上皮生长因子对鼠肺细胞动力学的调节及其对放射保护的意义(Ⅰ)

Modulating Effect of Keratinocyte Growth Factor on Cellular Kinetics of Murine Lung( I )

背景与目的:上皮生长因子(keratinocyte growth factor,KGF)引起肺Ⅱ型细胞增殖,并保护肺免受外界多种刺激。以往,对慢反应正常肺组织的动力学参数难以测定,然而,近年来发展的技术使得准确测定正常组织细胞动力学指标成为可 能。BrdUrd掺入DNA后,用流式细胞仪分析可测定组织中细胞的增殖状况,本研究的目的是采用上皮生长因子刺激后,探讨鼠肺正常组织细胞增殖动力学改变的情况。方法:采用KGF(5 mg/kg)或生理盐水C_3Hf/Kam小鼠气管内注射,并分别在0,1,2,3,4,5,及7天予以处死,处死前20分钟或6小时,腹腔内给予BrdUrd(60 mg/kg),肺组织切下后,固定于60％酒精,经消化处理形成单个细胞核,在流式细胞仪分析前行BrdUrd及总DNA含量标记染色,计算出标记指数(LI),细胞S期时间(Ts)及倍增时间(T_(pot))等细胞动力学参数;免疫荧光染色技术用于鉴定增殖细胞的类型。结果:(1)建立了上皮生长因子(5 mg/kg)刺激肺Ⅱ型细胞增殖的方法及气管内给药的途径;(2)肺组织LI由正常值0.5％升至给药后第三天的5.5％,第七天回至正常;(3)细胞T_(pot)由75.5天降至4.7天;生理盐水治疗组增殖参数没有变化。结论:上皮生长因子可引起肺Ⅱ型细胞的增殖,主要表现为LI的升高及T_(pot)降低。这种增殖效应短暂,第七天回至正常。

BACKGROUND &OBJECTIVE: Keratinocyte growth factor (KGF) causes the proliferation of type II pneumocytes in the lungs and confers protection against many external stimulation in the lung. Historically, the kinetic parameters, especially of slowly proliferating normal tissues, such as the lung, were difficult to measure. However, recently developed techniques made it possible to measure accurately the cellular kinetics in normal tissues. Flow cytometric techniques following bromodeoxyuridine (BrdUrd) incorporation into DNA of cells allow the accurate measurement of cellular proliferation. The purpose of this study was to measure the changes of the dynamic kinetics of normal lung tissue after treatment with KGF so as to build up the basis to prevent the occurrence of radiation-induced pneumonitis. METHODS: C3Hf/Kam mice were treated intratracheally (i. t. ) with KGF (5 mg/kg) or the control (saline) and were sacrificed at 0, 1, 2, 3, 4, 5, and 7 days. The mice were labeled intraperitoneally (i. p. ) with BrdUrd (60 mg/kg) at 20 minutes or 6 hours before sacrifice. Lungs were excised, fixed in 60% ethanol, digested to produce nuclei; and BrdUrd as well the total DNA content were labeled for flow cytometric analysis. The kinetic parameters including the labeling index (LI), duration of S-phase (Ts), and potential doubling time (Tpot) were measured by novel analytical methodology. Immunofluorescence staining was used to identify the specific cell type that was proliferating. RESULTS: (1 )An optimum route for the administration (i.t. ), dose (5 mg/kg), and time course of KGF to stimulate proliferation of type II pneumocytes in the lungs was established. (2) Lung LI control values (0. 5% ) rose to a maximum (5. 5% ) at 3 days after KGF treatment and returned to normal level on the 7th day. (3) Of the lung tissue, there is a dramatic reduction in Tpot from 75. 5 days to 4. 7 days in the KGF-treated mice, while the saline-treated control mice exhibited no change in proliferative parameter values. CONCLUSION: KGF caused the proliferation of type II pneumocytes, followed with the elevated LI and reduced Tpot. This proliferative effect was transient and levels returned to normal level by the 7th day. The data obtained from this study would lay the groundwork for future investigation of KGF as a possible radioprotector of the lung in the field of radiation oncology.