肝细胞生长因子促大鼠神经干细胞增殖的作用

目的:研究肝细胞生长因子(HGF)对大鼠神经干细胞(NSCs)增殖的作用以及PI3K/Akt信号途径的影响.方法:分离培养大鼠NSCs,通过向神经培养基中添加HGF(10,30,60 ng/ml),计数干细胞克隆形成率、四甲基偶氮唑蓝(MTT)比色法以及5-溴-2'-脱氧尿苷(BrdU)标记检测NSCs的增殖,Annexin V/FITC免疫荧光显色测定NSCs的凋亡;免疫印迹法检测细胞磷酸化Akt蛋白表达的变化.结果:与对照组相比,HGF各剂量组NSCs克隆率明显增加,生长速度增快,BrdU 阳性细胞增加,NSCs凋亡率显著减少.此外,HGF可上调磷酸化Akt蛋白的表达,HGF的效应可被PI3K/Akt通路抑制剂LY294002阻断.结论:HGF可促进大鼠NSCs增殖,抑制其凋亡,其作用机制可能与激活NSCs的PI3K/Akt信号转导通路有关.     

Hepatocyte growth factor effects on mesenchymal stem cells: proliferation, migration, and differentiation

Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin promoting migration, proliferation, and survival in a wide spectrum of cells, can also modulate different biological responses in stem cells, but the mechanisms involved are not completely understood so far. In this context, we show that short-term exposure of mesenchymal stem cells (MSCs) to HGF can induce the activation of its cognate Met receptor and the downstream effectors ERK1/2, p38MAPK, and PI3K/Akt, while long-term exposure to HGF resulted in cytoskeletal rearrangement, cell migration, and marked inhibition of proliferation through the arrest in the G1-S checkpoint. When added to MSCs, the K252A tyrosine kinase inhibitor prevented HGF-induced responses. HGF's effect on MSC proliferation was reversed by p38 inhibitor SB203580, while the effects on cell migration were abrogated by PI3K inhibitor Wortmannin, suggesting that HGF acts through different pathways to determine its complex effects on MSCs. Prolonged treatment with HGF induced the expression of cardiac-specific markers (GATA-4, MEF2C, TEF1, desmin, alpha-MHC, beta-MHC, and nestin) with the concomitant loss of the stem cell markers nucleostemin, c-kit, and CD105.     

Hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice

Although it is known that the lung undergoes compensatory growth after pulmonary resection, mechanisms by which lung cells exhibit compensatory proliferation are not well defined. We investigated the involvement of hepatocyte growth factor (HGF) in postpneumonectomy compensatory lung regeneration in mice, because HGF has mitogenic and morphogenic actions on lung epithelial cells. Following left pneumonectomy, alveolar and airway epithelial cells underwent compensatory DNA synthesis, reaching maximal levels 5 d after the surgery. Before changes in DNA synthesis in lung epithelial cells, expression of HGF mRNA and protein levels in the remaining lung, liver, and kidney were changed in response to left pneumonectomy, and these changes were associated with postoperative increases in plasma HGF levels. c-Met/HGF receptor expression was localized predominantly in alveolar type II and airway epithelial cells, whereas c-Met/HGF receptor mRNA expressions were transiently upregulated before the peak in lung DNA synthesis. Neutralization of endogenous HGF by an antibody in pneumonectomized mice suppressed the compensatory DNA synthesis in lung epithelial cells, whereas administration of recombinant HGF to pneumonectomized mice stimulated DNA synthesis in lung epithelial cells. These results strongly suggest that HGF has a role as a pulmotrophic factor in postpneumonectomy compensatory lung regeneration.     

成纤维细胞生长因子信号调控早期鸡胚胎神经嵴细胞的迁移

目的 利用Sprouty2基因阻断成纤维细胞生长因子（FGF）信号,探讨FGF在早期鸡胚胎发育过程中对神经嵴细胞迁移的影响及其机制。方法 通过体内培养的方法孵育鸡胚至HH9期,通过显微注射的方法将Sprouty2-绿色荧光蛋白（GFP）质粒注射入神经管腔内。实验侧使用电穿孔转染的方法转染胚胎半侧神经管,另一侧正常神经管设为对照侧。采用神经嵴细胞特异标记物HNK1免疫荧光的方法检测Sprouty2基因阻断FGF信号后是否影响胚胎头部和躯干部神经嵴细胞的迁移过程。随后,进一步通过检测神经细胞钙黏分子N-Cadherin的表达来观察细胞之间黏附作用的改变。结果 HNK1免疫荧光检测结果显示,Sprouty2转染侧即阻断FGF信号通路后,HNK1在早期鸡胚胎的头部和躯干部的表达量均比对照侧的表达量增多;而神经细胞钙黏分子N-Cadherin检测结果表明,Sprouty2转染侧和正常对照侧N Cadherin在头部和躯干部神经管上表达量的差异均无显著性。结论 Sprouty2基因阻断FGF信号后,促进了早期鸡胚胎神经嵴细胞的迁移,但是FGF信号对此过程的影响可能不是由神经钙黏分子N-Cadherin介导的。     

Transgelin is a marker of repopulating mesangial cells after injury and promotes their proliferation and migration

Mesangial cell (MC) migration is essential during glomerular repair and kidney development. The aim of the study was to identify marker/player for glomerular progenitor/reserve cells migrating into the glomerulus after MC injury and during glomerulogenesis in the rat. Experimental mesangial proliferative nephritis was induced in Sprague Dawley rats by intravenous injection of OX-7 antibody. We investigated mRNA expression profiles in isolated glomeruli from on days 0, 1, 2, 3, and 5 after induction of anti-Thy1 nephritis using Affymetrix microarray technology. Using self-organizing maps, transgelin was identified as a new marker for repopulating glomerular cells. Expression of transgelin during anti-Thy1 nephritis was investigated by northern blot, real-time PCR, western blot, and immunohistochemistry. Migration and proliferation assays using isolated MCs after transgelin knockdown by siRNA were performed to investigate the potential role of transgelin during glomerular repopulation. Transgelin mRNA was not detected in healthy glomeruli. It was strongly upregulated during the repopulation process starting on day 1, continued to be increased until day 5 and disappeared on day 7. Transgelin was specifically expressed at the edge of the migratory front during glomerular repopulation as indicated by transgelin/OX-7 double staining. Transgelin expression was similar in migrating vs non-migrating MCs in vitro. Blocking of transgelin expression by siRNA treatment resulted in inhibition of MC migration and proliferation. Transgelin was also expressed in MCs during glomerulogenesis and in biopsies from patients with IgA nephritis. In conclusion, transgelin in the kidney is upregulated in repopulating MCs in vivo and supports their migratory and proliferative repair response after injury.     

Geminin is essential for the development of preimplantation mouse embryos

Replication of DNA is strictly controlled to ensure that it occurs only once per cell cycle. Geminin has been thought to serve as a central mediator of this licensing mechanism by binding to and antagonizing the function of Cdt1 and thereby preventing re-replication during S and G2 phases. We have now generated mice deficient in geminin to elucidate the physiologic role of this protein during development. Lack of geminin was shown to result in preimplantation mortality. A delay in the development of homozygous mutant embryos was first apparent at the transition from the four- to eight-cell stages, concomitant with the disappearance of maternal geminin protein, and development was arrested at the eight-cell stage. The mutant embryos manifest morphological abnormalities such as dispersed blastomeres with nuclei that are irregular both in size and shape as well as impaired cell-cell adhesion. DNA replication occurs but mitosis was not detected in the mutant embryos. The abnormal blastomeres contain damaged DNA and undergo apoptosis, likely as a consequence of the deregulation of DNA replication. Our results suggest that geminin is essential for cooperative progression of the cell cycle through S phase to M phase during the preimplantation stage of mouse development.     

Cerebral endothelial derived vascular endothelial growth factor promotes the migration but not the proliferation of oligodendrocyte precursor cells in vitro

In gray matter, cerebral endothelium is known to provide trophic support for neighboring cells such as neurons. However, signaling from cerebral endothelium to white matter cells remains to be elucidated. Here, we show that vascular endothelial growth factor (VEGF-A) secreted from cerebral endothelial cells promotes the migration but not the proliferation of oligodendrocyte precursor cells (OPCs). Cultured OPCs were obtained from newborn rat cortex, and treatment with conditioned culture media of cerebral endothelial cells increased the OPC proliferation and migration. Importantly, co-treatment with anti-neutralizing antibody for Flk-1 (VEGF-receptor2) inhibited OPC movement but did not affect OPC propagation. Western blot and flow cytometry analyses confirmed that our cultured cerebral endothelial cells produced VEGF-A and our cultured OPCs expressed Flk-1. Taken together, our current data suggest that cerebral endothelium is supportive for oligodendrocyte lineage cells and VEGF-A may participate in the endothelium–OPC cell–cell signaling. This phenomenon may be important for white matter homeostasis.     

肝细胞生长因子基因转染对人脐静脉内皮细胞增殖、迁移的影响

目的:观察HGF基因对人脐静脉内皮细胞增殖、迁移的影响。方法:从已有质粒pRc/CMV-HGF中扩增出HGF基因,将其克隆到含增强型绿色荧光蛋白的真核表达载体中,构建重组质粒pEGFP-HGF,酶切及测序鉴定正确后,用脂质体将重组质粒pEGFP-HGF转染到人脐静脉细胞株ECV304中,G418筛选获得稳定表达细胞克隆,采用荧光显微镜观察、RT-PCR、免疫细胞化学方法检测鉴定重组质粒的表达情况;酶联免疫吸附法(ELISA)检测稳定表达细胞中HGF的含量;再以MTT法检测转染重组质粒后细胞增殖的改变,Transwell Migration实验检测细胞迁移能力的改变。结果:所构建重组质粒经酶切图谱分析和序列测定证实构建成功;荧光显微镜下观察到有绿色荧光;RT-PCR证实HGFmRNA在转染阳性细胞高表达;免疫细胞化学证实转染pEGFP-HGF质粒的细胞有HGF蛋白的表达;ELISA检测细胞培养基中HGF含量可达112.3 ng/ml,MTT法、TranswellMigration测定转染pEGFP-HGF阳性细胞的增殖、迁移能力明显高于对照组(P<0.01)。结论:重组质粒pEGFP-HGF能够在内皮细胞株ECV304转录、表达;表达的活性蛋白HGF刺激ECV304的增殖、迁移,为进一步应用于基因治疗奠定实验基础。     

Insulin-like growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos.

We present evidence that insulin-like growth factor II (IGF-II) mediates growth in early mouse embryos and forms a pathway in which imprinted genes influence development during preimplantation stages. mRNA and protein for IGF-II were expressed in preimplantation mouse embryos, but the related factors IGF-I and insulin were not. IGF-I and insulin receptors and the IGF-II/mannose-6-phosphate receptor were expressed. Exogenous IGF-II or IGF-I increased the cell number in cultured blastocysts, but a mutant form of IGF-II that strongly binds only the IGF-II receptor did not. Reduction of IGF-II expression by antisense IGF-II oligonucleotides decreased the rate of progression to the blastocyst stage and decreased the cell number in blastocysts. Preimplantation parthenogenetic mouse embryos expressed mRNA for the IGF-II receptor but not for either IGF-II ligand or the IGF-I receptor, indicating that the latter genes are not expressed when inherited maternally. These data imply that some growth factors and receptors, regulated by genomic imprinting, may control cell proliferation from the earliest stages of embryonic development.     

角质细胞生长因子促进大鼠胰腺导管上皮细胞体外增殖

目的 探讨不同浓度的角质细胞生长因子(KGF)对大鼠胰腺导管上皮细胞增殖的影响,寻求最佳浓度.方法 免疫细胞化学染色及RT-PCR方法鉴定SD大鼠胰腺导管上皮细胞;不同浓度的KGF刺激胰腺导管上皮细胞增殖,寻求最佳浓度.结果 大鼠胰腺导管上皮细胞表达Nestin和CK19,不表达Insulin及Glucagon;不同浓度KGF刺激胰腺导管上皮细胞2d后,细胞均有不同程度增殖,20 μg/L KGF作用2d时,细胞数为0.35±0.03,显著高于对照组的0.27±0.02(P ＜0.01).结论 KGF在本实验的剂量范围内可促进胰腺导管上皮细胞增殖,20 μg/L为促进增殖的最佳浓度.     

Onset of gastrulation, morphogenesis and somitogenesis in mouse embryos displaying compensatory growth

This is a study on the ability of mouse embryos to compensate for a loss of cells and to develop with body parts of normal size and normal proportions during postimplantation development. Micro-manipulations were performed on 4-cell pre-implantation mouse embryos to reduce the number of cells by 25% (3/4 embryos) or 50% (2/4 embryos). Blastocysts developed from these embryos showed a preferential loss of inner cell mass population, and fewer of them formed viable embryos after implantation. The size of post-implantation 3/4 embryos was initially smaller than controls of the same gestational age, but compensatory growth, achieved by increasing cell numbers at above the normal rate and beyond the normal duration, took place between 6.5 and 11.5 days, resulting in a complete restoration of body size. During compensatory growth the 3/4 embryos rescheduled events of gastrulation and morphogenesis in keeping with cell number or body size appropriate for each developmental stage. The formation of the correct number of somites was accomplished by changing the rate of somite segmentation and by an adjustment of the size of individual somites and somitomeres proportional to the available amount of precursor tissues. Morphogenesis and pattern formation in embryos recovering from earlier cell losses are therefore regulated in accordance to tissue volume (or cell number) instead of chronological age or some intrinsic cellular clock.     

PKN2 is essential for mouse embryonic development and proliferation of mouse fibroblasts

PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell–cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2^?/? embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2^?/? embryos at E9.5 failed to grow. Cre‐mediated ablation of PKN2 in PKN2^flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S‐phase population. Our results show that PKN2 is essential for mouse embryonic development and cell‐autonomous proliferation of primary MEFs in culture. Comparison of the PKN2^?/? phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.     

Fertilization and early embryology: Microsurgical correction of partially degenerate mouse embryos promotes hatching and restores their viability

We have evaluated the effects of degeneration of blastomereson the developmental fate of mouse embryos. Micro-manipulationtechniques were used first to destroy one or two blastomeresof a 4-cell embryo (thereby creating three-quarter and halfembryos), and later to repair the anomaly by removing the degeneratematerial. The embryos were either cultured in protein-free orprotein-supplemented medium. When cultured in protein-supplementedmedium, three-quarter embryos hatched at the same rate as intactembryos (84 and 91%, respectively), but this rate was reduced(54%; 67/125) when the embryos were cultured in a protein-freeenvironment. Destruction of two blastomeres of a 4-cell embryoand culture in protein-free medium was detrimental, as only3.2% (4/124) hatched. By supplementing the culture medium withprotein, some of these half embryos were rescued, as shown bya 34% (32/95) hatching rate. A more dramatic increase in hatchingwas achieved, however, after repair of the half embryos by microsurgicalremoval of the degenerate material. In this case, 72% (78/109)of the repaired embryos were able to hatch. These findings mayhave implications for human in-vitro fertilization where partialembryonic degeneration or fragmentation often leads to embryonicdemise and reduced implantation. Moreover, these observationsmay provide important clues to mechanisms of mammalian embryonichatching.     

Vascular endothelial growth factor immobilized in collagen scaffold promotes penetration and proliferation of endothelial cells

A key challenge in engineering functional tissues in vitro is the limited transport capacity of oxygen and nutrients into the tissue. Inducing vascularization within engineered tissues is a key strategy to improving their survival in vitro and in vivo. The presence of vascular endothelial growth factor (VEGF) in a three-dimensional porous collagen scaffold may provide a useful strategy to promote vascularization of the engineered tissue in a controlled manner. To this end, we investigated whether immobilized VEGF could promote the invasion and assembly of endothelial cells (ECs) into the collagen scaffolds. We conjugated VEGF onto collagen scaffolds using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride chemistry, and measured the concentrations of immobilized VEGF in collagen scaffolds by direct VEGF enzyme-linked immunosorbent assay. We demonstrated that immobilized VEGF (relative to soluble VEGF) promoted the penetration and proliferation of ECs in the collagen scaffold, based on results of cell density analysis in histological sections, immunohistochemistry, XTT proliferation assay, glucose consumption and lactate production. Furthermore, we observed increased viability of ECs cultured in scaffolds with immobilized VEGF relative to soluble VEGF. This research demonstrates that immobilization of VEGF is a useful strategy to promote the invasion and proliferation of ECs into a scaffold, which may in turn lead to a vascularized scaffold.     

RAGE expression in rhabdomyosarcoma cells results in myogenic differentiation and reduced proliferation, migration, invasiveness, and tumor growth

Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEDeltacyto) (TE671/RAGEDeltacyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH(2) terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEDeltacyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential.