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Objective@#To construct and identify a mouse model with conditional knockout (cKO) of p75 neurotrophin receptor (p75NTR-cKO) gene in epidermis cells by Cre-loxP system.@*Methods@#Five p75NTRflox/flox transgenic C57BL/6J mice (aged 6-8 weeks, male and female unlimited, the age and sex of mice used for reproduction were the same below) and five keratin 14 promotor-driven (KRT14-) Cre+ /- transgenic C57BL/6J mice were bred and hybridized via Cre-loxP system. Five p75NTRflox/+ ·KRT14-Cre+ /- mice selected from the first generation of mice were mated with five p75NTRflox/flox mice to obtain the second generation hybrids. After the second generation mice were born 20-25 days, the parts of the mice tail were cut off to identify the genotype by polymerase chain reaction method. Four p75NTR gene complete cKO mice (6 weeks old) and 4 wild-type mice (6 weeks old) were selected and sacrificed respectively. The abdominal skin tissue and brain tissue were excised to observe the expression of p75NTR in the two tissue of two types of mice by immunohistochemical staining. The abdominal skin tissue of two types of mice was obtained to observe the histomorphological changes by hematoxylin and eosin staining.@*Results@#(1) Twenty second generation mice were bred. The genotype of 4 mice was p75NTRflox/flox·KRT14-Cre+ /-(p75NTR-/-), i. e. p75NTR gene complete cKO mice; the genotype of 5 mice was p75NTRflox/+ ·KRT14-Cre+ /-, i. e. p75NTR gene partial cKO mice; the genotype of 5 mice was p75NTRflox/flox·KRT14-Cre-/-, and that of 6 mice was p75NTRflox/+ ·KRT14-Cre-/-, all of which were wild-type mice. (2) The expression of p75NTR was negative in skin epidermis tissue of p75NTR gene complete cKO mice, while numerous p75NTR positive expression was observed in skin epidermis tissue of wild-type mice. Abundant p75NTR positive expression was observed in brain tissue of both wild-type mice and p75NTR gene complete cKO mice. (3) There was no abnormal growth of skin epidermis tissue in both wild-type mice and p75NTR gene complete cKO mice, with intact hair follicle structure.@*Conclusions@#Applying Cre-loxP system can successfully construct a p75NTR-cKO mice model in epidermis cells without obvious changes in skin histomorphology.
ABSTRACT
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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ObjectiveTo observe the expression of nerve growth factor(NGF)and its receptors (TrkANGFRand P75NTR) in hepatocytes and to explore the biological effects of exogenous P75NTR protein on hepatocytes.MethodsL02 hepatocytes were cultured in vitro.The expression of NGF,TrkANNGFR and P75NTR in L02 cells were assessed by immunocytochemistry and fluorescent quantitation polymerase chain reaction (PCR). The effects on L02 cell proliferation by exogenous P75NTR,NGF,NGF+ P75NTR,anti-TrkANGFR and anti-P75NTR were detected by XTT assay.The effect of exogenous P75NTR on L02 cell apoptosis was measured by flow cytometry (Annexin V/PI) and the effect of exogenous P75NTR on L02 cell cycle was determined by flow cytometry (PI).ResultsL02 cell proliferation was promoted by P75NTR and in dose-dependent manner.The A value of NGF group and NGF+ P75NTR group was 0.4916±0.0565 and 0.5839 ± 0.0733,respectively,and there was statistical significance compared with control group (0.3601 ± 0.0310,P＜0.05).The A value ot anti-TrkANGFR group was 0.2689±0.0229,and there was statistical significance compared with control group (P=0.003).The A value of anti P75NTR was 0.3524 ± 0.0312,and there was no statistical significance compared with control group (P=1.000). Exogenous P75NTR had anti-apoptosis effect on L02 cells,the antiapoptosis effect was strongest when 100 ng/ml P75NTR was added and the expression quantity was 3.70 ±0.26.However there was no statistical significant compared with control group (4.10 ± 0.62,P=1.000).P75NTR affected the cell cycle S phase of L02 cells and in dose-dependent manner,which was inverted U shaped curve.The effect was strongest when the concentration was 100 ng/ml (25.60 ±0.40) and there was statistical significance compared with control group (20.10 ±1.00,P=0.000).Exogenous NGF,P75NTR and NGF + P75NTR up regulated the gene expression of NGFmRNA,TrkANGFRmRNA and P75NTR mRNA in L02 cells and there was statistical significance compared with control group (P＜0.05).There was no significant difference in the gene expression of NGFmRNA,TrkANGFFR mRNA and P75NTR mRNA between anti-TrkANGFR,anti-P75NTR groUp and control group (P＞0.05).Conclusion NGF and its receptors TrkANGFR and P75NTR were expressed in L02 cells.The appropriate dose of exogenous P75NTP protein promoted L02 cells proliferation via TrkANGFR/P75NTR heterodimer signal pathway.
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Objective To investigate the effects of artemin and its receptor GFRα - 3 on the invasion and metastasis of pancreatic cancer cells. Methods Human pancreatic cancer cell line MIA PaCa -2 was used in this study. Transwell cell culture chamber assay in vitro was used to detect the ability of invasion and metastasis of MIA PaCa -2 cells. The influence of artemin and GFRα -3 on the protein expression of MMP-2 and E-cadherin was investigated by Western blot and quantitative real time polymerase chain reaction-analyses (Q-RT-PCR). Results As the increase of artemin and GFRα -3, the invasion and metastasis of MIA PaCa- 2 was markedly increased [ 150 ng / ml concentration: Artemin group: 107.4 ± 11.4;GFRα3 group:94. 4 ± 9. 3 ;control group:34. 6 ± 7. 3, P < 0. 01 ]. With 150 ng / ml artemin and GFR-3,the synthesis of MMP-2 in MIA PaCa 2 cells was significantly increased than that in control group[ Artemin grou: (2. 17 ± 0. 05 ) × 108; GFRα3 group: (2. 02 ± 0. 03 ) × 108; control group: ( 1.02 ± 0. 02 ) × 108, t =6. 35,7. 32 ], while E-cadherin significantly decreased [ Artemin group: ( 0. 65 ± 0. 04 ) × 108; GFRα3 group: (0. 74 ± 0. 01 ) × 108; control group: ( 1. 36 ± 0. 03 ) × 108, t = 4. 27,5.61 ], the difference was statistically significant ( P <0. 01 ). Conclusions Artemin and its receptor GFRα3 could promote pancreatic cancer cell invasion and metastasis. This effect may be related to the up-regulated expression of MMP-2 and down regulated expression of E-cadherin.
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Nerve growth factor (NGF) and its high affinity receptor tyrosine kinase (TrkA)are indis-pensable for the development of nervous system. Latest researches reveal that over-expression of NGF and TrkA is related to better outcome in neuroblastoma and medulloblastoma. While in glioma, the over-expression and nuclear translocation of NGF and TrkA are indicators of malignant degree. Study of the expression and sub-cel-lular cocalization of NGF and TrkA can provide new way for the diagnosis and therapy of nervous system tumors.
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Objective To investigate the effects of high-affinity tyrosine kinase receptors TrkA, TrkC and the low-affinity neurotrophin receptor p75 (p75~ NTR) protein in laser-induced retinal injury. Methods The Wistar rats were anesthetized and exposed to frequency doubling Neodymium:Yttrium,aluminum garnet(ND:YAG,?=532nm) laser for 100 plus per eye, which were sacrificed on 12 hours,1,3,7,14 and 28 days after laser exposure and eyeball was taken out. We investigated retinal histology by hematoxylin and eosin staining, and the expression of TrkA, TrkC and p75~ NTR protein was studied by means of immunohistochemistry. Results Immunohistochemistry results showed a differential distribution for these three neurotrophin receptors in the laser injuried retina. TrkA was enhanced in nerve fiber layer (NFL), ganglion cell layer(GCL) and inner nuclear layer(INL) ,which is the most nuclei of the ganglion cells, proximal M?ller cell end feet and a little cells in the innermost part of the INL, its peak of up-regulation was after day 1-3, after day 28 there was sustained. Whereas TrkC and p75~ NTR expression was enhanced in the outer nuclear layer(ONL), which is distal M?ller cell processes, TrkC up-regulated after 12 hours, its peak was on day 1-3, after day 28 there was sustained, p75~ NTR up-regulated after 24 hours, its peak was on day 3, on day 14-28 there was sustained in the GCL, which is proximal M?ller cell processes. Conclusion The expression of TrkA, TrkC and p75~ NTR participated in the course of laser injuried retinal pathology.