Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.
Animals ; Apoptosis ; Body Patterning ; Bone Morphogenetic Proteins/biosynthesis* ; Developmental Biology ; Ectoderm/metabolism* ; Extremities/embryology* ; Fibroblast Growth Factor 10/metabolism* ; Fibroblast Growth Factors/biosynthesis* ; Gene Expression Regulation ; Hedgehog Proteins/biosynthesis* ; Homeodomain Proteins/biosynthesis* ; Mesoderm/metabolism* ; Mice ; Signal Transduction ; Wnt Proteins/biosynthesis*

miR-200c has been shown to regulate the epithelial-mesenchymal transition (EMT) by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software (miRanda) was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.
Biomarkers, Tumor ; Breast Neoplasms ; genetics ; metabolism ; Cell Movement ; genetics ; Epithelial-Mesenchymal Transition ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; HMGB1 Protein ; genetics ; Homeodomain Proteins ; biosynthesis ; Humans ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; genetics ; Neoplasm Metastasis ; genetics ; pathology ; Repressor Proteins ; biosynthesis ; Transcription Factors ; biosynthesis ; Zinc Finger E-box Binding Homeobox 2 ; Zinc Finger E-box-Binding Homeobox 1

To study the function and potential application of nkx2.5, a critical gene for heart development, we constructed a recombinant adenovirus overexpressing nkx2.5 gene (Ad-Nkx2.5) with the AdEasy system. To evaluate the effect and mechanism of Ad-Nkx2.5 against oxidative injury, the H9c2 myocardial cells were infected with the recombinant adenoviruses Ad-Nkx2.5 or Ad-EGFP, and subsequently exposed to H2O2 to induce apoptosis. The anti-apoptotic potential of Ad-Nkx2.5 was validated by MTT assay for cell viability, Hoechst33342 staining for cellular morphology, and immunoblotting for caspase-3 activity. Ad-Nkx2.5 infection led to an increased survival rate of H9c2 cells and decreased the amount of caspase-3 in an active form. Additionally, overexpression of Nkx2.5 inhibited the release of cytochrome C from the mitochondria into the cytosol. Mechanismic studies showed that Nkx2.5 upregulated bcl-2 gene expression and significantly repressed H2O2-induced expression of bax detected by Real-time PCR. Additionally, H2O2 treatment did not affect the nuclear localization of Nkx2.5. These findings indicate that adenovirus-mediated nkx2.5 gene transfer exerted a protective effect on H9c2 cells against H2O2-induced apoptosis via mitochondrial pathway, and the Nkx2.5-mediated expression modulation of apoptosis-associated genes could be involved in this event.
Adenoviridae ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; Genetic Vectors ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; biosynthesis ; genetics ; Hydrogen Peroxide ; pharmacology ; Myocytes, Cardiac ; cytology ; Oxidative Stress ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; metabolism

This study was to explore the induced differentiation of human mesenchymal stem cells (MSCs) modified by pancreatic and duodenal homeobox factor 1 (Pdx1) gene into insulin-producing cells in vitro. After recombined adenovirus vector with Pdx1 gene infected MSCs for 7 d, cells were induced by induction factors. The genes' expressions related to islet beta cells such as Pdx1, insulin, glucose transporter-2 (Glut2), were detected with RT-PCR, immunocytochemistry and Western blot. The levels of insulin and C peptide secretion were examined with chemiluminescence immunoassay. Insulin(+) cell rate was detected by flow cytometry. After infected by recombined adenovirus with Pdx1 and combined with induction factors, MSCs were aggregated and islet-like cell clusters formed. Dithizone staining of these cells was positive. The genes' expression related to islet beta cells, such as Pdx1, insulin, Glut2, could be detected. After induction, the islet-like cell clusters secreted insulin and C peptide. The levels of insulin and C peptide secretion increased with glucose stimulation. Insulin(+) cell rate was (11.61 +/- 4.83)%. It could be concluded that Pdx1 gene modified MSCs from human umbilical cord could be induced to differentiate into islet beta-like cells.
Adenoviridae ; genetics ; metabolism ; Cell Differentiation ; genetics ; Cells, Cultured ; Genetic Vectors ; genetics ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Islets of Langerhans ; cytology ; Mesenchymal Stromal Cells ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Trans-Activators ; biosynthesis ; genetics ; Umbilical Cord ; cytology

OBJECTIVETo investigate the expression pattern of FHL2, which is an intracellular signaling transcription molecule during mineralization in cultured human periodontal ligament cells (hPDLCs) in vitro.

METHODShPDLCs were cultured in vitro. Test group was cultured with mineral induction media while control group without induction media. 0, 14, 28 days after culture, alizarin red staining was used to measure the mineral nodules formation. Immunocytochemistry was used to examine the expression of FHL2 protein 0 day and 14 days after mineral induction. Meanwhile, mRNA expression level of FHL2 was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) on the 0, 14, 28 days after induction.

RESULTS14 and 28 days after cultivation, mineral nodules formed and were stained positively with alizarin red staining in test group while no mineral nodule formed in control group. Immunocytochemical results indicated that hPDLCs in test group expressed FHL2 positively. According to RT-PCR results, 14 and 28 days after mineral induction, the expression levels of FHL2 both increased significantly when compared with 0 day (P<0.01), and the expression level at 14 days was 1.4 folds of 0 day.

CONCLUSIONFHL2 protein is found to be involved in the in vitro mineralization of hPDLCs. FHL2 protein may play a role in the differentiation and mineralization of hPDLCs.

Cell Differentiation ; Cells, Cultured ; Humans ; In Vitro Techniques ; LIM-Homeodomain Proteins ; biosynthesis ; Muscle Proteins ; biosynthesis ; Periodontal Ligament ; RNA, Messenger ; Transcription Factors ; biosynthesis

To study the chemosensitivity and the mechanisms of recombinant adenovirus vector expressing ING4 and IL-24 (Ad-ING4-IL-24) on lung adenocarcinoma in vitro and in vivo, the expression of ING4 and IL-24 in A549 cells was detected by RT-PCR and Western blotting. The growth inhibition, apoptosis rate and apoptosis body were measured by MTT, flow cytometry and Hoechst staining respectively. For in vivo study, we first established the A549 tumor model by grafting A549 cells in athymic nude mice; and then injected Ad-ING4-IL-24 into the tumors. Two weeks after injection, we killed the mice, removed the tumors, weighted and calculated the ratios of tumor-suppression. We also detected the expressions of ING4, IL-24, bax, bcl-2, VEGF with immunohistochemistry. The results indicated that ING4 and IL-24 were proved successfully transcription and expression in A549 cells. More interestingly, the joint group inhibited the growth of A549 cells and induced apoptosis. The in vivo data showed that the joint group suppressed the tumor growth conspicuously through up-regulating the expression of bax, and down-regulating the expression of bcl-2, VEGF. The study proved that Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug DDP in lung adenocarcinoma, which may related with cell apoptosis and antiangiogenesis.
Adenocarcinoma ; drug therapy ; metabolism ; Adenoviridae ; genetics ; metabolism ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Lung Neoplasms ; drug therapy ; metabolism ; Mice ; Mice, Nude ; Neoplasms, Experimental ; drug therapy ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics

The expression of NF-kappaB is considered to be involved in the progress of neurodegeneration. It has been reported that Nanog can suppress the expression of NF-kappaB. To inspect and verify this finding, we constructed lentivirus (LV) vector that overexpressed the Nanog gene, infected mouse mesenchymal stem cells (mMSCs), and examined the influence of Nanog overexpression on NF-kappaB gene expression. The plasmid pNL-Nanog-IRES2-EGFP was constructed by double digestion and genetic recombination. Sequencing results confirmed that our cloned Nanog gene in the PNL-Nanog-IRES2-EGFP plasmid was consistent with the sequence reported in the GenBank. The three plasmids: pNL-Nanog-IRES2-EGFP, HELPER, and VSVG were cotransfected into 293T cells to produce LV particles. After co-transfection of the three lentiviral plasmids, green fluorescence was observed confirming successful transfection. The mMSCs were infected by the LV and the expression of Nanog was then also verified by the presence of green fluorescence. Nanog expression levels in the mMSCs were examined using Western blotting. Expression of NF-kappaB was also examined using RT-PCR and Western blotting, and in addition with fluorescent microscope after immunocytochemical staining. The levels of Nanog protein expression in Nanog-mMSCs were significantly increased, and the levels of NF-kappaB mRNA and protein expression in Nanog-infected mMSCs were significantly lower than those of Mock-mMSCs and the mMSCs control groups. Our findings suggest that mMSCs genetically modified to overexpress Nanog can lead to the suppression of NF-kappaB expression. This suppression of NF-kappaB could have important implications for the treatment of neurodegeneration, and hence further scientific investigations of these interactions will have significant impact on future clinical attempts to attenuate disease progression.
Animals ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; metabolism ; Homeodomain Proteins ; biosynthesis ; genetics ; Lentivirus ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; genetics ; metabolism ; Nanog Homeobox Protein ; Neurodegenerative Diseases ; therapy ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

We constructed a His-tagged prokaryotic expression vector of WUSCHEL gene of Arabidopsis thaliana, pET-31b(+)-WUS-His(6). The induction condition of the fusion protein expression in Escherichia coli was optimized. After purified by affinity chromatography, the recombinant WUS protein was resolved by renaturation of gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The rabbit anti-WUS antibody titer and specificity were analyzed and confirmed by agarose immunodiffusion testing; the antiserum sensitivity was assayed by dot blot and Western blotting. The results showed that the A. thaliana WUS prokaryotic expression vector was successfully constructed, and the optimized protein expression induction condition in E. coli was 0.5 mmol/L IPTG (isopropy-beta-D-thiogalactoside) at 28 degrees C for 10 hours. The purity of the affinity purified protein was higher than 96%, and the prepared polyclonal antibody was with high specificity and sensitivity, it was able to detect protein antigen at ng level.
Amino Acid Sequence ; Animals ; Antibodies ; isolation & purification ; metabolism ; Arabidopsis ; genetics ; Arabidopsis Proteins ; biosynthesis ; genetics ; Chromatography, Affinity ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Homeodomain Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVE: To generate and identify the induced pluripotent stem cells (iPSCs) from human embryonic fibroblast cells (hEFs) by introducing 4 defined factors. METHODS: We introduced 4 factors (Oct4, Sox2, Nanog,and Lin28) into hEFs by lentivirus infection. The iPSCs generated from this method were analyzed in many aspects, including surface antigens, gene expression, and telomerase activity, differentiation ability in vivo and in vitro and the patterns of short tandem repeat (STR). RESULTS: The human iPSCs generated from this study were positive for alkaline phosphatase (AKP) staining, expressed hESCs-specific surface antigens, and exhibited high telomerase activity. They also possessed the ability to differentiate into 3-germ layers both in vivo and in vitro. STR analysis indicated that the human iPSCs were derived from the donor material. CONCLUSION The iPSCs lines can be obtained from human embryonic fibroblast by introducing 4 factors. Such human iPSCs should be useful in the study of epigenetic reprogramming and pluripotency maintenance.
Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Fibroblasts ; cytology ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Lentivirus ; genetics ; metabolism ; Nanog Homeobox Protein ; Octamer Transcription Factor-3 ; biosynthesis ; genetics ; RNA-Binding Proteins ; biosynthesis ; genetics ; SOXB1 Transcription Factors ; biosynthesis ; genetics ; Transfection

HOXB4, a member of homeobox gene family, is closely related to the self-renewing and proliferative ability of primitive hematopoietic stem/progenitor cells (PHSC/PHPC). This study was aimed to investigate the self-renewing level of cord blood progenitor cells (CBPC) expanded in vitro. The HOXB4 expression at mRNA level was assayed by using real time RT-PCR. The results indicated that as culture prolonged, the total cells, CD34(+) cells greatly increased, however the HOXB4 expression gradually declined, even down to undetectable level similar to that of mature lymphocytes. Meanwhile, it was shown that CD34(+) cells co-cultured with bone marrow mesenchymal stem cells (BM-MSC) could abate the decline of HOXB4 expression. It is concluded that the self-renewing potential of CD34(+) cells gradually decreased during expansion in vitro, co-culture with BM-MSC was helpful to CD34(+) cell expansion and slowed the loss trend of its self-renewal.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Mesenchymal Stromal Cells ; cytology ; Transcription Factors ; biosynthesis ; genetics