Study on Dose-effect Relationship and Mechanism of Protective Effects of Total Asiaticoside on Gastrointestinal Motility and Enteric Nervous System in Aged Functional Dyspepsia Model Rats / 中国药房

OBJECTIVE：To explo re the dose-effect relationship and mechanism of protective effects of total asiaticoside （TA） on gastrointestinal motility and enteric nervous system （ENS）in aged functional dyspepsia （FD）model rats. METHODS ：Aged male SD rats of 16 months old were randomly divided into blank control group ，model group ，TA low dose ，medium dose and high dose groups （15，30，60 mg/kg），with 8 rats in each group. FD model was established by tail-stimulation combined with irregular diet for 4 weeks. The next day after modeling ，administration groups were given relevant doses of TA solution intragastrically ； control group and model group were given constant volume of normal saline intragastrically ，once a day ，for consecutive 15 d. Gastric emptying rate and small intestinal propulsion rate of rats were examined. ELISA were used to detect serum contents of MTL and VIP. Immunofluorescence and immunohistochemistry were proposed to measure the expression of ENS marker （S100β and GDNF）in gastric antrum tissue. The protein expression of S 100β，GFAP，PGP9.5，GDNF，p-MEK and p-ERK 1/2 in gastric antrum tissue were measured by Western blotting assay. RESULTS ：Compared with blank control group ，gastric emptying rate and small intestinal propulsion rate ，serum MTL content and protein expression of PGP 9.5 in gastric antrum tissue of model and TA low，medium dose group were decreased significantly ，while serum VIP content ，protein expressions of S 100β，GFAP，GDNF， p-MEK and p-ERK 1/2 in gastric tissue were increased significantly （P＜0.05）. Compared with model group ，gastric emptying rate and small intestinal propulsion rate of TA groups were increased significantly （P＜0.05）；except for GFAP protein in TA low dose group（P＞0.05），the serum MTL content and the expression of PGP 9.5 protein in gastric antrum tissue of rats in TA groups were increased significantly ，while serum VIP content ，protein expression of S 100β，GFAP，GDNF，p-MEK and p-ERK 1/2 in gastric antrum tissue were decreased significantly （P＜0.05）. Some or most of the content of gastrointestinal motility indexes and related factor protein expression were significantly different among TA groups （P＜0.05），and the indexes in TA high dose group could recover to the levels which were not significantly different with blank control group （P＞0.05）. CONCLUSIONS ：TA can dose-dependently improve the gastrointestinal motility deficiency and ENS dysfunction in aged FD model rats ，especially in high dose（60 mg/kg）of TA group. Its mechanism may be related with promoting the release of endogenous MTL ，inhibiting the secretion of VIP ，expression of GDNF and the activation of downstream signaling pathway ，and promoting the repair of ENS and intestinal neurons.

Hypericum perforatum chronic treatment affects cognitive parameters and brain neurotrophic factor levels

Objective: To evaluate the effects of Hypericum perforatum (hypericum) on cognitive behavior and neurotrophic factor levels in the brain of male and female rats. Methods: Male and female Wistar rats were treated with hypericum or water during 28 days by gavage. The animals were then subjected to the open-field test, novel object recognition and step-down inhibitory avoidance test. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell-line derived neurotrophic factor (GDNF) levels were evaluated in the hippocampus and frontal cortex. Results: Hypericum impaired the acquisition of short- and long-term aversive memory in male rats, evaluated in the inhibitory avoidance test. Female rats had no immediate memory acquisition and decreased short-term memory acquisition in the inhibitory avoidance test. Hypericum also decreased the recognition index of male rats in the object recognition test. Female rats did not recognize the new object in either the short-term or the long-term memory tasks. Hypericum decreased BDNF in the hippocampus of male and female rats. Hypericum also decreased NGF in the hippocampus of female rats. Conclusions: The long-term administration of hypericum appears to cause significant cognitive impairment in rats, possibly through a reduction in the levels of neurotrophic factors. This effect was more expressive in females than in males.

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

The temporal profile of astrocytes and Jak-STAT signal pathway after spinal ischemia and reperfusion injury in rabbits / 中华胸心血管外科杂志

Objective To observe the evolution of astrocytes,GDNF,BDNF and Jak-STAT signal pathway after spinal cord ischemia-reperfusion injury in rabbits.Methods Spinal cord ischemia was induced by means of balloon occlusion of the infrarenal aorta for 22 minutes in 54 male New Zealand white rabbits.We assigned rabbits to 9 groups (n =6),one sham group,eight operation groups.The operation process in the sham group was the same as the operation group except the ischemia reperfusion of the spinal cord.At 0 h,1 h,2 h,3 h,8 h,24 h,48 h and 72 h after reperfusion,animals were sarcrificed and the spinal cord was removed for histologic,immunohistochemical study and western blotting.Results Normal neurons were decreased with the extension of reperfusion time.Levels of GFAP increased at 3 h and reached a peak at 48 h after reperfusion.GDNF was increased reaching two peaks after injury,the first peak was at 3 h,the second was at 72 h.BDNF level was increased and peaked at 24 h after reperfusion.The expression of p-STAT3 showed a biphasic pattern which peaked at 1h and 48 h.GFAP,GDNF,BDNF were rare and the level of p-STAT3 could be neglected in sham group.Conclusion Spinal cord ischemia-reperfusion injury could induce the activation of astrocytes,the expression of GDNF,BDNF and the activation of JakSTAT signal pathway.They showed different expression rules in this study.

Effect of water temperature on the recovery of spinal cord injury in zebrafish / 中国实验动物学报

Objective Using zebrafish to analyze the effect of water temperature on the recovery of spinal cord in-jury. To detect the cell proliferation and changes of gene expression at the injury site during the process of recovery. Meth-ods Surgical operation was performed to induce spinal cord injury ( SCI) on adult fish. Water at a series of temperature was applied to culture the fish. Swimming ability was adopted to observe the recovery of spinal cord injury following surger?y. Vibration sections and immunohistochemistry were performed to observe the cell number post SCI at different stages. The changes of gdnf and nos gene expression were determined by real?time PCR. Results The water temperature changes from 28℃ to 32℃ did not affect the swimming ability of non?injured and sham?injured fish ( P>0. 05 ) . The swimming ability recovered mostly in 8 weeks post spinal cord injury. At 32℃, the swimming ability recovered faster than at 28℃ or at 30℃(P<0. 05). The cell proliferation increased obviously following spinal cord injury (P<0. 05). The proliferation of cells surrounding the spinal cord in jury was more extensive in SCI fishes incubated in 32℃ water than in 28℃ or 30℃ water ( P<0. 05). Real?time PCR assay showed that gdnf was up?regulated in all groups post SCI at 24 h, and 7 and 14 days (P<0. 05). The nos expression was up?regulated in all groups following SCI in 24 h (P<0. 05) and 7 days. There was no sig?nificant difference between the SCI group and sham?injury group (P<0. 05), while after 14 days, the expression of nos was reduced in the SCI group compared with the sham?injury group (P<0. 05). Conclusions A slight increase of incu?bating water temperature can accelerate the recovery of spinal cord injury in zebrafish.

The expression of GDNF and GFRα1 in childhood nephroblastoma and its clinical significance / 天津医药

Objective To investigate the expressions of GDNF and GFRα1 in childhood nephroblastoma, and therefore its clinical significance. Methods The expression levels of GDNF and GFRα1 were examined by immunohistochemistry of 30 parrafin samples from nephroblastoma section as well as 16 parrafin samples of peritumor renal tissue. Results The pos?itive expression rate of GDNF and GFRα1 in nephroblastoma were higher than those in normal renal tissue(66.7%vs 6.3%;63.3%vs 6.3%). GDNF and GFRα1 were strongly expressed in epithelium and embryo bud, but weakly expressed in lobus intermedius of nephroblastoma. There was no correlation between the expressions of GDNF and GFRα1 with histological types, clinical stage and metastasis of nephroblastoma of patients. Conclusion High expression of GDNF and GFRα1 in childhood nephroblastoma might indicate its role in tumor development.

Roles of panaxtrial saponins in cognition and memory of diabetic rat and in activity of astrocytes / 天津医药

Objective To explore protective effects of panaxtrial saponins (PTS) on cognition and memory of diabetic rats and to reveal its mechanism by which might involve regulating activity of astrocytes. Methods SD rats (n=24) were ran?domly assigned into control, diabetic and PTS-treated groups (n=8 in each group). Rat diabetic model was induced through streptozotocin injection intraperitoneally. Rats in control group were native rats, and rats in PTS-treated group were diabetic rats that were administered with PTS. Body weight and blood glucose were monitored through the experiments. Three months later, state of cognition was examined by methods of water maze. Hippocampal astrocyte morphology were detected by immu?nohistochemistry, and the expression of glial cell line-derived neurotrophic factor (GDNF) and glial fibrillary acidic protein (GFAP) in hippocampus were revealed by Western blot. Results Compared with control group, diabetic group showed cog?nitive dysfunction, atrophic astrocyte soma, shrinked astrocyte processes, and down-regulation of hippocampal GFAP and GDNF (P<0.05). Compared with diabetic group, PTS-treated group exhibited improved cognition and morphology of hippo?campal astrocyte, and reversed expression of GFAP and GDNF in diabetic hippocampus (P<0.05). Conclusion PTS re?versed astrocytic reactivity as well as expression of GDNF and GFAP in diabetic hippocampus and ameliorated diabetic cog?nitive dysfunction.

Regeneration and repair after ChABC,GDNF and Nogo-A Ab combination treatment in experimental spinal cord injury model / 西安交通大学学报(医学版)

Objective To investigate regeneration and repair effect after ChABC,GDNF and Nogo-A Ab combination treatment for experimental spinal cord injury model.Methods Rat (T7-8 )complete spinal cord injury crosscutting animal model was established.The SD rats were randomly divided into 6 groups:normal group, sham operation group,simple transection group,A (ChABC)group,G (GDNF)group,N (Nogo-A antibody) group,and AGN (ChABC+GDNF+Nogo-A antibody)group.At 24 w after spinal cord injury,BDA tracer,NF-200,GAP-43,and GFAP immunohistochemistry were evaluated.Results BDA tracer of A group,G group and N group showed dye light,the proximal end of damaged zone showed the blue tracer particles,while damaged zone showed few blue regenerated nerve fibers.AGN group showed visible blue nerve fibers through the damaged zone and the distal segment in the damaged zone;the central zone of injury vacuolar degeneration showed the blue dyed fibers.NF-200 immunohistochemical staining showed NF-positive staining in A group,AGN was stronger than that in control group and simple transection group (P 0.05 ).SEP wave was detected in control group and AGN group,while the latency time was longer in AGN group than in control group.Conclusion ChABC,GDNF,and anti-Nogo-A antibody used alone or in combination can improve spinal cord injury and nerve cell function,and the joint application could improve regeneration after spinal cord injury than any monotherapy.

Experimental study on expression of TH and GDNF bi-gene of lentivirus by improved Tet-On system for a rat model of Parkinson’ s disease / 中国药理学通报

Aim To investigate TH and GDNF genes expression and regulation of lentivirus ( Lv-TH-GDNF ) based on improved Tet-On system and the effect of the Lv-TH-GDNF intrastriatal transfer on a rat Parkinson’ s disease( PD) model. Methods 1. HeLa cells were infected by obtained Lv-TH-GDNF and rtTA2 s-M2 vi-rus. The expression of tyrosine hydroxylase ( TH ) and glial cell line-derived neurotrophic factor( GDNF) genes was induced by doxycycline( Dox) which was examined by Western blot. 2. The Lv-TH-GDNF together with rtTA2 s-M2 viruses were injected into lesion-side stria-tum of a rat PD model, and the expression of GDNF and TH genes was induced by Dox. Then, the effects of Lv-TH-GDNF were evaluated by the apomorphine-induced rotational behavior, the number of dopaminer-gic neurons in substantia nigra,DA and DOPAC levels in the lesion-side striatum. In addition, Western blot was performed to check the expression of TH and GD-NF genes in the transplanted striatum. Results 1. In vitro studies on HeLa cells, Western blot showed clear protein bands of TH and GDNF in the Dox-positive group, but not in the Dox-negative group. 2. In vivo experiments in animals, the results showed that, 4 weeks after transplantation, the apomorphine-induced turning effect was significantly improved ( P<0 . 01 ) , the number of TH-positive cells in the lesion-side sub-stantia nigra pars compacta as well as the content of DA and DOPAC, the protein level of GDNF and TH genes in the lesion-side striatum was significantly in-creased ( P<0 . 01 ) , each of which was only in Lv-TH-GDNF+rtTA2 s-M2+Dox-treated rats as compared with PBS-treated rats. Conclusion The expression of TH and GDNF genes in Lv-TH-GDNF based on im-proved Tet-On system is effectively regulated by tetra-cycline antibiotics without basal activity in vitro, and the intrastriatal transfer of which has certain therapeutic effect on PD rats.

Naringin: A Protector of the Nigrostriatal Dopaminergic Projection

Parkinson's disease is the second most common neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons and a biochemical reduction of striatal dopamine levels. Despite the lack of fully understanding of the etiology of Parkinson's disease, accumulating evidences suggest that Parkinson's disease may be caused by the insufficient support of neurotrophic factors, and by microglial activation, resident immune cells in the brain. Naringin, a major flavonone glycoside in grapefruits and citrus fruits, is considered as a protective agent against neurodegenerative diseases because it can induce not only anti-oxidant effects but also neuroprotective effects by the activation of anti-apoptotic pathways and the induction of neurotrophic factors such as brain-derived neurotrophic factor and vascular endothelial growth factor. We have recently reported that naringin has neuroprotective effects in a neurotoxin model of Parkinson's disease. Our observations show that intraperitoneal injection of naringin induces increases in glial cell line-derived neurotrophic factor expression and mammalian target of rapamycin complex 1 activity in dopaminergic neurons of rat brains with anti-inflammatory effects. Moreover, the production of glial cell line-derived neurotrophic factor by naringin treatment contributes to the protection of the nigrostriatal dopaminergic projection in a neurotoxin model of Parkinson's disease. Although the effects of naringin on the nigrostriatal dopaminergic system in human brains are largely unknown, these results suggest that naringin may be a beneficial natural product for the prevention of dopaminergic degeneration in the adult brain.

Effect of subarachnoid transplantation of bone marrow origin stem cell united with TCM on the gilial cell-line derived neurotrophic factor in brain of cerebal ischemai rats / 国际中医中药杂志

Objective Observe the influence on the Gilial cell-line derived neurotrophic factor (GDNF) after treatment stroke rats with Bone marrow mesenchymal stem cells (BMSCs) or Bone marrow mononuclear cells (BMNCs)combined with traditional Chinese medicine (TCM).Methods Separating and cultivating BMSCs and BMNCs were.140 Wistar rats were divided into 7 groups randomly,normal group,pretended surgery group,model group,BMNC group,BMNC+TCM group,BMSC group,BMSC+TCM group.The other five groups were performed for 2 hours middle cerebral arterial occlusion (MCAO) except normal group and pretended surgery group.Intervention methods in each group after 24 hours of MCAO:model group:Subarachnoid injection of 100 μl 0.01M PBS,BMNC group and BMNC + TCM group,Subarachnoid injection of 2 × 107 BMNCS,BMSC group and BMSC+TCM group,Subarachnoid injection of 2 × 107 BMSCS,BMNC +TCM group and BMSC+TCM group were treated united with TCM on the transplantation day (po.Qd.).GDNF level in all groups' rat brain were analyzed by Enzyme-linked immuno sorbent assay (ELISA) method at the 4th day and the 28th day after transplantation.Results The GDNF level of model group [(62.60±4.05) pg/ml] is higher than normal group's [(53.46 ± 3.91)pg/ml] at the 28th day (P＜ 0.05).The GDNF levels of BMNC group [(194.21 ±39.56)pg/ml,(67.70±4.73)pg/ml] and BMSC group [(169.83±28.84)pg/ml,(82.66±32.23)pg/ml] are higher than model group's at the 4th and 28th day(P＜0.05).The GDNF level of BMSC group is higher than BMNC group's at the 28th day(P＜0.05).The GDNF levels of group BMNC+TCM group[(560.61 ± 194.84) pg/ml,(265.83 ±93.58) pg/ml and BMSC+TCM group[(370.93 ±46.19) pg/ml,(247.34±98.02)pg/ml] are higher significantly than BMNC group's or BMSC group's at the 4th and 28th day(P＜0.05).At the 4th day the GDNF level of the BMNC+TCM group is higher than BMSC+TCM group's(P＜0.05).Conclusion Subarachnoid transplantation of BMNCs or BMSCs will increase the GDNF level in brain of MCAO rats.The transplantantion combined with TCM can inprove the capability of the enhance.That reflect the advantage of transplantantion bone marrow origin stem cell united with TCM.

Medullary Sponge Kidney Detected in the Pediatric Age

PURPOSE: Medullary sponge kidney (MSK) is a rare congenital disease characterized by diffuse ectasia or dilatation of precalyceal collecting tubules. MSK incidence and prevalence in the general population is uncertain and only a few patients are reported especially in the pediatric age. There has been increasing reports of patients with MSK who have other malformative disorders. Also several case reports concerning about etiological association of some genes. METHODS: Collaborative study through nation-wide survey was done to investigate the incidence and etiological association of some genes such as GDNF gene, ATP6V1B1, ATP6V0A4 gene in developing MSK in Korean children. RESULTS: Four cases of MSK who have various other malformative disorders were collected. There are no mutations of GDNF gene, ATP6V1B1, ATP6V0A4 gene in all patients. CONCLUSION: MSK is one of the very rare diseases in pediatric age. The etiological association of GDNF gene , ATP6V1B1, ATP6V0A4 gene in developing MSK in Korean children is not proved.

Effect of GDNF on pancreatic cancer cell proliferation and chemotaxis / 中华肝胆外科杂志

Objective To investigate the effect of GDNF on pancreatic cancer cell proliferation and chemotaxis.Methods The cell counting, MTT and flow cytometry were employed to investigate whether the neurotrophic factor GDNF can stimulate the proliferation of pancreatic carcinoma cells.Meanwhile, the trans-well invasion chamber was used to observe the chemotactic effect of GDNF on pancreatic cancer cells.Results The cells were exposed to incremental concentrations of human re-combinant GDNF (0-120 ng/mL).We found that the proliferation of pancreatic cancer cells was stim-ulated by GDNF in a dose-dependent manner and the number of cells in "S" phenotype was increased;The count of cells was increased by GDNF in a dose-dependent manner.Conclusion GDNF can stimu-late the proliferation and invasion of pancreatic cancer cells in a dose-dependent manner.

Ape1/Ref-1 Stimulates GDNF/GFR alpha1-mediated Downstream Signaling and Neuroblastoma Proliferation

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor alpha1 (GFR alpha1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRalpha1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFR alpha1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRalpha1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFR alpha1-specific RNA experiments demonstrated that the downregulation of GFR alpha1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLC gamma-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFR alpha signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

Expressions of Glial Cell Line-derived Neurotrophic Factor and Its Receptors in Cortex of Rats after Closed Traumatic Cerebral Injury / 中国康复理论与实践

@#Objective To investigate the expressions of glial cell line-derived neurotrophic factor(GDNF)and its receptors,GFRα-1 and Ret,in cortex of rats after closed traumatic cerebral injury.Methods Male Sprague-Dawley rats were divided into normal control,sham and injury groups.The rats of injury groups were subjected to Marmarou's closed traumatic cerebral injury and then were subdivided into 1 h,2 h,4 h,8 h,12 h,24 h,48 h,72 h and 5 d groups according to the time elapsed after injury.The expression of GDNF and its receptors were determined with immunohistochemistry.Results Mild expression of GDNF and its receptors were observed in cortex of rats in control groups.The number of GDNF positive neurons reached the peak level in cortex 2 h after injury,and that of GFRα-1 and Ret positive neurons reached the peak level 4 h after injury.Conclusion The expressions of GDNF and its receptors increased significantly at the early time in cortex of rats after injury,as well as its receptors.It suggests that GDNF and its receptors play an important role after traumatic cerebral injury.

Expressions of Glial Cell Line-derived Neurotrophic Factor and Its Receptors in Brain Stem of Rats after Brain Injury / 中国康复理论与实践

@#Objective To investigate the effect of traumatic brain injury on the expressions of glial cell line-derived neurotrophic factor (GDNF) and its receptors in brain stem of rats.Methods 55 male Sprague-Dawley rats were divided into the normal control group, sham surgery group and injury groups. The rats of injury groups were subjected to Marmarou's closed traumatic brain injury and then were subdivided into 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 5 d groups according to the time elapsed after injury. The expressions of GDNF and its receptors (GFRα-1 and Ret) were tested with immunohistochemistry.Results Mild expressions of GDNF and its receptors were observed in brain stem of rats in the normal control group and sham surgery group. The number of GDNF positive neurons reached the peak level at 2 h in brain stem after injury, and that of GFRα-1 and Ret positive neurons reached the peak level at 4 h after injury.Conclusion The expressions of GDNF and its receptors increase significantly at the early time in brain stem of rats after injury. The similar temporal patterns of expressions of GDNF and its receptors are observed in brain stem after brain injury.

Expression and construction of an adeno-associated virus vector containing dual genes-GDNF and TH / 基础医学与临床

Objective Rat glial cell derived neurotrophic factor(GDNF) gene and tyrosine hydroxylase(TH) gene were widely used in gene therapy of Parkinson's Disease(PD).The present study was designed to investigate whether the two target genes GDNF and TH could express simultaneously in one adeno-associated virus(AAV)vector and to discuss the probability of using it in gene therapy of PD.Methods RNA of GDNF and TH in HEK293 packaging cells were examined by reverse transcription-polymerase chain reaction(RT-PCR).Expressions of GDNF and TH in infected bone marrow stromal cells(BMSCs) and rat brain coronal sections were examined with immunocytochemistry and immunohistochemistry assay.Results RNA of GDNF and TH were detected in HEK293 packaging cells.Expressions of this two genes were observed in both bone marrow stromal cells and brain sections of rats.Theexpression efficiency of AAV-LacZ was about 50% in HEK293 packaging cells and approximately 15% in infected BMSCs and the expressions of TH and GDNF on the AAV-GDNF/TH virus injected points both had significant differences(P

Analysis of GDNF(△N39)-R9 Fusion Protein Delivery Across The Cellular Membrane,Blood Brain Barrier and Biological Function / 生物化学与生物物理进展

In order to study the application of glial cell line-derived neurotrophic factor(GDNF)in clinic,gene mutation,fusion protein expression in E.coli and purification methods have been used to obtain the fragments of GDNF,GDNF(△N39),GDNF(△N39)-R9.Using primary cultured dopaminergic neurons and PC12 cells with transfected with GFR?1 and Ret to observe their biological function and cytotoxicity.Using B-Endo3 cells and Transwell method to analyze their delivery across the cellular membrane and blood brain barrier.The results show that GDNF(△N39)-R9 has the same neurotrophic function with wild GDNF and nearly no cytotoxicity to dopaminergic neurons and PC12-GFR?1-Ret cells and can get through effectually the cellular membrane and simulacrum of blood brain barrier with matrigel and B-Endo3.

Changes of Expression in GDNF and GDNFR-?1 of Long-term Denervation of Posterior Cricoarytenoid Muscles / 听力学及言语疾病杂志

Objective To investigate the changes of the expression of GDNF and GDNFR?1 of long-term denervation of posterior cricoarytenoid muscles(PCAMs).Methods 38 patients with vocal paralysis were grouped into four according to their denervated period of time while 12 normal PCAMs as control group.Using double immunofluorescence stain,changes of GDNF and GDNFR1 expression were observed in myofibers at different time points after denervation.Results Double immunofluorescence stain with antibodies against GDNF and GDNFR1 showed no staining in the control group,and study groups.However,after the muscle denervation lasted for 6-12 months and 1-2 years,noted was a significant accumulation of GDNF and GDNFR1 protein in cytolemma and endochylema of myofiber.The mean grey scales and positive region ratios were compared using the image analysis system.The results revealed the levels of GDNF and GDNFR1 protein expression in 6-12 months group,1-2yr group changed significantly(P0.05).Conclusion The changes in expression of GDNF and the acceptor GDNFR-?1,a powerful neurotrophic factor,implied that a good nervous regenerated microenvironment in PCAMs within 2 years.This experiment indicated that denervated posterior cricoarytenoid muscles are able to regain their functions through reinneration within 2 years.

Expression of mRNAs for Neurotrophic Factors in Human Neural Stem Cells Derived from Fetal Telencephalon

OBJECTIVE: Cellular diversity in the mammalian central nervous system is originated from precursor cells present in the neural ectoderm. The multipotent neural stem cells(NSCs) rapidly proliferate to give rise to transiently dividing progenitors that eventually differentiate into several cell types of neural cells. The authors investigate whether NSCs could differentiate neurons and glia and express neurotrophic factor. METHODS: To establish human neural cell lines, we isolated neural stem cells from human fetal telencephalon. Secondly, to investigate the expression of neurotrophic factor, basic fibroblast growth factor(bFGF), brain-derived neurotrophic factor(BDNF) and glial derived neurotrophic factor(GDNF) in rat and human cell, mRNA expressions of bFGF, BDNF and GDNF were detected by the reverse transcripted polymerase chain reaction(RT-PCR) analysis. RESULTS: In the NSCs cultures of embryonic rat striata and human fetal telencephalon, we demonstrated that bFGF induces the proliferation of stem cell, which give rise to spheres of undifferentiated cell that generate neurons and glia. Also, neurotrophic factor transcripts were identified using PCR in rat and human NSCs. CONCLUSION: These results demonstrate that human NSCs derived from human fetal telencephalon could differentiate neurons and glia and express neurotrophic factors. Therefore, NSCs may be an important key for the therapeutic application of neurotrophic factors.