温州医科大学学报
 
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2015 Vol. 45, No. 12
Published: 2015-12-25

 
 
859 Effects of propofol on voltage-gated sodium channels and single action potention of S1 neurons in rats
HE Jiongce1, ZHANG Yu2, LIU Xingkui2, YU Tian2
Objective: The aim of this study is to investigate the effects of propofol on voltage-gated sodium channels and single action potention (AP) of primary S1 neurons in rats. Methods: Brain slices were prepared from SD rats of 7 to 14 days old, and whole-cell patch clamp technique was used to record currents and voltages. After application with propofol at different concentrations (10-300 μmol/L), the sodium currents (INa) were recorded. Data were used to make current-voltage curve, steady-state activation curve, steady-state inactivation curve, deinactivation curve and account characteristic parameters. AP was activated by a 30 mspulse of 50 pA, the data of each group were taken for analysis. Results: Propofol inhibited INA of S1 neurons in rats in a dose-dependent manner (10-300 μmol/L). It also inhibited the inactivation of sodium channels, shifted the inactivation curve towards the hyperpolarzating potential, and prolonged the recovery of sodium channels from their deinactivation, however it did not affect activation of sodium channels. Propofol inhibited the overshoot of single AP of S1 neurons in rats in a dose-dependent manner (10-100 μmol/L, P<0.01), and also depressed the overshoot (100 μmol/L, P<0.01), there was no AP be activated at 300 μmol/L. Conclusion: Propofol shows an inhibitory effect on voltage-gated sodium channels and AP of S1 neurons in the thalamocortical circuit, which may play a role in the mechanisms of propofol-induced general anesthesia
2015 Vol. 45 (12): 859- [Abstract] ( 654 ) HTML (1 KB)  PDF (2849 KB)  ( 1173 )
864 The role of RAGE-ROS axis in the apoptosis of gingival fibroblast in diabetic periodontitis
Shengbin, DAI Panpan, MAO Yixin, LI Xumin, MA Jianfeng.
Objective: To investigate the role of RAGE-ROS axis in the apoptosis of gingival fibroblast in diabetic periodontitis. Methods: Thirty wistar rats (male, six weeks old) were randomly divided into two groups: normal control group (N) and diabetic periodontitis group (D). Four weeks later, animals were sacrificed; meanwhile, the alveolar bone loss and apoptosis of gingival fibroblast were detected while the expression of RAGE and oxidative stress marker were also investigated. Results: Compared to N group, more severe alveolar bone loss, along with more apoptosis of gingival fibroblast appeared in D group (P<0.05). The expression of RAGE and oxidative stress marker increased significantly in gingival tissue in D group (P<0.05). Conclusion: RAGE-ROS axis plays an important role in the apoptosis of gingival fibroblast in diabetic periodontitis.
2015 Vol. 45 (12): 864- [Abstract] ( 728 ) HTML (1 KB)  PDF (10201 KB)  ( 644 )
869 Association between CACNB2 gene polymorphism and stroke of ethnic Han Chinese in Wenzhou region#br#
WANG Xin1,4, CHEN Jing1, WANG Qian1, LIN Yan2, NIU Yuxin3, WANG Xiaoou1, NI Li1, DU Bing1, HU
Objective: To explore the association between genetic polymorphism of CACNB2 and stroke of ethnic Han Chinese in Wenzhou region. Methods: Four hundred and thirteen cases of essential hypertension (EH), 418 cases of high blood pressure with ischemic stroke, 377 cases of high blood pressure with hemorrhagic stroke and 355 normal controls were analyzed via cross-sectional case-control. Six SNP loci of genotypes of rs11014166, rs10764319, rs7099380, rs7069292, rs2228645, rs2357928 of CACNB2 were typed by MALDI-TOFMS. Results: Compared to the control, the frequency of the CT genotype and allele frequency C of rs7069292 in high blood pressure group, high blood pressure with ischemic stroke group and high blood pressure with hemorrhagic stroke group were significantly higher (the frequency of the CT genotype 6.05% vs 1.97%, 6.94% vs 1.97%, 6.36% vs 1.97%, allele frequency C 3.03% vs 1.0%, 3.47% vs 1.0%, 3.18% vs 1.0%, P<0.05). There was no significant difference in the other 5 SNPs, P>0.05. Conclusion: There is significant association between polymorphisms of CACNB2 and people with essential hypertension (EH) in Wenzhou region. T>C may be a factor that affected the gene expression of CACNB2 and lead to high blood pressure. Mutation of T>C has no relevance with stroke by correcting the influence of high blood pressure.
2015 Vol. 45 (12): 869- [Abstract] ( 632 ) HTML (1 KB)  PDF (2011 KB)  ( 896 )
875 Expression and significance of aquaporin in actinic keratosis, Bowen’s disease and invasive squamous cell carcinoma
WANG Xiaoyong, TAO Chengjun, REN Jinping, YUAN Chengda, WANG Minlei, YING Hangyu.
Objective: To explore the expression and significance of aquaporin (AQP) 1, 3, 9, 10 in lesions of actinic keratosis, Bowen’s disease and invasive squamous cell carcinoma. Methods: Immunohistochemistry was utilized to measure the expression of AQP 1, 3, 9, 10 in tissue samples from normal human skin, actinic keratosis, Bowen’s disease and invasive squamous cell carcinoma. Results: The expression of AQP 3, 9, 10 in tissue samples from actinic keratosis, Bowen’s disease and invasive squamous cell carcinoma all increased significantly compared with the normal skin tissue. The high staining of AQP1 was observed in vascular inner membrance and endothelial cells of invasive squamous cell carcinoma. The expression of AOP9 and AQP10 in tissue samples from invasive squamous cell carcinoma increased significantly compared with actinic keratosis. Conclusion: The upregulated expression of AQP1, 3, 9, 10 may correlated with the carcinogenesis and progression of invasive squamous cell carcinoma.
2015 Vol. 45 (12): 875- [Abstract] ( 591 ) HTML (1 KB)  PDF (7577 KB)  ( 640 )
879 Predictive value of multi-mode CT scan in delayed cerebral infarction after spontaneous subarachnoid hemorrhage
DUAN Yuxia1, CHEN Weijian1, GUO Xianzhong1, LI Gang2, LI Rui1, LIN Yi1, ZHANG Yue1, HU Zilong1
Objective: To investigate the value of Multi-mode CT scan in predicting delayed cerebral infarction (DCI) after spontaneous subarachnoid hemorrhage (SSAH). Methods: Eighty-two patients were scanned with head Multi-mode CT in 72 hours after SSAH attacking, including plain CT scan, computer tomography angiography (CTA) scan and computer tomography perfusion (CTP) scan. All of the 82 patients received plain CT scan again in 14 days. A total of 82 patients were classified into two groups: DCI group (n=30) and NDCI group (n=52). Results: After quantitative comparison of CTP of both groups, the average rCBF was 0.82 and 1.02 (P<0.01), the average rCBV was 0.90 and 1.03 (P<0.01), and the average rMTT was 1.24 and 0.97 (P<0.01). Brain hemodynamics began to change in early days in SAH patients. The sensitivity, specificity, false negative rates and false positive rates of CTP and CTA’s value in predicting DCI were 100.0%, 42.3%, 0, 57.7% and 20.0%, 88.5%, 80.0%, 11.5%, respectively. All of these were calculated at the base of plain CT scan. Conclusion: Brain CTA combined with CTP will be helpful for clinician to comprehend the shape of cerebral vessel and the perfusion of brain tissue in patients with SAH. The brain blood flow changes can be known quantitatively. Also it is meaningful in early predicating DCI and its range, and guiding the early intervention of the DCI.
2015 Vol. 45 (12): 879- [Abstract] ( 566 ) HTML (1 KB)  PDF (9491 KB)  ( 643 )
883 Effect of mitochondrial aldehyde dehydrogenase 2 genotype on cardio-protection in patients with ischemia-reperfusion injury
ZHANG Yujian1,2, JIN Zhousheng1, CHEN Hongfei1, WU Yiquan1, XU Xuzhong1.
Objective: To investigate the protection of human mitochondrial aldehyde dehydrognase 2 gene mutations for patients undergone cardiopulmonary bypass (CPB). Methods: A prospective cohort of TOF patients (n=71) was recruited to investigate the influence of the ALDH2*2 allele on cardio-protection after surgical repair. The patients were divided into 2 groups: ALDH2*2 (n=45) and ALDH2*1 (n=26). The right atrial appendage was harvested. ALDH2 activity, MDA and GSH were analysed. The cTnI was tested 20 hours later after the surgery. The time in hospital were recorded. Results: ALDH2*2 carriers showed highter GSH. ALDH2*2 carriers showed lower MDA, cTnI, and shorter postoperative length of hospital stay. Conclusion: ALDH2*2 allele has a myocardial protection effect after ischemic reperfusion injury, it may associated with greater expression of GSH level.
2015 Vol. 45 (12): 883- [Abstract] ( 634 ) HTML (1 KB)  PDF (1431 KB)  ( 897 )
887 Expression of nectin-4 in hepatocellular carcinoma and its clinical significance
SUN Guangzheng, WU Yilei, ZHANG Haifeng, LI Jinhai.
Objective: To study the nectin-4 expressions in hepatocellular carcinoma (HCC) and its clinical significance. Methods: The nectin-4 expression in 36 pairs of HCC tissues, and 10 samples of normal liver tissues were detected with immunohistochemical techniques. The relations of nectin-4 expression with the clinicopathologic parameters were analyzed. Results: The IOD and area of nectin-4 were 2.55±0.64 and 8.96±1.57 in hepatocellular carcinoma tissues, which were significantly higher than those in the normal liver tissues (P<0.01). The expression of nectin-4 was not correlated with patients demographics (P>0.05), and the protein expression was correlated with histopathologic grade (P<0.01) and lymph metastasis (P<0.05). Conclusion: The high expression of nectin-4 in hepatocellular carcinoma tissues suggests that its high expression may be correlated with the malignant degree of the carcinoma, nectin-4 can be considered as a reference index of differentiation, metastasis and prognosis in hepatocellular carcinoma.
2015 Vol. 45 (12): 887- [Abstract] ( 711 ) HTML (1 KB)  PDF (2789 KB)  ( 824 )
890 Report a pedigree of familial lung cancer
LU Yao1, CHEN Bicheng2, WU Xiuling3, YE Junru1, OUYANG Jinsheng1, CHEN Chengshui1, LI Yuping1.1.Department of Respiratory Medicine, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou
Objective: To investigate the clinical iconographic characteristic, molecular patbologic test results and probable inherit susceptibility gene in a pedigree of familial aggregative lung cancer. Methods: According to family history of the proband, the pedigree was surveyed and the clinical characteristics of five cases of lung cancer in two generations who were admitted to our hospital was retrospectively analyzed. Four tumor samples and three normal lung tissues after surgery were send for molecular pathology test. A peripheral blood samples of 9 persons also send for molecular pathology examination. Results: Four persons of the second generation were diagnosed as lung cancer. And the first generation, mother was non-smoking and was confirmed with advanced adenocarcinoma and died. Three of children were diagnosed as adenocarcinoma in early stage and underwent surgery, another patient who had been in foreign country for more than 15 years and smoking, was diagnosed as small cell lung cancer, and died. Tumor samples from proband was tested with EGFR21 L858R mutation, Tumor samples from II1 and II2 had EGFR21 E868D mutation, other samples and blood samples were negative of T790M. Conclusion: As the literature reports, the mother suffering from lung cancer, the incidence of lung cancer increased, adenocarcinoma is the most common pathologic type, without germline EGFR gene T790M mutations.
2015 Vol. 45 (12): 890- [Abstract] ( 1008 ) HTML (1 KB)  PDF (7279 KB)  ( 761 )
894 Construction of red and blue fluorescent cells
WANG Zhen, SHI Xinyan, CHEN Yu.
Objective: To construct the eukaryotic expression plasmid of BFP-H2B and express it in the B16 mouse melanoma cells which can already express red fluorescence protein (RFP) in the cytoplasm, and make the cells’ nucleus show blue light while the plasmid show red light when activated by suitable light wave. Methods: The blue fluorescence protein (BFP) was acquired with PCR from a constructed PAAV3, and H2B gene was obtained with RT-PCR from cultured 293 cell’s total RNA. The sequence encoding H2B and the sequence encoding BFP were inserted into pRetroX to construct the eukaryotic expression vector pRetroX-BFP-H2B. After the constructed plasmid was confirmed by enzymatic digestion and PCR digestion, the recombination plasmid was transfected into B16 mouse melanoma cells which could already express RFP in the cytoplasm by the lipofectamine transfection technology for observation of the fusion protein’s expression. Results: The transfection of double fluorescent cells could present a double fluorescent effect, and stably expresssd batches after successfully constructed the eukaryotic expression plasmid of BFP-H2B. It provided a new reference for the construction of double fluorescent cells and the selection of the fluorescent protein species. Conclusion: In this study we establish an available survey system for the study of the two color fluorescence cells and the selection of fluorescence proteins used.
2015 Vol. 45 (12): 894- [Abstract] ( 696 ) HTML (1 KB)  PDF (16516 KB)  ( 810 )
899 Interrelation between insulin resistance and intracanial arterial stenosis in patients with cerebral infarction
WANG Xiangyun, YIN Weiyong, ZHOU Chengye.
Objective: To evaluate the relationship between insulin resistance and intracanial arterial stenosis in patients with cerebral infarction. Methods: Two groups were studied: ①161 patients with potential intracanial arterial stenosis (grade≥50%, at least one vessel); ②81 subjects without intracanial arterial stenosis (grade<50%). Insulin resistance was determined by the steady state model evaluation method, special tool was used to calculate the evaluation index of insulin resistance. MRI and MRA was performed in all subjects. The severity of intracanial arterial stenosis was determined by the Warfarin-Aspirin Symptomatic Intracranial Disease Study (WASID). Results: The homeostasis model assassment 2-insulin resistance (HOMA2-IR) was significantly higher in patients with intracanial arterial stenosis compared with control subjects. Based on logistic regression model, high HOMA2-IR was one of the risk factors. Conclusion: These results suggest that insulin resistance is a risk for intracranial arterial stenosis.
2015 Vol. 45 (12): 899- [Abstract] ( 633 ) HTML (1 KB)  PDF (1434 KB)  ( 701 )
902 A retrospective analysis of endovascular interventional treatment for 45 cases of iliac vein compression syndrome
HUANG Chongqing1, YU Guanfeng1, HUANG Jingyong1, JIN Hao2.
Objective: To investigate the clinical effect of endovascular interventional treatment for iliac vein compression syndrome (IVCS). Methods: The data of 45 patients with IVCS, underwent interventional treatment from April 2008 to April 2015 in our hospital, were analyzed retrospectively. Among these patients, 38 cases complicated with lower extremity varicose veins, underwent stenting in 37 cases and iliac venous balloon dilation in 1 case. Complicated with post-thrombotic syndrome (PTS) in 6 cases, underwent stenting in 5 cases and iliac venous balloon dilation in 1 case. Complicated with acute deep venous thrombosis (DVT) in 3 cases, underwent catheter directed thrombolysis (CDT) and then stenting in 2 cases, underwent CDT with balloon dilatation in 1 case. All the stents were self-expanding stent. The stenosis rate of iliac vein and the perimeter difference between the suffered limb and healthy one were measured before and after operation. The patients were followed up with Duplex ultrasound in 1-, 3-, 6-month, 1-, and 2- year postoperatively, respectively. Results: A total of 47 stents were placed in 45 patients. The diameter and the length of stent was 6 to 14 mm (mean 12.7±2.7 mm) and 40-200 mm (mean 64.3±39.4 mm), respectively. CDT after inferior vena cava filtration in 3 cases with DVT, using urokinase with a dose of 240 to 480 (mean 400±138.6) million U for 4 to 8 (mean 6.7) days, without the recurrence of pulmonary embolism. The difference of iliac venous stenosis was statistically significant [89.8%±8.2% (70.0%-100.0%) vs 4.0%±12.1% (0-70.0%), P<0.01]. Perimeter differences of thigh and calf between the suffered limb and healthy one were significant before and after operation, too (P<0.001). The patency rates of 1-, 3-, 6- months, 1- and 2- year were 95.5%, 93.2%, 93.2%, 90.6% and 90.6%, respectively. Four patients (8.9%) suffered minor bleeding at puncture site and were treated with compression successfully. The only 1 patients (2.2%) underwent stenting of right iliac vein, complicated migration stent to the right ventricle, and arrested by Amplatz Goose Neck Snare and Kit, successfully. The incidence of PTS was 8.9% (4/45) during the follow-up of 1 to 24 (mean 16.8) months, caused by iliac venous occlusion in different periods. Conclusion: Interventional therapy for patients of IVCS is safe and effective. It is satisfactory of stent placement for IVCS.

 

2015 Vol. 45 (12): 902- [Abstract] ( 596 ) HTML (1 KB)  PDF (7252 KB)  ( 698 )
907 The screening value of 3D-CTA in determining the etiology of spontaneous ICH
CHEN Yongchun, YANG Yunjun, XU Huazhi, GUO Xianzhong.
Objective: To assess the diagnostic accuracy of 64-slice three dimensional computed tomographic angiography (3D-CTA) for the etiology of spontaneous intracranial hemorrhage (ICH). Methods: From November 2013 to September 2014, 253 consecutive patients with ICH admitted to emergency department were included in this study and successively underwent 3D-CTA and DSA. All of the images of 3D-CTA and DSA were independently analysed by two neuroradiologists blinded, respectively. Then evaluate the accuracy of 3D-CTA for the etiology of ICH, compared with DSA as reference standard. Results: Among 253 patients, 27 patients were parenchymal hematoma, 12 patients were hemorrhage in ventricle system, and 214 patients were subarachnoid hemorrhage. DSA showed intracranial arteriovenous malformation in 17 patients, moyamoya disease in 13 patients, and intracranial aneurysm in 178 patients. The sensitivity and specificity of 64-slice 3D-CTA in the detection of the causes of ventricle system hemorrhage were both 100.0%, while in detection of the causes of parenchymal hematoma, the sensitivity and specificity were 90.9% and 100.0% respectively. Two patients with intracranial arteriovenous malformation less than 2 mm in diameter were omission. However, 3D-CTA missed 4 aneurysms, and misdiagnosed a small aneurysum in patients with subarachnoid hemorrhage, which made the sensitivity and specificity of 3D-CTA in detection of the causes of SAH lower (97.8% and 97.8% respectively). Conclusion: 3D-CTA has a high sensitivity and specificity for intracranial vascular disease, and can be used as the first choice of screening suspected intracranial aneurysms in emergency department. Moreover, 3D-CTA plays an important supplementary role in whole brain digital subtraction angiography for guiding aneurysm treatment.
2015 Vol. 45 (12): 907- [Abstract] ( 584 ) HTML (1 KB)  PDF (9821 KB)  ( 612 )
911 Observation on the curative effect of intestinal obstruction catheter complex combined with octreotide in the treatment of adhesive intestinal obstruction
ZHAO Yefang, MA Liang
Objective: To observe the curative effect of intestinal obstruction catheter complex combined with octreotide in the treatment of adhesive intestinal obstruction. Methods: A total of 48 adhesive intestinal obstruction patients were collected and randomly divided into the combination group (16 patients), the intestinal obstruction catheter group (16 patients) and the octreotide group (16 patients). Three groups were all treated with the routine comprehensive therapy, while the combination group added intestinal obstruction catheter and octreotide, the intestinal obstruction catheter group added intestinal obstruction catheter and the octreotide group added octreotide and nasogastric tube. Clinical symptoms and curative effect of there groups were observed and compared.The experimental data were analyzed by statistical methods. Results: After therapy, the bloating pain relief time and recovery exhaust time in the combination group were shorter than that in other two groups. The amount of first 24 h gastrointestinal decompression of the combination group was less than the intestinal obstruction catheter group and more than the octreotide group (P<0.05 or P<0.01). While the total effective rate of the combination group (100%) was higher than that of the other two groups (P<0.05 or P<0.01). Conclusion: The intestinal obstruction catheter complex combined octreotide in treatment of adhesive intestinal obstruction can reduce the gastrointestinal decompression amount, relieve the clinical symptoms probiotics in the adhesive intestinal obstruction patients fastly, and increase patient treatment efficiency.
2015 Vol. 45 (12): 911- [Abstract] ( 632 ) HTML (1 KB)  PDF (1422 KB)  ( 952 )
914 Endovascular treatment of posterior circulation aneurysms
ZHOU Jiang, ZHU Guangyao, CHEN Maosong,
Objective: To discuss the indications, techniques and complications of endovascular treatment of posterior circulation aneurysms. Methods: The clinical data of 33 cases of posterior circulation aneurysms treated by endovascular treatment was retrospectively reviewed. Results: Thirty-three cases were treated successfully. According to Raymond grading for the immediate occlusion of the aneurysm, grade I was obtained in 23, grade II in 6 and grade III in 4 cases. Of the 33 patients, 29 recovered well, 2 were mildly disabled, 2 were severely disabled and no one died after the treatment. All patients were in stable condition and no newly-developed neurological dysfunction was observed within 3~36 months except 1 rebleeding. Conclusion: Endovascular treatment is a minimally invasive, safe and effective method for posterior circulation aneurysms.
2015 Vol. 45 (12): 914- [Abstract] ( 482 ) HTML (1 KB)  PDF (6804 KB)  ( 787 )
918 The examination on five kinds of microelements within the venous blood of juveniles in Rui’an City
CHEN Lianping1, CHEN Hong2, ZHANG Xinming1, DENG Tongyang2.
Objective: To understand the status of the nutrition of the five microelements with the whole blood of juveniles in Rui’an so as to provide basis for the nutrition improvement strategies of this group of people. Methods: The flame atomic absorption method was taken to test the five kinds of elements within the venous bloods of the 3 561 juveniles and analyze the contents of the five kinds of elements in different age phases and between different genders. Results: The contents of copper and calcium within the whole blood of juveniles tend to descend with the growth of ages while those of zinc and iron tend to ascend, but the content of magnesium tends to be remaining stable. The differences of the contents of zinc, calcium and iron in different age phases had all of statistical meaning (P<0.05); differences exist in the contents in some age phases (P<0.05); and it was quite common that among the five kinds of elements lack of zinc and iron exists in various age phases, especially in the babyhood and infancy when the lack rate of zinc reached 31.3% and 26.1% respectively, the lack rates of iron were 42.7% and 26.3% respectively; while in the babyhood and infancy there was over-nutrition of calcium at the rates of 4.1% and 6.3% respectively. Conclusion: The distribution levels of copper and magnesium in the venous blood of juveniles in Rui’an tends to be reasonable, and lacked zinc and iron to different degrees exists, while over-nutrition of calcium exists in the babyhood and infancy, so health care instructions on the lacking of zinc and iron should be intensified and blind supplement of calcium should be prevented.
2015 Vol. 45 (12): 918- [Abstract] ( 514 ) HTML (1 KB)  PDF (2116 KB)  ( 902 )
922 The experience of surgical treatment of total anomalous pulmonary venous connection
WU Ji, HUA Qingwang, LIN Zhiyong, HU Xingti, XIA Jie, ZHAO Qifeng
Objective: To sum up the experience of surgical treatment of total anomalous pulmonary venous connection (TAPVC), to improve the successful rate of operation. Methods: The information of 76 patients with TAPVC undergone surgical correction in the hospital from January 2000 to December 2014 was analyzed retrospectively. In all patients, there were 45 males and 31 females aged 5 days to 8 years old. The weight ranged from 2.7 to 26.0 kg. Echocardiography, chest X-ray and electrocardiogram were used to confirm the diagnosis. CT angiography and three-dimensional reconstructions before operation was undergone in 31 cases. Supracardiac, intracardiac, infracardiac and mixed TAPVC were 39, 26, 8, 3, respectively. They were undrerwent TAPVC correction and repair of atrial septal defect with the superficial hypothermic CPB, except 1 patient with deep hypothermic circulatory arrest. Sixty-three cases of elective surgery, 13 cases of emergency operation were performed.  Results: All patients were weaned from CPB with the time of circulation 72.5±14.4 (range, 48 to 156) min, the time of aortic cross clamping 51.6±12.8 (range, 29 to 93) min. Eight patients died early after operation (the mortality rate, 10.5%), 68 cases were cured. Forty-six cases were followed up with duration from 3 months to 5 years. 1 case of infracardiac TAPVC was died after 6 months of discharge due to severe pneumonia. One patient with postoperative anastomotic stenosis involved reoperation, the others recovered well. Conclusion: Once TAPVC confirmed should be operated as early as possible. Surgical treatment of TAPVC may be safe and satisfactory result. Through the improvement of various surgical techniques, the key to successful operation lies on the smaller trauma and the unobstructed flow from pulmonary vein to left atrium.
2015 Vol. 45 (12): 922- [Abstract] ( 600 ) HTML (1 KB)  PDF (1438 KB)  ( 850 )
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