【摘要】目的研究卡氏肺孢子虫肺炎(PCP)引起的肺泡表面活性蛋白A和D(SP-A、SP-D)的变化，以探讨其在PCP发病机制中的作用和治疗中的意义。方法采用清洁级SD大鼠每周2次皮下注射醋酸可的松方法建立PCP模型为实验组，并设正常对照组，阴性对照组，细菌性肺炎组。8～12周分批处死动物，收集支气管肺泡灌洗液(BALF)，细胞计数分类；采用免疫斑点法测定BALF中SP-A、SP-D含量。结果根据实验设计和所诱导模型分组，A组：正常对照组，n=6，健康SD大鼠；B组：阴性对照组，n=6，使用醋酸可的松(CA)8周以上未发生肺部感染的大鼠；C组：细菌性肺炎组，n=11，CA诱导8周以上发生细菌性肺炎而无其他肺部感染；D组：PCP组，n=14，CA诱导8周以上出现PCP而无其他肺部感染。BALF中SP-A含量在PCP为(45.1±22.1)μg/ml，明显高于阴性对照组的(16.2±9.9)μg/ml和细菌性肺炎组的(16.2±5.6)μg/ml(P＜0.05)，同样SP-D相对含量(24 249±4 780灰度值)明显高于细菌性肺炎组(11 989±2 750灰度值)和阴性对照组(13 384±2 887灰度值)(P＜0.001)。结论PCP引起肺表面活性蛋白A和D的显著升高，且该变化较细菌性肺炎明显，应在PCP的防治中引起重视。

Alteration of surfactant protein A and D in bronchoalveolar lavage fluid in rats with pneumocystis carinii pneumonia

QU Jieming, RONG Zhaohui, HE Lixian

（Department of Pulmonary Medicine, Zhongshan Hospital, Institute of Respiratory Diseases, Shanghai Medical University, Shanghai 200032, China）

【Abstract】ObjectiveTo study the alteration of surfactant protein A and D (SP-A, SP-D) resulting from pneumocystis carinii pneumonia (PCP) and investigate its implication in the pathogenesis of PCP. MethodsSD rat models of PCP were induced by subcutaneous injection of 25 mg cortisone acetate, normal control and negative control as well as bacterial pneumonia group were set up for comparison. During 8～12 weeks, bronchoalveolar lavage fluid (BALF) of rats was collected. Total nucleate cells of BALF were counted and differentiated as well as the concentrations of surfactant protein A (SP-A) and surfactant protein D (SP-D) were measured by immunoblotting assay. ResultsThe rats were divided into three immunosuppressive groups, plus a normal control group. Group A: normal control (n=6) consisted of healthy SD rats; group B: negative control (n=6) employed rats with cortisone acetate injection over 8 weeks without lung infection; group C: bacterial pneumonia (n=11), rats were injected with cortisone acetate over 8 weeks and resulted in bacterial pneumonia without other pathogens isolated; group D (n=14): rats were injected with cortisone acetate during 8～12 weeks and resulted in PCP without other pathogens isolated. During PCP infection, the total cell counts and the percentage of polymorphonuclears (PMNs) in BALF were significantly increased (P＜0.01), but were lower than those in the bacterial pneumonia group. The concentration of SP-A of BALF in PCP (45.1 μg/ml±22.1 μg/ml) was significantly increased in comparison with that in negative control group (16.2 μg/ml±9.9 μg/ml, P＜0.05) and that in bacterial pneumonia group (6.2 μg/ml±5.6 μg/ml, P＜0.001). We also found that the relative content of SP-D was significantly higher in PCP (24 249±4 780 grey values) than that in both negative control (13 384±2 887 grey values, P＜0.001) and bacterial pneumonia group (11 989±2 750 grey values, P＜0.001). SP-A and SP-D were also higher in moderate to severe group of PCP than those seen in mild group (P＜0.01, P＜0.001). ConclusionThere was obvious increase of SP-A and SP-D in PCP rats, and particularly, the change of which was greater than that in bacterial pneumonia. Therefore, the alteration of SP-A and SP-D may be of implication in the prevention and management of PCP.

【Key words】Pneumocystis carinii pneumonia; Bronchoalveolar lavage fluids; Surfactant protein A, D

卡氏肺孢子虫肺炎(PCP)是艾滋病(AIDS)等免疫损害宿主最常见的呼吸系统感染性疾病^［1］，卡氏肺孢子虫(PC)进入肺泡腔与肺泡上皮相互作用，导致肺泡表面衬层发生改变，肺泡腔内充满大量渗出物。表面活性蛋白A和D(Surfactant Protein A、D；SP-A、SP-D)是肺泡腔内大量渗出物中主要的成分之一。SP-A、SP-D是结构相似的糖蛋白，由肺泡clara细胞及Ⅱ型上皮细胞合成分泌，在调节表面活性磷脂代谢及肺防御功能方面起重要作用。近年来有部分研究发现AIDS合并PCP时肺内SP-A、SP-D水平增高。我们应用醋酸可的松(Cortisone Acetate, CA)诱导SD大鼠建立PCP动物模型，研究SP-A、SP-D的变化，并与CA诱导的免疫抑制大鼠的细菌性肺炎、阴性对照组及正常对照比较，以探讨SP-A、SP-D在PCP发病机制中的作用及治疗中的意义。

材料和方法

一、PCP动物模型的建立

清洁级雄性SD大鼠(由上海医科大学实验动物部提供)50只，体重180～220 g。每2～3只置于1只鼠笼。饲养于空气层流室，室温20～24℃，环境清洁，鼠笼及垫料定期更换、消毒。喂正常饲料，饮含0.5 g/L盐酸四环素的冷开水，自由摄食和饮水。每周2次皮下注射醋酸可的松25 mg造成免疫抑制状态。喂养过程中死亡鼠去除，共8～12周。

二、支气管肺泡灌洗(BAL)方法

腹腔内注射1%戊巴比妥钠40 mg/kg麻醉动物后，固定，置于超净工作台内。用新洁尔灭消毒颈胸部，铺无菌洞巾。抽尽心脏血使动物死亡。无菌条件下进行气管插管，用20 cm H₂O压力将8 ml无菌生理盐水灌入单侧肺内，然后回收获取支气管肺泡灌洗液(BALF)。

三、标本制备

取BALF及少许肺组织作细菌培养。取病变较明显的肺组织制成肺印片，自然干燥后用甲醇固定，行姬姆萨(Giemsa)染色查找PC虫体。未行肺泡灌洗的另一侧肺组织浸入10%福尔马林缓冲液中固定，石蜡包埋、切片，作HE染色观察病理改变及哥氏银(Grocott-Gomori)染色查找PC虫体。

四、BALF的处理

(一) 细胞计数第一管BALF 2 000 r/min离心15 min，沉渣中加0.7 ml生理盐水稀释混匀，取1滴，加白细胞稀释液9滴，混匀，在普通显微镜下计数白细胞。用公式：白细胞数/L=四大格总数/4×10×10⁶/L，计算出白细胞浓度。

(二) 离心涂片分别取BALF沉渣稀释液2滴，用离心涂片机(LTP-A型，中国军事科学院实验仪器厂生产)涂片，自然干燥后用甲醇固定，分别予以瑞氏染色作白细胞分类及Giemsa染色找PC虫体。

(三) BALF储存取离心后的BALF上清液，分装于4只小冻存管，-20℃冻存待测。

五、染色方法

Giemsa染色及哥氏银染色按实验室常规技术程序操作。

六、诊断标准

(一) PCP具备下列条件之一者：(1) BALF沉渣离心涂片或肺组织印片Giemsa染色，光镜下见PC虫体(滋养体及囊内小体)；(2) 肺组织石蜡切片哥氏银染色见PC包囊。根据国际常用的随机计数高倍视野每100个肺泡中受PC累及的肺泡数区分PCP的严重程度^［2］，即以≥25个肺泡受累及/100肺泡者为中重度PCP，＜25个肺泡受累及/100肺泡者为轻度PCP。

(二) 细菌性肺炎同时具备下列2条：(1) BALF或肺组织细菌培养阳性；(2) 肺组织石蜡切片HE染色见炎症改变。

(三) 无肺部感染同时排除以上2种情况，且BALF沉渣涂片光镜下检查未发现真菌及其它病原体，肺组织石蜡切片HE染色示肺组织正常。

七、实验动物分组

实验中死亡鼠除外，根据实验结果去除不满足下述各组条件的动物，将剩余动物分成3组；另用6只清洁级雄性SD大鼠(体重200 g±)作为正常对照组。A组：正常对照组，6只。B组：阴性对照组，6只，CA诱导8周以上无肺部感染。C组：细菌性肺炎组，11只，CA诱导8周以上出现细菌性肺炎，而无其他肺部感染。D组：PCP组，14只，CA诱导8周以上出现PCP，而无其他肺部感染。

八、表面活性蛋白A、D的测定方法

SP-A含量测定采用免疫斑点法^［3］。将标本及标准品通过斑点吸引装置点在硝酸纤维素膜(NC)上，按0.1 ml/cm² NC加入杂交液(0.2 mol PBS，5%脱脂奶粉)，室温摇动1 h，加入SP-A抗体(SP-A抗体及SP-A标准品由上海医科大学儿科研究所制备)，于4℃过夜。然后用含Tween-20的0.2 mol PBS(pH 7.2)摇洗10 min 3次，0.05 mol Tris-HCl (pH 7.5)摇洗10 min。用含5%脱脂奶粉的Tris-HCl封闭，加二抗(羊抗兔IgG)，室温摇动1 h，再用Tris-HCl摇洗10 min 4次，加显色液(NBT，BCIP)，显色满意后用EDTA终止反应。用MAIS300<