RCMV感染星形胶质细胞对神经干细胞分化的影响

探讨大鼠巨细胞病毒（rat cytomegalovirus,RCMV）感染大鼠星形胶质细胞后,对神经干细胞分化的影响。原代分离培养新生大鼠星形胶质细胞和胚胎海马神经干细胞,将星形胶质细胞感染RCMV后和神经干细胞在Transwell24孔共培养体系下进行共培养,同时设对照组;用免疫荧光染色等方法检测神经干细胞与感染RCMV的星形胶质细胞共培养后,其分化细胞中神经元微管相关蛋白（microtubule-associated protein 2,MAP2）和星形胶质细胞胶质纤维酸性蛋白（glial fibril—lary acidic protein,GFAP）的表达。结果发现,感染RCMV的星形胶质细胞与神经干细胞共培养时,神经干细胞分化减慢,分化成的神经元和星形胶质细胞比率低于对照组,提示星形胶质细胞感染RCMV后可抑制神经干细胞的分化,可能与RCMV影响星形胶质细胞合成和分泌各种营养因子,干扰了神经干细胞的分化进程有关。     

肿瘤坏死因子α和β对电离辐射诱导细胞凋亡的效应

为探讨肿瘤坏死因子（tumor necrosis foctor)α和β（TNFα和β）对电离辐射诱发细胞凋亡的效应及其机理,采用DNA琼脂糖凝胶电泳和FACS分析等方法,观察了人肿瘤坏死因子α（hTNFα)和β（hTNFβ)对^60Co-γ射线诱发细胞凋亡的形态学,生化学变化。结果显示：hTNFα或hTNFβ均可明显抑制^60Co-γ射线诱发正常人胚肺二倍体细胞（2BS）的凋亡,而相同剂量的hTNFα能促进^60Co-γ射线诱发的人体肺腺癌细胞系A549细胞凋亡,而对另一株人体肺癌SPC细胞的效应比A549降低1倍;hTNFβ能分别增强A549和SPC的细胞凋亡频率。由此认为,hTNFα和hTNFβ均可通过调节细胞的生理生化反应来改变细胞对电离辐射的敏感性,可保护正常细胞免受辐射损伤,而增加某些肿瘤细胞对辐射的敏感性。     

少突胶质细胞发育分化的转录调控

少突胶质细胞(OL)的成熟分化是中枢神经髓鞘形成的关键环节.在少突胶质细胞形成、增殖、分化、成熟的发育过程中,骨形态发生蛋白(BMP)、sonic hedgehog (Shh)、Notch等三种信号通路发挥了重要的调控作用.这些通路最终激活或抑制下游一系列特异性转录因子(TFs)而完成对OL谱系特异性标志基因表达的激活.各种TFs在特定条件下的时空表达和相互作用是OL发生、发育、分化的重要调控方式.     

肿瘤坏死因子α对大鼠成骨样细胞增殖与甲状旁腺素受体表达的影响

大鼠成骨样细胞ＲＯＳ１７／２．８经肿瘤坏死因子α处理后,发现其ｃ－ｍｙｃ,ｃ－ｆｏｓ,ｃ－ｊｕｎ等原癌基因ｍＲＮＡ的表达降低,细胞增殖明显受到抑制;同时,其ＰＴＨ受体的表达以及ＰＴＨ经受体刺激后引起的细胞内ｃＡＭＰ的增加也受到抑制。提示ＴＮＦα可抑制大鼠在骨要细胞ＲＥＯＳ１７／２．８的增殖和分化。     

神经干细胞向少突胶质前体细胞的定向分化诱导

本研究采用神经胶质瘤细胞株(B104 neuroblatoma cells,B104 cells)培养上清(B104CM)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),将冷冻复苏的大鼠胚胎脊髓神经干细胞(neural stem cells,NSCs)定向诱导为少突胶质前体细胞(oligodendrocyte precusor cells,OPCs)。形态学和免疫组化的结果显示,诱导后95％以上的细胞具有双极或多极突起的典型OPCs形态,并表达A285和血小板源生长因子受体-α(platelet derived growth factor receptor-α,PDGFR-α等0PCs标志,所有PDGFR-α阳性的OPCs均不表达β-Tublin Ⅲ,其中仅少量细胞表达胶质原纤维酸性蛋白(glia fibrillary acidic protein,GFAP)。在B104CM和bFGF共存的培养条件下,悬浮培养的OPCs可大量增殖形成少突胶质细胞球,该细胞球可通过传代继续扩增,且扩增的OPCs仍能维持其特有的形态和自我增殖的特性。撤去bFGF和B104CM后,OPCs能进一步分化为成熟的少突胶质细胞(oligodendrocytes,OLs)或Ⅱ型星形胶质细胞。实验表明,诱导NSCs产生的OPCs在形态、增殖以及分化格局等方面均与已报道的存在于胚胎脑区的O-2A前体细胞相类似。该培养系统可为实验性细胞移植的研究提供丰富的细胞来源。     

白介素-6及肿瘤坏死因子α对新生儿低出生体重的影响

研究表明,在子宫内感染如绒毛膜羊膜炎发生时,脐血内的细胞因子浓度产生很大的变化,其中细胞因子白介素-6(IL-6)及肿瘤坏死因子α(TNF-α)水平都有显著性升高。有人提出这些细胞因子正是分娩发动的原因,它们通过促进前列腺素PGS的合成导致分娩。与此同时,由于母体的血液循环,母体血清中IL-6、TNF-α的浓度也有显著性变化,可作为检测宫内感染的诊断指标加以应用。     

肿瘤坏死因子-α对小鼠骨髓间充质干细胞成骨分化影响研究

目的:探讨肿瘤坏死因子-α(Tumor Necrosis Factor alpha, TNF-α)对小鼠骨髓间充质干细胞(Bone Marrow-derived Mesenchymal Stem Cells, BM-MSCs)成骨分化的影响。方法:以在促成骨分化培养基(Osteogenic media, OS)中培养的小鼠BM-MSCs作为对照,用含有TNF-α的OS处理小鼠BM-MSCs。第7天进行碱性磷酸酶染色,或培养第21天进行茜素红染色和定量分析。第7天时用一份细胞提取得到的mRNA通过实时定量RT-PCR测定Runx2和Osterix的mRNA表达,用另一份细胞得到的细胞裂解液通过SDS-PAGE来测定Runx2和Osterix的蛋白质表达。结果:TNF-α抑制碱性磷酸酶活性;TNF-α抑制矿化骨节的形成; TNF-α抑制Runx2和Osterix的mRNA和蛋白质表达。结论:TNF-α对小鼠BM-MSCs成骨分化的抑制,可能部分是通过抑制成骨分化中两个关键的转录因子Runx2和Osterix的mRNA和蛋白质表达来实现的。     

低氧对少突胶质前体细胞增殖的影响

目的：观察低氧／复氧和间歇性低氧时少突胶质前体细胞（O2A）增殖的影响。方法：取混合培养4d的胶质细胞。按实验分为低氧／复氧、间歇性低氧和常氧3组,低氧／复氧组细胞置低氧环境（3％O2）中分别培养1h、2h、3h、4h、8h、12h、24h、48h和72h后取出,恢复常氧培养至9d。间歇性低氧组每天上午定时将细胞置于低氧环境中分别培养1h、2h、3h、4h后取出。恢复常氧培养。每天一次,分别重复1～4次后。常氧培养至9d。然后同时取出各组细胞,进行O2A细胞的分离纯化和细胞计数。结果：低氧／复氧1h、2h、3h组和间歇性低氧1h、2h、3h组,O2A细胞数均较常氧组明显增多。结论：低氧／复氧和间歇性低氧都可促进体外培养的O2A细胞明显增殖。其机制尚有待于进一步研究。     

Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co-cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki-67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top-layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co-culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co-culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch-related genes using RT-PCR showed that after co-culturing with MSCs for 24 h, NSCs expressed much higher levels of ki-67, Notch1, and Hes1 than did NSCs co-cultured with NIH3T3 cells. Treatment with DAPT decreased ki-67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.     

Tumor necrosis factor-alpha stimulates proliferation of rat ovarian theca-interstitial cells.

Tumor necrosis factor-alpha (TNF-alpha) is a potent modulator of ovarian function, affecting steroidogenesis of both granulosa and theca-interstitial (T-I) cells. Women with polycystic ovary syndrome (PCOS) have increased levels of serum TNF-alpha. The present study evaluated the effects of TNF-alpha on T-I cell proliferation. Purified rat T-I cells were cultured for 48 h with or without TNF-alpha (0.001-1 nM), insulin-like growth factor I (IGF-I; 10 nM), and/or insulin (10 nM). Proliferation was measured by [(3)H]thymidine incorporation assay and by counting the steroidogenically active (stained positive for 3beta-hydroxysteroid dehydrogenase; 3beta-HSD) and inactive (3beta-HSD negative) cells. TNF-alpha stimulated thymidine incorporation in a dose-dependent fashion (up to 3.2-fold; P < 0.01). Insulin and IGF-I stimulated T-I proliferation (respectively, by up to 2.4- and 3.1-fold; P < 0.001). TNF-alpha potentiated effects of insulin and IGF-I in a dose-dependent and additive fashion (up to 6.7-fold; P < 0.001). TNF-alpha (1 nM) increased total cell count (by 80%, P < 0.05) and the proportion of 3beta-HSD-positive cells (by 19%, P < 0.05). Flow cytometry DNA analysis revealed that TNF-alpha (1 nM) increased the proliferative index by up to 16% (P = 0.05). The present findings demonstrate that TNF-alpha stimulates mitotic activity of T-I cells by increasing the proportion of actively dividing cells and preferentially increasing the number of steroidogenically active cells. The effects of TNF-alpha appear to be independent of those induced by insulin and IGF-I. We postulate that TNF-alpha may play a pathophysiologic role in disorders of the T-I compartment, such as PCOS.     

Enhancement of human B cell proliferation and differentiation by tumor necrosis factor-alpha and interleukin 1

The role of tumor necrosis factor-alpha (TNF-alpha) in human B cell responses was examined and compared with that of interleukin (IL) 1 by assessing the ability of each cytokine to support proliferation and differentiation. Recombinant TNF-alpha (rTNF-alpha) and recombinant IL-1 (rIL-1) each enhanced the generation of immunoglobulin-secreting cells (ISC) in cultures of pokeweed mitogen-stimulated B cells incubated with T cells. To examine the direct effect of rTNF-alpha and rIL-1 on the responding B cell, highly purified peripheral blood B cells were stimulated with Cowan I Staphylococcus aureus (SA). In the absence of T cell factors, proliferation was minimal and there was no generation of ISC. Recombinant IL-2 (rIL-2) supported both responses. Although rTNF-alpha alone did not support SA-stimulated generation of ISC, it did increase SA-stimulated B cell DNA synthesis by two- to eightfold. In addition, rTNF-alpha augmented B cell proliferation in rIL-2 supported SA-stimulated cultures. Moreover, rTNF-alpha enhanced the generation of ISC stimulated by rIL-2 alone or rIL-2 and SA. rIL-1 also augmented DNA synthesis and generation of ISC by B cells stimulated with SA and rIL-2. However, rTNF-alpha enhanced proliferation and ISC generation in SA + rIL-2-stimulated cultures even when they were supplemented with saturating concentrations of rIL-1. Utilizing a two-stage culture system, it was found that the major effect of rTNF-alpha was to enhance responsiveness of SA-activated B cells to rIL-2, whereas it exerted only a minimal effect during initial stimulation. These results indicate that TNF-alpha as well as IL-1 augment B cell responsiveness. Moreover, the ability of rTNF-alpha to enhance B cell responsiveness was not an indirect effect resulting from the induction of Il-1 production by contaminating monocytes, but rather resulted from the delivery of a signal by rTNF-alpha directly to the responding B cell that promoted both proliferation and differentiation after initial activation. The data therefore indicate that human B cell responsiveness can be independently regulated by the action of two separate monocyte-derived cytokines.     

Tumor necrosis factor-alpha inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells

We have investigated the effects of TNF-alpha on FSH-induced LH receptor expression, cAMP and progesterone production in cultured rat granulosa cells. TNF-alpha (0.5-100 ng/ml) inhibits the stimulating action of FSH on LH receptor formation in a dose-dependent manner with an IC50 of 1 ng/ml and an almost complete suppression of LH receptor induction for 50-100 ng/ml TNF-alpha. The inhibitory effect of TNF-alpha is not due to variations in cell number or viability but rather to a reduction of the LH receptor content per cell with no change in binding affinity (KD = 0.8 x 10(-10)M). TNF-alpha also inhibits the FSH-induced cAMP production but at a lower extent, with a maximum reduction of 60% for 100 ng/ml TNF-alpha. Moreover, TNF-alpha impairs the LH receptor formation induced by forskolin, cholera toxin or 8-Bromo-cAMP, indicating that the cytokine also acts at a step distal to FSH receptor and to cAMP formation. Finally, TNF-alpha decreases dramatically the progesterone synthesis that is stimulated by FSH, with a reduction to undetectable levels on and after 10 ng/ml TNF-alpha. These results suggest that TNF-alpha may drastically reduce the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the physiological ovarian follicular maturation. Such anti-gonadotropic action of TNF-alpha on granulosa cell differentiation may be also relevant to the alteration of ovarian function during physiopathological processes like inflammatory or infection diseases.     

Effects of tumor necrosis factor-alpha on cell proliferation, prostaglandins and matrix-metalloproteinases production in rat endometrial stromal cells cultured in vitro

Tumor necrosis factor-alpha (TNF-alpha) is known as a pluripotent cell mediator, and it is implicated in the control of uterine cell growth, differentiation and function during estrous cycle and pregnancy. In this study, we investigated the effect of TNF-alpha on endometrial stromal cells derived from rat uterus (rat endometrial stromal cells, RES). RES were isolated from rat endometrium at day 5 of pregnancy. Proliferation activities of RES were measured by using bromodeoxyuridine (BrdU) labeling kit, the productions of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) were measured by enzyme immunoassay kits and the production of matrix metalloproteinases (MMPs) was analyzed by gelatin-zymography. TNF-alpha, as well as epidermal growth factor and fibroblast growth factor-2, significantly increased the proliferation activity of RES (P<0.05). TNF-alpha selectively stimulated the production of PGE2 in RES (P<0.05), but not the production of PGF2alpha. Additionally, TNF-alpha did not stimulate the production of MMPs in RES at the concentration of 5 ng/mL, compared with the control groups (P>0.05). In conclusion, this study demonstrates several regulational functions of TNF-alpha on RES using in vitro culture system. The effects of TNF-alpha on proliferation and MMP production of RES have been shown for the first time. We believe that these results demonstrate part of the functions of TNF-alpha in endometrium and contribute to the better understanding of endometrial functions.     

神经干细胞体外增殖分化的钙成像研究

神经干细胞具有广阔的应用前景,但对于其增殖和分化的内源机制、外部环境信号还并不十分了解。研究表明,钙信号很可能在其中起到了调控作用。利用钙离子成像技术,观察神经干细胞的单细胞体外增殖和分化过程,记录了在细胞分裂过程中钙信号变化的曲线。发现细胞增殖和分化过程中都会产生钙浓度的变化,但在细胞分裂后期两者钙信号的模式却存在差别。实验结果提示,胞内钙水平的波动只是细胞增殖的伴随产物,但却是细胞分化的必要条件。由此提出钙信号对神经干细胞分化调控机制的假设,并指出其对今后研究的意义。     

川芎嗪对体外培养的胎鼠神经干细胞增殖和分化的影响

目的：探讨川芎嗪（TMP）在体外神经干细胞（NSCs）增殖与分化中的作用。方法：原代提取孕14 d雌性大鼠的胎鼠大脑皮层分离培养,并作免疫荧光染色鉴定,取传代培养第3代的NSCs进行实验。实验分为对照组、β-巯基乙醇阳性对照组、TMP诱导组和TMP+EGTA组（n=4）。采用BrdU法和MTT法观察川芎嗪对NSCs增殖数量的影响,采用蛋白免疫印迹法检测NSCs的分化表达情况。结果：实验成功分离纯化原代NSCs,培养3~5 d可见部分神经球形成,具备典型的NSCs形态并表达NSCs特异抗原巢蛋白;BrdU法和MTT法结果均显示,与对照组和β-巯基乙醇阳性对照组相比,TMP组NSCs增殖数量明显增多（P<0.05）;蛋白免疫印迹结果显示,TMP组和TMP+EGTA组NSCs的神经元分化率明显增高,TMP+EGTA组分化率增高更明显（P<0.05）。结论：TMP能显著增强NSCs的增殖和神经元分化率。减少细胞外Ca²⁺可促进TMP诱导NSCs向神经元分化,Ca²⁺信号在TMP诱导NSCs向神经元分化过程中起重要作用。     

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.     

Effect of tumor necrosis factor-alpha on intracellular Staphylococcus aureus in vascular endothelial cells.

Although tumor necrosis factor-alpha (TNF-alpha) is an important host factor against intracellular bacteria, little is known about the effect of TNF-alpha on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-alpha influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-alpha, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10(6) CFU well(-1)) into HUVECs for 1 h. TNF-alpha (1 ng mL(-1)) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-alpha plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-alpha preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-alpha helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-alpha preferentially induces apoptosis of endothelial cells infected by S. aureus.