【摘要】：目的:改進(jìn)人肺動脈內(nèi)皮細(xì)胞(human pulmonary artery endothelial cells,HPAECs)-人肺動脈平滑肌細(xì)胞(human pulmonary artery smooth muscle cells,HPASMCs)接觸式共培養(yǎng)方法 ,為更好模擬體內(nèi)兩種細(xì)胞間相互作用提供研究平臺。方法:采用微孔聚碳酸酯膜為載體,將HPAECs和HPASMCs接種于微孔膜兩側(cè),建立HPAECs-HPASMCs聯(lián)合培養(yǎng)模型以模擬正常血管壁兩種細(xì)胞的結(jié)構(gòu)關(guān)系,通過調(diào)整細(xì)胞種植密度、培養(yǎng)時(shí)間、培養(yǎng)液體系、培養(yǎng)基種類、血清濃度等,倒置相差顯微鏡、Hoechst染色及流式細(xì)胞術(shù)比較不同條件下接觸式共培養(yǎng)模型中細(xì)胞貼壁、生長和凋亡的差異,免疫熒光染色觀察優(yōu)化共培養(yǎng)條件后細(xì)胞標(biāo)記物的表達(dá)變化。結(jié)果:(1)明膠預(yù)包被Transwell膜可促進(jìn)內(nèi)皮細(xì)胞的黏附和生長;(2)滲透壓升高增加內(nèi)皮細(xì)胞凋亡;(3)內(nèi)皮細(xì)胞培養(yǎng)基較DMEM、RPMI1640更有利于接觸式共培養(yǎng)模型的建立;(4)內(nèi)皮細(xì)胞生長因子為1%、胎牛血清為2%時(shí)兩種細(xì)胞生長穩(wěn)定;(5)優(yōu)化共培養(yǎng)條件對兩種細(xì)胞的自身特性無顯著影響。結(jié)論:當(dāng)培養(yǎng)體系為內(nèi)皮細(xì)胞培養(yǎng)基、維持1%內(nèi)皮細(xì)胞生長因子及2%胎牛血清組分條件,可建立穩(wěn)定的HPAECs-HPASMCs接觸式共培養(yǎng)模型,為體外模擬人肺動脈血管壁結(jié)構(gòu)功能和研究兩種細(xì)胞間相互作用構(gòu)建了良好平臺。
[Abstract]:Aim: to improve the contact co-culture method of human pulmonary artery endothelial cells (human pulmonary artery endothelial cells,HPAECs) and human pulmonary artery smooth muscle cells (human pulmonary artery smooth muscle cells,HPASMCs) in order to provide a research platform for better simulating the interaction between two kinds of cells in vivo. Methods: microporous polycarbonate membrane was used as carrier, HPAECs and HPASMCs were inoculated on both sides of microporous membrane. HPAECs-HPASMCs co-culture model was established to simulate the structural relationship between the two kinds of cells in normal vascular wall. The differences of cell adhesion, growth and apoptosis in contact co-culture model under different conditions were compared by inverted phase contrast microscope, Hoechst staining and flow cytometry, including culture medium system, type of culture medium, serum concentration, inverted phase contrast microscope, flow cytometry and so on. Immunofluorescence staining was used to observe the expression of cell markers after optimizing the co-culture conditions. Results: (1) Transwell membrane precoated with gelatin could promote the adhesion and growth of endothelial cells, (2) the increase of osmotic pressure increased the apoptosis of endothelial cells, (3) the culture medium of endothelial cells was more favorable to the establishment of contact co-culture model than that of DMEM,RPMI1640. (4) when the endothelial growth factor was 1% and the fetal bovine serum was 2%, the growth of the two kinds of cells was stable, and (5) the optimal co-culture conditions had no significant effect on the self-characteristics of the two kinds of cells. Conclusion: stable HPAECs-HPASMCs contact co-culture model can be established by maintaining 1% endothelial growth factor and 2% fetal bovine serum components in the medium of endothelial cell culture. A good platform was constructed for simulating the structure and function of human pulmonary artery wall in vitro and studying the interaction between two kinds of cells.
【作者單位】： 南京醫(yī)科大學(xué)第一附屬醫(yī)院呼吸與危重癥醫(yī)學(xué)科;
【基金】：國家自然科學(xué)基金(81273571) 江蘇高校優(yōu)勢學(xué)科建設(shè)工程資助項(xiàng)目(JX10231802)
【分類號】：R563
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本文編號：2442350