Protective effects of TAT-tCNTF fusion protein on SH-SY5Y cells induced by β-amyloid peptide(25-35) / 中国药理学通报

Aim To determine TAT-Tcntf penetration ability and to investigate the effects of the fusion protein on SH-SY5Y cells against toxicity induced by β-amyloid peptide 25-35(Aβ_(25-35) ).Methods The conjugate(TAT-tCNTF)of TAT(47-57)of HIV-1 and the truncated human CNTF active fragment was genetic engineered and expressed in E.Coli.Immunofluorescence was used to identify cell permeation ability across membrane.MTT assay was used to measure the survival of SH-SY5Y cells injured by Aβ_(25-35).And Hoechst 33342/PI double staining was used to observe the morphology of cell apoptosis and necrosis.LDH was measured by spectrophotometric method.Results The expression vector of pBV220-TAT-tCNTF was constructed successfully.Western blot showed the recombinant fusion protein could bind specifically with CNTF antibody.The immunofluorescence assay clearly demonstrated that TAT-tCNTF did penatrate into the cells while little rhCNTF pass across the cells.Double staining and LDH release assay demonstrated that TAT-tCNTF could promote significantly the survival of the cells.Conclusion sTAT-tCNTF with high activities and effective transmembrane ability is obtained for the first time.The fusion protein protects SH-SY5Y cells from death after Aβ25-35 exposure.

The Neuroprotective Effect of Intravitreally Injected CNTF on Rat Retinal Ganglion Cell in Optic Nerve Crush Injury Model

PURPOSE: To make an optic nerve crush injury model and to investigate the neuroprotective effect of intravitreally injected ciliary neurotrophic factor (CNTF) on rat retinal ganglion cells (RGCs) in the model. METHODS: The optic nerves of 12 Sprague-Dawley rats were crushed at 3 mm posterior to the eyeball for 1 minute using aneurysm clip (110 g). Two micrograms of CNTF in 2 micro liter of vehicle was injected intravitreally in one group (n=6) and 2 micro liter of PBS was injected in the control group (n=6) at 4, 7, and 10 days after the optic nerve injury. After 2 weeks, the retrograde labeling of the RGCs was done by the dextran tetramethylrhodamine. Twenty-four hours after the labeling, the retina was wholly mounted and the labeled RGCs were counted under the fluorescence microscope. RESULTS: The death of RGCs in this model began at 1 week and continued for 3 weeks. The number of labeled RGCs in CNTF-injected group (510+/-139/mm2) were significantly higher than that in control group (345+/-87/mm2)(p<0.05). CONCLUSIONS: The optic nerve crush injury model was established by use of aneurysm clip. In this model, the intravitreally injected CNTF had a neuroprotective effect on the rat RGCs.

Research about the expression changes of CNTF and CGRP in the rabbits' model of neuroma-in-continuity / 中华显微外科杂志

Objective In view of being short of the mammalian model in neuroma-in-continuity,the experiment injured the part of peroneal nerve to the formation of the neuroma-in-continuity and was applied to the foundation of farther research.Methods Twelve New Zeland rabbits were selected as experimental sub- jects randomly.One lateral peroneal nerves of twelve rabbits were resected,the damaged nervous tissues' slice were showed to the typical pathological changes of neuroma by the stain of HE,luxol fast blue after six weeks. As compared with the health sides of six model rabbits,the methods of real-time PCR and Western blot were used to evaluate the expression of CNTF,CGRP mRNA and protein in injured nerves and L_7、S_1 dorsal root ganglions respectively.Results The injured nerve formed the typical pathological changes of neuroma at six weeks.Compared with hea|thg side the expression of CNTF mRNA and protein was down-graded at the lateral of neuroma(P＜0.05),and the expression of CGRP mRNA and protein was up-graded(P＜0.05).Con- clusion The method of partly injuring the peroneal nerve could effectively set up the model of the neuroma-in- continuity,furthermore,resulted to the expression changes of the CNTF,CGRP mRNA and protein.

Protective effects of TAT-tCNTF fusion protein on SH-SY5Y cells induced by ?-amyloid peptide(25-35) / 中国药理学通报

Aim To determine TAT-tCNTF penetration ability and to investigate the effects of the fusion protein on SH-SY5Y cells against toxicity induced by ?-amyloid peptide 25-35(A?25-35 ).Methods The conjugate(TAT-tCNTF)of TAT(47-57)of HIV-1 and the truncated human CNTF active fragment was genetic engineered and expressed in E.Coli.Immunofluorescence was used to identify cell permeation ability across membrane.MTT assay was used to measure the survival of SH-SY5Y cells injured by A?25-35.And Hoechst 33342/PI double staining was used to observe the morphology of cell apoptosis and necrosis.LDH was measured by spectrophotometric method.Results The expression vector of pBV220-TAT-tCNTF was constructed successfully.Western blot showed the recombinant fusion protein could bind specifically with CNTF antibody.The immunofluorescence assay clearly demonstrated that TAT-tCNTF did penatrate into the cells while little rhCNTF pass across the cells.Double staining and LDH release assay demonstrated that TAT-tCNTF could promote significantly the survival of the cells.Conclusions TAT-tCNTF with high activities and effective transmembrane ability is obtained for the first time.The fusion protein protects SH-SY5Y cells from death after A?25-35 exposure.