Effects of CNTF Antibody Block to the Bone Marrow Mesenchymal Stem Cells Transplantated by Abdominal Aortic on the Downstream Signaling Pathways STAT3/Caspase-9 in Spinal Cord Ischemic Reperfusion Injuried Rats / 昆明医科大学学报

Objective To investigate the functional recovery and the effect of CNTF antibody block to the bone marrow mesenchymal stem cells (BMSCs) transplantated by Abdominal aortic on the downstream signaling pathways STAT3/Caspase-9 in spinal cord ischemic reperfusion injury in rats.Methods Adult female SD ratswere assigned randomly to 4 groups. The neurological functional status of the animals was assessed with BBB scores, the Motor evoked potentials (MEP) and Cortical somatosensory evoked potentials (CSEP).The IHC were used to detect the the expressional changes of CNTF, then Western blot and RT-PCR were used to detect the expressional changes of STAT3、p-STAT3、CNTF and Caspase-9 in the ischemic segments of spinal cord.Results Compared with the sham group, in the SCIRI rats, the BBB scores were markedly decreased at all time points (P＜0.01), the latency and the amplitude of MEP and CSEP was longer and lower at 14 d post operation (P＜0.01), and this change was the most significant in the control group the second in the CNTF block group, and the last in the transplantation group, Resutts between each two groups were statistically significant (P＜0.05). At 7 d post operation, compared with the sham group, the immunoreactive products of CNTF were decreased in the CNTF block group (P＜0.05), but were increased (P＜0.05) in the control group and the transplantation group (P＜0.05), and results in the transplantation group were higher than in the control group (P＜0.05). At 7 d post operation, compared with the sham group, the m RNA and protein level of CNTF、STAT3、 p-STAT3 were decreased obviously in CNTF block group (P＜0.05), the levels were increased in the control group and the transplantation group (P＜0.05), and the levels in the transplantation group were higher than that in the control group (P＜0.05); but the m RNA and protein level of Caspase-9 were only decreased in the transplantation group (P＜0.05), the level was increased in the CNTF block group and the control groups (P＜0.05), and the level in the CNTF block group was more significantly increased than that in the control group (P＜0.05). At 14 d post operation, in CNTF block group, the m RNA and protein level of CNTF、STAT3、p-STAT3 were significantly higher than that in the sham group and the control group (P＜0.05), and the m RNA and protein level of caspase-9 was higher than that in the sham group (P＜0.05), but lower than that in the control group (P＜0.05), there were not statistically different in the level of each factor compared with transplantation group (P＞0.05). Conclusions BMSCs, transplanted by the abdominal aorta, can promote the expression of CNTF in the injuried spinal cord and significantly improve the hind limb function recovery by CNTF-mediated signaling pathway downstream of STAT3/Caspase-9 SCIRI in rats, but the role of BMSCs can be weakening by CNTF block that inhibited STAT3/Caspase-9 signaling pathway.

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.

Activity Study of Ciliary Neurotrophic Factor(CNTF)Mutant / 中国生物工程杂志

To study still further the activity of CNTF mutant designed by computer molecular modling,the methods of dissociated cultures of chick dorsal root ganglion、TF-1 prolification and the normal mice'weight loss tests weve used.The results indicated that the mutant protein promoted the survival of dorsal root ganglion、induced TF-1 prolification and made the normal mice lose weight,decrease appetite and reduce fat index.The weight loss effect was dependant with its administration dosage,ED50 was 150.986?g/kg/d.To TF-1,the specific activity reached 2.0?106U/mg against international reference reagent.In a word,CNTF mutant had excel bioactivity.So it provided clues for its development and application.

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.

Polymorphism of the Ciliary Neurotrophic Factor (CNTF) Gene in Korean / 체질인류학회지

Ciliary neurotrophic factor (CNTF) is one of the important factors in neuronal survival. Mutation in CNTF gene was found first in Japanese. We analyzed the CNTF genotype of 187 healthy volunteer of Korean, and compared the frequency difference in CNTF gene mutation among Korean, Japanese, and other nations or race. The number of normal homozygote was 138 (73.8%), mutant homozygote 47 (25.1%), and mutant homozygote was 2 (1.1%). The mutated allele frequency of CNTF gene in Korean was 13.6%, which was not significantly different from that of other nations or race (10~20%).

Immunohistochemical Localization of Nerve Growth Factor, Glial Fibrillary Acidic Protein and Ciliary Neurotrophic Factor in Mesencephalon, Rhombencephalon, and Spinal Cord of Developing Mongolian Gerbil

The distribution of the nerve growth factor (NGF), the glial fibrillary acidic protein (GFAP) and the ciliary neurotrohic factor (CNTF) was performed in coronal sections of the mesencephalon, rhombencephalon and spinal cord in the developing Mongolian gerbils. Generally, NGF specifically recognizes neurons with the NGF receptor, whereas GFAP does the glia, and CNTF does the motor neurons. The receptor expression was examined separately in gerbils between embryonic days 15 (E15) and postnatal weeks 3 (PNW 3). The NGF-IR was first observed in the spinal cord at E21, which might be related to the maturation. The GFAP reactivity was peaked at the postnatal days 2 (PND2), while the highest CNTF-reaction was expressed at PNW 2. The GFAP stains were observed in the aqueduct and the spinal cord, which appeared to project laterally at E19. The CNTF was observed only after the birth and found in both the neurons and neuroglia of the substantia nigra, mesencephalon, cerebellum and the spinal cord from PND1 to PNW3. These results suggest that NGF, GFAP and CNTF are important for the development of the neurons and the neuroglia in the central nervous system at the late prenatal and postnatal stages.

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.

The influence of chronic stress on the level of CNTF and CNTF mRNA in hippocampal neurons of rats / 中国药理学通报

AIM To study the influence of chronic stress on the level of CNTF and CNTF mRNA in hippocampal neurons of rats. METHODS The chronic stress model was established by chronic unpredictable mild stress, openfield test was performed to detect the behavior of rats. Immunohistochemistry and in situ hybridization were used to observe the level of CNTF and CNTF mRNA. RESULTS Compared to control group, the CNTF like immunoreactivity and signals of CNTF mRNA in situ hybridization in the hippocampal neurons of chronic stress group were significantly decreased. CONCLUSION These results show that chronic stress can significantly decrease the level of CNTF and CNTF mRNA in the hippocampal neurons of rats.

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.

CNTF GENE EXPRESSION AND EXPRESSIVE VARIETY DURING POSTNATAL DEVELOPMENT IN SPINAL CORD OF RAT / 解剖学报

Objective To observe CNTF gene expression and expressive variety during postnatal development in rat spinal cord. Methods The spatial expression of CNTF mRNA in spinal cord was examined by in situ hybridization with dig\|CNTF cDNA probe.Reverse transcription\|polymerse chain reaction(RT\|PCR),as hemi\|quantitative method was used to investigate the expressive variety of CNTF mRNA levels in spinal cord during postnatal development. Results Hybridized signals of CNTF mRNA was found only in part of glial cells on the peripheral region of the ventral and lateral white funiculi of spinal cord,but could not be detected in the gray matter of spinal cord.The expression of CNTF mRNA in spinal cord appeared with a lower level on the first postnatal day.It increased significantly at postnatal 15th days,the expression of CNTF mRNA reached the peak at 30th days,and it began to decrease on the 60th days. Conclusion This study indicated that CNTF mRNA might be expressed in part of glial cells in the white matter.There was expression in spinal cord on the 1st day after birth and the expression levels of CNTF mRNA possessed the changeable character during spinal cord development.