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目的探讨5溴脱氧尿苷(B rdu)和端粒酶逆转录酶(TERT)标记肺干细胞特点及其增殖、分化在肺发育和高氧肺损伤修复中的作用。方法(1)3 d新生鼠分为高氧组(95%左右氧气7 d)、高氧组siRNA干预组(高氧同时TGF-β1 siRNA经腹腔注入,隔日1次,共3次)和正常对照组,处死前自腹腔注入B rdu。(2)免疫组化法检测B rdu和TERT阳性显色细胞,并分别使用碱性磷酸酶和辣根过氧化氢酶两种显色系统作B rdu/TERT和SPC抗体免疫双染。(3)分离新处死肺细胞,立即涂片,做B rdu和TERT免疫标记,计数阳性细胞百分比。结果(1)高氧组可见肺泡壁较薄、结构简单化、肺泡大小不均,有些肺泡融合、体积增加,肺泡腔有较多脱落的AEC II。(2)肺组织B rdu阳性显色部位主要在各级支气管黏膜下和肺间隔,支气管黏膜上皮及肺泡壁有散在分布,阳性细胞数量较少,多呈立方形、核大;TERT阳性显色在外周肺组织肺间隔和肺泡壁部位,数量明显少于B rdu[表达积分(1.61±0.83)vs.(0.62±0.55),P<0.05],且阳性显色呈不对称性,即多集中在肺某一区域;高氧肺组织B rdu和TERT阳性标记细胞稍高于正常对照组[(1.43±0.85)vs.(1.61±0.83);(0.62±0.55)vs.(0.83±0.84),P>0.05]。(3)SPC分别和B rdu、TERT双染,可见少量阳性细胞。(4)肺细胞SPC免疫显色部位在胞浆,呈棕褐色,阳性细胞大小不一,高氧组和正常组显色细胞数无明显区别,阳性细胞百分比分别为80.3%、78.6%。(5)肺细胞B rdu阳性显色细胞明显少于SPC阳性细胞,体积明显大于未显色细胞,核形态主要为致密圆形,个别呈杆状;高氧组阳性细胞数略多于正常组,阳性细胞百分比分别为28.5%、21.4%。(6)TERT阳性显色细胞更为少见,阳性细胞体积多较小,核呈多种形态包括致密圆形、疏松圆形、杆状、分叶状;正常组和高氧组阳性细胞百分比分别为1.5%、2.3%。结论(1)B rdu和TERT可作为不同分化能力的肺干细胞标记,其中TERT更能反映肺干细胞特征或是成体肺干细胞标记更特异性的指标,B rdu标记从干细胞增殖分化尚保留干细胞特征的TAC。(2)高氧肺损伤时肺干细胞出现有限增殖分化。
Objective To investigate the characteristics of lung stromal cells labeled with BrdU and telomerase reverse transcriptase (TERT) and the role of proliferation and differentiation in lung development and hyperoxia - induced lung injury. Methods (1) Three days old neonatal rats were divided into hyperoxia group (95% oxygen for 7 days), hyperoxia group (normoxia and TGF-β1 siRNA injected intraperitoneally every other day for 3 times) and normal In control group, B rdu was injected intraperitoneally before sacrifice. (2) Brudu and TERT positive cells were detected by immunohistochemistry. Two kinds of chromogenic system of alkaline phosphatase and horseradish peroxidase were used respectively to immunoblot the Bdu / TERT and SPC antibodies. (3) Separate the newly-killed lung cells, smear them immediately, make B rdu and TERT immune markers and count the percentage of positive cells. Results (1) The hyperoxia group showed thinner alveolar wall, simple structure, uneven alveolar size, some alveolar fusion, increased volume, and more AEC II with alveolar septa. (2) The positive staining area of B rdu in lung tissue mainly distributed in bronchial submucosa, interspace of lung, bronchial mucosa epithelium and alveolar wall at all levels. The number of positive cells was small, mostly cubic and nucleus; TERT positive The numbers of lung space and alveolar wall in peripheral lung tissue were significantly less than B rdu [(1.61 ± 0.83) vs. (0.62 ± 0.55), P <0.05], and the positive color was asymmetric In the lung region, the positive cells of B rdu and TERT in hyperoxia group were slightly higher than those in the normal control group [(1.43 ± 0.85) vs. (1.61 ± 0.83); (0.62 ± 0.55) vs. (0.83 ± 0.84) P> 0.05]. (3) SPC and B rdu, TERT double staining, showing a small amount of positive cells. (4) SPC immunopositive sites in the cytoplasm of the lung cells were tan, and the positive cells were of different sizes. There was no significant difference in the number of the chromogenic cells between the hyperoxia group and the normal group, with the percentages of positive cells being 80.3% and 78.6% respectively. (5) The number of B rdu positive cells in lung cells was significantly less than that in SPC positive cells, the volume was significantly larger than the non-color cells, , The percentage of positive cells were 28.5% and 21.4% respectively. (6) The TERT positive cells were more rare, the positive cells were smaller, the nucleus showed various forms including dense round, loose round, rod, lobulated; the percentage of positive cells in normal group and hyperoxia group were 1.5%, 2.3%. Conclusions (1) B rdu and TERT can be used as lung stem cell markers with different differentiation ability. TERT can reflect the characteristics of lung stem cells more specifically or more specific markers of adult lung stem cells. The B rdu marker can proliferate and differentiate from stem cells while retaining the characteristics of stem cells TAC. (2) Limited proliferation and differentiation of lung stem cells during hyperoxic lung injury.