BACKGROUND: An important initial step in developing a tissue engineering artificial salivary gland organoid is to find an ideal scaffold. To find other new biomaterials should to be further studied.OBJECTIVE: To obtain an acellular matrix from tracheae of rabbits and SD rats, and to investigate its biocompatibility as a primary step toward developing a tissue engineering artificial salivary gland organoid.DESIGN, TIME AND SETTING: A randomized controlled animal study, which was performed at the Key Laboratory of Transplantation Engineering and Immunology of Ministry of Health, West China Hospital of Sichuan University from February 2003 to May 2005.MATERIALS: A modified detergent and enzyme link extraction procedure was performed to remove cells from SD rats and rabbits tracheae. The histology, topography of inner-surface and biocompatibility were studied on both acellular tracheae.METHODS: Eighteen SD rats were randomly divided into 2 groups. One group was planted acellular trachea of SD rats. Other group consisted of acellular trachea of rabbits. On the third generations, submandibular gland cells were inoculated on two acellular tracheae and cultured on PGA membrane; while, cells in the control group were inoculated on 12-well culture plate, and cell/scaffold complex was cultured at the same time.MAIN OUTCOME MEASURES: The structure and topography of inner-surface of the acellular tracheae matrixes were observed both by light and scanning electron microscopy. The inflammatory response of the tissue around acellular tracheae implanted under the skin of cheek was evaluated at 1, 4, 12 weeks. The numbers of cells grown on the acellular tracheae and PGA film were counted at 1, 2, 3, 4, 5, 6, 7 days. At 1, 3, 5, 7 days, the mean values of metabolic activity test and the amylase activities of supernatants of the cells/scaffold complexes were examined.RESULTS: The cells were completely removed from both tracheae. No evident inflammatory response was found in tissues around two kinds of acellular tracheae implanted under the skin of cheek. The number of submandibular gland cells (SSG) grown on the two kinds of naturally derived biomaterials was much more than grown on PGA (P<0.05). The mean values of metabolic activity test and the amylase activities of supernatants containing cell/acellular matrixes were much higher than that of cell/PGA (P<0.05).CONCLUSION: The acelhilar tracheae matrixes made by our laboratory can be used as scaffold in the study of tissue engineering artificial salivary gland organoid.

BACKGROUND: Seed cells with good proliferation and enough amounts are need in reconstructing artificial salivary gland in vitro. However, adult stem cells are difficult to be isolated from normal submandibular gland.OBJECTIVE: To in vitro isolate submandibular gland stem/progenitor cells for cloning culture using animal models of damaged gland tissue.DESIGN, TIME AND SETTING: Cytological in vitro experiment was performed at the Gu.izhou Provincial Key Laboratory of Cell Tissue Engineering, Zunyi Medical College from March 2006 to January 2007.MATERIALS: A total of 10 male Sprague Dawley rats aged 8 weeks were supplied by the Animal Center, Third Military Medical University of Chinese PLA.METHODS: The model of tissue damaged submandibular gland in 10 rats was made by deligation. One week later, the gland tissue was obtained to harvest submandibular gland stem/progenitor cells by enzyme digestion in vitro. Following 10-14 days of primary culture, small round cells were collected, purified and subcultured for monoclonal culture.MAIN OUTCOME MEASURES: Immunocytochemical staining and immunofluorescence staining results were measured in submandibular gland stem/progenitor cells. Growth curve was drawn to analyze the proliferation of submandibular gland stem/progenitor cells in vitro.RESULTS: Cells expressing laminin showed stem cell characteristics. Positive expression of CD29 suggested high-adherent and high-proliferative stem cell properties. Positive expression of keratin-19 indicated epithelium-derived submandibular gland stem/progenitor cells. Growth curve was near to "S" shape, and in vitro culture and proliferation was active.CONCLUSION: Submandibular gland stem/progenitor cells had the characteristics of tissue stem cells. They might be as a kind of seed cells for tissue engineered artificial salivary gland in further research.

Objective To evaluate the clinical value of fiberoptic ductoscopy in diagnosis and treatment of patients with galactophoritis or mammary duct ectasia. Methods From November 2005 to March 2008, fiberoptic ductoscopy were performed in 120 women with nipple discharge. The duct of 95 cases as non-malignant lesion were insufflated and perfusioned with entamycin and dexamethasone. Results Ninty-five of 120 cases were non-malignant disease,which contained one side 81 and two sides 14; the discharge was bloody,ivory, stramineous in 21, 17, 57 patinents, respectively; and the dignosis were 17 mammary duct ectasia, 53 galactophoritis, and 25 mammary duct ectasia with galactophoritis. Of the 95 cases, hich were intradutal insufflated and perfusioned with gentamycin and dexamethasone, the nipple discharge were decreased or disappeared in 81 cases, the effective rate was 85.3%. Conclusion Fiberoptic ductoscopy is a convenient,safe, accurate method in diagnosis and treatment of patients with galactophoritis or mammary duct ectasia.

Objective To develop a duplex fluorescence RT-PCR assay for detection of scavenger receptor class B, typeⅠ(SRBⅠ) knockout mice. Methods Primers and probes were designed according to knockout region of SRBⅠgene and related substituted sequence. DNA samples were extracted from tails of mice and performed amplification using real-time PCR. SRBⅠgenotypes of mice were analyzed according to amplification curves of FAM and CY5 channels. Finally, the sensitivity of the method was detected and the accuracy was verified by the direct sequencing. Results The homozygous SRBⅠwild genotype showed an amplification curve only in FAM channel. When the homozygous SRBⅠknockout genotype was present, the typical S amplification curve appeared only in the CY5 channel. Heterozygous genotype showed two typical S amplification curves in both FAM and CY5 channels, respectively. The results showed that the sensitivity reached 4×101 copies/μL, and there was complete concordance between this method and direct DNA sequencing. Conclusion The new method is simple, rapid and accurate, which is suitable for genotyping SRBⅠknockout mice.

Objective To investigate the role of Ras-related C3 botulinum toxin substrate 1/mitogen-activated protein kinase/extracellular signal-regulated kinase (Rac 1/MAPK/ERK) signal pathway in rats with ventilator induced lung injury (VILI) and its mechanism.Methods Thirty Sprague-Dawley (SD) rats were randomly divided into spontaneous respiration group,normal tidal volume (VT) group and high VT group with 10 rats in each group.The rats in spontaneous respiration group were kept their spontaneous breathing.The rats in normal VT group and high VT group were performed tracheal intubation after tracheostomy,and underwent mechanical ventilation on bilateral lungs with 6 mL/kg and 40 mL/kg VT respectively with maintenance anesthesia.After 4-hour ventilation,heart blood,bronchoalveolar lavage fluid (BALF) and lung tissues were harvested.The levels of interleukins (IL-1β,IL-6),tumor necrosis factor-α (TNF-α),myeloperoxidase (MPO) and macrophage inflammatory protein-2 (MIP-2) in serum and BALF were determined by enzyme linked immunosorbent assay (ELISA).Lung wet/dry radio (W/D) was determined.The lung tissues were stained with hematoxylin and eosin (HE),and pathological changes were observed,and pathological scores were evaluated.The ultra structure changes in type Ⅱ alveolar epithelial cells (AEC Ⅱ)were observed with transmission electron microscope.The positive expressions of phosphorylation of extracellular signal-regulated kinase (p-ERK) were determined by immunohistochemistry,and those of Racl and F-actin were determined by immunofluorescence.The mRNA expressions of ERK and Rac1 were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR),and protein expressions of Rac-1,p-ERK and F-actin were determined by Western Blot.Results ① Compared with spontaneous breathing group,lung W/D in both mechanical ventilation groups was significantly increased,with more significant increase in the high VT group (6.64 ± 0.88 vs.1.79 ± 0.36,P ＜ 0.01).② There was no obvious pathological changes in the lung tissue and AEC Ⅱ of the spontaneously breathing group.In the normal VT group,there was slight edema and infiltration of inflammatory cells;AEC Ⅱ had less lamellar bodies and uniform distribution of the villi of the alveolar epithelium.In the high VT group,the edema of the lung tissue,the widening of the pulmonary septum,the alveolus congestion,the infiltration of inflammatory cells,and alveolar structure disorder were found;and AEC Ⅱ was irregular,the number of lamellar bodies in the plastids was decreased and was unevenly distributed.The pulmonary histopathological score in the high VT group was significantly higher than that in the spontaneous breathing group and the normal VT group (12.00 ± 2.00 vs.6.00 ± 1.51,8.50 ± 0.53,both P ＜ 0.01).③ Compared with spontaneous breathing group,IL-1β,IL-6,TNF-α,MPO,and MIP-2in serum and BALF in both mechanical ventilation groups were significantly increased,with more siguificant increase in the high VT group [serum IL-1 β (ng/L):104.2 ± 15.1 vs.20.3 ± 8.3,IL-6 (ng/L):46.6 ± 11.5 vs.22.7 ± 7.5,TNF-α (ng/L):39.4±6.5 vs.5.4± 1.9,MPO (ng/L):0.66±0.24 vs.0.06±0.03,MIP-2 (ng/L):109.2±25.8 vs.22.8±8.4;BALF IL-1 β (ng/L):121.5 ± 25.6 vs.24.0 ± 7.5,IL-6 (ng/L):136.7 ± 32.7 vs.31.4 ± 10.5,TNF-α (ng/L):98.0 ± 14.8vs.10.1 ±2.6,MPO (ng/L):0.80±0.31 vs.0.08±0.04,MIP-2 (ng/L):144.4±28.9 vs.41.2±20.7;all P ＜ 0.01].④ There were only a few p-ERK,Rac1 and F-actin positive expressions in the spontaneous breathing group.The positive expressions in normal VT group were increased.In high VT group,the positive expression of p-ERK was significantly increased;Rac1 and F-actin were mainly distributed in the cell membrane and cytoplasm respectively,the positive expressions were further enhanced.⑤ The gene expressions of ERK and Rac1,and protein expressions of p-ERK,Rac1 and F-actin in the high VT group were significantly higher than those in the spontaneous breathing group and normal VT group [ERK mRNA (2-△△Ct):8.23±2.83 vs.1,3.02± 1.38,p-ERK protein (gray value):1.15±0.36 vs.0.61 ±0.23,0.88±0.22;Rac1 mRNA (2-△△Ct):4.45 ±2.26 vs.1,1.22±0.39,Rac1 protein (gray value):0.91 ±0.16 vs.0.48±0.11,0.55 ± 0.10;F-actin protein (gray value):0.70± 0.09 vs.0.49 ± 0.08,0.55 ± 0.04;all P ＜ 0.01].Conclusion F-actin expression in lung tissue was up-regulated in rats with VILI,which resulted in reconstruction of AEC Ⅱ cyto-skeleton,and variation of cell membrane permeability through Rac 1 /MAPK/ERK sigualing pathway during VILI.

Totally 79 obese children and 64 children with normal body weight were included in the present study.Serum apolipoprotein A5 (ApoA5) and leptin levels were determined by ELISA and fasting insulin by RIA.The clinical data including height,body weight,waist circumference,blood pressure,blood lipid,blood glucose,etc,were collected.Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated.The results showed that compared with normal weight children,both serum leptin and insulin levels were significantly raised in obese children [19.15 (13.01 ～ 25.08) ng/ml vs 3.29 (1.45 ～ 6.02) ng/ml and 15.44 (12.05 ～ 20.26) μg/L vs 10.12 (8.60 ～ 12.60) μg/L,both P＜0.01],while ApoA5 level was significantly lowered [134.5 (105.9 ～ 172.7) ng/ml vs 2005.9(164.3 ～ 265.3) ng/ml,P＜0.01].Serum ApoA5 was negatively correlated with serum leptin and insulin (both P＜0.01).

Objective To establish a dual real-time fluorescence quantitative polymerase chain reaction (dual real-time PCR) assay to detect human vitamin D receptor (VDR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Methods GAPDH gene was used as the internal control. The specific primers and TaqMan probes were designed by Primer Premier 5.0 software, which were applied to detect the VDR/GAPDH mRNA levels. The obtained PCR products were puri-fied to construct the VDR/GAPDH recombinant plasmid, which was taken as the standard to analyze the sensitivity and re-peatability of the method. Results The amplification products were confirmed as the specific fragment of VDR/GAPDH by DNA sequencing instrument. The results showed that the sensitivity, linear range, the determinate coefficient, the amplifica-tion efficiency, the intra-assay and inter-assay coefficient of variation were 40 copies/μL, 4.00 × 101-4.00 × 105 copies/μL, 0.998, 96.10%, 0.09%-1.21%, 0.17%-0.51%for VDR, and 40 copies/μL, 4.00 × 101-4.00 × 105 copies/μL, 0.999, 85.15%, 0.35%-0.88%, 0.51%-2.46% for GAPDH, respectively. Conclusion These results demonstrate that the dual real-time PCR assay with high sensitivity and specificity can detect the relative expressions of human VDR by single reaction tube, which can effectively shorten the time and reduce the experimental error.

OBJECTIVETo obtain an evidence-based treatment for an adolescent patient with temporomandibular osteoarthritis.

METHODSThe detailed history of an adolescent patient with temporomandibular osteoarthritis was analysed. Clinical Evidence (to Dec 2010), National Guideline Clearinghouse (2000-Dec 2010), Cochrane Library (Issue 4, 2010), MEDLINE(OVID, 1950-Dec 2010) and China Biology Medicine Database (1978-Dec 2010) were searched to obtain evidence such as clinical guidelines, systematic reviews and randomized controlled trials related to surgery or conservative treatment to temporomandibular osteoarthritis to find a personal treatment strategy for the patient.

RESULTSFive articles were finally included, i.e. 1 clinical guideline, 3 systematic reviews and 1 randomized controlled trials. These evidence showed that: Conservative treatment like intra-articular injection instead of surgery should be adopted for adolescent patient; hyaluronate is the drug with sufficient evidence in supporting its use in treating temporomandibular disorders; inferior temporomandibular joint cavity injection or both upper and lower cavity injection has better effect than that of superior cavity injection only; and there was some evidence to support the use of glucosamine to treat temporomandibular disorders. Considering the situation of the case and the clinical evidence, an individual treatment plan of hyaluronate injection into the upper and lower cavity and glucosamine take orally was established. A long-term follow-up of 6 months showed a good treatment outcome.

CONCLUSIONThrough the evidence-based methods and the use of clinical evidence, an individual treatment plan could be established for each patient with temporomandibular disorders, and this will provide strong supporting to the treatment of temporomandibular disorders. Up to now, it is clear that hyaluronate injection into the upper and lower cavity with glucosamine administration is effective in treating temporomandibular osteoarthritis.

China ; Evidence-Based Medicine ; Humans ; Hyaluronic Acid ; Injections, Intra-Articular ; Osteoarthritis ; Temporomandibular Joint ; Temporomandibular Joint Disorders ; Treatment Outcome

OBJECTIVEThree-dimensional finite method was used to analyze stress and strain distributions of periodontal ligament of abutments under dynamic loads.

METHODSFinite element analysis was performed on the model under dynamic loads with vertical and oblique directions. The stress and strain distributions and stress-time curves were analyzed to study the biomechanical behavior of periodontal ligament of abutments.

RESULTSThe stress and strain distributions of periodontal ligament under dynamic load were same with the static load. But the maximum stress and strain decreased apparently. The rate of change was between 60%-75%. The periodontal ligament had time-dependent mechanical behaviors. Some level of residual stress in periodontal ligament was left after one mastication period. The stress-free time under oblique load was shorter than that of vertical load.

CONCLUSIONThe maximum stress and strain decrease apparently under dynamic loads. The periodontal ligament has time-dependent mechanical behaviors during one mastication. There is some level of residual stress left after one mastication period. The level of residual stress is related to the magnitude and the direction of loads. The direction of applied loads is one important factor that affected the stress distribution and accumulation and release of abutment periodontal ligament.

Denture, Partial, Fixed ; Finite Element Analysis ; Humans ; Incisor ; Mandible ; Mastication ; Periodontal Ligament ; Tooth

OBJECTIVETo investigate the effect of fas gene transfection and monoclonal anti-fas antibody on tumorigenicity and proliferation of transplanted tumor of Tca8113 cell.

METHODSPlasmid including fas gene was transfected into Tca8113 cell by lipofectamine kit. Some transfected cells were treated by monoclonal anti-fas antibody after 48 hours since transfection. Untransfected cell (control), fas-tansfected cell and fas-transfected cell treated with antibody were transplanted to nude mice subcutaneously. Growth of transplanted tumor was observed and recorded regularly. Animals were sacrificed and tumor samples were harvested at the end of experiment. Fas expression in each neoplasm was assessed by RT-PCR. Apoptosis, proliferation and expression of fas protein in tumor tissue were measured by flow cytometry (FCM).

RESULTSTumor occurred much later in fas-transfected group and fas-transfected plus antibody treated group. Growth arrest was found in them. RT-PCR and FCM suggested that fas-transfection up-regulated the expression of fas mRNA and protein, increased apoptosis index (AI). But no effect on proliferation index (PI) was observed. Monoclonal anti-fas antibody did not effect the expression of fas mRNA and protein, but increased AI and decreased PI.

CONCLUSIONFas-transfection suppressing tumorigenesis of Tca8113 cell transplanted in nude mice might be caused by up-regulation of expression of fas gene and enhancement of apoptosis. However, anti-fas antibody suppressing tumorigenesis might be associated with activation of apoptosis and repression of proliferation.

Animals ; Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Fas Ligand Protein ; biosynthesis ; genetics ; pharmacology ; Humans ; Mice ; Mice, Inbred BALB C ; Mouth Neoplasms ; metabolism ; pathology ; RNA, Messenger ; genetics ; Transfection