【摘要】：目的:研究?jī)?nèi)皮祖細(xì)胞培養(yǎng)上清(endothelial progenitor cell-conditioned medium,EPC-CM)對(duì)高氧暴露新生大鼠肺損傷時(shí)肺泡結(jié)構(gòu)的改善作用及其機(jī)制。方法:從新生SD大鼠骨髓中獲取內(nèi)皮祖細(xì)胞并鑒定,收集第3代細(xì)胞的培養(yǎng)上清備用。另取新生SD大鼠40只隨機(jī)分為4組,即空氣組:仔鼠在空氣(21%O_2)中喂養(yǎng)21天;高氧組:仔鼠在85% O_2中喂養(yǎng)21天;內(nèi)皮細(xì)胞基礎(chǔ)培養(yǎng)基(endothelial cell basal medium,EBM)干預(yù)組:仔鼠在85%O_2中喂養(yǎng)至第14天時(shí),經(jīng)氣道給予100μL EBM,然后喂養(yǎng)至第21天;EPC-CM干預(yù)組:仔鼠在85% O_2中喂養(yǎng)至第14天,經(jīng)氣道給予100μL EPC-CM,喂養(yǎng)至第21天。第21天處死小鼠,左肺用4%多聚甲醛固定,留作石蠟切片,隨后HE染色進(jìn)行肺組織病理形態(tài)學(xué)觀察,并做輻射狀肺泡計(jì)數(shù)(radical alveolar count,RAC)及肺泡平均線性截距(mean linear intercept,MLI)測(cè)量;免疫組織化學(xué)方法對(duì)血管內(nèi)皮細(xì)胞FVIII染色,計(jì)數(shù)肺組織微血管密度;右肺留作實(shí)時(shí)熒光定量PCR檢測(cè)肺組織KGF、VEGF、SP-A和SP-C的mRNA表達(dá)。結(jié)果:培養(yǎng)所得細(xì)胞具有典型的EPCs形態(tài)改變,能結(jié)合異硫氰酸熒光素標(biāo)記的荊豆凝集素1并攝取Di I熒光標(biāo)記的乙�；兔芏戎鞍�。高氧組及EBM干預(yù)組的仔鼠體重、RAC、MLI和微血管密度較空氣組顯著降低(P0.05),EPC-CM干預(yù)組的RAC和微血管密度較高氧組和EBM干預(yù)組明顯增加(P0.05),而體重和MLI的變化無明顯差異,但有增高的趨勢(shì)。高氧組和EBM干預(yù)組肺組織KGF、VEGF、SP-A和SP-C的mRNA表達(dá)較空氣組顯著降低(P0.05),EPC-CM干預(yù)組的表達(dá)顯著高于高氧組和EBM干預(yù)組(P0.05)。結(jié)論:EPC-CM可改善高氧暴露新生大鼠的肺泡化和肺血管發(fā)育,可能與促進(jìn)肺內(nèi)KGF和VEGF mRNA的表達(dá)相關(guān)。
[Abstract]:Objective: To study the effect and mechanism of endothelial progenitor cell-conditioned medium (EPC-CM) on pulmonary alveolar structure in neonatal rats with hyperoxia exposure. Methods: to obtain endothelial progenitor cells from the bone marrow of newborn SD rats and identify the third generation cell culture supernatant. Another newborn SD rat was taken. 40 rats were randomly divided into 4 groups, that is, the air group: the young rats were fed in the air (21%O_2) for 21 days; the high oxygen group was fed for 21 days in 85% O_ 2; the endothelial cell basal medium (endothelial cell basal medium, EBM) intervention group: when the offspring were fed in 85%O_ 2 for fourteenth days, the channel was given 100 u L EBM, and then fed to twenty-first days; EPC-CM intervention group: Offspring Rats For fourteenth days in 85% O_ 2, 100 mu L EPC-CM was given through the airway and twenty-first days were fed for twenty-first days. The left lung was fixed with 4% polyformaldehyde and left for paraffin section. Then, the lung tissue was observed by HE staining, and the radiated alveolar count (radical alveolar count, RAC) and the mean linear intercept of pulmonary alveolus (mean linear inter) Cept, MLI) measurement; immunohistochemical method for FVIII staining of vascular endothelial cells, counting the microvascular density of lung tissue, and right lung for real-time fluorescence quantitative PCR to detect the mRNA expression of KGF, VEGF, SP-A and SP-C in lung tissue. Results: the cultured cells have typical EPCs morphologic changes and can be combined with fluorescein isothiocyanate labeled agglutinin 1. The body weight, RAC, MLI and microvascular density in the high oxygen group and the EBM intervention group were significantly lower than those in the air group (P0.05). The RAC and microvascular density in the EPC-CM intervention group increased significantly (P0.05) in the EPC-CM intervention group (P0.05), but there was no significant difference in weight and MLI, but there was an increased trend in the EPC-CM intervention group. The expression of KGF, VEGF, SP-A and SP-C in the lung tissue of the hyperoxic group and the EBM intervention group was significantly lower than that in the air group (P0.05). The expression of the EPC-CM intervention group was significantly higher than that of the hyperoxia group and the EBM intervention group (P0.05). Conclusion: EPC-CM can improve the alveolation and the development of pulmonary blood tube in the newborn rats with hyperoxia exposure, which may be related to the promotion of the expression of lung KGF and the expression of the lungs.
【作者單位】： 復(fù)旦大學(xué)附屬兒科醫(yī)院;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81270727)
【分類號(hào)】：R722.6

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