[Show abstract][Hide abstract] ABSTRACT: The poor survival of cells in ischaemic myocardium is a major obstacle for stem cell therapy. Exendin-4 holds the potential of cardioprotective effect based on its pleiotropic activity. This study investigated whether Exendin-4 in conjunction with adipose-derived stem cells (ADSCs) could improve the stem cell survival and contribute to myocardial repairs after infarction. Myocardial infarction (MI) was induced by the left anterior descending artery ligation in adult male Sprague-Dawley rats. ADSCs carrying double-fusion reporter gene [firefly luciferase and monomeric red fluorescent protein (fluc-mRFP)] were quickly injected into border zone of MI in rats treated with or without Exendin-4. Exendin-4 enhanced the survival of transplanted ADSCs, as demonstrated by the longitudinal in vivo bioluminescence imaging. Moreover, ADSCs adjuvant with Exendin-4 decreased oxidative stress, apoptosis and fibrosis. They also improved myocardial viability and cardiac function and increased the differentiation rates of ADSCs into cardiomyocytes and vascular smooth muscle cells in vivo. Then, ADSCs were exposed to hydrogen peroxide/serum deprivation (H2O2/SD) to mimic the ischaemic environment in vitro. Results showed that Exendin-4 decreased the apoptosis and enhanced the paracrine effect of ADSCs. In addition, Exendin-4 activated signal transducers and activators of transcription 3 (STAT3) through the phosphorylation of Akt and ERK1/2. Furthermore, Exendin-4 increased the anti-apoptotic protein Bcl-2, but decreased the pro-apoptotic protein Bax of ADSCs. In conclusion, Exendin-4 could improve the survival and therapeutic efficacy of transplanted ADSCs through STAT3 activation via the phosphorylation of Akt and ERK1/2. This study suggests the potential application of Exendin-4 for stem cell–based heart regeneration.
Full-text · Article · Apr 2014 · Journal of Cellular and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: In this study, the effects of forsterite and clinoenstatite powder extracts on the proliferation and osteogenic differentiation of rat adipose-derived stem cells (ASCs) were investigated and compared with the β-tricalcium phosphate (β-TCP) powder extracts. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and live-dead staining were performed to evaluate the viability and proliferation of rat ASCs. Osteogenic differentiation of rat ASCs were assayed by alkaline phosphatase (ALP) staining and ALP activity test. The expression of osteogenic marker genes (alkaline phosphatase (ALP), runt related transcription factor 2 (Runx2), collagen type Iα1 (Col1α1), secreted phosphoprotein1 (Spp1, osteopontin), integrin binding sialoprotein (Ibsp), bone gla protein (Bglap)) were detected by real-time polymerase chain reaction (PCR). The MTT assay and the live-dead staining showed that all the three ceramics possessed good cytocompatibility with rat ASCs. Furthermore, forsterite and clinoenstatite promoted the proliferation of rat ASCs compared with β-TCP. The results of the ALP activity test and the real-time PCR demonstrated that forsterite and clinoenstatite promoted the osteogenic differentiation of rat ASCs. These results suggested that forsterite and clinoenstatite are bioactive ceramics that may be used for preparation of bone tissue engineering (BTE) scaffolds.
Full-text · Article · Aug 2013 · Chinese Science Bulletin
[Show abstract][Hide abstract] ABSTRACT: Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.
[Show abstract][Hide abstract] ABSTRACT: Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC-derivates), iPSC-derivated cardiomyocyte (iPSC-CMs) were subcutaneously injected into the back of nude mice. Non-invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC-derivates and iPSC-CMs survived in receipts. Both iPSCs and iPSC-derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC-CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC-derivates transplanted mice, but not in iPSC-CM transplanted ones. In vitro study showed the long-term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC-like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage-specific differentiation is necessary prior to therapeutic use of iPSCs.
Preview · Article · May 2013 · Journal of Cellular and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Telocyte (TC) as a special stromal cell exists in mammary gland and might play an important role in the balance of epithelium-stroma of mammary gland. Considering that different types of breast interstitial cells influence the development and progression of breast cancer, TCs may have its distinct role in this process. We here studied the roles of TCs in the self-assembly of reconstituted breast cancer tissue. We co-cultured primary isolated TCs and other breast stromal cells with breast cancer EMT-6 cells in collagen/Matrigel scaffolds to reconstitute breast cancer tissue in vitro. Using histology methods, we investigated the immunohistochemical characteristics and potential functions of TCs in reconstituted breast cancer tissue. TCs in primary mammary gland stromal cells with long and thin overlapping cytoplasmic processes, expressed c-kit/CD117, CD34 and vimentin in reconstitute breast cancer tissue. The transmission electron microscopy showed that the telocyte-like cells closely communicated with breast cancer cells as well as other stromal cells, and might serve as a bridge that directly linked the adjacent cells through membrane-to-membrane contact. Compared with cancer tissue sheets of EMT-6 alone, PCNA proliferation index analysis and TUNEL assay showed that TCs and other breast stromal cells facilitated the formation of typical nest structure, promoted the proliferation of breast cancer cells, and inhibited their apoptosis. In conclusion, we successfully reconstituted breast cancer tissue in vitro, and it seems to be attractive that TCs had potential functions in self-assembly of EMT-6/stromal cells reconstituted breast cancer tissue.
Full-text · Article · Dec 2012 · Journal of Cellular and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Transplantation of mesenchymal stem cells (MSCs) has been reported a great therapeutic potential for acute kidney injury (AKI). However, the therapeutic benefits are limited due to the low retention and survival of transplanted cells within target sites. In this study, thermosensitive chitosan chloride (CSCl) hydrogel was explored as injectable scaffold for adipose-derived MSCs (ADMSCs) delivery into ischemia/reperfusion (I/R) induced acute kidney injury (AKI). Thermosensitive CSCl hydrogels with/without ADMSCs were injected into the I/R site of rat AKI models. Dihydroethidium staining was used to detect the number of ROS in vivo. In order to track ADMSCs in vivo, ADMSCs were transfected with firefly luciferase and monomeric red fluorescent protein reporter genes (fluc-mrfp). The retention and survival of ADMSC were assessed using bioluminescence imaging, differentiation behaviors of ADMSCs were investigated using immunofluorescent and immunohistochemical staining. Proliferation and apoptosis of host renal cell in vivo were characterized by PCNA and TUNEL staining. Results suggested that CSCl hydrogels could improve the retention and survival of grafted ADMSCs, moreover, CSCl hydrogels could enhance the proliferation activity and reduce apoptosis of host renal cells. At 4 weeks, significant improvement of the renal function, microvessel density and tubular cell proliferation were observed in CSCl hydrogels with ADMSCs groups. Therefore, the application of thermosensitive CSCl hydrogel as scaffold for ADMSCs delivery into renal region could resolve the main obstacle of cell transplantation for acute kidney injury (AKI). Therefore, CSCl hydrogel is a potential cell carrier for treatment of AKI.
[Show abstract][Hide abstract] ABSTRACT: Microencapsulated hepatocytes might solve immunological rejection, broadening a new perspective for the treatment of fulminant hepatic failure (FHF). However, the transplantation of microcapsulated hepatocytes is limited by low cell viability. Nevertheless, the co-microencapsulation of hepatocytes and human umbilical vein endothelial cells (HUVECs) may make the treatment of FHF more promising.
We prepared the microcapsules using the high-voltage electrostatic droplet spray method, transplanted the empty microcapsules, isolated hepatocytes, microcapsulated hepatocytes, and co-microencapsulated hepatocytes and HUVEC intraperitoneally into rat models of FHF induced by D-aminogalactose (D-gal). After 1, 3, and 7 days, and 2, 3, and 4 weeks posttransplantation, we calculated the mortality and assessed alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) levels in the serum of the model; evaluated the integrality and recovery of microcapsules; and stained with hematoxylin and eosin (H&E) the recovered microcapsules as well as the liver of the FHF rats.
Hepatocyte-specific functions, including the levels of ALT, AST, and ALB in the serum of the co-microencapsulation group, were significantly better than those in the other groups (p<0.05) from 2 to 4 weeks after transplantation. Moreover, cotransplantation of the microencapsulated hepatocytes and HUVECs decreased the mortality rate of the FHF rats. The recovered microcapsules were intact, and recovery was up to 90%. H&E staining showed that the microencapsulated cells were still alive, and the liver tissues had started to recover after 4 weeks posttransplantation.
The microcapsules have good biocompatibility and immunoprotection to protect the hepatocytes from immunological rejection. Cotransplantation of the microencapsulated hepatocytes and HUVECs could decrease mortality rates and improve liver function in FHF.
No preview · Article · Apr 2012 · The International journal of artificial organs
[Show abstract][Hide abstract] ABSTRACT: One challenge of cellular cardiomyoplasty for myocardial infarction (MI) is how to improve MI microenvironment to facilitate stem cell engraftment, survival and homing for myocardial repair. The application of injectable hydrogels is an effective strategy. However, it has not been thoroughly investigated on the role of the injectable scaffolds, in improving MI microenvironment, providing space and guidance for cell survival, engraftment and homing. We explored an injectable chitosan hydrogel for stem cell delivery into ischemic heart and investigated the beneficial effects and mechanisms of the hydrogel. In vitro, H(2)O(2)-treatment was used to mimic reactive oxygen species (ROS) microenvironment. The influence of ROS and protection of chitosan components on adipose-derived mesenchymal stem cells (ADSCs) was analyzed too. In vivo, MI was induced by the left anterior descending artery ligation in SD rats. PBS, chitosan hydrogel, ADSC/PBS and ADSC/chitosan hydrogel were injected into the border of infracted hearts respectively. Multi-techniques were used to assess the beneficial effects of chitosan hydrogel after transplantation. We observed that ROS generated by ischemia would impair ADSC adhesion molecules, including integrin-related adhesion molecules integrin αV and β1, focal adhesion-related molecules p-FAK and p-Src, and corresponding ligands of host myocardium ICAM1 and VCAM1. Chitosan hydrogel could rescue these molecules through ROS scavenging and recruit key chemokine for stem cell homing, such as SDF-1. The results suggest that chitosan hydrogel could improve MI microenvironment, enhance stem cell engraftment, survival and homing in ischemic heart through ROS scavenging and chemokine recruitment, contributing to myocardial repair.
[Show abstract][Hide abstract] ABSTRACT: Cell migration is an early-stage and critical step for cancer metastasis. The most common approach to monitor this process is wound-healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding "cell islands" and "pseudopodia" during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.
[Show abstract][Hide abstract] ABSTRACT: Celastrol is an active ingredient of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo; however, the lack of information on the target and mechanism of action of this compound have impeded its clinical application. In this study, we sought to determine the mode of action of celastrol by focusing on the processes that mediate its anticancer activity.
The downregulation of heat shock protein 90 (HSP90) client proteins, phosphorylation of c-Jun NH2-terminal kinase (JNK), and cleavage of PARP, caspase 9 and caspase 3 were detected by western blotting. The accumulation of reactive oxygen species (ROS) was analyzed by flow cytometry and fluorescence microscopy. Cell cycle progression, mitochondrial membrane potential (MMP) and apoptosis were determined by flow cytometry. Absorption spectroscopy was used to determine the activity of mitochondrial respiratory chain (MRC) complexes.
Celastrol induced ROS accumulation, G2-M phase blockage, apoptosis and necrosis in H1299 and HepG2 cells in a dose-dependent manner. N-acetylcysteine (NAC), an antioxidative agent, inhibited celastrol-induced ROS accumulation and cytotoxicity. JNK phosphorylation induced by celastrol was suppressed by NAC and JNK inhibitor SP600125 (SP). Moreover, SP significantly inhibited celastrol-induced loss of MMP, cleavage of PARP, caspase 9 and caspase 3, mitochondrial translocation of Bad, cytoplasmic release of cytochrome c, and cell death. However, SP did not inhibit celastrol-induced ROS accumulation. Celastrol downregulated HSP90 client proteins but did not disrupt the interaction between HSP90 and cdc37. NAC completely inhibited celastrol-induced decrease of HSP90 client proteins, catalase and thioredoxin. The activity of MRC complex I was completely inhibited in H1299 cells treated with 6 μM celastrol in the absence and presence of NAC. Moreover, the inhibition of MRC complex I activity preceded ROS accumulation in H1299 cells after celastrol treatment.
We identified ROS as the key intermediate for celastrol-induced cytotoxicity. JNK was activated by celastrol-induced ROS accumulation and then initiated mitochondrial-mediated apoptosis. Celastrol induced the downregulation of HSP90 client proteins through ROS accumulation and facilitated ROS accumulation by inhibiting MRC complex I activity. These results identify a novel target for celastrol-induced anticancer activity and define its mode of action.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that histone deacetylase inhibitors (HDACis) can kill cancer cells. In addition, HDACis can induce mitotic catastrophe in cancer cells due to insufficient localization of chromosomal passenger complex (CPC) to the centromere. However, the mechanisms behind these phenomena remain unclear. In this study, we found that a HDACi, FK228, affected multiple epigenetic modification characteristics of the centromere, including enhanced acetylation of histone H3 lysine 9 (H3K9), decreased trimethylation of H3K9, and decreased phosphorylation of histone H3 serine 10 (H3S10) and centromere protein A (CENP-A). These epigenetic changes implied that H3K9 hyperacetylation inhibits the CPC recruitment, induces impaired centromere assembly and function, and eventually leads to aberrant mitosis. These data suggested that hypoacetylation of histone in the pericentromere is the most important landmark for recruiting CPC and leading to the mitotic catastrophe in HDACi-induced killing of cancer cells.
Preview · Article · Oct 2010 · Acta Biochimica et Biophysica Sinica
[Show abstract][Hide abstract] ABSTRACT: Tumor metastasis is a multistep process with many genes involved in. A novel gene MAG-1, identified by suppression subtractive hybridization from lung cancer cells was found to be associated with tumor metastasis. The aims of this work are to investigate the metastasis related effects of MAG-1 on human lunggiant-cell line PLA801, and to compare the expression rate of MAG-1 in cancer tissue from lung cancer patients with different metastatic status.
Sense and anti-sense expressing vectors of MAG-1 were constructed and transfected into PLA801C and PLA801D respectively. Colony forming, adherence assay, MTT assay and Transwell experiments were used to evaluate the alterations of clone forming, cell-matrix adherence, proliferating and invasion of the stable transfected cell strains. Western blot was employed to detect the proteins levels of CD44, and MMP-2 in cell strains and mRNA state of MAG-1 in lung cancer tissue from patients with or without pathological metastasis were also analyzed by RTPCR.
MAG-1 could increase cell-ECM (Extracellular Matrix) adhesive capacity, promote invasion, enhance cell proliferation and had no effects on clone forming ability of PLA801 cells in soft agar. MAG-1 was also found have positive effects on the protein level of CD44 and MMP-2 in PLA801 cells, and the detection rate of MAG-1 mRNA was much higher in cancer tissue from metastatic patients (7/12) than that in non-metastatic patients (2/12).
MAG-1 could promote lung cancer metastasis and might be a metastasis associated gene of lung cancer.
Full-text · Article · Feb 2009 · Zhongguo fei ai za zhi = Chinese journal of lung cancer
[Show abstract][Hide abstract] ABSTRACT: Both tumor proliferation and metastasis are multistep processes with many genes involved. A novel gene MAG-2, which may have close correlation with lung cancer metastasis, was identified in our lab through approach of suppression subtractive hybridization using lung cancer cell strains with same origin but different metastatic potential as models. The objective of our works presented here is to investigate the proliferation related effects of MAG-2 on human lung-giant-cell line PLA801, and to explore the underlying molecular mechanism.
Sense and anti-sense expressing vector of MAG-2 were constructed and transfected into PLA801C and D cell respectively and the change of expressing levels of target gene in stable transfected cell strains was assayed by RT-PCR. The alteration in terms of proliferation and cytoplasm Ca(2+) of the stable transfected cell strains and vector controls were tested by the methods of MTT and Fluo-3-Am staining. The proteins levels of p53 and mRNA levels of PCNA of different cell strains were analyzed by Western blot and RT-PCR.
The stable transfected cell strains of CMAG2+, DMAG2- and their vector controls Cvector and Dvector were obtained. The MAG-2 mRNA levels of CMAG2+ and DMPAG2- were 7 and 0.6 times higher or lower comparing those of vector controls. MAG-2 could increase the mRNA level of PCNA and down-regulated the p53 protein in PLA801C strain. In consistent with PCNA's mRNA elevating, the proportion of S-phase cell in the mass was also increased. But the significant difference was not observed on MTT assay between CMAG2+ and its control, the reason might be due to the mutated p53, which had strong proliferation promoting effects, was also down-regulated by MAG-2's over-expression in PLA801C, and the levels of cytoplasm calcium in CMAG2+ had 1.05 times more than that of vector control. As to the PLA801D strain, the decreased expression of MAG-2 result in lower level of cytoplasm calcium (0.64 time of control's) and the cell proliferation was also restrained by means of MTT assay.
MAG-2 has taken part in the proliferation regulation of lung cancer cells PLA801, and the mechanism of its proliferation-related function maybe lie in its effects on cytoplasmic free calcium.
Full-text · Article · Aug 2008 · Zhongguo fei ai za zhi = Chinese journal of lung cancer
[Show abstract][Hide abstract] ABSTRACT: Depsipeptide FR901228 (FK228) is a new kind of histone deacetylase inhibitors (HDACi) that induces growth arrest and cell death in a variety of tumor cells. Though its effects on oncogene expression and degradation have been documented, the detailed mechanism of FK228-induced cytotoxicity is still undefined. In this study, a differential proteomic analysis was performed to identify proteins associated with FK228-induced cytotoxicity in human lung cancer cells. Two-dimensional gel electrophoresis (2-DE) revealed a distinct protein profile of H322 cells in response to FK228 treatment, and 45 protein spots with significant alteration were screened. In total, 27 proteins were identified by mass spectrometry and involved in signal transduction, transcriptional regulation, metabolism, cytoskeletal organization, and protein folding, synthesis and degradation, consistent with multiple effects of FK228 on tumor cells. Notably, a novel target protein, thioredoxin reductase (TrxR), was identified, which is downregulated in FK228-sensitive cancer cells, but upregulated in resistant cells. The expression level of TrxR was negatively correlated with ROS accumulation, DNA damage and apoptosis, implicating TrxR in FK228-induced apoptosis and HDACi sensitivity in cancer cells. Thus, proteomic analysis provides new information about target proteins important for FK228-induced cytotoxicity in cancer cells.
Full-text · Article · Aug 2008 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: To isolate and identify the genes related to cancer metastasis by comparison of two cell strains with different metastasis potentials subcloned from human lung giant cell carcinoma cell line.
Suppression subtractive hybridization (SSH) was used to compare the levels of gene expression between the two cell strains and SSH library was constructed. After screening the library by gene chip, the expressed sequence tags (ESTs) with different expressing level were sequenced and blasted with GenBank.
Seventy-nine genes were obtained that were expressed much higher in PLA-801D than in PLA-801C, including two full-length cDNA. GenBank Accession numbers of the two cDNA, named MAG-1 and MAG-2, were BC006236 and BC002420, the 8.5 kb MAG-1 gene was composed of four exons and located on the chromosome of 4q21. The MAG-2 gene, which was made up by 9 exons, had a length of 5.2 kb and its location was 2q35. Both sequences had open reading frames (ORF) and promoters before the theoretical transcription start points. Using special software, the secondary structure of theoretical products of the two cDNAs was prognosticated, α-helix was the main proportion, but β-pleated sheet and random coil were also included.
The expression of MAG-1 and MAG-2 has significant differences in these two cell strains, so they might impact tumor metastasis in some ways that are still uncharted.
No preview · Article · Dec 2003 · Zhongguo fei ai za zhi = Chinese journal of lung cancer