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1.
Chinese Journal of Contemporary Pediatrics ; (12): 60-66, 2023.
Artículo en Chino | WPRIM | ID: wpr-971040

RESUMEN

OBJECTIVES@#To study the factors influencing the short-term (28 days) efficacy of initial adrenocorticotropic hormone (ACTH) therapy for infantile epileptic spasms syndrome (IESS), as well as the factors influencing recurrence and prognosis.@*METHODS@#The clinical data were collected from the children with IESS who received ACTH therapy for the first time in the Department of Pediatric Neurology, Xiangya Hospital of Central South University, from April 2008 to January 2018 and were followed up for ≥2 years. The multivariate logistic regression analysis was used to evaluate the factors influencing the short-term efficacy of ACTH therapy, recurrence, and long-term prognosis.@*RESULTS@#ACTH therapy achieved a control rate of seizures of 55.5% (111/200) on day 28 of treatment. Of the 111 children, 75 (67.6%) had no recurrence of seizures within 12 months of follow-up. The possibility of seizure control on day 28 of ACTH therapy in the children without focal seizures was 2.463 times that in those with focal seizures (P<0.05). The possibility of seizure control on day 28 of ACTH therapy in the children without hypsarrhythmia on electroencephalography on day 14 of ACTH therapy was 2.415 times that in those with hypsarrhythmia (P<0.05). The possibility of recurrence within 12 months after treatment was increased by 11.8% for every 1-month increase in the course of the disease (P<0.05). The possibility of moderate or severe developmental retardation or death in the children without seizure control after 28 days of ACTH therapy was 8.314 times that in those with seizure control (P<0.05). The possibility of moderate or severe developmental retardation or death in the children with structural etiology was 14.448 times that in those with unknown etiology (P<0.05).@*CONCLUSIONS@#Presence or absence of focal seizures and whether hypsarrhythmia disappears after 14 days of treatment can be used as predictors for the short-term efficacy of ACTH therapy, while the course of disease before treatment can be used as the predictor for recurrence after seizure control by ACTH therapy. The prognosis of IESS children is associated with etiology, and early control of seizures after ACTH therapy can improve long-term prognosis.


Asunto(s)
Niño , Humanos , Lactante , Hormona Adrenocorticotrópica/uso terapéutico , Espasmos Infantiles/tratamiento farmacológico , Resultado del Tratamiento , Convulsiones , Electroencefalografía/efectos adversos , Espasmo/tratamiento farmacológico
2.
Chinese Journal of Contemporary Pediatrics ; (12): 724-729, 2021.
Artículo en Chino | WPRIM | ID: wpr-888472

RESUMEN

OBJECTIVE@#To study the clinical features and recurrence factors of myelin oligodendrocyte glycoprotein (MOG) antibody disease in children and the effect of recurrence prevention regimens.@*METHODS@#A retrospective analysis was performed on the medical data of 41 children with MOG antibody disease who were hospitalized in the Department of Pediatric Neurology, Xiangya Hospital of Central South University, from December 2014 to September 2020. According to the presence or absence of recurrence, they were divided into a monophasic course group (@*RESULTS@#For these 41 children, acute disseminated encephalomyelitis was the most common initial manifestation and was observed in 23 children (56%). Of the 41 children, 22 (54%) experienced recurrence, with 57 recurrence events in total, among which optic neuritis was the most common event (17/57, 30%). The proportion of children in the recurrence group who were treated with corticosteroids for less than 3 months in the acute phase was higher than that in the monophasic course group (64% @*CONCLUSIONS@#More than half of the children with MOG antibody disease may experience recurrence. Most children with recurrence are treated with corticosteroids for less than 3 months in the acute phase. Rituximab and azathioprine may reduce the risk of recurrence.


Asunto(s)
Niño , Humanos , Autoanticuerpos , Glicoproteína Mielina-Oligodendrócito , Neuritis Óptica , Recurrencia , Estudios Retrospectivos
3.
Journal of Medical Postgraduates ; (12): 844-849, 2020.
Artículo en Chino | WPRIM | ID: wpr-823280

RESUMEN

ObjectiveUsing Chromium-51 release assay, lactate dehydrogenase release assayand other methods to detect the cytotoxicity of CD19 CAR-T cells is cumbersome, with low repeatability and poor stability. This study aims to establish a label-free and real-time method for detectingspecific cytotoxicity of CD19 CAR-T cells.MethodsIn order to establish target cell models for cytotoxic assay of CD19 CAR-T cells by using Real Time Cellular Analysis (RTCA) system,the adherent human breast cancer cells were infected with lentiviral vectors encoding CD19. CD19 expression on the transduced cells was detected by flow cytometry. The cellsexpressing CD19 stably werethen sorted by fluorescence activated cell sorting (FACS).With such cells as target cells, CD19 CAR-T cells and BCMA CAR-T cells as effector cells, RTCAsystem was used to evaluate the cytotoxicity of CAR-T cells against target cells.ResultsMDA-MB-231 and SKBR3cells with stable expression CD19were obtained in this study.The results of flow cytometry showed that positive expression rate of CD19 in MDA-MB-231/CD19 cells and SKBR3/CD19 monoclonal cells were 99.03% and 98.91%,respectively.RTCA results showed that with MDA-MB-231 and MDA-MB-231/CD19 cells as target cells,CD19 CAR-T cells showed significant cytotoxicity to MDA-MB-231/CD19 cellsat the effector-target ratio of 5∶1, 1∶1 and 1∶5,but not to MDA-MB-231 cells. With SKBR3 and SKBR3/CD19 cells as target cells, CD19 CAR-T cells showed significant cytotoxicity to SKBR3/CD19 cellsat the effector-target ratio of 5∶1and 1∶1. When the effector-target ratio was 1∶5, there was no obvious cytotoxicity.The data of MDA-MB-231/CD19 or SKBR3/CD19 as target cells and CD19 CAR-T as effector cells were analyzed separately, showing that when the number of target cells was the same, the cytotoxicity detected by RTCA increased as the number of CD19 CAR-T cells increased.The cytotoxic assays of CD19 CAR-T cells showed specificity and dose-response relationship of CD19 CAR-T cytotoxicity against the target cells.ConclusionThis study established a method for evaluating cytotoxicity of CD19 CAR-T cells that is real-time, label-free, simple and convenient.

4.
Chinese Journal of Contemporary Pediatrics ; (12): 595-601, 2020.
Artículo en Chino | WPRIM | ID: wpr-828700

RESUMEN

OBJECTIVE@#To study the clinical features of the diseases associated with aminoacyl-tRNA synthetases (ARS) deficiency.@*METHODS@#A retrospective analysis was performed of the clinical and gene mutation data of 10 children who were diagnosed with ARS gene mutations, based on next-generation sequencing from January 2016 to October 2019.@*RESULTS@#The age of onset ranged from 0 to 9 years among the 10 children. Convulsion was the most common initial symptom (7 children). Clinical manifestations included ataxia and normal or mildly retarded intellectual development (with or without epilepsy; n=4) and onset of epilepsy in childhood with developmental regression later (n=2). Some children experienced disease onset in the neonatal period and had severe epileptic encephalopathy, with myoclonus, generalized tonic-clonic seizure, and convulsive seizure (n=4); 3 had severe delayed development, 2 had feeding difficulty, and 1 had hearing impairment. Mutations were found in five genes: 3 had novel mutations in the AARS2 gene (c.331G>C, c.2682+5G>A, c.2164C>T, and c.761G>A), 2 had known mutations in the DARS2 gene (c.228-16C>A and c.536G>A), 1 had novel mutations in the CARS2 gene (c.1036C>T and c.323T>G), 1 had novel mutations in the RARS2 gene (c.1210A>G and c.622C>T), and 3 had novel mutations in the AARS gene (c.1901T>A, c.229C>T, c.244C>T, c.961G>C, c.2248C>T, and Chr16:70298860-70316687del).@*CONCLUSIONS@#A high heterogeneity is observed in the clinical phenotypes of the diseases associated with the ARS deficiency. A total of 14 novel mutations in 5 genes are reported in this study, which enriches the clinical phenotypes and genotypes of the diseases associated with ARS deficiency.


Asunto(s)
Niño , Humanos , Aminoacil-ARNt Sintetasas , Genética , Epilepsia , Mutación , Fenotipo , Estudios Retrospectivos
5.
Chinese Journal of Applied Physiology ; (6): 235-239, 2020.
Artículo en Chino | WPRIM | ID: wpr-827811

RESUMEN

To investigate the effects of exogenous NaHS on myelin basic protein (MBP) and learning and memory of hippocampal neurons in mice with spinocerebellar ataxia type 3 (SCA3) and its therapeutic significance. Twelve male normal mice were randomly selected as normal control group (NC Group), and 48 SCA3 mice were randomly selected as SCA3 model group (M Group), low dose group (NL Group, 10 μmol/kg), medium dose group (NM Group, 50μmol/kg) and high dose group (NH Group, 100 μmol/kg), 12 rats in each group. The drug treated groups were injected with NaHS intraperitoneally once a day for 4 weeks. The changes of learning and memory ability of SCA3 mice before and after the intervention of different doses of NaHS were determined by Morris water maze, the content of hydrogen sulfide (HS) in hippocampus was measured by spectrophotometry, the expression of MBP was detected by immunohistochemistry, and the morphological changes of neuron myelin sheath were observed by electron microscope. Compared with the control group, the learning and memory ability of SCA3 mice was decreased significantly (P<0.05), and the content of HS in hippocampus was decreased (P<0.05). After different doses of exogenous NaHS treatment, the learning and memory ability was improved in different degrees (P<0.05), and the contents of HS and MBP in hippocampus of SCA3 mice were also improved in different degrees (P<0.05). Exogenous NaHS may increase the contents of HS and MBP in the hippocampus of SCA3 mice, which may have a protective effect on the neurons, and then improve the learning and memory ability of SCA3 mice, and provide a new idea for the treatment of SCA3.

6.
Chinese Medical Journal ; (24): 1533-1540, 2019.
Artículo en Inglés | WPRIM | ID: wpr-771225

RESUMEN

BACKGROUND@#Advanced technology has become a valuable tool in etiological studies of intellectual disability/global developmental delay (ID/GDD). The present study investigated the role of genetic analysis to confirm the etiology in ID/GDD patients where the cause of the disease was uncertain in central China.@*METHODS@#We evaluated 1051 ID/GDD children aged 6 months to 18 years from March 2009 to April 2017. Data concerning basic clinical manifestations were collected, and the method of etiology confirmation was recorded. Genome-wide copy number variations (CNVs) detection and high-throughput sequencing of exons in the targeted regions was performed to identify genetically-based etiologies. We compared the incidence of different methods used to confirm ID/GDD etiology among groups with differing degrees of ID/GDD using the Chi-square or Fisher exact probability test.@*RESULTS@#We recruited 1051 children with mild (367, 34.9%), moderate (301, 28.6%), severe (310, 29.5%), and profoundly severe (73, 6.9%) ID/GDD. The main causes of ID/GDD in the children assessed were perinatal factors, such as acquired brain injury, as well as single gene imbalance and chromosomal gene mutation. We identified karyotype and/or CNVs variation in 46/96 (47.9%) of cases in severe ID/GDD patients, which was significantly higher than those with mild and moderate ID/GDD of 34/96 (35.4%) and 15/96 (15.6%), respectively. A total of 331/536 (61.8%) patients with clear etiology have undergone genetic analysis while 262/515 (50.9%) patients with unclear etiology have undergone genetic analysis (χ = 12.645, P < 0.001). Gene structure variation via karyotype analysis and CNV detection increased the proportion of children with confirmed etiology from 51.0% to 56.3%, and second-generation high-throughput sequencing dramatically increased this to 78.9%. Ten novel mutations were detected, recessive mutations in X-linked genes (ATPase copper transporting alpha and bromodomain and WD repeat domain containing 3) and dominant de novo heterozygous mutations in X-linked genes (cyclin-dependent kinase like 5, protocadherin 19, IQ motif and Sec7 domain 2, and methyl-CpG binding protein 2) were reported in the study.@*CONCLUSIONS@#The present study indicates that genetic analysis is an effective method to increase the proportion of confirmed etiology in ID/GDD children and is highly recommended, especially in ID/GDD children with uncertain etiology.

7.
Chinese Medical Journal ; (24): 1533-1540, 2019.
Artículo en Inglés | WPRIM | ID: wpr-802552

RESUMEN

Background@#Advanced technology has become a valuable tool in etiological studies of intellectual disability/global developmental delay (ID/GDD). The present study investigated the role of genetic analysis to confirm the etiology in ID/GDD patients where the cause of the disease was uncertain in central China.@*Methods@#We evaluated 1051 ID/GDD children aged 6 months to 18 years from March 2009 to April 2017. Data concerning basic clinical manifestations were collected, and the method of etiology confirmation was recorded. Genome-wide copy number variations (CNVs) detection and high-throughput sequencing of exons in the targeted regions was performed to identify genetically-based etiologies. We compared the incidence of different methods used to confirm ID/GDD etiology among groups with differing degrees of ID/GDD using the Chi-square or Fisher exact probability test.@*Results@#We recruited 1051 children with mild (367, 34.9%), moderate (301, 28.6%), severe (310, 29.5%), and profoundly severe (73, 6.9%) ID/GDD. The main causes of ID/GDD in the children assessed were perinatal factors, such as acquired brain injury, as well as single gene imbalance and chromosomal gene mutation. We identified karyotype and/or CNVs variation in 46/96 (47.9%) of cases in severe ID/GDD patients, which was significantly higher than those with mild and moderate ID/GDD of 34/96 (35.4%) and 15/96 (15.6%), respectively. A total of 331/536 (61.8%) patients with clear etiology have undergone genetic analysis while 262/515 (50.9%) patients with unclear etiology have undergone genetic analysis (χ2 = 12.645, P < 0.001). Gene structure variation via karyotype analysis and CNV detection increased the proportion of children with confirmed etiology from 51.0% to 56.3%, and second-generation high-throughput sequencing dramatically increased this to 78.9%. Ten novel mutations were detected, recessive mutations in X-linked genes (ATPase copper transporting alpha and bromodomain and WD repeat domain containing 3) and dominant de novo heterozygous mutations in X-linked genes (cyclin-dependent kinase like 5, protocadherin 19, IQ motif and Sec7 domain 2, and methyl-CpG binding protein 2) were reported in the study.@*Conclusions@#The present study indicates that genetic analysis is an effective method to increase the proportion of confirmed etiology in ID/GDD children and is highly recommended, especially in ID/GDD children with uncertain etiology.

8.
Chinese Journal of Contemporary Pediatrics ; (12): 857-861, 2016.
Artículo en Chino | WPRIM | ID: wpr-340605

RESUMEN

A 9-year-old boy was admitted to Xiangya Hospital due to pain after trauma in the left lower limb for 5 days and fever with generalized pain for 2 days. The results of X-ray of the left lower limb were normal. Pulmonary computed tomography (CT) showed multiple pulmonary nodules in both lungs. Adrenal CT showed marked enlargement of the left adrenal gland. The patient also experienced generalized herpes and intermittent delirium and had a blood pressure up to 155/93 mm Hg. He was transferred to our hospital with a suspected diagnosis of pheochromocytoma. On admission, the patient had a blood pressure of 86/44 mm Hg, sporadic maculopapule and herpes, touch-evoked pain, exposure of superficial veins, white pus coating on the right side of the tongue, and tension in the abdominal muscle. No skin damage was observed in the left lower limb, and the patient was forced to be in the extending position and experienced significant swelling below the knees. Laboratory examination showed a reduction in platelet count, hypoproteinemia, a significant increase in creatase, a C-reactive protein level of 348 mg/L, and a procalcitonin level of >100 ng/mL. Thoracoabdominal and pelvic CT showed multiple patchy and nodular lesions in both lungs, which had an undetermined nature, as well as an enlarged spleen. The tests of puncture fluid from the left knee joint and the periosteum of the left tibia, blood culture, and bone marrow culture all showed methicillin-resistant Staphylococcus aureus. The patient was given anti-shock treatment, anti-infective therapy with vancomycin, debridement and continuous irrigation/drainage of osteomyelitis lesions in the left tibia, but the patient still experienced recurrent shivering and severe fever and increased subcutaneous and pulmonary nodules. Linezolid was added on day 8 after admission, and the patient's body temperature returned to normal on day 24 after admission. Subcutaneous and pulmonary nodules were gradually reduced and disappeared. The patient was treated for 2 months and then evaluated as cured.


Asunto(s)
Niño , Humanos , Masculino , Fiebre , Staphylococcus aureus Resistente a Meticilina , Nódulos Pulmonares Múltiples , Dolor , Infecciones Estafilocócicas , Quimioterapia , Tomografía Computarizada por Rayos X
9.
Chinese Journal of Contemporary Pediatrics ; (12): 133-137, 2013.
Artículo en Chino | WPRIM | ID: wpr-236853

RESUMEN

<p><b>OBJECTIVE</b>To observe the expression of dynamin-1 and phosphor-dynamin-1 in the hippocampus of children and rats with mesial temporal lobe epilepsy (MTLE) and to investigate the roles of dynamin-1 and phosphor-dynamin-1 in the development of MTLE.</p><p><b>METHODS</b>Male Sprague-Dawley rats (aged 25 days) were randomly divided into acute control (AC), acute seizure (AS), latent control (LC), latent seizure (LS), chronic control (CC) and chronic spontaneous seizure (CS) groups. Lithium chloride-pilocarpine was used to induce a rat model of MTLE. The hippocampus samples of 5 children with a pathologically confirmed hippocampal sclerosis who received surgical operation were collected as a human model (HM) group, and the hippocampus samples of 4 dead children (without organic lesion of the hippocampus) were collected by autopsy as a human control (HC) group. The expression of dynamin-1 and phosphor-dynamin-1 in the hippocampus of children and rats with MTLE was measured by Western blot and immunohistochemistry.</p><p><b>RESULTS</b>The Western blot showed that the expression of phosphor-dynamin-1 was significantly lower in the AS and CS groups than in the corresponding control groups (AC and CC groups) (P<0.05). The expression of phosphor-dynamin-1 was significantly lower in the HM group than in the HC group (P<0.05). There were no significant differences in the expression of dynamin-1 among the AS, LS and CS groups and between the HM and HC groups (P>0.05). The immunohistochemical results showed that phosphor-dynamin-1 was highly expressed in the cytoplasm of hippocampal neurons of AC, CC and HC groups, but its expression was significantly reduced in the AS, CS and HM groups (P<0.05).</p><p><b>CONCLUSIONS</b>The expression of phosphor-dynamin-1, not dynamin-1, is downregulated in the hippocampus of children and rats with MTLE during seizures, which suggests that the phosphorylation/dephosphorylation of dynamin-1 may be involved in the development of MTLE.</p>


Asunto(s)
Animales , Niño , Femenino , Humanos , Masculino , Ratas , Western Blotting , Dinamina I , Metabolismo , Epilepsia del Lóbulo Temporal , Metabolismo , Hipocampo , Química , Metabolismo , Inmunohistoquímica , Fosforilación , Ratas Sprague-Dawley
10.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 487-491, 2011.
Artículo en Chino | WPRIM | ID: wpr-282568

RESUMEN

<p><b>OBJECTIVE</b>To investigate the roles of mitogen-activated protein kinases (MAPK) on silica-induced cell cycle changes.</p><p><b>METHODS</b>After cells were treated with 200 microg/ml silica, Western blot and Immunofluorescence assays were utilized to detect the expression of cyclin D1, CDK4 and E2F-4, Flow cytometry was used to detect cell cycle progression, the dominant negative mutants techniques were used to investigate silica-induced signal pathway and the effects of which in silica-induced cell cycle changes.</p><p><b>RESULTS</b>After cells were exposed to 200 microg/ml silica 24 h, the results of present study showed the proportion of cells in G1 phases was decreased. Silica-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutants of ERK or JNK, but not p38. It was found that ERK and JNK were involved in silica-induced cyclin D1 and CDK4 overexpression and the decreased expression of E2F-4.</p><p><b>CONCLUSION</b>ERK and JNK, but not p38, mediated silica-induced cell cycle changes in human embryo lung fibroblasts.</p>


Asunto(s)
Humanos , Ciclo Celular , División Celular , Células Cultivadas , Fibroblastos , Biología Celular , Patología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos , Metabolismo , Cuarzo , Toxicidad
11.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 812-815, 2011.
Artículo en Chino | WPRIM | ID: wpr-282503

RESUMEN

<p><b>OBJECTIVE</b>To investigate the roles of cyclin D1 and CDK4 in the cell cycle changes of human embryonic lung fibroblasts (HELFs) exposed to silica.</p><p><b>METHODS</b>HELFs were divided into 4 groups: control group, curcumin (20 µmol/L for 1 h) group, silica (200 µg/ml for 24 h) group and curcumin plus silica group, i.e. after exposure to 20 µmol/L curcumin for 1h, the HELFs were treated with 200 µg/ml silica for 24 h. Western blot and Immunofluorescence assays were utilized to detect the expression levels of cyclin D1, CDK4 and E2F1/4. Flow cytometry was used to detect the cell cycle progression, the RNA transfection technique was used to investigate the silica-induced signal pathway and the roles of which in silica-induced cell cycle changes.</p><p><b>RESULTS</b>The expression levels of cyclin D1 and CDK4 significantly increased and the expression level of E2F-4 decreased obviously, but the expression level of E2F-1 did not significantly change in silica group. The proportion of G1 phase cells obviously decreased and the proportion of S phase cells significantly increased in silica group, as compared with control group (P < 0.05). When suppressing the expression of cyclin D1 or CDK4, the proportions of cells in G1 phase in anti-D1 plus silica group and anti-K4 plus silica group did not obviously change, as compared with control group. When suppressing AP-1 activity, the cyclin D1 and CDK4 expression levels decreased and the E2F-4 expression level increased in curcumin plus silica group, as compared with silica group.</p><p><b>CONCLUSION</b>The results of present suggested that 200 µg/ml silica could induce the high expression of cyclin D1 and CDK4 and the low expression of E2F-4, resulting in the cell cycle changes by AP-1/cyclin D1 pathway in HELFs.</p>


Asunto(s)
Humanos , Ciclo Celular , Células Cultivadas , Ciclina D1 , Metabolismo , Quinasa 4 Dependiente de la Ciclina , Metabolismo , Factor de Transcripción E2F4 , Metabolismo , Fibroblastos , Biología Celular , Metabolismo , Fase G1 , Cuarzo , Factor de Transcripción AP-1 , Metabolismo , Transfección
12.
Chinese Journal of Contemporary Pediatrics ; (12): 908-911, 2011.
Artículo en Chino | WPRIM | ID: wpr-272436

RESUMEN

<p><b>OBJECTIVE</b>To study the influence of lipopolysaccharide (LPS) on the permeability of rat brain microvascular endothelial cells (BMECs) and possible molecular mechanism.</p><p><b>METHODS</b>Monolayers of primary rat BMECs were separated and cultured, and then treated with (LPS group) or without LPS (control group). The barrier integrity was measured by transendothelial electrical resistance (TEER) assay. The degrees of RhoA activation were determined by Pull-down assay. The expression levels of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins were detected by Western blot analysis.</p><p><b>RESULTS</b>The average TEER values of rat BMECs in the LPS group were 108.3±4.2 Ω•cm2 and 85.4±2.5 Ω•cm2 respectively 3 and 12 hrs after LPS treatment, which were significantly lower than that in the control group (159.0±8.6 Ω•cm2). Compared with the control group, the activity of RhoA started to increase 5 minutes after LPS treatment, and the expression of p115RhoGEF protein started to increase 1 hr after LPS treatment and the cellular protein levels of ZO-1, occludin and claudin-5 decreased significantly 3 hrs after LPS treatment in the LPS group (P<0.05).</p><p><b>CONCLUSIONS</b>LPS may activate the p115RhoGEF/RhoA pathway and decrease protein expression of ZO-1, occludin and claudin-5, resulting in an increased permeability of rat BMECs.</p>


Asunto(s)
Animales , Ratas , Encéfalo , Permeabilidad Capilar , Impedancia Eléctrica , Células Endoteliales , Metabolismo , Factores de Intercambio de Guanina Nucleótido , Lipopolisacáridos , Farmacología , Ratas Sprague-Dawley , Factores de Intercambio de Guanina Nucleótido Rho , Uniones Estrechas , Química , Proteína de Unión al GTP rhoA
13.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 241-245, 2011.
Artículo en Chino | WPRIM | ID: wpr-272632

RESUMEN

<p><b>OBJECTIVE</b>To study the roles of DNA dependent protein kinase (DNA-PK)in silica-induced cell cycle changes and expressions of CyclinE and CDK2 in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>The expressions of Ku80 and DNA-PKcs proteins were inhibited by siRNA plasmids, respectively. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression levels of CyclinE and CDK2 after cells were exposed to 200 microg/ml silica for 0, 3, 6, 12, 24 h.</p><p><b>RESULTS</b>The proportion of G1 phases in negative control cells decreased from 83.53% +/- 2.24% to 69.11% +/- 3.12% after exposure to silica; the proportion of G1 phases in H-Ku80 and H-PKcs cells exposed to silica decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% and from 75.06% +/- 2.23% to 58.32% +/- 1.35%, respectively (P < 0.05). The exposure to silica resulted in the increasing protein expression levels of CyclinE and CDK2 in negative control cells, and the expression levels of CyclinE were obviously suppressed in H-Ku80 and H-PKcs as compared with control cells. However, the expression level of CDK2 protein did not change significantly.</p><p><b>CONCLUSION</b>DNA-PK might play a role in silica-induced alternations of cell cycle and regulate silica-induced overexpression of CyclinE in human embryo lung fibroblasts.</p>


Asunto(s)
Humanos , Ciclo Celular , Células Cultivadas , Ciclina E , Metabolismo , Quinasa 2 Dependiente de la Ciclina , Metabolismo , Proteína Quinasa Activada por ADN , Genética , Metabolismo , Fibroblastos , Biología Celular , Metabolismo , Pulmón , Biología Celular , Proteínas Nucleares , Genética , Metabolismo , Proteínas Oncogénicas , Metabolismo , Dióxido de Silicio , Farmacología
14.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 330-333, 2011.
Artículo en Chino | WPRIM | ID: wpr-272608

RESUMEN

<p><b>OBJECTIVE</b>To study the roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>Ku80 siRNA expression vectors were transfected into HELF by lipofectamine. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression level of Ku80, p53 and p21 proteins or the phosphorylation levels of p53-ser15 after cells were exposed to silica.</p><p><b>RESULTS</b>The expression levels of Ku80 protein increased in concentration-dependent and time-dependent manners after cells were exposed to silica. The proportion of G1 phases in H-NC cells (controls) decreased from 89.28% +/- 2.19% to 68.93% +/- 3.79% after exposure to silica, and the proportion of G1 phases in HELF cells (H-Ku80) decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% after exposure to silica (P<0.05). The expression levels of Ku80, p53 proteins or p21 proteins or phosphorylation level of p53-ser15 were obviously suppressed in H-Ku80, as compared with H-NC.</p><p><b>CONCLUSION</b>Ku80/p53 pathway plays a role in the cell cycle charges induced by silica in human embryo lung fibroblasts.</p>


Asunto(s)
Humanos , Antígenos Nucleares , Metabolismo , Ciclo Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Metabolismo , Proteínas de Unión al ADN , Metabolismo , Fibroblastos , Biología Celular , Metabolismo , Citometría de Flujo , Autoantígeno Ku , Pulmón , Biología Celular , Metabolismo , Fosforilación , Cuarzo , Toxicidad , Transducción de Señal , Proteína p53 Supresora de Tumor , Metabolismo
15.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 241-245, 2010.
Artículo en Chino | WPRIM | ID: wpr-275741

RESUMEN

<p><b>OBJECTIVE</b>To study the role of Phosphatidylinositol 3 kinase (PI3K) in silica-induced DNA double strand break repair in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>Control HELF cells and DN-Deltap85 (HELF transfected with Dominant negative mutant of PI3K) were treated with 200 microg/ml silica for different times. The expression levels of phosphor-H2AX (H2AX), Ku70, Ku80 and DNA-PKcs were determined by Western blot. Furthermore, DNA double strand breaks were measured by neutral comet assay after cells were treated with 200 microg/ml silica for 0, 12 and 24 h.</p><p><b>RESULTS</b>After treatment with 200 microg/ml silica for different times, the levels of H2AX were increased in a time-dependent manner and the expression levels of H2AX were obviously suppressed in DN-Deltap85 compared with control cells. The levels of Ku70 and Ku80 were also significantly suppressed in DN-Deltap85 (0.37 +/- 0.14, 0.55 +/- 0.17) compared with control cells (0.58 +/- 0.09, 0.95 +/- 0.21) after treatment with 200 microg/ml silica for 12 h (P < 0.05). Both the percentage of tail DNA in HELF and DN-Deltap85 increased significantly at 12 h (9.78 +/- 1.15, 11.79 +/- 4.90) compared with groups without treatment with silica (2.40 +/- 0.69, 3.31 +/- 1.35) and then decreased at 24 h (4.19 +/- 0.47, 7.58 +/- 4.32), but only the decrease of HELF at 24 h was significant compared with HELF at 12 h (P < 0.05). DNA repair competence of HELF was 75.74% and that of DN-Deltap85 declined to 49.64%.</p><p><b>CONCLUSION</b>Silica dust can induce DNA double strand breaks in human embryo lung fibroblasts. PI3K might play a role in silica-induced DNA double strand break repair by regulating the expression levels of Ku70 and Ku80.</p>


Asunto(s)
Humanos , Antígenos Nucleares , Metabolismo , Proteínas de Unión al Calcio , Metabolismo , Células Cultivadas , Ensayo Cometa , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , Metabolismo , Fibroblastos , Histonas , Metabolismo , Autoantígeno Ku , Pulmón , Biología Celular , Fosfatidilinositol 3-Quinasa , Metabolismo , Dióxido de Silicio , Toxicidad
16.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 246-249, 2010.
Artículo en Chino | WPRIM | ID: wpr-275740

RESUMEN

<p><b>OBJECTIVE</b>To study the role of p53 in silica-induced cell cycle alternation and DNA double strand breaks repair in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>Neutral comet assay was applied to detect silica-induced DNA double strand breaks. According to the neutral comet experimental result, the DNA repair competence was calculated. The expression levels and phosphorylation of protein in HELF were determined by Western blot. Cell cycle changes were identified by flow cytometry in HELF.</p><p><b>RESULTS</b>After treatment with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h), the expression levels and phosphorylation of p53 increased in a time-dependent manner, reaching maximum at 12 h and then decreasing at 24 h. After treatment with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h, the expression levels and phosphorylation of p53 increased in concentration-dependent manner. After p53 expression was inhibited, silica-induced DNA damage repair competence was markedly increased (DRC = 87.68%), compared with the negative control cell induced by silica (DRC = 57.19%). Silica increased the percentage of S phase (31.8 +/- 1.1)% compared with the controls (24.3 +/- 3.8)% (P < 0.05). When p53 expression was inhibited, the number of S phase cells was significantly increased, (41.4 +/- 0.6)% compared with the controls (25.4 +/- 1.9)% (P < 0.05).</p><p><b>CONCLUSION</b>The silica dramatically increases the expression levels and phosphorylation of p53. The increased expression of p53 mediates silica-induced cell cycle change and inhibits silica-induced DNA double strand breaks repair.</p>


Asunto(s)
Humanos , Ciclo Celular , Línea Celular , Ensayo Cometa , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Fibroblastos , Biología Celular , Metabolismo , Pulmón , Biología Celular , Dióxido de Silicio , Toxicidad , Proteína p53 Supresora de Tumor , Metabolismo
17.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 2-6, 2009.
Artículo en Chino | WPRIM | ID: wpr-347259

RESUMEN

<p><b>OBJECTIVE</b>To study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>Two stable transfectants, HELF transfected with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC), were established. HELF cells were treated with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h and with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h). HELF-PKcs and HELF-NC were treated with 200 microg/ml silica for 0, 12 and 24 h. The expression levels of DNA-PKcs and phosphor-H2AX (H2AX) were determined by Western blot. DNA double strand breaks were measured by neutral comet assay.</p><p><b>RESULTS</b>After treatment with different doses of silica for 12 h, the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner. After treatment with 200 microg/ml silica for different times, the levels of H2AX increased in a time-dependent manner. The percentages of tail DNA increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h. The expression level of DNA-PKcs was suppressed in HELF-PKcs. After treatment with silica at 12 h, the level of H2AX was lower in HELF-PKcs than in HELF-NC, and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells, but no significant difference was found in the percentages of tail DNA between them. The percentages of tail DNA decreased markedly in silica-treated HELF-NC and was significantly lower than in HELF-PKcs at 24 h (P < 0.05).</p><p><b>CONCLUSION</b>Silica can induce DNA double strand breaks in human embryo lung fibroblasts. DNA-PKcs might play a major role in silica-induced DNA double strand break repair. Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.</p>


Asunto(s)
Humanos , Línea Celular , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteína Quinasa Activada por ADN , Genética , Metabolismo , Fibroblastos , Fisiología , Histonas , Metabolismo , Fosforilación , Dióxido de Silicio , Farmacología , Transfección
18.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 3-6, 2008.
Artículo en Chino | WPRIM | ID: wpr-338907

RESUMEN

<p><b>OBJECTIVE</b>To investigate the alteration of activator protein-1 (AP-1) luciferase activity in human embryo lung fibroblasts (HELF) after exposed to silica, and the role of mitogen activated protein kinase (MAPK)/AP-1 pathway on silica-induced cell cycle changes.</p><p><b>METHODS</b>After HELF cells were treated with 200 microg/ml silica, immunofluorescence assays were employed to detect the translocation and the phosphorylation level of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK), flow cytometry was used to detect the distributions of cell cycle, the dominant negative mutant of ERK, JNK and p38 were applied to detect the upstream or downstream relationship of signaling pathways.</p><p><b>RESULTS</b>After HELF-AP-1 cells were exposed to 200 microg/ml silica 6, 12, 24 h respectively, silica exposure lead to AP-1 activation in a time-dependent manner, inducing significant AP-1 activation at 6 h, reaching a maximum activation at 12 h, and having a little decrease at 24 h. After silica exposure 1 h, phosphorylation level of ERK and JNK increased mainly in cytoplasm, however, after exposure 2 h, they translocated to nucleus. The proportion of cells in G1 phases was decreased from (63.80 +/- 9.57)% to (32.23 +/- 7.22)%, and the proportion of cells in S phases was increased from (35.17 +/- 10.33)% to (66.00 +/- 8.07)% after exposed to silica 24 h. Curcumin, a chemical inhibitor of AP-1, impaired the decrease of cells in G1 phases. Furthermore we found expression of dominant-negative mutant of ERK and JNK impaired silica-induced AP-1 activation, whereas, dominant-negative mutant of p38 did not show the effect.</p><p><b>CONCLUSION</b>These result suggested that 200 microg/ml silica exposure can induce AP-1 activation, induce cell cycle changes through ERK, JNK/AP-1-dependent pathway.</p>


Asunto(s)
Humanos , Ciclo Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular , Metabolismo , Fibroblastos , Biología Celular , Metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Metabolismo , Pulmón , Biología Celular , Cuarzo , Farmacología , Transducción de Señal , Factor de Transcripción AP-1 , Metabolismo
19.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 391-394, 2008.
Artículo en Chino | WPRIM | ID: wpr-347280

RESUMEN

<p><b>OBJECTIVE</b>To study the expression level of cyclin D1-CDK4 protein in human embryonic lung fibroblasts (HELF) induced by quartz, and to study whether the expression level of cyclin D1-CDK4 protein mediated by mitogen activated protein kinase (MAPK)/(AP-1) signaling pathways.</p><p><b>METHODS</b>Cells were harvested after stimulation 2 h for the detection of cytokines. Cyclin D1 and CDK4 (cyclin dependent kinase 4) proteins was measured by immunocytochemistry (IC) and Western blot (WB).</p><p><b>RESULTS</b>The exposure of HELF to crystalline quartz for 2 hours could cause the decrease of cyclin D1 and cyclin dependent kinase 4 (CDK4) protein expression level, (7.91 +/- 0.29) x 10(3) and (5.17 +/- 0.28) x 10(4) respectively, which was lower than that of the HELF group (P < 0.05). AG126 (chemical inhibitor of the extracellular signal regulated protein kinase (ERK) signaling pathway) and the dominant negative mutant of ERK2 (molecular inhibitor of ERK2), prevented the decrease of cyclin D1 and CDK4 protein expression level. The chemical inhibitor of c-Jun NH2-terminal amino kinase (JNK), SP600125, could prevent both cyclin D1 and CDK4 protein expression level decrease. But SB203580, the chemical inhibitor of p38, prevented neither cyclin D1 nor CDK4 protein expression level decrease. Curcumin could prevent CDK4 protein expression level decrease but not cyclin D1 protein.</p><p><b>CONCLUSION</b>ERKs and JNKs, but not p38, are responsible for the decrease of cyclin D1 and CDK4 protein expression level in HELF induced by quartz. AP-1 is responsible for the decrease of CDK4 expression level but not that of cyclin D1.</p>


Asunto(s)
Humanos , Células Cultivadas , Ciclina D1 , Metabolismo , Quinasa 4 Dependiente de la Ciclina , Metabolismo , Fibroblastos , Metabolismo , Pulmón , Biología Celular , Embriología , Proteínas Quinasas Activadas por Mitógenos , Metabolismo , Cuarzo , Toxicidad , Factor de Transcripción AP-1 , Metabolismo
20.
Chinese Journal of Industrial Hygiene and Occupational Diseases ; (12): 72-76, 2008.
Artículo en Chino | WPRIM | ID: wpr-304057

RESUMEN

<p><b>OBJECTIVE</b>To investigate the roles of activated protein 1 (AP-1) in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo (a) pyrene [B (a) P], and relationships between AP-1 and cyclin D1/CDK4-E2F-1/4.</p><p><b>METHODS</b>Cells transfected with AP-1 luciferase reporter plasmid (AP-H) were cultured with serum-free RPMI1640 for 48 h, and treated with 2 micromol/L B (a) P for 24 h. AP-1 relative activity was detected by luciferase assay. Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometer and Western blot assay.</p><p><b>RESULTS</b>After B (a) P was treated for 24 h, the ratio of G1 phase cells (71 +/- 2)% was decreased to (48 +/- 3)% (P < 0.05), and an increase was observed in the ratio of S phase. AP-1 activity and cyclin D1/E2F-1 expression were increased significantly, but CDK4/E2F-4 expression did not change after B (a) P treatment. When AP-1 activity was inhibited by curcumin, decreases of G1 phase in response to B (a) P treatment were blocked, and overexpression of cyclin D1/E2F-1 was attenuated, but CDK4/E2F-4 expression was not changed significantly.</p><p><b>CONCLUSION</b>AP-1 is involved in B (a) P induced cell cycle changes, and is the upstream signals of cyclin D1/E2F-1, but not CDK4/E2F-4.</p>


Asunto(s)
Humanos , Benzo(a)pireno , Toxicidad , Ciclo Celular , Células Cultivadas , Ciclina D1 , Metabolismo , Quinasa 4 Dependiente de la Ciclina , Metabolismo , Factor de Transcripción E2F1 , Metabolismo , Factor de Transcripción E2F4 , Metabolismo , Fibroblastos , Biología Celular , Metabolismo , Factor de Transcripción AP-1 , Genética , Metabolismo , Transfección
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