CAJ | 학술논문

目的观察宫内注射脂多糖(LPS)对围产期大鼠肺内天然免疫相关的Toll样受体4(TLR4)信号转导通路的影响,探讨天然免疫在宫内感染中的免疫调节能力及对肺发育的影响.方法将30只孕17d的SD大鼠随机分为LPS组和生理盐水对照组,LPS组宫内注射LPS 10μl(40μg/ml),对照组宫内注射等体积的灭菌生理盐水.分别留取胎龄18、20、22 d(E18、E20、E22)的胎鼠肺组织、胎盘组织标本以及生后1、3、7d(P1、P3、P7)新生鼠肺组织标本,HE染色观察病理改变,RT-PCR技术检测TLR4、髓样分化因子88(MyD88)和白介素1 β(IL-1β)mRNA表达,免疫组织化学技术检测肺组织TLR4、MyD88的表达分布情况.实验数据采用单因素方差分析和q检验进行统计学分析.结果 (1)LPS组孕鼠胎盘组织有大量中性粒细胞浸润,宫内感染模型建立成功;(2)在E18、E20和E22时,LPS组胎鼠肺组织无明显病理学改变,以后逐渐出现改变,于P7时可见肺泡数量减少,肺泡腔变大,间隔变薄,但未见明显结构紊乱;(3)LPS组TLR4、MyD88和IL-1β mRNA水平于E20和E22均高于对照组,差异有统计学意义(P<0.05),且均于E22表达达高峰,后缓慢下降;(4)免疫组织化学结果显示E18时两组肺组织内均未见明显TLR4和MyD88阳性染色,后均逐渐表达增加,且主要在细支气管和肺泡上皮细胞表达.结论 (1)宫内注射LPS可导致胎鼠和早产鼠肺组织TLR4、MyD88表达在一定范围内增加,后逐渐回复正常水平,同时肺组织的病理改变和炎症反应较为温和,推测在围产期胎肺天然免疫系统可以调节LPS诱导的炎症反应强度;(2)该实验在一定程度上证实宫内感染激活的信号转导通路是MyD88依赖性途径.
목적 관찰궁내주사지다당(LPS)대위산기대서폐내천연면역상관적Toll양수체4(TLR4)신호전도통로적영향,탐토천연면역재궁내감염중적면역조절능력급대폐발육적영향.방법 장30지잉17d적SD대서수궤분위LPS조화생리염수대조조,LPS조궁내주사LPS 10μl(40μg/ml),대조조궁내주사등체적적멸균생리염수.분별류취태령18、20、22 d(E18、E20、E22)적태서폐조직、태반조직표본이급생후1、3、7d(P1、P3、P7)신생서폐조직표본,HE염색관찰병리개변,RT-PCR기술검측TLR4、수양분화인자88(MyD88)화백개소1 β(IL-1β)mRNA표체,면역조직화학기술검측폐조직TLR4、MyD88적표체분포정황.실험수거채용단인소방차분석화q검험진행통계학분석.결과 (1)LPS조잉서태반조직유대량중성립세포침윤,궁내감염모형건립성공;(2)재E18、E20화E22시,LPS조태서폐조직무명현병이학개변,이후축점출현개변,우P7시가견폐포수량감소,폐포강변대,간격변박,단미견명현결구문란;(3)LPS조TLR4、MyD88화IL-1β mRNA수평우E20화E22균고우대조조,차이유통계학의의(P<0.05),차균우E22표체체고봉,후완만하강;(4)면역조직화학결과현시E18시량조폐조직내균미견명현TLR4화MyD88양성염색,후균축점표체증가,차주요재세지기관화폐포상피세포표체.결론 (1)궁내주사LPS가도치태서화조산서폐조직TLR4、MyD88표체재일정범위내증가,후축점회복정상수평,동시폐조직적병리개변화염증반응교위온화,추측재위산기태폐천연면역계통가이조절LPS유도적염증반응강도;(2)해실험재일정정도상증실궁내감염격활적신호전도통로시MyD88의뢰성도경.
Objective To investigate the influence of intrauterine infection caused by lipopolysaccharide on Toll-like receptor 4 (TLR4) signaling pathway in fetal and neonatal rat lungs in order to explore immunomodulating activity of innate immunity responding to intrauterine infection and its effect on lung development. Methods On day 17 of pregnancy, 30 pregnant Sprague-Dawley (SD) rats were randomly divided into two groups: LPS group and saline group. For LPS group, LPS (10 μl, 40 μg/ml) was intrauterine injected between every two embryonic sacs of the pregnant rats, while the rats in the control group were injected with the same volume of pyrogen-free saline. Lung tissues of fetal rats and corresponding placental tissues were collected on the embryonic day 18 (E18), E20, and E22. Neonatal lung tissues were also harvested on postnatal day 1(P1), P3, and P7. Lung sections and placental tissues were stained with hematoxylin and eosin for histological examination. Reverse transcription quantitative polymerase chain reaction (RT-PCR) analysis was performed to test mRNA expression for TLR4, myeloid differentiation 88 (MyD88) and IL-1β, while immunohistochemistry was used to evaluate TLR4 and MyD88 expression in lung tissues. All data were analyzed with one-way analysis of variance (ANOVA) and q teal Results (1) Placental hematoxylin-eosin staining showed a great number of neutrophils infiltration, obvious interstitial hyperplasia and narrow capillaries in placental tissues in the LPS group which indicated that intrauterine infection occurred. However, there were no obvious inflammatory cells in the control group. (2) On E18, E20 and E22, the lung of LPS group showed no obvious pathological changes, and there were no apparent neutrophils infiltrated in alveoli, then some structural changes appeared. On P7, we found that the number of alveoli decreased, space of alveoli was Larger than ever, septa thickened, but without significant constructive disorder. (3) In the LPS group, the TLR4, MyD88 and IL-1β mRNA levels increased compared with control group, higher than control group at E20 and E22 (P<0.05), and peaked at E22.Then the expression levels of these substances decreased slowly. (4) The result of immunohistechemistry showed that in lung tissues of the two groups at E18, there was no remarkable positive staining of TLR4 and MyD88, which mainly expressed in cytoplasm of bronchiole and alveolar epithelial cells, then positive cells increased slowly. Conclusion (1) For perinatal rat lungs, intrauterine LPS infusion can induce an increased expression levels of TLR4 and MyD88 to a certain extent, which then returned to normal level gradually. At the same time, lung tissues showed a mild pathological change and inflammatory reaction. We propose that innate immune system of fetal lungs controls the magnitude of the LPS-induced cytokine response during the perinatal period. (2) The findings confirmed that LPS-activated signaling transduction pathway was the MyD88-dependent pathway.