In vitro Proliferation of Human Bone Marrow Mesenchymal Stem Cells Employing Donor Serum and Basic Fibroblast Growth Factor

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10⁵ nucleated cells/cm²) with DMEM medium containing 10% serum (FCS or donor serum) and basic fibroblast growth factor, and incubated at 37 °C under a 5% CO₂ atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic fibroblast growth factor (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45^- CD105⁺ cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic fibroblast growth factor was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to 17 population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.     

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.     

Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation

Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, βIII-tubulin and GFAP were the most highest in the DPSCs?+?bFGF?+?NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF?+?NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.     

碱性成纤维细胞生长因子在苜蓿中的表达

为了降低bFGF(basic fibroblast growth factor)的生产成本,结合植物生物反应器的优点,就bFGF在转基因苜蓿中的表达进行了探索.将bFGF插入植物表达载体pBⅡ21中,获得了含有bFGF基因的植物表达pBIcbFGF,再将pBIcbFGF利用冻融法转到农杆菌中.利用农杆菌介导法将基因转化保定苜蓿,转基因苜蓿在TM-1培养基+20 mg/L卡那霉素(Kan)+200 mg/L特美汀(Tim)中诱导分化,在生根培养基中生根,获得再生植株.再生植株通过PCR检测、RT-PCR及Western blot证实外源基因已经在苜蓿中成功表达.获得含有目的蛋白的阳性植株.为苜蓿作为植物生物反应器生产bFGF奠定了理论及技术基础.     

Localization of Basic Fibroblast Growth Factor in Bovine Adrenal Chromaffin Cells

Abstract: We have investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.     

Keratinocyte Growth Factor Gene Delivery via Mesenchymal Stem Cells Protects against Lipopolysaccharide-Induced Acute Lung Injury in Mice

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality, and have no specific therapy. Keratinocyte growth factor (KGF) is a critical factor for pulmonary epithelial repair and acts via the stimulation of epithelial cell proliferation. Mesenchymal stem cells (MSCs) have been proved as good therapeutic vectors. Thus, we hypothesized that MSC-based KGF gene therapy would have beneficial effects on lipopolysaccharide(LPS)-induced lung injury. After two hours of intratracheal LPS administration to induce lung injury, mice received saline, MSCs alone, empty vector-engineered MSCs (MSCs-vec) or KGF-engineered MSCs (MSCs-kgf) via the tail vein. The MSCs-kgf could be detected in the recipient lungs and the level of KGF expression significantly increased in the MSCs-kgf mice. The MSC-mediated administration of KGF not only improved pulmonary microvascular permeability but also mediated a down-regulation of proinflammatory responses (reducing IL-1β and TNF-α) and an up-regulation of anti-inflammatory responses (increasing cytokine IL-10). Furthermore, the total severity scores of lung injury were significantly reduced in the MSCs-kgf group compared with the other three groups. The underlying mechanism of the protective effect of KGF on ALI may be attributed to the promotion of type II lung epithelial cell proliferation and the enhancement of surfactant synthesis. These findings suggest that MSCs-based KGF gene therapy may be a promising strategy for ALI treatment.     

VEGF通过调节黏着斑促进间充质干细胞的黏附及铺展

间充质干细胞（mesenchymalstemcells,MSCs）具有多向分化潜能并能在体外趋化剂或细胞因子的作用下进行定向迁移,体内移植后可趋向迁移至脑瘤病灶区。细胞黏附是细胞迁移的首要条件,了解细胞黏附及其调控有助于细胞迁移机制的研究。细胞黏附及铺展涉及到黏着斑（f0－caladhesions,FAs）的动态变化以及细胞骨架的重排。细胞铺展面积在黏附过程中逐渐增大,黏附初期形成的小的黏着复合物逐渐成熟,聚集在一起形成较大的FAs。肌动蛋白（F—actin）聚集形成的螺线圈样微丝结构逐渐被应力纤维代替,细胞也由圆形变为具有极性的梭形或多角形。黏着斑激酶（focal adhesion kinase,FAK）和桩蛋白（paxillin）具有调节FAs聚合及骨架重排的作用,其中,Y397－FAK和Y31／Y118－paxillin的磷酸化活性在细胞铺展过程中不断变化。FAs组装时,Y397－FAK的磷酸化活性升高;FAs成熟后,Y397．FAK的磷酸化活性下降。活化的FAK能够磷酸4LY31／Y118-paxillin,激活paxillin参与调节细胞骨架的形成和排列。血管内皮生长因子（vascular endothelial growthfactor,VEGF）诱导~SMSCs黏附过程中,细胞面积变大,完全铺展的时间缩短,黏着斑及细胞骨架的形成均提前。另外,VEGF诱导的细胞铺展过程中形成的FAs形态细长,数量较多。该研究表明,VEGF通过调节黏着斑和细胞骨架促L~MSCs的黏附与铺展,提示vEGF可以通过调节黏着斑进而调控MSCs的定向迁移,为细胞迁移行为的研究提供理论基础。     

Light-Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells

Abstract: The signal pathway for light-induced expression of c- fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c- fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c- fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca²⁺ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c- fos /SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser¹³³ and CaM kinase II in the neural retina.     

Growth Factor Regulation of Prostaglandin-Endoperoxide Synthase 2 (Ptgs2) Expression in Colonic Mesenchymal Stem Cells

We previously found that a population of colonic stromal cells that constitutively express high levels of prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox-2) altered their location in the lamina propria in response to injury in a Myd88-dependent manner (Brown, S. L., Riehl, T. E., Walker, M. R., Geske, M. J., Doherty, J. M., Stenson, W. F., and Stappenbeck, T. S. (2007) J. Clin. Invest. 117, 258–269). At the time of this study, the identity of these cells and the mechanism by which they expressed high levels of Ptgs2 were unknown. Here we found that these colonic stromal cells were mesenchymal stem cells (MSCs). These colonic MSCs expressed high Ptgs2 levels not through interaction with bacterial products but instead as a consequence of mRNA stabilization downstream of Fgf9 (fibroblast growth factor 9), a growth factor that is constitutively expressed by the intestinal epithelium. This stabilization was mediated partially through a mechanism involving endogenous CUG-binding protein 2 (CUGbp2). These studies suggest that Fgf9 is an important factor in the regulation of Ptgs2 in colonic MSCs and may be a factor involved in its constitutive expression in vivo.     

Production of 1,2-Diacylglycerol in PC12 Cells by Nerve Growth Factor and Basic Fibroblast Growth Factor

The addition of nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) to PC12 cells prelabeled with [3H]inositol and preincubated for 15 min in the presence of 10 mM LiCl stimulated the production of inositol phosphates with maximal increases of 120-180% in inositol monophosphate (IP), 130-200% in inositol bisphosphate (IP2), and 45-50% in inositol trisphosphate (IP3) within 30 min. The majority of the overall increase (approximately 85%) was in IP; the remainder was recovered as IP2 and IP3 (approximately 10% as IP2 and 5% as IP3). Under similar conditions, carbachol (0.5 mM) stimulated about a 10-fold increase in IP, a sixfold increase in IP2, and a fourfold increase in IP3. The mass level of 1,2-diacylglycerol (DG) in PC12 cells was found to be dependent on the incubation conditions; in growth medium [Dulbecco's modified Eagle's medium (DME) plus serum], it was around 6.2 mol %, in DME without serum, 2.5 mol %, and after a 15-min incubation in Dulbecco's phosphate-buffered saline, 0.62 mol %. The addition of NGF and bFGF induced an increase in the mass level of DG of about twofold within 1-2 min, often rising to two- to threefold by 15 min, and then decreasing slightly by 30 min. This increase was dependent on the presence of extracellular Ca2+, and was inhibited by both phenylarsine oxide (25 microM) and 5'-deoxy-5'-methylthioadenosine (3 mM). Under similar conditions, 0.5 mM carbachol stimulated the production of DG to the same extent as 200 ng/ml NGF and 50 ng/ml bFGF. Because carbachol is much more effective in stimulating the production of inositol phosphates, the results suggest that both NGF and bFGF stimulate the production of DG primarily from phospholipids other than the phosphoinositides.     

人碱性成纤维细胞生长因子突变体的高效表达

用PCR法将人碱性成纤维细胞生长因子(hbFGF)基因中编码第25、69和92位的半胱氨酸(Cys)密码子突变为丝氨酸(Ser)密码子,将突变的hbFGFcDNA片断与表达质粒pET3c连接,构建重组质粒pET3chbFGFSer25,69,92。hbFGFSer25,69,92在大肠杆菌BL21(DE3)中的表达量大于30%。通过阳离子交换和肝素亲和层析两步纯化,得到纯度大于95%的hbFGFSer25,69,92。MTT法测定纯化的产物活性表明,hbFGFSer25,69,92突变体促Balb/c细胞增殖的活性与野生型hbFGF相当,为下一步对hbFGFSer25,69,92突变体进行定点化学修饰打下了基础。     

Regulation of Tyrosine Hydroxylase Gene Expression in IMR-32 Neuroblastoma Cells by Basic Fibroblast Growth Factor and Ciliary Neurotrophic Factor

Abstract: The actions of basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF) on tyrosine hydroxylase (TH) gene expression were studied using IMR-32 neuroblastoma cells. Treatment of these cells with bFGF for 3 days induced the expression of detectable levels of immunoreactive TH protein and TH mRNA. In contrast, CNTF did not affect TH expression unless bFGF was present. In the presence of saturating amounts of bFGF, CNTF increased TH protein and mRNA levels of TH two- to threefold over those found in bFGF-treated cultures. The effects of CNTF on TH expression diminished with increasing culture time, and after 6 days of incubation CNTF no longer enhanced TH levels. The requirement for bFGF as cofactor in the effects of CNTF on TH was specific, as CNTF did not affect TH when it was coadministered with 8-(4-chlorophenylthio)-cyclic AMP, another agent that stimulates TH development in this cell line, and bFGF was not required for CNTF to stimulate the development of choline acetyltransferase. Moreover, cotreatment with bFGF reduced the ability of CNTF to enhance choline acetyltransferase. These results demonstrate that bFGF and CNTF can enhance expression of TH and that bFGF can modify the effects of CNTF on neurotransmitter phenotype.     

碱性成纤维细胞生长因子对牙周膜细胞内核心蛋白多糖基因表达的影响

利用外源性碱性成纤维细胞生长因子(Basic Fibroblast Growth Factor,bFGF)刺激体外培养的人正常牙周膜细胞.采用逆转录-聚合酶链式反应(RT-PCR)检测细胞内decorin的基因表达的变化,研究bFGF对体外培养的人牙周膜细胞内核心蛋白多糖(decorin)的作用,进一步探讨bFGF抑制Ⅰ型胶原的作用机制.发现bFGF刺激牙周膜细胞后能促进牙周膜细胞的增殖,bFGF抑制decorin的合成是bFGF促进牙周膜细胞增殖的重要调节因素之一.     

Fibroblast Growth Factor Signaling Is Essential for Self-renewal of Dental Epithelial Stem Cells

A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.     

重组人碱性成纤维细胞生长因子(bFGF)融合蛋白的高效表达及鉴定

碱性成纤维细胞生长因子（ｂＦＧＦ）参与了许多细胞生长和分化的调控过程。本文采用重组ＤＮＡ技术在大肠杆菌中高效表达了人ｂＦＧＦ。首先将编码人ｂＦＧＦ基因克隆到ｐＸＴ表达载体中与其上游的一短Ｓ导肽共一阅读框架,ｂＦＧＦ基因的表达受强的Ｔ７启动子调控。采用ＢＬ２１（ＤＥ３）大肠杆菌作为宿主菌,用ＩＰＴＧ诱导ＢＬ２１（ＤＥ３）细菌合成的Ｔ７ＲＮＡ聚合酶,后者可催化高水平的ｂＦＧＦ基因表达,其ｂＦＧＦ产量可占总菌体蛋白的４２.５％。采用肝素Ｓｅｐｈａｒｏｓｅ一步亲和层析法直接从诱导后的细菌裂解产物中得到纯化的重组人ｂＦＧＦ蛋白。经Ｗｅｓｔｅｒｎ印迹分析证明该蛋白可被人ｂＦＧＦ特异性单克隆抗体所识别。进一步研究证明该蛋白具有刺激ＮＲ６Ｒ－３Ｔ３成纤维细胞增殖的生物学活性,并且这一活性可被人ｂＦＧＦ特异性中和抗体所中和。     

Enhancement of Apoptosis in Developing Chick Neural Retina Cells by Basic Fibroblast Growth Factor

Abstract: To evaluate the role of various growth factors in naturally occurring cell death during development of the neural retina, we examined the effects of such factors on the nuclear morphology and the size of DNA in cultured chick embryonic neural retina cells. Basic fibroblast growth factor (bFGF) increased internucleosomal cleavage of DNA and nuclear fragmentation in a time- and dose-dependent manner. The effect was inhibited by anti-bFGF antibody, suramin, and cycloheximide. Epidermal growth factor, platelet-derived growth factor, nerve growth factor, tumor necrosis factor-α, and dexamethasone had no effect. These results provide evidence that bFGF may eventually act as a lethal factor inducing apoptotic cell death during the development of the neural retina in chick embryo.