CN114699430A - 线粒体及其在胰腺炎中的应用和应用方法 - Google Patents
线粒体及其在胰腺炎中的应用和应用方法 Download PDFInfo
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Abstract
本发明涉及胰腺炎技术领域,公开了游离线粒体在胰腺炎中的应用以及一种功能化线粒体在胰腺炎及胰腺炎引起的其它器官损伤中的应用,所述功能化线粒体由外源细胞膜与线粒体通过共孵育利用脂质融合得到,可应用于胰腺炎以及胰腺炎相关的其他器官损伤中。本发明将外源细胞膜融合的功能化线粒体单独或联合其他物质用于治疗胰腺炎中,通过静脉给药或局部给药的方式靶向作用于疾病发生部位,功能化线粒体可中和趋化因子和炎性因子,抑制炎性细胞的迁移、侵袭,吸附并中和炎症因子,并促进线粒体进入受损细胞改善胞内应激反应,从而阻止胰腺炎的发生与发展。
Description
技术领域
本发明涉及胰腺炎技术领域,具体而言,涉及线粒体及其在胰腺炎中的应用和应用方法。
背景技术
急性胰腺炎患者约20%会进一步发展为重症急性胰腺炎(severe acutepancreatitis,SAP),SAP伴有持续的器官功能衰竭,如合并感染病死率极高。临床治疗方案以补液、抑酸、抑酶和器官功能维护等为主,多为缓解症状,尚缺乏有效治疗方案。
胰腺腺泡细胞内持续活化的炎症信号通路在胰腺炎的疾病发生和进展中起关键作用。胰腺炎病理过程中,NF-κB信号通路激活调控多种因子包括细胞因子、趋化因子和黏附分子参与免疫细胞浸润受损组织和炎症反应。受损的胰腺腺泡细胞和(或)导管细胞释放损伤相关分子和炎性介质,进一步促进免疫细胞包括巨噬细胞及中性粒细胞浸润和炎症信号通路的激活,促发炎性级联反应,介导系统性炎症反应及远端器官功能衰竭。多器官功能衰竭是急性胰腺炎最严重的全身并发症,也是重症急性胰腺炎致死的主要原因。
目前研究发现,利用外源细胞膜与外援线粒体通过细胞融合的方式可制备具有更高生物活性的功能化线粒体,解决从细胞或机体组织分离提取得到的有生物活性的游离线粒体生物活性不稳定的问题。然而,上述融合修饰得到的功能化线粒体目前仅应用于肝脏细胞线粒体功能障碍疾病中。因此,我们通过对不同器官特定的生命活动进行研究,以得到上述功能化线粒体在更多领域的应用。
发明内容
本发明所要解决的技术问题:
目前,临床治疗急性胰腺炎的方案大多只能缓解症状,缺少可治愈的有效方案,导致病情进一步恶化至重症急性胰腺炎,引起持续性器官功能衰竭等病症。
本发明采用的技术方案:
本发明提供了游离线粒体在胰腺炎中的应用以及一种功能化线粒体在胰腺炎以及胰腺炎相关的其他器官损伤中的应用,功能化线粒体由外源细胞膜与线粒体通过共孵育利用脂质融合得到,还可联合其他物质共同应用于胰腺炎以及胰腺炎相关的其他器官损伤中。具体地,将外源细胞膜与外源线粒体通过共孵育,利用脂质融合获得外源细胞膜融合修饰的线粒体。当机体出现胰腺局部或全身炎性反应时,通过静脉给药或局部给药的方式,使功能化线粒体靶向地到达疾病发生部位,中和趋化因子和炎性因子,抑制炎性细胞的迁移、侵袭,吸附并中和炎症因子,并促进线粒体进入受损细胞改善胞内应激反应,从而阻止胰腺炎的发生与发展。
通过上述游离线粒体治疗胰腺炎或功能化线粒体治疗胰腺炎或胰腺炎相关的其他器官损伤,可降低血清脂肪酶含量;可修复损伤的胰腺组织,使其恢复正常结构;可有效作用于重症急性胰腺炎,减少重症急性胰腺炎的病死率。
附图说明
图1为游离线粒体以及功能化线粒体治疗对生化指标胰脂肪酶的影响;
图2为功能化线粒体治疗对胰腺组织病理学的影响;
图3为功能化线粒体治疗对炎症因子IL-6的影响;
图4为功能化线粒体治疗对急性胰腺炎小鼠存活率的影响;
图5为功能化线粒体治疗对胰腺炎相关肺损伤的影响;
图6为功能化线粒体联合治疗对生化指标胰脂肪酶的影响。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
本发明提供了游离线粒体以及一种功能化线粒体在胰腺炎以及胰腺炎相关的其他器官损伤中的应用,功能化线粒体由外源细胞膜与线粒体通过共孵育利用脂质融合得到,还可联合其他物质共同应用于胰腺炎以及胰腺炎相关的其他器官损伤中。具体地,将外源细胞膜与外源线粒体通过共孵育,利用脂质融合获得外源细胞膜融合修饰的功能性线粒体,其中,外源细胞膜可选择中性粒细胞、单核细胞、淋巴细胞、血小板、细菌、凋亡细胞中的任意一种。将得到的功能性线粒体通过静脉给药或局部给药的方式,使其靶向地到达疾病发生部位,中和趋化因子,抑制炎性细胞活化与趋化,吸附并中和炎症因子,并促进线粒体进入受损细胞改善胞内应激反应,从而阻止胰腺炎的发生与发展。
本发明中,游离线粒体应用于胰腺炎,通过静脉给药或局部给药的方式靶向作用于疾病发生部位。
本发明中,功能化线粒体应用于胰腺炎并发症以及有胰腺炎引起的肺部炎症等胰腺炎相关的其他器官损伤,通过静脉给药或局部给药的方式靶向作用于疾病发生部位;功能化线粒体由外源细胞膜与所述游离线粒体通过共孵育利用脂质融合得到;其中,外源细胞膜包括中性粒细胞、单核细胞、淋巴细胞、血小板、细菌、凋亡细胞中的任意一种。
本发明中,功能化线粒体可联合蛋白酶抑制剂或钙通道抑制剂应用于治疗胰腺炎;其中蛋白酶抑制剂可以选择乌司他丁,钙通道抑制剂可以选择咖啡因。
<实施例>
实施例1
(1)分别建立模型组、对照组、实验组进行分组实验
模型组
构建重症急性胰腺炎动物模型。
Balb/c小鼠(25±2g)实验前禁食12h。用3%戊巴比妥钠麻醉小鼠,小鼠麻醉后,用碘伏对手术部位进行消毒,于剑突下两手指宽度处剖腹,看到肝脏后往下切口,寻找胰胆管和十二指肠乳头;在对准乳头方向用1mL注射器针头在十二指肠上穿孔,将软管沿着乳头插入胰胆管中,用动脉血管夹将肝脏下方总胆管闭合,采用微量注射泵将3.5%牛磺胆酸钠以2mL/h流速缓慢泵入,剂量为2μL/g。牛磺胆酸钠注射完毕后,关闭微量注射泵,留置针继续停留在胰胆管内,保持胆总管闭合3min,取下血管动脉夹,还纳十二指肠,双层缝合腹腔。随后将小鼠置于有电加热板的鼠笼中保温,注射皮下注射适量生理盐水,注意观察小鼠呼吸和恢复情况。多数小鼠术后0.5-1h苏醒,继续监测小鼠情况至少4h。若小鼠情况严重,有异常痛苦及呼吸困难等情况则注射过量麻醉剂使其安乐死,其余无特殊情况的小鼠即作为重症急性胰腺炎动物模型。
对照组
小鼠经麻醉剖签将十二指肠随棉签翻出置于无菌纱布后还纳十二指肠,双层缝合腹腔它操作同模型组。腹腔缝合后分别于30min尾注射与受试药物组相同量的生理盐水。
实验组1
小鼠经麻醉剖腹后,按上述方法造模,腹腔缝合后分别于30min尾静脉注射0.4mg/kg游离线粒体。
实验组2
小鼠经麻醉剖腹后,按上述方法造模,腹腔缝合后分别于30min尾静脉注射0.8mg/kg游离线粒体。
实验组3
小鼠经麻醉剖腹后,按上述方法造模,腹腔缝合后分别于30min尾静脉注射0.4mg/kg功能化线粒体。
实验组4
小鼠经麻醉剖腹后,按上述方法造模,腹腔缝合后分别于30min尾静脉注射0.8mg/kg功能化线粒体。
其中,功能化线粒体通过中性粒细胞膜与线粒体通过共孵育利用脂质融合得到;
具体地,包括如下步骤:
S1:采用C57BL/6J小鼠骨髓通过Solarbio的小鼠骨髓中性粒细胞分离液试剂盒分离提取中性粒细胞,再通过探头超声法破碎中性粒细胞,冷冻干燥后制备得到外源中性粒细胞膜碎片;
S2:采用C57BL/6J小鼠心肌组织通过碧云天的细胞线粒体分离试剂盒分离提取外源线粒体;
S3:将分离的外源线粒体与外源中性粒细胞膜碎片,按蛋白质的质量比1:1混合于适量的0.01M PBS溶液,于4℃水浴超声2min,3500g离心10min,弃掉上清液,用0.01MPBS溶液洗涤沉淀2次,去掉未结合的外源中性粒细胞膜碎片;再于4℃,3500g离心10min即制得功能化线粒体。
(2)观察实验结果,测定实验数据
于造模后24h收集小鼠血液和组织样品。腹腔注射3%戊巴比妥钠溶液(1μL/g)麻醉小鼠,心脏取血,离心获取血清,用于检测胰胰脂肪酶和炎性因子等指标。
打开腹腔,选取胰腺头部将胰腺组织沿十二指肠剪下,置于相对组织10倍体积的中性福尔马林溶液中,于室温用脱色摇床晃动过夜固定,作HE染色和免疫组化等相关研究;
打开胸腔,剪开隔膜,用1mL注射器吸取中性福尔马林,从肺门处注入左肺,至肺部呈鼓泡状,剪取1cm左右的肺组织,放入包埋盒并置于相对组织10倍体积的中性福尔马林溶液中,于室温用脱色摇床晃动过夜固定,作HE染色研究。
(3)统计分析实验数据
1.胰脂肪酶的测定
胰脂肪酶常用于评价急性胰腺炎严重程度,血清脂肪酶水平≥正常值上限3倍以上是临床急性胰腺炎诊断的三项标准之一。取50μL血清用双蒸水稀释至200μL,用南京建成脂肪酶和淀粉酶检测试剂盒测定血清中胰脂肪酶水平。
如图1所示,与对照组相比,模型组在牛黄胆酸钠造模后,小鼠胰脂肪酶大幅升高;尾静脉注射游离线粒体(Free Mito)或功能化线粒体(NEM-Mito)治疗后,血清中的脂肪酶含量均显著下降。其中,高剂量0.8mg/kg游离线粒体和0.8mg/kg功能化线粒体作用更明显,且与游离线粒体相比,功能化线粒体对急性胰腺炎小鼠的生化指标改善作用更为突出。
2.胰腺组织病理学研究
如图2所示,功能化线粒体治疗后,损伤的胰腺组织得到了很大程度的修复。具体地,胰腺组织较模型组更加致密,无充血现象,腺泡细胞完整,坏死腺泡细胞减少,浸润胰腺的炎性细胞减少。其中,高剂量0.8mg/kg改善作用更为明显。实验结果表明,功能化线粒体治疗减轻了胆管逆流注射引起的胆源性重症急性胰腺炎,改善了胰腺的组织病理损伤,恢复了胰腺组织的正常结构。
3.IL-6含量的测定
IL-6是诱导胰腺炎发生时胰腺和其它器官损伤的主要炎性因子,血清IL-6水平是评价AP严重程度的可靠指标,抑制IL-6信号转导是改善AP及相关组织损伤的重要手段。小鼠IL-6含量检测采用武汉云可控科技股份有限公司IL-6检测试剂盒,根据说明书操作,检测血清IL-6含量。
如图3所示,实验组与对照组相比,小鼠血清中炎症因子IL-6的聚集显著减少。结合图2中的功能化线粒体减少胰腺组织中炎性细胞的浸润,表明功能化线粒体能够显著抑制胰腺炎致病因素导致的局部和全身性炎性反应。
4.小鼠存活率的测定
采用胰胆管逆流注射牛磺胆酸钠构建重症急性胰腺炎动物模型,手术30min后,尾静脉注射0.4mg/kg或0.8mg/kg线粒体,观察24h内小鼠死亡情况。
如图4所示,模型组小鼠存活率较低,而实验组小鼠存活率明显提高。因此,说明功能化线粒体治疗可有效降低重症急性胰腺炎小鼠死亡率,具有较好的改善重症急性胰腺炎病死率的作用。
5.胰腺炎相关肺损伤研究
全身炎症反应综合症是急性胰腺炎最常见的全身并发症。全身炎症反应综合症持续存在会增加急性胰腺炎发生器官功能衰竭的风险,肺是最易累及的远端器官。
如图5所示,对照组小鼠的肺组织基本正常,无肺泡壁水肿和炎性细胞浸润(图5Control组)。模型组小鼠在胰胆管注射3.5%牛磺胆酸钠后,肺泡壁明显增厚并伴有炎性细胞浸润。实验组小鼠在尾静脉注射功能化线粒体后,肺泡壁增厚和炎性细胞浸润程度均明显减弱,其中高剂量0.8mg/kg改善作用更明显。因此,上述实验结果说明线粒体治疗后,重症急性胰腺炎引起的肺损伤有明显改善。
综上,测试结果表明,在重症急性胰腺炎小鼠模型中,功能化线粒体明显提高重症急性胰腺炎小鼠存活率,并改善胰腺炎相关生化和病理指标。与模型组的重症急性胰腺炎模型小鼠相比,尾静脉注射0.4mg/kg或0.8mg/kg功能化线粒体可降低死亡率,改善生化指标(如:胰脂肪酶),并改善胰腺和肺组织病理损伤。与模型组相比,实验组小鼠在功能化线粒体治疗后,生化指标胰脂肪酶明显下降,炎性因子IL-6含量减少,胰腺组织坏死,炎性细胞浸润和胰腺水肿均有明显改善,肺的水肿和炎性也有明显改善,其中高剂量0.8mg/kg改善作用更明显。
实施例2
如图6所示,本实施例与实施例1的区别在于,分别将功能化线粒体联合钙通道抑制剂(咖啡因)、功能化线粒体联合蛋白酶抑制剂(乌司他丁)应用于急性胰腺炎动物模型,并检测作用后的脂肪酶水平。对比发现,经过功能化线粒体联合钙通道抑制剂或功能化线粒体联合蛋白酶抑制剂作用的小鼠体内脂肪酶水平降低更明显,即说明功能化线粒体联合钙通道抑制剂或功能化线粒体联合蛋白酶抑制剂可应用于治疗急性胰腺炎。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.游离线粒体在胰腺炎中的应用。
2.一种功能化线粒体,其特征在于,所述功能化线粒体由外源细胞膜与权利要求1所述的游离线粒体通过共孵育利用脂质融合得到。
3.根据权利要求2中所述的功能化线粒体,其特征在于,所述外源细胞膜包括中性粒细胞、单核细胞、淋巴细胞、血小板、细菌、凋亡细胞中的任意一种。
4.一种如权利要求2所述的功能化线粒体的应用,其特征在于,所述功能化线粒体可应用于胰腺炎并发症以及胰腺炎相关的其他器官损伤。
5.根据权利要求4所述的功能化线粒体的应用,其特征在于,所述功能化线粒体可应用于由胰腺炎引起的肺部炎症反应综合症。
6.一种如权利要求2所述的功能化线粒体的应用,其特征在于,所述功能化线粒体联合蛋白酶抑制剂、或所述功能化线粒体联合钙通道抑制剂可应用于治疗胰腺炎。
7.根据权利要求6所述的功能化线粒体的应用,其特征在于,所述蛋白酶抑制剂为乌司他丁。
8.根据权利要求6所述的功能化线粒体的应用,其特征在于,所述钙通道抑制剂为咖啡因。
9.一种如权利要求1所述的游离线粒体的应用方法,其特征在于,所述游离线粒体通过静脉给药或局部给药的方式靶向作用于疾病发生部位。
10.一种如权利要求4至8中任意一项所述的功能化线粒体的应用方法,其特征在于,所述功能化线粒体通过静脉给药或局部给药的方式靶向作用于疾病发生部位。
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