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 investigate the protein and mRNA expression of pancreas/duodenal homeobox-1 (PDX-1), a transcription factor as a marker for pancreatic stem cells, in pancreatic ductal cells of rats after partial (90%) pancreatectomy and evaluated the significance of the PDX-1 expression. Western blot and Reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PDX-1 protein and mRNA respectively. PDX-1 protein was only faintly detected in pancreatic ductal cells on the day 1 after partial pancreatectomy. On the day 2 and 3 after operation in operation group, a 2-3 fold increased PDX-1 protein was observed, corresponding to the characteristic 42-kD protein in Western blot. There was significant difference between operation group and sham-operation group (P<0.05). PDX-1 protein expression on the day 5 and 7 after operation had already been no difference from control group (P>0.05). RT-PCR revealed the PDX-1 mRNA expression showed no significant difference between operation group at various time points and sham-operation group (P> 0.05). These results indicate that there was overexpression of PDX-1 in the cells of pancreatic epithelium during the regeneration of remnant pancreas after partial pancreatectomy in adult rats, suggesting the pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells was regulated posttranscription.
Epithelial Cells/metabolism ; Homeodomain Proteins/*biosynthesis ; Homeodomain Proteins/genetics ; Pancreatectomy/methods ; Pancreatic Ducts/cytology ; Pancreatic Ducts/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Rats, Sprague-Dawley ; Trans-Activators/*biosynthesis ; Trans-Activators/genetics

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*

To investigate the protein and mRNA expression of pancreas/duodenal homeobox-1 (PDX-1), a transcription factor as a marker for pancreatic stem cells, in pancreatic ductal cells of rats after partial (90%) pancreatectomy and evaluated the significance of the PDX-1 expression. Western blot and Reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PDX-1 protein and mRNA respectively. PDX-1 protein was only faintly detected in pancreatic ductal cells on the day 1 after partial pancreatectomy. On the day 2 and 3 after operation in operation group, a 2-3 fold increased PDX-1 protein was observed, corresponding to the characteristic 42-kD protein in Western blot. There was significant difference between operation group and sham-operation group (P<0.05). PDX-1 protein expression on the day 5 and 7 after operation had already been no difference from control group (P>0.05). RT-PCR revealed the PDX-1 mRNA expression showed no significant difference between operation group at various time points and sham-operation group (P> 0.05). These results indicate that there was overexpression of PDX-1 in the cells of pancreatic epithelium during the regeneration of remnant pancreas after partial pancreatectomy in adult rats, suggesting the pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells was regulated posttranscription.
Animals ; Epithelial Cells ; metabolism ; Homeodomain Proteins ; biosynthesis ; genetics ; Pancreatectomy ; methods ; Pancreatic Ducts ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Trans-Activators ; biosynthesis ; genetics

To investigate the mechanisms of the inhibition of osteoblastic differentiation by dexamethasone (DEX), the effects of different doses of DEX on the activity of alkaline phosphatase (ALP), the synthesis of osteocalcin (OC) and the expression of collagen type I were observed in the cultured rat osteoblasts. The LIM mineralization protein-1 (LMP-1) mRNA, a positive regulator of osteoblasts, was semi-quantified by RT-PCR. The results showed that a lower dose (10(-9) mol/L) of DEX could enhance the activity of ALP, the synthesis of OC and the expression of collagen type I. However, a higher dose (10(-7) mol/L) of DEX inhibited them and down-regulated the expression of LMP-1 mRNA in osteoblasts. It is suggested that DEX stimulates osteoblast differentiation at lower dose, while at higher dose it inhibits osteoblast differentiation. The inhibitory action of DEX on osteoblast differentiation might be mediated by the down-regulation of LMP-1 mRNA.
Animals ; Animals, Newborn ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dexamethasone ; administration & dosage ; pharmacology ; Dose-Response Relationship, Drug ; Down-Regulation ; Homeodomain Proteins ; biosynthesis ; LIM-Homeodomain Proteins ; Osteoblasts ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; Rats ; Transcription Factors

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

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Female ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Estrogen ; metabolism ; Transcription Factors ; biosynthesis ; genetics

OBJECTIVETo investigate the expression of transcription regulator LMO4 mRNA in the developing mouse molar and compare the expression pattern of LMO4 with that of Shh signaling molecule.

METHODSWild-type embryos used in this study (E11.5-P1.5) were generated by mating Kun-Ming mice. The expression pattern of LMO4 during organ development was carried on by whole-mount in situ hybridization. The expression patterns of LMO4 and Shh mRNA during molar development were analysed by section in situ hybridization. Immunohistochemical staining of PCNA was carried on by SP method.

RESULTSLMO4 mRNA was widespread at early embryonic stages (E11.5) with positive hybridization signal in the mandibular reason, limb bud, brain, epidermis and somites revealed by whole-mount in situ hybridization. Section in situ hybridization showed that LMO4 was expressed in the tooth bud, the two tips of the enamel organ and the cervical loop from E13.5 to E16.5. While Shh was localized in the enamel knot on E14.5. On E18.5-P1.5, LMO4 transcripts were distributed in the ameloblast and the stratum intermedium. On E13.5-E16.5, the tooth bud cells and the cervical loop cells were PCNA positive. These were the same regions that showed LMO4 mRNA expression.

CONCLUSIONSLMO4 was confined to the dental epithelium and had spatial temporal expression patterns during tooth morphogenesis. The expression patterns of LMO4 and Shh were similar. In early tooth development, LMO4 might regulate cell proliferation. In late tooth development, it might participate in the ameloblast differentiation.

Adaptor Proteins, Signal Transducing ; Animals ; Animals, Newborn ; Female ; Gene Expression Regulation, Developmental ; Homeodomain Proteins ; biosynthesis ; LIM Domain Proteins ; Mice ; Mice, Inbred Strains ; Morphogenesis ; genetics ; Pregnancy ; Tooth Germ ; metabolism ; Transcription Factors ; biosynthesis ; Transcription, Genetic

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

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