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The extracellular domain (p75ECD) of p75 neurotrophin receptor (p75NTR) antagonizes Aβ neurotoxicity and promotes Aβ clearance in Alzheimer's disease (AD). The impaired shedding of p75ECD is a key pathological process in AD, but its regulatory mechanism is largely unknown. This study was designed to investigate the presence and alterations of naturally-occurring autoantibodies against p75ECD (p75ECD-NAbs) in AD patients and their effects on AD pathology. We found that the cerebrospinal fluid (CSF) level of p75ECD-NAbs was increased in AD, and negatively associated with the CSF levels of p75ECD. Transgenic AD mice actively immunized with p75ECD showed a lower level of p75ECD and more severe AD pathology in the brain, as well as worse cognitive functions than the control groups, which were immunized with Re-p75ECD (the reverse sequence of p75ECD) and phosphate-buffered saline, respectively. These findings demonstrate the impact of p75ECD-NAbs on p75NTR/p75ECD imbalance, providing a novel insight into the role of autoimmunity and p75NTR in AD.
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Mice , Animals , Alzheimer Disease/pathology , Receptor, Nerve Growth Factor , Amyloid beta-Peptides , Autoantibodies , Mice, TransgenicABSTRACT
Recently p75 neurotrophin receptor (p75NTR) has been found to play a critical role in the pathology of neurodegen¬erative! diseases including Alzheimer's disease (AD) , Parkin¬son' s disease ( PI)), Huntington's disease ( HI)) , amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).This arti¬cle reviews the research progress of p75NTR in regulating neuron apoptosis, axon degeneration and cognitive impairment, explo¬ring the application of p75NTR as a potential therapeutic target for the treatment of neurodegenerative diseases.
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Objective: Our knowledge of human neural crest stem cells (NCSCs) is expanding, owing to recent advances in technologies utilizing human-induced pluripotent stem cells (hiPSCs) that generate NCSCs. However, the clinical application of these technologies requires the reduction of xeno-materials. To overcome this significant impediment, this study aimed to devise a novel method to induce NCSCs from hiPSCs without using a feeder cell layer.Materials and Methods: hiPSCs were cultured in feeder-free maintenance media containing the Rho-associated coiled-coil forming kinase inhibitor Y-27632. When the cells reached 50–70% confluence, differentiation was initiated by replacing the medium with knockout serum replacement (KSR) medium containing Noggin and SB431542. The KSR medium was then gradually replaced with increasing concentrations of Neurobasal medium from day 5 to 11.Results: Immunocytochemistry and flow cytometry were performed 12 days after induction of differentiation and revealed that the cells generated from hiPSCs expressed the NCSC markers p75 and HNK-1, but not the hiPSC marker SOX2.Conclusion: These findings demonstrate that hiPSCs were induced to differentiate into NCSCs in the absence of feeder cells.
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Abstract Mesenchymal and epithelial stem cells were identified in dental tissues; however, knowledge about the odontogenic stem cells is limited, and there are some questions regarding their temporo-spatial dynamics in tooth development. Objective Our study aimed to analyze the expression of the stem cell markers CD146 and p75NTR during the different stages of odontogenesis. Methodology The groups consisted of 13.5, 15.5, 17.5 days old embryos, and 14 days postnatal BALB/c mice. The expression of CD146 and p75NTR was evaluated by immunohistochemistry. Results Our results showed that positive cells for both markers were present in all stages of tooth development, and the number of positive cells increased with the progression of this process. Cells of epithelial and ectomesenchymal origin were positive for CD146, and the expression of p75NTR was mainly detected in the dental papilla and dental follicle. In the postnatal group, dental pulp cells were positive for CD146, and the reduced enamel epithelium and the oral mucosa epithelium showed immunostaining for p75NTR. Conclusions These results suggest that the staining pattern of CD146 and p75NTR underwent temporal and spatial changes during odontogenesis and both markers were expressed by epithelial and mesenchymal cell types, which is relevant due to the significance of the epithelial-ectomesenchymal interactions in tooth development.
Subject(s)
Animals , Mice , Mesenchymal Stem Cells , Odontogenesis , Stem Cells , Cell Differentiation , Receptors, Nerve Growth Factor , CD146 Antigen , Mice, Inbred BALB CABSTRACT
Objective: To investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. Methods: Three lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining. Results: After lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced ( P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups ( P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant ( P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased. Conclusion: Silencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.
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Objective: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. Methods: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. Results: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). Conclusion: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.
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BACKGROUND: P75 neurotrophin receptor (P75NTR) is widely expressed in nerve tissues and cells, and plays a dual role in promoting or inhibiting differentiation. P75NTR is also overexpressed in local tissues with fracture nonunion. Therefore, P75NTR is studied for the osteogenic differentiation of bone marrow mesenchymal stem cells, which is of great significance to provide important targets for the clinical treatment of fracture nonunion. OBJECTIVE: To elucidate the effect of P75NTR overexpression on osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro. METHODS: The bilateral femurs of Sprague-Dawley rats were selected, and the rat bone marrow mesenchymal stem cells were extracted by whole bone marrow separation and adherence method and subcultured in vitro. The P75NTR overexpression plasmid GV358-P75NTR expressing enhanced green fluorescent protein was constructed, and the P75NTR overexpression lentiviral vector was collected by empty lentiviral vector packaging. Rat bone marrow mesenchymal stem cells primary cultured in vitro for 10 days were selected, and seeded into culture plates after digestion. P75NTR overexpression lentivirus and related infection reagents were added for subsequent infection experiments. After 7 days of infection, the expression of green fluorescent protein was observed by fluorescence microscope and overexpression of P75NTR protein was detected by western blot. Transfected cells were cultured for 7 days in a conventional medium, followed by culture in the osteogenic differentiation medium. Alkaline phosphatase activity was quantified by enzyme linked immunosorbent assay on the 7th, 10th, and 14th days after osteogenic induction. Formation of mineralized nodules was observed by alizarin red staining on the 7th and 14th days after osteogenic induction. RESULTS AND CONCLUSION: P75NTR overexpression lentiviral vector-infected bone marrow mesenchymal stem cells expressed green fluorescent protein (infection efficiency was about 90%), and the expression of P75NTR protein was significantly increased, which was significantly different from that in the negative control group (P < 0.05). Cell model of P75NTR overexpression was successfully constructed. Compared with the negative control and blank control groups, the alkaline phosphatase activity of the P75NTR overexpression group was significantly decreased at the corresponding time point after osteogenic induction, the number of mineralized nodules was significantly reduced, and cell aggregation and distribution were significantly weakened (P < 0.05). To conclude, P75NTR overexpression negatively regulates the osteogenic differentiation of rat bone marrow mesenchymal stem cells cultured in vitro. Overexpression of P75NTR in local tissues inhibits the osteogenic differentiation of surrounding bone marrow mesenchymal stem cells, which may be an important factor for bone defects or fracture nonunion.
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BACKGROUND: P75 Neurotrophin receptor (P75NTR) is one of the receptors for nerve growth factor (NGF). P75NTR plays a dual role in promoting proliferation or apoptosis in various cell tissues, and is highly expressed at fracture nonunion sites. However, excessive NGF can shut down P75NTR receptor, thereby saving damaged cells. Therefore, the study regarding co-transfection of silenced P75NTR and NGF overexpression is of great significance for the proliferation of bone marrow mesenchymal stem cells and provides new ideas for clinical treatment of fracture nonunion. OBJECTIVE: To observe the effect of lentivirus-mediated silencing of P75NTR combined with NGF overexpression on the proliferation of bone marrow mesenchymal stem cells in Sprague-Dawely rats. METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawley rats were cultured to the third generation in vitro and were divided into blank control group, negative control group, silent p75NTR group, NGF overexpression group, and silent p75NTR combined with NGF overexpression group. Lentivirus-mediated silencing of P75NTR and overexpression of NGF were transfected into rat bone marrow mesenchymal stem cells to induce P75NTR silencing and NGF overexpression. Inverted fluorescence microscopy was used to observe changes in cell morphology on day 3 after transfection. Flow cytometry was used to detect transfection efficiency and western blot method was applied to detect the expression of P75NTR and NGF. Finally, the cell proliferation activity was detected by MTT method and cell counting kit-8 method. RESULTS AND CONCLUSION: Cell growth and distribution were good after co-transfection of lentivirus. The transfection efficiency of the double-gene lentiviral vector exceeded 70%. Compared with the blank control and negative control groups, the expression of P75NTR protein was significantly down-regulated, and the expression of NGF was profoundly up-regulated in the silent p75NTR combined with NGF overexpression group. Compared with the blank control and negative control groups, cell proliferation was significantly increased in the other three groups (P < 0.05), and the fastest proliferation was observed in the silent p75NTR combined with NGF overexpression group. To conclude, silencing P75NTR combined with NGF overexpression co-transfection can promote the proliferation of bone marrow mesenchymal stem cells from Sprague-Dawley rats.
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OBJECTIVE: Nerve growth factor (NGF) is a member of the neurotrophic factor family and plays a vital role in the physiological processes of organisms, especially in the nervous system. Many recent studies have reported that NGF is also involved in the regulation of tumourigenesis by either promoting or suppressing tumor growth, which depends on the location and type of tumor. However, little is known regarding the effect of NGF on interspinal schwannoma (IS). In the present study, we aimed to explored whether mouse nerve growth factor (mNGF), which is widely used in the clinic, can influence the growth of interspinal schwannoma cells (ISCs) isolated from IS in vitro.METHODS: ISCs were isolated, cultured and identified by S-100 with immunofluorescence analysis. S-100-positive cells were divided into five groups, and separately cultured with various concentrations of mNGF (0 [phosphate buffered saline, PBS], 40, 80, 160, and 320 ng/mL) for 24 hours. Western blot and quantantive real time polymerase chain reaction (PCR) were applied to detect tyrosine kinase A (TrkA) receptor and p75 neurotrophin receptor (p75(NTR)) in each group. Crystal violet staining was selected to assess the effect of mNGF (160 ng/mL) on ISCs growth.RESULTS: ISCs growth was enhanced by mNGF in a dose-dependent manner. The result of crystal violet staining revealed that it was significantly strengthened the cells growth kinetics when cultured with 160 ng/mL mNGF compared to PBS group. Western blot and quantantive real time PCR discovered that TrkA receptor and mRNA expression were both up-regualated under the condition of mNGF, expecially in 160 ng/mL, while the exoression of p75(NTR) demonstrated no difference among groups.CONCLUSION: From these data, we conclude that exogenous mNGF can facilitate ISC growth by activating both TrkA receptor and p75(NTR). In addition, patients who are suffering from IS should not be administered mNGF in the clinic.
Subject(s)
Animals , Humans , Mice , Blotting, Western , Fluorescent Antibody Technique , Gentian Violet , In Vitro Techniques , Kinetics , Nerve Growth Factor , Nervous System , Neurilemmoma , Physiological Phenomena , Protein-Tyrosine Kinases , Real-Time Polymerase Chain Reaction , Receptor, Nerve Growth Factor , Receptor, trkA , Receptors, Nerve Growth Factor , RNA, MessengerABSTRACT
Objective To investigate serum p75 neurotrophin receptor-extracellular domain (p75NTR-ECD) level in patients with chronic cerebral hypoperfusion-vascular cognitive impairment (CCH-VCI) and its relationship with tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6. Methods The clinical data of patients with CCH-VCI (n＝34) were collected from Changhai Hospital, Naval Medical University (Second Military Medical University) from Aug. to Dec. 2018. Enzyme linked immunosorbent assay was applied for detection of serum levels of p75NTR-ECD, TNF-α, IL-1β and IL-6; and the results were then compared with those of ischemic stroke participants (n＝34) and healthy controls (n＝36), who were all in the same age range. Spearman correlation analysis was used to analyze the relationship between serum p75NTR-ECD level and the above-mentioned inflammatory factors in CCH-VCI patients. Results The serum p75NTR-ECD level in the CCH-VCI group was significantly higher than those in the healthy control group and the ischemic stroke group (544.36 [440.88, 628.50] pg/mL vs 276.49 [262.59, 313.87] pg/mL and 366.87 [337.09, 450.43] pg/mL, U＝87.500 and 335.500, both P0.05). The serum levels of TNF-α, IL-1β and IL-6 were 196.02 (141.20, 280.35) pg/mL, 68.23 (60.79, 91.94) pg/mL and 51.04 (40.24, 65.26) pg/mL in the CCH-VCI group, respectively, and 218.67 (143.76, 281.28) pg/mL, 76.87 (59.10, 99.91) pg/mL and 64.45 (43.13, 86.76) pg/mL in the ischemic stroke group, respectively, which were all significantly higher than those in the healthy control group (73.71 [56.94, 79.81] pg/mL, 42.98 [34.52, 51.34] pg/mL and 14.97 [11.76, 21.19] pg/mL, respectively; U＝ 31.000 and 4.000, 106.000 and 132.000, and 48.000 and 13.000; all P0.05). Serum p75NTR-ECD level in the CCH-VCI patients was correlated with TNF-α level (r＝0.391, P＝0.022), but not with IL-1β or IL-6 levels (r＝0.032 and 0.164, P＝ 0.855 and 0.355). Conclusion Serum p75NTR level may be related to inflammatory factors (TNF-α) after chronic cerebral hypoperfusion, and they may jointly participate in the pathogenesis of CCH-VCI.
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To determine the inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction,the protein-protein interaction between human immunodeficiency virus type 1( HIV-1) integrase and lens epithelial growth factor p75 protein( LEDGF/p75) was used as a target. The homogeneous time-resolved fluorescence( HTRF) technique was used in the inhibitory activity assay. The results showed that eight endophytic fungi with anti-IN-LEDGF/p75 interaction activity were screened out from fifty-three strains with different morphological characteristic. Among them,106 strain showed strong inhibitory activity against HIV-1 IN-LEDGF/p75 interaction with IC50 value of 5. 23 mg·L-1,and was identified as a potential novel species of Magnaporthaceae family by the analyses of ITS-rDNA,LSU and RPB2 sequences data. This study demonstrated that potential natural active ingredients against the HIV-1 IN-LEDGF/p75 interaction exist in the endophytic fungi of D. versipellis. These results may provide available candidate strain resources for the research and development of new anti-acquired immunodeficiency syndrome drugs.
Subject(s)
Humans , Berberidaceae , Microbiology , Endophytes , Fungi , Chemistry , HIV Integrase , Metabolism , HIV-1 , Intercellular Signaling Peptides and Proteins , Metabolism , Protein BindingABSTRACT
OBJECTIVE: Nerve growth factor (NGF) is a member of the neurotrophic factor family and plays a vital role in the physiological processes of organisms, especially in the nervous system. Many recent studies have reported that NGF is also involved in the regulation of tumourigenesis by either promoting or suppressing tumor growth, which depends on the location and type of tumor. However, little is known regarding the effect of NGF on interspinal schwannoma (IS). In the present study, we aimed to explored whether mouse nerve growth factor (mNGF), which is widely used in the clinic, can influence the growth of interspinal schwannoma cells (ISCs) isolated from IS in vitro. METHODS: ISCs were isolated, cultured and identified by S-100 with immunofluorescence analysis. S-100-positive cells were divided into five groups, and separately cultured with various concentrations of mNGF (0 [phosphate buffered saline, PBS], 40, 80, 160, and 320 ng/mL) for 24 hours. Western blot and quantantive real time polymerase chain reaction (PCR) were applied to detect tyrosine kinase A (TrkA) receptor and p75 neurotrophin receptor (p75(NTR)) in each group. Crystal violet staining was selected to assess the effect of mNGF (160 ng/mL) on ISCs growth. RESULTS: ISCs growth was enhanced by mNGF in a dose-dependent manner. The result of crystal violet staining revealed that it was significantly strengthened the cells growth kinetics when cultured with 160 ng/mL mNGF compared to PBS group. Western blot and quantantive real time PCR discovered that TrkA receptor and mRNA expression were both up-regualated under the condition of mNGF, expecially in 160 ng/mL, while the exoression of p75(NTR) demonstrated no difference among groups. CONCLUSION: From these data, we conclude that exogenous mNGF can facilitate ISC growth by activating both TrkA receptor and p75(NTR). In addition, patients who are suffering from IS should not be administered mNGF in the clinic.
Subject(s)
Animals , Humans , Mice , Blotting, Western , Fluorescent Antibody Technique , Gentian Violet , In Vitro Techniques , Kinetics , Nerve Growth Factor , Nervous System , Neurilemmoma , Physiological Phenomena , Protein-Tyrosine Kinases , Real-Time Polymerase Chain Reaction , Receptor, Nerve Growth Factor , Receptor, trkA , Receptors, Nerve Growth Factor , RNA, MessengerABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive impairments with progressive loss of memory and behavioral disorder.Up to now,there is no effective therapy or drug to cure AD.Recent studies have shown p75 neurotrophin receptor (p75NTR) plays a critical role in the pathogenesis of AD,while the extracellular domain of p75 neurotrophin receptor (p75ECD) has neuroprotective effect and can attenuate the development and progression of AD.Therefore,p75ECD is a research-hotspot for prevention and treatment of AD.Here,recent studies are reviewed to learn about the advances of p75ECD in the prevention and therapy of AD and provide references for getting novel methods and drugs to treat AD.
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The p75 neurotrophic factor receptor is a low affinity receptor for neurotrophic factors and plays an important role in nerve growth, development and function integrity. It is closely related to dental development, oral and maxillofacial tumor, nerve repair and tissue engineering. It shows good prospect for application. In this paper, the research progress of p75 neurotrophic factor receptor in Stomatology is reviewed.
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AIM:To investigate the role of specific binding of precursor form of nerve growth factor(proNGF) to p75NTR(proNGF-p75NTR)in isoflurane-induced cognitive impairment in aging mice.METHODS: Aging C57BL/6J male mice were randomly divided into 3 groups:control group,isoflurane(Iso)group and p75NTR specific inhibitor 2-ami-no-3-methyl-pentanoic acid amide(LM11A-31,LM)+isoflurane(LM+Iso)group.Aging C57BL/6J mice in Iso group and LM+Iso group were exposed to isoflurane,and the mice in control group were exposed to air.The mice in LM+Iso group were treated with LM,which dissolved in normal saline water and was administered daily by oral gavage at 50 mg· kg-1· d-1for 1 month.The mice in control group and Iso group received an equivalent volume of normal saline by the same route.Arterial blood gas analysis was conducted to detect the physiological state of the mice after isoflurane exposure. Morris water maze was performed to evaluate the cognitive function.The protein levels of proNGF,p75NTR,phosphorylated p38 MAPK and cleaved caspase-3 were determined by Western blot.RESULTS: Compared with the control group, the protein expression of proNGF and p75NTR in the hippocampus of Iso group was significantly elevated(P<0.05).The phos-phorylation level of p38 MAPK was higher in Iso group than that in control group(P<0.05),which was reduced in LM +Iso group significantly(P<0.05).A significant increase in the protein level of cleaved caspase-3 was observed in Iso group as compared with control group(P<0.05), while LM11A-31 treatment reversed this elevation significantly(P<0.05).The escape latency prolonged and the number of crossing the original platform location reduced in Iso group com -pared with control group(P<0.05), which were reversed by LM11A-31 in LM+Iso group(P<0.05).CONCLU-SION:ProNGF-p75NTR probably plays an vital role in the apoptotic pathway activation and the cognitive dysfunction in ag -ing mice exposure to isoflurane,which provides a potential target for clinical intervention.
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AIM:To study the oxidative damage and mechanism of p75 NTR receptor in retinal pigment epithelial cells (RPE).METHODS:The NTR p75 receptor was used to transfer the retinal pigment epithelium cells as the experimental group,and the non transfected retinal pigment epithelium cells were used as control group.The BrdU test detected the proliferation of two groups of cells.The rate of apoptosis in two sets of apoptosis was measured by PI/in the V-FITC double dye method.The laser microscope detects the ROS levels within the cell.The flow cytometer detected the levels of ROS,mitochondrial markers,cytochrome C expression in RPE cells.The Western blot method detected the expression level of Fas,Caspase-3,and VEGF165 in RPE cells.RESULTS:The RPE cell proliferation activity was gradually decreasing (P＜0.05) with the extension of the p75 NTR receptor transfer time in experimental group.The RPE cell proliferation activity in each transfection point was significantly lower in experimental group than in the control group (P＜0.05).The percentage of RPE apoptosis was gradually increased with the extension of transfection time in experimental group(P＜0.01).The percentage of RPE cell apoptosis in the experimental group was significantly higher than the control group (P＜0.01).ROS fluorescence was significantly better in the experimental group than the control group.Flow cytometry instrument method,according to the results of the experimental group PRE ROS levels in the cell,cytochrome C was significantly higher than control group (P ＜ 0.01),RPE cell mitochondria marker levels significantly lower than the control group (P＜0.01).The results of the Western blot method showed that the expression levels of VEGF165,Fas and Caspase-3 were significantly higher in the experimental group than in the control group (P＜0.01).CONCLUSION:The over expression of p75 NTR receptor could lead to damage of mitochondria in retinal pigment epithelium cells,but it could also promote the apoptosis reaction,eventually it led to the formation of choroidal neovascularization,so it could be speculated that p75 NTR receptor is the damage factors of retinal pigment epithelium.
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Objective To investigate the effect and mechanisms of β hydroxybutyrate (βOHB) regulation on p75NTR expression in Alzheimer's disease (AD) model cells.Methods First,cultured SH-SY5Y cells were exposed to Aβ (final concentrations:10,20,40,80 μmol/L) with or without 5 mmol/L βOHB pretreatment,and sham-treated cells were used as the control.At 24 h after treatment,the viability of cells was determined by the MTT assay.Secondly,cultured cells were divided into four groups.Cells in the Aβ group were exposed to Aβ (final concentration:20 μ mol/L)with or without 5 mmol/L βOHB pretreatment.Cells in the βOHB group were treated only with 5 mmol/L βOHB,and sham treated cells were used as the control.At 6 h and 24 h after treatment,the expression of p75NTR,HDAC1/2 mRNA and its protein expression,and p65 protein expression were measured by qRT-PCR or Western blot.Finally,the expression of p75NTR mRNA and protein was analyzed in cultured cells after silencing HDAC1 / 2 with siRNA.Results The viability of cells with 40 μmol/L or 80 μmol/L treatment was lower than that in the control group (P＜0.01),and there was a significant increase (P＜0.01) in cell viability of the βOHB intervention group,compared with the Aβ group.At 6 h or 24 h after treatment,the expression of p75NTR mRNA,its protein expression,and p65 protein expression were clearly increased in the βOHB group (P＜0.05) and markedly decreased (P＜0.01) in the Aβ group,compared with the control.Additionally,the expression of HDAC1 / 2 mRNA and protein was higher (P＜0.01) in the Aβ group at 6h or 24h after treatment and lower(P＜0.05 or P＜0.01)in the βOHB group at 6 h after treatment than in the control group.Compared with the Aβ group,there were significant increases (P＜0.01) observed in p75NTR mRNA,its protein expression,and p65 protein expression,and a notable decrease (P＜0.05) in HDAC1 / 2 mRNA and protein expression in cells of the βOHB intervention group at 6 h and 24 h after treatment.The expression of p75NTR mRNA and protein increased in HDAC1 knock-down cells compared with the control (P＜0.05).However,no difference was found in p75NTR expression in HDAC2 knock-down cells (P＞0.05).Conclusions βOHB up-regulates p75NTR expression by inhibiting HDAC1 of βOHB.It also activates p65 and prevents the decrease of cell viability.
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Objective To investigate the expression of neurotrophins receptor 75NTR in human breast cancer resistant cell line and its correlation with multidrug resistance. Methods Western blot was used to detect the expression level of p75NTR protein in various breast cancer cell lines and multidrug resistant cell lines. The over-expression vector and siRNA vector of p75NTR were constructed by gene recombination method. Western blot was used to detect the expression levels of p75NTR protein in transfected p75NTR and p75NTR - siRNA breast cancer resistant cell line MDA-MB-231/ADR;the sensitivity of MDA-MB-231 and MDA-MB-231/ADR to different chemotherapeutic anticancer drug (PTX ,ADM ,GEM ,DDP ,OXA) and the multi-drug resistance effects in over-expression and knock-down p75NTR MDA-MB-231/ADR were detected by CCK-8 assay. Results Western blot result showed that the expressions of p75NTR protein in the multidrug-resistant breast cancer cell lines MDA-MB-231/ADR and MCF-7/5-FU were higher than that of other breast cancer cell lines. Over-expression of p75NTRcan up-regulate the expression of p75NTR protein in MDA-MB-231/ADR;the CCK-8 assay indicated that over-expression of p75NTR can effectively enhance the resistance of MDA-MB-231/ADR cells to ADM,GEM and OXA. Conclusion The expression of p75NTR in breast cancer resistant cell line is higher than that of its parental cell line;over-expression of p75NTR can reduce the sensitivity to chemotherapeutic drugs and promote its multidrug resistance.
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Objective To investgate the effect of nervous growth factor(NGF) on the proliferation of the limbal stem cells(LSCs) in vitro,and the relationship bewteen expression of its receptors and cell proliferation.Methods After primary cultured,LSCs were divided into the control group and the NGF group.Selected cells cultured of 1 d,3 d and 5 d in the two groups and examined the expression of p63,TrkA,p75 with immunohistochemistry.Results The average gray scale values of expression of p63,TrkA and p75 at 1 d,3 d and 5 d in NGF group were significant decreased compared with the corresponding data in the control group(P<0.05).Pearson's correlations analysis showed that the average gray scale values of expression of TrkA and p63 were of statistically significant differences(P<0.05).Conclusion These results highlight that NGF could maintain the stem cell properties of LSCs.LSCs could exepress the NGF receptors of TrkA and p75,and the expression of TrkA showed a correlation with LSCs proliferation.
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@#Objective To investigate the effect of Santong electroacupuncture (EA) on mRNA and protein expression of p75 neurotroph-in receptor (p75NTR) in rats with spinal cord injury (SCI). Methods A total of 72 female Sprague-Dawley rats were randomly assigned to sham operation group (group A, n=8) and model group (n=64). In the model group, Allen's method was used to make SCI rats model, in which 48 survived model rats were further subdivided into model control group (group B, n=12), EA group (group C, n=12), inhibitor Nogo extra cellular peptide residues 1-40 (NEP1-40) group (group D, n=12) and EA+inhibitor NEP1-40 group (group E, n=12) according to de-sign proposal. The treatment groups were electroacupunctured on Dazhui (GV14) and Yaoyangguan (GV3), bilateral Ciliao (BL32) and Zu-sanli (ST36) with loose-tight wave, for 20 minutes every day. After 7 and 14 days of treatment, injured spinal cord tissue was extracted for detecting. The mRNA and protein expression of p75NTR was detected by real-time fluorescent quantitative PCR, in situ hybridization and Western blotting respectively. The hind limb motor function was assessed with Basso-Beattie-Bresnahan (BBB) score. Results The BBB score increased in the treatment groups compared with group B, and was higher in group E than in groups C and D (P<0.05), as well as on the 14th day than on the 7th day in all the treatment groups (t>2.623, P<0.05). The mRNA and protein expression of p75NTR in spinal cord tissues decreased in the treatment groups compared with group B (P<0.05), and no significant difference was found among the treatment groups (P>0.05). Conclusion Santong elerctroacupuncture treatment could improve the hind limb motor function, which may associate with inhibition of the mRNA and protein expression of p75NTR in rats after SCI.