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目的建立肺孢子菌动物模型,并对肺孢子菌病原学和分子生物学检测技术进行研究。方法 SD和Wistar大鼠随机分为实验组和对照组,实验组大鼠皮下注射地塞米松,对照组皮下注射生理盐水。诱导8周后处死全部大鼠,收集其支气管肺泡灌洗液(BALF)和肺组织,分别做涂片和肺印片,染色后镜检。用FTA卡采集所有大鼠的BALF进行PCR检测。对PCR产物进行测序,利用BLAST软件将测序结果与Gen Bank数据库中的鼠源性肺孢子菌进行比对,确定菌种。结果共采集肺组织标本和BALF各34份,病原学检测显示实验组总感染率为29.2%(7/24),对照组感染率为0;其中SD和Wistar大鼠实验组感染率分别为25.0%(3/12)和33.3%(4/12),差异无统计学意义(P=0.31);实验组SD大鼠肺印片和BALF肺孢子菌阳性检出率分别为25.0%(3/12)和16.7%(2/12),差异无统计学意义(P=0.34)。实验组Wistar大鼠肺印片和BALF肺孢子菌阳性检出率分别33.3%(4/12)和16.7%(2/12),差异无统计学意义(P=0.24)。用PCR方法共检测28份大鼠BALF样本,实验组阳性检出率为91.7%(26/28),对照组均为阴性。对PCR产物进行测序分析,发现其与Gen Bank已登录的大鼠源肺孢子菌(JX499145、GU133622、EF646865)的同源性均为100%。结论通过皮下注射地塞米松可以建立肺孢子菌动物模型,SD大鼠与Wistar大鼠在作为肺孢子菌动物模型的选择上无显著差别。在早期感染阶段,适宜用PCR法进行肺孢子菌检测,病原形态学检测漏检率高。
Objective To establish an animal model of Pneumocystis and investigate the etiological and molecular biological detection techniques of Pneumocystis sp. Methods SD and Wistar rats were randomly divided into experimental group and control group. Rats in experimental group were subcutaneously injected with dexamethasone, while control group were injected with normal saline subcutaneously. After 8 weeks of induction, all the rats were sacrificed and their bronchoalveolar lavage fluid (BALF) and lung tissue were collected and smear and lung print respectively. BALF from all rats was collected by FTA card for PCR assay. The PCR products were sequenced, and the sequencing results were compared with the murine Pneumocystis sp. In the Gen Bank database using BLAST software to determine the strain. Results A total of 34 specimens of lung tissue and BALF were collected. The etiological test showed that the total infection rate was 29.2% (7/24) in the experimental group and 0 in the control group. The infection rates in the experimental group were 25.0 % (3/12) and 33.3% (4/12) respectively. There was no significant difference between the two groups (P = 0.31). The positive rates of pulmonary print and BALF pneumocystis in the experimental group were 25.0% (3 / 12) and 16.7% (2/12) respectively, with no significant difference (P = 0.34). The positive rate of lung print and BALF pneumocystis in the experimental group were 33.3% (4/12) and 16.7% (2/12), respectively, with no significant difference (P = 0.24). A total of 28 rat BALF samples were detected by PCR. The positive rate of the test group was 91.7% (26/28), while the control group was negative. The PCR products were sequenced and found to have 100% homology with GenBank accession Pneumocystis sp. (JX499145, GU133622, EF646865). Conclusion The animal model of Pneumocystis can be established by injecting dexamethasone subcutaneously. There is no significant difference between the model of Pneumocystis carinii in Wistar rats and SD rats. In early stage of infection, PCR method is suitable for detecting Pneumocystis sp., Pathogen morphology detection missed detection rate.