1.Deubiquitinating enzyme OTUB2 increases the formation of neutrophil extracellular trapping nets and promotes the viability and invasion of colorectal cancer cells by inducing DDX54 activity
Acta Universitatis Medicinalis Anhui 2024;59(3):463-472
		                        		
		                        			
		                        			Objective The purpose of this study was to investigate the effects of OTU deubiquitinase,ubiquitin al-dehyde binding 2(OTUB2)on the activity of DEAD-box helicase 54(DDX54)and its influence on the formation of neutrophil extracellular traps(NETs)and the vitality and invasion of colorectal cancer(CRC)cells.Methods Peripheral blood neutrophils were isolated from CRC patients and healthy controls,then stimulated with phorbol-12-myristate-13-acetate(PMA)or DNaseⅠfor 4 hours.Western blot analysis was performed to detect the expression of NETs markers,myeloperoxidase(MPO),and citrullinated histone H3(Cit-H3).The expression of OTUB2 or DDX54 was manipulated in SW480 cells,and they were co-cultured with neutrophils.The cells were divided into the following groups:Control group(without any treatment),NETs group,vector group,OTUB2 group,NETs+si-OTUB2 group(SW480 cells,SW480 cells transfected with vector,OTUB2 overexpression plasmid and si-OTUB2 were co-cultured with PMA-treated neutrophils,respectively),OTUB2+DNase Ⅰ group(SW480 cells transfected with OTUB2 overexpression plasmid co-cultured with neutrophils treated with DNase Ⅰ),si-OTUB2group,OTUB2+si-NCgroup,OTUB2+si-DDX54group(SW480 cells transfected with si-OTUB2,OTUB2 overexpressing plasmid and si-NC,OTUB2 overexpressing plasmid and si-DDX54 were co-cultured with neutro-phils,respectively).ELISA was used to measure the relative expression of MPO-DNA complexes and the concen-tration of Cit-H3 in the supernatant of SW480 cells.MTT and Transwell assays were performed to evaluate cell via-bility and invasive ability.RNA sequencing was conducted to screen downstream genes regulated by OTUB2.The interaction between DDX54 and OTUB2 was validated using quantitative real-time polymerase chain reaction(qRT-PCR),co-immunoprecipitation(Co-IP),Glutathione S-Transferase(GST)pull-down,and His-tag pull-down ex-periments.Results Compared to healthy individuals,there was an increased formation of NETs in the peripheral blood of CRC patients.The CRC cells in the NETs group exhibited increased cell viability and invasion compared to the control group(P<0.05).In comparison to the vector group,the OTUB2 group showed increased relative ex-pression of MPO-DNA complexes and Cit-H3,as well as increased cell viability and invasion(P<0.05).Howev-er,when compared to the OTUB2 group,the OTUB2+DNase Ⅰ group exhibited decreased relative expression of MPO-DNA complexes and Cit-H3,as well as decreased cell viability and invasion(P<0.05).Co-IP,GST pull-down,and His-tag pull-down experiments demonstrated the interaction between OTUB2 and DDX54.In comparison to the OTUB2+si-NC group,the OTUB2+si-DDX54 group showed decreased relative expression of MPO-DNA complexes and Cit-H3,as well as decreased cell viability and invasion(P<0.05).Conclusion deubiquitinating enzyme OTUB2 can increase the formation of NETs and promote CRC cell viability and invasion by up-regulating DDX54.
		                        		
		                        		
		                        		
		                        	
2.Reform and practice of Biopharmaceutical Technology Comprehensive Experiments in higher vocational colleges in the context of skills competition.
Yanbin SUN ; Junling LIANG ; Yang JIANG ; Liangjun DONG ; Chuan ZHANG
Chinese Journal of Biotechnology 2023;39(4):1825-1837
		                        		
		                        			
		                        			Comprehensive experiments course is a bridge for higher vocational students to integrate theoretical knowledge with production practice. The article introduces that our biological pharmacy department is committed to the principles of "promotion of teaching, learning and construction through skills competition so as to integrate education and training". By taking penicillin fermentation process as an example, reform has been made in several aspects including teaching objectives, teaching content and teaching methods. We integrate the practical operation of fermentation equipment with virtual simulation software to develop a two-way interactive course. By reducing the subjective dependence, the quantitative management and evaluation of fermentation process parameter control were put into place, which efficiently integrated the skills competition with practical teaching. Improved teaching performance has been achieved over recent years, which may facilitate the reform and practice of similar courses based on skills competition.
		                        		
		                        		
		                        		
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		                        			Learning
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		                        			Biological Products
		                        			
		                        		
		                        	
3.The iodine nutritional status of pregnant women in the third trimester in the Southwest of Shandong Province and its impact on their cardiac electrical activity
Xin LYU ; Xiaoyun HAN ; Sucheng LIU ; Liangjun LI ; Weiwei JIANG ; Quan LI ; Haitao GONG ; Guangxing WANG ; Rutao LIU
Chinese Journal of Endemiology 2023;42(9):742-747
		                        		
		                        			
		                        			Objective:To study the iodine nutritional status of pregnant women in the third trimester in the Southwest of Shandong Province, analyze its impact on their cardiac electrical activity, and provide a basis for scientific supplementation of iodine during pregnancy.Methods:From January 2021 to June 2022, a cross-sectional survey was conducted using cluster random sampling method. According to the inclusion and exclusion criteria, 200 pregnant women in the third trimester were selected from 3 tertiary hospitals in three cities in the Southwest of Shandong Province, and were divided into the third trimester group ( n = 600), and 100 non-pregnant women were selected as the control group ( n = 300). The urinary iodine content was detected by arsenic-cerium catalytic spectrophotometry, and the pregnant women in the third trimester group were subdivided into iodine deficiency subgroup [G1 subgroup, median urinary iodine (MUIC) < 150 μg/L] based on the MUIC, iodine excess subgroup (G2 subgroup, MUIC≥500 μg/L) and moderate iodine subgroup (G3 subgroup, 150 μg/L≤MUIC < 500 μg/L). Chemiluminescence immunoassay was used to measure the serum levels of thyroid-stimulating hormone (TSH), free thyroxine (FT 4) and free triiodothyronine (FT 3). The cardiac electrical indexes were detected by a 12-lead surface electrocardiogram (ECG) machine. Results:There was no statistically significant difference in urinary iodine levels between pregnant women in the third trimester group and non-pregnant women in the control group among the 3 tertiary hospitals in the Southwest of Shandong Province ( H = 3.63, 3.27, P > 0.05). In the third trimester group, the proportion of pregnant women in the G1, G2 and G3 subgroups was 27.67% (166/600), 6.83% (41/600) and 65.50% (393/600), respectively. There was a statistically significant difference in urinary iodine levels between the subgroups and the control group ( H = 11.56, P < 0.001). The serum FT 3 and FT 4 levels in the G2 subgroup were lower than those in the G1 and G3 subgroups ( P < 0.001), but there was no statistically significant difference in serum TSH levels among the three subgroups ( P > 0.05). The normal rates of ECG in the G1, G2, G3 subgroups, and the control group were 38.55% (64/166), 41.46% (17/41), 92.37% (363/393), and 95.33% (286/300), respectively. The difference between the groups were statistically significant (χ 2 = 461.25, P < 0.001), the normal rate of ECG in the G1 and G2 subgroups was lower than that in the control group ( P < 0.001). Short P-R intervals and ST-T changes were the most common abnormal ECG in the third trimester group. Conclusions:The incidence of iodine deficiency, iodine excess, and other abnormal iodine nutritional status in pregnant women in the third trimester of the Southwest of Shandong Province is relatively high. Short P-R intervals, ST-T changes, and other arrhythmia caused by this are more common. It is necessary to strengthen monitoring of iodine nutritional status and ECG during pregnancy, and adjust intervention strategies such as iodine supplementation in a timely manner.
		                        		
		                        		
		                        		
		                        	
4.The clinical characteristics and management strategy of non-dislocated hyperextension tibial plateau fractures
Liangjun JIANG ; Hanxiao ZHU ; Erman CHEN ; Qiang ZHENG
Chinese Journal of Orthopaedics 2020;40(18):1266-1274
		                        		
		                        			
		                        			Objective:To explore the epidemiological characteristics, clinical characteristics, treatment strategies and clinical results of non-dislocated hyperextension tibial plateau fracture.Methods:A total of 25 cases of non-dislocated hyperextension tibial plateau fracture patients were collected, including 12 males and 13 females, aged 27-79 years with an average age of 51±15 years. Causes of injury: 18 cases of traffic accident, 2 cases of heavy injury, 5 cases of falling injury. Schatzker classification of tibial plateau fracture: 4 cases of type II, 5 cases of type IV, 13 cases of type V, 3 cases of type VI. Three columns theoretical classification: 4 cases of simple lateral column, 5 cases of simple medial column, 7 cases of medial column+lateral columns, 9 cases of three columns. Preoperative tibial plateau posterior slope angle was -10-0 degrees, average-5.2 degrees. Preoperative MRI showed 5 cases of medial collateral ligament (MCL) injury, 3 cases of posterolateral complex (PLC) complex injury, 2 cases of PLC+ posterior cruciate ligament (PCL) injury and 10 cases of menisci injury. The change of tibial plateau posterior slope angle was more than 10 degrees in patients with ligament injury, 5 patients had a tibial plateau posterior slope angle change more than 15 degrees, 5 patients had a tibial plateau posterior slope angle change more than 10 degrees, and the patients with a tibial plateau posterior slope angle change less than 10 degrees had no ligament injury; 6 patients with simple lateral column or medial column fracture had a ligament injury, 2 patients with medial column+lateral column fracture had a ligament injury, and 2 patients with three column fracture had a ligament injury.Results:All patients were followed up for 12-24 months, with an average of 16.4 months. The operation time was 124±33 min (65-180 min), and the bleeding volume was 106±48 ml (20-200 ml). All patients were treated by open reduction and internal fixation. According to the characteristics of fracture, targeted reduction of articular surface and restoration of lower limb force line were carried out. The plate was placed on the anterior part of tibial plateau to fix the fracture fragment. Evaluation of postoperative fracture reduction: 20 cases were anatomic reduction, 5 cases were good reduction (between 2-5 mm articular surface collapse), and the excellent rate of fracture reduction was 100%. Internal fixation: 4 cases were treated with simple lateral plate, 2 cases with simple medial plate, 15 cases with medial+ lateral plate, 1 case with medial plate+ lateral anchor suture, 1 case with medial plate+ posterior screw, 1 case with lateral plate+ fibular screw, 1 case with medial plate + lateral plate+ lateral anchor suture. The meniscus was repaired in 8 patients, including 5 medial and 3 laterals. The fracture healing time was 3-6 months (mean 3.3 months). The postoperative knee Rasmussen score was 24.9±3.5 (18-29), and the postoperative knee joint mobility was 118°±9° (90°-130°). Superficial infection occurred in 2 patients.Conclusion:The main imaging characteristic of "non-dislocated hyperextension tibial plateau fracture" is the change of tibial plateau posterior slope angle. The injury of single anteromedial column/anterolateral column fracture is easy to combine with the "diagonal" injury, and when the tibial plateau posterior slope angle changes more than 10 degrees, it is easy to be associated with peripheral ligament injury. By using open reduction and internal fixation, restoring the joint articular surface and lower limb force line, repairing the soft tissue structure and ligament, and reconstructing the stability of knee joint, we can achieve satisfactory results.
		                        		
		                        		
		                        		
		                        	
5. Reoperation strategy of malunited tibial plateau fracture
Liangjun JIANG ; Hang LI ; Weixu LI ; Gang FENG ; Jianwei WANG ; Qiang ZHENG
Chinese Journal of Orthopaedics 2019;39(14):845-854
		                        		
		                        			 Objective:
		                        			To explore the revision strategy of the malunited tibial plateau fracture and to analysis the main points of four common revision operations and the clinical effect.
		                        		
		                        			Methods:
		                        			From January 2012 to December 2016, 18 patients (5 males and 13 females) aged 35-60 years (average 49.7 years) underwent tibial plateau revision surgery in our hospital were collected. The time from the second revision operation to the first operation was 2-24 months (average 10.4 months). Our revision strategies were as follows: Firstly, to determine whether there is an infection or not. If there was infection, we changed the original internal fixation to external fixator to control infection. Secondly, todetermine whether the patient could suffer re-reduction and internal fixation. If the patient was older (>65 years old) or with severe local bone defect, total knee arthroplasty should be performed. Thirdly, patients were divided into four operation modes according to the tibial plateau fracture malunion type: 1. the original fracture line osteotomy; 2. the tibial tubercle + original fracture line osteotomy; 3. tibial metaphyseal window-rod reduction; 4. the osteotomy of fibula head and original fracture line osteotomy.
		                        		
		                        			Results:
		                        			All patients were followed up for 12-30 months (average 16.8 months), and the operation time was 120-300 min (average 185 min). 2 cases were infected before operation and the original internal fixation were removed to instead of external fixator;1 patient underwent total knee arthroplasty; 3 cases were treated with metaphyseal open window-rod reduction and internal fixation; 6 cases were operated with the original fracture line osteotomy and internal fixation; 4 cases were treated with tibial tubercle osteotomy+original fracture line osteotomy and internal fixation; 2 patients underwent fibular head osteotomy+ original fracture line osteotomy and internal fixation. All patients achieved bony union at the last follow-up. The healing time was 3-6 months (mean 3.6 months). The postoperative knee Rasmussen score was 19-29 (average 22.9), compared with average 14.4 points before operation (
		                        		
		                        	
6.Value of semi-quantitative indices of 68Ga-PSMA-11 PET/CT in differential diagnosis of malignant and benign prostate lesions
Luping QIN ; Jie LYU ; Mingzhao LI ; Jianfang LI ; Liangjun XIE ; Yueming ZHA ; Yongluo JIANG ; Muhua CHENG
Chinese Journal of Nuclear Medicine and Molecular Imaging 2019;39(2):67-71
		                        		
		                        			
		                        			Objective To evaluate the value of semi-quantitative indices of 68Ga-prostate specific membrane antigen (PSMA)-11 PET/CT in differentiating malignant and benign prostate lesions.Methods From November 2017 to June 2018,30 patients (age:52-86 years) who underwent 68Ga-PSMA-11 PET/CT imaging in the Third Affiliated Hospital of Sun Yat-sen University were retrospectively analyzed,and the serum total prostate specific antigen (tPSA) and free prostate specific antigen (fPSA) were examined within 1 week before PET/CT imaging.Semi-quantitative indices of 68Ga-PSMA-11 PET/CT on prostate lesions were measured by automatic segmentation algorithm method,including PSMA-related lesion volume (VPSMA),maximum standardized uptake value (SUVmax),mean standardized uptake value (SUVmean),peak standardized uptake value (SUVpeak) and total lesion uptake value of PSMA (TLUPSMA).The indices were compared between malignant and benign prostate lesions,and the optimal cut-off values for differentiating malignant and benign prostate lesions were obtained by receiver operating characteristic (ROC) curve analysis.Results According to the pathological results,19 patients had malignant lesions and 11 were with benign diseases.The differences of tPSA,SUVmax,SUVmean SUVpeak and TLUPSMA between malignant and benign prostate lesions were statistically significant (u values:17.00-48.00,all P<0.05),but there were no significant differences of fPSA,fPSA/tPSA and VPSMA between 2 groups (u values:64.00-99.00,all P>0.05).The optimal cut-off value of tPSA was 18.30 μg/L for differentiating malignant and benign prostate lesions,with sensitivity of 13/17 (PSA of 2 patients were missing),specificity of 9/11 and area under curve (AUC) of 0.743.The optimal cut-off values of SUVmax,SUVmean and SUVpeak were 5.50,3.09 and 3.56,respectively,with all corresponding sensitivity of 18/19,all specificity of 9/11,and AUC of 0.902,0.907 and 0.919,respectively.The optimal cut-off value of TLUPSMA was 54.81 cm3,with sensitivity of 14/19,specificity of 9/11 and AUC of 0.804.Conclusion The semi-quantitative indices of 68Ga-PSMA-11 PET/CT are valuable for differentiating malignant and benign prostate lesions,in which SUVpeak is superior to other indices.
		                        		
		                        		
		                        		
		                        	
7.The treatment of early and delayed infection of intramedullary nails of lower limb
Deting XUE ; Hang LI ; Zhijun PAN ; Wei ZHANG ; Weixu LI ; Qiang ZHENG ; Gang FENG ; Yanbin TAN ; Xiang GAO ; Liangjun JIANG ; Zhanfeng ZHANG
Chinese Journal of Orthopaedics 2018;38(9):556-562
		                        		
		                        			
		                        			Objective To explore the treatment methods and prognosis of early infection and delayed infection after intramedullary nail fixation.Methods Data of 22 cases of postoperative infections after intramedullary nail from January 2013 to August 2017 were retrospectively analyzed.There were 18 males and 4 females aged from 20 to 72 years old,with an average age of 46.8 years.14 cases were tibias and 8 cases were femurs.In the early infection group,6 cases showed swelling,heat and pain in the affected area with drainage and pus.In the late infection group,12 cases showed sinus formation and 4 cases showed no sinus tract.According to whether the infection occurred within six weeks,it was divided into early infection and delayed infection groups.Of 6 patients in early infection group,there was 1 case of septic shock which underwent removal of intramedullary nails,debridement and antibiotic bone cement stick implantation.5 cases were retained intramedullary nail and underwent local debridement treatment.Late infection occurred in 16 patients.One patient with tibia infection was given partial dressing to heal the fracture.Then the intramedullary nail was removed and intramedullary debridement was performed.Two patients with poor general condition,the intramedullary nails were removed and debridement was performed.Calcium sulphate cement was implanted and fixed with external fixation.The remaining 13 cases were treated with debridement and antibiotic cement stick implantation.We compared the differences between early and late infections of internal fixation,infection control,fracture healing,and secondary fracture fixation.Results Of the 6 patients with early infection,1 patient with septic shock removed intramedullary nails to control infection.After infection controlled,the fracture was treated with intramedullary nailing.Of the 5 patients with retained intramedullary nails,2 patients' infection were controlled and 3 were uncontrolled.After removal of the intramedullary nails the infection was control.The success rate of retaining intramedullary nails was 33.3% (2/6).Late infection occurred in 16 cases and infection was all controlled.The fractures healed in 22 patients.The fracture healing time of 6 patients with early infection was 2-6 months,with an average of 3.67±2.08 months.The fracture healing time of 16 patients with late infection was 2-4 months (average 3.2±0.79) months.Conclusion Patients with early bone infections after femoral and tibial intramedullary nail surgery may attempt debridement therapy with retained intramedullary nails,but the failure rate is high.If the intramedullary nail fails to remain,follow the treatment of patients with delayed bone infection.For patients with delayed bone infection,because the fracture has not yet healed,thorough debridement is used after the removal of internal fixation,then calcium sulfate or antibiotic bone cement stick should be implanted and fixed with external fixation.For the second phase,we may choose plate,intramedullary nail or external fixation to fix the fractures according to the soft tissue condition.All of the fixation methods could provide good fracture healing.
		                        		
		                        		
		                        		
		                        	
8.Extended anterolateral approach for thetreatment of tibial plateau fractures with posterolateral bone fragment
Liangjun JIANG ; Qiang ZHENG ; Gang FENG ; Jianwei WANG ; Zhijun PAN
Chinese Journal of Orthopaedics 2018;38(19):1161-1169
		                        		
		                        			
		                        			Objective To investigate the differences and clinical effects of extended anterolateral approach in the treatment of simple/complex tibial plateau fractures with posterolateral bone fragment.Methods From January 2013 to December 2015,forty-six patients with posterolateral tibial plateau fractures treated in our hospital were included in our research.The posterolateral plateau fractures were reduced and fixed by the extended anterolateral approach.According to Schatzker fracture classification,patients with type Ⅱ fractures were treated as simple group and patients with type Ⅴ and type Ⅵ fractures were treated as complex group.According to tibial plateau three column classification,patients were divided into single column group,double column group and three column group.The operative time,bleeding volume,fracture healing time,postoperative Rasmussen score,knee mobility,reduction and fixation of fractures and postoperative complications were compared among the groups.Results All 46 patients were followed up for 23-45 months,with an average of 31.9 months.There were 24 cases in simple fracture group and 22 cases in complex fracture group.The mean operation time of simple group was 124±33.8 min,and that of complex group was 175±65.5 min,with significant difference (t=2.302,P=0.025);the mean bleeding volume of simple group was 118±93.5 ml,and that of complex group was 190± 149.4 ml,with significant difference (t=1.905,P=0.028).12 cases were treated with arthroscopy.Bone union was achieved in all patients at the last follow-up,and daily activities were not limited.The average healing time was 4.58 months in simple group and 5.54 months in complex group.The excellent rate of fracture reduction was 83.3% in simple group and 27.3% in complex group (x2=14.679,P=0.000).Posterolateral bone fragment was not completely fixed in 4 cases (16.7%) in simple group and 8 cases (36.4%) in complex group.The average Rasmussen function score was 26.8±2.1 and the range of motion of knee joint was 100°-120° (average 115.5°±6.2°) in simple group;The average Rasmussen function score was 23.5±3.4 points and the range of motion of knee joint was 95°-115° (average 106.3°±7.4°) in complex group,neither with no significant differences.According to the three-column fracture classification system,there were 6 cases in the single-column group,24 cases in the double-column group and 16 cases in the three-column group.There were significant differences in the operative time (F=5.039,P=0.000),bleeding volume (F=5.215,P=0.000) and the excellent rate of fracture reduction (x2=7.003,P=0.030) between these three groups.But there was no significant difference in the time of fracture healing,un-fixation rate of posterolateral bone fragment,knee joint Rasmussen score and knee jointactivity.The excellent rate of fracture reduction was 83.3% in arthroscopy assisted cases and 47.1% in arthroscopy un-assisted cases respectively (x2=4.749,P=0.002).Postoperative wound infection occurred in 1 patient.Conclusion Extended anterolateral approach could provide good reduction and fixation of tibial plateau fractures with posterolateral bone fragment with satisfactory clinical outcomes obtained in both simple and complex fractures.The operation was relatively simple and posterolateral bone fracture could get better reduction and fixation in simple tibial plateau fractures,and arthroscopy treatment was helpful to improve fracture reduction.
		                        		
		                        		
		                        		
		                        	
9.Pulmonary embolism complicated with abnormal spinal cord embolism due to lack of protein S activity: a case report
Xingli TAN ; Qingxi FU ; Liangjun JIANG ; Naiyong GAO
Chinese Journal of Neurology 2018;51(11):911-913
		                        		
		                        			
		                        			Spinal vascular disease is rare in clinic, especially in cases of spinal cord embolism caused by abnormal embolism. It is rare in clinic and has not been reported in China. We report a case of pulmonary embolism complicated with abnormal spinal cord embolism due to lack of protein S activity and analyze its clinical data in order to improve clinical understanding of the disease.
		                        		
		                        		
		                        		
		                        	
10.The value of magnetic resonance imaging of the mandibular nerve using a micro surface coil and three-dimensional double-echo steady-state with water excitation sequence
Guixun HONG ; Qianqian WANG ; Jianping CHU ; Li JIANG ; Zhuhao LI ; Shaofu HE ; Liangjun RAO ; Zhiyun YANG
Chinese Journal of Radiology 2018;52(6):421-425
		                        		
		                        			
		                        			Objective To explore the value of small surface coil combined with three-dimensional double-echo steady-state with water excitation (3D-DESS-WE) sequence in displaying mandibular nerves and their branches. Methods The maxillofacial regions of 52 volunteers were scanned unilaterally by a small surface coil combined with 3D-DESS-WE sequence. According to the Gray's anatomy atlas,MPR and CPR was done according to the anatomic position of mandibular nerve and the branches,the signal characteristics of the nerves and neighboring structures were analyzed. The displaying rate of the mandibular nerves and their branches were evaluated.The image quality and display of the nerve were subjectively scored. The signal intensity(SI) of the nerve and adjacent lateral pterygoid muscle were measured. The signal intensity ratio(SIR) of mandibular nerve/lateral pterygoid muscle (SIRN/M) was calculated. Results Compared with the muscles, the nerve showed iso-signal intensity. The subjective scores for all small coil 3D-DESS-WE images were 3.02 ± 0.82. The displaying rates of V3 trunk,anterior trunk,buccal nerve, posterior trunk,inferior alveolar nerve and lingual nerve were all 100%. V3 posterior trunk,inferior alveolar nerve and lingual nerve showed the best quality scores,which were all about 3 points,and the SIRN/M were about 1. The auricular temporal nerve and masseter muscle nerve were also basically satisfied. The displaying rate of mylohyoid muscle nerve,medial pterygoid nerve and lateral pterygoid nerve was low. Conclusion Small surface coil combine with 3D-DESS-WE sequence performed high-quality in mandibular nerves and their branches MR imaging using MPR,CPR post-processing technology,which are suitable for clinical application.
		                        		
		                        		
		                        		
		                        	
            

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