BACKGROUND: The transcription factor, hypoxia-inducible factor-1al pha (HIF-1α), is the key regulator that controls the hypoxic response of mammalian cells. However, the role of HIF-1α in the therapeutic efficacy of chemo-radiotherapy in oral squamous cell carcinomas (OSC) is poorly understood. OBJECTIVE: To investigate the effect of HIF-α on the susceptibility of OSC cell lines against chemotherapeutic drugs and radiation. DESIGN: AN observational comparative experiment. SETTING: Beijing Chaoyang Hospital Affiliated to Capital Medical University. MATERIALS: OSC-2, OSC-4, OSC-5, and OSC-6 cell lines were estab lished from oral squamous cell carcinoma (The cell lines were from the De partment of Oral Oncology, Kochi Medical School, Japan). METHODS: The experiments were completed in Beijing Chaoyang Hospi tal affiliated to Capital Medical University from September 2004 to August 2006. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin. ① Total cell lysates were extracted from untreated OSC cells and those treated with 30 Gy γ-rays, 100 μmol/L cis-dichlorodiamine plat inum (CDDP), or 100 μmol/L 5-fluorouracil (5-FU). The expressions of HIF-1α protein and mRNA were determined with western blotting analysis and real-time polymerase chain reaction (PCR). ② Inhibition of cell prolif eration after different interventions: OSC cells were seeded in 96-well mi croplates (1.0×104) and cultured for 48 hours after the irradiation with 30 Gy γ-rays or in the presence of 100 μmol/L CDDP or 100 mmol/L 5-FU, and then the inhibition of cell proliferation was detected with 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ③ Apoptosis after different interventions: OSC cell lines were cultured for 24 hours after the irradiation with 30 Gy γ-rays or in the presence of 100 μmol/L CDDP or 100 μmol/L 5-FU. The cells were then stained with annexin V-FITC and propidium iodide, and the numbers of apoptotic cells were analyzed by a FACScan cytometer (only for the OSC cells treated with γ-rays and CDDP). ④ Plasmid construction and transfection: Human HIF 1α cDNA was cloned into the pcDNA3.1/V5-His TOPO expression vector. OSC-2 cells were temporarily transfected with HIF-1α cDNA. OSC-5 cells were temporarily transfected with HIF-1α siRNA (Sense sequence is 5' CUGAUGACCAGCAACUUGAtt 3'). The blank control group was also set, the inhibition of cell proliferation was observed, and the apoptosis was ana lyzed (only in the CDDP-treated group) by using the methods mentioned above. MAIN OUTCOME MEASURES: ① Expressions of HIF-1α protein and mRNA in different OSC cell lines; ② Inhibition of proliferation and the apoptosis of OSC cell lines after different interventions; ③ Overexpression and knockdown of HIF-1α by expression vector and siRNA; ④ Influence of overexpression and knockdown of HIF-1α on the susceptibility of OSC cell lines against γ-rays and chemotherapeutic drugs. RESULTS: ① Expressions of HIF-1α protein and mRNA in different OSC cell lines: The relative expression levels of HIF-1α mRNA were 1.0, 2.2, 4.3 and 4.0 in OSC-2, OSC-4, OSC-5 and OSC-6 cells, respectively. Simi larly, the expression of HIF-1α total cell lysates proteins and nuclear proteins were much lower in OSC-2 and OSC-4 cells than in OSC-5 and OSC6 cells. ② Inhibition of proliferation and the apoptosis in OSC cell lines after different interventions: After the treatment with g-rays, CDDP, and 5-FU, the cell numbers decreased obviously to (39.5±3.2)%, (39.2±1.2)%,and(47.9±3.6)% in OSC-2 cells, (53.9±6.6)%, (54.3±1.4)%, (54.8±3.8)%in OSC-4 cells. Those decreased to (74.1±3.8)%, (76.5±9.1)%, (69.6±7.7)%in OSC-5 cells and (71.4±7.4)%, (84.4±8.8)%, (82.0±4.5)% in OSC-6 cells.After the OSC cells were treated of with CDDP, the numbers of apoptotic cells were (50.9±1.3)%, (67.3±2.2)%, (12.2±0.8)% and (38.6±0.9)% in OSC-2, OSC-4, OSC-5 and OSC-6 cells. G-rays induced the apoptosis were (21.2±1.1)%, (14.6±0.9)%, (9.7±1.0)% and (10.4±0.8)% in OSC-2, OSC-4,OSC-5 and OSC-6 cells. ③ Inhibition of proliferation and the apoptosis in the transfected cell after different interventions: The cell numbers of HIF-1 α-transfected OSC-2 cells treated with γ-rays, CDDP and 5-FU were much more than those in the control group (t=-4.693 ,-8.617,-6.721, P ＜ 0.01),whereas the cell numbers of OSC-5 cells transfected with siRNA were obviously fewer than those in the control group (t=5.800, 5.595, 4.253, P ＜ 0.05-0.01). After the incubation of HIF-1α-transfected OSC-2 cells with CDDP for 24 hours, apoptosis was detected in (34.0±1.9)% of the cells, which was lower than that in the control group [(49.6±3.4)%, t=6.937, P ＜ 0.01]. After the incubation of HIF-1α siRNA transfected OSC-5 cells with CDDP for 24hours, apoptosis was detected in (27.7±2.3)% of the HIF-1α siRNA transfected OSC-5 cells, which was obviously higher than that in the blank control group [(11.4±2.1)%, t=-8.941, P ＜ 0.01].CONCLUSION: The expression level of HIF-1α correlates negatively with the susceptibility of OSC cells against chemotherapeutic drugs and radiation. The down-regulation of HIF-1α expression enhances the susceptibility of OSC cells against chemotherapeutic drugs and radiation. Therefore, the down-regulation of HIF-1α may be a potentially effective strategy for cancer treatment.

Objective To investigate the clinical effects of synthetic medical calcium phosphate and calcium sulfate as the bone replacements for bone defects.MethodsA total of 85 bone trauma patients combined with bone defects treated between February 2000 and October 2011 were randomized to calcium phosphate group ( n =43 ) and calcium sulfate group ( n =42 ) according to bone substitutes.The patients had orthopedic conventional treatment and then received external fixation ( external fixators,plasters) or internal fixation (plates,needles).Forty-three patients in the calcium phosphate group were all treated with medical calcium phosphate and 42 patients in the calcium sulfate group were all treated with medical calcium sulfate.Postoperative complications,fracture healing time and bone substitute absorption time of the two groups were observed and compared.ResultsAfter artificial bone filling for bone defects,all fractures were healed,with average healing time of 12.5 weeks ( range,9-17 weeks) in the calcium phosphate group and 11.3 weeks (range,7-15 weeks) in the calcium sulfate group.No complications or abnormal reactions occurred in the calcium sulfate group,whereas four patients with incision exudation was founded in the calcium phosphate group,and was cured after dressing change.The average follow-up period was 54.5 weeks (range,14-70 weeks).Average absorption time of bone substitute for the calcium phosphate group was 7.3 weeks,longer than that for the calcium sulfate group (P ＜ 0.05 ).Conclusions Medical calcium phosphate and calcium sulfate artificial bones can both be used as bone substitutes and are characterized by convenience,safety and good healing under the auxiliary of internal and external fixators.The calcium sulfate is sturdier and has relatively stronger supporting ability than the calcium phosphate.As the bone substitute,the calcium sulfate has more precise clinical effects,fewer complications and shorter healing time than the calcium phosphate.

Objective To explore the feasibility of Ilizarov technique in managing large tibia defects combined with soft tissue defects.Methods A total of 24 patients with large tibial defects combined with soft tissue defects caused by compound open tibial fractures were fixed with Ilizarov technique from September 2003 to September 2010.All patients belonged to open tibial fractures,including 20 patients with Gustilo type Ⅲ B and four with Gustilo type Ⅲ C.After debridement,the soft tissue defect areas was 10 cm ×6 cm and the bone defect was(8 ±4)cm.Fifteen patients with tibial defects ＜5 cm were treated with one stage debridement,fibula resection and tibial defect end compression.The other nine patients with tibial defect ＞ 5 cm were managed by one stage debridement,bone transport and bone lengthening.Then,15 patients were treated with one stage debridement,wound closure or wound reduction,bone grafting treatment and second stage cleansing of the incarcerated skin and fracture end.Results All patients were followed up for average 14 months(10-24 months),which showed reconstruction of the bone defects,restoration of the limb length,fracture healing and less than 2 cm difference between health limb and contralateral limb.One patient experienced common peroneal nerve palsy after operation,but recovered three months later.Of all,19 patients recovered without extra surgery,three restored with skin graft and two received skin flap.Conclusion Ilizarov technique is an effective option for treating the tibial defects combined with soft tissue defects at one stage.

BACKGROUND:Because human cells for culturing alveolar bone cell line are from alveolar bone, which is in oral cavity,and easily polluted, so laboratory study is often unsuccessful. Because the samples are from adults, so cell division index and the successful rate of culture are low.OBJECTIVE: To compare the biological characteristics of survived cell line established through passage,cryopreservation and revitalization following in vitro culturing the alveolar bone tissue obtained from normal persons and patients with chronic periodontitis accompanied with osteoporosis in aseptic operation; To compare the biological characteristics of two kinds of cells so as to provide theoretical and related experimental evidence for defect, repair and treatment of alveolar bone.DESIGN: Controlled observation.SETTING: Department of Stomatology, Beijing Chaoyang Hospital, Capital Medical University; Institute of Orthopaedics,General Hospital of Chinese PLA.MATERIALS: Alveolar bone tissue obtained from normal persons and patients with chronic periodontitis confirmed in clinic was used in aseptic operation.METHODS: Alveolar bone tissue from normal persons and chronic periodontitis accompanied with osteoporosis were cultured in vitro. In the four cell lines (H-171, H-258, 261, 262) cultured primarily, cell lines H-171 and H-258 were chosen from periodonitis patients group and normal group respectively, and stained with histochemical and immunohistochemical methods. Cell morphology was observed. Doubling time and division index of two kinds of cells were calculated with cytometry. After several circles of passage, cryopreservation and revitalization, growth and aging rule of cells were compared.MAIN OUTCOME MEASURES: Passage and biological characteristics of two groups of cell lines.RESULTS: ①In the abnormal alveolar bone group, there was one successful primary culture and cells presented short-spindle shape. There were 3 times of cryopreservation and 3 times of revitalization. Its doubling time was 53.4 hours. The average division index was about 4‰. Cells well grew after 20 times of passages. ②In the normal alveolar bone group, there were 26 cases of cell lines cultured primarily, but passage was found in only 3 cases of cell lines due to various causes. There were 10 passages and the cells presented long-spindle shape. After two circles of cryopreservation and revitalization, the survival and growth rate of cells were inferior as compared with cell line H-171.Doubling time was 65.9 hours and the average division index was 3.5‰. ③Both two kinds of cells adhered the wall, with the characteristics of osteoblasts: AKP, toluidine blue, PAS, tetracycline-labeled mineralized nodus, type Ⅰ collagen and BMP-2 immunohistochemical staining all presented positive.CONCLUSION: Both two kinds of cultured cells have the characteristics of osteoblasts. The growth speed of cell line H-171 is faster than that of cell line H-258. No obvious mutation is found in 20 passages. In the 8th generation of H-258,aging appears and growth speed becomes slow.

Objective To investigate feasibility and effect of external fixator plus steel plate in treatment of open tibiofibular fractures combined with tibial defect.Methods The study involved 21 patients with open fractures of tibia and fibula (15 patients with type Gastilo ⅢA,five with type Gastilo ⅢB and one with type Gastilo ⅢC) with concurrent tibial defect of 2-6 cm.External fixator plus fibular steel plate was performed at the first stage,followed by iliac bone grafting for bone defect at the second stage.Results All patients showed successful reconstruction of the tibial defects with length difference between affected and healthy extremities less than 2 cm in follow-up for (14.0 ± 10.5) months (range,8-24 months).Meanwhile,no talipes equinovarus existed.Conclusions External fixator plus steel plate is an effective method for treating open tibiofibular fractures combined with tibial defect.The length and function of the extremities of patients with open tibiofibular fractures combined with tibial defect of less than 6 cm can be successfully restored.

Objective To study the expression of PIWIL1, PIWIL3 and PIWIL4 in human colon cancer and its clinical significance. Methods We collected cancerous tissues and its adjacent tissues of 106 patients with colon cancer, two tissue microarrays were constructed, with 62 and 150 points respectively. We studied the expression of PIWIL1, PIWIL3 and PIWIL4 through immunohistochemistry. Results The expression of PIWIL1, PIWIL3 and PIWIL4 were significantly higher in cancerous tissues than those in adjacent tissues (P＜0. 01). In cancerous tissues and its adjacent tissues, postive correlation were seen among PIWIL1, PIWIL3 and PIWIL4 expression (P＜0. 01). PIWIL1 expression was significant higher in low differentiation group than that in high differentiation group (t =- 2. 840, P＜0.01 ). PIWIL3 expression was higher in high clinical stage than that in low clinical stage (F= 3. 112, P＜0.05). The expression of PIWIL3 and PIWIL4 were significantly higher in patients with colon cancer with distant metastasis than those without distant metastasis (t= -3. 349, P＜0.01 ; t = - 2. 168, P＜0. 05). PIWIL3 and PIWIL4 expression were correlated with occurring of colon cancer (P＜0. 01). Conclusions The expressions of PIWIL1,PIWIL3 and PIWIL4 in colon cancer were correlated with the differentiation, clinical stage and distant metastasis of colon cancer. PIWIL3 and PIWIL4 expression were two independent related factors of occurring of colon cancer, which would be furtherly investigated to be served as novel markers for early diagnosis and promising molecular targets for colon cancer therapy.

Objective To investigate the changes in Wnt/β-catenin,bone morphogenic protein (BMP),estrogen receptor (ER) and insulin-like growth factor (IGF) signaling pathways in bone marrow mesenchymal stem cells (BMSCs) differentiation to the osteoblasts after spinal cord injury (SCI) and understand the mechanism of osteoporosis after SCI.Methods Forty 6-week-old male rats were divided into SCI group (n =20) and control group (n =20) according to the random number table.Rats in SCI group were submitted to laminar osteotomy at T10-12 and given lower thoracic cord sharp transection.In control group,rat lower thoracic cord was only exposed without transaction.Femoral bone marrow density (BMD) of rat right side was determined at postoperative 3 months.Femoral bone marrow was harvested from rat left side.After BMSCs osteoblast differentiation,cells were harvested and used for examining expression of genes associated with the signaling pathways in the two groups using microarray technology and real-time PCR analysis.Results BMD in SCI group was significantly lower in the ephiphyses and metaphyses[(0.176 ± 0.017)g/cm2 and (0.170 ±0.016)g/cm2] compared to that in control group [(0.257 ± 0.023) g/cm2 and (0.196 ± 0.013) g/cm2,P ＜0.05].Microarray and PCR analysis revealed Wnt/β-catenin (eg.Wnt1,Wnt3a,Wnt5a,Lrp5,Ctnnb1,Lef1 and Axin),BMP (Tgfb1 and Bmpr1),IGF -1 (eg.IGF1 R,c-fos and c-Jun),and ER (eg.Esr1) signaling pathways in osteoblasts were significantly down-regulated in SCI group compared to these in control group (P ＜ 0.05).Conclusions The Wnt/β-catenin,BMP,ER,and IGF-1 signaling pathways in osteoblasts are significantly down-regulated after SCI,resulting in profound BMD loss.This indicates that these signaling pathways are implicated in the osteoporosis after SCI.

OBJECTIVETo investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli.

METHODSPrimary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining.

RESULTSPrimary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4 per thousand. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3 per thousand. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast.

CONCLUSIONSPrimary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

OBJECTIVETo evaluate the method of adenovirus vector-mediated herpes simplex virus-thymidine kinase gene (ADV-TK)/ganciclovir(GCV) system and photodynamic therapy(PDT) for treating the oral malignant tumor.

METHODSTen patients who were suffering from oral malignant tumor of the different positions were selected, and injected with the hematoporphyrin derivative (HPD), irradiated by picking the corresponding laser frequency, and injected the ADV-TK gene into the tumor body and periphery. By the imaging and the hemodynamics analysis, the clinical efficacy after infusing vein with GCV was assessed.

RESULTSAfter the combination method treatments, patients' tumors appeared clear shrinkage or complete extinction. There was obvious fall of the blood flow volume in the tumor body. Imaging results showed significantly differences. So it was a perfect and effective treatment.

CONCLUSIONThere are many advantages to apply the ADV-TK/GCV system and PDT treatment on the oral malignant tumor, minimal side effects and greater clinical security. It is a safe and credible therapy which can be offered for curing the oral malignant tumor systematically.

To explore the mechanism for the role of autophagy in endometriosis, and to provide a theoretical basis for prevention and treatment of endometriosis.
 Methods: The endometrial CRL-7566 cells were treated with ATG5 siRNA, autophagic activator rapamycin and autophagic inhibitor 3-MA, respectively. The cell proliferation and invasion were detected by clonal formation, cell growth curve and MTT assay. The clinical specimens of endometriosis were collected from 20 cases. The expression of autophagy marker LC3II and autophagy substrate protein P62 were detected.
 Results: Rapamycin inhibited the proliferation and clonal formation of CRL-7566 cells, while autophagy inhibitor 3-MA and ATG5 siRNA showed opposite effect. Moreover, rapamycin inhibited filopodia growth in endometriosis, whereas overexpression of filopodia-relevant protein fascin-1 inhibited the decrease in invasiveness caused by rapamycin. In clinical samples, we also found a significant decrease of LC3II while an increase in P62 compared with the control group.
 Conclusion: Autophagy inhibition may contribute to an increase in endometrial cell proliferation and invasiveness. Autophagy activation could be a potential strategy for endometriosis therapy.
Autophagy ; drug effects ; genetics ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; drug effects ; Endometriosis ; physiopathology ; Endometrium ; cytology ; Female ; Gene Expression Regulation ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Microtubule-Associated Proteins ; genetics ; RNA-Binding Proteins ; genetics ; Sirolimus ; pharmacology