Ischemia preconditioning protects rat submandibular glands from ischemia/reperfusion injuries.

To investigate the effects of ischemia/reperfusion on rat submandibular glands without denervation and the possible protective effects of ischemia preconditioning on the glands that experienced ischemia/reperfusion, in-situ ischemia/reperfusion and ischemia preconditioning experimental models of submandibular glands of healthy male Wistar rats were conducted. For ischemia/reperfusion groups, the glands were subjected to 90 min of ischemia without denervation, followed by 1, 12, 24, or 72 h of reperfusion. Ischemia preconditioning was achieved by 3 min of ischemia following 3 min of reperfusion, performed three times before ischemia/reperfusion. Salivary secretion, histological changes, alterations of tight junctions, myeloperoxidase activity, cellular apoptosis, and reactive oxygen species levels were detected. In ischemia/reperfusion glands, rising acute-inflammation responses, reduced tight-junction width, and increased myeloperoxidase activity, reactive oxygen species levels, and apoptotic cell numbers were observed, along with secretory dysfunction, especially at 1 and 12 h post-reperfusion, which seemed to gradually return to normal by 72 h post-reperfusion. In contrast, ischemia preconditioning showed the potential to ameliorate the injury-stress responses caused by ischemia/reperfusion. Our study revealed that ischemia/reperfusion could cause a series of injury-stress responses and ultimately lead to hyposecretion, independently of the parasympathetic nerve supply, which might play an important role in the early-phase dysfunction of the transplanted glands. Ischemia preconditioning could protect the involved glands and improve ischemia/reperfusion-induced hyposecretion.

Inhibitor of differentiation 1 is a candidate prognostic marker in multicentric Castleman's disease.

Castleman's disease (CD) is a benign lymphoproliferative disorder characterized by dysfunctional lymphatic node hyperplasia. Lymphatic node hyperplasia is associated with elevated levels of inhibitor of differentiation 1 (ID1) in many human tumors. To assess the possible role of ID1 expression as a prognostic marker in multicentric CD (MCD), intra-lymph node ID1 expression was analyzed and related to clinical characteristics and outcomes in 48 patients. Furthermore, the correlation between ID1 and possible signaling molecules such as interleukin-6 (IL6), phosphorylated extracellular response kinase (p-ERK), and vascular endothelial growth factor C (VEGFC) was explored on six fresh MCD surgical specimens. Immunohistochemistry revealed that the patients with extensive ID1 expression had significantly poorer prognosis, compared to those with localized ID1. In addition, ID1 was positively associated with levels of IL6, p-ERK, and VEGFC. We conclude that ID1 may ultimately be a prognostic marker in MCD and that the IL6/ERK/VEGFC pathway is involved in the progress of this disease.

Stable gene-silence of Kif2a synergistic with 5-fluorouracil suppresses oral tongue squamous cell carcinoma growth in vitro and in vivo.

OBJECTIVE: Squamous cell carcinoma of the oral tongue (SCCOT) is one of the most common malignant carcinomas in the head and neck. Recurrence and/or metastasis often results in failure of treatment and decreases the survival of the patients. The purpose of this study is to investigate the effect of gene-silence of Kif2a on SCCOT in viro and in vivo. STUDY DESIGN: Plasmid-mediated expression of Kif2a-siRNA (pGPU6/GFP/Kif2a) was employed to silence the expression of Kif2a in Tca8113 cells at both mRNA and protein levels. Tca8113 cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and growth of Tca8113 tumors was determined by intra-tumor injection of pGPU6/GFP/Kif2a in nude mice. RESULTS: Gene-silence of Kif2a suppressed Tca8113 cell proliferation. pGPU6/GFP/Kif2a synergized the tumor suppression effect of 5-Fluorouracil (5-Fu) on Tca8113 cells. CONCLUSIONS: Our data support that Kif2a is a potential molecular target for the therapeutics of recurrent and metastatic SCCOT.

Galectin-3 gene silencing inhibits migration and invasion of human tongue cancer cells in vitro via downregulating ß-catenin.

AIM: Galectin-3 (Gal-3) is a member of the carbohydrate-binding protein family that contributes to neoplastic transformation, tumor survival, angiogenesis, and metastasis. The aim of this study is to investigate the role of Gal-3 in human tongue cancer progression. METHODS: Human tongue cancer cell lines (SCC-4 and CAL27) were transfected with a small-interfering RNA against Gal-3 (Gal-3-siRNA). The migration and invasion of the cells were examined using a scratch assay and BD BioCoat Matrigel Invasion Chamber, respectively. The mRNA and protein levels of ß-catenin, Akt/pAkt, GSK-3ß/pGSK-3ß, MMP-9 in the cells were measured using RT-PCR and Western blotting, respectively. RESULTS: Transient silencing of Gal-3 gene for 48 h significantly suppressed the migration and invasion of both SCC-4 and CAL27 cells. Silencing of Gal-3 gene significantly decreased the protein level of ß-catenin, leaving the mRNA level of ß-catenin unaffected. Furthermore, silencing Gal-3 gene significantly decreased the levels of phosphorylated Akt and GSK-3ß, and suppressed the mRNA and protein levels of MMP-9 in the cells. CONCLUSION: Our data suggest that Gal-3 mediates the migration and invasion of tongue cancer cells in vitro via regulating the Wnt/ß-catenin signaling pathway and Akt phosphorylation.

[Gene expression of transforming growth factor beta receptor II in the epidermis of pathological scar].

OBJECTIVE: To study the gene expression of transforming growth factor beta receptor II (TbetaR II) in pathological scar. METHODS: Twenty samples of pathological scar were collected from 20 burn or trauma patients hospitalized in the General Hospital of Ji'nan Military Command from 2007 to 2009. Twenty specimens of epidermal layer were obtained from the middle portion and the edge of pathological scars. Twenty normal skin specimens which were located more than 10 cm away from the lesion sites of 20 patients were collected as self-controls. Serum from 1-2 mL whole blood were obtained from each of the 20 patients for second self-control. Eight normal skin specimens from 8 patients without pathological scar, discarded from un-related operations, were also collected as negative-control. Positive expressions of TbetaR II in three different skin specimens were determined with biotin-streptavidin-peroxidase staining. Gene expressions of TbetaR II in all specimens were compared with PCR-single strand conformation polymorphism analysis and gene sequencing. Data were processed with Fisher's exact test. RESULTS: Positive expression of TbetaR II in pathological scar epidermis was lower than that in normal skin specimen of patients with pathological scar or normal skin specimen of patients without pathological scar, and TbetaR II was mainly located in the basal layer of epidermis. Positive expressions of TbetaR II were seldom found in acanthocytes, granular cells, and cuticle or even non-existing. No abnormality of TbetaR II was found in normal skin epidermis or serum samples of pathological scar patients or normal skin epidermis of patients without pathological scar. TbetaR II expressing in 8 specimens of epidermis of pathological scar showed abnormal electrophoresis pattern at poly A fragments hand and loss of one A base in DNA fragment (P = 0.044). CONCLUSIONS: There may he abnormal gene expression of TbetaR II in pathological scar epidermis. Replantation of epidermis of scar may increase the risk of scar recurrence, while replantation of normal skin of patients with scar on wound may not increase the risk of scar recurrence.

New treatment strategy for granulomatous epulis: intralesional injection of propranolol.

Epulis is a relapsable lesion in gingiva without specific treatment for its unexplained pathogenesis. Nowadays, surgical excision is the most popular method of treatment. To prevent recurrence, it is necessary to resect diseased tissues thoroughly, and even to remove the involved teeth. However, this may cause functional and cosmetic deformities. Therefore, it is urgent to find a new therapy without severe side effects. Infantile hemangioma is a common benign pediatric tumor which shares many features with epulis, such as rich vascularity, high incidence of female patients, high hormone level and similar treatments. A recent study showed that propranolol, a beta adrenergic receptor (ß-AR) antagonist, was effective as treatment for infantile hemangioma. Our preliminary work showed that mRNA and protein levels of ß2-AR were higher in epulis than in adjacent tissue. Therefore, we hypothesize that intralesional injection of propranolol may be useful as epulis treatment. Further work need to be done to confirm the safety and therapeutic effect of the treatment. After that, this specific ß2-AR antagonist may be the first choice for epulis treatment.

Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells improves survival of ultra-long random skin flap.

BACKGROUND: Random flap is one kind of the most widely used skin flaps in reconstructive surgery; however, partial necrosis of its distal end remains a significant problem now. The aim of this study was to evaluate the effect of hypoxia preconditioned bone marrow mesenchymal stem cells (HpBMSCs) transplantation on ultra-long random skin flap survival in rats. METHODS: Normoxic bone marrow mesenchymal stem cells (nBMSCs) were cultured under normoxia (20% O2) and HpBMSCs under hypoxia (1% O2) for 48 hours before transplantation. Thirty Sprague-Dawley rats were randomly divided into control group, nBMSCs group and HpBMSCs group with each consisting of 10 rats. Survival area of ultra-long random skin flap on the dorsal of rats was measured seven days after flap surgery and cell transplantation. Cell survival in vivo, microvessel density and vascular endothelial growth factor (VEGF) were evaluated by histological examination and enzyme-linked immunosorbent assay. RESULTS: Compared with other two groups, flap survival area in HpBMSCs group was significantly larger (P < 0.05). Microvessel density in HpBMSCs group (36.20 ± 8.19) was higher than that in nBMSCs group (30.01 ± 5.68) and control group (17.60 ± 4.19) (P < 0.05). VEGF in HpBMSCs group ((300.05 ± 50.41) pg/g) was higher than those in nBMSCs group ((240.55 ± 33.64) pg/g) and control group ((191.65 ± 32.58) pg/g) (P < 0.05). CONCLUSION: HpBMSCs transplantation improves ultra-long random skin flap survival via promoting angiogenesis of more survival cells.

The possible roles of OPN-regulated CEACAM1 expression in promoting the survival of activated T cells and the apoptosis of oral keratinocytes in oral lichen planus patients.

Oral lichen planus is a chronic inflammatory disorder of the oral mucosa that represents T cell-mediated autoimmune diseases. The regulation and roles of carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), a novel immune molecule, in the immunopathogenesis of T cell-mediated autoimmune diseases remain unclear. In the current paper, CEACAM1 was found to be overexpressed in peripheral T cells and epithelial cells in oral lichen planus patients. A fraction of infiltrating inflammatory mononuclear cells in the lamina propria of the oral lichen planus mucosa also expressed CEACAM1. Importantly, for the first time, CEACAM1 expression in T cells and in normal human oral keratinocytes was demonstrated to be regulated differently by osteopontin in vitro. Furthermore, the apoptosis of oral keratinocytes and activated T cells can be markedly suppressed by CEACAM1-specific monoclonal antibodies. In conclusion, OPN-regulated CEACAM1 expression may play a critical role in the immunopathogenesis of oral lichen planus.

[Effects of silencing inhibitor of DNA binding-1 gene on the growth and invasiveness of adenoid cystic carcinoma cells].

OBJECTIVE: To investigate the role of inhibitor of DNA binding-1 (Id-1) gene in adenoid cystic carcinoma cell growth and invasion behavior. METHODS: With salivary adenoid cystic carcinoma cell lines ACC-M and ACC-2, dedected Id-1 gene expression was screened with immunofluorescence assay. After Id-1 mRNA knocking-down using small interfering RNA, RT-PCR and Western blot were used to detect the different expressions before and after interference, and the growth of cells before and after interference was deceted using the MTT assay, and the cell invasion ability was checked with the use of Transwell chamber assay. RESULTS: Id-1 were both expressed in the ACC-M and ACC-2, and the expression in ACC-M was higher than that in ACC-2. After Id-1 RNA interference, the growth and invasiveness of ACC-M and ACC-2 were inhibited with the restrained degree in ACC-M much stronger than that in the ACC-2. CONCLUSION: In view of the important role of Id-1 in the behavior of growth and invasion in ACC cell, interfering the expression of Id-1 gene is expected to be a novel and effective means for the treatment of adenoid cystic carcinoma.

Over-expression of Gadd45a enhances radiotherapy efficacy in human Tca8113 cell line.

AIM: To investigate the effect of the growth arrest- and DNA damage-inducible Gadd45a gene on the radiosensitivity of human tongue squamous cell carcinoma cell line to ionizing radiation (IR). METHODS: Short interfering ribonucleic acid (si-RNA) targeting Gadd45a or an irrelevant mRNA (nonsense si-RNA) was chemically synthesized. The constructed si-RNAs were transfected into Tca8113 cells and Gadd45a expression was determined using quantitative real-time PCR and Western-blot. After 24-h exposure to IR at a dose rate of 4 Gy/min, apoptosis of Tca8113 cells was detected using flow cytometry, and radiosensitivity was measured using MTT assays. RESULTS: IR apparently increased the expression of Gadd45a at mRNA and protein levels in Tca8113 cells. The effect was efficiently inhibited by transfection with Gadd45a si-RNA (P<0.01). Furthermore, silencing Gadd45a gene significantly increased cell viability and decreased the percentage of apoptotic cells during irradiation, which indicated that IR-induced Gadd45a over-expression could increase the radiosensitivity of Tca8113 cells. CONCLUSION: These results indicated that targeting Gadd45a may have important therapeutic implications in sensitizing Tca8113 cells to IR.

OPN promotes survival of activated T cells by up-regulating CD44 in patients with oral lichen planus.

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa, which represents cell-mediated autoimmune diseases. Pathological study demonstrated that abundant T lymphocytes infiltrated the oral mucosa, in which the activated T cells that trigger apoptosis of oral epithelial cells is an important mechanism for OLP. However, to date the molecular mechanisms underlying the T lymphocytes infiltration and accumulation in OLP remain unclear. In this paper, we found that the levels of plasma OPN were elevated and were associated with the up-regulated expressions of CD44 in OLP patients. In vitro, the addition of exogenous OPN can suppress the apoptosis of activated CD8(+) T cells via CD44, and this T cell resistance to apoptosis may be attributed to the reduction of endogenous mature granzyme B. Our results suggested that the abnormally elevated levels of OPN may contribute to the abnormal infiltration and accumulation of the activated T cells by up-regulating CD44 in OLP.

Overexpression of Kif2a promotes the progression and metastasis of squamous cell carcinoma of the oral tongue.

The aim of this study was to examine the Kif2a expression and its role in tumor progression, invasion and metastasis in squamous cell carcinoma of the oral tongue (SCCOT). The study included 44 cases of primary tumor and the corresponding adjacent tissues, 20 cases of primary tumor with lymph node metastasis. Immunohistochemistry was used to observe the Kif2a expression and its correlation with clinicopathologic factors in oral tongue cancer. The immunohistochemistry showed that Kif2a expression was stronger in oral tongue cancer tissues than in paired adjacent tissues (P<0.01), and the higher expression of Kif2a was also significantly associated with lymph node metastasis (P<0.01), tumor clinical stage (P<0.01). In addition, in vitro results from transwell chamber assay showed that Tca8113 cells transfected with Kif2a-siRNA had a decreased migratory ability (P<0.01) compared to nonsense-siRNA-transfected cells. Therefore we speculate the overexpression of Kif2a might be involved in the progression, invasion and metastasis of SCCOT and Kif2a should be as a predictor for prognosis.

[Radiosensitization by double suicide gene system in ACC-2 cell].

PURPOSE: To investigate the radiosensitization by prodrug and CD-TK double suicide gene therapy system in adenoid cystic carcinoma cells (ACC-2). METHODS: The eukaryotic expression plasmids pIRES-CD and pIRES-TK were introduced into ACC-2 cells by electroporation. Then ACC-2 cells stably expressing CD and TK gene were obtained by 10-day positive selection with 400 micro g/mL G418 . The total RNA was extracted and the expression of the CD and TK gene in transfected ACC-2 cells was identified by RT-PCR. The positive transfected ACC-2 cells were treated with radiotherapy of different dose (0,2,4,6,8,10 Gy) and prodrug system in aerobic and anoxic condition. Then cell clone formation assay was used to study the radiosensitization by CD-TK double suicide gene therapy and prodrug system in ACC-2.The data was analyzed by multiple factor ANOVA using SPSS11.5 software package. RESULTS: RT-PCR analysis demonstrated that CD and TK genes were effectively expressed in ACC-2 cells. With the increased of X-ray dose, the colony forming rate dropped significantly after radiotherapy. In aerobic condition, the survival fraction of group ACC-2/CD-TK+prodrug were significantly lower than that of group ACC-2 and group ACC-2/CD-TK with the same dose (P<0.05). In anoxic condition, the survival fraction of group ACC-2/CD-TK+pro-drug was significantly lower than that of experimental group ACC-2 and group ACC-2/CD-TK with the same dose (P<0.05). The colony forming rate in aerobic condition was significantly lower than that in anoxic condition of the same cell group and dose. CONCLUSION: The radiosensitivity and the killing effect of X ray to ACC-2 cells can be increased by CD-TK double suicide gene therapy and the prodrug system.

CEACAM1 distribution and it's effects on angiogenesis and lymphangiogenesis in oral carcinoma.

To investigate the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its effects on angiogenesis and lymphangiogenesis in oral carcinoma. Immunohistochemistry was used to study the expression of CEACAM1, LYVE1 and CD31, double-labelling immunofluorescence was used to detect the co-expression of CEACAM1 and LYVE1, and double-labelling immunohistochemistry was performed to observe the co-expression of LYVE1 and CD31 in vessels. Membranous CEACAM1 was expressed in well-differentiated squamous cell carcinoma and cytoplastic CEACAM1 in poorly and moderately differentiated carcinoma (P<0.05). More CEACAM1-positive vessels were observed in CEACAM1-positive tumors with cytoplasmic expression than with membranous expression (P<0.001). Co-expression of CEACAM1 and LYVE1, LYVE1 and CD31 in vessels was more common in CEACAM1-positive tumors with cytoplasmic expression than with membranous expression (P<0.001). CEACAM1 has different distribution in oral carcinoma. Membranous CEACAM1 inhibits angiogenesis and lymphangiogenesis, but cytoplasmic CEACAM1 promotes angiogenesis, and even promotes lymphangiogenesis by mediating the transformation of vascular endothelial cells (VECs) into lymphatic endothelial cells (LECs).

[Ectopic bone induction in vivo after transplantation of skeletal satellite cells from green fluorescence protein transgenic mouse transfected by adenoviral vectors encoding bone morphogenetic protein-2].

OBJECTIVE: To observe the ability of induced ectopic bone using skeletal muscles satellite cells (SMSCs) from newborn green fluorescence protein (GFP) transgenic mice mediated by Ad-BMP2. METHODS: Transplantation of SMSCs transduced with Ad-BMP2 into back lamb muscles of subfascia in wildtype 129sv mice with a complex of collagen scaffords, then the tissue histologic examination, X ray plain film, fluorescence microscopy were used. RESULTS: Transplantation of SMSCs transfected with Ad-BMP2 into back lamb muscles of subfascia generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks and mature bone formation 4 weeks after transplantation. SMSCs non-transfected with Ad-BMP2 failed to induce ectopic bone formation. CONCLUSION: SMSCs retain differentiation potentitality into osteoblasts in response to Ad-BMP2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.

[Isolation, identification, culture and bionomics of skeletal muscles satellite cells from green fluorescent protein transgenic mouse in vitro].

OBJECTIVE: To investigate the green fluorescent protein (GFP) expression and the bionomics of skeletal muscles satellite cells (SMSCs) in vitro in GFP transgenic mouse. METHODS: The newborn transgenic mice were acquired to separate skeletal muscles satellite cells with enzyme digestion method. Cells were cultured and subcultured in vitro. Morphological observation, growth curve were investigated to evaluate the proliferation and differentiation characteristics of skeletal muscles satellite cells, fluorescence microscope was used to observe the GFP expression. The cells were identified by immunocytochemical stain. In the basis of identification of anti-sarcometric actin anti-body, the combination of anti-desmin antibody and DAPI (4, 6-diamidino-2-phenylindole) were used to detect the purification of skeletal muscles satellite cells. RESULTS: Immunocytofluorescence suggested the good retain of GFP fluorescence in skeletal muscles satellite cells. The cells showed strong proliferative ability and they were positive with immunocytochemical stain of anti-sarcometric actin antibody and anti-desmin antibody. The combination of anti-desmin and DAPI stain can be used to determine the purification of SMSCs. CONCLUSION: Skeletal muscles satellite cells cultured in vitro showed strong proliferation and differentiation ability. They are fit to construct the cell bank of tissure engineering and to be a useful tool to explore cells fate after transplantation since these cells retain the expression of GFP.

[Effect of different nanophase hydroxyapatite particles on osteoblasts metabolism].

PURPOSE: To compare two different structural nanophase hydroxyapatite(HA) particles on the proliferation activity and functions of osteoblasts. METHODS: Primary culture of osteoblasts from rat calvaria was established and then cultured on the coatings of different size of nano particles (groupI 20-40nm,group II 70-100nm), the HA coatings without nano-particles was used as control group. Proliferation activity, protein content and synthesis of alkaline phosphatase(ALP) by osteoblasts were examined by MTT assay, coomassic brilliant blue method and PNPP test, and statistical significance was assessed by multiple comparison (q test, SNK) in one-way analysis of variance (ANOVA) with SPSS10.0 software package. RESULTS: Osteoblasts grew well on HA coatings. MTT assay demonstrated that there was significant difference between group I and group II at 6th day and 8th day (P<0.05).At first half stage(5th day and 10th day) ALP activity test showed no significant difference between group I and groupII (P>0.05) and as the culture going on(15th day and 20th day), there was significant difference between the two groups (P<0.05). Coomassic brilliant blue method showed that there was significant difference between group I and group II from 5th day to 20th day (P<0.05). CONCLUSION: The diameter of nano-particles on the HA coatings could influence the proliferation activity and functions of the osteoblasts. The nano-particles of similar size with HA crystal in vivo showed better cytocompatibility.

[Effect of bone morphogenetic proteins-2 on mandibular distraction osteogenesis in rabbits].

OBJECTIVE: To investigate the Effect of recombinant adenovirus vectors containing human Bone morphogenetic proteins-2 (Ad-hBMP-2) on the for mandibular distraction osteogenesis (DO) in rabbits. METHODS: Twenty-four New Zealand white rabbits were randomly divided into experimental group, and control group and underwent mandibular distraction osteogenesis. After 5 days latency, the distracters were activated at a speed of 0.5 mm every 12 hours for 7 days, then on the first day in the consolidation period, the distraction gaps of experimental group were injected with 0.2 ml Ad-hBMP2 10(12) pfu/L, while the animals of control group were injected with 0.2 ml Ad-EGFP 10(12) pfu/L. At the 7 th and 28 th day of consolidation period, specimens were obtained, X-ray and histomorphology were performed. The bone density and the quantity of new bone formation in the distraction gaps were observed and compared between the two groups at different consolidation period. RESULTS: Ad-hBMP-2 treated specimens demonstrated an increased amount of new bone formation CONCLUSIONS: Adenovirally-mediated delivery of BMP-2 can locally increase bone deposition during DO, which may potentially shorten the consolidation period.