Bone morphogenetic proteins but not growth differentiation factors induce dopaminergic differentiation in mesencephalic precursors

Bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) are potential therapeutic molecules for the treatment of Parkinson's disease (PO). Here we compare the effects of BMP3, 5, 6, and 7 and GDF5 and 6 in a rat mesencephalic cell culture system that reflects the developmental stage of neurons around birth. High concentrations of BMP5, 6, and 7 and GDF5 and 6 induced astroglial cell fate and a depletion of oligodendrocytes. Only BMP5, 6, and 7, however, significantly increased the number of tyrosine hydroxylase (TH)-positive neurons and induced nuclear translocation of the phosphorylated BMP-restricted Smad in a substantial number of TH- and microtubule-associated protein 2(MAP2ab)-positive cells. None of the proteins protected TH-positive cells against 6-hydroxydopamine-induced oxidative stress. BMP3 was without any effect throughout the studies. We conclude that BMP5, 6, and 7 act directly and independently on precursors of the dopaminergic and astroglial lineage and induce their differentiation. In contrast, GDF5 and 6 primarily affect nonneuronal cells in mesencephalic cultures of this stage.     

Angiotensin II increases differentiation of dopaminergic neurons from mesencephalic precursors via angiotensin type 2 receptors

In addition to the well-known actions of the humoral renin-angiotensin system, all components of this system are present in many tissues, including the brain, and may play a major role in brain development and differentiation. We investigated the possible effects of angiotensin II on the generation of dopaminergic phenotype neurons from proliferating neurospheres of mesencephalic precursors. We observed immunoreactivity for both angiotensin type 1 and type 2 (AT(1) and AT(2)) receptors in the cell aggregates. Double immunolabeling studies revealed that both receptor types are located in neurons and astrocytes. Interestingly, neurons with a dopaminergic phenotype (i.e. tyrosine hydroxylase activity) showed double labeling for AT(1) and AT(2) receptors although the labeling for AT(2) was more intense. Treatment of the neurospheres with angiotensin II (100 nm) during the differentiation period induced a marked increase (about 400%) in the generation of dopaminergic neurons. This was not affected by treatment with the AT(1) antagonist ZD 7155 but was blocked by treatment with the AT(2) antagonist PD 123319. This suggests that AT(2) receptors mediate the stimulatory effect of angiotensin II on the generation of dopaminergic neurons. Apoptotic cell death studies and bromodeoxyuridine immunohistochemistry indicated that the increase in generation of dopaminergic neurons is not due to increased survival or proliferation of dopaminergic cells during treatment with angiotensin and suggested that angiotensin induces increased differentiation of mesencephalic precursors towards the dopaminergic phenotype. Manipulation of the renin-angiotensin system may be useful for increasing production of dopaminergic neurons for transplantation in Parkinson's disease.     

骨形态发生蛋白4和骨形态发生蛋白4/7基因转染对骨髓基质干细胞成骨活性差异的比较**☆

背景：有文献报道骨形态发生蛋白(bone morphogenetic proteins,BMPs)异源二聚体比同源二聚体的活性高,目前国内外尚无BMP-4/7异源二聚体与BMP-4同源二聚体间诱导成骨活性比较的报道。目的：观察不同基因BMP-4、BMP-4/7对骨髓基质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨活性的差异,探讨融合基因BMP-4/7的生物学活性。方法：经脂质体转染分离培养的兔BMSCs,G418筛选出稳定表达株,利用RT-PCR法证明目的基因在BMSCs中的表达情况;以BMP-4和BMP4/7融合基因修饰的AAV载体,转染兔BMSCs,MTT法检测不同基因转染细胞的增殖能力;以BMP-4单基因和BMP-4/7融合基因修饰的AAV载体转染兔BMSCs 7 d后,测定两组细胞内碱性磷酸酶及骨钙素水平,观察成骨活性的变化。结果与结论：实验成功构建高滴度的分别携带BMP-4单基因、BMP-4/7融合基因的重组腺相关病毒载体AAV-BMP-4、AAV-BMP-4/7;RT-PCR结果证明目的基因能够在BMSCs中得到稳定表达。AAV-BMP-4及AAV-BMP-4/7载体转染效率分别为68.20%、72.18%;转染BMSCs后,细胞增殖能力均明显提高,BMP-4/7融合基因的细胞增殖能力活性高于BMP-4单基因。以BMP-4和BMP-4/7融合基因修饰的AAV转染细胞7 d后,细胞内碱性磷酸酶活性明显上调,骨钙素水平升高,成骨活性均增强;两种基因比较,BMP-4/7融合基因成骨活性强于BMP-4单基因(P < 0.01)。提示BMP-4、BMP-4/7修饰的AAV载体转染效率高,对BMSCs均有成骨活性,其中BMP-4/7融合基因成骨活性强于BMP-4单基因。     

大鼠胚胎中脑多巴胺神经元的分离培养与鉴定*

背景：神经细胞移植治疗是最有希望治愈帕金森病的方法之一。 目的：探讨大鼠胚胎中脑多巴胺神经元分离、培养与鉴定的方法。 方法：解剖分离E14d SD大鼠胚胎中脑组织,制备单细胞悬液,以神经细胞培养液培养,观察其生长情况并进行RT-PCR和免疫组织化学鉴定。 结果与结论：在神经细胞培养液中,细胞生长良好。RT-PCR和免疫组织化学结果显示大多数细胞表达多巴胺神经元特异性分子标志。证实实验成功建立了大鼠胚胎中脑多巴胺神经元分离培养与鉴定的方法。     

骨形态发生蛋白7在骨科的应用*

背景：骨形态发生蛋白家族因具有独特的异位骨诱导活性而被发现并被命名为骨形态发生蛋白,与胚胎组织的发育、功能维持及组织的修复均密切相关,是诱导骨发生的主要活性因子。骨形态发生蛋白7是骨形态发生蛋白家族中的一员,主要在骨、肾组织中表达,与胚胎组织的发育、功能维持及组织的修复均密切相关。目的：全面了解骨形态发生蛋白7在骨发育和骨折愈合过程中的表达及作用。方法：电子检索和计算机Medline数据库1990/2010收录的骨形态发生蛋白相关综述和论文报告,并分析生物学作用及调控途径的研究进展。结果与结论：随着研究的深入,骨形态发生蛋白7可望在骨折愈合、退行性骨关节炎、相关骨病的防治中发挥重要的作用。骨形态发生蛋白7的生物学活性使其对骨折、骨缺损、延迟愈合,骨不连、骨质疏松等多种骨科疾病的治疗具有巨大的临床应用价值。已有证据表明骨形态发生蛋白局部应用或骨形态发生蛋白涂层有利于假体-骨界面的骨整合,包括假体周围骨缺损的治疗。关键词：骨形态发生蛋白;骨形态发生蛋白7;应用;进展;骨科     

Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells

We report on generation of dopamine neurons from long-term cultures of human fetal mesencephalic precursor cells. These CNS precursor cells were successfully expanded in vitro using the mitogens epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Incubation of these cultures in 3% atmospheric oxygen resulted in higher cellular yields than room air. Following incubation in differentiation media containing interleukin (IL)-1b (IL-1b), IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF), up to 1% of the precursor cells converted into cells immunoreactive for tyrosine hydroxylase (TH), a marker for dopamine neurons. The TH immunoreactive cells exhibited morphological and functional properties characteristic of dopamine neurons in culture. These precursor cells might serve as a useful source of human dopamine neurons for studying the development and degeneration of human dopamine neurons and may further serve as a continuous, on-demand source of cells for therapeutic transplantation in patients with Parkinson's disease.     

Angiotensin II and interleukin-1 interact to increase generation of dopaminergic neurons from neurospheres of mesencephalic precursors

Cultures of rat mesencephalic precursors treated with interleukin-1beta or angiotensin II contained significantly more dopaminergic neurons than controls. However, simultaneous treatment with angiotensin II and interleukin-1beta did not induce any further increase. Treatment with the angiotensin type-2 receptor antagonist PD 123319 precluded both the angiotensin- and the interleukin-induced increase. The present results indicate that angiotensin type-2 receptors and interleukin-1 cooperate to induce the dopaminergic phenotype.     

Elimination of serotonergic cells induces a marked increase in generation of dopaminergic neurons from mesencephalic precursors

Production of dopaminergic (DA) neurons from stem/precursor cells for transplantation in Parkinson's disease has become a major focus of research. However, the inductive signals mediating the production of DA neurons remain poorly understood, and the influence of other cell populations simultaneously generated within the cell aggregates has not been studied. We investigated whether DA phenotype (i.e. tyrosine hydroxylase-immunoreactive, TH-ir), serotonergic, floor plate (FP4-ir), and fibroblast growth factor 8 (FGF-8)-ir cells differentiate from proliferating cell aggregates obtained from rat mesencephalic precursors, and we also investigated the effects of serotonergic cells on differentiation of DA cells. We observed FP4-ir, FGF-8-ir, TH-ir and serotonergic cells within the aggregates. The TH-ir cells appeared within or in close proximity to a central FP4-ir core, and then concentrated peripherally forming a cap that surrounded the central FP4-ir area. The serotonergic cells and fibers formed a cap surrounding that of TH-ir neurons. Cell aggregates treated with an antibody against FGF-4 or with the serotonergic toxin 5,7-dyhydroxytryptamine or the serotonin synthesis inhibitor dl-p-chlorophenylalanine showed a marked decrease in the number of 5-HT-ir cells (10-20% of controls) and a marked increase in that of TH-ir neurons (700-900% of controls). The present results show that manipulation of other cell populations in the cell aggregates, particularly the serotonergic population, may be an effective method of increasing the production of DA neurons from stem/precursor cells.     

Rapid and efficient differentiation of dopaminergic neurons from mouse embryonic stem cells

We have developed a fast and effective method for the differentiation of dopaminergic neurons from mouse embryonic stem cells. Neuronal precursors are obtained by formation of embryonic bodies or neural stem spheres via free-floating culture in the presence of the mitogens basic fibroblast growth factor and epidermal growth factor together with L-ascorbic acid. Subsequent culturing of the precursor cells in medium containing epidermal growth factor, FGF8b, SHH and ascorbic acid induces cell proliferation, following withdrawal of the growth factors leads differentiation into predominantly dopaminergic neurons. Mature neurons are obtained within 10 days of replacing the proliferation to differentiation medium. Embryonic stem-derived dopaminergic neurons are purified by cell sorting and may serve as a convenient source for the study of molecular, genetic and cellular properties of dopaminergic neurons.     

小胎龄鼠的腹侧中脑神经前体细胞体外培养研究

目的体外培养小胎龄的胎鼠腹侧中脑神经前体细胞,根据多巴胺能神经元产量,确定较好的研究对象,为细胞移植治疗帕金森病奠定基础。方法取不同胎龄胎鼠(E10、E11、E12)的腹侧中脑神经前体细胞,体外加碱性成纤维细胞生长因子扩增7d后用含AA-2P的分化增养液培养7 d,行免疫荧光染色比较多巴胺能神经元形成情况。结果E10、E11、E12细胞经培养后细胞总数分别增加76.39倍、59.67倍、51.85倍。多巴胺能神经元占所有细胞的比例在E11、E10、E12依次显著降低。对于多巴胺能神经元占所有神经元的比例,E11显著高于E10和E12。结论来自胎龄11 d的胎鼠腹侧中脑神经前体细胞是较好的体外培养对象,可为细胞移植研究提供较为充足的细胞资源。     

Ascorbate-induced differentiation of embryonic cortical precursors into neurons and astrocytes

A specific role for ascorbate (AA) in brain development has been postulated based on a rise of AA levels in fetal brain (Kratzing et al., 1985). To evaluate the role of AA during CNS development, we analyzed the survival, proliferation, and differentiation of AA-treated CNS precursor cells isolated from rat embryonic cortex. Immunocytochemical analyses revealed that AA promoted the in vitro differentiation of CNS precursor cells into neurons and astrocytes in a cell density-dependent manner. Additionally, AA increased the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) of postmitotic neurons in primary neuronal cultures. Differential expression analysis of genes specific to neuronal or glial differentiation revealed an AA-dependent increase in the expression of genes that could potentially compound the effects of AA on cell differentiation. These data suggest that AA may act in the developing brain to stimulate the generation of CNS neurons and glia, thereby assisting in the formation of neural circuits by promoting the acquisition of neuronal synaptic functions.     

Effect of GDNF on differentiation of cultured ventral mesencephalic dopaminergic and non-dopaminergic calretinin-expressing neurons

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for ventral mesencephalic (VM) dopaminergic neurons. Subpopulations of dopaminergic and non-dopaminergic VM neurons express the calcium-binding proteins calbindin (CB) and calretinin (CR). Characterization of the actions of GDNF on distinct subpopulations of VM cells is of great importance for its potential use as a therapeutic molecule and for understanding its role in neuronal development. The present study investigated the effects of GDNF on the survival and morphological differentiation of dopaminergic and non-dopaminergic neurons in primary cultures of embryonic day (E) 18 rat VM. As expected from our results obtained using E14 VM cells, GDNF significantly increased the morphological complexity of E18 CB-immunoreractive (CB-ir), tyrosine hydroxylase (TH)-ir, and CR-ir neurons and also the densities of CB-ir and TH-ir neurons. Interestingly, densities of E18 CR-ir neurons, contrarily to our previous observations on E14 CR-ir neurons, were significantly higher after GDNF treatment (by 1.5-fold). Colocalization analyses demonstrated that GDNF increased the densitiy of dopaminergic neurons expressing CR (TH+/CR+/CB-), while no significant effects were observed for TH-/CR+/CB- cell densities. In contrast, we found that GDNF significantly increased the total fiber length (2-fold), number of primary neurites (1.4-fold), number of branching points (2.5-fold), and the size of neurite field per neuron (1.8-fold) of the non-dopaminergic CR-expressing neurons (TH-/CR+/CB-). These cells were identified as GABA-expressing neurons. In conclusion, our findings recognize GDNF as a potent differentiation factor for the development of VM dopaminergic and non-dopaminergic CR-expressing neurons.     

骨形态发生蛋白与骨缺损的修复

骨缺损发生后,骨的生理连续遭严重破坏,在利用外源性骨形态发生蛋白治疗时,必须有适合的载体,通过该载体将外源性骨形态发生蛋白及其它生长因子释放到骨质缺损部位,诱导新骨形成从而达到治疗目的。骨形态发生蛋白的载体材料很多,主要包括无机材料、高分子聚合材料、生物性材料以及这些材料的复合物,但各有不足之处。许多学者对其加以改进,使其更有利于发挥骨形态发生蛋白的诱骨活性。载体或局部基因释放的骨形态发生蛋白可有效修复骨缺损,为治疗骨缺损提供了新途径。     

骨髓间充质干细胞促进中脑前体细胞增殖及定向分化的实验研究

目的 观察BMSCs对胚胎腹侧中脑前体细胞(VMP)体外扩增和定向分化的影响,并分析其可能的营养机制.方法 分别取胎龄11d大鼠胚胎VMP、成年大鼠BMSCs进行体外培养,并建立二者的共培养体系.体外扩增7 d的VMP分为对照组、BMSCs分化液组、BMSCs+VMP共培养分化液组,分别加入普通分化液、BMSCs分化液、BMSCs+VMP共培养分化液进行诱导分化.观察细胞生长情况.分化期末行免疫荧光染色,分析比较3组细胞中酪氨酸羟化酶(TH)阳性细胞占总细胞数的比例.结果诱导分化7 d后,对照组、BMSCs分化液组和BMSCs+VMP共培养分化液组中细胞数分别较培养前扩增(44.13±4.75)倍、(60.63±5.25)倍、(64.00±7.63)倍,TH阳性细胞比例分别为(18.76±5.20)%、(23.49±4.10)%、(28.08±5.42)%,比较差异有统计学意义(P<0.05).结论 BMSCs能够通过分泌营养因子有效促进VMP增殖并定向分化为多巴胺能神经元.     

骨髓间充质干细胞促进中脑前体细胞增殖及定向分化的实验研究

Objective To explore the potential neurotrophic effect of bone marrow-derived mesenchymal stem cells (BMSCs) on the proliferation and differentiation of ventral mesencephalic precursors (VMPs).Methods VMPs from E 11 inbred rat embryos and BMSCs from adult rats were cultured in 2 separate groups.Moreover,a co-cultured group was also established.Afterwards,the 3 different differentiation mediums obtained from these 3 defined groups were used to influence the differentiation procedures of normal VMPs that had amplification in vitro for 7 d.The growth of cells was observed;immunocytochemical staining was performed on these cells at the late differentiation phase.Relative yields of TH+ cells were calculated and compared.Results Seven d after the inducing differentiation,the total cell numbers multiplied about 44.13±4.75,60.63±5.25 and 64.00±7.63 folds in the VMP control group,the BMSCs group and the VMP+BMSCs co-cultured group,respectively,as compared with those before the differentiation.Correspondingly, the ratios of TH+ cells in the total cell population were (18.76±5.20)%, (23.49±4.10)% and (28.08±5.42)% in the VMP control group,the BMSCs group and the VMP+BMSCs co-cultured group,respectively; significant differences betwwen each 2 groups were found (P<0.05).Conclusion BMSCs can secret nutritional factors to promote the proliferation and committed differentiation of VMPs into dopaminergic neurons.     

骨髓间充质干细胞促进中脑前体细胞增殖及定向分化的实验研究

Objective To explore the potential neurotrophic effect of bone marrow-derived mesenchymal stem cells (BMSCs) on the proliferation and differentiation of ventral mesencephalic precursors (VMPs).Methods VMPs from E 11 inbred rat embryos and BMSCs from adult rats were cultured in 2 separate groups.Moreover,a co-cultured group was also established.Afterwards,the 3 different differentiation mediums obtained from these 3 defined groups were used to influence the differentiation procedures of normal VMPs that had amplification in vitro for 7 d.The growth of cells was observed;immunocytochemical staining was performed on these cells at the late differentiation phase.Relative yields of TH+ cells were calculated and compared.Results Seven d after the inducing differentiation,the total cell numbers multiplied about 44.13±4.75,60.63±5.25 and 64.00±7.63 folds in the VMP control group,the BMSCs group and the VMP+BMSCs co-cultured group,respectively,as compared with those before the differentiation.Correspondingly, the ratios of TH+ cells in the total cell population were (18.76±5.20)%, (23.49±4.10)% and (28.08±5.42)% in the VMP control group,the BMSCs group and the VMP+BMSCs co-cultured group,respectively; significant differences betwwen each 2 groups were found (P<0.05).Conclusion BMSCs can secret nutritional factors to promote the proliferation and committed differentiation of VMPs into dopaminergic neurons.     

Neuroprotection with methylaminochroman and lazaroid of embryonic ventral mesencephalic tegmental dopaminergic neurons in cold storage.

Embryonic ventral mesencephalic tegmental (EVMT) neurons die off over time in cold storage at 4 degrees C in hibernation buffers (HB). Manipulation of HB conditions may improve the survival of neurons in cold storage. We examined the effect of lipid peroxidation inhibitors, a methylaminochroman (U83836E) and a lazaroid (U74389G) on the viability and survival of embryonic dopaminergic neurons in the co-culture system of embryonic striatal target (EST) cells and EVMT neurons that had been stored for 3 days at 4 degrees C in HB with or without U83836E or U74389G. One-way analysis of variance (ANOVA) was used for analysis of data. The density of tyrosine hydroxylase immunoreactive (THIR)-positive neurons was significantly higher in the groups stored in supplemented HB than in the control (HB alone; P < 0.001). The neuroprotective effect was concentration-dependent. We conclude that either U83836E or U74389G-conditioned HB exerted a concentration-dependent neuroprotective effect on embryonic dopaminergic neurons in cold storage for 3 days. Supplementation of U83836E and U74389G or other methylaminochromans and lazaroids in HB may be important for cold storage of donor neuronal cells.