苯丙酮尿症研究十八年

将自1984年以来在中日友好医院开展的有关苯丙酮尿症(PKU)的主要研究工作总结如下：(1)1984年10月-2002年9月,共诊治苯丙酮尿症(PKU)患者603例。经新生儿筛查发现并在出生后3个月内开始治疗者136例,195例在3-12个月时就诊,272例1岁以上就诊。晚发现的467例PKU患者均有PKU的症状和体征,119例合并癫痫。经低苯丙氨酸饮食治疗,早治患者的智能和体格发育正常,晚治者的异常行为明显改善、智能也有不同程度提高。22个PKU家庭作了产前基因诊断,防止了第二胎患病胎儿的出生。(2)采用高效液相色谱仪(HPLC)对369例高苯丙氨酸血症(HPA)患者进行尿液喋呤测定,22例诊断为四氢生物喋呤(BH4)缺乏症。对18例BH4缺乏症患者及其家系进行基因分析,发现6种基因突变,其中259C→T和286G→A为中国北方人的常见突变。18例患者接受了BH4、L—多巴及5—羟色胺联合治疗,疗效满意。(3)未经治疗的PKU患者的脑电图(EEG)及颅脑磁共振成像(MRI)异常均具有高发生率。脑电图异常所见主要为痫样放电,少数为背景活动异常。颅脑MRI异常以T2WI上脑白质内散在斑片高信号灶最为常见,其次为脑髓鞘化发育延迟及脑发育不良。经低苯丙氨酸饮食治疗后,绝大多数患者脑电图及脑MRI随之改善。(4)将29例晚治的经典型PKU患者的基因型与治疗前的智能障碍程度作一比较,22例基因型与智力表型相符,7例不符,提示晚治PKU患者的基因型与其治疗前的智力表型有较好的一致性。     

大颗粒淋巴细胞白血病细胞免疫学表型分析

目的:分析大颗粒淋巴细胞白血病(LGLL)细胞的免疫学表型.方法:取3例LGLL患者的外周血和骨髓液涂片,采用流式细胞术对其免疫学表型进行分析,同时用瑞-姬混合染色和髓过氧化物酶(MPO)、过碘酸-雪夫反应(PAS)等细胞化学染色对其进行观察.结果:3例白血病细胞形态均呈原始、幼稚淋巴细胞样,胞浆内有数量较多的或大或小的紫红色颗粒,MPO染色和PAS反应均为阴性.3例LGLL均表达CD56,其中1例还表达CD3,为T细胞型LGLL;另2例不表达CD3,为NK细胞型LGLL.T细胞型LGLL型患者白血病细胞同时表达B细胞抗原CD20.结论:通过免疫学表型分析可以区分T细胞和NK细胞型LGLL,LGLL细胞存在抗原表达紊乱的现象.     

人乳牙牙髓干细胞的体外培养和鉴定

目的 从健康儿童滞留的乳牙牙髓组织中分离出乳牙牙髓干细胞(SHED),体外培养观察,研究其生物学特性.方法 用酶消化法培养SHED,观察细胞形态、克隆形成能力,以免疫组化和流式细胞技术检测多种抗原表达情况及细胞周期,并进行体外分化实验,诱导SHED形成脂肪组织及矿化结节.结果 以酶消化法分离培养所得的细胞多形性明显,克隆形成效率为16~18/103细胞,其中富含有表达STRO-1表型的细胞.在一定条件下,所培养细胞具有定向分化能力.结论 以酶消化法分离培养获得的细胞中,包含自我更新能力强、有一定分化能力的干细胞,即SHED.     

热休克汗腺细胞诱导人骨髓间充质干细胞的表型转化

背景：未休克汗腺细胞与骨髓间充质干细胞的共培养和休克汗腺细胞与骨髓间充质干细胞的直接共培养均对骨髓间充质干细胞的表型改变无影响。目的：在体外建立热休克汗腺细胞模型和骨髓间充质干细胞与汗腺细胞间接共培养体系;分析共培养前后骨髓间充质干细胞的形态学、细胞表型的变化情况,探讨骨髓间充质干细胞向汗腺细胞分化的可行性。方法：体外分别分离培养、扩增并鉴定骨髓间充质干细胞和汗腺细胞,建立汗腺细胞体外热休克模型,继续孵育3-5d,收集上清液作为条件培养基对骨髓间充质干细胞分化诱导,对比共培养5,10d骨髓间充质干细胞形态学变化;应用免疫组织化学和流式细胞仪法检测对比共培养10d后骨髓间充质干细胞细胞表型的改变。结果与结论：①骨髓间充质干细胞表面标志CD29、CD44、CD105阳性,汗腺细胞表面标志CK7、CK8、CK18、CK19、CEA阳性。②骨髓间充质干细胞诱导分化后细胞表型的改变：被诱导的细胞中CEA、CK7、CK8和CKl9染色阳性,而对照组没有检测到CEA、CK7、CK8和CK19染色阳性的细胞;流式细胞仪检测CEA、CK7、CK8和CK19的阳性率分别是60．67％,53．34％,54．11％和58．62％。说明实验成功诱导骨髓间充质干细胞,并获得汗腺细胞表型;通过建立适当的体外汗腺诱导培养体系,中胚层的骨髓间充质干细胞可以通过跨胚层分化途径转变为汗腺细胞。     

ZNF816A基因多态性rs9304742与汉族人寻常型银屑病表型的相关性研究

目的 探讨ZNF816A基因多态性rs9304742与汉族人寻常型银屑病一些临床表型(性别、发病年龄、家族史及皮损临床类型)之间的相关性.方法 利用7 318例银屑病患者和11 290例对照的Illumina 610芯片基因分型数据,采用χ2检验比较各临床表型分组之间rs9304742等位基因和基因型频率分布的差异.结...     

人牙周膜体外分离培养及其成骨表型的研究

目的：体外培养人牙周膜细胞（human periodontal ligament fibroblasts hPDLFs）,并对其成骨表型特征进行研究。方法：采用胶原酶消化法获得大量成活率高的人牙周膜细胞。在DMEM培养基中进行原代及传代培养,免疫组化方法检测细胞波形丝蛋白及角蛋白的表达,鉴定细胞。使用矿化液诱导,然后应用碱性磷酸酶活性检测、Von Kossa染色等方法观察hPDLFs在矿化液作用下生物学特性的改变。结果：hPDLFs呈星形或长梭形,免疫组化波形丝蛋白阳性,角蛋白阴性,证实该细胞来源可靠。矿化诱导5d后,有部分hPDLFS转化为成骨样细胞,碱性磷酸酶活性显著升高,诱导20d后可见矿化结节形成。结论：酶消化法可以快速得到大量hPDLFs,且hPDLFs具有一定的成骨表型特征。     

Camellia (Camellia oleifera Abel.) Seed Oil Regulating of Metabolic Phenotype and Alleviates Dyslipidemia in High Fat-Fed Mice through Serum Branch-Chain Amino Acids

Camellia (Camellia oleifera Abel.) seed oil (CO) has been shown to effectively reduce the blood lipid level of its host due to its fatty acid content, but the specific molecular mechanism associated with the metabolic phenotype after digestion is not clear. Here, we further investigated the relationship between branched-chain amino acids (BCAA) and the metabolic phenotype that may exhibit the anti-dyslipidemia effect of CO on mice fed a high-fat diet for 30 day C57BL/6J male mice were allocated to three groups: the control group (Cont), the high-fat feed group (HFD), and a high-fat feed group with CO treatment (CO). A serum sample was collected to detect lipid biomarkers and BCAA concentration. Notably, Low-density lipoprotein (LDL), Total Cholesterol (TC), and Triglycerides (TG) showed a significant decrease, whereas High-density lipoprotein (HDL) increased in CO mice but not in the HFD group. The concentration of Isoleucine (Ile), leucine (Leu), and valine (Val) was similar between the Cont and CO groups compared with the HFD group, exhibiting an inhibition induced by CO in mice fed with a high-fat diet. A metabolic phenotype from serum examined by non-targeted metabolite analysis using UHPLC/MS showed most metabolites exhibited lipid and BCAA metabolism. The results indicated that CO treatment notably regulated the metabolism of arachidonic acid and steroid biosynthesis in response to HFD-induced dyslipidemia. In addition, the expression of PPARγ genes that correlated with the BCAA and serum lipid biomarkers were compared, and significant inhibition was noticed, which might lead to the potential exposure of the anti-dyslipidemia mechanism of CO in HFD-fed mice. In conclusion, the expression of PPARγ genes, serum lipid level, BCAA concentration, and the metabolic phenotype was significantly positive in correlation with a high-fat diet, whereas oral CO improved the biomarkers and metabolism of some specific serum metabolites in HFD-fed mice.     

Simultaneous Mass Spectrometry-Based Apolipoprotein Profiling and Apolipoprotein E Phenotyping in Patients with ASCVD and Mild Cognitive Impairment

Apolipoprotein E (apoE) occurs on the majority of plasma lipoproteins and plays a major role in the lipid metabolism in the periphery and in the central nervous system. ApoE is a polymorphic protein with three common isoforms, apoE2, apoE3 and apoE4, derived from respective alleles ε2, ε3 and ε4. The aim of this study was to develop a sample pretreatment protocol combined with rapid mass spectrometry (MS)-based assay for simultaneous apolipoprotein profiling and apoE phenotype identification. This assay was validated in 481 samples from patients with stable atherosclerotic cardiovascular disease (ASCVD) and applied to study association with mild cognitive impairment (MCI) in the LIFE Adult study, including overall 690 study subjects. Simultaneous quantification of 8–12 major apolipoproteins including apoA-I, apoB-100 and apoE could be performed within 6.5 min. Phenotyping determined with the developed MS assay had good agreement with the genotyping by real-time fluorescence PCR (97.5%). ApoE2 isoform was associated with the highest total apoE concentration compared to apoE3 and apoE4 (p < 0.001). In the subgroup of diabetic atherosclerotic cardiovascular disease (ASCVD) patients, apoE2 isoform was related to higher apoC-I levels (apoE2 vs. apoE3, p < 0.05), while in the subgroup of ASCVD patients under statin therapy apoE2 was related to lower apoB-100 levels (apoE2 vs. apoE3/apoE4, p < 0.05). A significant difference in apoE concentration observed between mild cognitive impairment (MCI) subjects and controls was confirmed for each apoE phenotype. In conclusion, this study provides evidence for the successful implementation of an MS-based apoE phenotyping assay, which can be used to assess phenotype effects on plasma lipid and apolipoprotein levels.     

The In Vitro Replication,Spread, and Oncolytic Potential of Finnish Circulating Strains of Herpes Simplex Virus Type 1

Herpes simplex virus type 1 (HSV-1) is the only FDA- and EMA- approved oncolytic virus, and accordingly, many potential oncolytic HSVs (oHSV) are in clinical development. The utilized oHSV parental strains are, however, mostly based on laboratory reference strains, which may possess a compromised cytolytic capacity in contrast to circulating strains of HSV-1. Here, we assess the phenotype of thirty-six circulating HSV-1 strains from Finland to uncover their potential as oHSV backbones. First, we determined their capacity for cell-to-cell versus extracellular spread, to find strains with replication profiles favorable for each application. Second, to unfold the differences, we studied the genetic diversity of two relevant viral glycoproteins (gB/UL27, gI/US7). Third, we examined the oncolytic potential of the strains in cells representing glioma, lymphoma, and colorectal adenocarcinoma. Our results suggest that the phenotype of a circulating isolate, including the oncolytic potential, is highly related to the host cell type. Nevertheless, we identified isolates with increased oncolytic potential in comparison with the reference viruses across many or all of the studied cancer cell types. Our research emphasizes the need for careful selection of the backbone virus in early vector design, and it highlights the potential of clinical isolates as backbones in oHSV development.     

星形细胞肿瘤微血管组织芯片的构建和微血管壁组成细胞研究

目的初步探讨人脑星形细胞肿瘤中新生微血管的管壁组成成分与微血管构筑异质性(tumor microvascular architecturphenotype heterogeneity,T-MAPH)之间的关系。方法收集45例人脑星形细胞肿瘤标本,选择其新生微血管形态不同的“热点区域”制备组织芯片;在此基础上采用免疫组织化学染色技术分别进行GFAP、CD34和α-SMA标记,观察微血管管壁组成细胞的免疫表型与其不同形态特征之间的关系。结果成功构建了含幼稚微血管、厚壁微血管、肾小球样微血管及薄壁、蛇行状微血管等各种形态特征的153点组织芯片;各种形态的微血管中均可检测到呈单层排列、位于微血管最内层的CD34阳性的内皮细胞;α-SMA阳性反应可见于内皮细胞外相当于周细胞处,其阳性表达范围及数目在不同形态的微血管中呈现多样性:幼稚微血管仅见少量表达,而在厚壁和“肾小球样”微血管中α-SMA阳性细胞多呈活跃增生的单层或多层。结论星形细胞瘤微血管形态构筑的多样性(异质性)是由内皮细胞和α-SMA阳性周细胞共同参与形成的,而后者的作用可能更为突出和重要。