BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

Objective To investigate the impact of forkhead box O1(FoxO1)on oxidative stress andcardiomyocyte apoptosis in diabetic cardiomyopathy(DCM)rats through Janus kinase(JAK)/signal transducer and activator of transcription 3(STAT3)pathway.Methods A total of 60 rats were divided into normal control group(NC),model group(Mod),FoxO1 agonist group(Res),FoxO1 inhibitor group(AS1842856)and FoxO1 inhibitor+AG490 group(AS1842856+AG490),with 12 rats in each group.DCM models were established in all groups except NC group.After successful modeling,they were given drugs,and the levels of superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GSH-PX)were tested.HE and Masson staining were used to detect the pathological damage of myocardial tissue.TUNEL staining was used to observe myocardial apoptosis.Western blot was used to detect FoxO1,Bim,Bcl-2 related X protein(Bax),p-JAK1,p-JAK2,p-STAT3,JAK1,JAK2 and STAT3 proteins.Results Compared with NC group,the expressions of FBG,MDA,cardiomyocyte apoptosis rate,FoxO1 protein,Bax protein and Bim protein were increased(P<0.05),while the expressions of HR,LVEF,LVFS,GSH-PX,SOD,p-JAK1/JAK1,p-JAK2/JAK2 and p-STAT3/STAT3 were increased in Mod group(P<0.05).Compared with Mod group,FBG,MDA,myocardial apoptosis rate,FoxO1 protein,Bax protein and Bim protein increased(P<0.05),while the expressions of HR,LVEF,LVFS,GSH-PX,SOD,p-JAK1/JAK1,p-JAK2/JAK2 and p-STAT3/STAT3 decreased in Res group(P<0.05).The expressions of HR,LVEF,LVFS,GSH-PX,SOD,p-JAK1/JAK1,p-JAK2/JAK2 and p-STAT3/STAT3 increased(P<0.05),while FBG,MDA,myocardial apoptosis rate,FoxO1 protein,Bax protein and Bim protein decreased in AS1842856 group(P<0.05).Compared with AS1842856 group,FBG,MDA,myocardial apoptosis rate,FoxO1 protein,Bax protein and Bim protein increased(P<0.05),and HR,LVEF,LVFS,GSH-PX,SOD,p-JAK1/JAK1,p-JAK2/JAK2,p-STAT3/STAT3 decreasedin AS1842856+AG490 group(P<0.05).Conclusions Inhibition of FoxO1 may exert a protective effect on DCM rats by activating the JAK2/STAT3 pathway.

Objective:To analyze the clinical characteristics of patients with different types of coronavirus disease 2019 (COVID-19).Methods:A total of 272 eligible COVID-19 patients who were admitted to Guangzhou Eighth People′s Hospital, Guangzhou Medical University from January 22 to February 15, 2020 were retrospectively enrolled. General characteristics, the first laboratory examination and imaging data of these patients were collected. According to the clinical classification, there were 236 cases in non-severe group (mild+ common type) and 36 cases in severe group (severe+ critical type). Comparisons between groups were performed by t test, chi-square test or rank-sum test when appropriate. Results:There were 23 males and 13 females in the severe group, 103 males and 133 females in the non-severe group, and the difference was statistically significant ( χ2=5.149, P=0.023). The age of severe group was (60.5±11.2) years, which was higher than that of non-severe group (46.8±15.7) years. The difference was statistically significant ( t=6.43, P<0.01). The lymphocyte (LYM) count, platelet (PLT) count and arterial partial pressure of oxygen (PaO 2) in the severe group were 0.90(0.55, 1.10)×10 9/L, 170.00(143.50, 198.00)×10 9/L and 73.50(69.70, 83.00) mmHg(1 mmHg=0.133 kPa), respectively, which were all lower than those in the non-severe group (1.42(1.09, 1.95)×10 9/L, 187.00(148.00, 230.00)×10 9/L and 96.00(83.20, 108.00) mmHg, respectively). The differences were all statistically significant ( Z=5.59, 2.00 and 5.00, respectively, all P<0.05). The levels of creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), C reaction protein (CRP) and procalcitonin (PCT) in the severe group were 123.00(79.00, 212.00) U/L, 32.10(27.00, 47.40) U/L, 305.50(216.00, 396.00) U/L, 37.02(23.92, 63.66) mg/L and 0.09(0.05, 0.19) μg/L, respectively, which were all higher than those in the non-severe group (68.00(48.00, 103.00) U/L, 20.10(16.70, 26.20) U/L, 179.00(150.00, 222.00) U/L, 26.55(18.11, 36.96) mg/L and 0.04(0.03, 0.06) μg/L respectively), and the differences were all statistically significant ( Z=3.89, 5.60, 5.12, 2.85 and 5.43, respectively, all P<0.01). No significant differences were observed in white blood cell count, creatine kinase isoenzyme and blood lactate between the two groups ( Z=1.53, 0.41 and 1.00, respectively, all P>0.05). Conclusion:Gender, age, LYM count, PLT count, PaO 2, CK, AST, LDH, CRP and PCT could be used to provide reference for clinical classification of COVID-19 patients.

Objective:To discusses effectiveness of CTA in the classification of first plantar metatarsal artery and its application value in thumb reconstruction.Methods:Thirty-six cases who underwent thumb reconstruction with free second toe or hallucis flap between December, 2015 and December, 2018 were retrospectively analyzed. Among these cases, 22 cases were injured by machine stranding, 7 cases by heavy objects, and 7 cases were injured by rolling. Exact first plantar metatarsal arteries of all these cases were evaluated by using CTA preoperatively. And compared with intraoperative findings at the donor sites. Free first or second toe flap for thumb reconstruction were designed preoperatively based on branching pattern of first plantar metatarsal arteries.Results:The origin, course, 3-dimensional (3D) anatomical relationship with surrounding tissues and branching pattern of all these feet first plantar metatarsal arteries of 35 cases (70 feet)were well displayed in CTA images, and 1 case (2 feet) were showed poor vascular continuity and artifacts in CTA (2.78%). According to the branching pattern of first plantar metatarsal arteries, 29 cases (58 feet, 80.56%) were ramifying type, 5 cases (10 feet, 13.88%) were main trunk type, and 1 case (2 feet, 2.78%) were tiny branch type. Preoperative CTA images and intraoperative findings at the donor site of 35 cases were remarkably consistent. According to CTA images, 27 cases underwent thumb reconstruction with hallucis flap, 8 cases underwent thumb reconstruction with second toe, and 1 case of poor vascular continuity and artifacts in CTA underwent thumb reconstruction with hallucis flap eventually. All these cases were followed-up for 6-24 (average 12) months, and all reconstructed thumbs survived. The clinical outcomes of all these reconstructed thumbs were good with satisfactory appearance, sensory recovery, excellent motion. The donor feet of all cases recovered well.Conclusion:High-quality 3D images of first plantar metatarsal arteries could be obtained by CTA, allowing preoperative assessment of blood supply and planning of donor site. Therefore, success rate of reconstructed operation could be improved with low disability rate of donor site.

Objective To evaluate the value of 3D printing technology to assist in the repair of severe damage to the radial side hand.Methods From March,2014 to September,2016,there were 9 cases suffered from varying degrees of metacarpal and phalangeal bones injuries with soft tissue destruction at the radial side of the hand.The donor and recipient areas were scanned by 64 row spiral CT before operation.Three dimensional images were obtained by CTA 3D reconstruction technology.Preoperative plan was designed by using Mirror image technology and Virtual surgery aided design technique of Mimics software virtual surgery aided design system.Nine cases were treated with first toe toenail flap with dorsal foot flap combined with second phalangeal bone transplantation to reconstruct thumb and anterolateral thigh perforator flap to repair the wound.The cutting area of the dorsalis pedis flap and anterolateral thigh perforator flaps ranged from 4.0 cm × 5.5 cm to 6.5 cm × 9.5 cm and from 9.5 cm ×17.0 cm to 12.0 cm × 25.5 cm,respectively.Postoperative followed-up was performed to evaluate the effect of the operation.Results All 9 thumbs and flaps survived after the operation.Postoperative followed-up time ranged from 8 to 16 months.The appearance of reconstructed thumb was close to normal,and the two-point discrimination was from 8.0 to 11.0 mm.According to the evaluation criteria of upper limb function of the Chinese Academy of Medical Sciences,7 cases were excellent and 2 cases were good.The survival flaps had soft texture,good blood circulation and satisfactory appearance at the repaired site.Conclusion 3D printing technology assists thumb reconstruction and wound repair for severe damage to the radial side of the hand with good clinical results.

Objective To investigate the human epidermal growth factor receptor?2 ( HER?2 ) expression in esophagogastirc junction adenocarcinoma ( Siewert type Ⅱ) and its clinical significance. Methods A total of 180 patients with esophagogastric junction adenocarcinoma ( Siewert type Ⅱ) were included in this study. The HER?2 expression was detected by immunohistochemistry ( IHC ) , and fluorescence in situ hybridization ( FISH) analysis was performed to assess the HER?2 gene amplification in the IHC?positive and IHC?weak positive cases. Results HER?2 overexpression ( 3+) , weak positive ( 2+) and negative (1+/0) was 11.7%(21/180), 8.9%(16/180), and 79.4%(143/180), respectively. The FISH analysis showed HER?2 gene amplification in 95.2%(20/21) of HER?2(3+) cases and 18.8% (3/16) of HER?2( 2+) cases. The concordance rate between IHC and FISH was 95. 2%. Overexpression of HER?2 (3+) was associated with the tumor differentiation (P<0.05), and irrelevant to age, sex, pT stage, pN stage, pM stage and pTNM stage ( P>0.05) . The median overall survival time ( OS) was 13 months in HER?2( 3+) patients, significantly shorter than the 21 months in HER?2 ( 2+) and HER?2 (+/-) cases ( P<0.01) . Conclusions Approximately 11. 7% of the patients with esophagogastric junction adenocarcinoma ( Siewert type Ⅱ) are HER?2?positive on IHC. HER?2 overexpression is associated with the tumor differentiation. IHC can be used as a screening test for the positive expression of HER?2 in the esophagogastirc junction adenocarcinoma ( Siewert typeⅡ) . However, FISH detection can be used as a more reliable detection method.

Objective To investigate the human epidermal growth factor receptor?2 ( HER?2 ) expression in esophagogastirc junction adenocarcinoma ( Siewert type Ⅱ) and its clinical significance. Methods A total of 180 patients with esophagogastric junction adenocarcinoma ( Siewert type Ⅱ) were included in this study. The HER?2 expression was detected by immunohistochemistry ( IHC ) , and fluorescence in situ hybridization ( FISH) analysis was performed to assess the HER?2 gene amplification in the IHC?positive and IHC?weak positive cases. Results HER?2 overexpression ( 3+) , weak positive ( 2+) and negative (1+/0) was 11.7%(21/180), 8.9%(16/180), and 79.4%(143/180), respectively. The FISH analysis showed HER?2 gene amplification in 95.2%(20/21) of HER?2(3+) cases and 18.8% (3/16) of HER?2( 2+) cases. The concordance rate between IHC and FISH was 95. 2%. Overexpression of HER?2 (3+) was associated with the tumor differentiation (P<0.05), and irrelevant to age, sex, pT stage, pN stage, pM stage and pTNM stage ( P>0.05) . The median overall survival time ( OS) was 13 months in HER?2( 3+) patients, significantly shorter than the 21 months in HER?2 ( 2+) and HER?2 (+/-) cases ( P<0.01) . Conclusions Approximately 11. 7% of the patients with esophagogastric junction adenocarcinoma ( Siewert type Ⅱ) are HER?2?positive on IHC. HER?2 overexpression is associated with the tumor differentiation. IHC can be used as a screening test for the positive expression of HER?2 in the esophagogastirc junction adenocarcinoma ( Siewert typeⅡ) . However, FISH detection can be used as a more reliable detection method.

OBJECTIVETo compare the value of applicability of the 7th edition of UICC-AJCC esophageal and gastric cancer TNM staging system in the prognostic prediction of adenocarcinoma of esophagogastric junction (EGJ).

METHODSDuring June 1, 2007 through Dec. 31, 2010, a total of 199 patients with adenocarcinoma of esophagogastric junction (Siewert type II) underwent R0-intent resection from June 1, 2007 to Dec 31, 2010 in our hospital. Their clinicopathological and survival data were retrospectively analyzed with Kaplan-Meier and Cox regression models. They were restaged according to the 7th edition of UICC/AJCC TNM stage systems for esophageal adenocarcinoma and gastric cancer, respectively. Then the likelihood ratio chi-square test related to the Cox regression model and Akaike information criterion (AIC) were used for measuring goodness of fit for both staging systems.

RESULTS199 patients with Siewert type II esophagogastric junction adenocarcinoma were identified in this study. Out of them, there were 162 males and 37 females. Their age range was from 38 to 79 years, with a median age of 62 years. 176 cases underwent transthoracic surgery, and other 23 cases underwent transabdominal surgery. TNM-EC and TNM-GC classified 4 patients to stage T1, 39 to T2, 139 to T3, and 17 to T4a, respectively, and classified 76 patients to stage N0, 58 to N1, 49 to N2, 16 to N3, respectively. The median follow-up period was 30 months. The 1-, 3-, and 5-year survival rates were 95.0%, 52.7% and 39.2%, respectively. Univariate analysis indicated that age at surgery (P = 0.009), surgical approach (P = 0.002), cell differentiation (P = 0.030), preoperative co-morbidity implications (P = 0.026), depth of tumor invasion (P < 0.001) and number of metastatic lymph nodes (P < 0.001) were significantly influencing factors of postoperative overall survival. Multivariate analysis showed that the independent prognostic factors for adenocarcinoma of esophagogastric junction were only T stage, N stage and preoperative co-morbidity and morbidities according to the 7th edition of esophageal cancer or gastric cancer TNM staging systems. The AIC value was 961.4 for the 7th edition of esophageal adenocarcinoma caner staging system, and 965.7 for the 7th edition of gastric cancer staging system.

CONCLUSIONSThe UICC/AJCC 7th edition of esophageal adenocarcinoma cancer TNM classification staging system is superior to the 7th edition of gastric cancer TNM staging system for adenocarcinoma of esophagogastric junction.

Adenocarcinoma ; diagnosis ; Adult ; Aged ; Aged, 80 and over ; Esophageal Neoplasms ; diagnosis ; Esophagogastric Junction ; Female ; Humans ; Lymph Nodes ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis ; Proportional Hazards Models ; Retrospective Studies ; Stomach Neoplasms ; Survival Rate

Objective To compare the applicability of the 7th and 6th editions of the UICC-AJCC esophageal cancer TNM staging systems for adenocarcinoma of esophagogastric junction (EGJ).Methods During June 2007 through December 2010,199 patients with EGJ adenocarcinoma(Siewert type Ⅱ) underwent R0-intent resection in our hospital.Their clinicopatholigical and survival data were retrospectively analyzed with Kaplan-Meier and Cox regression models.They were restaged according to the 7th and 6th UICC/AJCC TNM staging systems for esophageal cancer,respectively.Then the Akaike information criterion(AIC) was used for measuring goodness of fit of both staging systems.Results Among 199 patients,there were 162 males and 37 females.Univariate analysis indicated that age(P =0.009),surgical approach(P =0.002),cell differentiation (P =0.030),preoperative co-morbidity (P =0.026),depth of tumor invasion (P ＜ 0.000) and number of metastatic lymph nodes(P ＜ 0.000) were significant influencing factors on overall survival.Multivariate analysis demonstrated that the independent prognostic factors for EGJ adenocarcinoma were age,T stage,N stage and preoperative co-morbidity according to the 6th edition of esophageal cancer TNM staging system,and only T stage,N stage and preoperative co-morbidity according to the 7th edition of esophageal cancer TNM staging system.The AIC value was 961.4 for the 7th edition of esophageal cancer staging system and 972.4 for the 6th edition.Conclusion The 7th edition of UICC/AJCC esophageal cancer TNM classification is su perior to its 6th edition of esophageal cancer staging system for EGJ adenocarcinoma.