【作者】 彭丹；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 博士

【摘要】 目的:通过本研究拟了解: 1.慢性HCV感染患者与慢性HBV病毒感染患者、正常对照间认知功能的差异,明确慢性HCV感染患者认知功能损害的程度和模式;以及与慢性HBV感染患者比较,认知功能损害是否为慢性HCV感染患者的独特特征。2.慢性HCV感染患者的认知功能与抑郁症状的关系。3.设计并建立巢式荧光定量PCR(Nested-qRT-PCR)检测慢性丙型病毒感染患者CSF中HCV 5’NCR基因片段,进一步证实慢性HCV感染患者的CSF中是否存在HCV感染。4.初步探讨HCV对CNS的直接感染是否与慢性HCV感染患者认知功能的损害有关。方法:1、采用目前较为常用的神经心理测验工具对36例轻度慢性HCV感染患者、32例慢性HBV病毒感染患者及28例对照者进行测验,各组还采用Beck抑郁自评量表(BDI-II)进行抑郁症状的评定。2、根据GenBank提供的基因序列,针对HCV基因的保守区域(HCV 5’NCR基因),设计并合成特异性内外引物和荧光标记TaqMan探针。从重庆医科大学病毒性肝炎研究所惠赠的含有目的基因的质粒出发,利用PCR方法扩增获得目的基因,克隆至pGEM-T Easy载体进行体外转录,将得到的RNA纯化并使用RiboGreen方法进行定量后作为阳性标准品。分别建立正链和负链HCV RNA的巢式荧光定量RT-PCR方法并分析敏感性和特异性。3、应用Nested-qRT-PCR方法检测12例慢性丙型肝炎病毒感染患者外周血液单个核细胞和脑脊液HCV 5’NCR基因片段,并对脑脊液阳性样本进行分子克隆和测序分析。同时对这12例患者进行神经心理测验,比较脑脊液HCV阳性者与阴性者的测验成绩。结果:1、HCV感染患者在反映注意、记忆和言语功能的测验如数字符号、延迟逻辑记忆、言语流畅性测试重复数、连线B测验(TMT-B)和Stroop测验上与健康对照组有差异,尤其在数字符号、TMT-B和Stroop-cw测验上差异显著,而HBV患者与健康对照组各项测验值均无差异。2、本研究成功建立了正链和负链HCV巢式荧光定量RT-PCR检测方法,其最低检出限即敏感性分别为2.6×102copies/μl、2.6×101 copies/μl,标准曲线的定量范围分别为2.6×102～2.6×107copies/μl、2.6×101～2.6×107copies/μl,相关系数R2分别为0.9883和0.9969,不与其他病毒发生非特异反应。3、共对12例慢性丙型肝炎感染患者的PBMCs,脑脊液细胞中的正链和负链HCV RNA (positive-和negative- strands)进行了检测,10例PBMCs检测5’NCR基因片段为阳性,而其中3例CSF正链HCV RNA检测阳性(3/12),2例负链HCV RNA检测阳性(2/12)。4、12例慢性丙型肝炎病毒感染患者中脑脊液HCV检测阳性者(3例)与脑脊液HCV检测阴性者(9例)以及健康对照组的神经心理测验成绩比较,脑脊液HCV阳性者在数字符号和TMT-B测验上与脑脊液HCV阴性者以及正常对照组有统计学上的差异。结论:1、慢性HCV患者在需要持续注意和更复杂的注意力集中测试的视觉扫描,以及知觉运动速度,工作记忆上存在障碍,此认知功能损害与抑郁自评症状之间无显著相关性。在HBV感染患者中没有发现类似的认知损害,提示这种认知功能损害模式是慢性HCV患者所独特的。2、本研究构建了HCV 5’NCR基因核酸定量检测用的正链和负链RNA标准品,并建立了正链和负链HCV巢式荧光定量RT-PCR检测方法。该方法具有灵敏性和特异性高、简便快捷等有点,不易出现污染引起假阳性结果,适用于脑脊液等体液细胞相对较少、RNA浓度过低的定量检测。3、部分慢性HCV感染患者脑脊液中存在有HCV感染,HCV对CNS的直接感染可能与慢性HCV患者的认知功能损害有关。更多还原

【Abstract】 Objective: In present research we want to investigate: a) the difference of neuropsychological tests among mild chronic HCV infected patients, mild chronic HBV infected patients, and controls; b) the correlation between the cognitive function deficits and depression in mild chronic HCV infected patients; c) nucleic acid testing techniques for detecting hepatitis c virus (HCV) in cerebrospinal fluid using nested fluorescence quantitative reverse transcriptase polymerase chain reaction (Nested-qRT-PCR); d) cognitive impairment caused by HCV infection in the central nervous system.Methods1. 36 mild chronic HCV infected patients, 32 mild chronic HBV infected patients and 28 controls were assessed by a series of neuropsychological tests. Depression symptoms were also assessed by Beck Depression Inventory (BDI-Ⅱ).2. Specific primers and TaqMan probes were designed in the conserved region of virus genome 5’NCR. Conserved region gene contained in plasmids which is offered by Chongqing Medical University hepatitis virus laboratory was inserted into pGEM-T Easy vector and transcribed in vitro to produce RNA, which would be used as standards in PCR quantification. A nested fluorescence quantitative RT-PCR method to detect positive- and negative- strands HCV RNA is established. At the same time, sensitivity and specificity of method were assessed.3. The HCV 5’NCR fragment of peripheral blood mononuclear cells (PBMCs) and CSF obtained from 12 patients with chronic HCV infection were examined by Nested-qRT-PCR. Furthermore, molecular cloning and sequence analysis were carried out on positive PCR products. Neuropsychological tests were also performed on 12 patients with chronic HCV infection.Results1. There is a significant difference between chronic HCV infected patients and controls in neuropsychological tests. The scores of digit-symbol, delayed-logical- memory, TMT-B and Stroop tests in chronic HCV infected patients are significantly lower than those in controls. However, there is no significant difference between chronic HBV infected patients and controls in neuropsychological tests.2. The Nested-qRT-PCR methods for the detections of positive- and negative- strands HCV RNA were successfully established. The minimum detection limits of Nested-qRT-PCR for HCV are 2.6×102 copies/μl and 2.6×101 copies/μl respectively, the quantitative range of standard curve is 2.6×102 ~2.6×107 copies/μl (R2 0.9883) for positive-strand HCV RNA, and 2.6×101~2.6×107 copies/μl (R2 0.9969) for negative-strand HCV RNA. Nonspecific PCR reaction with other virus was not discovered.3. Nucleic acid detections for positive- and negative- strands HCV RNA were successfully performed on PBMCs and CSF samples from 12 patients with chronic HCV infection using Nested-qRT-PCR. Positive-strand HCV RNA was detected in three out of 12 cases of CSF and negative-strand HCV RNA in two out of 12 cases of CSF.4. Neuropsychological tests were further performed on both three HCV detected positive cases and 9 HCV detected negative ones in CSF. Significant difference was found in digit-symbol and TMT-B testing.Conclusions1. Chronic HCV infected patients perform significantly worse than healthy controls on complex visual scanning, psychomotor speed, attention and working memory. The cognitive deficits are not related to depression symptoms. Hepatitis B patients are not similarly impaired in cognitive function, suggesting that the observed abnormalities are specific.2. Nested-qRT-PCR methods for detecting N positive- and negative- strands HCV RNA has been established successfully. The methods are sensitive and reliable and allow rapid detection and quantitation of hepatitis c virus in CSF.3. The results have demonstrated that cognitive impairment in chronic HCV infected patients may be caused by the HCV infection in the central nervous system.更多还原