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1.
Genomics ; 112(2): 2092-2105, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31830526

RESUMO

MicroRNAs (miRNAs) are involved in a series of pathology of spinal cord injury (SCI). Although, locally expressed miRNAs have advantages in studying the pathological mechanism, they cannot be used as biomarkers. The "free circulation" miRNAs can be used as biomarkers, but they have low concentration and poor stability in body fluids. Exosomal miRNAs in body fluids have many advantages comparing with free miRNAs. Therefore, we hypothesized that the specific miRNAs in the central nervous system might be transported to the peripheral circulation and concentrated in exosomes after injury. Using next-generation sequencing, miRNA profiles in serum exosomes of sham and subactue SCI rats were analyzed. The results showed that SCI can lead to changes of serum exosomal miRNAs. These changed miRNAs and their associated signaling pathways may explain the pathological mechanism of suacute SCI. More importantly, we found some valuable serum exosomal miRNAs for diagnosis and prognosis of SCI.

3.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura Ambiente , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
5.
Genomics ; 111(4): 986-996, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31307632

RESUMO

The underlying mechanisms of macrophage polarization have been detected by genome-wide transcriptome analysis in a variety of mammals. However, the transcriptome profile of rat genes in bone marrow-derived macrophages (BMM) at different activation statuses has not been reported. Therefore, we performed RNA-Sequencing to identify gene expression signatures of rat BMM polarized in vitro with different stimuli. The differentially expressed genes (DEGs) among unactivated (M0), classically activated pro-inflammatory (M1), and alternatively activated anti-inflammatory macrophages (M2) were analyzed by using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. In this study, not only we have identified the changes of global gene expression in rat M0, M1 and M2, but we have also made clear systematically the key genes and signaling pathways in the differentiation process of M0 to M1 and M2. These will provide a foundation for future researches of macrophage polarization.


Assuntos
Ativação de Macrófagos/genética , Macrófagos/imunologia , Transcriptoma , Animais , Células Cultivadas , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA , Transdução de Sinais
7.
Neural Regen Res ; 14(3): 542-552, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30539825

RESUMO

In traumatic brain injury, absent in melanoma 2 (AIM2) has been demonstrated to be involved in pyroptotic neuronal cell death. Although the pathophysiological mechanism of spinal cord injury is similar to that of brain injury, the expression and cellular localization of AIM2 after spinal cord injury is still not very clear. In the present study, we used a rat model of T9 spinal cord contusive injury, produced using the weight drop method. The rats were randomly divided into 1-hour, 6-hour, 1-day, 3-day and 6-day (post-injury time points) groups. Sham-operated rats only received laminectomy at T9 without contusive injury. Western blot assay revealed that the expression levels of AIM2 were not significantly different among the 1-hour, 6-hour and 1-day groups. The expression levels of AIM2 were markedly higher in the 1-hour, 6-hour and 1-day groups compared with the sham, 3-day and 7-day groups. Double immunofluorescence staining demonstrated that AIM2 was expressed by NeuN+ (neurons), GFAP+ (astrocytes), CNPase+ (oligodendrocytes) and CD11b+ (microglia) cells in the sham-operated spinal cord. In rats with spinal cord injury, AIM2 was also found in CD45+ (leukocytes) and CD68+ (activated microglia/macrophages) cells in the spinal cord at all time points. These findings indicate that AIM2 is mainly expressed in neurons, astrocytes, microglia and oligodendrocytes in the normal spinal cord, and that after spinal cord injury, its expression increases because of the infiltration of leukocytes and the activation of astrocytes and microglia/macrophages.

8.
J Histochem Cytochem ; 66(3): 175-187, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29300519

RESUMO

Interferon-induced transmembrane protein 1 (IFITM1) is a member of the IFITM family that is associated with some acute-phase cytokine-stimulated response. Recently, we demonstrated that IFITM1 was significantly upregulated in the injured spinal cords at the mRNA level. However, its expression and cellular localization at the protein level is still unclear. Here, a rat model of spinal cord injury (SCI) was performed to investigate the spatio-temporal expression of IFITM1 after SCI. IFITM1 mRNA and protein were assessed by quantitative reverse transcription-PCR and western blot, respectively. IHC was used to identify its cellular localization. We revealed that IFITM1 could be found in sham-opened spinal cords and gradually increased after SCI. It reached peak at 7 and 14 days postinjury (dpi) and still maintained at a relatively higher level at 28 dpi. IHC showed that IFITM1 expressed in GFAP+ and APC+ cells in sham-opened spinal cords. After SCI, in addition to the above-mentioned cells, it could also be found in CD45+ and CD68+ cells, and its expression in CD45+, CD68+, and GFAP+ cells was increased significantly. These results demonstrate that IFITM1 is mainly expressed in astrocytes and oligodendroglia in normal spinal cords, and could rapidly increase in infiltrated leukocytes, activated microglia, and astrocytes after SCI.


Assuntos
Antígenos de Diferenciação/análise , Traumatismos da Medula Espinal/patologia , Medula Espinal/patologia , Regulação para Cima , Animais , Antígenos de Diferenciação/genética , Astrócitos/metabolismo , Astrócitos/patologia , Feminino , Leucócitos/metabolismo , Leucócitos/patologia , Microglia/metabolismo , Microglia/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/genética
9.
J Neurosci Res ; 96(7): 1265-1276, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29377294

RESUMO

Ceruloplasmin (Cp), an enzyme containing six copper atoms, has important roles in iron homeostasis and antioxidant defense. After spinal cord injury (SCI), the cellular components in the local microenvironment are very complex and include functional changes of resident cells and the infiltration of leukocytes. It has been confirmed that Cp is elevated primarily in astrocytes and to a lesser extent in macrophages following SCI in mice. However, its expression in other cell types is still not very clear. In this manuscript, we provide a sensible extension of these findings by examining this system within a female Sprague-Dawley rat model and expanding the scope of inquiry to include additional cell types. Quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that the Cp mRNA and protein in SCI tissue homogenates were quite consistent with prior publications. However, we observed that Cp was expressed not only in GFAP+ astrocytes (consistent with prior reports) but also in CD11b+ microglia, CNPase+ oligodendrocytes, NeuN+ neurons, CD45+ leukocytes, and CD68+ activated microglia/macrophages. Quantitative analysis proved that infiltrated leukocytes, activated microglia/macrophages, and astrocytes should be the major sources of increased Cp.


Assuntos
Astrócitos/enzimologia , Ceruloplasmina/biossíntese , Microglia/enzimologia , Traumatismos da Medula Espinal/patologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos Nucleares/metabolismo , Astrócitos/patologia , Antígeno CD11b/metabolismo , Ceruloplasmina/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/enzimologia , Leucócitos/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos , Microglia/patologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Neurônios/fisiologia , Oligodendroglia/enzimologia , Oligodendroglia/patologia , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/induzido quimicamente
10.
Neurochem Int ; 113: 23-33, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29196144

RESUMO

Hexokinase-3 (HK3) is a member of hexokinase family, which can catalyze the first step of glucose metabolism. It can increase ATP levels, reduce the production of reactive oxygen species, increase mitochondrial biogenesis, protect mitochondrial membrane potential and play an antioxidant role. However, the change of its expression in spinal cord after injury is still unknown. In this study, we investigated the spatio-temporal expression of HK3 in the spinal cords by using a spinal cord injury (SCI) model in adult female Sprague-Dawley rats. Quantitative reverse transcription-PCR and western blot analysis revealed that HK3 could be detected in sham-opened spinal cords. After SCI, it gradually increased, reached a peak at 7 days post-injury (dpi), and then gradually decreased with the prolonging of injury time, but still maintained at a higher level for up to 28 dpi (the longest time evaluated in this study). Immunofluorescence staining showed that HK3 was found in GFAP+, ß-tubulin III+ and IBA-1+ cells in sham-opened spinal cords. After SCI, in addition to the above-mentioned cells, it could also be found in CD45+ and CD68+ cells. These results demonstrate that HK3 is mainly expressed in astrocytes, neurons and microglia in normal spinal cords, and could rapidly increase in infiltrated leukocytes, activated microglia/macrophages and astrocytes after SCI. These data suggest that HK3 may be involved in the pathologic process of SCI by promoting glucose metabolism.


Assuntos
Regulação Enzimológica da Expressão Gênica , Hexoquinase/biossíntese , Traumatismos da Medula Espinal/enzimologia , Animais , Feminino , Hexoquinase/genética , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/patologia , Vértebras Torácicas/lesões , Fatores de Tempo
11.
Artigo em Chinês | MEDLINE | ID: mdl-26510360

RESUMO

OBJECTIVE: To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. METHODS: FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. RESULTS: SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. CONCLUSION: SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.


Assuntos
Frutose-Bifosfato Aldolase/genética , Proteínas Recombinantes/biossíntese , Schistosoma japonicum/enzimologia , Animais , Escherichia coli/genética , Frutose-Bifosfato Aldolase/biossíntese , Frutose-Bifosfato Aldolase/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/crescimento & desenvolvimento
12.
Zhonghua Wei Chang Wai Ke Za Zhi ; 10(1): 67-9, 2007 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-17253179

RESUMO

OBJECTIVE: To study the value of screening hereditary nonpolyposis colorectal cancer (HNPCC) kindreds by detecting the expressions of hMLH1/hMSH2 with tissue microarray. METHODS: A tissue microarray with 22 colorectal cancers from HNPCC families and 15 sporadic colorectal cancers was established, and the expressions of hMLH1/hMSH2 were detected by immunohistochemistry (IHC). RESULTS: The expressions of hMLH1 or hMSH2 were negative in 15 of 22 HNPCC and 1 of 15 sporadic colorectal cancers in routine IHC. The expressions of hMLH1 or hMSH2 were negative in 17 of 22 HNPCC and 2 of 15 sporadic colorectal cancers in tissue microarray. The examination of hMSH2 expression yielded same results between routine IHC and tissue microarray. There were no difference on the hMLH1 expressions between routine IHC and tissue microarray. CONCLUSION: Tissue microarray is a high-throughput way to detect the expressions of hMLH1/hMSH2 and is applicable to screen HNPCC kindreds.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/genética , Análise Serial de Proteínas/métodos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Metilação de DNA , Feminino , Frequência do Gene , Testes Genéticos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Linhagem
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