CN114107179B - 鸡胆汁外泌体在治疗鸡肝损伤注射药物中的用途及药物 - Google Patents
鸡胆汁外泌体在治疗鸡肝损伤注射药物中的用途及药物 Download PDFInfo
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Abstract
本发明公开了鸡胆汁外泌体在治疗鸡肝损伤注射药物中的用途及药物，属于免疫学领域，本发明发现鸡胆汁外泌体对LPS诱导的肝细胞损伤具有良好的保护作用，明显降低了LPS所致的细胞培养上清液的ALT、AST水平，降低了IL‑6、TNF‑α、iNOS、Caspase、Bax表达水平，提高了Bcl‑2表达水平。以LPS诱导肝损伤动物模型，观察腹腔注射鸡胆汁外泌体对LPS诱导的肝损伤的影响，明确了注射鸡胆汁外泌体一次性给药后可明显减轻LPS诱导的肝损伤，即鸡胆汁外泌体对肝损伤有治疗作用。为临床上鸡肝损伤的治疗提供了一种新的治疗方法。
Description
技术领域
本发明属于免疫学领域，具体地涉及鸡胆汁外泌体在治疗鸡肝损伤注射药物中的用途及药物。
背景技术
近年来，养鸡场出现了一种以肝脏肿大、质地变脆、腹腔积血为特征的疾病，暂称为肝破裂出血综合征(HRHS)。HRHS的发病原因和致病机理尚不明确，所涉品种广泛，发病阶段集中在育成中后期、产蛋上升期，发病无地域差别，也无明显的季节性，全年都可发生。HRHS极大地影响了鸡生产性能，给养殖业带来了严重的经济损失。由于病因和致病机制不明，目前没有有效的药物应用于肝脏损伤的治疗。组织病理学观察显示，肝组织中淀粉样蛋白沉积和纤维素渗出为HRHS中主要病变特征，提示过度的炎症反应。
脂多糖(LPS)是内毒素主要的毒性成分，不仅可以直接或间接造成肝细胞损伤，而且在诱导肝细胞坏死和肝炎中发挥重要作用，因此，临床上常用脂多糖制造肝损伤模型，进行抗损伤研究。LPS诱导产生各种炎性细胞因子的释放，是引发肝损伤的重要原因之一。炎性因子主要包括肿瘤坏死因子(TNF-α)、白细胞介素1(IL-1)、白细胞介素6(IL-6)等，炎性因子之间的相互作用可以诱发和加重肝损伤。研究证明，内毒素和各种炎性因子会刺激中性粒细胞和巨噬细胞等，导致iNOS表达水平升高，进而催化合成并释放大量NO，导致肝细胞死亡和损伤。
评价肝损伤最常用的生化指标为谷丙转氨酶和谷草转氨酶，这两种酶在正常生理情况下存在于肝细胞内，血清含量较少，当肝细胞受到损伤时，细胞膜通透性增加，这两种酶释放入血，导致血清中ALT、AST含量升高。
除了炎症，细胞凋亡也是评估肝细胞损伤的一个重要指标。细胞凋亡分为两种不同的信号通路：死亡受体途径和线粒体介导的途径。其中涉到两组相反功能的蛋白组：(1)抑制细胞凋亡蛋白质组包括Bcl-2，Bcl-xL等；(2)促凋亡蛋白质组包括Bax和Bak。在存在凋亡刺激时，促凋亡蛋白Bax的表达增加，抑制凋亡蛋白Bcl-2表达降低，导致细胞色素C释放到细胞质中，从而活化半胱氨酸天冬氨酸蛋白酶-9切割caspase-3，致使其表达量升高，细胞发生凋亡。
外泌体(exosome)是一种来源于活细胞晚期内含体的具有生物活性的小囊泡，在细胞间进行信息传递，参与多种生理和病理过程。外泌体也存在于各种体液中，其功能与来源细胞密切相关。胆汁外泌体由肝脏间质细胞、肝细胞和胆管上皮细胞分泌，70％的胆汁外泌体来自于肝细胞，在肝脏的生理功能和疾病过程中发挥重要作用。
发明内容
本发明要解决的技术问题在于提供一种鸡胆汁外泌体在抗肝损伤治疗药物中的用途及药物，具体地研究了鸡胆汁外泌体在肝损伤治疗药物中的用途。以LPS诱导肝损伤模型，观察肝细胞形态变化，确定腹腔注射鸡胆汁外泌体对LPS诱导的肝损伤的影响，明确了注射鸡胆汁外泌体给药后可明显减轻LPS诱导的肝损伤，即鸡胆汁外泌体对肝损伤有治疗作用。
本发明是通过如下技术方案来实现的：
一种鸡胆汁外泌体在制备鸡的治疗肝损伤注射药物中的用途，所述的肝损伤为LPS诱导的鸡的肝损伤。
进一步，所述鸡胆汁为泌体的制备方法为将新鲜胆汁经8层纱布过滤后，在4℃条件下分级离心，分别经1000g离心15min，4000g离心20min，10 000g离心30min，50000g离心30min，除去胆汁中的杂质，收集上清液；收集的上清在4℃条件下，100000g离心70min，收集沉淀；将得到的沉淀用PBS重悬，在4℃条件下100000g离心70min，洗涤沉淀；将得到的沉淀用PBS重悬，分装于-80℃保存备用。
一种治疗鸡肝损伤的注射药物，其中包含鸡胆汁外泌体。
本发明与现有技术相比的有益效果：
本发明所述的鸡胆汁外泌体具有细胞保护作用，所述鸡胆汁外泌体降低了IL-6、TNF-α、iNOS、Caspase、Bax表达水平，提高Bcl-2表达水平。
对LPS诱导的肝损伤鸡通过腹腔注射给药，剂量为200μg/kg，单次腹腔注射鸡胆汁外泌体24h具有治疗肝损伤的作用。
附图说明
图1为LPS诱导的肝损伤对鸡肝细胞形态学的影响；
图2为鸡胆汁外泌体对LPS诱导的鸡肝损伤肝细胞培养上清ALT、AST的影响；A:对ALT的影响；B、对AST的影响。
图3为鸡胆汁外泌体对鸡肝细胞中炎症和凋亡因子的影响；A:对凋亡相关因子Caspase、Bax、Bcl-2的影响；B、对炎性因子IL-6、TNF-α和氧化应激因子iNOS的影响。
图4为鸡胆汁外泌体对LPS诱导肝脏损伤鸡血清ALT、AST的影响；A、对ALT的影响，B、对AST的影响。
图5为鸡肝脏大体剖检图；A、正常组肝脏，B、注射鸡胆汁外泌体组，C、模型组肝脏也即LPS诱导的肝细胞损伤组；D、模型加注射鸡胆汁外泌体组；
图6为鸡胆汁外泌体对LPS诱导的肝脏损伤鸡肝组织形态学的影响；
图7为鸡胆汁外泌体对鸡肝脏中炎症反应、细胞凋亡和肝细胞生长因子的影响。A、对炎性因子IL-6、TNF-α和氧化应激因子iNOS的影响，B、对凋亡相关因子Caspase、Bax、Bcl-2的影响，C、对肝细胞生长因子的影响。
具体实施方式
下面通过实施例结合附图来对本发明技术方案的进一步理解，并不构成对本发明技术方案的限制。
通过实验发现，体外诱导肝细胞损伤，加入鸡胆汁外泌体共同孵育可显著降低鸡肝细胞上清ALT、AST的含量，减轻肝细胞炎性损伤，抑制细胞凋亡，促进肝细胞分裂增殖。单次给予鸡胆汁外泌体可显著降低LPS诱导的肝脏损伤鸡血清ALT、AST含量，减轻肝脏组织病理损伤，同时降低了IL-6、TNF-α、iNOS、Caspase、Bax表达水平，提高Bcl-2和肝细胞生长因子的表达水平，减轻了炎症反应，抑制了细胞凋亡，促进细胞的分裂再生。表明鸡胆汁外泌体具有治疗肝损伤作用，在此基础上完成了本发明。
为了更好地理解本发明的内容，下面结合具体的实施方法对本发明内容作进一步说明。但在进一步描述本发明具体实施方式之前，应理解，本发明保护的内容不局限于以下实施例。
实施例1鸡胆汁外泌体的提取
480mL新鲜胆汁经8层纱布过滤后，4℃条件下分级离心，分别经1000g离心15min，4000g离心20min，10000g离心30min，50000g离心30min，除去胆汁中的杂质，收集上清液。上清在4℃条件下，100000g离心70min，沉淀用PBS重悬，4℃条件下100000g离心70min，洗涤沉淀。3mL的PBS重悬沉淀，分装于-80℃保存备用。
实施例2细胞培养方法，细胞保护实验，RT-PCR分析
1.细胞培养方法
原代鸡肝细胞系，用DMEM培养基(含10％胎牛血清)置于5％的CO2、37℃培养箱中培养，每24h换液一次，取对数期生长的细胞，用0.25％胰酶消化，制备细胞悬液，密度调整至2×105/mL，接种到24孔板，培养24h。
2.细胞保护实验
取对数期生长的肝细胞，以2×105/mL的密度接种于24孔细胞培养板，贴壁生长达70～80％后，37℃预热的PBS清洗两次。分三组(n＝3)，对照组：用含有10％胎牛血清的DMEM培养基；LPS模型组：10％胎牛血清DMEM培养基加入终浓度为600μg/mL LPS；治疗组：在LPS模型组基础上，分别加入终浓度为2.5μg/mL、5μg/mL、10μg/mL、20μg/mL exosome，继续培养。
24h后于倒置显微镜下观察肝细胞形态并拍照。4℃，10000g离心3min，收集细胞上清，检测上清中ALT、AST浓度。
3.RT-QPCR
收集上述实验细胞，用TRNZOL法提取RNA，逆转录成cDNA。再以cDNA为模板进行PCR扩增，反应条件：预变性95℃30s，PCR反应95℃5s，60℃30s，共40个循环。结果以2-ΔΔCt方法分析基因的表达差异，β-actin作为内参，计算IL-6、TNF-α、iNOS、Caspase、Bax及Bcl-2的mRNA相对表达量。
检测结果(图1)，LPS浓度为600μg/mL时，肝细胞数量明显减少，部分肝细胞破裂、坏死并悬浮在培养基中，随着exosome浓度的增大，肝细胞数量明显增加，坏死的细胞数量减少，当exosome浓度达到20μg/mL时，出现明显的肝细胞分裂团。检测结果(图2)，模型组与对照组相比，细胞上清培养液中ALT、AST两种酶的含量显著上升，显示肝细胞损伤明显。治疗组与模型组相比，细胞上清培养液中ALT、AST含量均随着exosome浓度的增大而降低，说明不同浓度的exosome对肝细胞均有保护作用。检测结果(图3)，LPS可以提高肝细胞内IL-6、TNF-α、iNOS、Caspase、Bax的mRNA表达量，降低Bcl-2的表达水平，说明LPS刺激鸡肝细胞产生了炎症反应和细胞凋亡，而加入exosome可以使肝细胞内IL-6、TNF-α、iNOS、Caspase、Bax的mRNA表达水平降低，Bcl-2的mRNA表达水平升高。结合以上实验结果，证明鸡胆汁外泌体可明显抑制LPS诱导的炎症反应和细胞凋亡，具有保护肝细胞的作用。
实施例3动物实验，肝组织病理切片制作，RT-PCR分析
1.动物实验
SPF鸡40只，随机分为对照组、模型组、exosome组、治疗组；每组10只。对照组鸡腹腔注射等体积的生理盐水；模型组鸡腹腔注射LPS(6mg/kg)；exosome组鸡腹腔注射外泌体(200μg/kg)；治疗组鸡腹腔注射LPS(6mg/kg)30min后，腹腔注射exosome(200μg/kg)。
在注射LPS/exosome 24h后，鸡翅静脉采血，室温静置1h，4℃，3500g，离心10min，分离血清检测ALT、AST水平。
2.肝组织病理切片制作
采血后将鸡处死，取肝脏，一部分于RNA的提取，一部分置于4％福尔马林中固定，酒精梯度脱水，石蜡包埋后切片，苏木精-伊红染色，中性树脂封片，在光学显微镜下观察肝脏组织的病理变化。
3.RT-PCR分析
用TRNZOL法从新鲜的肝脏中提取RNA，逆转录成cDNA。再以cDNA为模板进行PCR扩增，反应条件：预变性95℃30s，PCR反应95℃5s，60℃30s，共40个循环。结果以2-ΔΔCt方法分析基因的表达差异，以β-actin作为内参，计算IL-6、TNF-α、iNOS、Caspase、Bax、Bcl-2和肝细胞生长因子(HGF)mRNA表达量。
检测结果(图4)显示，单次注射LPS/exosome 24h后，与对照组相比，模型组鸡血清ALT、AST水平显著升高；exosome组鸡血清ALT、AST水平无明显变化；治疗组鸡血清ALT、AST水平与模型组相比，鸡血清ALT、AST水平显著下降。剖检结果(图5)显示，A组正常肝脏大体形态、颜色、大小正常、质地柔软；B组exosome组与A组相比无明显差异；C组模型组肝脏表明有大量散在的灰白色、灰黄色坏死灶；D组治疗组较模型组肝脏表面灰白色坏死灶减少。检测结果(图6)显示，对照组鸡肝脏组织结构完整，肝细胞形状均匀，呈索状排列；模型组鸡肝脏轻度淤血，肝细胞索排列紊乱，中央静脉血管周围有大量炎性细胞浸润，肝窦内出现红细胞，部分肝细胞发生坏死；exosome组，与正常组相比无明显病理变化，未见炎性细胞；治疗组肝脏，肝静脉周围炎症反应减轻，炎性细胞数量明显减少，肝细胞索排列紊乱显著改善，炎性细胞减少的区域出现明显分裂再生的肝细胞团。检测结果(图7)显示exosome组与对照组相比，exosome对鸡肝脏没有明显不良影响；损伤组与对照组相比，IL-6、TNF-α、iNOS、Caspase、Bax的mRNA表达水平升高，Bcl-2mRNA表达水平下降；治疗组与损伤组相比，IL-6、TNF-α、iNOS、Caspase、Bax的mRNA表达水平显著降低，Bcl-2mRNA表达水平升高。综合以上结果表明，腹腔注射exosome对LPS诱导的肝脏损伤有显著治疗作用，降低炎性反应，抑制细胞凋亡，促进了肝细胞的分裂再生。
通过对LPS诱导的肝损伤鸡肝细胞细胞培养上清ALT、AST水平检测、肝细胞形态学观察、对LPS诱导的肝损伤血清ALT、AST水平检测、肝组织病理学观察，RT-PCR分析，明确鸡胆汁外泌体治疗肝损伤作用的关键在于鸡胆汁外泌体可以显著减轻肝细胞形态学损伤；降低LPS诱导的肝损伤细胞培养上清ALT、AST水平；也可以减轻肝脏病理组织学损伤，降低LPS诱导的肝损伤血清ALT、AST水平；降低了炎症因子IL-6、TNF-α、iNOS的mRNA表达水平，降低了凋亡相关因子Caspase、Bax的mRNA表达水平，提高了抑制凋亡蛋白Bcl-2和肝细胞生长因子(HGF)的mRNA表达水平，说明鸡胆汁外泌体对肝脏中的炎症因子和凋亡因子的表达有显著调节作用，可以提高鸡的抗炎能力和抑制凋亡能力。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种同等交换，这些同等变换均属于本发明的保护范围。另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合。为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。
Claims (2)
1.一种鸡胆汁外泌体在制备治疗鸡肝损伤腹腔注射药物中的用途，所述的肝损伤为LPS诱导的鸡肝损伤，治疗原理为防止肝细胞凋亡，促进肝细胞分裂再生。
2.根据权利要求1所述的用途，其特征在于所述鸡胆汁外泌体的制备方法为将新鲜胆汁经8层纱布过滤后，在4℃条件下分级离心，分别经1000g离心15min，4000g离心20min，10000g离心30min，50000g离心30min，除去胆汁中的杂质，收集上清液；收集的上清在4℃条件下，100000g离心70min，收集沉淀；将得到的沉淀用PBS重悬，在4℃条件下100000g离心70min，洗涤沉淀；将得到的沉淀用PBS重悬，分装于-80℃保存备用。
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