【摘要】目的探讨小鼠肺发育过程细胞增殖与凋亡的变化规律及凋亡相关基因p53、bcl-2在肺发育中的表达意义。方法不同发育天数的小鼠肺组织，采用免疫组织化学及TUNEL法，观察细胞增殖与凋亡及相关基因p53、bcl-2的表达。结果1.胎14和16 d肺PCNA阳性细胞数达到高峰；之后PCNA阳性率逐渐下降；生后4到14 d，又恢复低水平的细胞增殖；2.小鼠肺发育过程中出现两个细胞凋亡高峰：即胎14至16 d及生后7 d;3.bcl-2阳性表达开始于胎19 d延续至生后14 d，仅见于近端支气管上皮及血管平滑肌细胞；4.小鼠肺发育的各个时期均未见p53阳性细胞。结论小鼠肺发育过程中的细胞增殖与凋亡相伴存在，肺发育中的结构分化与两者的动态变化密切相关；肺发育中bcl-2的表达可能在于抑制细胞凋亡，细胞凋亡可能不依赖p53基因的调控。探讨小鼠肺发育过程细胞增殖与凋亡的变化规律及凋亡相关基因p53、bcl-2在肺发育中的表达意义。方法不同发育天数的小鼠肺组织，采用免疫组织化学及TUNEL法，观察细胞增殖与凋亡及相关基因p53、bcl-2的表达。结果1.胎14和16 d肺PCNA阳性细胞数达到高峰；之后PCNA阳性率逐渐下降；生后4到14 d，又恢复低水平的细胞增殖；2.小鼠肺发育过程中出现两个细胞凋亡高峰：即胎14至16 d及生后7 d;3.bcl-2阳性表达开始于胎19 d延续至生后14 d，仅见于近端支气管上皮及血管平滑肌细胞；4.小鼠肺发育的各个时期均未见p53阳性细胞。结论小鼠肺发育过程中的细胞增殖与凋亡相伴存在，肺发育中的结构分化与两者的动态变化密切相关；肺发育中bcl-2的表达可能在于抑制细胞凋亡，细胞凋亡可能不依赖p53基因的调控。

【中图分类号】R730.21【文献标识码】A【文章编号】0529-1356(2000)04-372

CELL PROLIFERATION， APOPTOSIS AND THE EXPRESSION OF

RELATED GENES IN THE DEVELOPING LUNG OF MOUSE

WANG QiangHUANG Zhong-xinXIA Chao-yong

（Department of Histology and Embryology, Medical College Jinan University, Guangzhou 510632, China）

【Abstract】Objective This study is to explore the dynamic trends of cell proliferation, apoptosis and the expression significance of involved genes as p53 and bcl-2 in developing lungs of mouse.Methods Lung tissues were taken from different developmental phases of Kunming mouse. Immunohistochemistry and TUNEL techniques were adopted to observe the variation of cell proliferation and apoptosis and the expression characteristics of p53 and bcl-2.Results 1.From the 14th to the 16th day of gestation the positive ratio of PCNA reached its peak. After that, it descended gradually. From the 4th day to the 14th day after birth, the proliferation maintained in low level; 2.Two peaks of apoptosis emerged during the development of lungs. The first one appeared from the 14th to the 16th day of gestation; the second one appeared at the 7th day after birth; 3.The expression of bcl-2 first appeared from the 19th day of gestation to the 14th day after birth, with constant expression sites in the epithelial cells lined in the proximal bronchia and the smooth muscle cells. 4.The expression of p53 was never seen in all phases during the developmental process.Conclusion 1.During the development of mouse lungs, cell proliferation was accompanied by apoptosis. The dynamic changes of both ratios might be correlated to the different structures appearing in different developmental phases; 2.The possible function of bcl-2 during the lung developmental process might be that it prevented the main frame structures of the bronchial tree and the capillary network from being destroyed by apoptosis; 3.Apoptosis during the development of lung might be independent of the regulation of p53.

【Key words】 Cell proliferation; Apoptosis; p53; bcl-2l; Fetal lung

细胞增殖和凋亡与机体众多生理及病理现象密切相关。在胚胎发育中两者的关系受各种生长因子或相关基因调控^［1］。研究胚胎发育中细胞增殖与凋亡的变化、相互关系及其相关基因的表达特点，对探讨胚胎正常发育过程中的相关机制很有意义。本课题研究小鼠肺发育过程中细胞增殖与调亡出现的部位及变化趋势，并探讨两者在肺结构和功能发育中的可能意义，同时分析凋亡相关基因p53及bcl-2在肺发育中的表达意义。

材料和方法

体重25～28 g的雌雄昆明小鼠同笼1晚，母鼠阴道口出现阴栓定为妊娠0 d。取胎14、16、18、19 d及生后2、4、7、14 d鼠肺组织，每组8例，4%中性福尔马林固定，常规石蜡包埋切片，片厚5μm，常规免疫组织化学ABC法染色，以TBS缓冲液替代一抗作阴性对照。一抗分别为：PCNA单抗(MO879，DAKO，工作浓度1∶100)；Bcl-2多抗(sc-492-G,Santa Cruz,工作浓度1∶100)；p53单抗(BMO521，Santa Cruz，工作浓度1∶50)；应用凋亡检测试剂盒(Apop-Tag in situ apoptosis detection kit, S7100-Kit, Onco公司)原位检测凋亡细胞。阳性对照为小鼠乳腺组织，阴性对照以TBS缓冲液代替TdT酶。每个样本高倍镜下随机选取5个视野，计算免疫组织化学阳性细胞数和视野总细胞数，原始数据经医学统计SPSS软件处理，求得实验各组细胞PCNA阳性率及凋亡率，并对其进行组与组之间卡方检验。

结果和讨论

1. 肺发育进程中的细胞增殖与凋亡

小鼠肺发育与人肺发育相似也经历4个时期，即假腺期(胎9.5～16.6 d)、小管期(胎16.6～17.4 d)、原始囊泡期(胎17.4 d至新生5 d)和肺泡期(新生5 d以后)^［2］。

本实验观察到，假腺期(胎14)，小鼠肺组织细胞增殖主要位于上皮细胞，此时间质细胞增殖处于较低水平(图1)。到小管期(胎16)，PCNA阳性细胞在上皮和间质均大量出现，总阳性率达到肺发育过程的顶点(图2)。原始囊泡期(胎18 d至新生5 d)，上皮细胞的增殖迅速减弱，间质细胞的增殖较强，间质内毛细血管增多，支气管远端逐渐分化为原始囊泡，上皮逐渐向肺泡上皮分化(图3)。肺泡期(新生5d 以后)，PCNA阳性率维持在较低水平。

肺发育在细胞增殖的同时，还必需将过度增殖或不再需要的细胞以细胞凋亡的形式加以清除。本实验发现，肺发育中细胞凋亡出现两个高峰：胎14和胎16 d的上皮及间质及生后7 d的肺泡间隔(图4～6)。静止组织的细胞凋亡率通常是0.1%～1%，而在肺发育过程中，细胞凋亡阳性率最高达到胎14 d的23.58%，说明细胞凋亡在肺发育中占有重要地位，是肺发育进程中的重要事件。

图1胎14 d，腺腔的立方上皮PCNA阳性表达明显多于间质细胞。PCNA(1∶100)×1 000

Fig.1 On the 14th day of gestation, the positive expression of PCNA in the cuboidal epithelial cells was apparently more than in the mesenchyma. PCNA(1∶100)×1 000

图2胎16 d，PCNA阳性表达广泛出现在各级支气管上皮及间质细胞。PCNA(1∶100)×1 000

Fig.2 On the 16th day of gestation, the positive expression of PCNA appeared extensively in bronchial epithelial cells and mesenchyma. PCNA(1∶100)×1 000

图3生后7 d，肺泡间隔内有散在分布的PCNA阳性细胞。PCNA(1∶100)×1 000

Fig.3 On the 7th day after birth, PCNA positive cells scattered in the septums of alveoli. PCNA(1∶100) ×1 000

图4胎14 d，凋亡细胞广泛出现在腺管上皮及间质中。TUNEL检测×400

Fig.4 On the 14th day of gestation, apoptotic cells appeared extensively both in the epithelium and in mesenchyma. TUNEL stain ×400

图5胎16 d，凋亡细胞大量出现于各级支气管上皮及间质。TUNEL检测×1 000

Fig.5 On th