| Objective:In order to establish SD pregnant rats amniotic fluid embolism model,hAMSCs were cultured to the 4th generation,and simulate the human amniotic and meconium fluid into the maternal circulation.Pregnant Rats were transplanted hAMSCs by abdominal venous,before and after injection of amniotic and fetal fluid,to study changes of complements C3a,MPO and NF-?B,and investigate the effect of hAMSCs in AFE in pregnant rats.Methods:The forth generation of hAMSCs were detected its phenotypes and vimentin expression by FCM and IHC respectively.The concentration of hAMSCs was adjusted to 1×10~5/mL.The specimen(Forty pregnant SD pregnant rats)were randomly divided into control group(NS group,n=10),amniotic fluid meconium group(MAF group,n=10),experimental group 1(n=10)and experimental group 2(n=10).hAMSCs were transplanted in experimental group 1 30 minutes in advance before establishment of amniotic fluid embolism model,while hAMSCs were transplanted immediately after amniotic fluid meconium infusion in experimental group 2.The aim to verify the amniotic fluid embolism model was successful or not.Right lung tissue through processed by HE staining and APM special dye,then detected CK16 expression via IHC method.Lung tissue quantify its value between wet weight and dry weight(W/D).IHC was used to detect the expression of NF-?B and C3a in the right lung of each group.The relative content of NF-?B in left lung was detected by Western-blot,the absorbance of left lung MPO was measured by spectrophotometry(A),and the level of serum C3a was detected by ELISA.All data used SPSS18.0 statistical analysis;each group was examined by variance test,including homogeneity test of variance checked by univariate analysis of variance,heterogeneity of variance detected by rank sum test,P<0.05 was statistically significant?Results:1.hAMSCs phenotypic identification:flow cytometry results show that hAMSCs high expression on CD34,CD90,CD105,CD73,CD45,almost no expression on CD11b,CD19and HLA-DR.2.The clinical symptom:There was no obvious abnormality in the control group;while MAF group performance shallow-fast breathing,rapid heart rate firstly then gradually slowed down,in addition few specimen present convulsions and incontinence during the process.In the experimental group 1,hAMSCs were transplanted in one group.The resuscitation of transient excretion of amniotic fluid resulted in a transient increase in the rate of respiration,And hAMSCs after transplantation,rapid respiration,fast heart rate,some pregnant rats convulsions,incontinence.3.Identification of AFE model:HE staining see the MAF group,the experimental group 1,the experimental group 2 lung tissue congestion to varying degrees,and pulmonary vascular see the presence of amorphous material,APM special to see the blue dye in the pulmonary vascular amniotic fluid mucus and pink silk IHC examination of CK16 revealed a yellowish-yellow flocculent or filamentous expression in the pulmonary blood vessels.4.The lung tissue W/D:control group(3.67±0.23),MAF group(10.03±0.60),experimental group 1(8.43±0.20),experimental group 2(8.27±0.22).The MAF group,the experimental group 1,the experimental group 2 were different from the control group(P<0.05),and the MAF group was different from the experimental group 1 and the experimental group 2(P<0.05).5.C3a expression:IHC staining around the pulmonary blood vessels,pulmonary bronchial lung space and lung tissue see the expression of brown granules,strong positive control group,MAF group weakly positive expression in experimental group 1,experimental group 2 positive expression.The OD value in each group was(0.36±0.02)in the control group,(0.142±0.027)in the MAF group,(0.25±0.15)in the experimental group 1,(0.27±0.02)in the experimental group 2.The levels of C3a:control group(27.23±1.06),MAF group(16.47±1.13),experimental group 1(21.61±0.72)and experimental group 2(22.52±0.83).The MAF group,the experimental group 1,the experimental group 2 were different from the control group(P<0.05),and the MAF group was different from the experimental group 1 and the experimental group 2(P<0.05).6.The expression of NF-?B:The IHC staining showed the yellowish-yellow granules around the pulmonary blood vessels and the lung bronchus.The expression of NF-?B was weakly positive in the control group and strong in the MAF group.The expression of NF-?B in experimental group 1 and experimental group 2 was positive.The OD value:control group(0.14±0.02),MAF group(0.39±0.05),experimental group 1(0.27±0.02)and experimental group 2(0.25±0.03).The expression of NF-?B:control group(0.09±0.04),MAF group(0.44±0.08),experimental group 1(0.15±0.03),experimental group 2(0.12±0.04).The MAF group,the experimental group 1,the experimental group 2 were different from the control group(P<0.05),and the MAF group was different from the experimental group 1 and the experimental group 2(P<0.05).7.MPO activity was measured by spectrophotometry:control group(1.22±0.13),MAF group(3.56±0.85),experimental group 1(2.26±0.12),experimental group 2(2.15±0.13).The MAF group,the experimental group 1,the experimental group 2 were different from the control group(P<0.05),and the MAF group was different from the experimental group 1 and the experimental group 2(P<0.05).Conclusion:1.Complement C3a plays a significant role in the process of AFE,while Neutrophil and NF-?B are important proinflammatory factors in AFE of acute lung injury.2.hAMSCs is promotive function in adjustment of C3a,MPO,NF-?B,which can reduce AFE-induced lung injury. |
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