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Biomedical subjects

Ying Chen

Publications and source records attributed to Ying Chen.

At least 145 records · Page 8Linked to original sources

Analysis of SAW properties in ZnO/AlxGa1-xN/c-Al2O3 structures.

Piezoelectric thin films on high acoustic velocity nonpiezoelectric substrates, such as ZnO, AlN, or GaN deposited on diamond or sapphire substrates, are attractive for high frequency and low-loss surface acoustic wave devices. In this work, ZnO films are deposited on AlxGa1-xN/c-Al2O3 (0 < or = chi < or = 1) substrates using the radio frequency (RF) sputtering technique. In comparison with a single AlxGa1-xN layer deposited on c-Al2O3 with the same total film thickness, a ZnO/AlxGa1-xN/c-Al2O3 multilayer structure provides several advantages, including higher order wave modes with higher velocity and larger electromechanical coupling coefficient (K2). The surface acoustic wave (SAW) velocities and coupling coefficients of the ZnO/AlxGa1-xN/c-Al2O3 structure are tailored as a function of the Al mole percentage in AlxGa1-xN films, and as a function of the ZnO (h1) to AlxGa1-xN (h2) thickness ratio. It is found that a wide thickness-frequency product (hf) region in which coupling is close to its maximum value, K(2)max, can be obtained. The K(2)max of the second order wave mode (h1 = h2) is estimated to be 4.3% for ZnO/GaN/c-Al2O3, and 3.8% for ZnO/AlN/c-Al2O3. The bandwidth of second and third order wave modes, in which the coupling coefficient is within +/- 0.3% of K(2)max, is calculated to be 820 hf for ZnO/GaN/c-Al2O3, and 3620 hf for ZnO/AlN/c-Al2O3. Thus, the hf region in which the coupling coefficient is close to the maximum value broadens with increasing Al content, while K(2)max decreases slightly. When the thickness ratio of AlN to ZnO increases, the K(2)max and hf bandwidth of the second and third higher wave modes increases. The SAW test devices are fabricated and tested. The theoretical and experimental results of velocity dispersion in the ZnO/AlxGa1-xN/c-Al2O3 structures are found to be well matched.

Journal Article↗

Histone deacetylase inhibitors decrease reelin promoter methylation in vitro.

We investigated the effects of agents that induce reelin mRNA expression in vitro on the methylation status of the human reelin promoter in neural progenitor cells (NT2). NT2 cells were treated with the histone deacetylase inhibitors, trichostatin A (TSA) and valproic acid (VPA), and the methylation inhibitor aza-2'-deoxycytidine (AZA) for various times. All three drugs reduced the methylation profile of the reelin promoter relative to untreated cells. The acetylation status of histones H3 and H4 increased following treatment with VPA and TSA at times as short as 15 min following treatment; a result consistent with the reported mode of action of these drugs. Chromatin immunoprecipitation experiments showed that these changes were accompanied by changes occurring at the level of the reelin promoter as well. Interestingly, AZA decreased reelin promoter methylation without concomittantly increasing histone acetylation. In fact, after prolonged treatments with AZA, the acetylation status of histones H3 and H4 decreased relative to untreated cells. We also observed a trend towards reduced methylated H3 after 18 h treatment with TSA and VPA. Our data indicate that while TSA and VPA act to increase histone acetylation and reduce promoter methylation, AZA acts only to decrease the amount of reelin promoter methylation.

Cell Adhesion Molecules, Neuronal↗

Reduction in fear of falling through intense tai chi exercise training in older, transitionally frail adults.

OBJECTIVES: To determine whether an intense tai chi exercise program could reduce fear of falling better than a wellness education (WE) program in older adults who had fallen previously and meet criteria for transitioning to frailty. DESIGN: Cluster-randomized, controlled trial of 48 weeks' duration. SETTING: Ten matched pairs of congregate living facilities in the greater Atlanta area. PARTICIPANTS: Sample of 291 women and 20 men, aged 70 to 97. MEASUREMENTS: Activity-related fear of falling using the Activities-Specific Balance Confidence Scale (ABC) and the Fall Efficacy Scale at baseline and every 4 months for 1 year. Demographics, time to first fall and all subsequent falls, functional measures, Centers for Epidemiologic Studies Depression Scale, medication use, level of physical activity, comorbidities, and adherence to interventions. RESULTS: Mean ABC was similar in both cohort groups at the time of randomization but became significantly higher (decreased fear) in the tai chi cohort at 8 months (57.9 vs 49.0, P<.001) and at study end (59.2 vs 47.9, P<.001). After adjusting for covariates, the mean ABC after 12 months of intervention was significantly greater in the tai chi group than in the WE group, with the differences increasing with time (mean difference at 12 months=9.5 points, 95% confidence interval=4.8-14.2, P<.001). CONCLUSION: Tai chi led to a significantly greater reduction in fear of falling than a WE program in transitionally frail older adults. The mean percentage change in ABC scores widened between tai chi and WE participants over the trial period. Tai chi should be considered in any program designed to reduce falling and fear of falling in transitionally frail older adults.

Accidental Falls↗

Ginsenoside Rg1 reduces MPTP-induced substantia nigra neuron loss by suppressing oxidative stress.

AIM: To investigate the effect of ginsenoside Rg1, an effective ingredient from ginsenoside, on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced substantia nigra neuron lesion. METHODS: C57-BL mice were given MPTP to prepare Parkinson disease mice model. Different doses of Rg1 (5, 10, and 20 mg.kg(-1).d(-1)) or N-acetylcystein (NAC) (300 mg.kg(-1).d(-1)) were given 3 d prior to MPTP in the pretreatment groups. Glutathione (GSH) level and total superoxide dismutase (T-SOD) activity in substantia nigra were determined by spectrophotometry. Nissl staining, tyrosine hydroxylase immunostaining, and TUNEL labeling were used to observe the damage and apoptosis of nigral neurons. Western blot analysis was used to detect the phospho-JNK and phospho-c-Jun levels in midbrain homogenates. RESULTS: Pretreatments of C57-BL mice with different doses of Rg1 or NAC were found to protect against MPTP-induced substantia nigra neurons loss. Rg1 or NAC prevented GSH reduction and T-SOD activation in substantia nigra, and attenuated the phosphorylations of JNK and c-Jun following MPTP treatment. CONCLUSION: The antioxidant property of Rg1 along with the blocking of JNK signaling cascade might contribute to the neuroprotective effect of ginsenoside Rg1 against MPTP.

Animals↗

Cloning, structural organization and chromosomal mapping of rat costimulatory molecule 4-1BBL.

4-1BBL (TNFSF9) is a member of the tumor necrosis factor (TNF) ligand superfamily, which is expressed on some activated antigen presenting cells and B cells. We isolated a rat cDNA clone encoding the rat homologue of the human 4-1BBL (GenBank accession No. AY259541). The deduced rat 4-1BBL protein, consisting of 308 amino acids with a molecular weight of 33,469 Da, was a typical type II transmembrane glycoprotein, the same as human and murine 4-1BBL. "SDAA" in the cytoplasmic domain of rat 4-1BBL was deduced to act as the phosphorylation site for casein kinase I ("SXXS" motif), which is present in the cytoplasmic domains of human and murine 4-1BBL, and all other TNF ligand family members known to utilize reverse signaling. The two introns of 4-1BBL were also cloned (GenBank accession No. AY332409). Rat 4-1BBL is much more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Rat 4-1BBL was expressed in all tested tissues: brain, lung, colon, liver, thymus, testicle, kidney, adrenal, stomach, spleen and heart. The chromosomal location of rat 4-1BBL was first identified by bioinformatics, then by fluorescence in situ hybridization at 9q11 (GenBank accession UniGene No. Rn.46783). Rat, murine and human 4-1BBL genes are evolved from a common gene. The identification and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biological function of this molecule.

4-1BB Ligand↗

Post-Amadori AGE inhibition as a therapeutic target for diabetic complications: a rational approach to second-generation Amadorin design.

Aminoguanidine and pyridoxamine (Pyridorintrade mark), two major inhibitors of advanced glycation end product (AGE) formation, have entered clinical trials for diabetic nephropathy. They share no structural similarity and are believed to inhibit AGE formation by entirely different mechanisms. Pyridoxamine is a post-Amadori AGE inhibitor-that is, an "Amadorin"-whereas aminoguanidine primarily scavenges reactive dicarbonyl precursors to AGEs. However, pyridoxamine also has a limited potential to react with dicarbonyls. We thus embarked on an effort to develop second-generation Amadorins with low nucleophilicity. Our hypothesis was that we could improve specificity for inhibiting the post-Amadori pathway by minimizing the potential for scavenging small dicarbonyl intermediates. This mechanism-based strategy has led to a rational drug design program that has successfully produced candidate Amadorins, among them the novel compound BST-4997. This Amadorin has greater post-Amadori potency than pyridoxamine but possess no dicarbonyl scavenging activity. Prototypical inhibitors like BST-4997 provide a unique tool to help identify relevant AGE pathways that contribute to diabetic complications. Targeting AGE inhibition differs significantly from traditional approaches to drug discovery and thus represents a new paradigm for the drug industry that should be recognized.

Animals↗

Environmental mycological study and allergic respiratory disease among tobacco processing workers.

This study presents the results of an investigation of respiratory symptoms, lung function, chest X-ray examinations, analysis of specific IgE antibodies and skin prick tests to fungi on 130 tobacco-processing workers and 112 control workers. Industrial hygiene survey and environmental mycological studies were also performed. The average dust concentrations ranged from 13.76 to 29.55 mg/m(3) in the tobacco processing workshops. Also, the numbers of fungi colonies in the processing environments were much higher than those in the control environments. The prevalences of chronic respiratory or nasal symptoms in exposed workers were significantly higher than those in control workers (p<0.05 or p<0.01). The prevalences of lung function abnormalities in the exposed workers were significantly higher than those in control workers (p<0.05). The positive prevalences of specific IgE reactions to fungi (26.92% for A.fumigatus and 51.54% for Rhizopus nigricans) in exposed workers were also significantly higher than those in control workers (p<0.01). The positive prevalences of the skin prick test showed that 18.46% of the exposed workers were positive to A. fumigatus and 23.85% were positive to Rhizopus nigricans. Our results suggested that tobacco processing workers may develop respiratory diseases related to tobacco dust and we consider that fungi might be one of the allergens causing allergic respiratory or nasal diseases in tobacco processing workers.

Adult↗

Decreased expression of pigment epithelium-derived factor is involved in the pathogenesis of diabetic nephropathy.

Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor. Previous studies have shown that decreased ocular levels of PEDF are associated with diabetic retinopathy. However, the implication of PEDF expression in diabetic nephropathy has not been revealed. In the present study, we demonstrated for the first time that the expression of PEDF was decreased at both the mRNA and protein levels in the kidney of diabetic rats, whereas transforming growth factor-beta (TGF-beta) and fibronectin levels were increased in the same diabetic kidneys. As shown by immunohistochemistry, the decrease of PEDF expression occurs primarily in the glomeruli. In vitro studies showed that high concentrations of glucose significantly decreased PEDF secretion in primary human glomerular mesangial cells (HMCs), suggesting that hyperglycemia is a direct cause of the PEDF decrease in the kidney. Toward the function of PEDF, we showed that PEDF blocked the high-glucose-induced overexpression of TGF-beta, a major pathogenic factor in diabetic nephropathy, and fibronectin in primary HMCs, suggesting that PEDF may function as an endogenous inhibitor of TGF-beta expression and fibronectin production in glomeruli. Therefore, decreased expression of PEDF in diabetic kidneys may contribute to extracellular matrix overproduction and the development of diabetic nephropathy.

Animals↗

[Survey of the utilization and adverse effects of antibacterial drugs in patients admitted for respiratory diseases].

OBJECTIVE: To evaluate the utilization of antibacterial drugs and their adverse effects in patients admitted for respiratory disease based on medical record review. METHODS: The medical records of 141 inpatients admitted in 2002 for respiratory diseases were reviewed. The drug utilization index (DUI) and daily cost of antibacterial drugs were calculated, with also assessment of the adverse effects in relation to the drug use. RESULTS: The DUI ranged from 0.9 to 1.1 in 66.7% of the patients, and the daily cost of the antibacterial drugs was 317.8+/-194.3 RMB, with a total of 14 cases with adverse drug effects recorded. CONCLUSIONS: The utilization of antibacterial drugs was basically adequate in Nanfang Hospital though with occasional abuse of cefoperazone and sulbactam, inappropriate combination and high cost of the antibacterial drugs. More careful application of these drugs in old patients is stressed.

Adolescent↗

[Identification of a novel HLA-A2-restrictive CTL epitope of an ovary cancer-associated antigen OVA66].

AIM: To identify a novel HLA-A2-restrictive CTL epitope of an ovary cancer-associated antigen OVA66. METHODS: Dendritic cells (DCs), induced from peripheral blood mononuclear cells(PBMCs) by cytokines, were confirmed by morphological observation and FACS. Mature DCs were pulsed with each of the two synthesized peptides which were selected as possible CTL epitopes by software analysis. The pulsed DCs were used to stimulate autologous CD8+ T cells from an HLA-A2+ healthy donor. One week later, the peptides-pulsed autologous PBMCs were used to stimulate the CD8+ T cells for another 3 times at weekly intervals. The stimulated CD8+ T cells were used as CTLs. The cytotoxicity of CTLs to target cells was detected by lactate dehydrogenase (LDH) release assay and the number of T cells secreting antigen-specific IFN-gamma in CTLs was analyzed by enzyme-linked immunospot assay (ELISPOT). RESULTS: The results of morphology observation and FACS indicated that mature DCs were induced from PBMCs. Of the two peptides, peptide L235(FLPDHINIV) induced peptide-specific CD8+ T cells that lysed HLA-A2+ T2 cells pulsed with L235 and OVA66+/HLA-A2+ SW480 cells. Compared with control peptide, L235 increased the number of IFN-gamma producing T cells. CONCLUSION: This novel OVA66-derived CTL epitope L235 can induce HLA-A2-restrictive CTL response, which lays the foundation for preparation of tumor-specific peptide vaccine.

Amino Acid Sequence↗

[Analysis of preoperative findings and ossicular condition in chronic suppurative otitis media].

OBJECTIVE: To discussion the relationship of preoperative findings and ossicular condition in chronic suppurative otitis media. METHOD: The correlation between the ossicular conditions and classification of tympanic membrane perforation, ear discharge, air conduction pure tone average, air-bone gap, pneumatization, complication, and cholesteatoma in 251 patients(288 ears) with chronic suppurative otitis media was analysed. RESULT: The air-conduction threshold and air-bone gap in patients with ossicular discontinuity are higher than that in patients with ossicular continuity. Ossicular discontinuity in patients with perforation of the pars flaccida of tympanic membrane, persistently draining ears, complications, and cholesteatoma occurred significiantly more frequently than those without these conditions. CONCLUSION: The air-conduction threshold and air-bone gap are the more reliable indications to identify the ossicular conditions in patients with chronic suppurative otitis media. There are significiant correlation between the ossicular conditions in patients with chronic otitis media and their classification of tympanic membrane perforation, ear discharge, complication, and cholesteatoma.

Adolescent↗

[Change of free hemoglobin content after mixing type B whole blood with different amounts of type O whole blood].

To investigate the changes of free hemoglobin (FHb) content after mixing type B whole blood with different amounts of type O whole blood at room temperature and at 37 degrees C, two lots of type B whole blood stored at 4 degrees C for 24 hours were randomly taken as recipient blood, and were packed as 60 ml respectively. Type O blood was taken as donor blood. 60 ml type B whole bloods were mixed with different amounts of type O whole blood, i.e. with 9, 12, 15 and 18 ml. The mixed blood was packed into 100 ml plastic blood bags and stored at 37 degrees C or room temperature, shaken once every 15 minutes. Free hemoglobin content was determined for the harvested samples at 1, 2, 4, 8 and 12 hours after store. The results showed that there was no significant elevation of FHb within 12 hours after mixing B whole blood with different amounts of type O whole blood. In another lot, there was obvious difference in FHb after 1 hour store along with the prolongation of store at either room temperature or 37 degrees C. In one lot, there was no difference of FHb (P > 0.05) during 1 - 8 hours of store at room temperature or 37 degrees C, but significant difference at 12 hours of store (P < 0.001). In another lot, there was no difference of FHb (P > 0.05) within 1 hour of store at room temperature and at 37 degrees C, but significant difference during 2 approximately 8 hours of store (P < 0.001). It is concluded that the FHb would not change significantly within 12 hours after type B blood was mixed with 1 200 ml of type O whole blood, but when the mixed blood was placed at room temperature or at 37 degrees C for 8 hours, the FHb content approaches, even exceeds 170.4 mg/L which was observed in the blood stored for 2 days. It suggests that freshly collected blood must be put into refrigerator of 2 approximately 4 degrees C for storing as soon as possible, so as to decrease the catabolism of erythrocyte and the releasing of FHb and other metabolites which are deleterious to the recipients.

ABO Blood-Group System↗

[The application and development of ultrasonic scalpel].

Ultrasonic scalpel has the advantages of less bleeding, less damage to surrounding tissue, early healing, etc. The applications of ultrasound in medical surgery include incision and hemostasis, phacoemulsification, tumor aspiration, fat aspiration, and bone cutting. In this paper, the principles, development, characteristic, key technology and clinical application of different ultrasonic scalpel in the world are introduced, and the domestic application and future development of ultrasonic scalpel are prospected.

Humans↗

Synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of SARS-associated coronavirus.

A novel severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. We previously isolated and characterized SARS-CoV and SARS-CoV-like viruses from human and animals, respectively, suggesting that SARS could be transmitted from wild/farmed animals to humans. Comparison of the viral genomes indicated that sequence variation between animal and human isolates existed mainly in the spike (S) gene. We hypothesized that these variations may underlie a change of binding specificity of the S protein to the host cells, permitting viral transmission from animals to humans. Here we report that four 20-mer synthetic peptides (S protein fragments), designed to span these sequence variation hotspots, exhibited significant antiviral activities in a cell line. SARS-CoV infectivity was reduced over 10 000-fold through pre-incubation with two of these peptides, while it was completely inhibited in the presence of three peptides. Molecular modelling of the SARS-CoV peplomer suggests that three of these antiviral peptides map to the interfaces between the three monomers of the trimeric peplomer rather than the heptad repeat region from which short peptides are known to inhibit viral entry. Our results revealed novel regions in the spike protein that can be targeted to inhibit viral infection. The peptides identified in this study could be further developed into antiviral drugs.

Amino Acid Sequence↗

[Surgical approaches to the skull base neoplasms].

OBJECTIVE: To discuss the best surgical approach to the skull base neoplasms. METHODS: Retrospective analysis the 79 skull base neoplasms cases treated with surgical resection in Qilu hospital of Shandong university from 1992 to 2002. Eleven surgical approaches including midfacial degloving, frontal coronal discission, nasal eversion, maxillary swing, partial maxillary resection, total resection of orbit, mandibular swing, combination of front, temple, preauricular, post aureum, neck, and transoral approaches were used to resect the tumor which involved fossae pterygopalatine, paranasal sinuses, nasopharynx, antero, meso and posterobasilar region, lobi frontalis and lobi temporalis of cerebrum. RESULTS: Seventy-nine skull base neoplasms were totally removed and no one died from the operation. Although 5 cases complicated with cerebrospinal fluid leak and all recovered within 1 week, no serious cranium-cerebrum complication occurred. In 29 patients with benign tumor including 11 cases of meningioma, 3 cases of chondroma, 1 case of hemangio-meningioma, 1 case of cavernous hemangioma, 2 cases of osteodysplasia fibromas, 9 cases of neurofibroma, 1 case of glomus jugular tumor, 1 case of neurilemmoma, 19 have survived over 5 years and the longest one has survived over 8 years. For 50 patients with malignant tumor including 3 cases of well-differentiated squamous cell carcinoma, 17 cases of moderately differentiated squamous cell carcinoma, 11 cases of poorly differentiated squamous cell carcinoma, 1 case of undifferentiated carcinoma, 2 cases of chondrosarcoma, 5 cases of canceration of papilloma, 2 cases of adenocarcinoma, 1 case of esthesioneuroblastoma, 2 cases of malignant fibrohistiocytoma, 1 case of fibrosarcoma, 2 cases of malignant mixed tumour, 3 cases of sarcoma survival rates of 3 and 5 years were 59.2% (29/49), 38.5% (10/26) respectively. CONCLUSION: In order to resect the tumor completely and reduce the complication and malformation as far as possible, different surgical approaches must be designed according to the pathological changes characters and involved area,and the surgeon should select the shortest approach, avoid to damage the important neurovascular structure, and resect the tumor through the natural anatomy space by the shelter incision.

Adult↗

[Effect of RNAi-mediated survivin gene silencing on apoptosis of ovarian cancer cell lines SKOV3 and SKOV3/ADM].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) has been widely used in tumor gene therapy, antivirus, and gene drug selection. Survivin gene is highly expressed in ovarian cancer cell line SKOV3 and drug-resistant cell line SKOV3/ADM, and is an ideal target of gene therapy for ovarian cancer. This study was to explore effect of RNAi-mediated Survivin gene silencing on apoptosis of SKOV3 and SKOV3/ADM cells. METHODS: Recombinant plasmid pshRNA-Survivin was transfected into SKOV3 and SKOV3/ADM cells. The expression of Survivin was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The apoptosis of SKOV3 and SKOV3/ADM cells after transfection were evaluated by AO/EB dyeing, TUNEL, and flow cytometry. RESULTS: After transfection of pshRNA-Survivin, mRNA and protein levels of Survivin gene in SKOV3 and SKOV3/ADM cells were obviously reduced; the apoptosis of SKOV3 and SKOV3/ADM cells were significantly higher in transfection group than in control group (P0.01); 48 h after transfection, the apoptosis rate was 14.05% in SKOV3 cells, and 21.02% in SKOV3/ADM cells, no obvious apoptosis was detected in control cells. CONCLUSION: pshRNA-Survivin could reduce the expression of Survivin gene, and induce apoptosis of SKOV3 and SKOV3/ADM cells.

Antineoplastic Agents↗

[Simultaneous determination of cimaterol, clenbuterol and salbutamol in feeds by capillary zone electrophoresis].

A method for simultaneous determination of cimaterol, clenbuterol and salbutamol in feeds was developed by capillary zone electrophoresis. The effects of the experimental conditions on the separation and determination of cimaterol, clenbuterol and salbutamol have been examined. Under the optimum conditions, all three compounds were completely separated in 8 min. The linear range of cimaterol, clenbuterol and salbutamol was 0.1-1.0 mg/L, and the detection limits (S/N = 3) were 0.02, 0.03 and 0.02 mg/L, respectively. The developed method has been used for the determination of spiked feed samples. The recoveries of cimaterol, clenbuterol and salbutamol were 89%-103%, 86%-91% and 83%-87%, respectively. The relative standard deviations for cimaterol, clenbuterol and salbutamol were 3.8%-4.3%, 3.6%-5.0% and 4.0%-5.6%, respectively. The proposed method is a sensitive, fast and simple method for the determination of cimaterol, clenbuterol and salbutamol in feeds.

Albuterol↗

[Clinical study of combined approach of tympanoplasty and mastoidectomy with close technique].

OBJECTIVE: To improve the surgery results and living quality of patients following the operation of the combined approach of tympanoplasty and mastoidectomy with close technique. METHODS: The clinical data and following-up results of 49 patients treated with combined approach of tympanoplasty and mastoidectomy with close technique and 82 patients treated with open mastoidectomy with tympanoplasty were retrospective analyzed. RESULTS: In the group treated with combined approach tympanoplasty, all patients got dry ear in 20 days and 31 patients' hearing level enhanced over 15 dB after the operation; but in the group treated with open mastoidectomy with tympanoplasty, only 13 patients got dry ear in 20 days and no patients' hearing level enhance over 15 dB after the operation. Although the recurrence rates between two groups were not significant difference, the patients of former group not only took a shorter recovery time and got a better hearing recovery, but also kept a normal external auditory meatus, at the same time, they need not to clear scab at fixed period all life long. CONCLUSIONS: When performed on carefully selected patients, combined approach tympanoplasty was a feasible surgical method to improve the surgery results and living quality of patients following the operation, however, the advanced equipment and perfect operation skill are necessary.

Adolescent↗