Dendritic branch typing and spine expression patterns in cortical nonpyramidal cells

To understand the dendritic differentiation in various types of cortical nonpyramidal cells, we analyzed quantitatively their dendritic branching and spine expression. The dendritic internode and interspine interval obeyed exponential distributions with type-specific decay constants. The initial branching pattern, internode interval and spine density at the light microscopic level divided nonpyramidal cells into three dendritic types, correlated with axonal, neurochemical and firing types. The initial branching pattern determined the overall vertical spread of dendrites. Basket cell subtypes with different firing and chemical expression patterns were distinct in the vertical and horizontal spatial spread, providing diverse input territories. Internode densities of dendritic spines, as well as those of axonal synaptic boutons, did not correlate with the tortuosities and intervals, suggesting a tendency to distribute synapses homogeneously over the arbor. Dendritic spines identified at the electron microscopic level were different in length and shape among subtypes. Although the density was lower than that of pyramidal cells, spines themselves were also composed of several morphological types such as mushroom and multihead ones, which were expressed differentially among subtypes. Correlation of dendritic branching characteristics with differences in spine structure suggests distinct ways to receive specific inputs among the subtypes.     

大鼠失神经靶肌肉注射异体不同细胞对神经再生影响的研究

目的研究注射异体不同细胞于大鼠失神经靶肌肉对神经再生的影响.方法 SD成年大鼠36只,雌性,体重120～150 g,随机分为4组,每组9只.无菌条件下切断左侧坐骨神经,一期神经外膜缝合.术后即于小腿三头肌处注射相应细胞,7天注射1次,共4次.A组注射单纯雪旺细胞1×106/ml 1 ml,B组注射雪旺细胞加成肌细胞(雪旺细胞∶成肌细胞为1∶1)1×106/ml 1 ml,C组注射肾内皮细胞提取液1 ml,D组注射无血清培养液1 ml作对照.术后3个月取手术侧坐骨神经及坐骨神经所支配的小腿三头肌,进行大体和组织形态学观察、神经鞘细胞密度及单位面积靶肌肉(小腿三头肌)运动终板计数.结果术后3个月,各组近端神经鞘细胞均数为A组0.134 5±0.029 8,B组0.093 1±0.025 6,C组0.072 4±0.023 7,D组0.187 7±0.054 2;A组∶D组P值<0.05,B组∶D组、C组∶D组及A组∶B组P值均<0.01,B组∶C组P值＞0.05.术后3个月各组远端神经鞘细胞密度均数为A组0.186 0±0.042 5,B组0.155 1±0.032 1,C组0.104 7±0.013 3,D组0.240 9±0.056 8;A组∶D组P值<0.05,B组∶D组、C组∶D组及B组∶C组P值<0.01,A组∶B组P值＞0.05.术后3个月各组靶肌肉运动终板个数均数为A组6.000±0.866,B组9.000±2.291,C组12.780±1.394,D组3.110±0.782;A组∶D组、B组∶D组及C组∶D组P值<0.01,A组∶B组、A组∶C组及B组∶C组P值<0.01.结论雪旺细胞、混合细胞、肾内皮细胞提取液均有促进神经再生作用,肾内皮细胞提取液优于雪旺细胞及混合细胞.     

尿嘧啶脱氧核苷及超顺磁性氧化铁双标记的神经干细胞活体移植后细胞迁徙及功能性分化的实验研究

目的观察尿嘧啶脱氧核苷(Brdu)及超顺磁性氧化铁(SPIO)双标记的神经干细胞活体移植后存活细胞的迁徙及分化。方法SPIO和Brdu对神经干细胞进行双标记,移植至大脑中动脉梗死模型的对侧脑组织,术后采用MRI监测。第6周MRI检查后取组织切片行免疫组织化学染色,观察神经干细胞的迁徙及分化。结果双标神经干细胞活体移植后第3周MRI开始显示细胞沿胼胝体迁徙,第6周脑组织免疫组织化学染色亦显示干细胞沿胼胝体向对侧迁徙,免疫组织化学荧光双标染色显示移植的神经干细胞可以分化为星形胶质细胞及神经元。结论移植存活的神经干细胞在迁徙过程中能够进行功能性分化。     

大鼠肝窦内皮细胞分离、培养及其生物学特性的初步研究

目的　建立大鼠肝窦内皮细胞 (SEC)的原代分离、培养方法 ,并研究其生物学特性。方法　胶原酶灌注结合Percoll密度梯度离心加选择性贴壁的方法分离SEC ;用光镜、扫描电镜观察培养SEC的形态学变化及超微结构 ;用Ⅷ因子和CD14免疫细胞化学染色及RECA 1单抗间接免疫荧光法观察SEC表面分子的表达。结果　分离培养的SEC得率为 (2 .0 6± 0 .35 )× 10 7/只大鼠 ,活力≥ 92 % ,纯度达 90 % ;光镜下细胞形态典型 ,SEM下可观察到特征性的窗孔结构 ;Ⅷ因子染色阴性而CD14染色阳性 ,RECA 1单抗间接免疫荧光染色阳性。结论　分离培养的SEC细胞得率、活力及纯度较高 ,形态典型且具有一般细胞所没有的窗孔结构及表面标志 ,为其功能和作用机制的深入研究奠定了基础。     

肝干细胞的研究进展及应用前景

目的阐述肝干细胞（liver stem cell，LSC）的研究进展并展望其应用前景。方法广泛查阅近年来相关文献，根据研究成果，综述LSC研究的最新进展。结果LSC向特定的细胞分化和增殖受多种因素影响。LSC活化、分离培养、筛选及鉴定等方法尚未成熟。结论随着研究不断地深入，LSC有望成为肝病治疗的新型种子细胞，但其研究有待进一步完善。     

骨质疏松小鼠模型骨髓间充质干细胞特性的研究

目的 探讨骨质疏松小鼠模型骨髓间充质干细胞（mesenchymal stem cells ,MSCs）生物学特性的改变。方法 通过皮下注射地塞米松构建小鼠骨质疏松动物模型（OP组）,同时设立对照（NC组）,分离OP组及NC组骨髓MSCs进行体外培养;通过流式细胞术检测细胞表面分化抗原;运用CKK-8实验检测细胞增殖活力;通过脱氧核苷酸末端转移酶介导的dUTP缺口末端标记法（TUNEL）检测细胞凋亡水平;运用碱性磷酸酶（ALP）活性检测、油红O染色及Western blot法检测两组细胞成骨分化及脂肪分化能力。结果 经过骨组织学检测证明模型建立成功,体外培养OP组及NC组骨髓MSCs细胞形态无差异。OP-MSCs细胞表面分化抗原Scal1及CD44为阳性, CD34及CD11b为阴性,与NC-MSCs相似,但OP-MSCs增殖活力下降（P<0.05）;此外两组细胞凋亡水平类似（P>0.05）。成骨分化诱导7 d后OP-MSCs的ALP活性显著低于对照组（P<0.01）,21 d后矿化小结明显减少（P<0.001）;脂肪分化诱导8 d后OP-MSCs形成更多脂滴（P<0.05）。Western blot结果显示OP-MSCs在成骨分化中低表达转录因子Runx2（P<0.05）及Osterix (P<0.001）,但在脂肪分化早期高表达PPARγ（P<0.001）及C/EBPα（P<0.01）。结论 骨质疏松小鼠骨髓MSCs的增殖及分化潜能显著下降。     

兔雪旺细胞的培养方法及形态学研究

利用兔坐骨神经组织进行体外细胞培养研究，获得了雪旺细胞。兔雪旺细胞的形态与其他文献报道的鼠或人的外周神经培养获得的雪旺细胞形态基本相似，典型的雪旺细胞形态特征为胞体较小、梭形、两侧突起纤长、核窄长。倒置显微镜下看，胞体四周常有亮边，细胞成极性生长排列。通过免疫组织化学染色证实具有典型形态的梭状细胞为雪旺细胞，而扁平状形态不规则的细胞为成纤维细胞     

Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model

INTRODUCTION: The phenotypic plasticity of the human prostate stem cell within human prostate tissue was examined to determine the response of the stem cell to changes in the androgenic environment. METHODS: Prostate xenografts were transplanted into athymic nu/nu mice implanted with testosterone pellets, allowed to establish for 1 month time point, the hosts were castrated and pellets removed, and following 1 month of androgen deprivation, the hosts were stimulated with androgen for 2 days to induce proliferation of the residual population of stem cells (2-month time point). RESULTS: Glands in benign xenografts harvested at the 1- and 2-month time points contained basal cell layers that expressed p63 and high molecular weight cytokeratin, and in which essentially all of the cellular proliferation was localized, consistent with the proposed localization of the prostate stem cell. Benign glandular structures in the xenografts were populated by basal, secretory epithelial, neuroendocrine (NE), or squamous cells overlaying the basal cell layer, whereas, adenocarcinoma glands in the xenografts resembled the original prostate cancer (CaP) tissue. CONCLUSIONS: In this human prostate primary xenograft model, the residual stem cell population that survives transplantation, or androgen deprivation, maintains significant pluripotentiality as demonstrated by the capacity to generate progeny that differentiate along multiple lineages in response to microenvironmental signals, particularly along the secretory epithelial lineage in response to androgen, and along the NE cell lineage in response to androgen deprivation.     

肿瘤干细胞的研究进展

肿瘤具有异质性的特征，随着研究的深入，越来越多的证据提示肿瘤组织中也存在少量具有干细胞性质的肿瘤干细胞，于是人们在此基础上提出了肿瘤干细胞学说，肿瘤干细胞已成为当今肿瘤研究领域的热点。目前，已经从白血病、脑肿瘤、乳腺癌、恶性黑色素瘤及前列腺癌等多种恶性肿瘤中初步分离鉴定出肿瘤干细胞。肿瘤干细胞学说的提出，使得靶向性杀伤肿瘤干细胞从而根治肿瘤成为可能。     

肝干细胞与肝癌关系的研究现状与临床价值

目的探讨肝干细胞与肝癌的关系及其临床应用前景。方法对国内、外相关研究成果进行回顾性分析。结果肝癌可能是肝干细胞分化不全或分化异常造成并由各种不同分化等级的细胞组成的。结论肝干细胞假说正逐渐得到广泛认同,对其的不断深入研究对揭示肝癌的细胞起源,研究肝癌的发生、转移机理等有重要意义。     

干细胞治疗勃起功能障碍研究进展

勃起功能障碍(ED)的发生与血管内皮功能障碍及相关神经的损伤有关。近年来,干细胞对阴茎勃起神经和海绵体血管内皮细胞修复保护作用的临床前研究已成为研究的热点。早期研究显示干细胞或基因修饰的干细胞对ED治疗持久有效,并有可能成功治愈ED。间充质干细胞、肌源性干细胞、胚胎干细胞、脂肪源性干细胞、内皮祖细胞等均具有不同的分化潜能,在内皮细胞的更新、修复及神经组织细胞的保护方面有各自的优势。干细胞有望用于人类ED的治疗。     

猪肾小球内皮细胞与系膜细胞的发育过程及相互关系

目的:明确中国实验用小型猪肾小球内皮细胞与系膜细胞的发育过程及相互关系。方法:采集不同时间点(胚胎28～112d及出生后1d、7d、14d、21d)中国实验用小型猪肾组织,应用免疫荧光技术检测胚肾发育不同阶段(帽状间充质、肾小囊体、逗号形体、"S"形体、毛细血管袢期肾小球及成熟肾小球)内皮细胞标志物CD31与系膜细胞标志物α-SMA的表达情况。结果:内皮细胞标志物CD31在胚肾早期呈散在性分布,继而围绕肾小囊体和逗号形体呈"环抱"状分布,然后进入"S"形体下端的血管裂隙内聚集形成无管腔的"前毛细血管束",最后表达于成熟肾小球毛细血管内皮细胞。系膜细胞标志物α-SMA在早期胚肾、肾小囊体和逗号形体阶段均无表达;"S"形体早期分布于"S"形体周围,后期进入"S"形体下端的血管裂隙;毛细血管袢期聚集在肾小球血管极根部;随着肾小球发育逐渐向肾小球内延伸,最终表达于成熟肾小球系膜区。CD31与α-SMA双重染色的结果显示,在毛细血管袢期CD31阳性的内皮细胞聚集形成无管腔的前毛细血管束,而α-SMA阳性的系膜细胞聚集在肾小球血管极的根部;随着肾小球发育,α-SMA阳性的系膜细胞逐渐由血管极根部向肾小球内迁移,同时CD31阳性的内皮细胞逐渐形成带有管腔的毛细血管丛。结论:中国实验用小型猪肾小球内皮细胞的发育开始于后肾间充质阶段,系膜细胞的发育开始于"S"形体阶段,即肾小球系膜细胞发育在内皮细胞之后;在肾小球血管丛形成过程中,内皮细胞与系膜细胞间可能存在重要的相互作用。     

睾丸间质干细胞分化和移植

睾丸间质细胞是男性体内合成雄激素的主要细胞,胚胎发育期中肾胚的间质细胞及生精小管周成纤维样细胞可能是睾丸间质细胞的干细胞。在胚胎期间质干细胞分化为胎儿型间质细胞;出生后间质干细胞经间质祖细胞、未成熟间质细胞分化为成熟间质细胞。老年期间质细胞数量可能不变,但雄激素合成下降。间充质干细胞及脂肪干细胞等干细胞经诱导可分化为分泌雄激素的睾丸间质细胞,因此,间质干细胞移植可望成为治疗男性性腺功能不全和中老年雄激素缺乏的创新方法,本文对睾丸间质干细胞的分化及移植方面研究进行综述。     

Effects of fatty acids and eicosanoid synthesis inhibitors on the growth of two human prostate cancer cell lines

Dietary fatty acids (FAs) may be involved in the carcinogenic process within the prostate gland and progression to clinically manifest disease. We have shown that growth of the androgen-unresponsive PC-3 human prostate cancer cell line is stimulated in vitro by the presence of linoleic acid (LA), an omega-6 polyunsaturated FA. The response was positively related to the FA concentration over the entire range examined (5-750 ng/ml). Conversely, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), two omega-3 FAs present in fish oils, inhibited PC-3 cell growth in a dose-dependent manner; both were equally effective, with an approximately 65% reduction in growth occurring at a concentration of 2.0 micrograms/ml (P less than 0.001). The DU 145 human prostate cancer cell line, which is also androgen-unresponsive, showed no growth response to LA and was less susceptible to growth inhibition when cultured in the presence of omega-3 FAs. Growth experiments with indomethacin, esculetin, and piroxicam, pharmacological inhibitors of eicosanoid biosynthesis with differing sites of action, indicated that human prostate cancer cell growth requires intact metabolic pathways for both leukotriene and prostaglandin production.     

三维及二维培养大鼠胰腺导管来源干细胞的体外对比实验研究

目的观察在不同培养模式下大鼠胰腺导管来源干细胞(PDSC)的生物学特性。方法以大鼠胰腺组织为材料,采用动力性三维培养及传统二维培养方法进行细胞培养,获得原代PDSC,并连续传代,纯化PDSC,比较动力性三维培养及传统二维培养细胞间连接及细胞凋亡周期的差异。结果动力性三维培养的大鼠PDSC在培养第2天即可沿三维支架贴壁生长,在第7天即可形成细胞团,并沿三维支架多向贴壁生长;二维培养的PDSC在培养第5天贴壁生长。三维培养与二维培养相比,二维培养的细胞间连接少见,三维培养的细胞连接结构丰富。三维培养与二维培养相比,三维培养的G1期细胞增多,G2期细胞相对减少(P<0.01),三维培养早期自发凋亡细胞比例相对减少(P<0.05),晚期自发凋亡与坏死细胞比例两种培养方式间的差异无统计学意义(P>0.05)。结论动力性三维细胞培养较传统二维细胞培养,在细胞贴壁时间、细胞连接以及细胞凋亡方面具有明显的优势。     

Antitumor Killer Lymphocytes in the Peripheral Blood of a Patient with Transitional Cell Carcinoma of the Bladder

Background: Peripheral blood lymphocytes (PBL) from patients with bladder cancer also contain cells possessing cytotoxic activity against autologous tumor cells. These cells are phenotypically heterogenous and include natural killer (NK) and cytotoxic T cells. This study investigated the role of cytotoxic lymphocytes directed against autologous bladder cancer cells.
Methods: PBL were obtained at intervals before and after surgery and analyzed for cytotoxic activity against autologous bladder cancer cells in 4-hour⁵¹ Cr release assay. PBL stimulated with autologous tumor cells were also transformed with human T-lymphotropic virus type-1, establishing a cell line (KB31) which was analyzed for phenotype and cytotoxic activity against the autologous tumor cells.
Results: PBL preoperative cytotoxic activity was low, but increased after surgery. Cytotoxic activity was found not only against autologous bladder cancer cells, but also against heterologous bladder cancer (KK-47) and myeloid leukemia (K562) cells, with the highest activity against the heterologous cell lines. The cytotoxic activity of KB31 was 40|X% against autologous tumor cells 6 weeks after initiation of the cell line, but decreased to 5|X% by 6 months. This activity was lower than that against the other cell lines, and was similar to that of PBL in short-term culture. Fluorescence-activated cell sorter (FACS) analysis demonstrated that in KB31 cells at 6 weeks, CD8+ cells were dominant, but CD56+ cells predominated at 6 months.
Conclusion: These results suggest that the presence of cytotoxic activity in the peripheral blood of the patient was due to both cytotoxic T cells and NK cells. The cytotoxic activity was lowest prior to surgery and increased postoperatively.     

不同接种密度对人表皮细胞培养融合的实验研究

目的:研究不同密度接种对人表皮细胞体外培养融合的影响。方法:外科切取无菌皮肤或包皮环切手术标本,经两步酶消化处理后,制备单个表皮细胞悬液。以不同细胞密度接种于含有3T3滋养细胞的培养瓶中,实验室体外原代培养扩增。结果:观察不同接种密度表皮细胞的体外培养增殖,结果表明人表皮细胞具较高的生长、增殖、融合能力。2×106cell/75cm2培养瓶密度接种人表皮细胞于含3T3滋养细胞培养瓶中,表皮细胞贴壁良好,细胞融合时间较短。结论:鼠3T3细胞能促进人表皮细胞贴壁,仍不失为较好的表皮细胞培养滋养层。适宜的表皮细胞接种密度为2×106cell/75cm2培养瓶。     

Stem cell‐mediated accelerated bone healing observed with in vivo molecular and small animal imaging technologies in a model of skeletal injury

Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose‐derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [¹⁸F]FHBG (9‐(fluoro‐hydroxy‐methyl‐butyl‐guanine)‐micro‐PET, [¹⁸F]Fluoride ion micro‐PET and micro‐CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC's traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro‐CT measurements showed increased bone healing in the cell‐injected group compared to the noninjected group at postoperative day 7 (p < 0.05). Systemically administered ADMC's traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue‐derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:295–302, 2009