Distribution of platelet-derived growth factor-alpha receptor expressing oligodendrocyte precursor cells in the adult rat brain

BACKGROUND： Studies have demonstrated that NG2-positive glial cells in the adult rats are predominantly located in the gray and white matter of the cerebral cortex and hippocampus. Platelet-derived growth factor-a receptor （PDGF-αR） cells are a subset of oligodendrocytes, which are not as mature as NG2-positive cells. Distribution and migration of PDGF-αR-positive cells in the rat brain remain poorly understood. OBJECTIVE： Using immunohistochemical methods, the distribution of oligodendrocyte precursor cells （PDGF-αR-positive） was analyzed in the adult rat brain. DESIGN, TIME AND SETTING： Immunohistochemical study was performed at the Department of Histology and Embryology of the Third Military Medical University from September 2007 to September 2008. MATERIALS： Rabbit anti-PDGF-αR polyclonal antibody was purchased from Santa Cruz Biotechnology, USA. Streptomycin-avidin-biotin complex immunohistochemistry kit was purchased from Zhongshan Goldenbridge Biotechnology, China. METHODS： Whole brains from 5 healthy, adult, Wistar rats were collected for immunohistochemistry, and the mean value of PDGF-αR-expressing cells was quantified. The absolute values were translated to ranked data of high, moderate, and low grades （high grade： 10 positive cells; moderate grade： 5-9 cells; low grade： 〈 5 cells in a 400 × visual field）. Based on the number of cell processes and branches, as well as the number of PDGF-αR-positive cells, in different regions, the cells were classified into three categories, i.e., type Ⅰ-Ⅲ. From type I to type Ill, the number of processes gradually increased. MAIN OUTCOME MEARSURES： The number and distribution of PDGF-αR-positive cells in different brain regions of adult rats. RESULTS： PDGF-αR-positive cells were located in the forebrain and midbrain, but not in the cerebellum or brainstem. In the olfactory bulb and hippocampus, a total of 60% PDGF-αR-positive cells were type Ⅰ and these cells were not mature as others. In the cerebral cortex, olfactory system, hippocampus, and optic chiasma, where neuronal bodies aggregated, approximately 40% of the PDGF-αR-positive cells were type Ⅱ, with few type Ⅲ cells. In the white matter, corpus callosum, basal nucleus, and thalamus, PDGF-αR-positive cell density was moderate. In the olfactory bulb and hippocampus, PDGF-αR-positive cell density was high. PDGF-αR-positive cells were not observed in the cerebellum or brainstem CONCLUSION： PDGF-αR-positive cells were aggregated in the olfactory bulb and hippocampus in the adult, rat brain, but few cells were detected in the cerebellum and brainstem.     

经尾静脉注入大鼠骨髓间充质多能成体祖细胞向脑缺血灶微环境中的移行及效应

BACKGROUND： Multipotent adult progenitor cells （MAPCs） from the bone marrow have been shown to differentiate into neurons.
OBJECTIVE： To observe migration, survival, and neuronal-like differentiation of MAPCs by tail vein injection. DESIGN, TIME AND SETTING： Randomized, controlled experiment of neural tissue engineering was performed at the Laboratory for Cardio-Cerebrovascular Disease, Hospital of Integrated Traditional and Western Medicine, Tongji Medical College of Huazhong University of Science and Technology between September 2006 and August 2007. MATERIALS： Eighty Sprague Dawley rats, 3-6 months old, underwent cerebral ischemia/reperfusion by thread technique, and were randomly divided into model and MAPCs groups （n = 40）. METHODS： Mononuclear cells were harvested from bone marrow using the FicolI-Paque density gradient centrifugation method. After removing CD45 and glycophorin A-positive cells （GLYA＋） with immunomagnetic beads, CD45 GLYA adult progenitor cells were labeled with bromodeoxyuridine （5-bromo-2-deoxyuridine, BrdU）. A total of 1 mL cell suspension, containing 5 × 10^6 MAPCs, was injected into the MAPCs group through the tail vein. A total of 1 mL normal saline was injected into the model rats.
MAIN OUTCOME MEASURES： After 60 days, BrdU and neuron-specific enolase double-positive cells were observed using immunofluorescence. Cell morphology was observed under electron microscopy, and nerve growth factor mRNA was measured through RT-PCR. In addition, rat neurological functions were measured with behavioral tests.
RESULTS： Immunofluorescence revealed that MAPCs positive for BrdU and neuron specific enolase were found surrounding the ischemic focus in the MAPCs group. Microscopic observation suggested that MAPCs-derived neuronal-like cells connected with other nerve cells to form synapses. Compared with the model animals, the level of nerve growth factor mRNA was significantly upregulated in rats injected with MAPCs （P 〈 0.05）. In addition, rats in the MAPCs group performed better in behavioral tests than the model group on days 28 and 60 （P 〈 0.05）.
CONCLUSION： Transplanted MAPCs migrated to the ischemic region, survived, and differentiated into neuronal-like cells, resulting in stimulation of nerve growth factor mRNA and improved neurological function in ischemic rats.     

Lewis鼠视网膜脉络膜巨噬细胞、MHC－Ⅱ类抗原阳性细胞的免疫组织化学研究

在视网膜平片、脉络膜－巩膜复合体平片及眼组织切片上进行了免疫组织化学研究，在平片上发现，视网膜和脉络膜内均有网状分介的巨噬细胞，脉络膜内尚有网状分布的ＭＨＣ－Ⅱ类抗原阳性细胞，细胞的形态、密度容易辨认和计算。切片则未能显示组织中细胞的分布、确切的形态特征及密度。说明在组织平片上进行免疫组化研究具有重要价值。本研究结果并提示：巨噬细胞、ＭＨＣ－Ⅱ类抗原阳性细胞在维持视网膜和脉络膜免疫环境的稳定性和防御感染等方面可能起重要作用．     

骨髓基质细胞向神经样细胞的分化

目的:有研究表明,在诱导剂β-巯基乙醇或二甲基亚砜与丁羟茴醚的作用下,80%骨髓基质细胞可诱导分化成神经样细胞,并表达神经元特异性蛋白,一些神经营养因子也能诱导骨髓基质细胞分化为神经样细胞.实验拟以三磷酸胞苷二钠为诱导剂,验证其诱导骨髓基质细胞分化为神经样细胞的可行性.方法:实验于2006-03/12在白求恩国际和平医院实验室完成.[1]实验材料:SD大鼠,体质量50～60g,雌雄不限,由河北医科大学实验动物中心提供,实验过程中对动物处置符合动物伦理学标准.[2]实验方法:分离大鼠骨髓基质细胞进行体外培养,传代至第3代时实验组加入三磷酸胞苷二钠诱导剂诱导,对照组不加诱导剂.[3]实验评估:观察细胞分化为神经样细胞的形态变化,并进行免疫组织化学染色鉴定,计算阳性细胞百分比.结果:刚分离的骨髓基质细胞呈圆形,胞体透亮.接种24h后,少量细胞贴壁,72h后大部分细胞贴壁,形态为梭形、圆形、多角形,8～10d细胞达到90%融合,细胞呈长梭形排列呈栅栏状,传代后的细胞约24h完全贴壁,3～7d基本铺满瓶底.诱导后的骨髓基质细胞胞体变圆,突起逐渐伸长,并互相连接成网状,10d后免疫组织化学染色鉴定实验组表达神经元特异性烯醇化酶、神经胶质纤维酸性蛋白阳性细胞百分比明显高于对照组.结论:三磷酸胞苷二钠在体外可诱导骨髓基质细胞分化为神经样细胞.     

人神经干细胞移植到缺氧缺血性脑损伤新生大鼠脑内的存活及分布

目的:新生儿缺氧缺血性脑病是导致脑性瘫痪的重要原因,至今缺乏有效疗法.将体外培养的人神经干细胞经脑室移植入缺氧缺血性脑损伤新生鼠,观察植入细胞在宿主脑内的存活、迁移及分化.方法:实验于2005-01/09在解放军海军总医院儿科实验室完成.①对象:神经干细胞来源于孕12周流产的人胎儿脑组织,孕妇签署知情同意书,符合医院伦理委员会规定.清洁级SD新生鼠80只,随机数字表法分为细胞移植组、模型对照组,40只/组,实验过程中对动物的处置符合动物伦理学标准.②实验方法:取人胚胎脑组织,机械分散法分离单个核细胞,接种于添加表皮生长因子、碱性成纤维细胞生长因子、白血病抑制因子的N2培养基中,获取生长旺盛的人神经干细胞球,制成单细胞悬液,浓度约为5.0×1011L-1,培养6 d后行PKH标记用于植入后示踪.两组新生鼠均建立缺氧缺血性脑损伤模型,造模后3 d,细胞移植组损伤侧脑室缓慢注入5 μL人神经干细胞悬液,模型对照组于相同部位注入等量生理盐水.③实验评估:取未经PKH标记的细胞球, 通过免疫细胞化学染色鉴定巢蛋白的表达及其向神经元、星形胶质细胞的分化情况.分别于细胞移植后1,2,4周及3个月,常规取脑组织,行免疫组织化学和荧光分析,观察植入后细胞存活及分布情况.结果:在造模及细胞移植过程中,因麻醉、出血细胞移植组新生鼠死亡5只,模型对照组死亡7只,存活率85%～90%.①神经干细胞的鉴定及分化:80%活细胞巢蛋白呈阳性表达,并可分化为神经元、星形胶质细胞.②神经干细胞植入后存活及分布情况:植入后1周,神经丝蛋白阳性细胞多位于损伤侧皮质及海马处,纹状体、脑干、小脑、嗅球也有少量分布.植入后2周,海马和皮质可见神经丝蛋白阳性细胞,胞体伸出的神经微丝更长,细胞数量与1周时基本相似.植入后4周及3个月时PKH阳性细胞数量明显减少.结论:在含有表皮生长因子、碱性成纤维细胞生长因子、白血病抑制因子的N2培养基中形成的人神经干细胞球,具有良好的增殖能力,可分化为神经元,移植至缺血缺氧新生鼠脑中能够向损伤区迁移,分布范围广.     

质粒PLXSN介导bcl-2基因对癫痫大鼠海马神经细胞凋亡的保护作用

选择28只雄性Wistar大鼠随机分为正常对照组、生理盐水组、空质粒组和bcl-2组.24 h后用红藻氨酸(KA)局部注射诱导癫痫模型,以TUNEL方法检测凋亡细胞在大鼠海马CA3区的表达,采用免疫组织化学方法和半定量RT-PCR技术检测bcl-2蛋白及bcl-2 mRNA的表达.结果 显示,生理盐水组与空质粒组CA3区有大量凋亡细胞,给予PLXSN-bcl-2基因后凋亡细胞数明显减少.与空质粒组比较,bcl-2组海马CA3区bcl-2阳性细胞百分比及bcl-2 mRNA表达均明显升高.认为质粒PLXSN介导bcl-2基因转入脑内,对KA致癫痫大鼠海马神经元凋亡有抑制作用,起到脑保护作用.     

Expression of Hsp70 and Caspase-3 in rabbits after severe traumatic brain injury

ObjectiveTo investigate the expression of Caspase-3 and Hsp70 in rabbits after severe traumatic brain injury (TBI) and to explore the feasibility of its application in estimation of injury time in forensic medicine.MethodsA rabbit model of heavy TBI was developed by high velocity impact on the parietal bone with an iron stick. Totally 8 healthy adult New Zealand white rabbits were randomly divided into control group (n=2) and injury group (n=6). Four hours after injury, tissue specimens from the parietal lobe, temporal lobe, occipital lobe, cerebellum and brainstem were harvested to detect the expression of Hsp70 and Caspase-3 by immunohistochemistry. Besides, the gray values of cells positive for Hsp70 and Caspase-3 were analyzed with an image analyzer.ResultsImmunohistochemistry staining demonstrated a low level of Caspase-3 and Hsp70 expression in normal control group. While in injury group, both the Caspase-3 and Hsp70 expression was significantly elevated (P<0.05). Positive cells gathered around the lesion focus. Occipital lobe and cerebellum had fewer positive cells while temporal and brainstem had the fewest.ConclusionThe expression of Caspase-3 and Hsp70 at an early stage following severe TBI is characteristic and can be applied to estimate the time of injury.     

c-IAP1 MCM2 P-gp对宫颈鳞癌新辅助化疗疗效的预测价值

宫颈癌是女性最常见的生殖系统恶性肿瘤,发病率逐年上升且年轻化趋势明显。宫颈癌新辅助化疗（NACT）,能有效缩小肿瘤体积和转移范围,降低肿瘤的临床分期,使原本没有手术机会的患者可能重新获得手术机会,从而保留患者的卵巢或阴道功能。同时可有效杀灭亚临床病灶的肿瘤细胞,减少转移和局部复发。但并非所有的患者通过化疗均能获得手术的机会,对于化疗效果不够理想,结束时不能达到手术要求的患者,宜尽快放疗。故寻找预测NACT疗效的理想指标,早期准确评估NACT的有效性颇为重要。