1.Expression and role of early growth response gene-1 in experimental silicosis of rat.
Ling CHU ; Xiang LI ; Yong-bin HU ; Jin-sheng WANG ; Hui ZHENG ; Qing-fu ZENG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2004;22(1):47-50
OBJECTIVETo study the expression and location of early growth response gene-1 (Egr-1), transforming growth factor-beta(1) (TGF-beta(1)), fibronectin (FN) in silicotic rat and to discuss the role of Egr-1 in the development of silicosis.
METHODSSilicotic animal model of rat was established, and the expressions of Egr-1, TGF-beta(1), FN in various lung cells of silicotic rat were analysed by using immunohistochemical technique (SP) and the image analysis.
RESULTSThe expressions of Egr-1 in bronchial epithelial cell, pulmonary macrophage, alveolar epithelium cell and interstitial cell in lung silicotic tissue (gray values: 118.58 +/- 5.65 - 168.52 +/- 5.67) were higher than those of controls (gray values: 166.23 +/- 5.23 - 188.12 +/- 8.35) during 1 - 28 days, and the expression was mainly in nucleus; the expressions of TGF-beta(1) in these cells (gray values: 123.49 +/- 5.65 - 170.24 +/- 3.56) were also higher than those of controls (166.53 +/- 6.25 - 198.56 +/- 4.53), and the expression was mainly in cytoplasm. The expressions of FN in bronchial epithelial cell, pulmonary macrophage and alveolar epithelial cell (gray values: 150.32 +/- 6.54 - 201.54 +/- 7.38) were lower, while those in interstitial cell (gray values: 121.43 +/- 5.65 - 167.55 +/- 6.35) were higher than those of controls. The changes of TGF-beta(1) and Egr-1 expression level in bronchial epithelial cell, pulmonary macrophage, alveolar epithelium cell and interstitial cell were synchronous during the experiment (1 - 28 days). Both of them were correlated with each other (r = 0.61, P < 0.01), while the expression of FN was not correlated with Egr-1, but correlated to TGF-beta(1) in interstitial cell (r = 0.46, P < 0.01).
CONCLUSIONSilicon dioxide could up-regulate the expression of nuclear transcription factor Egr-1 in several kinds of cell in lung. The activated Egr-1 may coordinate the expression of TGF-beta(1) and FN to regulate the development of silicosis.
Animals ; DNA-Binding Proteins ; analysis ; physiology ; Disease Models, Animal ; Early Growth Response Protein 1 ; Fibronectins ; analysis ; physiology ; Immediate-Early Proteins ; analysis ; physiology ; Immunohistochemistry ; Lung ; chemistry ; physiopathology ; Rats ; Silicosis ; etiology ; metabolism ; Transcription Factors ; analysis ; physiology ; Transforming Growth Factor beta ; analysis ; physiology
2.High glucose promotes the CTGF expression in human mesangial cells via serum and glucocorticoid-induced kinase 1 pathway.
Quansheng, WANG ; Ali, ZHANG ; Renkang, LI ; Jianguo, LIU ; Jiwen, XIE ; Anguo, DENG ; Yuxi, FENG ; Zhonghua, ZHU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2008;28(5):508-12
The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
Cells, Cultured
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Connective Tissue Growth Factor/genetics
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Connective Tissue Growth Factor/*metabolism
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Glucose/*pharmacology
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Immediate-Early Proteins/metabolism
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Immediate-Early Proteins/*physiology
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Mesangial Cells/cytology
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Mesangial Cells/*metabolism
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Protein-Serine-Threonine Kinases/metabolism
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Protein-Serine-Threonine Kinases/*physiology
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Signal Transduction/drug effects
3.RHPN2 is highly expressed in osteosarcoma cells to promote cell proliferation and migration and inhibit apoptosis.
Zhi Yu LIU ; Feng Zhu FANG ; Jing LI ; Guang Yue ZHAO ; Quan Jin ZANG ; Feng ZHANG ; Jun DIE
Journal of Southern Medical University 2022;42(9):1367-1373
OBJECTIVE:
To screen for aberrantly expressed genes in osteosarcoma cells and investigate the role of RHPN2 in regulating the proliferation, apoptosis, migration and tumorigenic abilities of osteosarcoma cells.
METHODS:
We used GEO2R to analyze the differential gene expression profile between osteosarcoma cells and normal cells in the GSE70414 dataset. RTqPCR and Western blotting were performed to detect RHPN2 expression in osteosarcoma cell lines MG-63, 143B and SAOS2. Two RHPN2-shRNA and a control NC-shRNA were designed to silence the expression of RHPN2 in 143B cells, and CCK8 assay, colony-forming assay, annexin V-FITC/PI staining and scratch assays were carried out to examine the changes in proliferation, apoptosis and migration of the cells. We also established nude mouse models bearing osteosarcoma xenografts derived 143B cells and RHPN2-shRNA-transfected 143B cells, and assessed the effect of RHPN2 silencing on osteosarcoma cell tumorigenesis using HE staining. Kaplan-Meier survival curves were used to analyze the correlation between RHPN2 expression and survival outcomes of patients with osteosarcoma.
RESULTS:
RHPN2 expression was significantly upregulated in osteosarcoma cell lines MG-63, 143B and SAOS2 (P < 0.01). Silencing of RHPN2 significantly inhibited the proliferation and migration of 143B cells in vitro, promoted cell apoptosis (P < 0.01), and suppressed tumorigenic capacity of the cells in nude mice. A high expression of RHPN2 was significantly correlated with a poor prognosis of patients with osteosarcoma (P < 0.05).
CONCLUSION
RHPN2 is highly expressed in osteosarcoma cells to promote cell proliferation and migration and inhibits cell apoptosis. A high expression of RHPN2 is associated with a poorer prognosis of the patients with osteosarcoma.
Adaptor Proteins, Signal Transducing/metabolism*
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Animals
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Apoptosis
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Bone Neoplasms/metabolism*
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Carcinogenesis
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Cell Line, Tumor
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Cell Movement/physiology*
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Cell Proliferation/physiology*
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Humans
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Immediate-Early Proteins
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Mice
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Mice, Nude
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Osteosarcoma/metabolism*
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RNA, Small Interfering/genetics*
4.The potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.
Shuai WANG ; Jing LONG ; Chun-fu ZHENG
Protein & Cell 2012;3(5):372-382
Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.
Herpes Simplex
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virology
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Herpesvirus 1, Human
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genetics
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physiology
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Humans
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Immediate-Early Proteins
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metabolism
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Intranuclear Inclusion Bodies
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metabolism
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virology
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Leukemia, Promyelocytic, Acute
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metabolism
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Ubiquitin-Protein Ligases
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metabolism
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Virus Latency
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physiology
5.Effects of antisense oligonucleotides on the expression of connective tissue growth factor gene and on the collagen synthesis in the cultured human keloid fibroblasts.
Jian-Yi LIU ; Shi-Rong LI ; Shu-Xing JI
Chinese Journal of Burns 2004;20(2):72-75
OBJECTIVETo study the role of connective tissue growth factor (CTGF) in the pathogenesis of human keloid.
METHODSHuman keloid fibroblasts (HKF) were isolated from human keloid and cultured in vitro. The cells were then divided into 3 groups according to different processing, i.e. ASODN treatment (AT), in which phosphorothioate CTGF antisense oligonucleotides (ASODN) labeled by fluorescent isothiocyananate were transfected into the HKFs by liposome; liposome control (LC, with liposome only); control groups (without liposome or ASODN). The distribution of CTGF ASODN in all groups of cells was observed under fluorescent microscope. The CTGF mRNA index (RI) of HKF was assessed by reverse transcription polymerase chain reaction method (RT-PCR). The collagen synthesis of HKF was assessed by (3)H-proline incorporation method.
RESULTSA large amount of fluorescence could be observed in the cytoplasm of HKFs in AT 12 hours after transfection, but not in LC and C groups. The CTGF mRNA index of HKF in AT group 48 hours after transfection was significantly lower than that in LC and C groups (0.12 +/- 0.62 vs 0.51 +/- 0.18 vs 0.54 +/- 0.35, P < 0.01). The (3)H-proline incorporation rate in AT group (108.96 +/- 79.05) was lower than that in LC and C groups (P < 0.01).
CONCLUSIONThe expression of CTGF gene and collagen synthesis of the cultured HKF could be inhibited by CTGF ASODN, implying that CTGF played a role in the development of excessive fibrosis of human keloid.
Collagen ; biosynthesis ; Connective Tissue Growth Factor ; Fibroblasts ; metabolism ; Humans ; Immediate-Early Proteins ; antagonists & inhibitors ; genetics ; physiology ; Intercellular Signaling Peptides and Proteins ; genetics ; physiology ; Keloid ; etiology ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Transfection
6.Pressure-induced expression of immediate-early gene product c-Jun of the common carotid arteries in rats.
Zhiqiang YAN ; Zonglai JIANG ; Yan ZHANG ; Xiangqun YANG
Journal of Biomedical Engineering 2005;22(1):35-37
In order to investigate the mechanism of mechanical stress-mediated arterial remodeling, we studied the pressure-induced expression of immediate-early response gene product c-Jun in common carotid arteries in rats. The common carotid arteries were perfused with both high pressure (160 mmHg) and normal pressure (80 mmHg) for 0.5, 1, 3 and 6 hours. Expression of immediate-early response gene product c-Jun in the arteries was examined by immunohistochemistry and computer image processing. c-Jun was weakly expressed at 1 h, then increased at 3 h and 6 h after exposure of the arteries to normal pressure. Positive immunohistochemical product of c-Jun appeared in the arteries at 0.5 h after the onset of high pressure, then it increased markedly till 6 h. There was significant difference between the two groups. These results indicate that expression of c-Jun of the arteries can be induced by pressure, which may play an important role in mechanical stress-mediated arterial remodeling.
Animals
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Biomechanical Phenomena
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Carotid Artery, Common
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cytology
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metabolism
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physiology
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Genes, Immediate-Early
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Male
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Muscle, Smooth, Vascular
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cytology
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metabolism
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Pressure
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Proto-Oncogene Proteins c-jun
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biosynthesis
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genetics
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Rats
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Rats, Sprague-Dawley
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Stress, Mechanical
7.Aldosterone Upregulates Connective Tissue Growth Factor Gene Expression via p38 MAPK Pathway and Mineralocorticoid Receptor in Ventricular Myocytes.
Young Sam LEE ; Jeong A KIM ; Koung Li KIM ; Hyung Suk JANG ; Jeong Min KIM ; Jae Young LEE ; In Soon SHIN ; Jung Sun LEE ; Wonhee SUH ; Jin Ho CHOI ; Eun Seok JEON ; Jonghoe BYUN ; Duk Kyung KIM
Journal of Korean Medical Science 2004;19(6):805-811
The effect of aldosterone on connective tissue growth factor (CTGF) was examined in rat embryonic ventricular myocytes. Upon aldosterone treatment, CTGF expression was significantly increased in a dose and time-dependent manner. To explore the molecular mechanism for this upregulation, we examined the role of mineralocorticoid receptor. Pre-treatment of an antagonist (spironolactone) at 5-fold excess of aldosterone blocked the CTGF induction by aldosterone, suggesting that the upregulation was mediated by mineralocorticoid receptor. Aldosterone treatment resulted in activation of ERK1/2, p38 MAPK, and JNK pathways with a more transient pat-tern in p38 MAPK. Blocking studies using pre-treatment of the inhibitor of each path-way revealed that p38 MAPK cascade may be important for aldosterone-mediated CTGF upregulation as evidenced by the blocking of CTGF induction by SB203580 (p38 MAPK inhibitor), but not by PD098059 (ERK1/2 inhibitor) and JNK inhibitor I. Interestingly, JNK inhibitor I and PD098059 decreased the basal level of CTGF expression. On the other hand, pre-treatment of spironolactone abrogated the p38 MAPK activation, indicating that mineralocorticoid receptor mechanism is linked to p38 MAPK pathway. Taken together, our findings suggest that aldosterone induces CTGF expression via both p38 MAPK cascade and mineralocorticoid receptor and that cross-talk exists between the two pathways.
Aldosterone/*pharmacology
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Animals
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Cells, Cultured
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Dose-Response Relationship, Drug
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Gene Expression Regulation/drug effects/physiology
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Heart Ventricles/drug effects/embryology/metabolism
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Immediate-Early Proteins/*metabolism
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Intercellular Signaling Peptides and Proteins/*metabolism
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Myocytes, Cardiac/*drug effects/*metabolism
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Rats
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Receptors, Mineralocorticoid/*metabolism
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Research Support, Non-U.S. Gov't
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Signal Transduction/drug effects/physiology
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Spironolactone/pharmacology
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Up-Regulation/drug effects/physiology
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p38 Mitogen-Activated Protein Kinases/*metabolism
8.The Role of Hepatitis C Virus Core Protein on Liver Fibrogenesis: A Study Using an In Vitro Co-culture System.
Ju Yeop SHIN ; Seung Kew YOON ; Jin Sang WANG ; Wonhee HUR ; Jong Soon RYU ; Si Hyun BAE ; Jong Young CHOI ; Jin Mo YANG ; Se Hwan YANG ; Young Chul SUNG ; Kyu Won CHUNG ; Hee Sik SUN
The Korean Journal of Gastroenterology 2003;42(5):400-408
BACKGROUND/AIMS: The study of liver fibrogenesis by hepatitis C virus (HCV) has been limited due to the lack of an efficiency in vitro culture systems. In the present study, we investigated whether or not HCV core protein is directly related to liver fibrogenesis through stimulation of hepatic stellate cells (HSC). METHODS: Human and rat HSC were isolated and we established an in vitro co-culture system of a stable HepG2-HCV core cell line which was transfected with HCV core gene and primary HSC. We performed immunocytochemical staining and Western and Northern blot analysis in the stimulated HSC by HCV ocre protein to identify the expression of transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbeta R II), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF). The expression of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) in the culture media were measured by zymogram and ELISA, respectively. RESULTS: The expression of TGF-beta1 and CTGF was significantly higher in the stable HepG2-HCV core cell line than in HepG2 cells. Furthermore, the makers related to fibrosis such as alpha-SMA, TGF-beta1, Col I, TGFRII and MMP-2 were highly experssed in the co-culture of stable HepG2-HCV core with HSC. CONCLUSIONS: HCV core protein may play a direct role in the fibrogenesis of chronic liver disease with HCV infection.
Actins/metabolism
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Animals
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Cell Line, Tumor
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Coculture Techniques
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Connective Tissue Growth Factor
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Fibrosis
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Hepatitis C Antigens/*physiology
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Humans
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Immediate-Early Proteins/metabolism
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Immunoblotting
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Immunohistochemistry
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Intercellular Signaling Peptides and Proteins/metabolism
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Liver/metabolism/*pathology
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Protein-Serine-Threonine Kinases
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Rats
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Rats, Sprague-Dawley
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Receptors, Transforming Growth Factor beta/metabolism
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Transforming Growth Factor beta/metabolism
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Transforming Growth Factor beta1
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Viral Core Proteins/*physiology
9.The effect of human cytomegalovirus on the formation of CFU-MK in vitro.
Junxia YAO ; Sanjun SONG ; Lihua HU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(2):134-137
To investigate the mechanism and the suppressive effect of human cytomegalovirus (HCMV) on colony forming unit-megakaryocyte (CFU-MK), semi-solid culture system was used to observe the effect of HCMV AD169 strain on CFU-MK's growth of 18 cord blood samples. HCMV DNA and immediate early (IE) protein mRNA in CFU-MK was detected by PCR and reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that HCMV AD169 significantly suppressed the formation of CFU-MK in vitro. Compared with the mock group, the CFU-MK colonies decreased by 21.6%, 33.8% and 46.3%, respectively, in all the 3 infected groups (P<0.05), suggesting the suppression and the titer of the virus was dose-dependent. Both HCMV DNA and the expression of HCMV IE protein mRNA were positively detected in the colony cells of viral infected group. It is concluded that HCMV AD169 strain could inhibit the differentiation and proliferation of CFU-MK by directly infecting their progenitors. There was early transcription of HCMV IE protein in CFU-MK infected by virus.
Antigens, CD34
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metabolism
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Antigens, Viral
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biosynthesis
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genetics
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Cell Division
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drug effects
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Cells, Cultured
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Cytomegalovirus
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genetics
;
physiology
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Cytomegalovirus Infections
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DNA, Viral
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biosynthesis
;
genetics
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Humans
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Immediate-Early Proteins
;
biosynthesis
;
genetics
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Megakaryocytes
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cytology
;
virology
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Molecular Sequence Data
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Stem Cells
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cytology
;
virology
10.SiRNA targeting ICP4 attenuates HSV-1 replication.
Yu-tao LIU ; Bo SONG ; Ya-lun WANG ; Yu-ming XU ; Zhi-qiang HAN ; Xin-yu ZHAO ; Li-jie JIA
Chinese Journal of Virology 2010;26(3):163-169
HSV-1, a neurotropic virus, always leads to severe nervous symptoms. It is hard to completely eradicate the latent viruses after conventional therapy so that recurrence is inevitable. ICP is a key regulator for HSV replication and transcription that determines the cytolytic infection or latent state. In search of new anti-virus strategy targeting HSV-1ICP4, two pairs of siRNA were designed, and a recombinant eukaryotic lentiviral expression plasmid pLKO-puro(r)-hU6-siRNA was constructed. Vero cells were transfected with the designed siRNAs by Lipofectamine 2000 and four stable monoclonal cell lines were established by puromycin screening method. The ICP4 expression at mRNA level was detected with real-time PCR, and the HSV-1 replication was measured with TCID50 assay. SiRNA was shown as an effective way to inhibit the expression of ICP4 in monoclonal cell lines. Meanwhile, HSV-1 replication was significantly inhibited when ICP4 was shut down by siRNA. We conclude that siRNA targeting ICP4 attenuates HSV-1 replication. Further more, multi-site siRNAs show stronger inhibitory effect on viral replication, which may be an effective and feasible approach for biological anti-viral drugs.
Animals
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Base Sequence
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Cercopithecus aethiops
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Genetic Therapy
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Genetic Vectors
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genetics
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Herpesvirus 1, Human
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physiology
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Humans
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Immediate-Early Proteins
;
deficiency
;
genetics
;
RNA Interference
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RNA, Messenger
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genetics
;
metabolism
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RNA, Small Interfering
;
genetics
;
Vero Cells
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Virus Replication
;
genetics