高氧致慢性肺疾病早产鼠的肺组织CDK4、p21动态变化及其意义
Dynamic expression and significance of CDK4、p21 in the lung of premature rats with hyperoxia induced chronic lung disease

慢性肺疾病(CLD)是早产儿长时间吸入高浓度氧后最常见的并发症,严重影响早产儿远期生活质量[1]，因此探寻CLD发生机制对降低早产儿死亡率、改善早产儿预后具有深远的意义。尽管CLD发病机制仍未清楚，但最终发生肺组织纤维化的病理结局已被大量临床和动物研究所证实，其病理过程为在CLD各种致病因素下（如高氧、机械通气、感染等）肺上皮细胞受损、损伤后组织重塑和肺间质细胞增生[2]。细胞周期是细胞生命活动的基本过程，其发展受细胞周期检控点的严格控制，而检控点活化则需细胞周期依赖蛋白激酶（cyclin dependent kinase, CDK）如CDK2、CDK4及细胞周期蛋白依赖激酶抑制因子（cyclin dependent kinase inhibitor, CKI）如p53、p21的相互协调作用。国内、外很少有人从细胞周期调控角度探讨早产儿CLD肺纤维化发病机制。因此，本文以高氧诱导早产鼠CLD模型为对象，研究CDK4及p21动态表达规律及其对CLD肺纤维化的影响作用，试图阐明早产儿CLD肺纤维化的细胞周期调控机制，对完善CLD发生机制及研究新的防治策略将有重要意义。
Chronic lung disease (CLD) is the most common complication accompanying the long duration of hyperoxia treatment in premature infants, which severely affects long-term quality of life of the premature infants [1]. Therefore, it is of profound significance to explore the pathogenesis mechanism of CLD in order to reduce the death rate and improve the prognosis of premature infants. Although the pathogenesis mechanism of CLD has been unknown, the pathological outcome of pulmonary interstitial fibrosis has been proved by many clinical and animal researches. The pathological process is injury of lung epithelial cells under the pathogenic factors of CLD (such as hyperoxia, mechanical ventilation and infection), tissue remodeling after injury, and pulmonary interstitial fibrosis [2]. The cell cycle is the basic process in cell life and its development is strictly controlled by the cell cycle checkpoints. The activation of checkpoints needs the coordination of cyclin dependent kinase (CDK), such as CDK2 and CDK4 and cyclin dependent kinase inhibitor (CKI), such as p21 and p53. In China and abroad, few researches of the pathogenesis of CLD pulmonary interstitial fibrosis of premature infants are conducted from the perspective of cell cycle control. This article takes hyperoxia induced premature rats with CLD as the object, studies the dynamic expression of CDK4 and p21 and the influence on pulmonary interstitial fibrosis of CLD, in order to clarify the control mechanism of the cell cycle in the pulmonary interstitial fibrosis of the premature infants. It is significant to perfect the pathogenesis mechanism of CLD and study new strategies of prevention and treatment of CLD.

材料与方法
Materials and method

华译网北京翻译公司翻译过大量有关高氧致慢性肺疾病早产鼠的肺组织CDK4、p21动态变化及其意义的文件资料,Beijing Chinese Translation Service Company has translated many technical documents about Dynamic expression and significance of CDK4、p21 in the lung of premature rats with hyperoxia induced chronic lung disease

一、实验对象及分组  孕21d SD大鼠（由中国医科大学实验动物部提供）剖宫取出的鼠仔即为早产鼠[1]。依据吸氧浓度（FiO2）随机分为实验组（FiO2为90%）和对照组（FiO2为21%）,每组均为40只。平均体重分别为（4.84±0.13）g、（4.83±0.12）g，雌雄不限。
One. Object and Grouping The mice taken from pregnant 21d SD rats (provided by the Department of Experimental Animals of China Medical University) by cesarean section are premature rats [1]. They were randomly assigned into 2 groups: the Model group with CLD induced by hyperoxia (90% FiO2, n=40), and the Control group (21% FiO2, n=40). The average body weight is （4.84±0.13）g and（4.83±0.12）g, including both male and female.
二、动物模型制备  实验组将早产SD大鼠（连同母鼠）生后即置于氧箱中，持续输入氧气，维持FiO2为90%(用测氧仪监测)，CO2浓度<0.5%（用钠石灰吸收CO2），温度25℃~27℃，湿度50%~70%，每24 h定时开箱30 min，添加水、饲料及更换垫料，并与对照组交换母鼠以避免其因氧中毒致喂养能力下降。对照组FiO2为21%，具体方法及实验控制因素同实验组[3]。
Two. Preparation of Animal Model In the Model group, the premature rats (including the mother rats) were put into the oxygen box after birth. The oxygen gas is input continuously. FiO2 was maintained at 90% (monitored by oxygen measuring meter), concentration of CO2 <0.5% (absorbed by soda lime), temperature was 25℃~27℃, and humidity was 50%~70%. The box was opened every 24h to add water and feed and change bedding. The mother rats were exchanged with those in the Control group to prevent the declining of feeding ability due to oxygen toxicity. FiO2 in the Control group was 21% and the method and control factors of the experiment were the same with those in the Model group [3].
三、研究方法  1．标本采集：每组分别于实验后的1、3、7、14和21d随机选取8只麻醉后处死。将肺组织置于4%多聚甲醛中固定，石蜡包埋，制备切片。2．肺组织纤维化评分[2]：石蜡切片经苏木素-伊红染色后，于10倍光镜下依肺纤维化程度进行双盲评定，分为0~8分，1分：肺泡或细支气管壁少许纤维化；2~3分：中度纤维化，无肺泡结构改变；4~5分：有明确的肺泡结构变化（肺泡壁明显增厚）和纤维小结；6~7分：严重的肺泡结构改变和大片纤维化；8分：完全纤维化。3．肺组织CDK4及p21蛋白表达检测：分别以1：100稀释的兔抗大鼠CDK4及p21蛋白抗体为一抗（武汉博士德生物制品公司），按SABC方法（武汉博士德生物制品公司）进行染色，以细胞浆有棕黄色颗粒沉着为阳性细胞。结果判定：应用美国Universal Imaging  Porporation 图像分析系统，以平均灰度值代表阳性产物的表达强度。平均灰度值越低，其阳性反应产物表达强度越强，表明蛋白的含量越高。
Three. Research Method 1. Collection of samples: on the 1st, 3rd, 7th, 14th and 21st days after the experiment, eight rats were selected randomly from the two groups to be executed after anesthesia. The lung tissue was put in 4% paraformaldehyde to be fixed and paraffin embedded to be sliced.2. Scoring of pulmonary interstitial fibrosis[2]：after hematoxylin-eosin (HE)staining, the paraffin sections were evaluated double-blind under 10x light microscope according to the fibrosis of the lung. The scoring range was 1-8. 1:  a little fibrosis in the alveolus or the wall of bronchiole; 2-3: moderate fibrosis, no change of the structure of alveolus; 4-5: clear change of the structure of alveolus (obvious thickening of wall of alveolus) and fibrosis nodule; 6-7: serious change of the structure of alveolus and fibrosis in a large area; 8: complete fibrosis. 3. Determination of expression of CDK4 and p21: rabbit antirat CDK4 and p21 protein antibody (Wuhan Bo Shi De Biological Product Company) diluted into 1:100 were taken as the first antibody, and they were stained with SABC (Wuhan Bo Shi De Biological Product Company) method. The positive cells were those that had deep yellow particles in the cytoplasm .Determination of the result: American image analysis system of Universal Imaging Porporation was applied and the average gray value represented the expression strength of the positive product. The lower the average gray value was, the stronger the expression strength of the positive product was, which shows the protein contents were higher.
四、统计学处理  应用SPSS11.5统计软件，所有数据均以 ±S表示，组间比较应用t检验，相关分析采用Spearman分析。
Four. Statistical Treatment The statistical software of SPSS 11.5 was applied and all the data were expressed as  ±S. The comparison between the two groups was checked by t and Spearman analysis was used for relative analysis.