Icariin contributes to healing skull defects in rabbit model.

OBJECTIVE: To investigate the efficacy of icariin for healing skull defects in rabbit models. METHODS: Thirty-six 3-month-old female New Zealand white rabbits, weighing 1.8-2.0 kg each, were randomly divided into either the control or experimental group. Skull defect models were constructed in both groups, with the experimental group receiving oral icariin afterwards. At 4, 8 and 12 postoperative weeks, the rabbits were euthanized, and X-rays and samples were taken. Tissue sections were stained using hematoxylin and eosin, followed by additional processing using histological and bone metrological methods for observing the rate and quality of the bone formation. RESULTS: Histologically, additional mature lamellar bone formed in the defect area in the experimental group compared with that of the control group. Bone metrological methods showed that the bone mass, trabecular bone width and number of osteoblasts in the experimental group were significantly higher than those of the control group (P < 0.01). The number of osteoclasts did not significantly differ between the two groups (P > 0.05). CONCLUSION: Icariin increased the bone mass and improved the condition of the defect area in the rabbit skulls.

Treatment of obstructive sleep apnoea-hypopnea syndrome by mandible advanced device reduced neuron apoptosis in frontal cortex of rabbits.

Objective: To investigate effects of mandible advanced device (MAD) therapy for obstructive sleep apnoea-hypopnea syndrome (OSAHS) on the neuron apoptosis and acetylcholine esterase activity in frontal cortex. Materials and methods: Thirty male New Zealand white rabbits were randomly divided into three groups (n = 10 in each group): group OSAHS, group MAD, and control group. Hydrophilic polyacrylamide gel was injected into soft palate of the animals to induce OSAHS in group OSAHS and group MAD. The group MAD animals wore MAD to relief the obstructiveness. The control group was not given any treatment. Computed tomography (CT) examination of the upper airway and polysomnography (PSG) recordings were performed in supine position. All rabbits were induced to sleep in a supine position for 4 to 6 hours every day and were observed for consecutive 8 weeks. The frontal cortices of three groups were dissected and the neuron apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and flow cytometry. Acetylcholine esterase (AchE) activity in the frontal cortex was measured by spectrophotometry. Results: The group OSAHS exhibited high neuron apoptosis rate and low AchE activity than those of group MAD and control group. The blood oxygen saturation was negatively correlated with neuronal apoptosis rate and positively correlated with AchE activity. Applying MAD in OSAHS animals significantly improve the neuronal damage and function deficits by apnoea-hypoxia caused by narrowed upper airway. Conclusion: This study provided evidence that MAD therapy for OSAHS can significantly decrease neuronal apoptosis and increase AchE activity in the frontal cortex.

Fundamental research on the action mechanism of the 800 nm semiconductor laser on skin blackheads and coarse pores.

The aim of the study was to determine the mechanism of action of the 800 nm semiconductor laser on skin blackheads and coarse pores. A total of 24 healthy purebred short-haired male guinea pigs, weighing 350-400 g, were selected and smeared with 0.5 ml coal tar suspension evenly by injector once daily. Treatment was continued for 14 days to form an experimental area of 8×3 cm on the back of the guinea pigs. The animals were divided into the following groups: Normal control group (NC), low-dose laser treatment group (L-LS), high-dose laser treatment group (H-LS), and Q-switched Nd:YAG treatment group (QC). Samples were extracted 1, 7 and 14 days after surgery and hematoxylin and eosin staining was used to identify the following: Epidermis, dermis, sebaceous gland change and hair follicle damage; the expression of proliferating cell nuclear antigen (PCNA) of sebaceous gland cells using immunohistochemistry; sebaceous gland cell apoptosis using TUNEL; and the protein expression of caspase-3, Bax and Bcl-2 using western blot analysis. With the extension of time, we observed inflammatory cell infiltration, an increase in hair follicle distortion and necrosis of the surrounding hair follicles. The expression levels of PCNA of the L-LS, H-LS and QC groups decreased with time. Regarding the respective time points, the NC group was highest, the L-LS and H-LS groups were next highest and the H-LS group was lowest. The difference was statistically significant (P<0.05). The apoptotic rate of the L-LS, H-LS and QC groups increased with time. With regard to the respective time points, the NC group was lowest, the L-LS and QC groups were next lowest and the H-LS group was highest. The difference was statistically significant (P<0.05). The protein expression of caspase-3, Bax and Bcl-2 of the L-LS, H-LS and QC groups increased with time. Regarding the respective time points, caspase-3 and Bax protein expression of the NC group was lowest, the L-LS and QC groups were next lowest and the H-LS group was highest. Bcl-2 protein expression of the NC group was highest, protein expression of the NC group was next highest and the H-LS group was lowest. The difference was statistically significant (P<0.05). In conclusion, the low-dose 800 nm semiconductor laser is an effective treatment on skin blackheads and coarse pores, and promotes hair follicle cell apoptosis without reducing the expression of PCNA.

The effects of increasing occlusal vertical dimension on the deep masseter of rat at different ages.

OBJECTIVE: To investigate the influence of increasing the occlusal vertical dimension (iOVD) on the fibre-type distribution and ultrastructure of deep masseter of rat at different ages. DESIGN: A total of forty-eight male Wistar rats were divided into two groups according to age: 'teenage' group (n=24, 1.5 months) and 'young adult' group (n=24, 8 months). Both the teenage and the young adult rats were then randomly divided into the control group (n=12) and the experimental group (n=12). The occlusal vertical dimensions of the rats in the experimental groups were increased by placing composite resin on all maxillary molars. The fibre-type distribution and ultrastructure of the deep masseter were subsequently observed on day 7 and day 14 after iOVD. RESULTS: In the teenage experimental group, the proportion of type IIa fibres increased, while the proportion of type IIb and type IIx fibres decreased by day 7 after iOVD (P<0.05). However, no significant fibre phenotype transformation was observed in the young adult experimental group until day 14 after iOVD. In addition, the proportion of type IIa in the teenage experimental group was higher than that of the young adult experimental group on day 7 and 14 (P<0.05). Under the transmission electron microscope, muscle fibre reconstruction and the compensatory increase in the number and volume of mitochondria appeared earlier in the teenage experimental group. The cellular traumatic reaction was less than that in the young adult experimental group. CONCLUSION: The teenage rat alters masseter muscle structure to a slower phenotype earlier and to a greater degree than that of the young adult rat when increasing the occlusal vertical dimension.

Effects of a mandibular advancement device on genioglossus in obstructive sleep apnoea hypopnea syndrome.

OBJECTIVE: To investigate effects of mandibular advancement device (MAD) therapy for obstructive sleep apnoea hypopnea syndrome (OSAHS) on the genioglossus contractile properties and fibre-type distribution. MATERIALS AND METHODS: Thirty 6-month old male New Zealand white rabbits were randomised into three groups: OSAHS, MAD, and controls. Rabbits in Group OSAHS and Group MAD were established as OSAHS models by injection, at a dose of 2 ml hydrophilic polyacrylamide gel, via the submucous muscular layer of soft palate. Spiral computed tomography (CT) showed a significant reduced retropalatal upper airway, and apnoeas happened with an increase of Apnea Hypopnea Index (AHI) and a decrease of blood oxygen saturation during polysomnography (PSG), which indicated the OSAHS model developed successfully. OSAHS rabbits in Group MAD were fitted with a MAD made from self-curing composite resin, at 30 degrees to the upper incisors, and the mandible was guided forward 3 to 4mm. Further, spiral CT and PSG suggested MAD was effective. Rabbits in 3 groups were induced to sleep for 4-6 hours per day for 8 weeks, after which the genioglossus was removed, mounted in a tissue bath, and stimulated through platinum electrodes; maximal twitch tension, contraction time, half-relaxation time, force-frequency relationship, and fatigability were recorded. The percentage of Type I and Type II fibres was quantified. RESULTS: The fatigability and percentage of Type II fibres of genioglossus increased in Group OSAHS compared with controls; this abnormality was corrected by MAD. CONCLUSION: MAD therapy for OSAHS could prevent genioglossus fatigue and abnormal fibre-type distribution of genioglossus in OSAHS.

[Establishment of the craniofacial three-dimensional finite element models with the sutures defined alone].

OBJECTIVE: To develop a child craniofacial three-dimensional (3D) finite element model (FEM) with sutures defined alone. METHODS: The CT data for this study was developed from sequential computed tomography scan images taken at 0.625 mm intervals of an 8 years children skull. Data set was imported into Mimics 10.0 and processed with Geomagic 9.0, and exported as initial graphics exchange specification(IGES) files. The IGES files were then imported into Ansys 13.0 to set up two FEM with or without the median palatine suture being opened. The FEM contained nine craniofacial sutures and eight teeth which were defined alone.For simulating orthopedic maxillary protraction, three forces (F1-F2) were loaded on FEM.F1(1 N) was loaded at 1 cm above the geison. F2(1 N) was loaded at articular fossa of temporal bone. F3(2 N) was directed anteriorly and paralleled with occlusal plane near the canine. The stress distribution and the values distributed in each point gained in the two models were compared. RESULTS: Two craniofacial 3D FEM of the child were developed with the median palatine suture opened or not .With median palatine suture being opened or not, the two models showed the similar von Mises stresses (VMS). The distribution of the VMS was in the bridge of the nose and dextro-ala nasi.When the median palatine suture was opened, the max VMS value was 18916.00×10(-4) MPa which appeared in the nose point and the min VMS value was 1.61×10(-4) MPa which appeared in the maxillary central incisor point. At the same time, the max stress value at the direction Y was -3985.30×10(-4) MPa and appeared in the frontomaxillary suture point, and the min Y value was 0.08×10(-4) MPa which appeared in the maxillary central incisor point. When the median palatine suture was not opened, the max VMS value was 19 244.00×10(-4) MPa and appeared in the nose point. The min VMS value was 1.62×10(-4) MPa and appeared in the maxillary central incisor point. At the same time, the max stress value at the direction Y was -4258.20×10(-4) MPa and appeared in the frontomaxillary suture point, and the min Y value was 0.08×10(-4) MPa which appeared in the maxillary central incisor point. CONCLUSIONS: To define the sutures as entities alone contributed to develop child craniofacial 3D FEM which consist nine sutures. There was tiny difference in stress distribution in both the VMS and in Y direction with the median palatine suture being opened or not.

[The effect of mini-implant lengths on stress distributions in peri-implant surface].

OBJECTIVE: To observe the effect of mini-implant lengths on stress distribution in peri-implant surface. METHODS: The 3D finite element analysis mandible and mini-implant models with diameter of 1.6 mm, lengths of 6, 8, 10 and 12 mm were established. The mini-implants were inserted into designed site of mandibular vertically, respectively. A force of 1.96 N were applied mesioly and 45 degrees tilted mesio-vertically in models. The stress distribution under every condition was recorded and analyzed. RESULTS: When load was applied mesially, the maximum stress varied from 3.500 to 3.765 MPa, the maximum displacement varied from 1.266 to 1.288 microm. When load was applied 45 degrees tilted mesio-vertically, the maximum stress varied from 4.075 to 4.510 MPa, the maximum displacement varied from 1.668 to 1.694 microm. All of the maximum stress and displacement of loading mesially were lower than loading mesio-vertically. CONCLUSION: The change of the mini-implant length within 6-12 mm don't show much influence on the stress distribution. The loading type is an important factor influencing stress and displacement of peri-implant bone.

[Relationship between proteoglycans and proliferation of human salivary adencid cystic carcinoma].

OBJECTIVE: To investigate the effect of down-regulated proteoglycans on the proliferation of human salivary adenoid cystic carcinoma (SACC). METHODS: The short hairpin RNA (shRNA) plasmid silencing human xylosyltransferase-I (XT-I) gene was constructed and named shRNA-WJ3. Adenoid cystic carcinoma cells with high metastatic tendency (ACC-M) were transfected by shRNA-WJ3. The plasmid shRNA-HK not targeting any human gene was transfected into ACC-M cells used as negative control. After 48 h of transfection, the positive cells were screened by G418 to isolate the stable transfected cells. Real-time PCR and Western blotting were used to test the gene silence, and the proteoglycans contents of the cells were detected. The stable cell line silenced XT-I was named ACC-M-WJ3. MTT assay was performed to detect the cell proliferation. The cell cycle was analyzed by flow cytometry. RESULTS: ShRNA-WJ3 showed powerful RNA interference and gene silence of XT-I. The inhibition rate was 83.70% of mRNA expression and 79.60% of protein expression respectively. The content of proteoglycans in ACC-M-WJ3 was down-regulated by 49.71%-54.59%. The results of MTT assay showed that the cell growth was inhibited significantly. S phrase decreased and G1-G0 phrase increased in group ACC-M-WJ3 compared with that of group ACC-M-HK (P < 0.05). CONCLUSIONS: The down-regulated proteoglycans could inhibit the proliferation of human ACC-M cells.

[Effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells to osteoblasts].

OBJECTIVE: To investigate the effects of traditional Chinese medicine on the differentiation of mesenchymal stem cells (MSC) to osteoblasts. METHODS: Six male Beagle dogs weighed 10-15 kg each were divided into three groups, group A: medicine serum group, group B: non-medicine serum group and group C: bovine serum group. The serum of group A was obtained from the femoral artery of 2 Beagle dogs drinking equivalent dose of traditional Chinese medicine according to body surface area for 7 continuous days. The serum of group B was collected from the femoral artery of 2 Beagle dogs fed with equal volume of normal saline for 7 days. The serum of group C was fetal bovine serum. The tibia marrow was harvested from another 2 Beagle dogs and MSC were isolated and purified by density gradient centrifugation. MSC were cultured in DMEM solution with fetal bovine serum. After MSC were digested by trypsin, MSC were cultured in DMEM solution with the osteogeneic inducer, which contained dexamethasone, antiscorbutic and beta-glycerophosphate. Morphological and histological changes of the MSC were observed under an inverted microscope. Alizarin monosulfonate and nitric acid argentum staining was performed to observe the calcium deposition. MSC were curtured in DMED solution with medicine serum (group A), non-medicine serum (group B) and bovine serum (group C) respectively. The growth curve was detected by the methyl thiazolyl tetrazolium (MTT). The alkaline phosphatase (ALP) activities were detected to observe the differentiation of MSC. RESULTS: The original MSC were observed as fibroblast-like cell shapes. After the osteogeneic inducer was added, MSC were polygon cells with a few polyprocess. Calcium deposition appeared during 10-14 days and alizarin monosulfonate and Von Kossa staining presented positive. MTT results showed that the number of adherent cells of group A was more than that of group B and that of group C significantly after 6 days (P < 0.05). ALP detection proved that ALP activity of group A was more than that of group B and that of group C significantly after 5 days (P < 0.05). CONCLUSIONS: The traditional Chinese medicine promotes the differentiation of MSC to osteoblasts and osteogenesis.

[Therapeutic effect of emodin in experimental periodonititis rats].

AIM: Through observing the change of Nitric Oxide (NO) in the peripheral blood and gingival tissue, to explore the mechanism of Emodin (EMD) in the therapy of periodontal disease. METHODS: SD rats were randomly divided into four groups of thirty (group N, group P, group PL and the group PH). The periodontitis model was made and EMD was administered at PL and PH. At the 4th, 6th, 8th weeks after the animal models were established, the rats were killed respectively and the maxilla and the peripheral blood were collected. A side of maxilla was stained with HE for histological examination and the other side of gingival tissue surround ligated tooth was collected for the measurement of NO. Also, it was measured in the peripheral blood. GLM method using SPSS was used to compare changes in each of the variables over time. RESULTS: The levels of NO in the peripheral blood and gingival tissue in PL and PH groups were significantly lower than that in P groups(P<0.05). CONCLUSION: Emodin can take effect on periodontitis by reducing the levels of NO in the peripheral blood and gingival tissue.

[Effect of maxillary protraction with or without rapid palatal expansion in treating early skeletal Class III malocclusion].

OBJECTIVE: To evaluate the effectiveness of treatment with maxillary protraction with or without rapid palatal expansion (RPE) for skeletal Class III malocclusion in mixed dentition. METHODS: A total of 31 children with Class III malocclusion in mixed dentition were selected, and 15 (group A) received maxillary protraction treatment with RPE, the other 16 (group B) received maxillary protraction without RPE. Cephalometric films were taken before and after treatment, and traditional and Pancherz analysis were used. RESULTS: The average duration of treatment was 10.14 months in group A and 9.77 months in group B respectively (P>0.05). According to Pancherz analysis, maxillary basal bone moved forwards by 2.99 mm in group A and 3.33 mm in group B respectively (P>0.05), mandibular basal bone moved backwards by 0.07 mm in group A, while forwards by 0.80 mm in group B (P>0.05), the overjet increased by 4.51 mm in group A and 6.37 mm in group B respectively (P<0.05), and the molar relationship improved by 4.97 mm in group A and 4.73 mm in group B respectively (P>0.05). The effects were clinically satisfactory in the both groups. Lower molar moved forwards by 1.18 mm in basal bone in group A, while backwards by 1.20 mm in group B (P<0.05). Traditional cephalometric analysis showed no statistic differences between the two groups except that upper incisior showed greater procline in group B than in group A (P<0.05). CONCLUSION: The study shows that maxillary protraction treatment, with or without RPE, is clinically satisfactory to correct early skeletal Class III malocclusion.

[Rapid canine distalization through distraction of the periodontal ligament after reducing interseptal bone resistance].

OBJECTIVE: To investigate the effect of rapid canine distalization through distraction of the periodontal ligament after reducing interseptal bone resistance. METHODS: Twenty canines in 11 patients who needed first premolar extractions were involved. A tooth-borne, custom-made distractor was bonded right after the first premolar extraction and the interseptal bone resistance reduction. Three days post-operatively, the distractor was activated 0.1 mm three times a day. Orthodontic models, panoramic radiographs, periapical radiographs, electrical vitality test were assessed pre- and post distraction procedure and 3 months after the completion of the procedure. RESULTS: The distraction procedure was completed in 18 to 35 days [mean (25.6 +/- 4.7) days], with the distal displacement of the canines ranging from 3.53 to 8.29 mm [mean (5.56 +/- 1.32) mm]. The canines showed a mean of 12.20 degrees distal tipping and 18.53 degrees rotation. The anchorage teeth showed an average of (0.76 +/- 0.75) mm mesial movement. The mesial contact point of incisors showed a mean of (0.67 +/- 0.55) mm lingual movement. There was no significant root resorption or long-time change on pulp vitality after distraction. CONCLUSIONS: The canine distalization through distraction of the periodontal ligament after reducing interseptal bone resistance was an effective approach to move canines rapidly.

[Effect of shRNA-mediated silence of H-ras gene on proliferation of human SACC-M cells].

OBJECTIVE: To examine the effects of H-ras gene silence on cell cycle, proliferation and apoptosis of salivary adenoid cystic carcinoma -M (SACC-M) cell lines. METHODS: The plasmid H-ras-shRNA, containing the sequence of shRNA targeting H-ras, and HK-shRNA (without interfering effect) were constructed and transfected into SACC-M cells. The cell line with shRNA plasmid stable expression was isolated by G418. The expression levels of H-ras were detected by RT-PCR and protein immunofluorescent assay; cell cycle and cell apoptosis were analyzed by flow cytometry (FCM). The proliferation of cell was also determined by subcutaneous tumor formation in nude mice. RESULTS: After transfection of H-ras-shRNA plasmid, the mRNA expression of H-ras in SACC-M cells was down-regulated by 61.80% and protein expression of H-ras was inhibited by 62.76%; the cell proliferation was inhibited obviously; the G0G1 phase cells were increased. The cell apoptosis rate of H-ras-shRNA group was significantly higher than that of HK-shRNA group (P <0.05). The volume of subcutaneous tumor in nude mice was significantly smaller in Hras-shRNA group than in control group. CONCLUSIONS: The recombinant plasmid HRAS-shRNA could efficiently down-regulate the expression of H-ras gene and protein, induce apoptosis of SACC-M cells and simultaneously inhibit proliferation of these cells in vitro and in vivo.

[Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-I].

AIM: To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity. METHODS: The predominant epitope of XT-I gene was predicted by the DNAssist software. The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli ER2566 and the fusion protein GST-XT was induced and isolated. The purified fusion protein was used to immunize New Zealand rabbits. The antibody titer was determined by ELISA. Purified polyclonal antibody was obtained through affinity chromatography column and the specificity of the purified antibody was characterized by Western blot. RESULTS: The amino acid 175-205 of XT-I (QKHQPELAKKPPSRQK-ELLKRKLEQQEKGKG) was selected as an antigen epitope. The synthesized DNA fragment of XT-I was successfully inserted into pGEX-4T-2 vector and the protein GST-XT was expressed. The purified fusion protein GST-XT was used as the immunogen to immunize rabbits and the polyclonal antibody against XT-I protein was obtained. The result of ELISA showed that the antibody titer was 1:640 000. Western blot analysis showed that the antibody had a good specificity. CONCLUSION: The rabbit polyclonal antibody against human XT-I protein has been successfully prepared, which lays the foundation for further study on the biosynthesis of PG by neoplastic myoepithelial cells in salivary tumors.

[An experimental study on porcine fibroblasts transfected by dental matrix protein-1 gene].

OBJECTIVE: To evaluate the expression of dental matrix protein-l (DMP1) in porcine oral mucosa fibroblasts (POMF) transfected by DMP1 and the influences of the transfection. METHODS: The full length of porcine DMP1 cDNA was linked into an eukaryotic expression vector pEGFP-C1. POMF and mesenchymal stem cells (MSC) were transfected with the pEGFP-DMP1. The expression of DMP1, dental sialoprotein (DSP), amelin and ameloblastin (Ambn) gene of transfected POMF and MSC were detected by RT-PCR. The expression of DMP1 and DSP protein was examined by immunocytochemical staining. The formation ratio of mineralized nodules of transfected cells was compared with un-transfected ones after mineralized induction. The formation of mineralized nodules of three-dimensional pellet transfected cells was compared with un-transfected ones after hematoxylin and eosin staining. RESULTS: The constructed pEGFP-DMP1 could produce 4.7 kb and 1.5 kb fragments. DMP1 gene, DSP gene and Ambn gene were expressed by POMF after transfection. Immunohistochemical staining and the quantitative analysis of protein showed that DMP1 and DSP protein was positive in transfected POMF and MSC. The formation ratio of mineralized nodules of transfected POMF and MSC was higher than that of un-transfected ones (P < 0.05). CONCLUSIONS: The expression of porcine DMP1 in POME after gene transfection can induce the expression of tooth-development-associated gene Ambn and DSP and enhance the formation of mineralized nodules.

[Study on effect of snore guard to upper airway structure of normal occlusion people by magnetic resonance imaging].

OBJECTIVE: To investigate the imagery changes of the upper airway and the surrounding soft tissues of local adults with non-apnea who used snore guard and to provide experimental data for the clinical diagnosis and treatment of obstructive sleep apnea syndrome (OSAS). METHODS: Thirty students with non-apnea from Hebei medical university were chosen, and magnetic resonance imaging (MRI) was used to measure the changes of the upper airway and the surrounding soft tissues after snore guards were used. SPSS 105 software was used to analyze statistically. RESULTS: After the snore guard was put into oral cavity, the change of the average section and volume of the nasopharynx, the palatopharynx, the hypopharynx and the glossopharynx were statistically significant. The average sagittal size, the average horizontal size of the nasopharynx, the palatopharynx, the hypopharynx and the glossopharynx were increased statistically. The ratio of sagittal size, the horizontal sizand the in the hypopharynx and the glossopharynx changed statistically important. There was a decrease of the soft palate, the shape, the height, and the length of the tongue, the difference was statistically significant. The results demonstrated that snore guard affected the upper airway mainly by changing the volume and the shape of the upper airway, there was an obvious increase of the pharynx. The results also showed that snore guard could increase the width (both sagittal and horizontal) of the upper airway and could change the shape of the surrounding soft tissues, which caused air way more smooth. Snore guard could make the indexes of soft palate and tongue change decreasingly, resulted in the straight stand up of the tongue and the forwardness of the soft palate. CONCLUSION: Snore guard is an effective and convenient instrument for treating the patients with OSAS.

[wt-p53 gene repair for p53 mutations of salivary pleomorphic adenoma cells].

OBJECTIVE: To analyze the original mutated codon of p53 gene of salivary pleomorphic adenoma (SPA) and to evaluate the repair effects of wt-p53 on SPA cells. METHODS: Four cases of SPA were obtained from clinical fresh samples and SPA cells were separated and cultured, and then the cells were transduced by Ad-wt-p53. The cells and the corresponding tumor tissue DNA were extracted, PCR and single strand conformational polymorphism (SSCP) and DNA sequencing analysis were performed. RESULTS: PCR-SSCP analysis showed 3 out of 4 SPA with abnormal exon 8 and abnormal exon 6. DNA sequencing analysis showed that exon 6 point mutation was found at codon 203 (GTG-->GCG), poly-codon mutations were found in exon 8 at codon 272 (GTG-->GT square), 275 (TGT-->T square T), 276 (GCC--> square CC) and at codon 290 (CGC-->CGCC). After transduced by Ad-wt-p53, all of the mutated codons were repaired. CONCLUSIONS: p53 gene mutation involved many codons that occurred frequently in the tumorigenesis of SPA. Exogenous wt-p53 could compensate and repair all the mutated p53 codons of SPA cells. SPA cells transduced by Ad-wt-p53 showed the typical apoptosis.

[Expression of p53 gene with DNA polbeta and CMV promoter in salivary adenoid cystic carcinoma cells].

OBJECTIVE: To evaluate the activity of DNA polbeta promoter on p53 gene in salivary adenoid cystic carcinoma (SACC) cells. METHODS: The luciferase activity was examined and used to evaluate the activity of DNA polbeta promoter on SACC-83 cells. Eukaryotic expression plasmids of p53 gene were constructed and stably transfected into SACC-83 cells. RT-PCR was used to assess the expression of p53 gene. The SACC-83 cells were subjected to the treatments of H2O2, ultraviolet radiation, Bleocin, and affected p53 mRNA and protein level in SACC-83 cells were characterized with RT-PCR and Western blotting. RESULTS: The result of luciferase activity proved that the activity of DNA polbeta promoter in SACC-83 cells was much higher than that of CMV promoter. The results of RT-PCR suggested that p53 gene with different promoters were all expressed effectively, but the expression efficiency was different. It was greater in DNA polbeta group than in CMV group. After DNA damage, p53 gene expression increased and DNA polbeta promoter could enhance the expression of p53 gene more than CMV promoter. The results of Western blotting indicated that the expression of P53 protein between the two groups did not show any difference. CONCLUSION: In SACC cells, the activity of DNA polbeta promoter was increased and DNA polbeta promoter could enhance the expression of p53.

[Study of p16INK4/CDKN2 gene homozygous deletions and point mutation in squamous cell carcinoma of buccal mucosa].

OBJECTIVE: To explore the correlation between homozygous deletions and mutation of p16 gene and the carcinogenesis and progression of squamous cell carcinoma of buccal mucosa. METHODS: Thirty buccal cancers, 10 leukoplakias and 8 buccal mucosas were involved. DNA was extracted from the tissues. PCR was used to analyses homozygous deletion of p16 gene. PCR-SSCP-DNA sequencing was performed to detect the point mutation of p16 gene. Immunohistochemical techniques were used to detect the expression of P16 protein. RESULTS: Gene deletions and point mutations were not found in leukoplakia and normal buccal mucosa. Gene deletions were found in 7 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (23.3%), while point mutations were found in 5 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (16.7%). Sequencing analysis showed that 5 cases point mutations were missense mutations, occurred on exon 2. Three cases occurred in the same point, codon 99 (GAT --> AAT). The result of immunohistochemical stains showed that 11 out of 12 cases gene inactivation did not expressed P16 protein. CONCLUSION: Homozygous deletion and point mutation of p16 were the main pattern of gene inactivation in squamous cell carcinoma of buccal mucosa. There was a closely correlation between p16 gene inactivation and the carcinogenesis of squamous cell carcinoma of buccal mucosa.

[The relationship between methylation of p16 INK 4/CDKN2 gene in promoter region and progress of squamous cell carcinoma of buccal mucosa].

OBJECTIVE: To determine the correlation between methylation of p16 gene in promoter region and the carcinogenesis and progression of squamous cell carcinoma (SCC) of buccal mucosa. METHODS: Methylation of pl6 gene in SCC and leukoplakia of buccal mucosa was investigated by MSP and pl6 protein was analyzed by Western blot. RESULTS: The methylation of p16 gene was found in 15 of 30 cases SCC and 1 of 10 cases of leukoplakia of buccal mucosa (P < 0.05). Methylation of p16 gene in SCC of buccal mucosa was not related with age, sex, cell differentiation and clinical stage. But methylation of p16 in the cases with lymph node-metastasis was higher than that in the cases without lymph node-metastasis protein (P < 0.05). Meanwhile Methylation of p16 gene was positively correlated with no-expression of p16 protein (P < 0.01). CONCLUSIONS: The methylation of p16 gene leaded to the inactivation of p16 gene and was related with the carcinogenesis and progress of SCC of buccal mucosa.