缺氧对体外培养的肺动脉平滑肌细胞形态及增殖的影响

EFFECTS OF HYPOXIA ON THE MORPHOLOGY AND PROLIFERATION OF CULTURED PULMONARY ARTERIAL SMOOTH MUSCLE CELL

本实验应用形态学、免疫组化染色、３Ｈ－ＴｄＲ掺入、流式细胞等技术探讨缺氧（＜１％Ｏ２和２．５％Ｏ２）对肺动脉平滑肌细胞（ＰＡＳＭ）形态及增殖的影响。结果表明：缺氧２４ｈ使ＰＡＳＭ表型从收缩型向合成型转换，胞浆内ＳＭ－αａｃｔｉｎ减少，线粒体和粗面内质网增多，缺氧４８ｈ以后线粒体肿胀、空泡化，内质网扩张，胞浆内出现髓鞘样结构。流式细胞分析显示缺氧ＰＡＳＭＧ２／Ｍ期细胞比例明显增多（Ｐ＜０．００１），无氧（＜１％Ｏ２）１２ｈ末Ｓ期细胞比例明显增多（Ｐ＜０．０５），之后Ｓ期细胞比例明显减少（Ｐ＜０．００１），缺氧ＰＡＳＭＧ０／Ｇ１期细胞比例明显减少（Ｐ＜０．００１），细胞内总蛋白含量有所降低（Ｐ＜０．００１）。低氧６ｈ使ＰＡＳＭ３Ｈ－ＴｄＲ掺入减少（Ｐ＜０．０５），无氧６ｈＰＡＳＭ的３Ｈ－ＴｄＲ掺入显著减少（Ｐ＜０．００１），而缺氧１２ｈ则促进其掺入，至２４ｈ末２．５％Ｏ２组和＜１％Ｏ２组３Ｈ－ＴｄＲ参入均较常氧对照组明显增多（Ｐ＜０．０５）。无氧２４ｈ使ＰＡＳＭ增殖细胞核抗原（ＰＣＮＡ）阳性反应颗粒明显增加，其含量是常氧的２．７倍。表明缺氧早期ＰＡＳＭ增殖受抑制，１２ｈ以后ＰＡＳＭ则发生表型转换和增殖，推测缺氧?

The effects of hypoxia (2 5%O 2 and/or <1%O 2 ) on bovine pulmonary arterial smooth muscle cell(PASM) proliferation and phenotype change were investigated using immunocytochemical analysis and 3 H TdR incorporation. The results showed that hypoxia initiated the change of PASM from contractile phenotype to synthetic phenotype.The synthetic phenotype smooth muscle cell was characterized by increase in endoplasmic reticulum and mitochondria,and by decrease in α actin and muscle fiber at the end of 24h hypoxia. Then the endoplasmic reticulum became dilated , the mitochondria became swollen and myelin figure appeared after 48h hypoxic exposure. PASM 3 H TdR incorporation was decreased at the end of 6h hypoxia ( P <0 05),then increased gradually at the end of 12h. PASM DNA synthesis was significantly stimulated by 24h hypoxia ( P <0.05). Flow cytometric DNA analysis revealed that hypoxia induced significant enhancement of G 2/M phase of PASMs, decreased G 0/G 1 phase of PASMs( P <0. 001) , and only increased S phase of PASMs in 12h anoxia group ( P <0.05), but decreased S phase of PASMs from 24h to 48h ( P <0.001). Immunocytochemical reaction of proliferating cell nuclear antigen(PCNA) in PASM under anoxic condition was more stronger than that of normoxic PASM at 24h. These results suggest that hypoxia may at first inhibit and then stimulate PASM proliferation and induce phenotype change,which may lead to the development of hypoxic pulmonary hypertension