【作者】 张莉；

【导师】 余舰；

【作者基本信息】 遵义医学院 ， 法医学， 2017， 硕士

【摘要】 目的:利用h AMSCs的免疫调节作用和减轻肺损伤的特点,将h AMSCs经静脉注入到孕鼠羊水栓塞模型体内,从炎症角度初步探讨羊水栓塞时对肺损伤可能的保护机制。方法:1.SD孕鼠40只,随机分为对照组、羊水胎粪组、实验组1、实验组2(每组10只),从孕鼠子宫中抽取羊水与胎鼠胎粪制成99:1的羊水胎粪混合液,沿腹主静脉:对照组注入生理盐水,羊水胎粪组注入羊水胎粪混合液,实验组1输注h AMSCs后输注羊水胎粪混合液,实验组2输注羊水胎粪混合液后注入h AMSCs。观察1小时后处死孕鼠,取动脉血3ml,肺组织称重后,左肺-80℃保存备用,右肺固定于10%甲醛溶液中,常规包埋切片用于HE染色、APM染色及免疫组化。2.检测第四代h AMSCs是否培养成功,用流式细胞仪检测h AMSCs,免疫细胞化学染色观察h AMSCs波形蛋白表达。3.各组进行肺系数计算,右肺组织均进行HE染色、APM染色和CK16免疫组织化学法鉴定,验证羊水栓塞模型是否建立成功。4.四组均采用免疫组织化学法检测右肺组织NF-κB的表达;蛋白印迹法检测左肺组织NF-κB的含量;酶联免疫吸附试验检测血清中TNF-α、IL-8的浓度。结果:1.临床表现:对照组输注生理盐水后呼吸平稳并无太大变化;羊水胎粪组表现出明显的呼吸急促,心跳加快、爪湿冷、大小便失禁、四肢抽搐等症状;实验组1、实验组2呼吸加快后又逐渐平稳,偶有四肢抽搐的症状,爪稍湿冷。其中对照组、实验组1、实验组2观察1小时后孕鼠均无死亡案例发生,羊水胎粪组有1例孕鼠死亡,其羊水粘稠。2.肺系数:对照组(3.083±0.256),羊水胎粪组(3.805±0.691),实验组1(3.043±0.457),实验组2(3.180±0.436),羊水胎粪组与对照组、实验组1、实验组2比较均有统计学意义(P<0.05)。肺组织HE染色:对照组未见异常;羊水胎粪组可见不同程度的肺水肿,充血,肺间隔增宽、肺组织中有巨噬细胞、单核细胞、中性粒细胞等炎性细胞浸润,肺血管内见疑似羊水粘液成分及角化鳞状上皮等羊水成分;实验组1与实验组2肺血管内也可见疑似羊水成分及角化的鳞状上皮等,但病理改变轻于羊水胎粪组,可见少许水肿,充血,少量炎性细胞浸润。肺组织APM染色:对照组肺组织血管内未见异常染色;羊水胎粪组、实验组1与实验组2肺组织血管内可见细丝状角化的鳞状上皮,呈桃红色,同时可见亮染蓝色的羊水粘液成分。免疫组织化学检测肺组织CK16:羊水胎粪组、实验组1、实验组2均可见棕黄色絮状或丝状角化的鳞状上皮。3.h AMSCs免疫细胞化学染色:第四代h AMSCs波形蛋白表达阳性。流式细胞仪检测分析h AMSCs的免疫表型:第四代h AMSCs高表达CD73、CD90、CD105,不表达或低表达CD34、CD11b、CD19、CD45、HLA-DR。4.免疫组织化学检测肺组织NF-κB:对照组肺组织表达少量棕黄色颗粒;羊水胎粪组肺组织有大量棕黄色颗粒,呈强阳性表达,实验组1与实验组2呈弱阳性表达;各组平均光密度值:对照组(0.405±0.059),羊水胎粪组(0.453±0.036),实验组1(0.376±0.060),实验组2(0.405±0.047)。羊水胎粪组与对照组、实验组1、实验组2比较均有统计学意义(P<0.05)。蛋白印迹法检测NF-κB的浓度:羊水胎粪组、实验组2、实验组1、对照组条带颜色依次减弱,各组灰度值:对照组(0.539±0.131),羊水胎粪组(0.678±0.228),实验组1(0.522±0.169),实验组2(0.553±0.200)。羊水胎粪组与对照组、实验组1、实验组2比较均有统计学意义(P<0.05)。酶联免疫吸附试验:IL-8:对照组(123.169±13.615),羊水胎粪组(158.346±47.682),实验组1(79.316±46.035),实验组2(109.637±28.287),羊水胎粪组与对照组、实验组1、实验组2比较均有统计学意义(P<0.05);TNF-α:对照组(36.169±4.469),羊水胎粪组(39.408±2.406),实验组1(30.640±4.486),实验组2(35.550±2.962),羊水胎粪组与对照组、实验组1、实验组2比较均有统计学意义(P<0.05)。以上数值经统计学分析,羊水胎粪组与对照组、实验组1、实验组2比较均有统计学意义(P<0.05)。结论:1.通过HE染色、APM染色及免疫组化CK16的表达确认羊水栓塞模型制备成功;2.h AMSCs可以通过下调IL-8、TNF-α的水平、抑制肺组织NF-κB的活化干预孕鼠羊水栓塞,达到减轻羊水栓塞炎症反应及减轻肺损伤的目的。更多还原

【Abstract】 Objective: Utilizing the immunomodulatory effect of h AMSCs and alleviating lung injury,h AMSCs were injected intravenously into the amniotic fluid embolism model of pregnant rats,and the protective mechanism of amniotic fluid embolism leading to lung injury was preliminarily explored from the perspective of inflammation.Methods:1.Forty pregnant SD rats were randomly divided into control group,amniotic fluid meconium group,experimental group1 and experimental group2(10 in each group).The uterus from the pregnant rats to extract amniotic fluid and fetal rat meconium made of amniotic fluid meconium mixed solution of 99: 1.Along the abdominal main vein :control group injected saline;amniotic fluid meconium group inject amniotic fluid meconium mixture;experimental group1 injected amniotic fluid meconium mixture after injection of h AMSCs;experimental group2 was injected h AMSCs after injection of amniotic fluid meconium mixture.Collect 3ml arterial blood and kill the pregnant rats in each group after one hour observation.The left lung was freezed at-80 degrees in low temperature and the right lung was put in ten percent formaldehyde solution,conventionally embedded sections for HE staining and APM staining and immunohistochemistry.2.In order to detect whether the fourth generation of h AMSCs cultured successfully,h AMSCs was detected by flow cytometry and the vimentin expression of h AMSCs was dectected by immunocytochemical.3.The lung coefficient of each group was calculated,right lung tissue were stained with HE,APM staining and immunohistochemistry of CK16 to verify whether the amniotic fluid embolism model was established successfully.4.IHC was used to detect the expression of NF-κB in the right lung tissue in all four groups.The content of NF-κB in the left lung tissue was detected by Western blotting.The concentrations of TNF-α and IL-8 were detected by Enzyme-linked immunosorbent assay.Results:1.Clinical manifestations: control group after infusion of saline did not change much.The amniotic fluid meconium group showed obvious shortness of breath,rapid heartbeat,claw wet and cold,incontinence,convulsions and other symptoms.Experimental group 1 and experimental group 2 accelerated after the gradual smooth breathing,occasional limbs convulsions,claws slightly wet.In control group,experimental group1 and experimental group2,no death occurred in the pregnant rats one hour after the observation.In amniotic fluid meconium group,a pregnant rat died and its amniotic fluid was thick.2.Lung coefficient: control group(3.083±0.256),amniotic fluid meconium group(3.805±0.691),experimental group1(3.043±0.457),experimental group2(3.180±0.436).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).HE staining of lung tissue:HE staining of lung tissue: No abnormalities in the control grop.Amniotic fluid meconium group,there were different pulonary edema,hyperemia and increased lung septum.There were macorophages,monocytes,neutrophils and other inflammatory cell infiltration in lung tissues.There were suspected amniotic fluid components and keratinized squamous cell amniotic fluid composition in pulmonary vascular.Experimental group 1 and experimental group 2 also showed pulmonary vascular suspicious amniotic fluid composition and keratinized squamous epithelium,but the pathological changes were lighter than amniotic fluid meconium group,showing a small amount of edema,congestion,a small amount of inflammatory cell infiltration.APM staining of lung tissue: There was no abnormal staining in the pulmonary vascular of the control group;amniotic fluid meconium group,experimental group 1 and experimental group 2 showed fine filamentous keratinized squamous epithelium in the pulmonary vascular,stained blue amniotic fluid mucus composition.Immunohistochemical detection of lung tissue CK16: Brown yellow keratinized squamous epithelium were seen in the amniotic fluid meconium group,experimental group1 and experimental group2.3.Immunocytochemical staining of h AMSCs: The fourth generation of h AMSCs had positive vimentin expression.The immunophenotypes of MSCs were analyzed by flow cytometry.The fourth generation h AMSCs overexpressed CD73,CD90 and CD105,but did not express CD34,CD11 b,CD19,CD45 and HLA-DR.4.IHC of lung tissue NF-κB: control group of alveoli,bronchial,perivascular low expression of light brown particles.amniotic fluid meconium group pulmonary vascular,bronchial,alveolar area has a large number of brown particles,was strongly positive expression.The experimental group1 and the experimental group2 showed a weak positive expression,experimental group2 lighter than experimental group1.The average optical density of each group: control group(0.405±0.059),amniotic fluid meconium group(0.453±0.036),experimental group1(0.376±0.060),experimental group2(0.405±0.047).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).Western blotting to detect the concentration of NF-κB:amniotic fluid meconium group,experimental group1,experimental group2,control group,the color of the bands weakened in turn.The gray value of each group: control group(0.539±0.131),amniotic fluid meconium group(0.678±0.228),experimental group1(0.522±0.169)and experimental group2(0.553±0.200).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).Enzyme-linked immunosorbent assay: IL-8:control group(123.169±13.615),amniotic fluid meconium group(158.346±47.682),experimental group1(79.316±46.035)and experimental group2(109.637±28.287).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).TNF-α:control group(36.169±4.469),amniotic fluid meconium group(9.408±2.406),experimental group1(30.640±4.486)and experimental group2(35.550±2.962).The amniotic fluid meconium group was significantly different from the control group,experimental group 1,and experimental group 2(P<0.05).The above values were statistically analyzed and there was statistically significant difference between amniotic fluid meconium group and control group,experimental group 1,and experimental group2(P<0.05).Conclusion:Through the HE staining and APM staining and immunohistochemical expression of CK16 confirm the amniotic fluid embolism model was successfully prepared;human amniotic mesenchymal stem cells can intervene amniotic fluid embolism in pregnant rats by down-regulating the levels of IL-8 and TNF-α and inhibiting the activation of NF-κB in lung tissue,so as to reduce the amniotic fluid embolism inflammatory reaction and relieve the lung injury.更多还原