Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

Plants are known to be efficient hosts for the production of mammalian therapeutic proteins. However, plants produce complex N-glycans bearing β1,2-xylose and core α1,3-fucose residues, which are absent in mammals. The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy. In this study, we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L. cv Bright Yellow 2 (BY2), which is a well-established cell line widely used for the expression of therapeutic proteins. The expression of the endogenous xylosyltranferase (XylT) and fucosyltransferase (FucT) was downregulated by using RNA interference (RNAi) strategy. The xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines. However, these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype. Therefore, this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.

Three-dimensional finite element analysis for different directions distraction at midface / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the biomechanical changes with different directions distraction at midface.</p><p><b>METHODS</b>An anteriorly directed 500 g force was applied to the floor of apertura piriforms in different directions to the occlusal plane. Three-dimensional finite element analysis was used to evaluate the biomechanical change of craniofacial complex.</p><p><b>RESULTS</b>As the force direction was moved downward, the sagittal distraction length of the craniofacial complex decreased and vertical movement changed from upward to downward. The craniofacial complex was moved anteriorly when the downward force was applied about 20-30 degrees to the occlusal plane. The forces could generate the uniform stress distribution in the craniofacial sutures and avoid counterclockwise rotation of the maxilla.</p><p><b>CONCLUSIONS</b>The craniofacial complex can be effectively distracted anteriorly when the downward force is applied to the floor of aperture piriforms in direction of 20-30 degrees to the occlusal plane.</p>

Changes in proliferative activity of myoblasts and expression of Akt in skeletal muscle of rats after severe burn injury / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate changes in proliferative activity of myoblasts in skeletal muscle and potential role of phosphorylated Akt on it, so that a better understanding in mechanisms of skeletal muscle atrophy after burn injury will be got.</p><p><b>METHODS</b>One hundred and twenty Wistar rats were randomly divided into 2 groups: control and severe thermal injury group. Rats in severe thermal injury group were subjected to a 40% total body surface area full-thickness scald injury, and Tibialis Anterior (TA) muscles were collected on 0, 1, 4, 7, 10, 14 days post-injury. After muscle mass determined, immunohistochemical double staining was used for detection of Proliferative Cell Nuclear Antigen (PCNA) of myoblasts. Protein expression of total Akt and phosphorylated Akt was determined by Western Blot.</p><p><b>RESULTS</b>Burn injury induced significant reduction of TA muscle mass and maximal reduction of it appeared by 4 days after injury (P < 0.01). Proliferative activity of myoblasts decreased significantly from the first day post-injury (P < 0.01) and increased slowly to basal level of controls after 7 days post-injury. The phosphorylated Akt was undetectable in both of controls and injured samples before 4 days but increased significantly after 7 days post-injury (P < 0.01), though total Akt expression had no significant alteration at any time points (P > 0.05).</p><p><b>CONCLUSIONS</b>Decrease in proliferative activity of myoblasts may be one of the contributors of significant atrophy of skeletal muscle after burn injury. Effect of phosphorylated Akt on proliferation attenuated in early stage and increased significantly in later stage after burn injury may partly explain the changes in proliferative activity of myoblasts.</p>

Application of thoracic skin flap with multiple blood supply in repair of tissue defects and deformities in jaw and neck / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore an appropriate measure to repair tissue defects and deformities in mandibulo-cervical region.</p><p><b>METHODS</b>Eighteen cases with severe tissue defects and deformity in jaw and neck were repaired with thoracic skin flap with multiple blood supply system in our unit from Jan. 2006 to Nov. 2008. Anterior cutaneous branch of transverse cervical artery, intercostal branch of internal thoracic artery and lateral thoracic artery were included in the pedicles.</p><p><b>RESULTS</b>All skin flaps survived, except in one patient in whom a small belb appeared at the distal end of the island flap with anterior cutaneous branch of transverse cervical artery, and it was healed after a few dressing changes. The functions and appearances were satisfactory after 6-month to 2-year follow-up, without showing secondary deformity.</p><p><b>CONCLUSIONS</b>The blood supply of thoracic skin flap is abundant and constant, which is an ideal method for repair of tissue defects and deformities in jaw and neck after taking into account some factors, such as the demand of the patient, general physical condition, and the size of the defect.</p>

Effect of intensive insulin therapy on apoptosis-related ligands in serum in rats with severe scald / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate changes in apoptosis-related ligands in serum in rats with severe scald and the effect of intensive insulin therapy on the changes.</p><p><b>METHODS</b>One hundred and fifty Wistar rats were randomly divided into 3 groups: sham burn (SB), scald (S) and treatment (T) groups. Rats in S and T groups were inflicted with 40% TBSA full-thickness burn, followed by intraperitoneal injection with 40 mL/kg of isotonic saline for resuscitation. Rats in T group were subcutaneously injected insulin in a dose of 0.25 U/100 g 24 hours after burn injury, and every 12 hours for 5 days (0.25, 0.50, 0.75, 1.00, 1.25 U/100 g each day, respectively) to control the level of blood glucose between 3 and 6 mmol/L. Rats in SB group were sham scalded at 37 degrees C without resuscitation. Blood was drawn from abdominal aorta on 1, 4, 7, 10, 14 post burn day (PBD) for determination of serum levels of TNF-alpha, soluble Fas ligand (sFasL) and soluble Fas receptor (sFas) by enzyme-linked immunosorbent assay (ELISA), and insulin by radioimmunity assay (RIA).</p><p><b>RESULTS</b>The serum level of TNF-alpha in S group peaked on 1 PBD (30.9 +/- 8.7) ng/L, which showed statistically significant difference when compared with that of SB and T groups (12.7 +/- 2.8) ng/L, (16.8 +/- 4.7) ng/L, respectively, P < 0.01), then lowered gradually to become similar to that of SB group on 7 PBD. The level of TNF-alpha in T group increased gradually, but was obviously lower than that of S group on 1, 4, 7 PBD (P < 0.01). The level of sFasL in S (on 7-14 PBD) and T (4-10 PBD) groups was significantly higher than that in SB group (P < 0.05), then lowered to normal level. The levels of sFas on 4-10 PBD in T group were obviously higher than that in S and SB group (P < 0.05). Ratio of sFasL to sFas in serum of S group was higher than that in SB group on 7, 10 PBD, which was higher than that in T group on 7 PBD (P < 0.05). There was significant decrease in serum level of insulin in S group compared with that of SB group on 4-10 PBD (P < 0.05). The level of insulin in T group increased on 1 PBD, peaked on 4 PBD (327 +/- 15 microU/mL), which was significantly higher than that in SB and S groups (42 +/- 15, 28 +/- 10 microU/mL, respectively, P < 0.01), then decreased gradually to normal level.</p><p><b>CONCLUSIONS</b>Insulin may inhibit apoptosis after burn by down-regulating secretion of apoptotic ligands.</p>

Construction of tissue-engineering skin with carrier of active composite dermal matrix / 中华烧伤杂志

<p><b>OBJECTIVE</b>To construct of tissue engineering skin including active composite dermal matrix.</p><p><b>METHODS</b>The human fibroblasts and bovine collagen with type I were inoculated on the surface of porcine acellular dermal matrix (PADM) for construction of active dermal substitute, then epidermal cells were inoculated on the dermal matrix for gas-liquid interface culture. The tissue-engineering skin was observed by histological examinations.</p><p><b>RESULTS</b>The structure of fibroblasts in collagen was intact, which was used to construct composite dermal matrix with PADM. The epithelial structure of tissue-engineering skin was similar to that of normal skin with good cell differentiation. Some phenomena were showed in epidermis: basic layer, stratum spinosum, granular layer and stratum corneum, desmosomes.</p><p><b>CONCLUSION</b>Fibroblasts-Collagen-PADM can be an optimal dermal matrix for construction of tissue-engineering skin.</p>

Study on preparation of laser micropore porcine acellular dermal matrix combined with split-thickness autograft and its application in wound transplantation / 中华烧伤杂志

<p><b>OBJECTIVE</b>To prepare a porcine acellular dermal matrix (PADM), and to optimize the interpore distance between PADM and co-grafted split-thickness autologous skin.</p><p><b>METHODS</b>Porcine skin was treated with trypsin/Triton X-100 to prepare an acellular dermal matrix. Micropores were produced on the PADM with a laser punch. The distance between micropores varied as 0.8 mm, 1.0 mm, 1.2 mm and 1.5 mm. Full-thickness defect wounds were created on the back of 144 SD rats. The rats were randomly divided into 6 groups as follows, with 24 rats in each group. Micropore groups I -IV: the wounds were grafted with PADM with micropores in four different intervals respectively, and covered with split-thickness autologous skin graft. Mesh group: the wounds were grafted with meshed PADM and split-thickness autograft.</p><p><b>CONTROL GROUP</b>with simple split-thickness autografting. The gross observation of wound healing and histological observation were performed at 2, 4, 6 weeks after surgery. The wound healing rate and contraction rate were calculated.</p><p><b>RESULTS</b>Two and four weeks after surgery, the wound healing rate in micropore groups I and II was lower than that in control group (P < 0.05), but no obvious difference was between micropore groups I , II and mesh group (P > 0.05) until 6 weeks after grafting( P <0.05). The wound contraction rate in micropore groups I and II ([(16.0 +/- 2.6)%, (15.1 +/- 2.4)%] was remarkably lower than that in control group 4 and 6 weeks after grafting (P < 0.05), and it was significantly lower than that in mesh group [(19.3 +/- 2.4)%] 6 weeks after surgery (P <0.05). Histological examination showed good epithelization, regularly arranged collagenous fibers, and integral structure of basement membrane.</p><p><b>CONCLUSION</b>Laser micropore PADM (0.8 mm or 1.0 mm in distance) grafting in combination with split-thickness autografting can improve the quality of wound healing. PADM with laser micropores in 1.0 mm distance is the best choice among them.</p>

Growth activity of epidermal cells from different parts of human body / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most epidermal cells used in skin tissue engineering are obtained from the skins of fetuses or prepuces, which can not be widely used in culturing and transplanting autologous epidermis for patients with extensive burn wounds. To solve the problem, in this study, we cultured epidermal cells from different parts of human body in vitro, and detected their growth activity.</p><p><b>METHODS</b>Normal epidermal cells obtained from the prepuce, scalp, and axilla of male patients, were cultured and passaged. Their growth characteristics including adherent rate and growth activity were compared. Data were analyzed by homogeneity test of variance.</p><p><b>RESULTS</b>In primary culture, the growth of epidermal cells from the prepuce was significantly faster than that of the epidermal cells from the scalp and axilla. In the cells obtained from the prepuce, 80% confluence was achieved on day 12, while on day 16 and day 20 in the cells from the scalp and axilla, respectively. However, no significant difference was detected in their growth and proliferation in the second passage.</p><p><b>CONCLUSIONS</b>Although the growth of epidermal cells obtained from the scalp and axilla is slower than that from the prepuce in primary culture, stable cell line can be established and used in preparation of auto-epidermal grafts for patients with extensive burn wounds. Therefore, the scalp and axillary skin should be considered as important sources of epidermal cells other than the prepuce.</p>

Characteristics of and strategies for patients with severe burn-blast combined injury / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Severe burn-blast combined injury is a great challenge to medical teams for its high mortality. The aim of this study was to elucidate the clinical characteristics of the injury and to present our clinical experiences on the treatment of such cases.</p><p><b>METHODS</b>Five patients with severe burn-blast combined injuries were admitted to our hospital 77 hours post-injury on June 7, 2005. The burn extent ranged from 80% to 97% (89.6% +/- 7.2%) of TBSA (full-thickness burns 75% - 92% (83.4% +/- 7.3%)). All the patients were diagnosed as having blast injury and moderate or severe inhalation injury. Functions of the heart, liver, kidney, lung, pancreas and coagulation were observed. Autopsy samples of the heart, liver, and lungs were taken from the deceased. Comprehensive measures were taken during the treatment, including protection of organ dys function, use of antibiotics, early anticoagulant treatment, early closure of burn wounds, etc. All the data were analyzed statistically with t test.</p><p><b>RESULTS</b>One patient died of septic shock 23 hours after admission (four days after injury), the others survived. Dysfunction of the heart, liver, lungs, pancreas, and coagulation were found in all the patients on admission, and the functions were ameliorated after appropriate treatments.</p><p><b>CONCLUSIONS</b>Burn-blast combined injury may cause multiple organ dysfunctions, especially coagulopathy. Proper judgment of patients' condition, energetic anticoagulant treatment, early closure of burn wounds, rational use of antibiotics, nutritional support, intensive insulin treatment, timely and effective support and protection of organ function are the most important contributory factors in successful treatment of burn-blast combined injuries.</p>