5溴脱氧尿苷和端粒酶逆转录酶标记肺干细胞在新生鼠高氧肺损伤中的作用

Role of pulmonary stem cells labeled with bromodeoxyuridine and telomerase reverse transcriptase in hyperoxic lung injury in neonatal rats

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 characteristics of pulmonary stem cells labeled with bromodeoxyuridine （Brdu） and telomerase reverse transcriptase （TERT） in lung tissue, as well as the effects of proliferation and differentiation of the stem cells on lung development and repair of pulmonary injury. Methods A model of hyperoxia in neonatal rats was made by exposing the rats to 95% O2 for 7 d. Before sacrificing the rats, Brdu was injected through peritoneum, and immune staining positive cells were analyzed after the rats were sacrificed. TERT positive cells were stained by an immunohistochemical method. At the same time, the double staining for surfactant protein C （SPC） and Brdu or SPC and TERT were performed. Lung histologic study was done on HE stained tissue slices. Results （1）The lung with hyperoxic injury had thinner walls of alveoli, simple alveolar structure, fewer and larger alveoli, expanded and shrunken alveoli, and there were many fell-off alveolar epithelial cells in the alveolar cavities as well. （2）The cells positively stained with Brdu located in septa, mucosa and submucosa of various bronchi,scattering in epithelium of bronchi, and the number of positive cells was low, having a large nucleus. The TERT-positive cells were apparent in the septa and alveolar walls of peripheral lung tissue, characterized by uneven distribution in the lung lobes, the number of positive cells was less than that of Brdu-positive cells [integral of expression （1.61 ±0.83） vs. （0.62 ±0.55） ,P〈0.05]. The number of Brdu- and TERT-positive cells had no significant difference in hyperoxic rats compared to that in controls [ integral of expression （1.43±0.85） vs. （1.61±0.83）;（0.62±0.55） vs. （0.83±0.84）, P〉0.05]. （3）After double staining, a few positive cells were found in double-stained tissues with SPC and Brdu or TERT. （4） The cells positively stained with SPC antibody had different size. The percentage of positive cells was not significantly different between the hyperoxia group （ 80. 3 % ） and control group （78. 6 % ）. The Brdu positive staining located in nucleus of cells that had larger size than the cells not stained, round nucleus with intense staining（ seldom, pole-shaped）and the number of such cells was less than that of the SPC positive cells. The percentage of positive cells was not significantly different between the hyperoxia group （28.5 % ） and control group （21.4%）. （5）The TERT staining located in nucleus of cell that had smaller size than the cells not stained, various nuclear shape, including round intensively stained, round slightly stained, poleshaped and divided shape. The percentage of positive cells was not significantly different between the hyperoxia group （2.3%） and control group （1.5%）. Conclusions （1） Brdu and TERT, as markers of stem cells having different capability of differentiation, possess special characterisitics, respectively. The cells with Brdu could be transit amplifying cell （TAC） which retains characteristics of stem cells originated from differentiated stem cells, while, the cells stained with TERT especially reflects the characteristics of stem cells. （2） The proliferation and differentiation of pulmonary stem cells during hypoxic lung injury are limited and may be related with arrest of alveolization.