CN106620694B - Cornulin作为靶点在制备防治银屑病的药物中的应用 - Google Patents
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Abstract
本发明公开了Cornulin作为靶点在制备防治银屑病的药物中的应用,所述的药物是在基因水平和/或蛋白水平以Cornulin作为药物靶点。cornulin与银屑病的发展呈正相关,采用适当浓度的cornulin抗体治疗咪喹莫特小鼠银屑病模型,能够改善鳞屑和皮损厚度,并且通过抑制AKT信号来促成银屑病的进展以及抑制皮损恢复正常;cornulin抗体相比其它治疗银屑病药物,干预靶点更加明确,即针对银屑病发病的炎症通路中AKT信号相关分子通路。
Description
技术领域
本发明属于生物医药技术领域,涉及Cornulin在制备防治银屑病中的应用。
背景技术
银屑病是炎症性、免疫介导的慢性皮肤疾病,以角质形成细胞增生和异常分化、炎症细胞浸润为特征。银屑病的临床特征是上覆在红斑基础上的银白色鳞屑,长期的疾病发展严重影响了患者的生活质量。典型银屑病的组织病理为:表皮角化不全,常伴有角化过度;颗粒层消失,棘层肥厚,表皮突延伸,下端增宽,可与邻近表皮突相吻合,真皮乳头上延,其上方棘层变薄。基地细胞可有轻度海绵形成,甚至空泡形成或水肿变性。乳头内毛细血管迂曲扩张充血,周围淋巴细胞、中性白细胞浸润。角化不全区域常可见中性白细胞聚集,称为Munro氏小脓肿。
银屑病的治疗方法有很多种,治疗银屑病的药物包括皮质类固醇类、维A酸类、维生素D3衍生物类、钙调神经磷酸酶抑制剂等药物,但是由于该病病因不清,目前许多治疗方法的疗效并不能使患者满意,并且该病容易复发,由于治疗银屑病的药物不良反应或者药物价格等方面,而限制了这些药物在临床上的使用。
Cornulin(CRNN),也称为clorf10(Chromosome 1open reading frame 10)和SEP53(Squamous Epithelial induced stress Protein of 53kDa),最初是Xu等(Xu Z,Wang MR,Xu X,et al.Novel human esophagus-specific gene clorf10:cDNA cloning,gene structure,and frequent loss of expression in esophagealcancer.Genomics.2000.69(3):322-30.)于2000年在食管上皮细胞中发现的。由于cornulin在热休克后合成明显增多,并且编码了一个分子量53kDa的钙结合蛋白,故Yagui-Beltran等又称为SEP53[7]。Clorf10产物是蛋白质,并且它的结构特征与表皮分化复合体的“融合基因”家族成员相似,在表皮分化中起作用,故Contzler称之为cornulin。
Cornulin是分子量为53kDa的编码495个氨基酸且它的N末端有约90个氨基酸残基的钙结合基序和60个氨基酸的保守的连续重复序列,cornulin是编码在表皮分化复合体的1q21染色体上,主要定位在细胞质和核周区。除食管上皮细胞发现cornulin外,还在口腔鳞状上皮细胞,宫颈鳞状上皮细胞,表皮鳞状上皮细胞,睑板腺上皮细胞和甲状腺滤泡等也被鉴定出了cornulin。除在鳞状细胞肿瘤cornulin表达降低外,还在甲状腺癌和湿疹等cornulin表达也降低。
发明内容
本发明解决的问题在于提供Cornulin作为靶点在制备防治银屑病的药物中的应用,以Cornulin作为干预靶点对防治银屑病作用更加明确,而且安全性良好。
本发明是通过以下技术方案来实现:
Cornulin作为靶点在制备防治银屑病的药物中的应用。
所述的药物是在基因水平和/或蛋白水平以Cornulin作为药物靶点。
所述的药物是针对Cornulin的抗体。
所述的药物是阻碍Cornulin表达或转录的DNA或RNA。
所述的药物是阻碍或抑制Cornulin活化AKT信号的药物。
针对Cornulin的抗体在制备防治银屑病的药物中的应用。
siRNA敲除Cornulin的载体在制备防治银屑病的药物中的应用。
与现有技术相比,本发明具有以下有益的技术效果:
本发明提示cornulin与银屑病的发展呈正相关,采用适当浓度的cornulin抗体治疗咪喹莫特小鼠银屑病模型,能够改善鳞屑和皮损厚度,并且通过抑制AKT信号来促成银屑病的进展以及抑制皮损恢复正常,因此提出制备防治银屑病的药物时新的药物作用靶点-cornulin。
cornulin抗体相比其它治疗银屑病药物,干预靶点更加明确,即针对银屑病发病的炎症通路中AKT信号相关分子通路。
cornulin抗体安全性良好,cornulin抗体特异性结合cornulin,可以来源于兔的多克隆抗体,以及其辅料BSA都对身体无毒副作用,具有良好的安全性。
附图说明
图1为不同浓度的CRNN外用治疗咪喹莫特小鼠的临床照片,其中对照组为0.1%叠氮钠,治疗组分别为0.5,1,2ng/ml的CRNN。
图2为外用0.5ng/ml的CRNN治疗咪喹莫特小鼠皮损和对照组小鼠皮肤组织蛋白的Western blot,其中veh为对照组0.1%叠氮钠,CRNN为0.5ng/ml的CRNN。
图3为0.5ng/ml的CRNN治疗咪喹莫特小鼠的PASI评分。其中A为总的PASI评分,B为红斑的PASI评分,C为鳞屑的PASI评分,D为皮肤厚度的PASI评分。
图4为人体正常皮肤组织和银屑病皮损组织中cornulin免疫组化及半定量对比图。
图5为人体正常皮肤组织和银屑病皮损组织中cornulin western blot及其相对表达。
图6为在HaCaT细胞用不同浓度的M5刺激后24小时,提取细胞总RNA,real-timePCR测cornulin mRNA的相对表达变化。
图7为在HaCaT细胞应用si-negative、si-crnn1和si-crnn2敲除cornulin后48小时,提细胞总蛋白,检测相关分子的western blot。
图8为在HaCaT细胞应用si-negative、si-crnn1和si-crnn2敲除cornulin后24小时、48小时及72小时,应用MTT法检测对HaCaT细胞增殖的影响。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。
本发明发现在银屑病患者皮损和咪喹莫特小鼠皮损中cornulin表达升高,应用M5(IL-17A,IL-22,IL-1a,OSM和TNF-a混合物)刺激HaCaT和HEKa细胞模拟银屑病细胞水平后,cornulin表达升高;这些提示cornulin与银屑病的发展呈正相关,下面对本发明进一步详细的说明。
1)咪喹莫特小鼠模型的建立
购买6-7周BALB/c小鼠,2%水合氯醛麻醉后,剃去背部毛约2*2cm大小,外用咪喹莫特乳膏62.5mg,每天一次,外用不同浓度(0.5ng/ml,1ng/ml和2ng/ml)的CRNN抗体(catalog number:11799-1-AP)40ul,每天一次,每天拍照并PASI评分,共7天,第八天脱颈椎处死小鼠后,取皮损,一部分做免疫组化,一部分提蛋白做WB。
不同浓度的CRNN外用治疗咪喹莫特小鼠的临床照片结果如图1所示,其中对照组为0.1%叠氮钠,治疗组分别为0.5,1,2ng/ml的CRNN;
0.5ng/ml的CRNN治疗咪喹莫特小鼠的PASI评分结果如3所示,其中A为总的PASI评分,B为红斑的PASI评分,C为鳞屑的PASI评分,D为皮肤厚度的PASI评分。
咪喹莫特小鼠皮损对照实验结果表明经过常规的治疗银屑病药物外用和cornulin抗体治疗后,小鼠皮损症状均明显改善。
2)CRNN治疗咪喹莫特小鼠皮损的Western blot检测
检测方法如下:
(1)细胞总蛋白的提取
a)提前按照100∶1的比例将RIPA裂解液和PMSF混合均匀,放置于冰上;
b)弃去六孔板或1.5cm培养皿中的培养基,用PBS洗两遍;
c)每孔加入150μl的裂解液,冰上裂解20min;
d)冰上充分刮细胞,将其转移至EP管中;
e)用液氮反复冻融3次;
f)4℃12000rpm离心15min,小心吸取上清,转移至另一个EP管中;
g)BCA法测蛋白浓度:试剂盒A液和B液按照50∶1的浓度混合均匀,向96孔板的每个孔中加入98μl,再加入2μl所提蛋白或者标准蛋白品(0μg/μl,0.25μg/μl,0.5μg/μl,1μg/μl,2μg/μl),振荡器上震荡混合均匀后,放入37℃恒温箱中30min,然后在分光光度仪上测定OD562nm,根据标准曲线,计算出蛋白浓度。
h)蛋白内加入1/4体积的5×上样缓冲液,95℃煮5min后-20℃保存备用。
(2)SDS-PAGE电泳
a)配制聚丙烯酰氨凝胶电泳的下层胶,待胶凝固后,倒入上层胶,插入洗净干燥的梳子,确保无气泡,静置20-30min待浓缩胶聚合;
b)将胶固定于蛋白电泳槽中,根据蛋白浓度,调整上样量,保证每孔上相同含量蛋白(约20-25μg),同时加入预染蛋白分子量marker 5μl以确定待检蛋白的分子量;
c)电泳:加样后,于15mA电泳30min后用25mA电泳约60min,至溴酚兰至分离胶底部停止电泳;
d)取下凝胶,置于转膜缓冲液中。
(3)转膜
a)准备2张滤纸和1张PVDF膜,尺寸和SDS-PAGE凝胶的大小相近;
b)PVDF膜需先在甲醇中浸泡1min,然后浸泡于转移缓冲液中备用;
c)转膜装置上从阴极向阳极依次放置滤纸-凝胶-PVDF膜-滤纸,注意各层间无气泡;
d)恒流230mA转膜1.5-2h。
(4)封闭
用TBST配置的5%脱脂奶粉或BSA的封闭液室温封闭PVDF膜1h。
(5)抗体孵育
a)将PVDF膜按照抗体的分子量大小才成适当宽度的条带;
b)用TBST漂洗一下后,加入按要求浓度配制好的一抗,4℃孵育过夜;
c)回收一抗,用TBST将膜漂洗3次,每次10min;
d)加入按要求浓度配好的二抗,温孵育1h;
e)再用TBST将膜漂洗3次,每次10min,然后倒尽液体。
(6)显影(暗室中操作)
a)取等体积的化学发光试液A液和B液,用前临时混合均匀;
b)将配置好的发光液均匀铺于PVDF膜上,反应1min后滤纸吸干发光液,放置胶片,根据曝光效果调整曝光时间;
c)取出胶片浸入显影液中,清水漂洗后定影,晾干后进行扫描采图与分析。
(7)膜再生strip法
a)发光后的PVDF膜用TBST漂洗5min;
b)加入适量的膜再生液,室温孵育30min;
c)再用TBST漂洗5min×3遍;
d)重新封闭、抗体孵育、显影,同步骤(4)(5)(6)。
CRNN治疗咪喹莫特小鼠皮损和对照组小鼠皮肤组织蛋白的Western blot检测结果如图2所示,可以看到治疗效果与CRNN含量正相关,治疗效果更好的对照组的CRNN含量更高。
3)正常皮肤组织和银屑病皮损组织中cornulin检测,包括免疫组织化学染色和western blot
1)免疫组织化学染色
(1)临床标本的处理
将手术过程中得到的标本用生理盐水彻底冲洗,去除血液和污染物,随后将组织块置于4%多聚甲醛中,固定24h。
(2)载玻片的处理
a)将载玻片洗洁精超声清洗后,放入重铬酸钾-浓硫酸混合液中浸泡24h;
b)酸缸中取出后用自蒸馏水冲洗至完全干净,置于烤箱中60℃烘干过夜;
c)为防止组织掉片,载玻片需经3-氨丙基-3-乙氧基甲硅烷(3-Aminopropyl-Triethoxysilane,APES)处理。将APES原液与丙酮1∶50稀释成工作液,将洗净的玻片放入新配置的APES工作液中停留30s后取出;
d)取出玻片,停顿10s钟,再放入丙酮溶液30s,涮去未结合的APES;
e)再置于烤箱中烘干2h,装盒后备用。
(3)石蜡包埋和组织切片
a)包埋组织:先在模具中加入一些液态石蜡,稍微冷却后,将待包埋的组织置于石蜡之中,并排列整齐,再将塑料模具盒盖上,最后加入少许液体石蜡,冷却使其变成固态;
b)切片:将包埋好的组织从模具上取下来,置于石蜡切片机上,调节切片的厚度4μm,连续切片;
c)切片置于60℃烤箱中烤90min,然后转入37℃烤箱中过夜。
(4)免疫组织化学染色步骤
a)将组织切片置于二甲苯中脱蜡10min×2次;
b)再依次置于无水酒精、95%酒精、90%酒精、80%酒精、70%酒精各10min;
c)置于PBS中水化5min×3次;
d)滴加新鲜配制的3%H2O2,室温孵育20min,以阻断内源性过氧化物酶,然后用PBS洗5min×3次;
e)将切片放入盛0.01M枸橼酸钠缓冲溶液(pH6.0)的高压锅中,加热至压力阀开始喷气时开始计时2min,然后离开热源,冷却后取下气阀,取出切片,蒸PBS洗5min×3次;
f)滴加5%山羊血清37℃封闭20min,甩除勿洗;
g)加一抗:滴加稀释好的一抗工作液(CRNN的稀释比例为1∶100),4℃过夜;
h)室温复温30min,用PBS洗5min×3次;
i)加二抗:滴加生物素标记二抗,37℃孵育30min,PBS洗5min×3次;
j)滴加辣根过氧化酶标记的链霉卵白素,37℃孵育20min,PBS洗5min×3次;
k)按照说明书配制DAB显色液,滴加于切片上,显色3-5min,显微镜下观察显色反应;
l)清水冲洗后,用苏木素复染2min,再用清水冲洗;
m)将切片依次置于70%酒精、80%酒精、90%酒精、95%酒精、无水酒精各3min,二甲苯透明10min×2次;
n)中性树胶封片,阴性对照以PBS代替一抗,其余步骤相同,在显微镜下观察、拍照并对结果进行分析。
(5)免疫组化染色结果的判定
由两位医师分别双盲阅片。阳性表达为细胞质和(或)细胞核内出现黄色、棕黄色或褐色颗粒。在400倍镜下随机挑选10个视野,观察细胞染色强度,计算阳性细胞百分率。阳性率≤5%为0分,6%~25%为1分,26%~50%为2分,51%~75%为3分,>75%为4分;染色强度分为4级,无着色为0分,淡黄色为1分,棕黄色为2分,棕褐色为3分。每个视野阳性细胞百分率与染色强度相乘为该视野得分,最终得分为5个视野得分的平均值。0分为阴性(-),1~4分为弱阳性(+),5~8分为中等程度阳性(++),9~12分为强阳性(+++)。
检测结果分别如图4、图5所示,结果表明在皮损组织中CRNN的含量较正常组织提升明显。以上结果表明CRNN与银屑病的发展呈正相关。
4)应用M5刺激HaCaT和HEKa细胞模拟银屑病细胞的检测
应用M5(IL-17A,IL-22,IL-1a,OSM和TNF-a混合物)刺激HaCaT和HEKa细胞模拟银屑病细胞水平,然后提取cornulinRNA进行检测;具体操作如下:
(1)细胞复苏
a)将细胞冻存管从液氮罐中取出,立即置于37℃水浴箱中,使其在1min内迅速融化;
b)加入事先准备好的含有5ml 10%FBS的DMEM培养基的中,800rpm离心5min,弃去上清;
c)加入5ml含10%FBS的DMEM培养基重悬细胞,接种到25cm2培养瓶中,置37℃、5%CO2培养箱中培养,次日更换培养基。
(2)细胞培养
细胞常规培养于37℃恒温、5%CO2培养箱中,每2d更换一次培养基。
(3)细胞传代
a)当细胞覆盖率达到80%以上时,可以进行传代。先弃去旧的培养基,用PBS洗2次;
b)加入2ml 0.25%胰酶消化,37℃放置约5min,在倒置显微镜下观察,可见细胞变圆,细胞间隙变大,此时立刻加入4ml含10%FBS的DMEM培养基终止消化,用移液枪轻轻吹打细胞悬液,转入离心管中;
c)将细胞悬液移至离心管,800rpm离心5min,弃去上清,加入新鲜的培养基重悬,重新接种于新的培养瓶中继续培养。一般传代比例为1∶2-4。
(4)细胞冻存
a)当细胞覆盖率达到80%以上且状态较好时时,可进行细胞冻存。先弃去旧的培养基,用PBS洗2次;
b)用0.25%胰酶消化,含10%FBS的DMEM培养基终止消化,转移到离心管中,800rpm离心5min;
c)弃去上清,加入1ml细胞冻存液,吹打均匀,转移至冻存管中;
d)将冻存管放入冻存盒中后,放入-80℃冰箱过夜,最后转移到液氮罐中进行长期保存。
细胞总RNA的提取及反转录成cDNA
(1)细胞总RNA的提取
a)提前用DEPC浸泡实验所需的枪头、枪头盒、EP管等过夜,以除去RNA酶;
b)弃去六孔板或1.5cm培养皿中的培养基,用PBS洗两遍;
c)加入1ml Trizol静置2min,用加样枪吹打数下,转移至1.5ml EP管中,轻柔颠倒10次,冰上放置5min
d)加入200μl氯仿,上下颠倒10次,使其充分混匀呈乳白状,冰上放置5min,然后4℃12000rpm离心15min;
e)小心地将上层无色透明的上清液(约300μl)转移至新的1.5ml EP管中,加入等体积的异丙醇,轻柔颠倒10次,然后冰上放置20min;
f)4℃12000rpm离心15min,可见管底有白色沉淀,小心弃去上清;
g)加入预冷的75%乙醇1ml洗涤沉淀,然后4℃7500rpm离心5min;
h)弃去上清,室温干燥5-10min,可见底部白色沉淀物变为透明,加入适量的(30-50μl)DEPC水,弹匀并放置在55-60℃10min,使RNA充分溶解;
i)RNA纯度和浓度的测定:吸取1μl提取的RNA加入99μl DEPC水中(稀释100倍),于分光光度仪上测OD260nm和OD280nm值,OD260/OD280在1.9-2.0之间说明所提取的RNA纯度较高。总RNA浓度(μg/ml)=A260nm×40×稀释倍数,以备其后反转录为cDNA实验所需,余RNA产物放于-80℃冰箱保存备用。
(2)反转录反应
a)在冰上向PCR管中按照如下反转录体系加入试剂,混匀之后短暂离心;
5×RT super mix 4μl
RNA 2μg
DEPC水 加至总体积20μl
b)放入PCR仪中,反转录条件:25℃,10min;42℃,30min;85℃,5min;
c)反转录产物放入-20℃保存备用。
4)普通PCR
(1)PCR扩增反应
a)向反转录好的20μl cDNA中加入80μl DEPC水(即稀释至5倍);
b)在冰上向PCR管中按照如下体系加入试剂,混匀之后短暂离心。引物序列见表3-2;
表3-2PCR引物序列
b)放入PCR仪中,反应条件如下所示:
(2)琼脂糖凝胶电泳
a)配2.5%的琼脂糖凝胶,室温冷却20-30min待其凝固成凝胶;
b)取各个RNA样品10μl,Marker 6μl混匀,加入加样孔中;
c)电泳:110V电压下电泳20-30min,用凝胶紫外分析仪观察并拍照。
5)荧光实时定量PCR(qRT-PCR)
(1)在冰上向PCR管中按照如下体系加入试剂,混匀之后短暂离心;
(2)放入实时定量PCR仪中,反应条件如下所示:
95℃ | 10min | |
95℃ | 30s | |
58℃ | 30s | 40个循环 |
72℃ | 30s | |
60℃ | 1min | |
95℃ | 30s | 熔解曲线 |
61.5℃ | 15s |
(3)结果判定:用2-ΔΔCt的方法对CT值进行分析,计算该基因的相对表达量。
real-timePCR测cornulin mRNA的相对表达变化检测结果如图6所示,结果表明刺激HaCaT和HEKa细胞模拟银屑病细胞水平后,cornulin表达升高
5)对M5刺激HaCaT和HEKa细胞应用siRNA敲除cornulin后进行检测
针对cornulin的siRNA为si-negative、si-crnn1和si-crnn2,其序列分别为:
siRNA转染过程
a)转染前24h将约1.5-2×105个细胞接种至6孔板,用含10%FBS DMEM培养基在37℃、5%CO2的温箱中孵育24h,转染时细胞融合度需要达到60-70%;
b)将Lipofectamine 2000以5μl/孔加入250μl/孔的Opti-MEM减血清培养基中,轻轻上下颠倒混匀后室温孵育5min;
c)分别取5μl/孔的CRNN-siRNA和阴性对照siRNA分别加入250μl/孔的Opti-MEM减血清培养基中混合均匀;
d)将b)和c)中的液体混合,轻柔颠倒混匀,室温孵育20min,以便形成siRNA与Lipofectamine 2000的转染复合物;
e)把相应的siRNA与Lipofectamine 2000的混合液滴加到各孔中,用纯DMEM培养基补充至每孔的液体总量为2ml,轻柔地摇晃6孔板以混匀,做好标记后将6孔板放入细胞培养箱;
f)常规培养6h后,更换为含10%FBS和1%双抗的普通DMEM培养基。
(3)转染效率的验证
a)转染24h后提取RNA进行qRT-PCR实验检测CRNN mRNA表达水平;
b)转染48h后提取蛋白进行Western Blot实验检测CRNN蛋白表达水平。
图6为在HaCaT细胞应用si-negative、si-crnn1和si-crnn2敲除cornulin后48小时,提细胞总蛋白,检测相关分子的western blot。
结果表明在cornulin被抑制表达之后AKT表达提升,提示cornulin通过活化AKT信号来促成银屑病的进展。
6)MTT法检测敲除cornulin对HaCaT细胞增殖的影响
(1)取对数生长期的细胞,用胰酶消化收集细胞后,用含10%FBS的DMEM培养基制成单个细胞悬液,调整细胞密度为2.5×104个/ml;
(2)96孔板中每孔加入200μl细胞悬液(5×103个细胞/孔),每组设5个复孔,同时设置空白对照组只加培养基,37℃、5%CO2细胞培养箱中常规培养;
(3)24h后行转染(各种试剂用量按照6孔板/96孔板培养基体积换算);
(4)于转染后24h进行检测。测时每孔加入0.5mg/ml的MTT溶液20μl,置于培养箱中继续孵育4h,弃去液体,每孔加入150μl DMSO,室温震荡10min溶解结晶,在波长490nm测定吸光度(OD)值;
(5)转染后48h、72h分别重复步骤(4);
(6)以无细胞完全培养液为本底对照,绘制细胞生长曲线(以平均OD值为纵坐标,以时间为横坐标)。
在HaCaT细胞应用si-negative、si-crnn1和si-crnn2敲除cornulin后24小时、48小时及72小时的细胞增殖如图8所示,结果表明cornulin被抑制表达后影响到所模拟的银屑病细胞的增殖,提示cornulin促进细胞银屑病细胞的增殖。
综上实验检测表明,cornulin与银屑病的发展呈正相关,适当浓度的cornulin抗体治疗咪喹莫特小鼠银屑病模型,能够改善鳞屑和皮损厚度,并且通过抑制AKT信号来达到抑制银屑病发展以及抑制皮损恢复正常。
鉴于此,Cornulin可以作为靶点在制备防治银屑病的药物中的应用。
而且可以在基因水平和/或蛋白水平以Cornulin作为药物靶点。比如通过阻碍Cornulin表达或转录的DNA或RNA,或者是阻碍或抑制Cornulin作用AKT信号的药物。
相应的针对Cornulin的抗体在制备防治银屑病的药物中的应用。
siRNA敲除Cornulin的载体在制备防治银屑病的药物中的应用。
以上给出的实施例是实现本发明较优的例子,本发明不限于上述实施例。本领域的技术人员根据本发明技术方案的技术特征所做出的任何非本质的添加、替换,均属于本发明的保护范围。
Claims (1)
1.针对Cornulin的抗体在制备治疗银屑病的药物中的应用。
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