摘要
可传播性海绵状脑病(Transmissible spongiform encephalopathies, TSEs)也称做prion病,是一组罕见的以脑内出现海绵状病理改变为特征的致死性神经系统退行性疾病。该类疾病包括人类的克雅氏病(Creutzfeldt-Jakob disease, CJD)、GSS综合症(Gerstmann-Straussler-Scheinker syndrome, GSS)、Kuru病以及致死性家族型失眠症(Fatal familial insomnia, FFI);绵羊和山羊的瘙痒病(Scrapie),牛的海绵状脑病(Bovine spongiform encephalopathy, BSE)、鹿的慢性消耗性疾病(Chronic wasting disease, CWD),可传播性貂脑病(Transmissible Mink encephalopathies, TME)、猫科动物海绵状脑病(Feline Spongiform encephalopathies, FSE)以及捕获的野生反刍动物的海绵状脑病。人类的克雅氏病根据其发病机制的不同分为散发型、家族遗传型和医源型三种。本文分为三部分,第一部分是根据我国克雅氏病监测系统的数据,将2006～2007年的监测结果进行了总结研究,分析我国近两年克雅氏病的发病特点,并且对两例分别为G114V突变及T188K突变的遗传型克雅氏病病例进行了诊断分析。第二部分中采用免疫组织化学方法、Western blot方法、RT-PCR方法、Real-time PCR方法及蛋白双向电泳方法对一例G114V突变的遗传型克雅氏病病例脑组织区域的神经病理学改变包括海绵样空泡样变性、PrPSc的沉积、星型胶质细胞的增生及PrP蛋白的mRNA转录水平、PrPSc的分子特点进行分析,进一步对遗传型克雅氏病的发病机制及PrPSc的分子特点进行研究。第三部分中应用免疫组织化学方法、Western blot方法、透射电镜观察的方法对羊瘙痒小鼠适应株139A及ME7跨种属感染仓鼠产生新的毒株特征进行研究,对原有毒株是否经过跨种属感染后具有与新的宿主毒株相似的分子特点进行分析。
第一部分：2006～2007年我国克雅氏病的监测及两例遗传型克雅氏病病例的发现
在WHO对克雅氏病的监测系统框架下,我国在2002年开始了对中国克雅氏病的监测。为了了解我国在近两年内克雅氏病的发病特点,我们根据我国克雅氏病监测系统的数据,将2006～2007年的监测结果进行了总结研究。通过对病例的一般流行病学资料分析,发现临床及疑似诊断的克雅氏病病例最高的发病年龄段均为60～69岁；临床诊断的克雅氏病病例男性与女性的比例为35：16,疑似诊断的克雅氏病病例的男性与女性的比例为20：10；临床及疑似诊断的克雅氏病病例居住地分布在全国23个省,地域分布比较广泛；病例职业分布也较广泛,包括工人、农民、教师、干部、自由职业者及退休在家人员；病例的报告分布于全年各个月份,没有时间的聚集性。
通过对其临床资料的分析,发现最常见的首发症状为进行性痴呆,其它还包括精神相关症状、小脑症状、锥体和锥体外系症状等；在最常见的临床症状中,最常见的依次为锥体和锥体外系症状、肌阵挛、视觉或小脑障碍及无动性缄默,这些常见症状的出现在临床及疑似诊断的克雅氏病病例中没有统计学差异；另外,同时具备其它两个、三个或者四个主要临床表现的频率在临床及疑似诊断的克雅氏病病例中没有统计学差异。
通过对临床诊断的克雅氏病病例的临床症状及实验室检测结果分析,同时出现14-3-3蛋白检测阳性及脑电图检测阳性的病例似乎会出现更多典型的临床症状,经统计学分析,不同的组在视觉与小脑障碍、锥体及锥体外系症状、无动性缄默这些症状上都没有统计学差异,但肌阵挛的症状在不同的组之间具有统计学差异。对于血液标本PRNP基因的测序分析发现,大部分为129位甲硫氨酸纯合子,占97.0%,4例为PRNP基因129位甲硫氨酸／缬氨酸杂合子,占3.0%。
对一例G114V突变的遗传型克雅氏病病例及其家系的流行病学研究发现,该病例具有进行性痴呆等典型的克雅氏病临床症状,但临床脑电图及MRI检测无特征性改变；PRNP基因测序分析发现341位碱基由鸟嘌呤(G)变为胸腺嘧啶(T),导致114位氨基酸由甘氨酸(G)变为缬氨酸(V),129位为甲硫氨酸纯合子；经Western blot方法检测该病例中脑、丘脑、小脑、额叶、顶叶、枕叶和颞叶脑区有PrPSc的分布,并且以单糖基化的形式为主,是Ⅰ型PrP分子；HE染色及免疫组织化学检测可以观察到严重的分布广泛的海绵样空泡变性及PrPSc的沉积。对该病例家系的调查发现先证者的外祖母、哥哥、姐姐都曾出现相似的临床症状并且死亡；先证者的外甥经PRNP基因测序分析具有G114V突变,出现了记忆力丧失等临床症状,目前仍存活。
对一例T188K突变的遗传型克雅氏病病例研究发现,该病例以白天出现逐渐增加的持续睡眠为首发症状,并逐渐出现典型的临床症状；经Western blot检测脑脊液14-3-3蛋白为阳性；血液PRNP基因核苷酸563位碱基由胞嘧啶(C)变为腺嘌呤(A),导致PrP蛋白188位氨基酸由苏氨酸(T)突变为赖氨酸(K),129位为甲硫氨酸纯合子。
第二部分：一例G114V突变的遗传型克雅氏病病例各脑区病理及致病特征分析
在2007年克雅氏病的监测体系中发现了我国首例G114V突变的遗传型克雅氏病病例,这个病例是世界上第二例G114V突变的克雅氏病病例。此研究分析了该病例各脑组织区域的神经病理学特征、PrPSc蛋白的分子特征及PrP蛋白的mRNA转录水平等。通过HE染色,在该病例脑组织的各个区域均发现典型的空泡样变性,在皮质区域比较严重,空泡比较大而且互相融合；通过免疫组织化学检测,发现在大脑的皮质区域包括额叶、顶叶和枕叶可以都看到颗粒状分布的PrPSc阳性信号,只有极微少的PrPSc在小脑部位沉积；而且在各个脑区都发现有大量的胶质细胞增生,但是增生的特点有所不同,大脑皮质区域的星型胶质细胞比较大,并且伴随着广泛分布的空泡样变性,在延髓、胼胝体和小脑的星型胶质细胞相对较小并且同时有大量的棕色颗粒及纤维；在脑桥、中脑和丘脑同时具有以上两种特点。
用Western blot方法检测不同脑区总PrP及具有蛋白酶K抗性的PrPSc,结果显示在没有进行PK消化时,所有脑区都可以看到具有三种糖基化形式的PrP蛋白,而且皮质区域的PrP要相对多于其它部位。经过PK消化后,在大脑皮质区域都可以看到明显的PrP信号；在中脑、丘脑及小脑可以观察到较弱的PrP信号；在延髓、脑桥和胼胝体观察不到抵抗PK的PrP信号。对PrP蛋白糖基化特点的分析发现在没有出现PrPSc的区域包括延髓、脑桥、胼胝体,以及PrPSc沉积很少的小脑,总PrP的糖基化形式以双糖基化为主；其它PrPSc沉积相对比较多的区域总PrP的糖基化形式以单糖基化为主。
通过RT-PCR方法发现该病例各个脑区的转录水平相差不大,但皮质区域转录水平相对较高一些；Real-time PCR方法得到同样的结果,各个脑区Ct值基本一致；以小脑部位的Ct值为基准,PRNP基因的cDNA相对含量从小脑的1.00E+00到枕叶的5.54E+00,皮质区域的值要相对高一些,这些数据再一次证实大脑各个区域的PRNP基因的转录不会因为PrPSc的沉积及空泡样变性而减弱,相反,PrP的转录水平与PrP蛋白的变化相一致。
通过定量Real-time PCR方法,发现该病例脑组织中存在的野生型PRNP基因的拷贝数是突变型PRNP基因的拷贝数的2 2.78倍。突变及野生型PRNP基因cDNA的相对值分别为1.00E+00及6.86E+00；通过RT-PCR实验也进一步表明野生型PRNP基因的转录水平要高于突变型PRNP基因的转录水平。
为了了解脑组织中PrPSc的更多分子特征,皮质区域的脑组织匀浆在经过PK消化后进行了蛋白双向电泳的分析。结果发现双糖基化及单糖基化的PrPSc分子大部分主要分布在pH值较低的酸性区域及中性区域,而无糖基化的PrPSc主要分布在pH值较高的碱性区域；这些数据说明,脑组织中的PrPSc分子是个分子的混合体,具有不同的生化特性,不同的分子间因糖基化特点、分子量及蛋白分子所带电荷的不同而有所差异。
第三部分：羊瘙痒小鼠适应株139A及ME7跨种属感染仓鼠后毒株特征的研究
基于不同的发病潜伏期、神经病理学特征、中枢神经系统PrPSc的沉积、PrPSc的电泳特征及糖基化特征,目前已经有超过20种不同的与TSE疾病相关的毒株被报道。在本研究中,将羊瘙痒小鼠适应株139A及ME7感染仓鼠,经过长的潜伏期之后出现了典型的海绵状脑病症状。因此,对新毒株感染的仓鼠脑组织进行神经病理学的检测,进一步分析新毒株的特征。
仓鼠接种了139A及ME7毒株后,经过分别平均为395±8.5天、496.25±27.22天的潜伏期后开始出现典型的临床症状,被139A毒株感染后最突出症状是发痒,被ME7毒株感染后,出现严重消瘦及行动迟缓,这些结果证实羊瘙痒小鼠适应株可以跨种属屏障感染仓鼠。通过在动物接种后的潜伏期中定期抽取一定数量的仓鼠进行检测发现,SAF的形成及PrPSc的出现都要早于临床症状的出现,而且SAF的出现比PrPSc的出现还要早一些。
分别对263K、139A、ME7感染的仓鼠脑组织中出现的SAF形态学特点进行观察发现,三个毒株形成的羊瘙痒纤维都出现许多长的分枝且表面粗糙,但是感染139A及ME7毒株的SAF相对短一些；免疫胶体金染色实验发现羊瘙痒纤维中主要的成分是PrP蛋白。神经病理学检测发现,典型的海绵样空泡在263K毒株感染的大脑皮质区比较严重,139A及ME7感染仓鼠的大脑皮质区可以看到中度的空泡分布；空泡样变性在小脑部位相对比较少；大脑皮质区星型胶质细胞的免疫组织化学检测发现三种毒株感染的仓鼠脑组织都出现典型的星型胶质细胞的增生。
139A及ME7感染仓鼠后形成的PrPSc与263K毒株具有相似的电泳及糖基化形式,以双糖基化条带占最多数,之后是单糖基化及无糖基化条带,而139A、ME7感染小鼠PrPSc糖基化形式以单糖基化的PrPSc条带为主；经糖苷酶的消化,三个毒株在相同的位置均出现单一条带,具有与263K相似的糖基化特征；PrP特异性抗体包括3F4、6D11、6H4、8H4的Western blot检测显示三个毒株具有同样的电泳及糖基化特征,并且观察到了经过切割以后形成的C2片段。
三个毒株的PrPSc对蛋白酶K的抵抗性比较一致,263K-ha及ME7-ha在不同的PK作用下基本保持稳定,但是139A-ha在200μg/ml PK作用时有所减弱；对于构象稳定性的检测发现,三个毒株在经GdnHCl作用后具有了相似的PK抗性,而且263K毒株在小于5M的GdnHCl作用下都可以出现蛋白条带,139A-ha及ME7-ha毒株在小于4M的GdnHCl作用下可以出现蛋白条带；说明羊瘙痒小鼠适应株139A、ME7跨种属感染后在新的宿主中显示了与仓鼠适应株263K相似的特性。
Transmissible spongiform encephalopathies (TSEs) are rare degenerative neurological disorders that afflict human beings (Creutzfeldt-Jakob disease, CJD; Gerstmann-Straussler-Scheinker syndrome, GSS; Kuru; and fatal familial insomnia, FFI), sheep and goat (scrapie), cattle (bovine spongiform encephalopathy), elk (Chronic wasting disease, CWD), mink (Transmissible Mink encephalopathies, TME), Feline(Feline Spongiform encephalopathies, FSE) and other animals. Creutzfeldt-Jakob disease may have a sporadic, inherited or transmissible origin. This study contains three individual parts including the surveillance for Creutzfeldt-Jakob disease in China from 2006 to 2007, the pathological and pathogenic characteristics in various brain regions from a Chinese patient with G114V genetic CJD and mouse-adapted scrapie strains 139A and ME7 overcome species barrier to induce experimental scrapie in hamsters and changed their pathogenic features.
Part I:Surveillance for Creutzfeldt-Jakob disease in China from 2006 to 2007 and two genetic CJD cases
In China, the CJD surveillance system was established under the framework of the surveillances for communicable diseases led by Chinese Center for Disease Control and Prevention (CCDC) since 2002 and became broader in the past sereval years. The paper collects the surveillance data from 2006 to 2007. The highest incidences were in the group of 60-69 year-old in both probable and possible sCJD cases. The male to female ratio was 35:16 in probable sCJD and 20:10 in possible sCJD. The resident places of probable and possible sCJD cases were diffused in 23 different provinces in China. The occupations of probable and possible sCJD cases were also various, including workers, farmers, teachers, officials, self-employees and house-wives. Additionally, the suspected CJD cases were reported around all year, without seasonal specificity.
The familiar foremost symptoms of the reported sCJD cases was progressive dementia and followed by mental-related syndromes, cerebellum-related syndromes, pyramidal or extrapyramidal disfunction and slow progressive dementia and so on. The most common clinical symptom was pyramidal or extrapyramidal disfunction, followed by myoclonus, visual or cerebellar disturbance, akinetic mutism. The appearance of common symptoms between probable and possible sCJD had no statistical difference. The distributing frequency of the main clinical symptoms between probable and possible sCJD were quite similar, without statistical difference.
Comparative analyses of the clinical manifestations showed that the patients with both 14-3-3 positive and EEG change seem to have more clinical symptoms. By statistical analyses, there was no difference in visual or cerebellar disturbance, pyramidal or extrapyramidal disfunction and akinetic mutism symptoms, but significant difference in myoclonus symptom among the three groups. Analyses of PRNP revealed again that most cases (131,97.0%) were methionine homozygous genotype at codon 129, four (3.0%) were methionine/valine heterozygosity and none was valine/valine homozygous.
Under the framework of the China CJD surveillance system, we found a Chinese female with G114V mutation suffering from progressive dementia, tiredness, lethargy and mild difficulty in falling asleep for two years. EEG and MRI findings more like as sporadic CJD. Obviously PK-resistant PrP-specific signals were detected in midbrain, thalamus, cerebellum, frontal lobe, temporal lobe, parietal lobe and occipital lobe with predominance of monoglycosylated PrPSc which was similar to that of PrPSc typeⅠ. Severe and extensive vacuolization was frequently identified in the tested tissues and PrPSc immunoblots were widely distributed in the tested tissues. Information on the pedigree was collected by interviews with family members. One of them was hospitalized for progressive memory impairment at the age of 32-year-old. During hospitalization, he showed mild ataxia of limbs and tremor. Neurological examination showed capability decline of memory, calculation and comprehension, and left limb Babinski sign positive. He is still alive.
In addition, we reported a 58 year-old Chinese female with mutation in codon 188 (T188K) of the PRNP gene, while the codon 129 was a methionine homozygous genotype. The patient displayed 4-year long slowly progressive sleeping disturbance and rapid exacerbation of neurological status after other neurological manifestations appeared. Cerebral spinal fluid (CSF) 14-3-3 protein was positive.
Part II:The pathological and pathogenic characteristics in various brain regions from a Chinese patient with G114V genetic CJD
Previously we have reported a Chinese gCJD case with a substitution of valine (V) for glycine (G) at codon 114. To investigate the detailed neuropathological and pathogenic characteristics of G114V gCJD, ten different brain regions were thoroughly analyzed. Neuropathological tests revealed the typical vacuolations in brains, far more intense and severe in the cortex regions, characterized with larger vacuoles that often fused each other. Widespread granular PrPSc positive-signals were observed in the sections of the brain cortex regions, while only tiny PrPSc deposits in the area of cerebellum. Abundant GFAP-positive stained cells and signals were observed in all tested regions while the profiles of GFAP-staining in various regions seemed to be diversity.
In line with the observations of total PrP, intensive PrPres signals were seen in all four cortex regions, while much weaker signals in midbrain, thalamus and, cerebellum. Analyses of the glycosylating profiles of total PrP in various brain regions revealed a predominance of diglycosylated PrP in the regions without PrPres or with mild deposit of PrPres and a predominance of monoglycosylated PrP in the regions with severe deposit of PrPres.
Assays of the transcriptions of PrP specific mRNA by RT-PCR and real-time PCR showed comparable levels in ten brain regions and lightly higher in the four cortex areas. Using the comparative Ct formula 2 (ΔCt1 [minus]ΔCt2), the relative quantitative difference of wild-type and mutant PRNP mRNA was 22.78 times at this experimental condition. The values of the relative amounts of the mutated and wild-type PRNP cDNA were 1.00E+00 and 6.86E+00 respectively.
Two-dimensional immunoblots showed that the large portions of the dots of the diglycosylated and monoglycosylated PrPSc appeared in the acidic region of the pH gradient and some in the neutral area, whereas the dots of the aglycosylated PrPSc mostly presented in the neutral and alkaline region of the pH gradient. This data suggests that the PrPSc in brains is a mixture of the molecules with different biochemical property, varying each other not only in glycosylating profiles and molecular masses, but also in the protein electric charges.
Part III:Mouse-adapted scrapie strains 139A and ME7 overcome species barrier to induce experimental scrapie in hamsters and changed their pathogenic features
To understand the potential pathogenic alterations, two mouse-adapted strains 139A and ME7 were cerebrally inoculated to hamsters. After long incubation times, animals infected with strain 139A and ME7 showed typical clinical symptoms. In line with the observation of agent 263K-infected hamsters, both SAFs and PrPSc were detected in the brain tissues earlier than the appearance of clinical symptoms.
Brain samples of each strain at the terminal clinical stage identified many long, ramose and roughly 25 nm in diameter fibrils, whose surfaces were shaggy. But the lengths of SAFs in 139A-ha and ME7-ha infected hamsters seemed shorter than that of hamster-adapted scrpaie agent 236K. Typical spongiform degeneration was more intensive and severe in the cortex infected by the strains and rarely observed in cerebellum regions. Abundant large GFAP-positively stained astrocytes were detected in cortex regions, in which the gliosis in the agent ME7-ha infected brains were comparably severe as that in agent 263K-infected one, while gliosis in the agent 139A-ha infected brains was significantly mild.
The PrPres from the hamster brains infected with strains 139A and ME7 showed identical electrophoretic and glycosylation profiles, in which the diglycosyl form of PrPres was the most predominant, followed by monoglycosyl and aglycosyl forms, showing similar profiles as that in the preparation of the hamsters infected with strain 263K and different with the characters of mouse-adapted scrapie strains. In addition, the PrPSc in hamsters'brains by infections of hamster-adapted agent 263K or mouse-adapted agents 139A and ME7 have similar PK-resistant features and conformational stabilities. The data conclude that mouse-adapted agent 139A and ME7 change their pathogenic characteristics during cross-species transmission on hamsters. The PrPSc formed in hamsters'brains obtain new molecular properties that show markedly hamster-specific.
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