CN116139279A - C1inh在治疗宫腔粘连中的应用 - Google Patents
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Abstract
本发明公开了C1INH在治疗宫腔粘连中的应用。本发明发现炎症因子预处理hUC‑MSCs显著降低IUA样小鼠子宫内膜炎症水平和CD301+巨噬细胞数量，从而抑制子宫内膜纤维化。体外结果显示，炎症因子预处理hUC‑MSCs分泌的C1INH显著高于对照hUC‑MSCs，且C1INH可以抑制炎症引起的CD301+巨噬细胞极化。我们首次发现C1INH可以抑制炎症引起的CD301+巨噬细胞极化，从而治疗子宫内膜纤维化，可以用于制备治疗宫腔粘连或者其他与子宫纤维化相关的疾病的药物。
Description
技术领域
本发明属于生物医药领域，涉及C1酯酶抑制物(C1INH)在治疗宫腔粘连中的应用。
背景技术
宫腔粘连(intrauterine adhesion,IUA)是一种纤维化疾病，其特征是多种因素引起的子宫内膜基底层受损，导致子宫内膜干细胞不足、血管稀疏、腺体再生障碍和子宫内膜纤维化。我们前期研究发现，IUA患者子宫内膜中存在炎症异常激活，且CD301+巨噬细胞是导致子宫内膜纤维化的主要细胞类型。IUA是继发性不孕的最常见的病因，在不孕妇女中占比高达25-30％，在我国，随着各种宫腔手术特别是人流刮宫术数量的上升，IUA的发生率明显增高。目前IUA的治疗金标准是宫腔镜下粘连松解术，其对轻中度的IUA尚有效，但对重度广泛内膜损伤效果有限，且轻中度IUA复发的风险将近30％，尤其是重度IUA复发率超过62％，组织再粘连仍然是一个挑战。因此亟需寻找一种有效的IUA治疗方法。
C1酯酶抑制物(C1INH)，又称C1抑制物(C1 inhibitor)，是目前唯一已知的既能通过补体经典途径，又能通过凝集素途径对补体系统进行调节的血浆蛋白酶抑制物，其在补体系统，激肽释放酶-激肽系统，纤溶系统和凝血系统中发挥重要的调节作用。C1INH缺乏可导致血管性水肿，从而导致疼痛，身体虚弱，甚至危及生命。除此之外，近年研究发现C1INH与细菌内毒素，粘附分子，E,P-选择素等结合发挥非蛋白酶抑制功能，因而广泛参与危重病领域，包括全身炎症反应综合征和脓毒血症，Covid-19感染，缺血再灌注损伤，移植，热损伤及神经脊髓炎等。但是C1INH是否能够调节子宫内膜的免疫微环境，从而治疗子宫内膜纤维化仍未见报道。
发明内容
本发明的目的是提供C1INH在制备治疗宫腔粘连的药物中的应用。
本发明的目的通过以下技术方案实现：
C1INH在制备治疗宫腔粘连的药物中的应用。
C1INH在制备治疗因子宫纤维化引起的疾病的药物中的应用。
本发明中所述的宫腔粘连指由于子宫内膜基底层损伤，功能层再生修复障碍，形成以内膜纤维化为特征的子宫壁间粘连。
本发明中所述的子宫内膜纤维化引起的疾病指宫腔粘连或子宫内膜疤痕化以及由此导致的子宫性不孕、反复流产及胎盘植入等。
炎症因子预处理hUC-MSCs在制备治疗宫腔粘连的药物中的应用。
炎症因子预处理hUC-MSCs在制备治疗因子宫纤维化引起的疾病的药物中的应用。
所述的炎症因子预处理hUC-MSCs通过以下方法制备：IL-1β10ng/ml+TNFα20ng/ml+IFN-γ20ng/ml在37℃，5％ CO2处理hUC-MSCs 24小时。
有益效果：
炎症因子预处理hUC-MSCs通过分泌C1INH降低小鼠子宫内膜炎症水平和CD301+巨噬细胞数量，从而抑制子宫内膜纤维化。我们首次发现C1INH可以抑制炎症引起的CD301+巨噬细胞极化，从而治疗子宫内膜纤维化，可以用于制备治疗宫腔粘连或者其他与子宫纤维化相关的疾病的药物。
附图说明
图1、A：Masson三色染色、α-SMA和Collagen 1组化染色分析小鼠子宫内膜组织纤维化水平；B：免疫组化和免疫荧光分析小鼠子宫内膜组织中CD301+巨噬细胞数量；
图2、A：炎症因子预处理hUC-MSCs和对照hUC-MSCs的差异基因热图；B：qPCR分析细胞中Serping1(C1INH)的表达量；C：WB分析细胞中C1INH的蛋白含量；
图3、A：流式检测CD301+巨噬细胞占比和CD301-APC平均荧光强度(MFI)；B：代表性流式细胞术点图；
具体实施方式
实施例1炎症因子预处理hUC-MSCs抑制子宫内膜CD301+巨噬细胞极化及纤维化1、材料，试剂，设备
1.1IUA样小鼠模型构建及hUC-MSCs治疗
8～10周龄雌性C57BL/6小鼠，在SPF条件下饲养，体重约20g，建立IUA样小鼠模型，在动情期进行刮宫和炎症损伤。吸入异氟醚麻醉后，开腹暴露小鼠子宫，用表面粗糙的7号针头在子宫侧壁仔细搔刮约50次，直至子宫充血，再将10μL LPS(1mg/ml；Sigma)注射入子宫角，用镊子夹闭5min。最后，将子宫轻轻返回腹腔，关闭腹壁。造模后第2天行二次开腹术，将装载于Matrigel(Corning)的炎症因子预处理hUC-MSCs(IL-1β(R&D system；10ng/ml)+TNFα(R&D system；20ng/ml)+IFN-γ(R&D system；20ng/ml)37℃，5％ CO2处理hUC-MSCs24小时)和对照hUC-MSCs(2×104/ul,20ul)分别注入子宫角，用镊子夹闭7min后还纳子宫关闭腹腔。PBS(20ul)和Matrigel(20ul)作为相应的对照。假手术组小鼠仅行开腹和关腹手术，未对子宫进行任何操作。分别于细胞治疗后第3和5天(IUA样模型建立后第5和7天)处死小鼠，收集子宫进行进一步研究。所有实验操作均经南京大学医学院附属鼓楼医院动物实验委员会批准。
1.2主要试剂
二甲苯、无水乙醇、95％、80％、75％乙醇、3％双氧水、Masson三色染色试剂盒(索莱宝)、柠檬酸修复液、EDTA修复液、α-SMA抗体(abcam)、Collagen 1抗体(proteintech)、CD301抗体(R&D Systems)、F4/80抗体(abcam)、DAB、苏木素。
1.3主要仪器
烘箱、显微镜(Leica)。
1.4主要方法
1.4.1子宫内膜组织免疫组化
60℃恒温箱烤片60min，二甲苯处理3次，每次5min，梯度酒精(100％乙醇5min，95％乙醇5min，80％乙醇5min，75％乙醇5min)处理后，水冲洗适当时间，3％H2O2浸泡15min(去除内源性过氧化物酶)，水冲洗适当时间；柠檬酸或EDTA修复后一抗37℃孵育2h，PBST冲洗3遍，每次5min，二抗孵育8min，PBST冲洗3遍，每次5min。DAB显色，苏木素染核，封片后显微镜观察并拍照。
1.4.2子宫内膜组织免疫荧光
冰冻切片室温复温15min，甲醛固定15min，PBST冲洗3遍，每次5min，2％BSA封闭30min，一抗37℃孵育2h，PBST冲洗3遍，每次5min，荧光二抗孵育1h，PBST冲洗3遍，每次5min。DAPI封片后显微镜观察并拍照。
2、结果
在IUA样小鼠模型中，与PBS、Matrigel和对照hUC-MSCs治疗组相比，炎症因子预处理hUC-MSCs治疗可以更加有效地抑制子宫内膜CD301+巨噬细胞极化及纤维化(图1)。实施例2炎症因子预处理hUC-MSCs分泌更多C1INH
1、材料，试剂，设备
1.1主要试剂
hUC-MSCs、炎症因子预处理hUC-MSCs、TRNzol(天根)、反转录试剂(雅酶)、SYBRGgreen(雅酶)、氯仿、异丙醇、C1INH抗体(Proteintech)、电泳液、转膜液、脱脂牛奶(bio-rad)、TBST、
1.2主要仪器
细胞培养箱、qPCR仪(Roche)、PCR仪(ABI)、摇床、PVDF硝酸纤维素膜、双垂直电泳槽、凝胶成像分析系统
1.3主要方法
1.3.1转录组测序分析
对hUC-MSCs和炎症因子预处理hUC-MSCs进行转录组测序分析(基迪奥)，通过DESeq2软件包计算两组数据间的FDR(错误发现率)和FC(差异倍数)，以FDR<0.05和FC>2倍筛选出差异表达基因。
1.3.2细胞RNA提取及实时荧光定量PCR
采用Trizol法提取总RNA。取1ug RNA反转录后得到cDNA，用适量体积的RNase-free水稀释后，SYBR Green方法进行荧光定量PCR检测，不同目标基因的表达量采用GAPDH作为内参标准的ΔΔCT值做统计。
1.3.3WB
弃去培基，用预冷的PBS洗3-4遍，加适量细胞裂解液，放至冰上裂解30min，吸至EP管中离心15min取上清，测蛋白浓度，按上样量30μg计算体积，加loading buffer，99℃金属水浴锅加热10-15min，10％胶跑至溴酚蓝到底，转膜1-2h，5％牛奶封闭1h；一抗4℃孵育过夜，TBST洗5min，洗3遍，二抗室温孵育1h，TBST洗5min，洗3遍；曝光。
2、结果
根据测序结果分析，结合qRT-PCR和WB检测，证实与未处理hUC-MSCs相比，炎症因子预处理hUC-MSCs中Serping1(C1INH)的mRNA水平和蛋白水平均显著上调(图2)，提示炎症因子预处理hUC-MSCs可能通过分泌更多的C1INH来发挥抗纤维化作用。
实施例3C1INH抑制促纤维化CD301+巨噬细胞的极化
1、材料，试剂，设备
1.1主要试剂
THP-1细胞系、RMPI 1640培基(Gibco)、血清(Gibco)、佛波醇肉豆蔻酸酯(PMA，Sigma)、LPS(Sigma)、IFN-γ(R&D Systems)、人重组C1INH蛋白(Peprotech)、胰酶(Gibco)、CD14-FITC(Biolegend)、CD301-APC(Biolegend)、FVS510(BD Biosciences)
1.2主要仪器
细胞培养箱、流式细胞仪(Beckman Coulter)
1.3主要方法
1.3.1细胞培养
THP-1细胞用5ng/ml PMA刺激48小时后分化为幼稚巨噬细胞，用100ng/ml LPS和20ng/ml IFN-γ刺激24小时，诱导细胞极化为CD301+巨噬细胞。之后撤去炎症刺激，加入100ng/ml人重组C1INH蛋白，继续处理24小时。
1.3.2流式细胞术
弃去培基，用预冷的PBS洗3-4遍，加适量胰酶消化并收集细胞。PBS洗涤一次后，加入100ul PBS重悬细胞，并加入FVS510、CD14-FITC和CD301-APC室温孵育15分钟。PBS洗涤一次后，加入200ul PBS重悬细胞，进行流式检测。
2、结果
LPS和IFN-γ刺激显著上调CD301+巨噬细胞的数量，人重组C1INH蛋白处理可以逆转CD301+巨噬细胞的增多，表明C1INH抑制促纤维化CD301+巨噬细胞的极化，进而参与抑制子宫内膜纤维化。
Claims (5)
1.C1INH在制备治疗子宫纤维化引起的疾病的药物中的应用。
2.根据权利要求1所述的C1INH在制备治疗宫腔粘连药物中的应用。
3.炎症因子预处理hUC-MSCs在制备治疗宫腔粘连的药物中的应用。
4.根据权利要求3所述的应用，其特征在于，炎症因子预处理hUC-MSCs在制备治疗因子宫纤维化引起的疾病的药物中的应用。
5.根据权利要求3或4所述的应用，其特征在于所述的炎症因子预处理hUC-MSCs通过以下方法制备：IL-1β10ng/ml+TNFα20ng/ml+IFN-γ20ng/ml在37℃，5％CO2处理hUC-MSCs 24小时。
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