CN111494609A - IFN-λ2在刚地弓形虫感染中的新用途 - Google Patents
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Abstract
本发明属于生物医疗技术领域,涉及IFN‑λ2在刚地弓形虫感染中的新用途。本发明公开了IFN‑λ2在制备治疗或预防刚地弓形虫感染的药物中的应用。本发明还公开了一种预防或治疗刚地弓形虫感染的组合物,其特征在于,所述组合物的活性成分为IFN‑λ2。本发明另外还一种组合物在制备预防或治疗刚地弓形虫感染的药物中的应用,其特征在于的,所述组合物包括IFN‑λ2和一种或多种药学上可接受的载体。本发明为刚地弓形虫感染的治疗提供新的途径和方法,解决现有技术中无法治疗刚地弓形虫感染所致不良妊娠的问题。
Description
技术领域
本发明属于生物医疗技术领域,涉及IFN-λ2在刚地弓形虫感染中的新用途,具体涉及IFN-λ2在制备治疗或预防刚地弓形虫感染的药物中的应用、一种预防或治疗刚地弓形虫感染的组合物、一种组合物在制备预防或治疗刚地弓形虫感染的药物中的应用。
背景技术
现有技术中对IFN-λ在治疗刚地弓形虫感染所致不良妊娠中的用途没有得到有效开拓,是本领域长期以来一直研究的课题。
刚地弓形虫(Toxoplasma gondii)是重要的机会致病性原虫。免疫力正常的个体感染弓形虫后多呈隐性感染状态。对于免疫功能损伤或免疫缺陷患者,如艾滋病、器官移植及恶性肿瘤患者,弓形虫感染是导致死亡的重要原因。孕妇感染可影响胎儿的发育,导致流产、畸胎、死胎、早产、出生缺陷等先天性弓形虫病。近年来,随着城市人口急剧增加,饲养宠物(尤其是猫)人数不断壮大,人们喜食野生动物等不良饮食习惯等原因,越来越多的人暴露在感染弓形虫的危险之下。据统计,全球约有10亿人感染了弓形虫,其中孕妇感染率为10%~27.5%,每年约有9万新生儿受到弓形虫感染的威胁。中国作为人口大国,如何有效地预防和治疗先天性弓形虫病是我们面临的巨大挑战。
干扰素(Interferon,IFN)主要分为3型:I型干扰素(IFN-α和IFN-β)、II型干扰素(IFN-γ)及III型干扰素(IFN-λ)。I型IFN(IFN-α和IFN-β)主要以抗病毒为主,通过相关信号通路诱导多种干扰素激活基因(IFN-stimulated gene,ISGs)表达,从而发挥抗病毒的作用。II型干扰素(IFN-γ)是抑制弓形虫增殖的重要的细胞因子。弓形虫速殖子侵入机体后,刺激巨噬细胞产生IL-12,进而激活NK细胞和T细胞,使之分泌IFN-γ。IFN-γ随之诱导IFN-γ-inducible genes表达,继而直接杀伤寄生在宿主细胞中的速殖子。但是,在妊娠期间,IFN-γ的产生及过量分泌是发生不良妊娠的主要因素。IFN-γ通过招募CD49b+NK,同时调节NK细胞Ly-49受体的表达,导致流产的发生。在大鼠流产模型中,IFN-γ上调tumornecrosis factor–α(TNF-α),同时下调Matrix metalloproteinases-2and-9(MMP-2andMMP-9)表达,进一步加重流产。在人类的妊娠中,IFN-γ导致不良妊娠的作用同样得到了证实,胎儿来源的T细胞通过分泌IFN-γ及TNF-α促进子宫收缩,从而导致早产的发生。因此,IFN-γ在妊娠期间无法保护母体抵抗弓形虫感染,维持正常妊娠。
IFN-λ(III型干扰素)具有相对独立和特异性的抵抗病原体感染的能力。IFN-λ受体由白介素28受体α(Interleukin-28receptor alpha,IL-28Rα)及白介素10受体β(Interleukin-10Receptor Beta,IL-10Rβ)组成,IL-10Rβ广泛存在于细胞和组织,IL-28Rα则主要表达在上皮细胞、中性粒细胞及肝细胞的表面。受体表达的局限性意味着,IFN-λ在某些组织发挥着相对独立和特异性的抗病原体的作用。人类IFN-λ家族由IFN-λ1、IFN-λ2、IFN-λ3和IFN-λ4组成,而小鼠只有两种功能性IFN-λ,即IFN-λ2和IFN-λ3。在轮状病毒的研究中,研究者发现IFN-λ2能显著抑制幼鼠肠道中的轮状病毒复制,IFN-λ受体敲除则促进小鼠体内轮状病毒增殖。Muir等研究者首次提出IFN-λ1a能抑制丙型肝炎病毒(hepatitis Cvirus,HCV)的增殖。IFN-λ1a能快速(12小时内)抑制HCV的复制,并在24小时内能达到与IFN-α相似的作用。但患者出现血小板减少症及嗜中性粒细胞减少症等并发症的比例明显下降。因此,IFN-λ1a有望在临床上取代IFN-α用于HCV的治疗。Rebecca L.等以烟曲霉(Aspergillus fumigatus,Af)为模型研究抗真菌免疫反应,发现CCR2+单核细胞的耗竭降低了中性粒细胞抑制侵袭性真菌生长的能力。IFN-λ2/3直接作用于中性粒细胞以激活其抗真菌反应,而中性粒细胞特异性缺失IFN-λ受体的小鼠则死于侵袭性曲霉菌病。通过过继转移CCR2+单核细胞或IFN-λ2/3均能有效治疗中性粒细胞CCR2耗竭的小鼠。因此,IFN-λ2/3是中性粒细胞的关键调节因子,发挥抗真菌的作用。微小隐孢子虫(Cryptosporidiumparvum,C.parvum)作为重要的机会性致病原虫,引起以腹泻为主的临床表现。外源性IFN-λ3可以减轻肠上皮细胞(Intestinal Epithelial Cells,IECs)内的虫荷数,恢复跨膜电阻(Transepithelial electrical resistance,TEER),抵抗C.parvum入侵。
尽管大量的实验数据支持IFN-λ2/3具有抑制病毒、真菌及微小隐孢子虫增殖的能力,但IFN-λ2能否抑制弓形虫的增殖,有待进一步研究。
发明内容
发明目的:本发明提供IFN-λ2在制备治疗或预防刚地弓形虫感染的药物中的应用、一种预防或治疗刚地弓形虫感染的组合物、一种组合物在制备预防或治疗刚地弓形虫感染的药物中的应用,以解决现有技术中的问题。
针对上述发明目的,本发明的实施例提供IFN-λ2在制备治疗或预防刚地弓形虫感染的药物中的应用。
进一步的,所述IFN-λ2干预上调胎盘中CK功能分子的表达及抑制刚地弓形虫的增殖。
本发明的实施例还提供一种预防或治疗刚地弓形虫感染的组合物,其特征在于,所述组合物的活性成分为IFN-λ2。
进一步的,所述组合物包括IFN-λ2和一种或多种药学上可接受的载体。
其中,所述载体包括药学上可接受的稀释剂、赋形剂、填充剂、粘合剂、促进吸收剂、表面活性剂和增效剂。
本发明的实施例另外还提供一种组合物在制备预防或治疗刚地弓形虫感染的药物中的应用,其特征在于的,所述组合物包括IFN-λ2和一种或多种药学上可接受的载体。
进一步的,所述载体包括药学上可接受的稀释剂、赋形剂、填充剂、粘合剂、促进吸收剂、表面活性剂和增效剂。
本发明的上述技术方案的有益效果如下:
(1)本发明的实施例通过制备多组孕鼠模型,从多组孕鼠模型实验证明IFN-λ2干预能显著降低弓形虫感染导致的流产率、减轻弓形虫感染所致的小鼠胎盘的损伤、上调胎盘中CK等功能分子的表达;同时,本发明通过弓形虫与JEG-3细胞共培养后,PCR检测各细胞培养模型组中弓形虫SAG1的表达,验证了IFN-λ2干预能抑制刚地弓形虫的增殖,从而本发明的实施例验证了IFN-λ2干预能够治疗刚地弓形虫所致不良妊娠、抑制刚地弓形虫的增殖,为治疗刚地弓形虫感染提供了理论基础。
(2)本发明提供IFN-λ2在抑制刚地弓形虫感染方面的应用,一种预防或治疗刚地弓形虫感染的药物,为刚地弓形虫感染的治疗提供新的途径和方法,解决现有技术中无法治疗刚地弓形虫感染所致不良妊娠的问题。
附图说明
图1为本发明的实施例1中孕鼠未感染刚地弓形虫组、孕鼠感染刚地弓形虫组、孕鼠经IFN-λ2预处理24h后,感染刚地弓形虫组的流产情况图;
图2为本发明的实施例2中孕鼠未感染刚地弓形虫组、孕鼠感染刚地弓形虫组、孕鼠经IFN-λ2预处理24h后,感染刚地弓形虫组的小鼠胎盘的损伤情况图;
图3为本发明的实施例3中孕鼠未感染刚地弓形虫组、孕鼠感染刚地弓形虫组、孕鼠经IFN-λ2预处理24h后,感染刚地弓形虫组的胎盘CK的表达情况图;
图4为本发明的实施例4中弓形虫与JEG-3细胞共培养后,细胞未感染刚地弓形虫组、细胞感染刚地弓形虫组、细胞经刚地弓形虫及IFN-λ2共同刺激组、细胞经预处理IFN-λ224h后,感染刚地弓形虫组、细胞与刚地弓形虫及IFN-γ共同处理组、细胞经预处理IFN-γ24h后,感染刚地弓形虫组的弓形虫SAG1表达情况图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合具体实施例进行详细描述。
实验材料
1.1、JEG-3细胞株购自Thermo Fisher Scientific公司。
1.2、Trizol试剂购自美国Invitrogen公司。
1.3、逆转录试剂购自美国Bio-Rad公司。
1.4、SYBR Green Master Mix,TaqMan Universal Master Mix购自美国ThermoFisher Scientific公司。
1.5、引物由美国Sigma合成。
1.6、胎牛血清购自Hyclone公司。
1.7、胰酶及MEM购自美国Thermo Fisher Scientific公司。
孕鼠实验准备
取至少24只公鼠和48只母鼠分成24个组,每个组包括2只母鼠和一只公鼠,2只母鼠与1只公鼠于前一晚下午5点合笼,次日7点检测白色的阴道栓。若检出阴道栓,则确定小鼠孕期为E0.5(embryonic day 0.5)。
对24组孕鼠分组,并制备成不同组的模型组,具体分组如下:
(1)孕鼠未感染刚地弓形虫组(正常妊娠组);
(2)孕鼠感染刚地弓形虫组;
(3)孕鼠经IFN-λ2预处理24h后,感染刚地弓形虫组;
本发明的实施例1-实施例3准备以上三组孕鼠,每组至少四只孕鼠(各孕鼠的孕期相同),进行以下实施例1-3的实验。
实施例1、IFN-λ2干预能显著降低弓形虫感染导致的流产率
孕鼠妊娠第9.5天(E9.5)注射IFN-λ2(2μg/ml),在E10.5天,孕鼠感染刚地弓形虫。随后,孕鼠每天注射IFN-λ2直至妊娠18.5天。监测孕鼠体重的变化,观察孕鼠的流产率、胎鼠的体重和胎鼠的大小。孕中期感染弓形虫,孕鼠体重与自身体重相比呈下降趋势。在E18.5天剖杀,通过顶臀长度(mm)×枕额径(mm2)以测量胎鼠的大小,同时观察流产率。收集母鼠及胎鼠体内的各个组织样本,进行后续实验。
结果如图1所示,与孕鼠未感染刚地弓形虫组(正常妊娠组)相比,孕鼠感染刚地弓形虫组中弓形虫感染导致58.9%左右的流产率。而IFN-λ2干预后,孕鼠经IFN-λ2预处理24h后,感染刚地弓形虫组中孕鼠的流产率显著下调。
这些结果提示:IFN-λ2能改善弓形虫感染导致的不良妊娠结局。
实施例2、IFN-λ2能减轻弓形虫感染所致的小鼠胎盘的损伤
孕鼠在E10.5天感染弓形虫后,于E18.5天收集小鼠胎盘,福尔马林固定进行HE染色以观察胎盘的结构变化。
其中,HE染色的方法如下:自来水冲洗玻片3min。蒸馏水冲洗1min。将玻片置于4℃预冷3%Triton X-100溶液中进行通透5min。苏木精染色10min。自来水冲洗30s。分化液(2%盐酸乙醇)20s。自来水冲洗蓝化5-10min。伊红染色30s-2min。50%、70%、80%、95%、无水乙醇梯度脱水1-3min。二甲苯I、II透明1-3min。中性树脂封片,显微镜下拍摄图像。
从HE染色结果可以看出,正常小鼠胎盘的正常结构分为蜕膜层(Decidua,DE),连接区(Junctional Zone,JZ)及迷路区(Labyrinth Zone,LZ)。与孕鼠未感染刚地弓形虫组(正常妊娠组)相比,孕鼠感染刚地弓形虫组中弓形虫感染破坏胎盘的正常结构,表现为蜕膜层及连接区变薄,伴随着胎盘的迷路区细胞数量明显减少,如图2所示,提示胎盘营养物质提供减少以及气体交换障碍。而通过IFN-λ2干预之后,孕鼠经IFN-λ2预处理24h后,感染刚地弓形虫组中,蜕膜层及连接区的厚度接近于正常小鼠胎盘,同时,迷路区的细胞数量明显增加。
这些结果表明,弓形虫感染能破坏小鼠胎盘的正常结构,而IFN-λ2能显著减轻胎盘的损伤。
实施例3、IFN-λ2干预能上调胎盘中CK等功能分子的表达
Cytokeratin(CK)是小鼠胎盘滋养层细胞的标志蛋白。
细胞免疫荧光检测的方法:收集小鼠胎盘组织,制成6-8微米切片。染片时,切片在室温晾干15分钟。然后置PBS中浸泡10分钟,以去除OCT;0.5%Triton X-100(PBS配制)室温通透20min;用含10%正常山羊血清的PBS室温封闭切片1h;将稀释的一抗滴在玻片上,置于4℃冰箱孵育过夜。PBS洗涤3次后,将稀释的荧光二抗滴在玻片上,室温避光孵育90min。PBS洗涤3次后,Hoechst染料滴在玻片上,于室温避光孵育15min。PBS洗涤3次后,50%的甘油封片。运用激光共聚焦拍摄图像。
在正常妊娠组(孕鼠未感染刚地弓形虫组),小鼠胎盘中观察到大量CK的表达,而在孕鼠感染弓形虫感染组,小鼠胎盘中CK数量显著下降,表明弓形虫能降低小鼠胎盘滋养层细胞的数量。如图3所示,孕鼠经IFN-λ2预处理24h后,感染刚地弓形虫组,即在进行IFN-λ2干预后,CK表达明显增加,提示IFN-λ2能减轻胎盘损伤,改善胎盘功能。
实施例4、IFN-λ2干预抑制刚地弓形虫的增殖
在本实施例中,采用细胞培养的体外实验验证IFN-λ2干预抑制刚地弓形虫的增 殖;其中,细胞模型组可分为以下六组:细胞未感染刚地弓形虫组、细胞感染刚地弓形虫组、细胞经刚地弓形虫及IFN-λ2共同刺激组、细胞经预处理IFN-λ224h后,感染刚地弓形虫组、细胞与刚地弓形虫及IFN-γ共同处理组、细胞经预处理IFN-γ24h后,感染刚地弓形虫组。
刚地弓形虫感染JEG-3细胞株的获得方法如下:将JEG-3细胞株培养于细胞培养瓶中,用含10%的胎牛血清的MEM完全培养基(含100μg/mL的链霉素以及100U/mL的青霉素),置于37℃5%CO2的细胞培养箱中培养。细胞隔天更换培养液,并在细胞达到80%左右的融合度时进行细胞传代或者进行后续实验。2×105/孔JEG-3细胞铺板于6孔板,细胞培养箱内培养24h后,刚地弓形虫以感染复数(multiplicity of infection,MOI)为2的比例感染JEG-3细胞株。
IFN-λ2预处理JEG-3细胞后,与刚地弓形虫共培养,real-time PCR检测SAG1的表达。检测刚地弓形虫的数量的方法如下:收集组织或细胞,取适当重量的组织或细胞加入Trizol裂解,提取RNA,用逆转录试剂盒,以Oligo(dT)18逆转录为cDNA。-80℃冻存。以10倍比稀释的刚地弓形虫感染JEG3细胞后,提取的RNA逆转录为cDNA,并以此为模板,在荧光定量PCR仪上扩增,制定标准曲线。反应条件:95℃3min预变性;40个循环:95℃,15s;60℃,1min。对应标准曲线,计算出的刚地弓形虫SAG1相对量。
IFN-λ2预处理JEG-3细胞后,与刚地弓形虫共培养,real-time PCR检测SAG1的表达。结果如下:IFN-γ在体外与刚地弓形虫共同作用于JEG-3细胞,共同培养48h后,收集细胞培养上清,用Real-time PCR法检测弓形虫SAG1的表达。结果发现,细胞与刚地弓形虫及IFN-γ共同处理组作为阳性对照组,其中的IFN-γ具有抑制弓形虫增殖的能力。而细胞经预处理IFN-γ24h后,感染弓形虫组(另一阳性对照组)中弓形虫增殖受到抑制。同样地,细胞经刚地弓形虫及IFN-λ2共同刺激组中IFN-λ2能显著降低SAG1的表达。而经IFN-λ2预处理后,如图4所示,细胞经预处理IFN-λ2 24h后,感染刚地弓形虫组中弓形虫SAG1的表达进一步下降。这些结果表明,IFN-λ2在体外能抑制刚地弓形虫增殖。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.IFN-λ2在制备治疗或预防刚地弓形虫感染的药物中的应用。
2.根据权利要求1所述的IFN-λ2在制备治疗或预防刚地弓形虫感染的药物中的应用,其特征在于,所述IFN-λ2干预上调胎盘中CK功能分子的表达及抑制刚地弓形虫的增殖。
3.一种预防或治疗刚地弓形虫感染的组合物,其特征在于,所述组合物的活性成分为IFN-λ2。
4.根据权利要求3所述的一种预防或治疗刚地弓形虫感染的组合物,其特征在于,所述组合物包括IFN-λ2和一种或多种药学上可接受的载体。
5.根据权利要求4所述的一种预防或治疗刚地弓形虫感染的组合物,其特征在于,所述载体包括药学上可接受的稀释剂、赋形剂、填充剂、粘合剂、促进吸收剂、表面活性剂和增效剂。
6.一种组合物在制备预防或治疗刚地弓形虫感染的药物中的应用,其特征在于的,所述组合物包括IFN-λ2和一种或多种药学上可接受的载体。
7.根据权利要求6所述的一种组合物在制备预防或治疗刚地弓形虫感染的药物中的应用,其特征在于,所述载体包括药学上可接受的稀释剂、赋形剂、填充剂、粘合剂、促进吸收剂、表面活性剂和增效剂。
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