,LL37诱导的皮肤增厚以及皮下炎症细胞侵润也被羟氯喹所抑制。取炎症皮损进行相关炎症因子的PCR分析,我们发现LL37明显诱导CAMP、IL1β、IL6、IL17A和TNFα的表达,而羟氯喹干预可以抑制上述炎症因子的升高。因此这些研究结果说明羟氯喹可以减轻LL37诱导的玫瑰痤疮样皮炎,并且改善皮下炎症的严重程度。
3.2羟氯喹抑制LL37诱导的皮下肥大细胞侵润和蛋白酶生成
肥大细胞在皮肤炎症中起到重要的作用,它通过脱颗粒释放蛋白酶并且诱导炎症因子的表达,促进血管扩张和神经免疫反应。特别是在玫瑰痤疮中,肥大细胞被认为是关键的致病细胞,肥大细胞释放的糜蛋白酶(CMA1)和胰蛋白酶(Tpsab1)可以激活KLK5,从而诱导局部皮肤组织活性LL37表达升高;研究也证实在玫瑰痤疮病人和小鼠模型皮损中都有明显的侵润。
本研究中,通过甲苯胺蓝染色我们发现LL37明显诱导肥大细胞在小鼠玫瑰痤疮样皮炎皮损中的侵润,并且羟氯喹干预可以抑制肥大细胞在皮肤组织的聚集。取皮损组织行免疫荧光和PCR检测,肥大细胞相关的蛋白酶,胰蛋白酶、糜蛋白酶和基质金属蛋白酶(MMP9)在皮损中都明显的升高,但羟氯喹可以抑制上述蛋白酶的表达。因此,通过上述实验,我们认为羟氯喹减轻玫瑰痤疮样皮肤炎症可能部分是基于其抑制肥大细胞局部侵润和蛋白酶释放。3.3羟氯喹抑制LL37诱导的肥大细胞激活
除了体内实验,我们还在体外对羟氯喹对肥大细胞的作用进行了研究。大量的研究已经证实LL37可以诱导肥大细胞的激活。通过PCR检测,LL37明显诱导皮肤肥大细胞Tpsab1、CMA1、MMP9、IL1β、IL17A、TNFα、CCL2、CXCL2的表达;而在羟氯喹干预后,除了CMA1其它的炎症因子表达均可被一定程度地抑制。
我们注意到,之前的研究也发现羟氯喹在体外也不能抑制肥大细胞CMA1的表达。于是,这些证据提示羟氯喹可以抑制LL37诱导的肥大细胞炎症因子的的释放。
3.4羟氯喹抑制LL37诱导的肥大细胞趋化、脱颗粒及钙离子内流
一些研究发现在体外LL37可以诱导肥大细胞趋化聚集并且可以促进肥大细胞脱颗粒。本研究中,我们也发现LL37可以诱导小鼠皮肤肥大细胞的趋化和脱颗粒;而羟氯喹可以抑制这种作用。目前的研究认为肥大细胞的激活与功能同钙离子关系密切,钙离子内流可以促进肥大细胞炎症因子的释放和脱颗粒,并且肥大细胞的趋化也受钙离子的影响。之前的研究也发现羟氯喹可以抑制T淋巴细胞中钙离子的内流,基于上述因果联系,我们检测了LL37和羟氯喹对肥大细胞钙离子内流的影响。研究发现羟氯喹可以明显抑制LL37诱导的肥大细胞钙离子内流。
3.5羟氯喹抑制肥大细胞激活可能通过抑制Kca3.1介导的钙离子信号
Mas相关基因X2(小鼠中为MrgprB2)和钙激活钾通道3.1(Kca3.1)在肥大细胞钙离子内流中起着重要的作用。基于前人的发现,我们推测羟氯喹抑制肥大激活可能是通过上述两个通道蛋白。通过体外刺激,LL37可以明显诱导肥大细胞MrgprB2和Kca3.1的表达,羟氯喹可以抑制Kca3.1的激活但对MrgprB2没有明显的影响。之前的研究也发现羟氯喹可以抑制Kca3.1通道,这与本研究的发现相似。
为了进一步验证,我们选用了Kca3.1通道的抑制剂TRAM-34进行后续的实验。通过肥大细胞趋化实验、脱颗粒实验和钙离子内流检测,我们发现TRAM-34同样也可以抑制LL37诱导的肥大细胞脱颗粒、趋化和钙离子内流。因此,这进一步提示羟氯喹抑制肥大细胞的功能部分是通过抑制了Kca3.1介导的钙离子信号所致。
结论
羟氯喹可以明显抑制小鼠玫瑰痤疮样皮炎的表型和炎症程度,关于潜在的药理机制,羟氯喹对玫瑰痤疮的治疗作用可能部分通过抑制Kca3.1介导的肥大细胞钙离子内流来抑制肥大细胞的激活而实现。
以上所述的仅是本发明的实施例,方案中公知的具体结构及特性等常识在此未作过多描述,所属领域普通技术人员知晓申请日或者优先权日之前发明所属技术领域所有的普通技术知识,能够获知该领域中所有的现有技术,并且具有应用该日期之前常规实验手段的能力,所属领域普通技术人员可以在本申请给出的启示下,结合自身能力完善并实施本方案,一些典型的公知结构或者公知方法不应当成为所属领域普通技术人员实施本申请的障碍。应当指出,对于本领域的技术人员来说,在不脱离本发明结构的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
序列表
<110> 中南大学湘雅医院
<120> 羟氯喹治疗玫瑰痤疮的模型构建方法
<130> 20200508
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Mouse TPSAB1
<400> 1
gccaatgaca cctactggat g 21
<210> 2
<211> 21
<212> DNA
<213> Mouse TPSAB1
<400> 2
gcttacggag ctgtactctg a 21
<210> 3
<211> 19
<212> DNA
<213> Mouse CMA1
<400> 3
cgcccctaca tggcctatc 19
<210> 4
<211> 23
<212> DNA
<213> Mouse CMA1
<400> 4
aggaggactg ttatagacct tcc 23
<210> 5
<211> 21
<212> DNA
<213> Mouse MMP9
<400> 5
ctggacagcc agacactaaa g 21
<210> 6
<211> 20
<212> DNA
<213> Mouse MMP9
<400> 6
ctcgcggcaa gtcttcagag 20
<210> 7
<211> 22
<212> DNA
<213> Mouse IL1β
<400> 7
gcaactgttc ctgaactcaa ct 22
<210> 8
<211> 21
<212> DNA
<213> Mouse IL1β
<400> 8
atcttttggg gtccgtcaac t 21
<210> 9
<211> 23
<212> DNA
<213> Mouse IL6
<400> 9
tagtccttcc taccccaatt tcc 23
<210> 10
<211> 21
<212> DNA
<213> Mouse IL6
<400> 10
ttggtcctta gccactcctt c 21
<210> 11
<211> 19
<212> DNA
<213> Mouse CXCL2
<400> 11
ccaaccacca ggctacagg 19
<210> 12
<211> 19
<212> DNA
<213> Mouse CXCL2
<400> 12
gcgtcacact caagctctg 19
<210> 13
<211> 21
<212> DNA
<213> Mouse IL17
<400> 13
tcagcgtgtc caaacactga g 21
<210> 14
<211> 21
<212> DNA
<213> Mouse IL17
<400> 14
cgccaaggga gttaaagact t 21
<210> 15
<211> 20
<212> DNA
<213> Mouse TNFα
<400> 15
ctgaacttcg gggtgatcgg 20
<210> 16
<211> 23
<212> DNA
<213> Mouse TNFα
<400> 16
ggcttgtcac tcgaattttg aga 23
<210> 17
<211> 23
<212> DNA
<213> Mouse CCL2
<400> 17
gcattagctt cagatttacg ggt 23
<210> 18
<211> 23
<212> DNA
<213> Mouse CCL2
<400> 18
ttaaaaacct ggatcggaac caa 23
<210> 19
<211> 21
<212> DNA
<213> Mouse KCA3.1
<400> 19
ccctacccga gaaggagtac c 21
<210> 20
<211> 21
<212> DNA
<213> Mouse KCA3.1
<400> 20
ggatgaccat gaccgacaca a 21
<210> 21
<211> 23
<212> DNA
<213> Mouse Mrgprb2
<400> 21
atcaagaatc taagcacctc agc 23
<210> 22
<211> 22
<212> DNA
<213> Mouse Mrgprb2
<400> 22
gaaagcaaaa tcatggcttg gt 22
<210> 23
<211> 21
<212> DNA
<213> Mouse GAPDH
<400> 23
aggtcggtgt gaacggattt g 21
<210> 24
<211> 23
<212> DNA
<213> Mouse GAPDH
<400> 24
tgtagaccat gtagttgagg tca 23
Claims (7)
1.羟氯喹治疗玫瑰痤疮的模型构建方法,其特征在于:包括以下步骤,
S1、选用7周大小的BALB/C实验鼠;
S2、随后将小鼠随机分为四组,分别为A组阴性对照组、B组羟氯喹处理组、C组玫瑰痤疮模型组和D组LL37加羟氯喹治疗组;
S3、对每组的造模时间均为一周,其中A组给予100μlPBS灌胃7天,B组给予100μlHCQ溶液灌胃连续7天,最后两天A、B组同时进行PBS皮内注射;C组给予100μlPBS灌胃7天,D组给予100μlHCQ溶液灌胃7天,最后两天C、D组同时予以LL37皮内注射;
S4、造模完成24小时之后,收取小鼠背部皮肤,将剪下的背部皮肤分别进行-80℃、液氮冷冻处理以及组织固定液中保存;
S5、对冷冻处理完成后的组织进行样本化处理,其中-80℃冷冻的组织进行免疫荧光和免疫组化检测,液氮冷冻的组织用来提取RNA,组织固定液中的用以HE或甲苯胺蓝染色;
S6、最后用新生鼠的肥大细胞构建体外的玫瑰痤疮细胞模型,并在体外进行验证,对比治疗效果。
2.根据权利要求1所述的羟氯喹治疗玫瑰痤疮的模型构建方法,其特征在于:所述S1中采用的小鼠为BALB/C小鼠,S3中任意组注射的LL37每次注射50μL,每日间隔12小时一次,连续注射两天。
3.根据权利要求2所述的羟氯喹治疗玫瑰痤疮的模型构建方法,其特征在于:在步骤S4中,将每一组的小鼠背部皮肤都剪下四块,一块放入于冻存管中置于液氮冷冻以备RNA提取;一块铺平贴于锡箔纸放置于-80℃冰箱冷冻,以备冰冻切片使用;两块铺平面贴于滤纸,放置于装有通用性组织固定液的1.5ml EP管中用以HE或甲苯胺蓝染色。
4.根据权利要求3所述的羟氯喹治疗玫瑰痤疮的模型构建方法,其特征在于:所述S6中肥大细胞培养和建模方法如下;
选取刚出生2-3天的胎鼠,酒精擦拭身体,减去头部和四肢取皮,取胎鼠皮肤经含有5X、2X、1X双抗的PBS溶液中清洗过后,在生物安全柜中用剪刀将其剪碎;
之后,取适量细胞分离液悬浮剪碎的皮肤组织,置于15ml离心管中,然后放置于37℃震荡箱中160转/分,震荡消化60分钟,待消化完毕后,用等量的含血清的培养基终止消化,然后经过8mm的分子筛滤过细胞;
含有细胞的浑浊液经800转/分离心10分钟后,弃上清,然后加入适量红细胞裂解液轻轻吹打混匀,静置5分钟裂解红细胞,之后再用800转/分离心5分钟,弃上清,用培养基重悬细胞后放入悬浮细胞培养皿中进行培养。
5.根据权利要求4所述的羟氯喹治疗玫瑰痤疮的模型构建方法,其特征在于:培养基选用肥大细胞完全培养基,培养基的成分含有500μl三抗,其中三抗包括100μg/ml青霉素、100μg/ml链霉素和0.25mg/ml两性霉素B,500μl非必需氨基酸100mmol/L,500μl丙酮酸钠100mmol/L,500mol/Lβ-巯基乙醇,10mg/mL重组鼠IL-3,10mg/mL重组鼠SCF和10%的胎牛血清;培养环境和时间为37℃、5%CO2,连续培养14天。
6.根据权利要求5所述的羟氯喹治疗玫瑰痤疮的模型构建方法,其特征在于:随后对肥大细胞进行纯化,将原代的肥大细胞培养14天后,取培养皿中悬浮细胞,800转/分、5分钟离心重悬后加入到40%的percoll分离液(经过聚乙烯吡咯烷酮(polyvinyl pyrolidone,PVP)处理的硅胶颗粒混悬液)(60%培养基加40%等渗percoll分离液),1600转/分、10分钟离心,弃上清并取最下层的细胞,即为原代皮肤肥大细胞,可用甲苯胺蓝染色鉴定细胞成熟度。
7.根据权利要求6所述的羟氯喹治疗玫瑰痤疮的模型构建方法,其特征在于:皮肤肥大细胞建模将2×106的肥大细胞置于30mm细胞培养皿中,模型组给予4μm的LL37刺激6小时;干预组给予10μm的HCQ或300nm的TRAM-34提前处理肥大细胞1小时,然后给予4μm的LL37刺激6小时;同时也需设置药物对照组,给予10μm的HCQ或300nm的TRAM-34,而空白对照组不给予任何处理,收集细胞RNA,置于-80℃冰箱中保存。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019136225A1 (en) * | 2018-01-05 | 2019-07-11 | Attillaps Holdings | Treating dermatological conditions with chloroquine and/or hydroxychloroquine |
CN110141563A (zh) * | 2019-05-08 | 2019-08-20 | 中南大学湘雅医院 | 一种改善玫瑰痤疮症状的药物及其动物模型和构建方法 |
CN110278919A (zh) * | 2019-07-30 | 2019-09-27 | 罗洋 | 一种构建蠕形螨致玫瑰痤疮样皮损动物模型的方法 |
-
2020
- 2020-05-11 CN CN202010391068.2A patent/CN111534490A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019136225A1 (en) * | 2018-01-05 | 2019-07-11 | Attillaps Holdings | Treating dermatological conditions with chloroquine and/or hydroxychloroquine |
CN110141563A (zh) * | 2019-05-08 | 2019-08-20 | 中南大学湘雅医院 | 一种改善玫瑰痤疮症状的药物及其动物模型和构建方法 |
CN110278919A (zh) * | 2019-07-30 | 2019-09-27 | 罗洋 | 一种构建蠕形螨致玫瑰痤疮样皮损动物模型的方法 |
Non-Patent Citations (2)
Title |
---|
JI LI 等: "Hydroxychloroquine is a novel therapeutic approach for rosacea", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
邢倩倩等: "玫瑰痤疮的发病机制及个体化治疗进展", 《华夏医学》 * |
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