| Background and ObjectivesNestin is a class Ⅵ intermediate filament protein mainly distribution in cytoplasm. Nestin is expressed in neural system, pancreas, myocardium, skeletal, muscle, dental, paranephros, testicular and tumors. In1985, Hockfield and Mckay firstly found that nestin is expressed in embryonic rat spinal cord neural tube neural precursor cells. Nestin was highly expressed in the early embryonic development such as the development of neural tube, gastrula and the formation of some organs. Upon differentiation, nestin expression is down-regulated and finally disappear and do not express in the adult cells, however, nestin expression is up-regulated in the adult under pathological conditions. Recently, more and more studies found that nestin not only expressed in the central nervous system tumors but also expressed in breast cancer, prostate cancer, thyroid cancer, adrenal carcinoma, pancreatic cancer and melanoma. Xiang Peng found that nestin also expression in angiosarcoma, pancreatic adenocarcinoma, gastrointestinal stromal tumor(GIST)and its higher levels of expression seem to be correlate with tumor malignant phenotype. Recent studies indicate that nestin has an important role of distribution and organization of cellular factors regulating cell proliferation, migration, and differentiation. Li-chun wei found that Ki67expression was not reduced in astrocytoma cells after nestin was knockdown, those findings indicate that nestin has not direct relation with cells proliferation. On the other hand, nestin was found to be bind the Cdk5/p35complex and plays a critical role of anti-apoptosis. Furthermore, a recent study showed that loss of nestin in zebrafish embryos cause a defects in brain and eye development due to increased apoptosis of NPCs. However, down-regulating nestin expression in the mice cerebral cortex markedly suppressed the formation of NPCs due to reduced cells proliferation. In addition, recent studies indicate that nestin may promote PI3K activity through a competitive binding with glucocorticoid receptor (GR) and through a series of enzymatic reaction by downstream promote cell into the proliferation cycle. Recent reports showed that growth factor signaling pathways regulates the expression of nestin and thereby it is essential for cell proliferation and/or survival. For example, PDGF, which promotes cell proliferation in mesangial cells, was shown to up-regulate nestin expression. Thrombin was also reported to increase nestin expression and concomitantly stimulate the growth of radial glial cells in vitro. These findings indicate that nestin effects on cell proliferation and survival regulated by growth factors signaling pathways. The above results show that the functional significance of nestin whether it is regulating of cells apoptosis and/or cells proliferation has not been fully elucidated.As a marker of neural stem cells, nestin expression mode is particularly. Nestin generally express in the differentiation of immature cells, upon differentiation, nestin expression is down-regulated and finally disappear. To this end, we propose a hypothesis that over-expression of nestin can maintain cell proliferation? Overexpression of nestin can influence cells normal apoptosis process in the development process?As an important role in the study of the gene functions, a gene over-expression technology is widely favored. So far, there is no relate reports for nestin over-expression transgenic mice functions in vivo. In order to examine the role of nestin in intact mammalian tissue, we used the nestin overexpression transgenic mice was reported previously. In the early stage study, we found that nestin over-expression can increase the proliferative capacity in the case of partial hepatectomy (PH). In this study, we performed to study the effects of nestin over-expression on mice embryonic development and further explore nestin function in the mammals intact.Contents and methods1.To determine the relative levels of nestin expression in the transgenic mice embryos major organsTissue expressed of nestin overexpression status in the transgenic mice embryos major organs (brain, heart, liver, lung, skin) was determined by PCR, Q-PCR and Western blotting analysis.2.The influence of nestin on transgenic mice embryonic growthWe chosen the male transgenic mice to be bred with female transgenic mice, and collected embryos range from E11.5to E18.5. Macroscopic analysis the morphological changes between the transgenic and wild type mice embryonic. Hematoxylin and eosin (H&E) stained analysis the morphological difference between the nestin overexpression embryos and the controls.3.The influnce of nestin on mice heart and brain proliferation and apoptosisTo examine whether the increase in heart and hindbrain size was due to cell proliferation increased and/or apoptosis reduced. The proportions of proliferating cells and apoptosis cells in the heart and forebrain region were examined using5-bromo-2’-deoxy-uridine (Brdu) and DeadEnd TUNEL System, respectively.To examine cell differentiation in the forebrain tissues, we performed GFAP staining in forebrain sections.To examine cell proliferation in the NSCs compartment, we also co-stained ventral telencephalic tissue sections with antibodies against SOX2, a marker for NSCs, and Ki67, a marker of cell proliferation.4.To detection the effect of nestin over-expression status on PI3K/Akt signaling pathwayActivation of PI3K and Akt in the heart and brain tissue was assessed by western blotting using phosphorylated antibody p-PI3K and p-Akt, respectively. In addition, western blot determine the change of apoptosis gene such as casepase3and BCL2in transgenic mice heart and brain.Results1.Nestin is highly expression in the embryos major organs of transgenic miceWe used PCR analysis determined the DNA levels of nestin in the transgenic mice, the results showed that compared with wild type mice, the transgenic mice at about500bp detected positive signal; We used Q-PCR method determined the mRNA levels of nestin in the transgenic mice embryonic major organs, Q-PCR analysis showed that the mRNA expression level of nestin was much higher in transgenic embryos major organs (brain, heart, liver, lung, skin) than those in wild type embryos. Agarose gel electrophoresis recycling product display that nestin gene expression was significantly increased in transgenic mice embryonic organs compared with the controls. Furthermore, western blotting analysis showed that the protein expression levels of nestin was also significantly increased in the embryos major organs (brain, heart, liver, lung, skin) of transgenic mice compared with wild type mice. 2.Nestin overexpression promotes an increase in the size of heart and forebrain in mouse embryosWe collected transgenic and wild type mice embryos range from E11.5to E18.5. The transgenic embryonic mice apparently looked normal at E11.5and E12.5similar to the non-transgenic mice. However, starting from E13.5, the transgenic embryos were appeared bigger than embryos from non-transgenic mice (as determined by crown-rump length), these distinguishable were most obvious in E13.5-E14.5embryonic period. These transgenic and control embryos were not displayed development defect, such as widespread hemorrhage, edema and pale, et al. To study the contribution of nestin overexpression on embryos development, we examined hematoxylin and eosin (H&E) stained sections of nestin overexpression embryos and the controls. We found that the transgenic embryos had a larger heart when compared to the wild type embryos at E13.5, E14.5and E15.5embryonic period. There were an striking increase in compact myocardium and trabecular myocardium thickness compared with hearts of wild type embryos. The cardiomyocytes of the transgenic embryos were larger in size than of wild type embryos. Myocardial cell division actively, we can determined the visible dual-core and naked nucleus. Furthermore, at E13.5, transgenic embryos had a larger hind-brain when compared to the wild type embryos. Transgenic embryos had an increase cortex thickness, larger brain cells in size, larger nucleus and nucleolus than of the wild type embryos. We also detected a striking increase in the thickness of the myocardum.At E13.5,the compact myocardium wall was51±9μm thick, an increase of more than2times compared to non-transgenic mice (23±6, n=6, P<0.05). The trabecular myocardium wall was also1.3times thicker,19±4μm versus14±2, of non-transgenic animals (n=6, P<0.05). In addition, cardiomyocytes were larger in transgenic mice,3698±1440arbitrary units compared to1050±452(n=50, P<0.0001). At E14.5, the compact myocardium wall was60±4μm thick, an increase of more than2times compared to non-transgenic mice (26±5, n=6, P<0.05). The trabecular myocardium wall was also1.3times thicker,20±4μm versus15±2, of non-transgenic animals (n=6, P<0.05). In addition, cardiomyocytes were larger in transgenic mice,3883±1163arbitrary units compared to1209±427(n=50, P<0.0001). At E15.5, the compact myocardium wall was73±8μm thick, an increase of more than1.8times compared to non-transgenic mice (42±3, n=6, P<0.05). The trabecular myocardium wall was also1.4times thicker,25±3μm versus18±1, of non-transgenic animals (n=6, P<0.05). In addition, cardiomyocytes were larger in transgenic mice,3900±1262arbitrary units compared to1274±479(n=50, P<0.0001). Transgenic E13.5, embryos also had a larger forebrain with a two fold increase in the thickness of the cerebral cortex (324±97μm versus157±82of non-transgenic mice; n=6, P<0.05) and larger cells (3688±2839arbitrary units versus753±334; n=50, P<0.0001).In all transgenic animals were viable, healthy, and did not exhibit premature death or sign of heart failure or neural tube defect after1year of observation.3.Overexpression of nestin causes cell proliferation increased and does not affect apoptosisAs we known, the size of an organ is determined by the coordinate regulation of cell proliferation and apoptosis during development. By balancing these processes, proper size of various organs and composition of cell types can be achieved. In order to determine whether nestin over-expression can increase the proliferation status in heart and brain. The proportions of proliferating cells in the heart were examined using5-bromo-2’-deoxy-uridine (Brdu) methods. Pregnant mice at E13.5, E14.5and E15.5were injected intraperitoneally with Brdu (120mg/kg body weight)6h before the embryos were collected by Caesarean section. Tissue sections were incubated with anti-Brdu monoclonal antibody (1:20dilution) overnight at4℃. Then, the sections were incubated in peroxidase-conjugated anti-mouse. Brdu-positive cells were counted against nuclei counterstained with DAB. The proliferation index was determined by counting the Brdu-positive cells in relation to total cells in every three sagittal section of the heart and brain under a x200objective for each group (transgenic and control) in each independent experiment. The Brdu assay showed that the number of Brdu-labeling cells in the trabecular and compact myocardium was increased in nestin transgenic embryos compared to wild type embryos.At E13.5, the number of proliferation cells in the heart was increased in nestin transgenic embryos (trabecular and compact myocardium,>13%,p=0.0024) compared to wild type embryos; At E14.5, the number of proliferation cells in the heart was increased in nestin transgenic embryos (trabecular and compact myocardium,>24%, p=0.0007) compared to wild type embryos; At E15.5, the number of proliferation cells in the heart was increased in nestin transgenic embryos (trabecular and compact myocardium,>5.5%,p=0.015) compared to wild type embryos. Additional, at E13.5, the total number of Brdu-positive cells in the hind-brain was significantly increased in nestin transgenic embryos (hind-brain,>32%, p=0.016) when compared to wild type embryos. In principle, the increased thickness of the cerebral cortex in transgenic mice could be the result of a widespread differentiation of astrocytes, resulting in a larger number of proliferating NSCs. To test this hypothesis, we performed GFAP staining in forebrain sections. The results indicate an almost complete absence of GFAP+cells, suggesting that nestin overexpression does not affect NSCs differentiation.In order to examine cell proliferation in the hindbrain was specifically neural stem cells (NSCs), we co-stained brain sections with anti-bodies against SOX2, a marker for NSCs, and Ki67, a marker for proliferation cells. We found that among SOX2+cells, the total number of Ki67+cells was significantly increased in nestin transgenic embryos when compared to wild type embryos. These results demonstrated that the increase proliferation cell in the hind-brain almost NSCs. Moreover, In order to determine whether nestin over-expression can effect on cells apoptosis in heart and brain. The apoptosis cells in embryonic heart and brain was detected by TUNEL assay. TUNEL assay showed that the percentage of apoptosis cells in the heart and hindbrain was comparable between wildtype and nestin transgenic embryos.4.Activation of PI3K-Akt signaling pathway in nestin transgenic embryosActivation of PI3K/Akt in the heart and brain tissue was assessed by western blotting using phosphorylated antibody p-PI3K and p-Akt, respectively. In addition, Western blot determine the changes of apoptosis gene such as casepase3and BCL2in transgenic mice heart and brain. In embryos brain, the amount of phosphorylated PI3K was increased and the amount of phosphorylated Akt was increased in the nestin transgenic embryos when compared to the wild type embryos. Furthermore, in embryos hearts, the amount of phosphorylated PI3K was also increased and the amount of phosphorylated Akt was increased in the nestin transgenic embryos when compared to the wild type embryos. These results indicate that over-expression of nestin promotes PI3K/Akt signaling pathway activity in the intact tissue. In addition, we also examined the activation of casepase3and BCL2, which are associated with cells apoptosis. In both embryos heart and brain tissue, there was no significantly difference in the amounts of casepase3and BCL2between wild type and nestin transgenic mice.Conclusion1.The expression levels of nestin was significantly increased in the embryos major organs (brain, heart, liver, lung, skin) of transgenic mice compared with wild type mice. These results demonstrated that the transgenic mice displayed major organs over-expression of human nestin gene. And this model can use to study of nestin is function in vivo.2.The transgenic embryos were appeared bigger than embryos from non-transgenic mice. The transgenic embryos had a larger heart and hind-brain when compared to the wild type embryos. There were an striking increase in compact myocardium and trabecular myocardium thickness compared with hearts of wild type embryos. Furthermore, the transgenic embryos had an increase cortex thickness, larger brain cells in size than of the wild type embryos. These results demonstrate that nestin is necessary and sufficient to promote heart and brain growth and development.2.The number of proliferation cells in the heart and hind-brain was increased in nestin transgenic embryos when compared to wild type embryos. There is no significantly differences apoptosis cells between wild type and nestin transgenic embryos. These results demonstrate that over-expression of nestin effect on mice heart and brain development through promoting the proliferation of myocardial cells and NSCs.4.The amount of phosphorylated PI3K and the amount of phosphorylated Akt was increased in the nestin transgenic embryos when compared to the wild type embryos. There was no significantly difference in the amounts of casepase-3and BCL-2between wild type and nestin transgenic mice. These results indicate that nestin may be one of a upstream regulatory genes of PI3K/Akt signaling pathway and nestin affect embryonic organs development and growth due to promotes PI3K/Akt signaling pathway activity in the intact tissue. |
PDF Full Text Request