禽流感病毒感染食蟹猴肺组织内抗原提呈细胞亚型分析

目的分析禽流感病毒感染的食蟹猴肺组织内抗原提呈细胞的不同亚型。方法收集中国医学科学院实验动物研究所病理室禽流感病毒H5N1亚型感染的食蟹猴肺组织蜡块标本,观察其病理学变化及运用免疫组织化学的方法进一步分析CD68、CD14及S-100在感染禽流感病毒食蟹猴肺组织中的表达。结果禽流感病毒感染的肺组织内肺间隔增宽伴巨噬细胞为主的炎细胞浸润,肺泡腔内可见血浆蛋白及纤维素渗出。部分肺泡间隔断裂、巨噬细胞及淋巴细胞浸润。肺组织内H5N1病毒抗原染色呈阳性。肺间隔及肺泡内可见大量CD68阳性细胞,局部可见散在CD14和S-100阳性细胞。结论禽流感病毒感染的食蟹猴肺组织内抗原提呈细胞主要以巨噬细胞为主。     

青少年食蟹猴糖尿病模型的肝脏病理生理学研究

目的通过对胰岛素用量不足条件下链脲佐菌素诱导的青少年食蟹猴1型糖尿病模型肝脏病理生理学的研究,探讨长期高血糖所致青少年食蟹猴肝损伤特点及机制。方法通过静脉注射68 mg/kg的链脲佐菌素,诱导4只3岁的食蟹猴成为1型糖尿病模型,然后经长期的血糖监测和静脉糖耐量实验来评价该模型的可靠性及稳定性,造模4年后,对模型猴进行血生化、PAS染色、苏丹III染色及普通病理和超微病理等指标的检测,另外选取4只健康与模型猴年龄匹配的猴作为正常对照组,同时进行相应的检测。结果与正常对照组比较,糖尿病猴血清学检测指标中总胆汁酸、尿素氮、谷丙转氨酶、谷草转氨酶、胆碱酯酶、乳酸脱氢酶、总胆固醇、甘油三酯和低密度脂蛋白胆固醇明显升高。组织化学染色结果显示,与正常猴比较,糖尿病猴中央静脉区肝实质细胞肿胀,肝细胞PAS染色（糖原染色）加深,苏丹Ⅲ染色（脂肪染色）阳性细胞增多;电镜结果显示糖尿病猴肝细胞内胞质糖原颗粒增多;线粒体电子密度显著增高,结构不清;窦周隙内含有大量脂滴的肝星状细胞明显增多。结论在长期胰岛素用量不足血糖控制不理想的条件下,青少年食蟹猴1型糖尿病模型肝脏特异性的病理改变是肝糖原贮积和含有大量脂滴的肝星状细胞增生,这些病理改变与非酒精性脂肪肝病的病变特点存在显著不同,但其机制目前尚不清楚。     

鉴别无色素性恶性黑色素瘤方法的探讨

无色素及少色素性恶黑鉴别诊断时,（１）组织化学铁反应,褪色素和黑色素银染对证实瘤细胞中黑色素是有帮助的,但不能从无色素性恶黑中检出黑色素。因此对无黑色素恶黑的诊断和鉴别诊断帮助不大。网状纤维染色良性病均有增加,而恶黑几乎没有增加或仅极少增加。因此网状纤维染色有助于良恶性的鉴别。（２）免疫组化Ｓ－１００１８例恶黑及６例良性痣均显示阳性,这对无色素恶黑的诊断是有价值的,但对色素痣的恶变帮助不大。１６例恶黑中１４例色素性与１０例无色素性恶黑ＨＭＢ４５均显示阳性,证明两者有共同抗原,总阳性率８７．５％,６例良性痣均为阴性,证明无ＨＭＢ４５抗原。结果提示ＨＭＢ４５免疫组化检测不仅对无色素及少色素性恶黑的诊断与鉴别诊断实用性大,还可以用于对恶黑与良性痣、良性痣恶变的鉴别。（３）本组４例无色素性恶黑电镜下均找到前黑色素小体。因此在其他方法诊断困难时,应用电镜检查对确诊具有决定性作用。     

H5N1禽流感病毒感染小鼠和两种灵长类动物的肺组织病理学比较

目的比较H5N1禽流感病毒感染小鼠、恒河猴及食蟹猴急性期肺组织的病理学变化。方法在麻醉状态下对BALB／c小鼠、恒河猴及食蟹猴进行H5N1病毒滴鼻接种,在感染急性期实施安死术,取肺组织运用H＆E结合免疫组化技术分析肺组织的病理变化。结果BALB／c小鼠感染急性期,肺组织以变质性炎为主,肺泡结构被广泛破坏,以单核细胞为主的炎细胞浸润,局部可见渗出性炎。而在恒河猴感染急性期肺组织病理改变以渗出性炎为主,同时可见变质性炎和增生性炎。在食蟹猴感染急性期肺组织病理改变以渗出性和变质性炎为主,同时亦可见上皮的新生。结论H5N1禽流感病毒感染小鼠与恒河猴、食蟹猴急性期肺组织的病理变化不同,这将为进一步认识禽流感的发病机制及研究针对性的治疗方法提供一些理论依据。     

SARS动物模型的研究

利用分离的SARS CoV毒株BJ 0 1,经滴鼻等途径感染大鼠、豚鼠、黑线仓鼠、白化仓鼠和雏鸡等 5个种属的动物 ,筛选对SARS易感的小动物。在此基础上 ,选择食蟹猴和恒河猴进行SARS的人工感染实验 ,评价其作为SARS动物模型的可能性。结果表明 ,大鼠、豚鼠、黑线仓鼠、白化仓鼠和雏鸡等动物对SARS均不易感 ,感染后未观察到任何的临床及病理学改变 ,不过从感染 2周后的大鼠和豚鼠的肺和咽等组织样本中检测到了的特异的核酸 ,提示SARS CoV能够在这两种动物的体内复制。从感染猴子的分泌物和脏器中分离出了病毒 ,证明SARS CoV也能够在猴子体内复制。临床和病理组织学检查结果显示 ,SARS病毒接种食蟹猴和恒河猴后 ,可以引起所有实验猴发生间质性肺炎 ,其病理学改变与人类感染SARS病毒后肺部病变近似 ,但病变的严重程度比较人类的轻得多 ,除此之外无任何其它的明显的临床表现及组织病理学改变 ,按照动物模型的指标判断食蟹猴和恒河猴并不是SARS的理想动物模型 ,不过在目前尚没有更理想的动物模型情况下 ,以间质性肺炎为病理学检查指标 ,恒河猴和食蟹猴可以作为评价抗SARS药物和疫苗的模型动物     

CD34与CD31显示78例非小细胞肺癌微血管密度的对比研究及意义

目的探讨血管内皮标记物CD34与CD31在显示非小细胞肺癌(NSCLC)微血管密度(MVD)中的差异,以及MVD与淋巴结转移的关系.方法采用微波免疫组化染色方法(LSAB)检测CD34与CD31在 78例NSCLS中的阳性表达.结果两种血管内皮标记物CD34与CD31显示的MVD分别是65.8±14.3条和63.5±17.9条,范围分别是18～112条和16～102条,二者差异无显著性(P>0.05);CD34与CD31微血管内皮标记物显示NSCLC的MVD,在转移组分别是69.5±15.7和65.3±14.5,在无转移组分别是44.3±14.3和40.5±12.6,转移组与无转移组间均有非常显著的差异性(P<0.01),而两种微血管内皮标记物之间显示的MVD无显著差异(P>0.05).结论 CD34、CD31相关抗原均能清晰地选择性地显示血管内皮细胞,CD34比CD31因子相关抗原更敏感,能突出显示较小的、不成熟的微血管或单一的内皮细胞;MVD与转移密切相关,可作为预测NSCLC病人预后的指标.     

体外诱导小鼠脂肪来源干细胞向内皮细胞分化的实验研究

目的：探索体外小鼠脂肪来源干细胞（Adipose—derivedstemcells,ASCs）诱导分化为内皮细胞（endothelialprogenitorcells,EPCs）的可行性。方法：利用I型胶原酶消化法和传代培养纯化法从小鼠脂肪组织中分离、培养及扩增ASCs,通过流式细胞仪检测ASCs特异性表面抗原CD29和CD44、CD105的表达;取第2代ASCs进行内皮诱导：即BD基质胶包被＋内皮细胞生长诱导培养基（M199＋10％血清＋10ng／mL血管内皮细胞生长因子VEGF＋10ng／ml碱性生长因子bFGF）。诱导两周左右,倒置显微镜观察诱导前后细胞的一般形态学特征;同时通过成血管实验观察成管腔能力;通过免疫荧光鉴定CD31表型分子的表达。结果：成功培养小鼠ASCs;CD29、CD44、CD90、呈阳性表达,而CD31、CD34、CD45呈阴性表达;诱导后的细胞形态呈三角形或多边形;HE染色观察可见明显的管腔样结构;免疫荧光法CD31表达阳性;MTT法成功记录EPCs增殖生长曲线。结论：成功的诱导小鼠脂肪来源干细胞分化为血管内皮细胞,为后续体内移植实验提供理论基础,同时也为临床受损组织或器官的重建再生提供实验基础。     

强直性脊柱炎食蟹猴膝关节及肠道组织病理变化分析

目的 强直性脊柱炎(ankylosing spondylitis, AS)是一种免疫介导的累及中轴关节、周围关节和肠道的慢性疾病,但外周膝关节和肠道的病因病机未明。本研究旨在研究AS食蟹猴膝关节和肠道主要病理变化及机制。方法 通过组织形态学分析及免疫组化实验,研究AS外周膝关节及肠道组织的关键特征并初步分析其发病机制。结果 AS食蟹猴外周膝关节病理特征在早期主要表现为关节表面软骨侵蚀、软骨下骨暴露、关节表面呈锯齿状;晚期主要表现为软骨表层肥大软骨细胞异位增生,通过软骨成骨和纤维成骨形成骨赘,软骨基本丢失。软骨和血管中MMP-3的表达上调,引起软骨破坏并刺激血管增生。AS食蟹猴小肠绒毛严重萎缩且隐窝增生明显,空肠和回肠的黏膜肠腺中可见大量γδT细胞。结论 本研究通过对AS食蟹猴膝关节和肠道的病理分析,获得了该自发模型膝关节与肠道组织的关键特征,并提出可能的发病机制。为探讨AS的骨骼病变与外周疾病间的潜在关联,以及AS的治疗提供新的见解。     

IL-10在人骨髓间充质干细胞移植治疗食蟹猴脑缺血中的作用

目的探讨食蟹猴脑缺血模型在人骨髓间充质干细胞（human bone marrow-derived mesenchymal stem cells,hBMSCs）移植后IL-10的表达及其对脑缺血损伤的保护作用。方法食蟹猴8只,在脑定位仪定位下,应用光化学法构建食蟹猴脑缺血模型,并将其随机分为高剂量治疗组、低剂量治疗组和模型组,分别在脑缺血部位附近注射高、低密度的hBMSCs和生理盐水。手术后,通过影像学、神经功能评分及组织病理学观察对hBMSC的治疗效果进行评价。并应用原位细胞凋亡检测的方法观察脑缺血周围神经细胞的凋亡情况。应用免疫组织化学、RT-PCR以及real-time PCR法检测检测脑损伤周围IL-10的表达水平。结果与模型组相比,hBMSC治疗组损伤部位周围细胞凋亡明显减少,免疫组织化学显示[L-IO阳性细胞的数量及染色强度均较模型组明显升高,IL-10在mRNA水平表达也明显升高。结论hBMSCs对食蟹猴脑缺血模型具有修复作用,其治疗机制可能与促进炎症抑制因子IL-10的表达有关。     

儿童皮肤肥大细胞瘤的临床病理分析

目的探讨儿童皮肤肥大细胞瘤的临床病理特征。方法对5例皮肤肥大细胞瘤进行临床病理观察和免疫组化检测。结果患者年龄3个月-8岁,3例出生时即有病变,1例在出生后半年内,另一例在7岁时发现病变,均无明显症状,免疫组化示CD43、CD117、LCA、VIM阳性,甲苯胺蓝染色见胞浆内有紫红色颗粒。结论皮肤肥大细胞瘤好发于儿童,大多数在出生时即有或6个月内发病,临床医生易诊断为脉管瘤。组织病理学上关键在于认识肥大细胞,结合免疫组化和甲苯胺蓝染色,并通过询问病史可以确诊。儿童皮肤肥大细胞瘤预后良好,在青春期会自动消退。     

Hypophysial and meningeal melanocytes in the Zucker rat.

Melanocytes have not been described in the pituitary of mammals or in the meninges of the rat. In this paper, we report the presence of the cluster of melanocytes in the intermediate lobe of the pituitary and around the median eminence of the hypothalamus forming an 'infundibulo-hypophysial circle', and also describe the characteristics of meningeal melanocytes in Zucker rats. In the leptomeninges, numerous melanocytes were found on the ventrolateral surface of cerebral hemisphere in the area of the middle cerebral artery. Pigment granules were also observed in the surrounding tissue outside the melanocytes as well as incorporated in the cytoplasm of neural and epithelial cells. Electron microscopy revealed that melanosomes in hypophysial and meningeal melanocytes were in different (II-IV) stages of maturity. In the leptomeninges of Zucker rats, HMB-45 immunoreactivity was found in round non-melanosome-containing cells, while no HMB-45 reaction was found in the leptomeninges of the albino rat. We conclude that both obese and lean Zucker rats possess functionally active melanocytes in the meninges and the pituitary and transfer pigment granules to neighboring cells. The distributions of melanocytes in proximity to blood vessels in the leptomeninges and in the 'infundibulo-hypophysial circle' suggest an endocrine secretory function.     

Hypophysial and Meningeal Melanocytes in the Zucker Rat

Melanocytes have not been described in the pituitary of mammals or in the meninges of the rat. In this paper, we report the presence of the cluster of melanocytes in the intermediate lobe of the pituitary and around the median eminence of the hypothalamus forming an ‘infundibulo-hypophysial circle’, and also describe the characteristics of meningeal melanocytes in Zucker rats. In the leptomeninges, numerous melanocytes were found on the ventrolateral surface of cerebral hemisphere in the area of the middle cerebral artery. Pigment granules were also observed in the surrounding tissue outside the melanocytes as well as incorporated in the cytoplasm of neural and epithelial cells. Electron microscopy revealed that melanosomes in hypophysial and meningeal melanocytes were in different (II-IV) stages of maturity. In the leptomeninges of Zucker rats, HMB-45 immunoreactivity was found in round non-melanosome-containing cells, while no HMB-45 reaction was found in the leptomeninges of the albino rat. We conclude that both obese and lean Zucker rats possess functionally active melanocytes in the meninges and the pituitary and transfer pigment granules to neighboring cells. The distributions of melanocytes in proximity to blood vessels in the leptomeninges and in the ‘infundibulo-hypophysial circle’ suggest an endocrine secretory function.     

Culture and characterization of sinusoidal endothelial cells isolated from human liver

Summary Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.     

The expression of platelet endothelial cell adhesion molecule-1 in mouse primordial germ cells during their migration and early gonadal formation

The platelet endothelial cell adhesion molecule-1 (PECAM-1), or CD31, a member of the immunoglobulin superfamily, is located on the plasma membrane of endothelial and hematopoietic cells and involved in vascular development and inflammation. In this study, by use of immunohistochemistry at light and electron microscopic levels in combination with enzyme histochemistry for alkaline phosphatase, we demonstrated that PECAM-1/CD31 is expressed in the mouse primordial germ cell (PGC). Up to 8 days postcoitum (dpc), PGCs with alkaline phosphatase activity showed no PECAM-1/CD31 immunoreactivity. At 9 dpc, PECAM-1/CD31 immunoreactivity was first detected with low intensity in some PGCs located in the hindgut. Between 10 and 11 dpc, intense immunoreactivity was shown on the entire surface of PGCs migrating along the dorsal wall. After arrival and settlement of PGCs in the genital ridges around 11.5 dpc, the intense immunoreactivity was maintained on the entire surface of PGCs. By electron microscopy, the immunoreactivity was localized exclusively on the plasma membrane of PGCs, being as strong at the portions adjacent to neighboring PGCs as those adjacent to somatic cells. As the male and female gonads began to differentiate, PECAM-1/CD31 immunoreactivity remained strong in germ cells until 13 dpc, after which it gradually decreased in intensity and disappeared by 16 dpc. These results suggested that cell-to-cell interaction through PECAM-1/CD31 plays roles in the development of PGCs during their migration on the dorsal wall and homing in the gonads.     

Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation

Recent research findings postulate that adipocytes and endothelial cells (EC) may share a common progenitor. However, the interlinking pathways between adipose tissue and endothelium, and the differentiation potential of cells to convert from one tissue into the other via progenitor cells have not been elucidated and are therefore the focus of this study. Stromal vascular fraction (SVF) cells were isolated from liposuction aspirates or excised adipose tissue and separated into CD31+ and CD31- populations by magnet-assisted cell sorting. Differentiation to fat tissue was induced in both CD31 fractions after expansion by insulin, dexamethasone, isobutylmethylxanthine, triiodothyronine, pioglitazone, and transferrin. Differentiation was assayed enzymatically and by cell counting. Maturation to endothelium was performed with vascular endothelial growth factor (VEGF), insulin-like growth factor-1 plus 2% fetal calf serum, and confirmed by flow cytometry and tube formation assays on Matrigel. Our results show that the SVF contains a CD31-, S100+ cell type that can differentiate into adipocytes and EC. The SVF also comprises CD31+ cells that, although they have an endothelial phenotype, can be converted into mature adipocytes. These findings demonstrate the potency of SVF cells to perform both adipogenic and endothelial differentiation. Further, they reveal the plasticity of mature cells of mesenchymal origin to undergo conversion from endothelium to adipose tissue and vice versa.     

Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina

The distributions of occludin and claudin-1, two tight junction–associated integral membrane proteins were investigated by immunohistochemical analysis of whole-mount preparations of the blood vessels in the myelinated streak of the rabbit retina. Light microscopy revealed that occludin and claudin-1 immunoreactivities were abundant along the interface of adjacent endothelial cells of all blood vessels. Electron microscopy revealed that both proteins were distributed in a regular pattern (at regular intervals of approximately 80 nm) along the length of tight junctions, probably in the regions of tight junction strands. No other structures or cell types expressed either of these two proteins in the myelinated streak. Whereas occludin immunoreactivity was concentrated only at the tight junction interface, claudin-1 immunoreactivity also extended into the cytoplasm of the endothelial cells, suggesting a different structural role for claudin-1 than for occludin at tight junctions. Retinal pigment epithelial cells expressed occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid. Also consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was expressed at only a low level in, and showed an irregular distribution along, the vessels of the choroid, a vascular bed that lacks blood-barrier properties. Further, the distribution of occludin was examined during formation and remodelling of the rat retinal vasculature. Occludin expression was evident at the leading edge of vessel formation and was found on all vessels in both the inner and outer vascular plexus. Numerous vascular segments at the early stage of vascular formation and regression lost occludin expression. The biological significance of this transient loss of occludin expression in terms of barrier function remains to be elucidated.     

Melanin protects melanocytes and keratinocytes against H2O2-induced DNA strand breaks through its ability to bind Ca2+

Reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H(2)O(2) in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 microM H(2)O(2) increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH(4)Cl and elevated l-tyrosine, H(2)O(2)-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H(2)O(2)-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca(2+)-chelator BAPTA. Thus, BAPTA reduced the level of H(2)O(2)-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca(2+) and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca(2+) binding capacity and, in addition, correlated inversely with H(2)O(2)-induced increases in intracellular Ca(2+). Our results show that melanin may have an important role in regulating intracellular Ca(2+) homeostasis and it is suggested that melanin protects against H(2)O(2)-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca(2+).     

THE FINE STRUCTURE OF NORMAL AND NEOPLASTIC MELANOCYTES IN THE SYRIAN HAMSTER, WITH PARTICULAR REFERENCE TO CARCINOGEN-INDUCED MELANOTIC TUMORS

The dermal melanocyte system of the Syrian hamster is particularly responsive to the melanogenetic and tumor-inducing effects of 7,12-dimethylbenz(a)anthracene (DMBA). The melanocytes of the hair follicles appear to be susceptible to the melanogenetic effect of DMBA but not to its tumor-inducing effect. The epidermal melanocytes are non-pigmented and are unresponsive to both melanogenetic and carcinogenic effects of DMBA. The pigmented granules of the dermal melanocytes of both the golden and the white hamster have an identical substructure and pattern of melanization which occurs in an orderly fashion on a delicate fibrillar component. The hair melanocytes have larger pigment granules with a more complicated fibrillar substructure. The epidermal melanocytes do not possess pigment granules but are recognized by their dendritic shape, the absence of desmosomes and tonofilaments, and the presence of racket-shaped or rod-shaped organelles. The melanin granules in neoplastic melanocytes of the golden hamster differ from corresponding normal melanocytes only in their larger size. In the white hamster, however, the melanin granules in tumors produced under identical experimental conditions are so bizarre and atypical that consideration was given to the possibility that a genetic difference in the melanization pattern between the two varieties becomes apparent in carcinogen-induced melanotic tumors. No definite conclusions could be reached as to the precise origin of the melanin granules in either normal or neoplastic melanocytes.