【摘要】 目的探索宫内炎性暴露对新生鼠的巨核细胞和血小板稳态的影响,为解析宫内感染致新生儿血小板减少症的分子机制提供动物实验依据。方法按怀孕时间将SD孕鼠共24只随机分为内毒素(LPS)组和对照组各12只。将LPS(0. 79mg/kg)和等量0. 9%氯化钠注射液分别在孕8d、10d和12d给LPS组和对照组孕鼠进行腹腔注射。在孕21d(预产期为22d)剖宫产娩出早产鼠,其中LPS组娩出118只早产鼠,对照组娩出144只,在两组中分别随机抽取早产鼠23只,纳入LPS早产鼠组和对照早产鼠组,在其出生后1h内采集外周血、骨髓、肺组织、肝脏及肾脏组织标本,再运用全血细胞分析和血浆凝集培养方式分别检测早产鼠循环中巨核细胞数和骨髓巨核细胞集落单位(CFU-MK)及运用流式细胞分析方法检测循环中血小板表面标志物CD62P的表达。运用ELISA分别检测早产鼠肝脏和肾脏血小板生成素(TPO)水平和TPO的表达。同时,通过电镜观察肺毛细血管血小板聚集现象。结果相对于对照早产鼠组(n=23),LPS早产鼠组的外周血小板计数较低[LPS早产鼠组和对照早产鼠组:(239.22±63.00)×10~9/L vs.(377.96±79.90)×109/L,P<0.001],循环中MK计数低[(5.78±1.70)/mL vs.(8.30±1.90)/mL, P <0.001]和CFU-MK[(9.52±2.80)/10~5 cell vs.(15.83±3.60)/10~5 cell,P<0.001];血小板表面标志物CD62P阳性率较低[(43. 05±6.30)%vs.(34. 91±12.10)%,P <0.001];血浆TPO水平显著增高[(482.44±91.20) pg/mL vs.(294.70±68.20) pg/mL,P<0.001],肝脏TPO mRNA表达增多,肾脏TPO mRNA表达显著减少。通过电镜在LPS早产鼠组可观察到肺毛细血管内血小板聚集现象,而在对照组早产鼠肺内没观察到类似现象。结论宫内炎性暴露可能损害了早产鼠巨核细胞和血小板稳态。其可能影响机制为:(1)损害了骨髓巨核细胞稳态;(2)增加血小板的活化;(3)反馈性促进了TPO的生成。更多还原

【Abstract】 Objective Prenatal inflammation is a risk factor for impaired fetal development. The aim of this study is to investigate the effect of prenatal Lipopolysaccharide( LPS)-induced inflammation on neonatal megakaryocytopoiesis and platelet homeostasis in a rat model. The project will be important,deeper analysis regulating molecular mechanism about thrombocytopenia of the newborn caused by inflammatory exposure. Methods The timed pregnant Sprague-Dawley( SD) rats(n=24) were randomly devided in to LPS group(n = 12) and control group(n=12). Lipopolysaccharide(0.79 mg/kg) and normal saline were intraperitoneally(i. p.) injected on gestational day 8, 10 and 12 to LPS group and control group respectively. Premature rats were delivered by cesarean section on the 21 st day of pregnancy(the preterm was 22 days), of which 118 were born in the LPS group and 144 were born in the control group, Resin the two groups randomly the offspring rats premature 23, into the LPS group and the control group, in their blood samples within 1 hour after birth, bone marrow, lung, liver, kidney tissue samples, Circulating megakaryocyte count and bone marrow megakaryocyte colony forming units(CFU-MK) were quantified by whole blood infiltration method and plasma clot culture system,respectively. Platelet activation marker, CD62 P was estimated by flow cytometry. Plasma thrompoietin(TPO) level and TPO expression in liver and kidney were detected by immunosorbent assays( ELISA)and southern blot respectively. Meanwhile, capillary platelet aggregation in lung was observed by electron microscopy. Results When compared to control group( n = 23), the prenatal LPS group had significantly lower peripheral platelet count [ LPS group vs. controls:(239.22 ± 63. 00) x 109/L vs.(377. 96 ± 79. 90) x 10~9)/L, P < 0. 001 ]; CD62 P expression on platelets [(43. 05 ± 6. 30)% vs.(34. 91 ± 12. 10) %,P <0. 001 ]; significantly increased plasma TPO level [(482. 44 ±91.20) pg/mL vs.(294.70 ±68.20) pg/mL, P < 0. 001 ], hepatic TPO mRNA expression, however, significantly weaker kidney TPO mRNA expression. Intra-pulmonary capillary platelet aggregation was observed in the LPS group but not in the controls by electronic microscope. Conclusions Prenatal inflammation may impair neonatal megakaryocytopoiesis and platelet homeostasis. The possible mechanism might be① impaired megakaryocytopoiesis in bone marrow;② increased platelet activation;③ elevated TPO production as a feed-back control.更多还原