【作者】 徐霞；

【导师】 陈锡慰； 张兆松；

【作者基本信息】 南京医科大学 ， 病原生物学， 2003， 硕士

【摘要】 核酸分子杂交作为分子生物学的基本技术已被广泛用于医学基础和临床研究。因其具有灵敏度高、特异性好和相对简易的优点，已被用于多种病原体的检测。 本研究采用醋酸泼尼松龙诱导建立卡氏肺孢子虫肺炎(Pneumocystis carinii pneumonia,PCP)大鼠模型，获得实验所需的基本材料。以随机引物法制备地高辛标记的非放射性探针，采用斑点杂交检测大鼠模型肺组织和患者临床标本中的卡氏肺孢子虫(Pneumocystis carinii,Pc)DNA。 1．PCP大鼠模型的建立、Pc检测及实验标本的采集 采用SD大鼠，醋酸泼尼松龙腹股沟皮下注射2个月，诱导建立PCP动物模型。无菌操作取出死亡大鼠的肺组织，切面做印片，分别进行吉氏染色和快速银染，显微镜下查找Pc。从每只大鼠的肺组织中分离Pc包囊和滋养体，提取DNA用于PCR反应。琼脂糖凝胶电泳后紫外灯下观察，见到约350bp大小的DNA条带为阳性。其余的肺组织保存于-20℃备用。 结果：病原学检查结果显示成功建立了PCP动物模型。检测结果显示，吉氏染色检出率为80％，快速银染检出率为50％，PCR检出率为95％，后者明显高于吉氏染色和快速银染法。PCP动物模型的诱导成功及肺组织的获得为后续的实验研究奠定了基础。 唐京医矜大学硕士学位技文2．PC特异性目白基因的获得及序列测定 以 Wakefield等设计的 MI 02－E和 PAZ 02－H为引物，采用常规 PCR扩增计的线粒体核糖体 RNA大亚基（LSU mt r伽A）基因。琼脂糖凝胶电泳后将含有约350hp大小DNA条带的凝胶切割，回收其中DNA片段。将所得基因片段测序，采用Blast软件将该段基因序列与GeneBank中已公布序列比对确认所得基因的来源。结果：测序结果显示所得基因片段长为357hP，与GeneBank中的D有序列比对；结果显示所得基因与 Sinclair等公布的 Pc LSU mt rRNA基因的同源性为 99儿 故确认是 PC特异的基因。该基因的获得及鉴定为探针的制备及检测提供了必需的材料，并保证了探针的特异性。3．PC特异性DNA探针的制备及其实验应用 以获得的PC特异性DNA为模板，采用随机引物法制备地高辛标记的非放射性探针。以已知浓度的地高辛标记的DNA为参照确定探针的浓度，随后检测该探针的特异性和敏感性。将该探针用于斑点杂交检测大鼠肺组织和肾移植并发PCP 患者支气管肺泡灌洗液 （bronchoalveolar lavage，BAL）标本中的 Pc DNA。结果：实验中所得探针浓度为30ng／ul，显色敏感性为…，杂交敏感性为沙，特异性检测显示该探针不与小鼠伯氏疟原虫、人恶性疟原虫、弓形虫、正常人白细胞和正常宿主（大鼠）肺组织DNA反应，仅与 PC DNA反应，杂交后显色出现紫红色斑点。将该探针用于斑点杂交检测大鼠模型肺组织中的几，检出率为 73．7％，肾移植并发PCP患者的 B队标本中未检测到八 DNA。将这些标本采用 PCR扩增后结合杂交重新检测，检出率为 94．7y，在肾移植并发 PCP患者的 BAL中检出 PC DNA。更多还原

【Abstract】 As a basic technology of molecular biology, nucleic acid molecule hybridization has been widely used for basic and clinical medicine research. Being highly sensitive, specific and easy to operate, it has been widely used for detection of many pathogens.In this research, the rat model of Pneumocystis carinii pneumonia(PCP) was established by injecting and oral administration of acetate hydroprednisone to acquire necessary materials for experiments. After preparation of non-radioactive digoxingenin labeled probe by random priming, dot slot hybridization was used to detect Pneumocystis carinii(Pc) DNA from the lungs of rats model and clinical specimens of patients.1 .Establishment of the rat model with PCP, detection of PC and collection of samplesThe rat model with PCP was successfully induced by injecting acetate hydroprednisone subcutaneously to the groin of SD rat for two months.The lungs were taken out from the dead rats aseptically, and made imprints with the cross-section of ones.The imprints were stained by Giemsa’s staining and improved Gomori’s methenamine silver nitrate staining(GMS), and detected for PC microscopically.The cysts andtrophozoits of PC were isolated from the lungs to extract DNA for thetemplate of PCR. The PCR product was identified by agarose gelelectrophoresis and a about 350bp DNA band occurring in the ultravioletlight was thought to be positive.Results: The results of pathogen examination showed that the rat modelwith PCP was established successfully. The positive rate of the PCinfection found by Giemsa’s staining, improved GMS and PCR was80%, 50 % and 95% respectively.The last was higher than the other both.The establishment of the rat model and the acquirement of the lungs laidbasis for the subsequent experiments.2.The acquirement of PC specific target DNA and sequencingRegular PCR was used to amplify the large subunit mitochondrial ribosomal RNA(LSU mt rRNA) gene of PC with the primers of PAZ102-E and PAZ102-H designed by Wakefield and his colleagues.The agarose gel comprising about 350bp DNA band was cut and recollected for the target DNA. After the purified DNA fragment being sequenced, software BLAST was used to align the acquired DNA sequence and those known sequences in the GeneBank.Results: The sequenced DNA fragment was 357bp long, and it shared 99% homology with the PC LSU mt rRNA gene announced by Sinclair and his colleagues. This result confirmed that the purified DNA fragment was specific for PC. The acquirement of the target DNA fragment was important and necessary for the preparation and detection of the probe, which was about to guarantee the specifity of the probe. 3.The preparation and experimental application of PC specific DNA probeDigoxingenin labeled non-radioactive probe was prepared by random priming, using the acquired targe DNA fragment specific for PC as the template. The density of the probe was confirmed through comparing with the density-known digoxingenin labeled control DNA, and thespecifity and sensitivity of the probe were identified by dot slot hybridization. Then the probe was used in dot slot hybridization to detect PC DNA from the lungs of rats model and clinical specimens of patients. Results: The density of the probe was 3 Ong/ul, and the lowest detectable amount of the probe was 2pg. The probe only reacted with the PC DNA, but did not react with the DNAs from Plasmodium berghei, P. falciparum, Toxoplasma gondii, human leukocytes and normal host lungs. So it showed that the probe was specific for PC. The dot slot hybridization could successfully detect PC DNA from 73.7% of the lungs of rats model, but not from the specimen (bronchoalveolar lavage, BAL ) of the kidney transplantation recipient with PCP. PCR combined with dot slot hybridization could detect PC from 94.7% of these samples, and PC DNA was detectable from the BAL of the patient.更多还原