树脂包埋半薄切片在视神经组织学和免疫组织化学研究中的应用

目的探讨半薄切片在视神经组织学和免疫组织化学研究中的应用,寻找一种视神经形态学研究和疾病诊断的有效方法。方法健康Sprague-Dawley大鼠视神经分别行树脂包埋制成半薄切片与石蜡包埋制成石蜡切片,分别采用HE、甲苯胺蓝染色及髓鞘碱性蛋白(myelin basic protein,MBP)免疫组织化学方法染色,光学显微镜下观察并比较半薄切片与石蜡切片染色结果的差异。结果石蜡切片HE和甲苯胺蓝染色均显示视神经髓鞘不着色,MBP免疫组织化学染色示MBP阳性着色较重,着色较紊乱,髓鞘着色不清晰。半薄切片HE及甲苯胺蓝染色均显示髓鞘呈环形,着色清晰,颜色对比鲜明;MBP免疫组化染色结果可见髓鞘呈环形,非特异性染色不明显,阳性对比显著,可清晰显示髓鞘结构。结论半薄切片在视神经组织学和免疫组织化学研究中优于石蜡切片,可用于视神经疾病的诊断和研究。     

缺氧缺血诱导对原代新生大鼠前脑混合细胞髓鞘形成和细胞骨架的影响

目的:该研究探讨了缺氧缺血诱导对新生大鼠前脑混合细胞髓鞘形成和细胞骨架的影响。方法:在原代培养的新生大鼠前脑混合细胞中,应用免疫组化染色髓鞘碱性蛋白(MBP)和劳克坚牢蓝(LFB)染色检测髓鞘和轴突的发育情况;Western印迹分析NF155蛋白的表达情况;免疫荧光分析参与细胞骨架调节的ERM-Rho GTP酶家族相关蛋白表达。结果:缺氧缺血对混合细胞的进一步分化和成熟起到抑制作用,MBP染色阳性率降低57%,髓鞘和轴突染色阳性率降低74%;NF155蛋白表达降低51%;Rho GTP酶家族成员Rac1、Cdc42分别降低81%和75%。结论:缺氧缺血使细胞突起的形成和骨架重组受到阻碍,继而影响髓鞘的发育和成熟,这一过程与ERM-Rho GTP酶细胞骨架通路有关。     

神经髓鞘染色新方法的建立及其原理探讨

在神经病理诊断和研究工作中,髓鞘染色是一种常用的方法。任何因素的神经纤维损伤,均可导致髓鞘的变性、崩解或脱失。普通染色中髓鞘不易着色,在正常或病理情况下均需要特殊染色法来观察髓鞘的损害程度。常规髓鞘特殊染色多采用传统的苏木素（如Wei1氏和Loyez氏法）及锇酸染色法。     

神经细胞尼氏体和神经髓鞘的双重组合染色法与应用

探讨了显示实验动物脊髓组织中的神经尼氏体和神经髓鞘等组织成分的双重组合染色法,通过分别选用孔雀石绿(Malachite green)和橙黄G、磷钨酸(Orange G,Phosphotungstic acid)组合染色(简称MG-OP法)。已能够显示狗脊髓神经尼氏体呈绿色,细胞核呈黄色。神经纤维髓鞘轴突呈黄色,神经膜和结缔组织纤维呈绿色,背景呈淡黄色。MG-OP法克服了原法成分单一,色彩效果差,所建立的双重组合染色法,对比清晰,色彩鲜艳,方法简便的较好染色方法。     

选择性敲除小鼠神经细胞adam10基因导致胼胝体和海马联合发育不全

目的探讨adam 10基因在小鼠神经系统发育的作用。方法将gfapcre转基因鼠和adam10loxlox转基因鼠杂交,得到神经细胞特异性adam10基因敲除鼠(gfapcre-adam10loxlox);在出生后第14d对小鼠大脑切片行HE染色和免疫组织化学染色,观察adam10基因敲除后神经系统发育情况。结果选择性敲除小鼠神经细胞adam10基因(gfapcre-adam10loxlox)后,小鼠可存活至出生后3w左右;HE染色发现胼胝体缺失,海马发育不全,髓鞘碱性蛋白(myelin basic protein,MBP)免疫荧光染色发现髓鞘发育异常。结论 Adam10基因在小鼠神经系统发育中具有重要作用,选择性敲除小鼠神经细胞adam10基因导致胼胝体和海马联合发育不全,髓鞘发育异常。     

髓鞘碱性蛋白片段69-85诱导建立大鼠自身免疫性脑脊髓炎模型

目的探索以大鼠髓鞘碱性蛋白片段69-85（MBP69-85）为单一抗原,建立Wistar大鼠实验性自身免疫性脑脊髓炎（EAE）模型,并观察其病理改变。方法MBP69-85以生理盐水溶解,与完全福氏佐剂（CFA）充分混匀制备免疫乳剂;雌性Wistar大鼠70只,留取10只作为正常对照组,余者根据免疫乳剂组分的不同随机分为A、B、C组（n=20）。A组：MBP69-85每只50μg＋CFA（含卡介苗6 mg）;B组：MBP69-85每只25μg＋CFA（含卡介苗6 mg）;C组：MBP69-85每只25μg＋CFA（含卡介苗12 mg）。脑和脊髓组织切片进行HE染色,MBP及神经微丝（neurofilament,NF）免疫组化检测,观察神经组织的病理改变。结果A组内部分大鼠在免疫后第12-16天发病,表现为尾部及四肢无力或麻痹、斜颈等,平均临床症状评分为2.38±1.89;B、C组均未见发病;HE染色可见神经组织内炎细胞浸润,血管＂袖套＂样病灶形成;MBP及NF免疫组化染色显示病变组织内白质脱髓鞘及轴突损伤明显。结论EAE模型建立与免疫抗原的剂量有一定的依赖关系;佐剂中的卡介苗含量不是诱导大鼠发病的主要原因;本模型具有多发性硬化的典型临床表现及病理改变,是研究多发性硬化发病机制及治疗的可靠的动物模型。     

Olig2在cuprizone介导的脱髓鞘动物模型中的表达变化

目的探讨Olig2在cuprizone诱导的急性脱髓鞘动物模型中的表达变化规律。方法应用含0.2%cuprizone饲料饲育小鼠,通过调控饲育时间,造成神经脱髓鞘及髓鞘再生,使用免疫荧光染色和实时定量PCR(qRT-PCR)的方法,观察模型髓鞘脱失后及髓鞘再生2周后Olig2、少突胶质细胞碱性髓鞘蛋白(MBP)及星形胶质细胞神经胶质酸性蛋白(GFAP)的表达变化。结果 Cuprizone饲育6周后,动物胼胝体白质内髓鞘脱失严重,在恢复正常饲料后,髓鞘逐渐恢复正常结构。正常小鼠大脑Olig2低水平表达。髓鞘脱失后Olig2、GFAP表达增高,并可见Olig2+/GFAP+细胞,MBP表达明显降低。髓鞘再生2周后Olig2表达降低,MBP、GFAP表达增高。结论 Olig2基因在cuprizone诱导的脱髓鞘模型中的表达变化,提示Olig2可能参与祖细胞向有活性的星形胶质细胞的分化过程,并与胶质瘢痕的形成有关。     

大鼠受损视神经早期保护的电镜观察

为了比较应用不同的干预手段对视神经及其髓鞘溃变速度的延缓作用,将成年SD雄性大鼠随机分为6组,AX组（实施视神经轴索切断术）,MP组（术后尾静脉注射甲泼尼龙）,OECs（术后移植嗅成鞘细胞）,SC组（术后移植坐骨神经）,OECs＋MP组,SC＋MP组,于术后14天、21天取材,进行常规切片染色。半薄切片甲苯胺兰染色显示,AX组髓鞘发生大量崩解变性,失去正常神经束膜的结构,束膜间的血管溃变;OECs＋MP组束膜形态较完整,可观察到完整的神经束膜,束膜间的血管较规则。     

新生期缺氧缺血大鼠脑白质损伤及脑内Fyn、p-ERK和MBP下调

目的检测新生大鼠缺氧缺血后脑内酪氨酸蛋白激酶Fyn、p-ERK及髓鞘碱性蛋白(myelin basic protein,MBP)表达的变化,探讨Fyn-ERK通路在缺氧缺血脑白质损伤中的作用。方法新生3日龄SD大鼠24只,随机分为对照组和缺氧缺血(HI)组。缺氧缺血后4周,应用HE染色法观察脑组织病理学改变;应用免疫荧光染色法和Western blot法观察Fyn、p-ERK及MBP在大鼠脑内的定位定量表达变化。结果 HE染色显示缺氧缺血大鼠出现缺氧缺血脑白质损伤病理改变;免疫荧光染色和Western blot检测显示脑内Fyn、p-ERK和MBP水平均明显下调。结论新生期缺氧缺血损伤4周后,可能通过Fyn与p-ERK水平的下调而使MBP减少,进而使少突胶质细胞成熟障碍,引起脑白质损伤。     

冰冻切片的H&E和LFB染色技术的优化及其在自身免疫疾病中的应用

该文旨在探讨基于冰冻切片样品进行苏木素–伊红(hematoxylin-eosin,HE)染色和快蓝(luxol fast blue,LFB)染色方法的优化及其在实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)组织病理分析中的应用。取正常及EAE建模后的C57BL/6小鼠脊髓样品各5份,每份脊髓样品一分为二,分别制成冰冻切片和石蜡切片样品,通过设置不同优化条件对这两组切片进行HE和LFB染色,比较两组切片染色的结果。石蜡切片经过HE染色和LFB染色后细胞的形态结构和组织结构清晰,冰冻切片HE染色和LFB染色后细胞结构清晰度几乎和石蜡切片一致,进一步将该方法应用于分析β-arrestin2基因敲除对于小鼠EAE发生中的病理变化,发现能够很好地重复出基因敲除加重EAE疾病这一现象。冰冻切片染色优化后,基于冰冻切片进行HE染色和LFB染色可以达到石蜡切片类似的效果,并且实验过程简洁快速,大大缩短了实验时间,提高了效率,可以较好地应用于分析自身免疫疾病病理变化,该优化方法将在自身免疫疾病特别是多发性硬化动物模型的研究中有广泛的应用。     

骨髓间充质干细胞移植对急性脊髓损伤脱髓鞘病变的保护作用

骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)已被广泛应用于治疗脊髓损伤,但目前对其治疗机制了解甚少。BMSCs被移植至脊髓钳夹损伤模型大鼠,以研究其保护作用。通过LFB(Luxol fast blue)染色、锇酸染色、TUNEL(Td T-mediated d UTP nick-end labeling)染色和透射电镜对白质有髓神经纤维进行观察。免疫印迹检测BMSCs移植对脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)和caspase 3蛋白表达的影响。通过脊髓损伤后1、7、14 d三个时间点移植BMSCs并进行后肢运动评分(Basso,beattie and bresnahan;BBB评分)和CNPase(2′,3′-cyclic-nucleotide 3′-phosphodiesterase)、髓鞘碱性蛋白(Myelin basic protein,MBP)、caspase 3蛋白水平的检测。免疫荧光观察BMSCs移植到受损脊髓后分化情况及CNPase-caspase 3~+共表达情况。骨髓间充质干细胞移植7 d后,部分移植的BMSCs可表达神经元和少突胶质细胞标记物,大鼠后肢运动能力和髓鞘超微结构特征均明显改善。骨髓间充质干细胞移植后BDNF蛋白表达水平增加,caspase 3蛋白表达水平则降低。相对于脊髓损伤后1 d和14 d,7 d移植BMSCs后MBP和CNPase蛋白表达水平最高;caspase 3蛋白表达水平则最低。骨髓间充质干细胞移植后CNPase-caspase 3~+细胞散在分布于脊髓白质。结果表明,急性脊髓损伤后,BMSCs移植到受损脊髓有分化为神经元和少突胶质细胞的倾向,并促进BDNF的分泌介导抗少突胶质细胞凋亡而对神经脱髓鞘病变有保护作用,且最佳移植时间为脊髓损伤后7 d。     

中国五种锄足蟾科无尾两栖动物的细胞遗传学研究

本文对中国５种锄足蟾科无尾两栖动物的骨髓细胞有丝分裂中期作了细胞遗传学研究,内容包括核型、Ａｇ－ＮＯＲｓ和Ｃ－带。研究结果表明景东角蟾：２ｎ＝２６（２０Ｍ＋４ＳＭ＋２ＳＴ）,５＋８,ＳＣ和Ａｇ－ＮＯＲｓ位于６ｐ＾ｐｅｒ,同时呈Ｃ－带正染。Ｃ－带以着丝点区域正染为主;粗皮角蟾：２ｎ＝２６（２０Ｍ＋６ＳＭ）,５＋８,Ａｇ－ＮＯＲｓ在６ｑ＾ｉｎｔｅｒ,以着丝点Ｃ－带为主,但Ｎｏｓ．１２,１３有明显的端位     

Myelin Basic Protein and Myelin Basic Protein Peptides Induce the Proliferation of Schwann Cells Via Ganglioside GM1 and the FGF Receptor

Myelin basic protein (MBP) and two peptides derived from MBP (MBP_1–44 and MBP_152–167) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of ¹²⁵I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of ¹²⁵I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP_1–44) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP_1–44 and MBP_152–167 associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.     

Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets

The plasma and serum of humans and various animal species exert an actin-depolymerizing activity. Human actin-depolymerizing factor (ADF) has been purified by ammonium sulfate fractionation, DEAE-cellulose and blue-Sepharose chromatography. It is a single polypeptide of approximately 90 kDa, with a pI between 6.0 and 6.5. ADF is heat and trypsin-sensitive, inactivated by EGTA, not stained by HIO4/Schiff on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and not retained on a concanavalin-A-Sepharose column. Incubation of ethanol-fixed cultured cells or unfixed cryostat tissue sections with ADF abolishes immunofluorescent actin staining, by a mechanism which involves extraction of actin from the preparations. ADF promotes fragmentation and depolymerization of actin filaments as shown by electron microscopy, differential ultracentrifugation and DNAse I inhibition assay. This depolymerized actin retains its mobility on SDS/PAGE and is able to repolymerize in the presence of EGTA. Human white blood cells and platelets (but neither human fibroblasts nor white blood cells and platelets from pig, rat and rabbit) contain a 90-kDa protein reacting with an antibody raised in rabbit against human ADF as judged by immunofluorescence and immunoblotting techniques. Immunoblots of human granulocyte subcellular fractions suggest that the protein reacting with ADF antibody is present in the soluble cytoplasmic fraction. ADF may play a role in solubilization of plasma actin and in the intracellular organization of actin, and should be useful for the evaluation of the relative stability of cytoplasmic actin filaments in various physiological and pathological processes.     

Hemodynamic effects of calcitonin in the normal rat.

Calcitonin (CT) was administered acutely (IV 4-8 U/kg) and chronically (SC 2 U/kg/day x 150 day) to normal male rats. Measurements included heart rate (HR), mean blood pressure (MBP), cardiac index (CI), peripheral vascular resistance (PVR), and stroke volume index (SVI). The MBP was higher in CT rats examined under pentobarbital anesthesia. Upon awakening from anesthesia, rats chronically on CT exhibited impaired recovery of CI and SVI. Hemodynamic effects were not seen in rats acutely treated with CT. Heart weight was unchanged in chronic treatment with CT. Therefore, CT had minimal hemodynamic effects in the normal male rat.     

Construction of a tagging system for subcellular localization of proteins encoded by open reading frames

We have previously characterized a monoclonal antibody (SC1D7) that is directed to maltose-binding protein (MBP) of Escherichia coli and other closely related enteric bacteria. SC1D7 does not cross-react with proteins in eucaryotes and appears to be a highly specific tool in immunochemical analyses. To better map the epitope, we took advantage of an available plasmid, pMAL-c2, that encodes the E. coli MBP-coding sequence and constructed plasmids to express MBP fragments. A construct containing the N-terminal portion of MBP does not react with SC1D7, whereas a second construct expressing glutathione S-transferase fused with the C-terminal half of MBP does react with SC1D7. To precisely define the epitope, random peptides displayed on M13 were used to react with SC1D7. Sequences of reactive peptides were aligned, and a consensus sequence of XDXRIPX was deduced. This sequence matches MBP with an amino acid stretch of KDPRIAA. To consolidate the mapping result, a sequence encoding this epitope was inserted into an expression vector and the resulting recombinant protein did react with SC1D7. Thereafter, this epitope was incorporated into a eucaryotic expression plasmid containing a previously defined hepatitis delta virus epitope for protein tagging. This two-epitope-tagging vector is useful in various molecular analyses. We demonstrate its usage for localization of a bacterial virulence factor in host cells. This vector should be applicable for high-throughput characterization of new open reading frames found in genome sequencing.     

Cell-mediated immunoreactivity of tumor-bearing mice with myelin basic protein and related peptides

Spleen cells (SC) from tumor-bearing mice and mice immunized with porcine myelin basic protein (MBP) reacted in vitro in E-rosette augmentation assays with MBP and certain of its constituent peptides. Peptides 1-115, 43-169, 64-83, 113-121, and 153-161 reacted significantly with both types of SC, while peptide 1-19 reacted only with SC from MBP-immunized mice. The phenomenon of specific inhibition of peptide reactivity by a moderate excess of a related protein was used to identify peptides as accessible epitopes of that protein. Peptide 113-121 was specifically inhibited by excess MBP when reacted with both types of SC, whereas peptide 64-83 was inhibited by excess MBP only when reacted with SC from MBP-immunized mice. These reactions suggest that the immunizing antigen in tumor-bearing mice is related to MBP but differs in epitopes associated with peptides 1-19 and 64-83.     

云南景东地区四川湍蛙的核型,C—带和Ag—NORs的研究

本文报道了云南景东四川湍蛙的核型：２ｎ＝２６（２０Ｍ＋６ｓＭ），ＮＦ＝５２，５＋８模式，次缢痕以及Ａｇ－ＮＯＲｓ均位于６ｐｉｎｔｅｒ，Ｃ－带阳性分布于全部染色体的着丝点区域和８ｑｉｎｔｅｒ，但６ｐｉｎｔｅｒ１２ｐｔｅｒ和１３ｑｔｅｒ等处间或也显示Ｃ－带正染。没有发现与性别相关的异形染色体。与采自四川宝兴地区的居群作了比较，并讨论了细胞地理学问题。