Experiment of oral mucosa epithelial cells cultured on small intestinal submucosa in vitro / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore an effective method to culture oral mucosa epithelial cells (OMECs) of canine in vitro, and to observe the biological characteristics of OMECs growing on small intestinal submucosa (SIS) in order to provide the experimental basis for epithelium tissue engineering.</p><p><b>METHODS</b>The primary OMECs were cultivated with DKSFM (defined keratinocyte serum free medium) containing 6% fetal bovine serum (FBS). The morphological characteristics and the growth curve of OMECs were observed. The expressions of OMECs marker (CK19) were examined by immunocytochemistry. The 2nd passage of OMECs were seeded on SIS, OMECs co-cultured with SIS were observed by hematoxylin-eosin staining, immunohistochemical staining, and scanning electron microscope (SEM).</p><p><b>RESULTS</b>OMECs were grown well in DKSFM. Immunohistochemical staining of the 2nd passage cultured canine OMECs with broadly reacting anti-cytokeratin anyibodies (CK19) was positive. OMECs formed a single layer on the surface of SIS, and eight days later the cells were polygong and arranged like slabstone.</p><p><b>CONCLUSION</b>Culture of canine OMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS has good biocompatibility, it is a kind of good bioscafold in the tissue-engineered epithelium.</p>

The possible role of Myosin light chain in myoblast proliferation

Skeletal muscles have the potential to regenerate by activation of quiescent satellite cells, however, the molecular signature that governs satellite cells during muscle regeneration is not well defined. Myosin light chains (Myls) are sarcomere-related proteins as traditional regulator of muscle contraction. In this report, we studied the possible role of Myl in the proliferation of skeletal muscle-derived myoblasts. Compared to diaphragm-derived myoblasts, the extraocular muscle-derived myoblasts with lower levels of Myl proliferated faster, maintained a longer proliferation phase, and formed more final myotubes. It was found that blockading Myl with anti-Myl antibody or knockdown of Myll by siRNA targeted against Myll could enhance the myoblast proliferation and delay the differentiation of myoblasts. Our results suggested that Myl, likely Myll, can negatively affect myoblast proliferation by facilitating myoblast withdrawal from cell cycle and differentiation.

Effects of hypoxia on human placental decidua basalis-mesenchymal stem cells proliferation, apoptosis and VEGF expression / 生理学报

Human placental decidua basalis-mesenchymal stem cells (PDB-MSCs) are multipotent cells from the human term placenta, which are ethically conducive, easily accessible and high-yielding source. PDB-MSCs can differentiate into adipogenic, osteogenic and neurogenic cells under appropriate conditions, which may be an attractive and alternative source of seed cells for tissue engineering. To investigate the effect of hypoxia (1% O2) on human PDB-MSCs and the expression of cytokine, PDB-MSCs were isolated from human placenta by density gradient centrifugation and cultured in the Dulbecco's modified Eagle's medium-high glucose (DMEM-HG) containing 10% fetal bovine serum (FBS), and the fifth passage of PDB-MSCs were taken. PDB-MSCs were divided into 4 groups according to the concentrations of O2 and FBS: 20% O2, 10% FBS; 20% O2, 0% FBS; 1% O2, 10% FBS; 1% O2, 0% FBS. The proliferation and apoptosis of PDB-MSCs were detected by MTT and flow cytometric analysis at the time points of 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, respectively. Vascular endothelial growth factor (VEGF) released from PDB-MSCs was detected by enzyme-linked immunosorbent assay (ELISA) at the same time points. The results showed that hypoxia enhanced the proliferation of PDB-MSCs at 12 h under the condition of 10% FBS, while at 24 h under the condition of 0% FBS (P<0.01, n=3). In normoxia, the cells cultured in 10% FBS displayed a significant proliferation compared to those cultured in 0% FBS. However, in hypoxia, the number of cells cultured in 0% FBS (serum deprivation) increased significantly compared to that cultured in 10% FBS at 24 h and 96 h respectively (P<0.05, P<0.01, n=3). With the flow cytometric analysis of cell apoptosis under the condition of hypoxia and serum deprivation, we found that hypoxia and serum deprivation did not induce PDB-MSCs apoptosis (P>0.05, n=3). This conclusion may relate to the expression of VEGF which needs further research. In conclusion, the results obtained indicate that PDB-MSCs are able to bear hypoxia and serum deprivation, suggesting that PDB-MSCs can be used as seed cells for ischemia related tissue engineering.

Current understanding of dystrophin-related muscular dystrophy and therapeutic challenges ahead / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To review the recent research progress in dystrophin-related muscular dystrophy includes X-linked hereditary Duchenne and Becker muscular dystrophies (DMD and BMD).</p><p><b>DATA SOURCES</b>Information included in this article was identified by searches of PUBMED and other online resources using the key terms DMD, dystrophin, mutations, animal models, pathophysiology, gene expression, stem cells, gene therapy, cell therapy, and pharmacological. Study selection Mainly original milestone articles and timely reviews written by major pioneer investigators of the field were selected.</p><p><b>RESULTS</b>The key issues related to the genetic basis and pathophysiological factors of the diseases were critically addressed. The availabilities and advantages of various animal models for the diseases were described. Major molecular and cellular therapeutic approaches were also discussed, many of which have indeed exhibited some success in pre-clinical studies but at the same time encountered a number of technical hurdles, including the efficient and systemic delivery of a functional gene and myogenic precursor/stem cells to repair genetic defects.</p><p><b>CONCLUSIONS</b>Further understanding of pathophysiological mechanisms at molecular levels and regenerative properties of myogenic precursor/stem cells will promote the development of multiple therapeutic strategies. The combined use of multiple strategies may represent the major challenge as well as the greatest hope for the therapy of these diseases in coming years.</p>

Ectopic osteogenesis of tissue engineered bone with slow-released bioactive factors / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the ectopic osteogenesis of tissue engineered bone with recombinant human bone morphogenetic protein/transforming growth factor-beta (rhBMP/TGF-beta) or WO-1 slow-released factors.</p><p><b>METHODS</b>Partial demineralized freeze-dried bone (PDFDB) of pig was used as scaffold material. rhBMP/TGF-beta or WO-1 were pre-coated on the surface of material by means of vacuum negative pressure absorption, and then coated with polylactic acid (PLA) to make slow-released material. There were six group: PDFDB material (group A); PDFDB combined with osteoblasts (group B); PDFDB material with rhBMP/TGF-beta slow-released system (group C); PDFDB material combined with rhBMP/TGF-beta slow-released system osteoblasts (group D); PDFDB with WO-1 slow-released system (group E); PDFDB material combined with WO-1 slow-released system and osteoblasts (group F) were implanted in bilateral lower limbs of 36 Newzealand rabbits respectively (6 rabbits in each groups). Histological, histochemical and biochemical analysis were detected 2, 4, 6, 8 weeks after operation.</p><p><b>RESULTS</b>Within the observation periods, no osteogenesis was observed in group A. The osteogenesis in group B, D, F were superior to that of group C and E (P < 0.05). The osteogenetic activity in group C and E was delayed. The quantity and quality of osteogenesis in group D and F were 2 weeks ahead of time compared with group B, and 4 weeks to that of group C and E. The newborn calcification content was superior to that of group A, C, and E (P < 0.05).</p><p><b>CONCLUSIONS</b>The osteogenesis of PDFDB materials with BMP/TGF-beta or WO-1 is slower than that of which combined with osteoblasts. Simple material PDFDB has no ectopic osteogenesis.</p>

Reconstitution of telomerase activity in human embryonic tendon cells transfected by ptsA58H plasmid / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.</p><p><b>METHODS</b>THETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).</p><p><b>RESULTS</b>The THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.</p><p><b>CONCLUSIONS</b>The life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.</p>