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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 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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BACKGROUND: Previous studies have found that nerve growth factors play an important role in the process of wound healing, but there is less research for the low-affinity nerve growth factor receptor p75 and sortilin in fibroblasts, and no reports on whether there are differences in expression of p75 and sortilin in the scar fibroblasts and normal skin fibroblasts. OBJECTIVE: To study the expression of low-affility nerve growth factor receptor p75 and sortilin in the normal human skin fibroblasts and the human keloid fibroblasts. METHODS: The keloid fibroblasts and normal hunman skin fibroblasts were cultured in vitro, and the immortalized epithelial cells HaCaT were used as the positive control. The real-time PCR was used to detect the mRNA expression of the p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts, and western blot and immunocytochemical staining were used to detect the protein expression of p75 and sortilin. RESULTS AND CONCLUSION: The real-time PCR and western blot results showed that in the protein and mRNA levels, p75 and sortilin showed positive expression in the keloid fibroblasts and normal human skin fibroblasts, and there was no significant difference in the expression of p75 between keloid fibroblasts and normal human skin fibroblasts, and the expressions of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were significantly lower than those in HaCaT. There was no significant difference of p75 expression between keloid fibroblasts and normal human skin fibroblasts, and the expression of sortilin in the keloid fibroblasts was significantly lower than that in the normal human skin fibroblasts (P < 0.05). Immunocytochemical staining result showed that the expression of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were distributed in the membrane and cytoplasm. Precursor nerve growth factor combined with high-affinity p75 receptor could promote the apoptosis of the cells with the help of sortilin, and the expression of sortilin in the keloid fibroblasts was significantly lower than that in the normal human skin fibroblasts, which may associated with the high proliferation of the keloid fibroblasts. The results provide a new target for the prevention and treatment of pathological scars.
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It is important to understand the mechanisms that enable peripheral neurons to regenerate after nerve injury in order to identify methods of improving this regeneration. Therefore, we studied nerve regeneration and sensory impairment recovery in the cutaneous lesions of leprosy patients (LPs) before and after treatment with multidrug therapy (MDT). The skin lesion sensory test results were compared to the histopathological and immunohistochemical protein gene product (PGP) 9.5 and the p75 nerve growth factor receptors (NGFr) findings. The cutaneous neural occupation ratio (CNOR) was evaluated for both neural markers. Thermal and pain sensations were the most frequently affected functions at the first visit and the most frequently recovered functions after MDT. The presence of a high cutaneous nerve damage index did not prevent the recovery of any type of sensory function. The CNOR was calculated for each biopsy, according to the presence of PGP and NGFr-immunostained fibres and it was not significantly different before or after the MDT. We observed a variable influence of MDT in the recovery from sensory impairment in the cutaneous lesions of LPs. Nociception and cold thermosensation were the most recovered sensations. The recovery of sensation in the skin lesions appeared to be associated with subsiding inflammation rather than with the regenerative activity of nerve fibres.
Subject(s)
Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , Young Adult , Leprosy/physiopathology , Nerve Regeneration/physiology , Peripheral Nervous System Diseases/physiopathology , Receptors, Nerve Growth Factor/physiology , Immunohistochemistry , Leprostatic Agents/therapeutic use , Leprosy/drug therapy , Leprosy/pathology , Peripheral Nervous System Diseases/pathology , Sensory Thresholds , ThermosensingABSTRACT
Objective To investigate the distribution of nitric oxide synthase (NOS), nerve growth factor receptor (NGFR) and interstitial cells of Caial (ICCs) in Hirschsprung's disease (HD). Methods The distribution of NOS,NGFR and ICCs was studied by using NADPH diaphoruse histochemistry, immanohistochemistry with a monoclonal antibody to human NGFR and the specific polycloual antibody against c-kit in 8 normal controls and 10 cases of HD.Results NOS and NGFR were abundantly present in the myenteric plexus and in the nerve fibers of musculature. ICCs were intensively distributed in the surface of circular musculature and around the myenteric plexus to form a network in normal control colon. In contrast, NOS and NGFR were scarce or absent in the myenteric plexus and in the nerve fibers of musculature, while the hypertrophic nerve trunks were NGFR positive, ICCs were scarcely distributed and the network was disrupted in the aganglionic colon in HD. Conclusion These findings suggest the Involvement of NOS,NGFR and ICCs in the patbophysiology of HD.
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Objective To observe the expression and distribution the low affinity receptor p75 of nerve growth factor in the traumatic neuroma and investigate its significance and relationship with the growth of peripheral nerve and the formation of the traumatic neuroma.Methods Fifty-one cases of traumatic neuro- ma were collected and were divided into four groups according to its course,i.e,groupⅠ(less than one month),groupⅡ(1～3 months),groupⅢ(3～6 months) and groupⅣ(more than six months).Fifteen cases of normal nerve samples were harvested as the control group(n=15).Immunohistochemical studies were performed to observe the expression of p75.the expression level was detected by the computer graph analysis system.Results There was no significant expression of p75 in the normal nerve while in the four studied groups,significant expression of p75 waspositively observed.The level of p75 was weak in the early time and achieved its peak in three months,but the high level state was maintained even after six months.The one-way ANOVA statistic analysis manifests that there were no significant differences of the expression of p75 among the groupⅡ,ⅢandⅣ(P＞0.05),but the differences among the groupⅠand the latter three groups were signifi- cant (P＜0.05).p75 was mainly distributed in the Schwans cell.Conclusion p75 is expressed in the traumatic neuroma and may play an important role in the modulation progress of the formation of traumatic neu- roma with an unknown mechanism.
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Numerous studies have demonstrated interactions between the nervous/endocrine and the immune system. Increasing evidence suggests that some members of neurotrophins such as nerve growth factor (NGF) are involved in the control of immune system. Recent studies have demonstrated that the TrkA receptor, which serves as the high affinity receptor for NGF and neurotrophin-3 (NT-3), is expressed in thymic epithelial cells. In the present study, we investigated the expression of the TrkA receptor in the rat thymus from a model of thymic involution and regeneration induced by cyclophosphamide. After single dose of cyclophosphamide (150 mg/kg) was administered to Sprague-Dawley rats by intraperitoneal injection, the rats were sacrificed at 3, 7 and 14 days. The immunocytochemical characterization of the thymus was carried out using cryostat-cut sections. We found an increased expression of TrkA immunoreactivity in the thymic epithelial cells, especially in the subcapsular epithelial cells in cyclophosphamide-treated rats. The cortical epithelial cells also showed an increased expression of TrkA immunoreactivity after cyclophosphamide treatment, although the expression level was lower than that of the thymic subcapsular epithelial cells. However, there was no significant alteration of TrkA immunoreactivity in the medullary epithelial cells of the thymus from cyclophosphamide-treated rats. In general, most of these phenomena disappeared two weeks after cyclophosphamide administration and thus, the immunohistochemical features became to be similar to those of normal thymus. In conclusion, it may be speculated that TrkA receptor via interaction with their ligands provides an important signal to the thymic epithelial cells, especially to the subcapsular epithelial cells, for the thymic regeneration during recovery from acute thymic involution. Thus, our results support the proposed immunoregulatory role of neurotrophins.
Subject(s)
Animals , Rats , Cyclophosphamide , Epithelial Cells , Immune System , Injections, Intraperitoneal , Ligands , Nerve Growth Factor , Nerve Growth Factors , Rats, Sprague-Dawley , Receptor, trkA , Regeneration , Thymus GlandABSTRACT
The immunohistochemical localization of epidermal growth factor receptor (EGFR) and nerve growth factor receptor (NGFR) in the submandibular gland of rats was investigated after chronic administration of isoproterenol (IPR) or phenylephrine (PEP). The weight of submandibular gland relative to body weight increased sharply by IPR administration for 14 days and reached twice of that in control, while no significant differences were observed after PEP administration. In PTAH staining, the intensity of duct compartments in rats exposed to IPR and PEP were paler than that of controls. But small secretory granules were observed in the GCT cells of IPR administrated groups. Acini showed characteristic features of hypertrophy, decreased in number of nuclei per unit area, after IPR administration, but not after PEP. EGFR immunoreactivities were distributed mainly in the duct compartments including GCT cells, intercalated duct cells and secretory duct cells. EGFR immunoactivities were more intense after both of PEP and IPR administration than those in controls. However, EGFR immunoactivities gradually decreased after IPR administration. NGFR immunoreactivities were distributed mainly in connective tissue cells surrounding ducts, but not in duct cells. Their intensities increased in the rat with PEP administration but decreased by IPR administration. These results demonstrated that EGFR or NGFR is localized mainly in the duct cells or the cells surrounding ducts, respectively, and that both population of EGFR and NGFR immunoreactive cells are altered by PEP and IPR. The results suggest that EGF and NGF may have some physiological roles by binding with their specific receptors in the submandibular gland as well as oral cavity.
Subject(s)
Animals , Rats , Body Weight , Connective Tissue Cells , Epidermal Growth Factor , Hypertrophy , Immunohistochemistry , Isoproterenol , Mouth , Nerve Growth Factor , Phenylephrine , ErbB Receptors , Secretory Vesicles , Submandibular GlandABSTRACT
Immunohistochemical changes of epidermal growth factor receptor (EGFR) and nerve growth factor receptor (NGFR) were investigated in the rat mandibular molar and incisors after submandibular sialadenectomy. In the sham operated rat, any EGFR immunoreactivity was not observed in the teeth but NGFR immunoreactivities were observed exclusively in the periodontal ligament and ameloblasts of incisor. In the sialadenectomized rat, EGFR immunoreac-tivities were observed in the odontoblasts of the mandibular first molar, periodontal ligament cells, ameloblasts of incisor and some cells of bone marrow. NGFR immunoreactivities were more intense and widely distributed in alveolar bone, periodontal ligaments and odontoblasts of the sialadenectomized rat than in the sham operated rat. Both of EGFR and NGFR immunoreactivities gradually increased in their intensities in a time-dependent manner after submandibular sialadenectomy. The results show that expression of EGFR and NGFR in the mandibular molar and incisor is enhanced by submandibular sialadenectomy. Therefore, it is suggested that EGF and NGF derived from submandibular gland may affect to the mandibular molar and incisors by direct and/or indirect mechanism.