CAJ | 학술논문

目的建立肺孢子虫感染大鼠模型并探讨ITS1-5.8S rDNA-ITS2巢式PCR检测卡氏肺孢子虫DNA的敏感性. 方法大鼠分为实验组和对照组,实验组从第1周开始用每周2次每次每只皮下注射地塞米松1 mg的方法诱导,共8周,并分别在首次注射后第2、4、6、8周解剖大鼠制作肺印片、肺组织匀浆液及支气管肺泡灌洗液(BAL)涂片,并进行六亚甲基四胺银(Gomori's methenamine silver,GMS)染色镜检;对照组不作激素注射,并分别在0周和第10周剖杀染色检查.同时分别提取大鼠BAL和肺组织的肺孢子虫DNA进行巢式PCR扩增,比较GMS法和ITS巢式PCR检测的敏感性. 结果实验组从第6周开始染色镜检,可见少量肺孢子虫包囊,第8周肺印片、肺组织匀浆液均检测到包囊,检出率为100%(10/10),BAL的检出率为80%(8/10),对照组则均未检出.用ITS1-5.8S rDNA-ITS2巢式PCR法均能检测出实验组第8周大鼠BAL和肺组织卡氏肺孢子虫DNA,阳性率为100%(10/10),对照组均为阴性.比较BAL标本、肺印片和肺组织匀浆液GMS染色法的检出率,BAL最低,肺组织匀浆液最高.其中20%(2/10)大鼠的BAL标本用GMS染色法未能检出,但用巢式PCR方法均能成功扩增.结论成功用地塞米松诱导法建立了肺孢子虫大鼠感染模型;ITS1-5.8S rDNA-ITS2巢式PCR检测卡氏肺孢子虫敏感性高、特异性强,可推广应用于临床诊断肺孢子虫肺炎.
목적 건립폐포자충감염대서모형병탐토ITS1-5.8S rDNA-ITS2소식PCR검측잡씨폐포자충DNA적민감성. 방법 대서분위실험조화대조조,실험조종제1주개시용매주2차매차매지피하주사지새미송1 mg적방법유도,공8주,병분별재수차주사후제2、4、6、8주해부대서제작폐인편、폐조직균장액급지기관폐포관세액(BAL)도편,병진행륙아갑기사알은(Gomori's methenamine silver,GMS)염색경검;대조조불작격소주사,병분별재0주화제10주부살염색검사.동시분별제취대서BAL화폐조직적폐포자충DNA진행소식PCR확증,비교GMS법화ITS소식PCR검측적민감성. 결과 실험조종제6주개시염색경검,가견소량폐포자충포낭,제8주폐인편、폐조직균장액균검측도포낭,검출솔위100%(10/10),BAL적검출솔위80%(8/10),대조조칙균미검출.용ITS1-5.8S rDNA-ITS2소식PCR법균능검측출실험조제8주대서BAL화폐조직잡씨폐포자충DNA,양성솔위100%(10/10),대조조균위음성.비교BAL표본、폐인편화폐조직균장액GMS염색법적검출솔,BAL최저,폐조직균장액최고.기중20%(2/10)대서적BAL표본용GMS염색법미능검출,단용소식PCR방법균능성공확증.결론성공용지새미송유도법건립료폐포자충대서감염모형;ITS1-5.8S rDNA-ITS2소식PCR검측잡씨폐포자충민감성고、특이성강,가추엄응용우림상진단폐포자충폐염.
Objective To establish the rats model of Pneumocystis carinii infection and study on the sensitivity of ITS1-5.8S rDNA-ITS2 nested PCR. Methods SD rats were divided as experiment group and control group at random. Each experiment rat was rendered immunedepressant by subcutaneous injection of dex-amethasone from the 1 st week with 1 mg per rat twice a week for 8 weeks, and the control rats with normal feeding. P. carinii of experiment group was examined microscopically in bronchoalveolar lavage (BAL) on lung impression smears and lung tissue smears stained with Gomorismethenamine silver (GMS) at the 2nd, 4th, 6th and 8th week after injection, respectively. Those of control group was examined at the beginning of experi-ment and the 10th week. The DNAs of P. carinii were extracted beth from the BAL and lung tissues of rats. ITS1-5.8S rDNA -ITS2 of P. carinii were amplified by nested PCR. The sensitivity was compared between GMS and nested PCR. Results P. carinii could be observed from the 6th week in the experiment group and at the 8th week with 100% (10/10) positive in lung impression smears and lung tissue smears, 80% (8/10) in BAL smear. The control group was negative. Nested PCR detected the DNAs of P. carinii both from BAL and lung tissue at the 8th week with 100% positive, and all negative in control group. Comparing the detect efficiency of P. carinii among BAL, lung impression smears and lung tissue smears by GMS method, BAL was the lowest, lung tissue smears was the highest. 20% specimens of BAL couldnt be detected by GMS method, while nested PCR worked. Conclusions The rats model of P. carinii infection was successfully established by injection ofdexamethasone. ITS1-5.8S rDNA-ITS2 nested PCR is a method with high sensitivity and specificity, whichcould be potentially used to detect Pneumocystis pneumonia in clinic.