[Objective]To study the difference in expression of RFC, GST-?, DHFRmRNA between human osteosarcoma U2-OS cell line and the MTX-resisitant variants U2-OS/R1-R3, and to investigate the significance in MTX resistance for human osteosarcoma. [Methods]Three resistant MTX human osteosarcoma cell lines were established by pulse exposure parental cell line(U2-OS) in gradually increased dose of MTX . The expression of RFC,GST-?,DHFRmRNA were assayed by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).[Results]Three MTX-resistant variants(U2-OS/R1-R3) were successfully established , the results of the FQ-PCR revealed that the MTX resistance was associated with the decreased expression of the RFC mRNA and increased expression of DHFR mRNA and GST-? mRNA.[Conclusion]The author investigated the MTX resistant mechanism of human osteosarcoma cell line at a gene level. The decreased expression of RFC mRNA and the increased expression of DHFR mRNA and GST-? mRNA participate in the MTX resistance in human osteosarcoma cell lines U-2 OS. This provides the evidence for exploring the MTX resistance mechanism in clinical osteosarcoma patients ,and helps to screen the patients who are insensitive to MTX chemotherapy.

[Objective]To explore the application value of vascularized fibular graft in the repairment of maxillofacial defects.[Methods] Forty-five patients with maxillofacial defects caused by various reasons were enrolled and treated by vascularized fibular graft.The related disease history,survival situation of blood vessels and tissue flaps of all the patients were recorded.[Results]All the patients were followed up for 3-6 months after surgery.All the patients could eat by mouth and without no obvious obstacle.There was no facial asymmetry,healthy side bite jaws deflection and so on.The maxillofacial appearance and functions almost returned back to normal.Oral panoramic radiography showed that the osseous healing of transplanted fibular and mandibular was good,and there was no bone nonunion,osteonecrosis,titanium plate rejection or fracture happened.Three-dimensional CT scàn revealed that the recovery of mandibular appearance was good.There was 1 case with leg wound drainage and 1 case with cervical wound effusion,which were all healed after the second phase by dressing change and necessary treatment.The rest of the patients were all healed at the first phase.There was no free tissue flap necrosis and calf obvious dysfunction.[Conclusions] Using vascularized fibular graft for the repairment of maxillofacial defects,the maxillofacial appearance and functions of patients almost return to normal after surgery.The surgery can satisfy the requirements of patients on postoperative oral and maxillofacial appearance and function with lower incidence of complications and hig her survival rate of free tissue flap.Therefore,it is worthy of clinical application.

Objective To investigate the relationship between serum soluble B7-H4(sB7-H4) expression and clinical pathological characteristics in patients with non small cell lung cancer(NSCLC),and its clinical significance.Methods 65 patients with NSCLC were selected as NSCLC group,58 patients with benign lung disease were selected as the benign lung disease group,another 45 healthy people of the same period in our hospital outpatient physical examination were selected as healthy control group.By enzyme-linked immunosorbent assay(ELISA) method,serum sB7-H4 and vascular endothelial growth factor(VEGF) were detected.The serum embryonic antigen(CEA) and cancer antigen-125(CA125) were detected by chemiluminescence method.The relationship between sB7-H4 and NSCLC and other pathological features,and the relationship of the tumor markers CEA,CA125,VEGF were analyzed.Results The level of serum sB7-H4 of the NSCLC group was (59.47±8.76)μg/L,which was significantly higher than (17.38±3.72)μg/L of the lung benign lesion group and (15.26±3.17)μg/L of the health control group,the differences were statistically significant(t=18.276,19.872,all P<0.01).The serum CEA,CA125,VEGF levels of the NSCLC group were higher than those of the lung benign lesion group and healthy control group,the differences were statistically significant(t=9.447,14.883,33.467;t=11.374,15.137,37.759,all P<0.01).The level of serum sB7-H4 of the NSCLC group after treatment was (35.47±7.46)μg/L,the difference was statistically significant compared with before treatment[(59.47±8.76)μg/L](t=4.372,P<0.01).The serum sB7-H4 level was correlated with NSCLC tissue differentiation degree,clinical stage,whether with lymph node metastasis (F=3.786,t=4.821,4.172,all P<0.01).In NSCLC patients,sB7-H4 level was positively correlated with serum CEA,CA125 and VEGF levels (r=0.643,0.727,0.586,all P<0.05).Conclusion The sB7-H4 may be involved in the occurrence and development of NSCLC,and it is expected to become a new target for NSCLC diagnosis and treatment.

Objective To compare the histological fates and the effects of autologous fat and fascia used as augmentative materials to treat unilateral vocal fold paralysis (UVFP). Methods Vocal fold augmentation with autologous fat and fascia was performed on 2 dogs (group A) to cure glottic insufficiency caused by left vocal fold paralysis. Autologous fat and fascia 0.4 ml were injected respectively into left and right vocal folds of 9 dogs (group B). The dogs of group B were killed after 1,3 and 5 months and the dogs of group A were killed after 8 months. The histology of both substances and the effect of treatment were evaluated. Results Surviving autologous fat grafts was found in the canine vocal fold after 8 months, but the absorption was too rapid to maintain the bulk of grafts, the effect could last no more than 5 months. Autologous fascia also survived after 8 months, Conclusion The results showed that these two augmentative materials were both effective in the treatment of IJVFP. Autologous fat can only be used as short - term augmentative material. However, autologous fascia has a potential use for long - term application to correct glottic insufficiency caused by UVFP and other diseases.

Objective To explore the effect of immunogenicity of freeze-dried bone allograft on different in vitro experimental models. Methods The lymphocytes were obtained respectively from 10 healthy young human volunteers, 10 Balb/c and 10 C57 mice and 10 New Zealand rabbits. The experiment was carried out in 6 groups: positive control group (PHA/ConA+lymphocyte), negative control group (Hydroxyapatite powder + lymphocyte), allogeneic bone group A (Freeze-dried bone powder 2. 0 g/L + lym-phocyte), allogeneic bone group B (Freeze-dried bone powder 1.0 g/L + lymphocyte), allogeneic bone group C (Freeze-dried bone powder 0.5 g/L + lymphocyte), and negative control group (culture solution + lym-phocyte). Lymphocyte transformation test (Alamarblue) was conducted to culture the 6 kinds of experimental materials in vitro. After 72 hours, samples were scanned with ELISA muhiscan at wave lengths 570 nm and 600 nm to fetal the light absorption value. Pearson analyses were performed 10 determine the relationships a-mong the 3 animals and 1 human groups and find out which animal would be highly correlated to human. Results In the human and Balb/c mice lymphocyte transformation tests, there was no significant difference (P > 0.05) between allogeneic bone groups A, B, C and negative control group (HA) ; but there was sig-nificant difference (P < 0.001) between allogeneic bone groups A, B, C and positive control group (PHA/ConA); there was no significant difference between the 3 allogeneic bone groups (P > 0.05). There was no significant difference among the 6 groups of C57 mice and New Zealand rabbits (P > 0.05). The coefficient r between Balb/c mice and human groups was 0.959, P = 0.003, showing a highly positive correlation. The coefficient r between C57 mice and human groups was 0.527, P = 0.283, while the coefficient r between New Zealand rabbits and human groups was 0.866, P =0.026. Conclusions The immunogenicity of freeze-dried bone powder in this experiment may not be sufficient enough to induce significanrt immunologic response. Balb/c mice may be preferable for immunogenicity related experiments.

Objective To explore the effect and adverse reaction of priming regimen CAG in treatment of acute myeloid leukemia in elderly.Methods 32 elderly patients with acute myelogenous leukemia were treated with CAG regimen and 25 cases with standard chemotherapy regimen( DA regimen). The effect and adverse reaction were compared between the two groups. Results There was no significant difference of the complete remission rate and effective rate between the two groups. The recovery time of bone marrow and death rate of CAG the regimen group were superior to those of the DA group. Conclusion CAG regimen was effctive and safe for elderly patients with acute myeloid leukemia.

Objective:To evaluate the role of nuclear factor erythroid 2-related factor/ heme oxygenase-1 (Nrf2/HO-1) signaling pathway in dexmedetomidine-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury to microglia.Methods:BV-2 microglia were cultured in high-glucose DMEM culture medium supplemented with 10% fetal bovine serum in an normal culture incubator at 37 ℃ (5%CO 2-21%O 2-74 %N 2). The cells were seeded in 96-well plates at a density of 1.5×10 4 cells/ml (200 μl/well) or 6-well plates at a density of 2×10 5 cells/ml (2 ml/well) and divided into 5 groups ( n=30 each) using a random number table method: control group (group C), dexmedetomidine group (group D), group OGD/R, OGD/R+ dexmedetomidine group (group OGD/R+ D) and OGD/R+ dexmedetomidine+ ML385 group (group OGD/R+ D+ ML). The cells in group C were continuously cultured in a normal culture incubator for 26 h. In group D, dexmedetomidine at the final concentration of 10 μmol/L was added, cells were incubated for 2 h, and then were continuously incubated in a normal culture incubator for 26 h. In OGD/R, OGD/R+ D and OGD/R+ D+ ML groups, the culture medium was replaced with glucose-free DMEM culture medium, cells were cultured for 2 h in an incubator at 37 ℃ (5%CO 2-1%O 2-94 %N 2), the culture medium was replaced with high-glucose DMEM culture medium containing 10% fetal bovine serum and then the cells were cultured for 24 h in a normal incubator.Dexmedetomidine at the final concentration of 10 μmol/L was added at 2 h before OGD in OGD/R+ D and OGD/R+ D+ ML groups.Nrf-2 inhibitor ML385 at the final concentration of 4 μmol/L was added at 30 min before dexmedetomidine was added in group OGD/R+ D+ ML.Cells in 6 wells in each group were selected randomly for assessment of cell viability (by methyl thiazolyl tetrazolium assay) and apoptosis (using flow cytometry), and for determination of the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 in the supernatant (using enzyme-linked immunosorbent assay), the expression of Nrf2 in nucleus, Nrf2 and HO-1(by Western blot ) and the expression of HO-1 mRNA (by real-time polymerase chain reaction). Results:Compared with group C, the cell viability was significantly decreased, cell apoptosis rate and concentrations of TNF-α, IL-6 and IL-10 in the supernatant were increased, and the expression of Nrf2 in nucleus, Nrf2, HO-1 and its mRNA was up-regulated in OGD/R and OGD/R+ D groups ( P<0.05), and no significant change was found in each parameter mentioned above in group D ( P>0.05). Compared with group OGD/R, the cell viability and IL-10 in the supernatant concentration were significantly increased, cell apoptosis rate and concentrations of TNF-α and IL-6 in the supernatant were decreased and the expression of Nrf2 in nucleus, Nrf2, HO-1 and its mRNA was up-regulated in group OGD/R+ D ( P<0.05), and no significant changes were found in the parameters mentioned above in group OGD/R+ D+ ML ( P>0.05). Compared with group OGD/R+ D, the cell viability and concentration of IL-10 in the supernatant were significantly decreased, cell apoptosis rate and concentrations of TNF-α and IL-6 in the supernatant were increased and the expression of Nrf2 in nucleus, Nrf2, HO-1 and its mRNA was down-regulated in group OGD/R+ D+ ML ( P<0.05). Conclusion:The mechanism by which dexmedetomidine alleviates OGD/R injury to microglia may be related to promoting the activation of Nrf2/HO-1 signaling pathway and inhibition of inflammatory responses.

AIM:To explore the effects of Gax gene transfection on expressions of c-fos and c-jun mRNA and proliferation of pulmonary arterial smooth muscle cells (PASMCs) in rat under hypoxia. METHODS: PASMCs were transfected with Gax gene by Ad-Gax. Under normal oxygentention (21% O2) or hypoxia (2.5% O2) for 12 h condition, expressions of Gax mRNA and protein in PASMCs were detected by RT-PCR and immunocytochemistry. The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR. [3H]-TdR incorporation was used to measure the PASMCs proliferation. RESULTS: The Gax overexpression in transfection group was confirmed by RT-PCR and immunocytochemistry. Under normal oxygentention or hypoxia, the c-fos and c-jun mRNA levels in transfection group were lower than those in the non-transfection group, respectively (P

Objective: To compare the effectiveness of proximal femoral nail anti-rotation (PFNA) in the treatment of intertrochanteric fracture of femur in traction bed supine position and non-traction bed lateral position. Methods: A retrospective analysis of 102 elderly patients with intertrochanteric fracture of femur who met the selection criteria between January 2013 and April 2018 was made. According to the different operative positions, the patients were divided into two groups: group A (50 cases, PFNA internal fixation in traction bed supine position) and group B (52 cases, PFNA internal fixation in non-traction bed lateral position). There was no significant difference in age, gender, fracture side, cause of injury, AO classification, complications, and time from injury to operation between the two groups ( P>0.05). The preoperative preparation time, incision length, operation time, intraoperative blood loss, intraoperative X-ray fluoroscopy times, fracture healing time, and complications were recorded and compared between the two groups, and the effectiveness was evaluated by Harris hip score at 1 year after operation. Results: There was no significant difference in incision length between groups A and B ( t=1.116, P=0.268). In addition, the preoperative preparation time, operation time, intraoperative blood loss, and intraoperative X-ray fluoroscopy times in group A were significantly greater than those in group B ( P<0.05). Both groups were followed up 12-14 months, with an average of 13 months. There were 3 postoperative complications in group A and group B respectively. In group A, there were 2 cases of hip joint pain and 1 case of local fat liquefaction (healed after dressing change); in group B, there were 2 cases of hip joint pain and 1 case of deep vein thrombosis in lower extremity; there was no significant difference in the incidence of postoperative complications between the two groups ( P=0.642). The patients of the two groups had a good result of fracture reduction and the internal fixation quality, and there was no main nail loosening, screw fracture, spiral blade cutting, withdrawal, and the nail breakage occurred, and no nonunion of bone, coxa vara, and other complications occurred. X-ray showed that the fracture healed in both groups, and there was no significant difference in fracture healing time between the two groups ( t=1.515, P=0.133). There was no significant difference in Harris hip score between the two groups at 1 year after operation ( t=0.778, P=0.438). Conclusion: Compared with the traction bed supine position, PFNA internal fixation for intertrochanteric fracture of femur in the non-traction bed lateral position has the advantages of short preparation time, short operation time, less intraoperative blood loss, less X-ray fluoroscopy times, and satisfactory postoperative recovery effect.

Objective To investigate the therapeutic effect of the sandwich method (medical glue +gelatin sponge+medical glue) in the repair of spinal dura mater to prevent the cerebrospinal fluid leakage.Methods From February 2007 to June 2011,54 patients with spinal subdural tumors underwent excision of tumor in our hospital.According to manner of repairing spinal dura mater,all patients were classified into two groups:routine group and sandwich group.There were 16 males and 7 females with an average age of 45.2±7.2 years in the routine group,while 19 males and 12 females with an average age of 44.2±6.4 years in sandwich group.In routine group,the spinal dura mater was repaired through running locked suture.In sandwich group,the spinal dura mater was repaired through running locked suture,painting medical glue around the dural incision,covering with gelatin sponge,and painting medical glue on the surface and margin of gelatin sponge successively.Results Compared with the routine group,the total volume of postoperative drainage in sandwich group decreased significantly on the very day,the first day,the second day,and the third day,and the incidence of cerebrospinal fluid leakage decreased significantly.Before discharge,hydrops happened in 3 cases in the routine group,and got well through aspiration,continuous pressure by sandbag,and prone position.Three months after operation,5 cases from the routine group got deep hydrops under the incision and no treatment was applied to them.There was no obvious abnormality in the sandwich group.Conclusion The sandwich method can improve the repair effect of spinal dura mater injury,reduce the volume of postoperative drainage,and decrease the incidence of cerebrospinal fluid leakage