卡氏肺孢子蟲肺炎動物模型實驗診斷的研究
發(fā)布時間：2018-10-18 18:46
【摘要】：卡氏肺孢子蟲是一種機會性致病的病原體,該蟲可在免疫功能低下的人群中誘發(fā)致命性的卡氏肺孢子蟲肺炎,尤其是CD4~+ T細胞功能缺陷者。隨著艾滋病病人在我國不斷的增加,以及為數(shù)眾多的惡性腫瘤化療、器官移植及接受免疫抑制劑治療的患者,而我國目前對卡氏肺孢子蟲實驗室診斷大都依賴病原學常規(guī)染色,其敏感性和特異性易受不同的操作人員影響波動較大,并且其敏感性總體偏低,因此,急需建立一套簡便、穩(wěn)定、敏感性和特異性均高的診斷體系。本研究擬探討改良的常規(guī)染色法和分子生物學方法在卡氏肺孢子蟲實驗診斷中的應用。 目的:采用瑞氏-吉姆薩染色法、寡核苷酸探針RNA原位雜交(RNA in situ hybridization)技術和巢式PCR法檢測卡氏肺孢子蟲,并探討這三種方法在卡氏肺孢子蟲診斷中的應用。 方法:Wistar雌性大鼠皮下注射地塞米松建立卡氏肺孢子蟲肺炎動物模型。采用改進的病原學染色法瑞氏-吉姆薩染色法檢測肺印片和BALF涂片。采用小亞單位RNA位點來源的寡核苷酸探針一條,地高辛加尾標記,對肺組織制備石蠟標本進行RNA原位雜交。采用Pc-ITS-PCR引物,對肺組織、BALF和血清標本進行巢式PCR法檢測。 結果:共檢測了43只實驗組大鼠,經瑞氏-吉姆薩染色后,在肺組織中查到的滋養(yǎng)體和/或包囊的陽性率為74.42%(32/43),BALF涂片陽性率為67.44%(29/43)。寡核苷酸探針RNA原位雜交法成功檢測到肺組織內的蟲體,實驗組大鼠在第3周即檢測到蟲體,7～8周時檢測到大量包囊,9～10周時滋養(yǎng)體大量出現(xiàn),而空包囊多出現(xiàn)在8～9周,蟲體分布于肺泡腔、肺泡壁及血管壁。大鼠實驗組肺組織切片陽性率為88.37%(38/43)。巢式PCR方法電泳結果出現(xiàn)550bp條帶清晰,無非特異條帶出現(xiàn),實驗組肺組織、BALF和血陽性率分別為86.05%(37/43)、83.72%(36/43)和76.74%(33/43)。上述三種方法,對照組陽性率均為0%(0/16)。經統(tǒng)計學分析處理,原位雜交對該蟲的檢出率高于瑞氏-吉姆薩染色法,差別有顯著統(tǒng)計學意義(p0.01),原位雜交法與PCR法比較差別無統(tǒng)計學意義(p0.05),PCR法對該蟲的檢出率高于瑞氏-吉姆薩染色法,差別有顯著統(tǒng)計學意義(p0.01)。 結論:寡核苷酸探針RNA原位雜交法顯示組織內蟲體清晰,準確,在形態(tài)學
[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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R-332
本文編號：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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R-332
【引證文獻】
相關博士學位論文 前1條
1 趙治國;我國駱駝斯氏副柔線蟲病傳播媒介的研究[D];內蒙古農業(yè)大學;2010年
，本文編號：2280044
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