卡氏肺孢子虫肺炎动物模型实验诊断的研究
发布时间：2018-10-18 18:46
【摘要】：卡氏肺孢子虫是一种机会性致病的病原体,该虫可在免疫功能低下的人群中诱发致命性的卡氏肺孢子虫肺炎,尤其是CD4~+ T细胞功能缺陷者。随着艾滋病病人在我国不断的增加,以及为数众多的恶性肿瘤化疗、器官移植及接受免疫抑制剂治疗的患者,而我国目前对卡氏肺孢子虫实验室诊断大都依赖病原学常规染色,其敏感性和特异性易受不同的操作人员影响波动较大,并且其敏感性总体偏低,因此,急需建立一套简便、稳定、敏感性和特异性均高的诊断体系。本研究拟探讨改良的常规染色法和分子生物学方法在卡氏肺孢子虫实验诊断中的应用。 目的:采用瑞氏-吉姆萨染色法、寡核苷酸探针RNA原位杂交(RNA in situ hybridization)技术和巢式PCR法检测卡氏肺孢子虫,并探讨这三种方法在卡氏肺孢子虫诊断中的应用。 方法:Wistar雌性大鼠皮下注射地塞米松建立卡氏肺孢子虫肺炎动物模型。采用改进的病原学染色法瑞氏-吉姆萨染色法检测肺印片和BALF涂片。采用小亚单位RNA位点来源的寡核苷酸探针一条,地高辛加尾标记,对肺组织制备石蜡标本进行RNA原位杂交。采用Pc-ITS-PCR引物,对肺组织、BALF和血清标本进行巢式PCR法检测。 结果:共检测了43只实验组大鼠,经瑞氏-吉姆萨染色后,在肺组织中查到的滋养体和/或包囊的阳性率为74.42%(32/43),BALF涂片阳性率为67.44%(29/43)。寡核苷酸探针RNA原位杂交法成功检测到肺组织内的虫体,实验组大鼠在第3周即检测到虫体,7～8周时检测到大量包囊,9～10周时滋养体大量出现,而空包囊多出现在8～9周,虫体分布于肺泡腔、肺泡壁及血管壁。大鼠实验组肺组织切片阳性率为88.37%(38/43)。巢式PCR方法电泳结果出现550bp条带清晰,无非特异条带出现,实验组肺组织、BALF和血阳性率分别为86.05%(37/43)、83.72%(36/43)和76.74%(33/43)。上述三种方法,对照组阳性率均为0%(0/16)。经统计学分析处理,原位杂交对该虫的检出率高于瑞氏-吉姆萨染色法,差别有显著统计学意义(p0.01),原位杂交法与PCR法比较差别无统计学意义(p0.05),PCR法对该虫的检出率高于瑞氏-吉姆萨染色法,差别有显著统计学意义(p0.01)。 结论:寡核苷酸探针RNA原位杂交法显示组织内虫体清晰,准确,在形态学
[Abstract]:Pneumocystis carinii (Pneumocystis carinii) is an opportunistic pathogen that can induce fatal pneumocystis carinii pneumonia especially in patients with CD4~ T cell dysfunction. With the increasing number of AIDS patients in China, as well as a large number of malignant tumor chemotherapy, organ transplantation and immunosuppressive therapy patients, the laboratory diagnosis of Pneumocystis carinii mostly depends on routine etiological staining. Its sensitivity and specificity are easily fluctuated by different operators, and its sensitivity is on the low side. Therefore, it is urgent to establish a simple, stable, sensitive and specific diagnostic system. The purpose of this study was to investigate the application of modified conventional staining and molecular biological methods in the experimental diagnosis of Pneumocystis carinii (Pneumocystis carinii). Objective: to detect Pneumocystis carinii by Ricker-Gimsa staining, oligonucleotide probe RNA in situ hybridization (RNA in situ hybridization) and nested PCR. The application of these three methods in the diagnosis of Pneumocystis carinii was discussed. Methods: the animal model of pneumocystis carinii pneumonia was established by subcutaneous injection of dexamethasone in Wistar female rats. Lung prints and BALF smears were detected by modified etiology staining method. A probe of oligonucleotide derived from RNA locus of small subunit was used to label paraffin tissue of lung tissue with digoxin in situ hybridization (RNA). Pc-ITS-PCR primers were used to detect lung tissue, BALF and serum samples by nested PCR. Results: the positive rate of trophozoites and / or cysts in lung tissue was 74.42% (32 / 43), BALF smear was 67.44% (29 / 43). In situ hybridization with oligonucleotide probe RNA was used to detect the parasites in lung tissue. In the experimental group, the parasites were detected at the third week, a large number of cysts were detected at 7 ~ 8 weeks, and a large number of trophozoites were found at 9 ~ 10 weeks. But the empty cyst mostly appeared at 8 ~ 9 weeks, and the worm was distributed in the alveolar cavity, alveolar wall and vascular wall. The positive rate of lung sections in the experimental group was 88.37% (38 / 43). The positive rates of 550bp, BALF and blood in the experimental group were 86.05% (37 / 43), 83.72% (36 / 43) and 76.74% (33 / 43), respectively. The positive rate of control group was 0% (0 / 16). By statistical analysis, the detection rate of in situ hybridization was higher than that of Rish-Jimsa staining. The difference was statistically significant (p0.01), and there was no significant difference between in situ hybridization method and PCR method (p0.05), PCR method was higher than Riesh-Gimsa staining method, the difference was statistically significant (p0.01). Conclusion: Oligonucleotide probe RNA in situ hybridization is a clear, accurate and accurate method in demonstrating the morphology of the insect in the tissue.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332
本文编号：2280044
[Abstract]:Pneumocystis carinii (Pneumocystis carinii) is an opportunistic pathogen that can induce fatal pneumocystis carinii pneumonia especially in patients with CD4~ T cell dysfunction. With the increasing number of AIDS patients in China, as well as a large number of malignant tumor chemotherapy, organ transplantation and immunosuppressive therapy patients, the laboratory diagnosis of Pneumocystis carinii mostly depends on routine etiological staining. Its sensitivity and specificity are easily fluctuated by different operators, and its sensitivity is on the low side. Therefore, it is urgent to establish a simple, stable, sensitive and specific diagnostic system. The purpose of this study was to investigate the application of modified conventional staining and molecular biological methods in the experimental diagnosis of Pneumocystis carinii (Pneumocystis carinii). Objective: to detect Pneumocystis carinii by Ricker-Gimsa staining, oligonucleotide probe RNA in situ hybridization (RNA in situ hybridization) and nested PCR. The application of these three methods in the diagnosis of Pneumocystis carinii was discussed. Methods: the animal model of pneumocystis carinii pneumonia was established by subcutaneous injection of dexamethasone in Wistar female rats. Lung prints and BALF smears were detected by modified etiology staining method. A probe of oligonucleotide derived from RNA locus of small subunit was used to label paraffin tissue of lung tissue with digoxin in situ hybridization (RNA). Pc-ITS-PCR primers were used to detect lung tissue, BALF and serum samples by nested PCR. Results: the positive rate of trophozoites and / or cysts in lung tissue was 74.42% (32 / 43), BALF smear was 67.44% (29 / 43). In situ hybridization with oligonucleotide probe RNA was used to detect the parasites in lung tissue. In the experimental group, the parasites were detected at the third week, a large number of cysts were detected at 7 ~ 8 weeks, and a large number of trophozoites were found at 9 ~ 10 weeks. But the empty cyst mostly appeared at 8 ~ 9 weeks, and the worm was distributed in the alveolar cavity, alveolar wall and vascular wall. The positive rate of lung sections in the experimental group was 88.37% (38 / 43). The positive rates of 550bp, BALF and blood in the experimental group were 86.05% (37 / 43), 83.72% (36 / 43) and 76.74% (33 / 43), respectively. The positive rate of control group was 0% (0 / 16). By statistical analysis, the detection rate of in situ hybridization was higher than that of Rish-Jimsa staining. The difference was statistically significant (p0.01), and there was no significant difference between in situ hybridization method and PCR method (p0.05), PCR method was higher than Riesh-Gimsa staining method, the difference was statistically significant (p0.01). Conclusion: Oligonucleotide probe RNA in situ hybridization is a clear, accurate and accurate method in demonstrating the morphology of the insect in the tissue.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332
【引证文献】
相关博士学位论文 前1条
1 赵治国;我国骆驼斯氏副柔线虫病传播媒介的研究[D];内蒙古农业大学;2010年
，本文编号：2280044
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2280044.html
最近更新
教材专著
