外源EGFP和hTH基因在恒河猴骨髓源神经干细胞内的稳定表达

目的构建携带人酪氨酸羟化酶(hTH)的荧光真核表达质粒-pEGFP-C2-hTH,转染骨髓基质细胞源神经干细胞(BMSCs-D-NSCs),观察外源EGFP和hTH基因在BMSCs-D-NSCs中的表达情况。方法应用基因重组技术,将pWAV2-TH中的TH目的基因亚克隆到荧光真核表达载体 pEGFP-C2,以酶切和测序鉴定重组质粒pEGFP-C2-hTH的正确性:pEGFP-C2-hTH经NucleofectorTM 核转染仪转染培养的恒河猴BMSCs-D-NSCs,24 h后观察绿色荧光蛋白的瞬时表达情况,10 d后行 TH单克隆抗体的免疫组化和TH基因的RT-PCR。结果 (1)酶切、PCR和DNA序列鉴定均证实插入片段的正确性;(2)细胞转染24 h后,荧光显微镜下可观察到绿色荧光蛋白(GFP)的表达,观察到 80％的转染细胞发出绿色荧光;转染10 d后细胞的RT-PCR检测到hTH基因的表达,TH单克隆抗体免疫组化结果显示转染细胞呈阳性染色,同时在荧光显微镜下观察到绿色荧光。结论构建的 hTH荧光真核表达重组质粒pEGFP-C2-hTH,经电转染方法转染至BMSCs-D-NSCs内,成功表达hTH 和EGFP,为BMSCs-D-NSCs基因治疗提供了实验基础。     

人类GAD67基因转染骨髓基质源性神经干细胞及初步检测

目的利用转基因技术将人类谷氨酸脱羧酶（GAD）67基因转染骨髓基质源性神经干细胞仍MSCs-D-NSCs），获得GAD67修饰的成体专能干细胞。方法以PCR、双酶切及测序鉴定pEGFP-C2-GAD67和pcDNA3.1-GAD67重组子；利用密度梯度离心法分离培养大鼠BMSCs并促其向NSCs方向分化，将编码人类GAD67基因的真核表达质粒pEGFP-C2-GAD67及pcDNA3．1-GAD67分别转染BMSCs-D-NSCs，荧光显微镜下观察并用流式细胞仪检测转染效率。结果pEGFP-C2-GAD67及pcDNA3.1-GAD6，均可扩增出约1800bp目的条带，经双酶切和基因测序证实。荧光显微镜下观察可见大量绿色荧光细胞，两种载体转染效率分别为39.3％和40.5％。结论pEGFP-C2-GAD67及pcDNA3.1-GAD67真核表达载体重组正确，利用脂质体介导人类GAD6，基因能有效转染BMSCs-D-NSCs，为进一步观察其GAD67基因和蛋白的表达提供了前提条件。     

重组大鼠质粒pEGFP-GDNF的构建及大鼠胚胎神经干细胞转染的研究

目的 探讨携带GDNF基因的神经干细胞表达载体的构建方法。方法 采用RT-PCR方法从大鼠胎脑组织总RNA中扩增出该基因的全序列cDNA，并克隆到增强型绿色荧光蛋白（EGFP）报告基因的真核表达载体pEGFP-C1中，经酶切鉴定及测序分析对重组质粒pEGFP-GDNF作进一步鉴定。采用阳离子脂质体将重组质粒pEGFP-GDNF转染至鼠胚胎神经干细胞。结果 大鼠GD-NF cDNA已正确地克隆到真核表达载体pEGFP-C1中，而构建成重组大鼠质粒pEGFP-GDNF，GDNF基因在细胞中可稳定表达。结论 神经干细胞可直接作为基因靶细胞，能被GDNF真核细胞表达载体pEGFP-GDNF有效的感染。     

帕金森病脑内移植基因工程细胞系的构建

目的 为进行帕金森病（PD）的转基因细胞治疗，在实验室构建人类酪氨酸羟化酶（hTH）基因表达工程细胞系。方法 重组质粒pcDNA3/hTH经亚克隆、纯化后，用脂质体法转染到人类神经母细胞瘤细胞系SH－SY5Y细胞内，经G418筛选、克隆，分离培养经免疫细胞化学分析。结果 转染pcDNA3/hTH的SH－SY5Y细胞免疫细胞化学染色明显，与仅呈非特异性染色的对照组SH－SY5Y细胞形成鲜明的对比。结论 转染pcDNA3/hTH的SH－SY5Y细胞能够有效地表达hTH，在PD治疗的非人类灵长目实验中具有潜在的应用价值。     

TH基因修饰细胞脑内移植治疗猴帕金森病的实验研究

目的 评价包囊化酪氨酸羟化酶（tyrosine hydroylase,TH）基因修饰的基因工程细胞脑内移植治疗帕金森病的疗效。方法 将pcDNA3/hTH质粒转染人神经母细胞瘤细胞系SYTY细胞，筛选出阳性克隆，微包囊化处理后的含有TH基因修饰细胞植入PD猴模型脑内，观察其行为、CSF中DA含量的变化，用免疫组化法检查移植细胞的存活情况。结果 （1）pcDNA3/hTH基因经亚克隆，提取纯化的质粒，经ECORI酶切后产生1.9Kb和5.5Kb的片段。转基因后的SY5Y细胞免疫细胞化学染色显示TH染色强阳性；（2）移植后PD猴症状明显改善，脑脊液中DA含量升高；（3）SABC免疫组化发现移植区存在大量TH阳／性细胞。结论 构建的TH基因工程细胞体外和体内均表达人类TH基因；微包囊化处理后的基因工程细胞在PD猴脑内存活并发挥治疗作用。     

大鼠骨髓间充质干细胞转染人TH基因的实验研究

目的评价酪氨酸羟化酶(tyrosine hydroxylase,TH)基因修饰骨髓间充质干细胞(mesenchymalstem cells,MSCs)后TH的表达情况及转染TH基因对MSCs的影响.方法构建含人TH基因的pCMV/hTH质粒,用脂质体转染原代培养的大鼠MSCs;Western blot及免疫细胞化学染色鉴定TH基因的表达及MSCs的分化情况;MTT比色法测定转染后的MSCs细胞活性,并与未转染的MSCs进行细胞活性比较.结果构建的pCMV/hTH质粒经ECoRI酶切后产生1.9okb和5.3kb的片段,与回收的目的基因及载体基因片段大小相符;转基因后的MSCs Western blot及免疫细胞化学染色显示TH染色阳性;转染后MSCs未见有NeuN,GFAP的表达;MTT比色法测定细胞活性,未转染与转染者差异无显著性.结论构建的TH基因能在体外培养的鼠MSCs中较好的表达,转染不会诱导MSCs向神经样细胞分化,对MSCs细胞活性无明显影响.     

SOX-9和胰岛素样生长因子1共同转染大鼠骨髓间充质干细胞

背景：单基因诱导虽能对骨髓间充质干细胞向软骨细胞分化产生积极影响,但作用有限,多种基因共同作用才符合机体真实内环境的需求,并有助于发挥多基因产物之间的协同作用,提高分化效果。 目的：验证应用SOX-9和胰岛素样生长因子1基因转染大鼠骨髓间充质干细胞后,目的基因分泌情况及向软骨细胞的分化效果。 设计、时间及地点：两样本观察,实验于2008-04/2009-02在中国医科大学细胞生物实验室完成。 对象：6周龄健康雄性Wistar大鼠2只用于骨髓间充质干细胞提取。 方法：扩增、提取质粒pcDNA3.1-IGF-1、pcDNA3.1-SOX-9。分离、纯化Wistar大鼠骨髓间充质干细胞。按转染情况分为5 组：①未转染组：仅加入双无(无血清无双抗)L-DMEM。②转染空载体组：双无L-DMEM +pcDNA3.1空载体和脂质体。③转染SOX-9组：双无L-DMEM+pcDNA3.1- SOX-9质粒和脂质体。④转染胰岛素样生长因子1组：双无L-DMEM分别+pcDNA3.1-IGF-1质粒和脂质体。⑤共转染组：双无L-DMEM分别+pcDNA3.1-IGF-1、pcDNA3.1-SOX-9质粒和脂质体,混合。 主要观察指标：对转染后细胞进行筛选,MTT法测定筛选后细胞的增殖活性,反转录-聚合酶链反应和Western blot法检测筛选后细胞目的基因和蛋白的表达。 结果：MTT法检测转染SOX-9组、转染胰岛素样生长因子1组和共转染组骨髓间充质干细胞增殖活性A值显著高于未转染组、转染空载体组(P < 0 01)。反转录-聚合酶链反应和Western blot检测目结果显示,SOX-9表达以转染SOX-9组最多;胰岛素样生长因子1表达以共转染组最多;软骨细胞特异性基质Ⅱ型胶原表达以共转染组最多。 结论：双基因转染在诱导骨髓间充质干细胞向软骨细胞转化的能力和细胞外基质分泌的能力上更明显高于单基质转染;另外结果间接说明胰岛素样生长因子1具有诱导骨髓间充质干细胞向软骨细胞分化的作用,但能力弱于SOX-9。     

人β-NGF及BDNF基因共转染对大鼠骨髓基质细胞分化的影响

目的 探讨人β-神经生长因子(β-NGF)和脑源性神经营养因子(BDNF)基因共转染对大鼠骨髓基质细胞(BMSCs)分化的影响. 方法 梯度离心法分离培养大鼠胫股骨骨髓中BMSCs,用Src癌基因同源区3抗体(SH3)的免疫细胞化学染色鉴定BMSCs.按照3μL脂质体/1μg(3 μL)pSVCEP NGF/BDNF-CAT质粒的比例共转染第一代BMSCs,并转染pEGFP-C1质粒作为转染的标记.BMSCs培养4周后,荧光显微镜观察增强型绿色荧光蛋白(EGFP)的表达并计算转染率,细胞免疫荧光染色后用激光扫描共聚焦显微镜观察表达产物以及BMSCs分化情况. 结果 体外培养能够获得纯化的原代和传代BMSCs,传代后仍然保持增殖和分化功能;免疫组化SH3染色阳性证实为纯化的BMSCs.人β-NGF/BDNF基因共转染BMSCs后,BMSCs能够稳定和持续表达NGF和BDNF;BMSCs也能表达Nestin、NSE、NF-M和GFAP. 结论 经脂质体介导的外源性目的 基因NGF/BDNF cDNA均能分别和共同在BMSCs中成功表达,基因转染后的BMSCs能诱导分化为神经前体细胞或神经样细胞.     

体外培养条件下人骨髓间充质干细胞碱性成纤维细胞生长因子基因的转染与鉴定

背景：骨髓间充质干细胞的定向分化需要合适生长因子的调控,利用细胞因子基因转染促进其生长、定向分化和加速骨缺损修复是近年来研究热点。 目的：探讨在体外培养条件下转染碱性成纤维细胞生长因子基因对人骨髓间充质干细胞生物学特性的影响。 方法：将含人pcDNA3.1-bFGF真核表达载体DH-5α菌扩增,用EndoFree质粒抽提纯化试剂盒抽提质粒,并对所提取的pcDNA3.1-bFGF重组表达质粒进行酶切鉴定和测序。利用脂质体将pcDNA3.1-bFGF质粒转染到生长良好的P3代骨髓间充质干细胞,G418筛选获得抗性克隆。采用荧光定量PCR、免疫组化、免疫荧光、Western-blot检测转染后骨髓间充质干细胞碱性成纤维细胞生长因子基因及其产物的表达,流式细胞仪检测细胞增殖周期。 结果与结论：脂质体介导pcDNA3.1-bFGF重组表达质粒转染骨髓间充质干细胞后,转染细胞确实表达碱性成纤维细胞生长因子,且表达部位主要位于胞浆。转染细胞增殖活力加强,处于增殖周期的细胞比例更高(P < 0.05)。提示采用脂质体转染法能将pcDNA3.1-bFGF成功导入体外培养的骨髓间充质干细胞,碱性成纤维细胞生长因子基因转染后可改善骨髓间充质干细胞的生存状态,促进其增殖。     

BDNF、EGFP基因修饰大鼠胚胎腹侧中脑神经干细胞的建立

目的建立脑源性神经营养因子（BDNF）、增强型绿色荧光蛋白（EGFP）基因修饰大鼠胚胎腹侧中脑神经干细胞（mNSCs）。方法以FuGENEHD转染试剂介导质粒pcDNA3-BDNF、pEGFPN1共转染第三代E14大鼠胚胎mNSCs。荧光显微镜观察EGFP表达情况；免疫细胞化学方法和Western blot鉴定BDNF的表达；体外诱导分化后免疫细胞化学鉴定其分化能力。结果基因转染12h后EGFP开始表达：免疫细胞化学、Western blot结果表明BDNF能在细胞中正确表达；体外诱导分化研究表明BDNF、EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立了BDNF、EGFP基因修饰大鼠胚胎mNSCs，可为进一步开展帕金森病的细胞移植治疗研究奠定基础。     

Place cells and silent cells in the hippocampus of freely-behaving rats

In the present study, nearly two-thirds of all hippocampal pyramidal units isolated under barbiturate anesthesia, which maximizes these cell's activity, were behaviorally silent. These "silent cells" showed no spontaneous firing activity in the awake, freely-behaving rat. Both reanesthetization and antidromic stimulation, however, activated these silent cells. More than 92% of the remaining spontaneously active hippocampal pyramidal cells recorded from freely-behaving rats were place cells; i.e., they exhibited spatially specific changes in firing activity in at least one environment. The firing rates of these place cells varied depending on the animal's location in this environment. Interestingly many of these place cells displayed low or no spontaneous activity and no spatial specificity in other, dissimilar environments; i.e., their lack of firing in some spatial environments mirrored the behavioral silence of the more numerous silent cells reported here. In complex information processing, such as the processing of spatial information by the hippocampus demonstrated here, neural silence may be as important a signal as neural activity.     

Uncoupling of horizontal cells alters the receptive fields of retinal bipolar cells

Effects of uncoupling of horizontal cells by 1-octanol, a non-specific gap junction uncoupling agent, on the receptive field organization of cone-dominant bipolar cells were investigated in isolated, superfused carp retina, using intracellular recording techniques. At 1 mM, 1-octanol increased responses of cone driven horizontal cells to light spots, but decreased those to light annuli, indicating a reduction of the receptive field size of these cells by uncoupling. Furthermore, 1-octanol eliminated the surround response of OFF type bipolar cells and increased their center response. Similar effects of 1-octanol on the center and surround responses were observed in ON type bipolar cells. These results suggest that uncoupling of horizontal cells can significantly alter the receptive field organization of retinal bipolar cells.     

Astroglial cells: glucocorticoid target cells in the brain

Glutamine synthetase (GS), an enzyme localized in astroglial cells in the brain, is directly implicated in brain detoxification. An ontogenic study of GS activity was performed in homogenates from four distinct brain areas in comparison with the respective astrocytes obtained in primary cultures. GS was induced by hydrocortisone in the astrocytes of all brain areas studied; only cerebellum and cerebral hemisphere astroglial cells had a higher specific activity when compared with the corresponding homogenates. N6O2-Dibutyryl adenosine 3',5'-cyclic monophosphate (dBc AMP), insulin, soluble brain factors, and noradrenaline (NA) were also able to modulate GS activity. Brain factors as well as dBc AMP interfered with hydrocortisone induction of GS. Regulation by hydrocortisone paralleled the variation in its concentration in brain during development. We conclude that astroglial cells are target cells for glucocorticoids, which may modulate ammonia detoxification in these cells.     

Immune cells of the human peripheral taste system: Dominant dendritic cells and CD4 T cells

Taste loss or alterations can seriously impact health and quality of life due to the resulting negative influence on eating habits and nutrition. Infection and inflammation are thought to be some of the most common causes of taste perception disorders. Supporting this view, neuro–immune interactions in the peripheral gustatory system have been identified, underlying the importance of this tissue in mucosal immunity, but we have little understanding of how these interactions influence taste perception directly or indirectly. This limited understanding is evident by the lack of even a basic knowledge of the resident immune cell populations in or near taste tissues. The present study characterized the distribution and population of the major immune cells and their subsets in healthy human anterior, lingual, fungiform papillae (FP) using immunohistochemistry. Dendritic cells (DCs) were the predominant innate immune cells in this tissue, including four subtypes: CD11c⁺ DCs, DC-SIGN+ immature DCs, CD83⁺ mature DCs, and CD1a⁺ DCs (Langerhans cells). While most DCs were localized beneath the lamina propria and only moderately in the epithelium, CD1a⁺ Langerhans cells were exclusively present within the epithelium and not in sub-strata. A small number of macrophages were observed. T lymphocytes were present throughout the FP with CD4⁺ T cells more prevalent than CD8⁺ T cells. Very few CD19⁺ B lymphocytes were detected. The results show that DCs, macrophages, and T lymphocytes are the constitutive guardians of human FP taste tissue, with DCs and CD4 T cells being dominant, while B lymphocytes are rare under normal, healthy conditions. These observations provide a basic anatomical foundation for the immune response in the healthy human tongue as a basis for subsequent disease-related studies, but none of the present data indicate that the immune cell populations identified are, in fact, altered in individuals with abnormal taste perception.     

嗅鞘细胞对脂肪干细胞诱导分化的影响

目的 比较2种不同方法共培养脂肪干细胞后神经元特异性核蛋白(neuron specific nuclear protein,NeuN)的表达.方法 以transwell小室为共培养载体,实验组中将脂肪干细胞接种于上室,嗅鞘细胞接种于下室,对照组下室无嗅鞘细胞,只加入嗅鞘细胞条件培养液,共培养12 d后观察脂肪干细胞NeuN的表达.结果 共培养12 d后2组都有部分脂肪干细胞表达NeuN,实验组NeuN阳性率为303/345(87.8%),而对照组为120/320(37.5%),差异有统计学意义(χ~2=181.6,P＜0.05).结论 脂肪干细胞在与嗅鞘细胞共培养时更容易向神经元样细胞分化.     

神经干细胞移植对视网膜节细胞再生的影响

目的探讨神经干细胞（NSCs）在视神经损伤后对视网膜节细胞轴突再生的作用及其在视神经内迁移和分化。方法实验动物分对照组（PBS组），实验组（NSCs组）；成年SD大鼠在眼球后1min处切断视神经。移植NSCs或PBS至视神经断端；4周后以霍乱毒素B亚基顺行标记观察轴突再生情况，并观察NSCs在视神经内的迁移及免疫组织化学法检测移植后的细胞表达神经丝蛋白（NF）、2，3-环核苷酸磷酸二酯酶（CNP）、胶质纤维酸性蛋白（GFAP）的情况。结果4周后视网膜节细胞再生轴突穿过视神经断端到达远端，移植的NSCs分化为星形胶质细胞并在视神经内迁移0．5～1min。免疫组织化学法检测NSCs部分呈GFAP阳性，未见NF、CNP表达。结论NSCs移植可促进视网膜节细胞轴突再生，能在视神经内迁移并在视神经周围分化为星形胶质细胞。     

The intercalated cells of the amygdala

The intercalated cell groups, or massa intercalata, of the amygdala have been studied in rodent brains with Golgi methods. They also have been examined in gallocyanin-chromalum-, AChE-, and Timm-stained rat brains. The Golgi data indicate that the intercalated cells are not confined to a series of isolated cell clumps but form a neuronal net that covers the rostral half of the lateral-basolateral nuclear complex, stretches across a major portion of rostral amygdala, and continues rostrally beneath the anterior commissure. There are two general types of intercalated neuron--medium and large neurons. The medium intercalated neurons are more common. They have round to elongate somata, 9-18 microns in diameter, and round to bipolar dendritic trees, depending on their location. Most of the dendrites are spine-bearing, as are 20% of the somata. Their axons often have locally ramifying collaterals. The parent axons apparently terminate in either the lateral-basolateral or central nuclei and some of them appear to enter the external capsule. There is a unique medium intercalated neuron that has nearly spine-free, varicose dendrites and an axon that is typical of short axon (Golgi II) cells. There are two varieties of large intercalated neuron-spiny and aspiny. Most of them are aspiny, although they usually have a few spines scattered along their dendrites. Both varieties have elongate, sometimes round, somata that can be as much as 60 microns long. Their dendrites are long, thick, and have few branch points. Only the initial part of the large aspiny cell axon has been impregnated. The large spiny cell axons have several local collaterals; the destination of the parent axons is unknown. The intercalated cells occur along fiber bundles, which are probably afferent to them. The axons that travel among the intercalated cells give off short collaterals and boutons en passant. The sources of these fibers are not known. From the published experimental data, it is likely that they originate in the piriform and entorhinal cortices, the lateral preoptic area, lateral hypothalamus, and ventral pallidum. Axon collaterals of basolateral nucleus pyramidal cells appear to terminate among the intercalated cells. It is suggested that the intercalated cells serve as sites for integration of the output of these various areas and, in turn, communicate it to the lateral-basolateral and central amygdaloid nuclei. The intercalated cells closely resemble neurons in the corpus striatum. Thus the question is raised and discussed of whether the intercalated cells are a ventral extension of the corpus striatum.