p62dok-PTB结构域样新基因片段的克隆

利用RT-PCR方法,从胎儿脾脏和肝脏组织中克隆出一个342bp的基因片段,并测定了其核苷酸序列.在基因库中未见相同序列报道,但该片段与p62dok-PTB结构域核苷酸序列有93%的同源性,提示它是一种新的p62dok-PTB结构域样基因片段.     

dystrophin基因常见易缺失外显子片段的克隆

背景：既往研究显示,dystrophin基因缺失集中在两个热点区域即外显子2~20和44~53,其主要缺失可以通过对一系列外显子的检测来发现。DNA微阵列可以将许多基因或基因的许多片段同时进行检测,为检测dystrophin基因缺失提供了一项新技术。 目的：对dystrophin基因18个常见易缺失外显子片段进行克隆、鉴定,以克隆产物作为核苷酸探针为研制dystrophin基因缺失检测微阵列作准备。 设计、时间及地点：分子生物学观察实验,于2002年在第四军医大学附属西京医院神经内科完成。 材料：细菌菌株E.coli JM109由全军基因诊断技术研究所保存,质粒载体pGEM®-T Easy Vector购自美国Promega公司,18对寡核苷酸引物由上海生物工程公司合成。 方法：以人类基因组DNA为模板,应用18对引物对dystrophin基因常见易缺失外显子片段进行PCR扩增。将扩增产物与pGEM-T Easy载体连接,转化E.coli JM109感受态细胞。通过平板培养,挑选阳性克隆。提取重组质粒,Not I酶切,获得完整的探针片段,并作测序鉴定。通过核酸序列数据库相似性检索工具验证序列的来源及其与GeneBank收录序列的相似性。 主要观察指标：①dystrophin基因外显子片段的PCR扩增。②扩增片段与pGEM-T Easy载体连接构成重组质粒并克隆。③重组质粒的Not I酶切及测序鉴定。④克隆片段的序列分析及同源性检索。 结果：PCR扩增出18个片段,与dystrophin基因预期扩增片段大小相一致。重组克隆质粒的酶切产物与PCR产物大小相近,与预期相一致。经测序获得18个克隆片段全序列,其核苷酸数量与预期基本一致,序列相似性检索分析证实了这些克隆片段与GeneBank收录的dystrophin基因片段具有极高的同源性。 结论：克隆产物确为dystrophin基因常见易缺失外显子片段。     

人脑胶质瘤中p16,p15基因分子的病理学研究

目的:探讨p16、p15基因改变、蛋白表达与脑胶质瘤组织病理的相互关系。方法:应用PCR、银染PCR-SSCP及免疫组化技术检测68例脑胶质瘤中p16、p15基因变化和蛋白表达情况。结果:显示脑胶质瘤中p16、p15基因缺失和P16蛋白丢失主要见于Ⅱ~Ⅳ级肿瘤中,而PCR-SSCP分析未见p16、p15基因点突变。结论:表明脑胶质瘤中p16、p15基因改变是以缺失为主,该基因功能的丧失促进了肿瘤细胞由良性向恶性的演进过程;P16蛋白表达状态可作为判断脑胶质瘤恶性程度及预后的指标。     

缺失型Duchenne型肌营养不良症基因连接片段的BAC克隆和断裂点的分子结构特征

目的　分析缺失型Duchenne型肌营养不良症 (DMD)缺失热区第 46号外显子缺失后形成的连接片段的断裂点的分子结构特点 ,并分析分子结构特点与内含子高不稳定性及外显子缺失的关系。方法　多重引物PCR法鉴定第 46号外显子缺失的DMD患者 ,用BAC载体克隆第 46号外显子缺失后形成的连接片段 ,测定断裂点侧翼的核苷酸序列。结果　第 46号外显子缺失后 ,5’端断裂点位于 45号内含子的AT富含区内。 3’端断裂点位于 46号内含子MER1类重复序列内。连接片段有两个bp的连接同源序列ta ,局部无小的缺失、插入和碱基的置换。第 46号外显子缺失后连接片段的断裂点的二级结构分析示断裂点均位于单链发夹环的非匹配区。结论　第 46号外显子缺失后形成的连接片段 ,其断裂点的共同特征是均位于重复序列 ,这些重复序列形成的单链发夹结构 ,使DNA结构具有不稳定性 ,易于断裂并导致外显子缺失。     

精神分裂症患者BDV—p24基因的扩增及其产物的测序鉴定

目的：扩增精神分裂症患PBMCs标本中博尔纳病病毒（Borna disease Virus,BDV)p24基因，并对基因扩增产物进行测序鉴定，分析其与标准株之间的差异。方法：用巢式RT－PCR方法检测黑龙江省精神分裂症患及正常人PBMCs中BDV－p24基因片段，对2例BDV－p24基因阳性的巢式RT－PCR产物进行测序，并与标准株比较。结果：9例精神分裂症患中有2例BDV－p24基因阳性，7例正常人标本中未发现BDV－p24基因阳性。测序结果进一步证实扩增产物为BDV－p24基因，其序列与标准株高度同源。结论：用巢式RT－PCR方法可以特异性扩增出BDV－p24基因，扩增产物序列与标准株高度同源，提示黑龙江省的精神分裂症的发生可能与BDV感染有关。     

阿尔茨海默病患者线粒体CO2基因片段的突变分析

20 0 0年 ,我们利用ＰＣＲ及巢式ＰＣＲ技术扩增了阿尔茨海默病 (ＡＤ)患者 2 9例老年人的编码 910ｂｐ细胞色素Ｃ氧化酶亚基Ⅱ的ｍｔＤＮＡ片段 (ＣＯ2 ) ,探讨其在ＡＤ发生发展中的作用。资料和方法 :ＡＤ组 9例 ,男 3例 ,女 6例 ,平均年龄 (76 1± 2 3)岁。全部病例是 1999年 3月至 2 0 0 0年 5月自江西省某军分区干休所 ,应用简明智力状况检查量表 (ＭＭＳＥ)、长谷川痴呆量表 (ＨＤＳ Ｒ)和克莱顿智能量表 (ＡＤＬ)进行痴呆筛选调查 ,采用美国国立神经病学、语言障碍以及卒中研究所(ＮＩＮＣＤＳ) ＡＤ及其有关疾病协会 (ＡＤＲＤＡ…     

重症肌无力P9—ZFD基因片段表达蛋白的研究

目的:探讨P9-ZFD基因片段在人骨骼肌中表达的蛋白质。方法:以MG骨骼肌RNA为模板,扩增编码P9-ZFD片段的cDNA,构建pFT24a-P9-ZFD表达载体,经BL21(DE3)诱导表达P9-ZFD蛋白和组氨酸亲和层析法进行纯化,并制备P9-ZFD抗体。Western blot鉴定MG和对照组骨骼肌中与P9-ZFD抗体产生特异性免疫反应的蛋白组分。结果:骨骼肌中与P9-ZFD抗体产生特异性免疫反应的蛋白质相对分子质量约40000,在伴胸腺增生或胸腺瘤MG骨骼肌中的表达水平明显高于对照组(P<0.001)。结论:骨骼肌中40 000的蛋白是P9-ZFD基因片段的编码产物,该蛋白在伴胸腺增生或胸腺瘤MG骨骼肌中的表达水平明显上调,可能具有重要的病理生理意义。     

应用差异显示技术克隆脑脓肿早期表达上调的新基因

本研究以大鼠脑脓肿模型为基础，利用mRNA荧光差异显示技术，比较正常组、对照组和脑脓肿组脑组织中基因表达的差异，以寻找脑脓肿发病的早期相关基因。     

脑胶质瘤p16基因CPG岛高甲基化与基因失活的研究

目的探讨脑胶质瘤中p16基因5'端CPG岛高甲基化与该基因失活的相关性.方法应用免疫组化检测50例脑胶质瘤中P16蛋白的表达;应用PCR技术检测脑胶质瘤中p16基因第1、2外显子缺失及第1外显子5'CPG岛高甲基化.结果免疫组化结果显示50例脑胶质瘤组织中27例恶性胶质瘤P16蛋白缺失,23例低级别胶质瘤P16蛋白阳性;9例恶性胶质瘤p16基因纯合性缺失;P16蛋白阴性的恶性胶质瘤中7例显示p16基因CPG岛高甲基化.结论恶性脑胶质瘤中P16蛋白缺失而没有p16基因纯合性缺失,是由于p16基因5'端CPG岛高甲基化后抑制该基因的转录所致.p16基因高甲基化也是该基因失活的重要机制之一.     

脑胶质瘤中全长新基因PKIβ的克隆与表达

目前世界上在不同组织或不同病理样本中获取差异表达基因的方法有很多种。在各种技术中诸如:差异显示技术,只适用于少量样本,同时跑电泳的工作相当辛苦,并受限于一次电泳样品数量,而     

大鼠代谢型谷氨酸受体1亚型基因片段的克隆及其在小脑的表达

目的：克隆大鼠代谢型谷氨酸受体1亚型（mGluR1)基因特异片段，制备cDNA探针。方法：从Wistar大鼠小脑中提取总RNA，以RT－PCR方法得到预期的599bp条带，将这一片段克隆到pGEM-T easy载体上，经酶切鉴定正确后送测序。将重组质粒经限制性内切酶酶切制备成线性模板，通过体外转录的方法 合成地高辛标记的mGluR1cRNA正义及反义探针。取成年Wistar大鼠小脑组织进行原位杂交实验，以检测探针的可靠性。结果：测序证实用RT－PCR的方法获得了mGluR1基因特异片段，成功地构建了pGEM-TmGluR1重组质粒。根据斑点杂交实验结果计算出正义、反义探针浓度分别为10ng/μl及30ng/μl。原位杂交实验的结果显示，用mGluR1反义探针进行杂交的阳性信号主要分布在大鼠小脑蒲肯野氏细胞胞浆，用正义探针杂交无阳性信号。结论：本实验克隆了mGluR1基因特异片段，并制备了cRNA探针，并用大鼠小脑进行的原位杂交实验显示，此探针灵敏度高，特异性好。     

急性脑梗死患者血小板表达血小板内皮细胞黏附分子-1、P选择素的改变及其意义

目的观察急性脑梗死(AC I)患者血小板表达血小板内皮细胞黏附分子-1(CD31)、P选择素(CD62p)的改变及其意义。方法采用全血流式细胞术测定53例AC I患者发病48 h内血小板CD31、CD62p的表达水平,并与有脑梗死易患因素组及健康对照组比较。结果AC I组血小板表达CD31、CD62p[(90.91±15.39)%,(7.00±2.96)%]明显高于易患因素组和健康对照组(均P<0.001);AC I组中合并高血压或糖尿病患者血小板CD62p表达高于无高血压和糖尿病的患者(均P<0.01);血小板CD31、CD62p的表达与脑梗死体积正相关(r=0.39,P<0.05;r=0.63,P<0.01)。结论AC I发病后血小板表达CD31、CD62p显著增高,其表达程度与脑梗死体积以及是否合并高血压或糖尿病有关。     

Photic injury promotes cleavage of p75NTR by TACE and nuclear trafficking of the p75 intracellular domain

The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily that paradoxically mediates neuronal survival and differentiation or apoptotic cell death. Cleavage of p75NTR by a constitutively active metalloprotease could result in shedding of its extracellular domain (p75ECD) and generation of a pro-apoptotic intracellular domain (p75ICD). In this study, we established that exposure of a transgenic mouse photoreceptor cell line to intense light upregulated the expression of p75NTR and of the disintegrin metalloprotease tumor necrosis factor-converting enzyme (TACE) and resulted in apoptotic cell death. Light damage promoted TACE cleavage of p75NTR resulting in shedding of the soluble p75ECD and nuclear translocation of the p75ICD. Overexpression of TACE and p75NTR-induced p75NTR cleavage and secretion of p75ECD, but not nuclear transport of p75ICD. Light-induced cleavage of p75NTR, nuclear localization of p75ICD, and apoptosis were inhibited by IC-3, a metalloprotease inhibitor. Increased levels of p75NTR and TACE were observed in photoreceptor cells of animals with photic injury. Our findings support a role for TACE in the proteolytic cleavage of p75NTR and light-induced apoptosis.     

CD62p、CD63联合血栓弹力图检测在氯吡格雷治疗复发性脑梗死的临床意义

目的 探讨初发脑梗死患者与复发脑梗死患者在氯吡格雷治疗后,血小板膜糖蛋白CD62p、CD63及血栓弹力图指标变化特点及其临床意义。方法 纳入2019年7月至2021年1月在该院住院的急性缺血性脑卒中患者103例,分为复发脑梗死组（43例）和初发脑梗死组（60例）。入院后服用氯吡格雷(75 mg/d),服药后第8天采用流式细胞术测定患者血浆中血小板膜糖蛋白CD62p和CD63表达率,并行血栓弹力图(TEG)检测,包括血小板ADP抑制率(%)、血细胞凝集块形成时间(K)、凝血反应时间(R)、血细胞凝集块形成速率(α角)和血凝块最大硬度或强度(MA)等指标,分析TEG各参数与CD62P、CD63表达率特点及其相关性。结果 初发组和复发组治疗后血小板糖蛋白CD62p、CD63表达阳性率较治疗前均下降,差异具有统计学意义(P<0.05)。与初发组相比,复发组治疗后CD62p、CD63的表达阳性率下降幅度更小,差异具有统计学意义(P<0.05)。与初发组相比,复发组治疗后第8天检测血小板ADP抑制率更低、K值更短、MA更高,差异具有统计学意义(P<0.05)。初发组与复发组治疗第8天TEG检测中凝血反应时间R与CD63表达阳性率均呈负相关;CD62p表达阳性率与ADP抑制率均呈负相关。复发组ADP抑制率与CD63表达阳性率呈负相关。结论 复发性脑梗死患者在氯吡格雷治疗后具有更高的血小板反应性、血小板活性和血凝状态,CD62p、CD63联合血栓弹力图指标检测有一定的临床意义。引用格式:471-475.]     

Implications of mitogen‐activated protein kinase signaling in glioma

Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen‐activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B‐RAF in the ERK, c‐Jun N‐terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)‐activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS‐mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK‐activated MAPK pathway during gliomagenesis and progression. © 2015 Wiley Periodicals, Inc.     

Cloning of a cDNA encoding a novel interleukin-1 receptor related protein (IL1R-rp2)

We have identified and isolated both the rat and human cDNAs for a novel putative receptor related to the interleukin-1 type 1 receptor. We have named this protein interleukin 1 receptor related protein two (IL1R-rp2). The rat cDNA for IL1R-rp2 was first identified using oligonucleotides of degenerate sequence in a polymerase chain reaction (PCR) paradigm with rat brain mRNA as the template. The protein encoded by both of these cDNAs are 561 amino acids long and exhibit 42% and 26% overall identity with the interleukin-1 type 1 and type 2 receptors, respectively. RNase protection assays from rat tissues revealed a predominant expression for IL1R-rp2 in the lung and epididymis with lower levels detected in the testis and cerebral cortex. By in situ hybridization we were able to determine that the expression in rat brain appeared to be non-neuronal and associated with the cerebral vasculature. When expressed transiently in COS-7 cells the receptor was incapable of high affinity binding to either [¹²⁵I]-recombinant human IL1α or [¹²⁵I]-recombinant human IL1β. Together, these data demonstrate the existence of a novel protein that is related to the interleukin-1 receptor but does not bind IL-1 by itself.     

Expression of mutant ubiquitin (UBB+1) and p62 in myotilinopathies and desminopathies

Protein aggregates in muscle cells are the morphological hallmark of myofibrillar myopathies, including myotilinopathies and desminopathies. The aim of the present study is to analyse the expression of mutant ubiquitin (UBB⁺¹), an aberrant form of ubiquitin which accumulates in certain disorders characterized by intracellular aggregates of proteins, and p62, a multimeric signal protein which plays an active role in aggregate formation, in muscle biopsies from patients suffering from myotilinopathy and desminopathy in order to gain understanding of the mechanisms leading to protein aggregation in these disorders. Single immunohistochemistry, and single- and double-labelling immunofluorescence and confocal microscopy for UBB⁺¹ and p62, has been performed in muscle biopsies from patients suffering from myotilinopathy and desminopathy. Strong UBB⁺¹ immunoreactivity, colocalizing with myotilin aggregates, was found in muscle fibres in myotilinopathies. UBB⁺¹ accumulation, colocalizing with desmin aggregates, also occurs in desminopathies. In addition, strong p62 immunoreactivity colocalizing with myotilin aggregates was observed in myotilinopathies. Similarly, p62 immunoreactivity colocalizing with desmin aggregates was found in desminopathies. The present findings suggest that accumulation of protein aggregates in myotilinopathies and in desminopathies may be related with UBB⁺¹/abnormal protein complexes which are resistant to proteasome degradation. Furthermore, these observations suggest a relationship between the presence of p62 and the formation of inclusions in different subtypes of myofibrillar myopathies.     

奥扎格雷对急性脑梗死患者血小板CD62p和CD63表达的影响及其疗效

目的观察奥扎格雷对急性脑梗死(ACI)患者血小板CD62p、CD63表达的影响及其疗效。方法将64例ACI患者随机分为奥扎格雷治疗组和血塞通治疗组(对照组),采用流式细胞术检测ACI患者治疗前后及正常人(正常组)血小板CD62p、CD63的表达;观察奥扎格雷治疗组和对照组的临床疗效并进行比较。结果ACI患者血小板CD62p、CD63表达水平明显高于正常组(均P<0.01);奥扎格雷治疗组与对照组治疗后血小板CD62p、CD63表达水平较治疗前均有明显下降(P<0.05～0.01),奥扎格雷治疗组又明显低于对照组,差异有显著性(均P<0.05)。奥扎格雷治疗组的基本痊愈率、显著进步率、总有效率明显高于对照组(均P<0.05)。结论ACI发病后血小板CD62p、CD63表达水平显著增高;奥扎格雷有明显抑制血小板表达CD62p、CD63的作用,对ACI的治疗效果显著。