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BACKGROUND: Recent studies show that the neurons in central nervous system have the regenerating ability under certain suitable environment such as elevating the c-AMP level of the neuron. But the mechanism is unclear.OBJECTIVE: To investigate the effect of protein kinase A inhibitor H-89and PI3-kinase inhibitor, wortmannin, on Cholera Toxin (CTx) in promoting the axon regeneration of retinal ganglion cells (RGCs) after distal axotomy of the optic nerve in adult hamsters.DESIGN: A randomized and controlled animal experiment SETTING: Department of Histology and Embryology of Guangzhou Medical College and Sun Yat-sen University of Medical Sciences MATERIALS: This experiment was conducted in the Guangzhou Medical College and Sun Yat-sen University of Medical Sciences between January 2000 and December 2001. Totally 25healthy adult male hamsters were chosen and randomly divided into 5 groups: model group; experimental control group; CTx group; CTx +protein kinase A inhibitor group; CTx +PI3-K inhibitor group, with 5 animals in each group.METHODS: A 2 cm segment of autologus sciatic nerve was removed and desheathed. The proximal end of the sciatic nerve was connected to the proximal stump of the optic nerve (ON). .The wound was enveloped with gelatin sponge. The remaining portion of sciatic nerve was placed on the top of the skull. Model group: a segment of autologus sciatic nerve was connected to the ON proximal stump. Experimental control group: Received an injection of Na2EDTA/NaCL solution introvitreally based on the treatment in control group. CTx group: CTx (1000 pg/eye)was injected introvitreally on the basis of the treatment in the control group. CTx+ protein kinase A inhibitor group: 3μL protein kinase A inhibitor H89 (60μmol/L)was injected introvitreally 30 minutes before operation, the other treatment was like mannin (1μmol/L) was injected introvitreally 30 minutes before operation,and the other treatment was like that of CTx group.⑤CTx+PI3-K inhibitor group: 3 μL PI3-K inhibitor wortmannin (1μmol/L) was injected introvitreally 30 minutes before operation,and the other treatment was like that of CTx group. Animals in each group survived for 4 weeks. Administration was given every other 5 days. H-89,wortmannin were given once five days and CTx was also given 30 minutes later every time. There were four times in total. 3 days before operation, a piece of gel foam soaked with 30g/L GB was applied to the proximal end of transected PN to label the RGCs conversely. Observation was performed under the fluorescence microscope and the number of GB-labeled cells was counted.MAIN OUTCOME MEASURES: The quantity of retrograde labeled axon regenerating RGCs in each groupRESULTS: There were fewer regenerating RGCs in the model group and experimental control group (2.6±0.87,2.4±0.95),and the number of them was significantly higher in the CTx group than in the control group and experimental group [(43.2±1.36),q=73.294 and 73.655,P ＜ 0.001]. There was no significant difference of the mean number of axon regenerating RGCs between CTx + protein kinase A inhibitor H-89 group and model group and experimental control group [(3.2±0.16), q=1.083 and 1.444, P＞ 0.05]; The mean number of axon regenerating RGCs in the three groups was significantly lower than that in the CTX group, with significant difference (q=72.211, P ＜ 0.001). The mean number of axon regenerating RGCs was higher in the CTx+PI3-K inhibitor group (9.6±1.85)than in the model group and experimental control group (q=12.637 and 12.998, P ＜ 0.05);but significantly lower than in the CTx group (q=60.657, P ＜ 0.001).CONCLUSION: CTx can promote the axon regeneration of RGCs after distal axotomy of the optic nerve; its promoting function can be blocked by H-89 and Wortmannin
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AIM: To investigate the potential of murine epidermal stem cell (ESC) differentiation after seeded in a biodegradable carrier and implanted subcutaneously into syngeneic recipient mice. METHODS: ES cells were induced in vitro to differentiate into ESCs. After stained with a fluorescent dye Hoechst 33342, these ESCs were seeded into a polyglycolic acid (PGA) net containing collagen gel, functioning as a cell carrier, and implanted subcutaneously into 129/J mice, which were syngeneic to these stem cells. RESULTS: The ESCs kept alive in the implant when observed under a fluorescent microscopy 3 weeks or longer after implantation, and could differentiate into hair follicle - like structure,glandular structure, and gave rise to additional structures displaying features resembling native dermis. No apparent rejection or severe side effects were observed at least 10 weeks post- implantation. CONCLUSION: It is feasible to use these ESCs as seed cells in the study to fabricate dermal equivalent having the potential to develop dermal appendages.
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Objective To explore the differentiation potency of ES cell-derived epidermal stem cells compounded by dermal analogs reconstructed with bone MSCs in hypodermis.Methods The dermal analogs were reconstructed with rat bone MSCs and compound gel-gelatin sponge.E14-ES cells,labeled with Hoechst 33342,were cocultured with human amnion.Four days later,epidermal stem cell clones were formed.The skin analogs,reconstructed with ES cell-derived epidermal stem cells and dermal analogs,were transplanted into 129 mice hypodermis.The differentiation tissue of skin analogs was sampled at 2,4,6,8 weeks.The sections were observed with HE staining,immunohistochemical and di-labeled immunofluorescence methods to test the expression of ?1 integrin,CK15,CK19,CEA,CK18.Results The sections were showed tubular or follicular like structures formed with simple or stratified epithelium at 2 and 4 weeks.Keratinized stratified squamous epithelium,sweat glands-like,sebaceous glands-like and hair follicles-like structures were observed at 6 week and 8 week after transplantation.The cells labeled by Hoechst 33342,formed tubular or follicular like structures,expressed?1 integrin,CK15,CK19,CEA and CK18positive respectively at 2 and 4 weeks.The sweat glands-like structure expressed CEA and CK18 positive respectively at 6 and 8 weeks.There were more sebaceous glands-like structures.Conclusion ES cell-derived epidermal stem cells compounded by dermal analogs reconstructed with bone MSCs can differentiate into keratinized stratified squamous epithelium,sweat glands-like,sebaceous glands-like and hair follicles-like structures in hypodermis.
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Objective Use three inhibitors to block three different signaling pathway to explore the mechanisms of 3-isobutyl-1-methylxanthine(IBMX) and 8 (4 Chlorophenylthio) cAMP(CPT-cAMP) on the regeneration of retinal ganglion cells. Method Fluorescent retrogratde tracing method and quantitative anatomical techniques were used to measure the numbers of RGCs in control one,control two group and experiment groups. Results 1
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Objective To investigate the effects of transplantation of sciatic nerve(SN) and injection of 8-(4-Chlorophenylthio)-cAMP(CPT-cAMP) into the viteous on axotomized retinal ganglion cells(RGCs) in adult hamsters. Methods Twenty adult golden hamsters were ramdomly divided into 4 groups.Optic nerve(ON) was transected and a segment of autologous sciatic nerve(attached graft,AG) was removed and sutured to the proximal stump of ON in regenerating control group(AG group).The animals in experimental groups were further treated with CPT-cAMP injection and/or implantation of a short of segment sciatic nerve(SN) intravitrously.Fluorescent retrograde labeling method and quantitative anatomical techniques were used to measure the number of RGCs. Results 1.In control (AG) retinas,the mean number of regenerating RGCs was 1*!428?284/retina.2.In AG+SN group,the mean number of regenerating RGCs was 2*!220?415/retina.3.In AG+cAMP group,the mean number of regenerating RGCs was 2*!175?364/retina.4.The mean number of regenerating RGCs in AG+cAMP+SN group were 4*!255?820/retina.AG+SN,AG+cAMP groups were significantly different from AG group(P
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Objective To investigate the differentiation of mesenchymal stem cells(rMSCs) of young rat into neuron-like cells with Ligustrazin hydrochloride. Methods rMSCs were separated from femurs marrow flushed out with DMEM(low glucose) by using a needle and syringe, then planted in plastic culture flask. Through expanded to 5 passages, rMSCs were induced to differentiate into neuron-like cells with Ligustrazin hydrochloride. Anti-neurofilament(NF-M), nestin, neuron-specific enolase(NSE), MAP-2,GAP-43 and glial fibrillary acidic protein(GFAP) antibodies were detected by immunohistochemistry. Results rMSCs were comprised a single phenotypic population and displayed a fibroblast-like morphology after 5 passage in culture. With 10*!?g/L bFGF pre-induce for 24*!h, then the medium was replaced with induction medium containing Ligustrazin hydrochloride. The induced-rMSCs exhibited neuronal morphological characteristics from the first half an hour to 5*!h. The neuron-like cells expressed NF-M, NSE, MAP-2,GAP-43 and nestin positive, but didn't express glial astrocyte marker GFAP.Conclusion rMSCs can be induced to differentiate into neuron like cells with Ligustrazin hydrochloride in vitro.
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【Objective】 To examine the regeneration of NPY-immuno reactivity (IR) retinal ganglion cells (RGCs) and the effects of cholera toxin ( CTx) injected or/and peripheral nerve implanted on regeneration of NPY-IR RGCs. 【Methods】 16 adult golden hamsters were ramdomly divided into 4 groups. Optic nerve (ON) was transacted and a segment of autologous sciatic nerve (attached g raft, AG) was removed and sutured to the proximal stump of ON in regenerating co ntrol group (AG group). The animals in experimental groups were further treated with CTx injection or/and implantation of a short of segment sciatic nerve (SN) intravitrously. By using the retrograde labeling with fluorogold (FG) combined w ith fluorescent immunocytochemistry, the regenerated NPY-IR RGCs were observed and counted under fluorescent microscope. 【Results】 At 4 weeks after surgery, the mean number of regenerated NPY-IR RGCs per retina in AG group was 58±22 wh ich constitutes (3.36±0.65)% of the total regenerated RGCs. In AG+CTx, AG+SN and AG+CTx+SN experimental groups, the mean numbers of regenerated NPY-IR RGCs per retina were 143±47, 125±37 and 437±77 ordinally which constitute (5.15± 0.89)%, (5.34±0.37)% and (8.11±0.85)% of the total regenerated RGCs respec tively, which were increased significantly compared with those in AG group. 【Co nclusion】 The results show that the axotomized NPY-IR RGCs have th e capability of regenerating their axons into the attached PN graft, CTx and/or SN could enhance the regeneration of the NPY-IR RGCs.
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Objective To explore the effects of neurotrophin-3 genetically modified Schwann cells(NT-3-SCs) and neural stem cells(NSCs) combinative transplantation to promote the neurons'survival and axonal regeneration of the spinal cord transected rat. Methods After the transected spinal cord injury(SCI) model was established in SD rats,NT-3-SCs or LacZ report gene modified Scwhann cells(LacZ-SCs) were combinative transplanted with NSCs into the transected site.60 days later,fluorogold(FG) was injected into the caudal spinal cord fo the transected site.7days after FG injected,the sacrifice,cryosection and morphology serious studies were performed to observe the FG labeled neurons in the rostral spinal cord(RSC) of transected site,red nuclei(RN) and the sensorimotor cortex(SMC);The survival neurons in the L_1 Clark's nuclei(CN),RN and SMC were couut,and regenerated axons within or near the transected spinal cord observed. Results From more to less,the order of groups of the survival neurons in the CN,RN and SMC was NT-3-SCS & NSCs group,LacZ-SCs & NSCs group,experimental control group.In the NT-3-SCs & NSCs group and LacZ-SCs & NSCs group,there were 5-HT,CGRP and SP positive axons within or near the transected spinal cord.And some FGlabeled cells were found in RSC,RN and SMC. Conclusion Combinative grafting NSCs and NT-3-SCs could promote the neurons'survival and axonal regeneration of the spinal cord injured rat.
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AIM: To investigate the differentiation of neural stem cells (NSCs) after transplanted into vitreous and the effects on the regeneration of retina ganglion cells (RGCs) after optic nerve microcrushed.METHODS: After optic nerve microcrushed in adult rat, 2?104/2 ?L NSCs or 2 ?L 0.1 mol/L PBS was injected into vitreous. Animals were divided into control group (MC group, MC+PBS group) and experiment group (MC+NSCs). Animals in each group were allowed to survive for 3, 4, 5 weeks, respectively. The regenerating RGCs were labeled retrogradely with granular blue, and the numbers of regenerating RGCs in each retina were observed under fluorescent microscope. In addition after 5 animals in MC+NSCs group survived for 4 weeks, rat eyeballs were removed and prepared as freezing microtome sections for observing the migration of NSCs and NF, GFAP, CNP immumodetection.RESULTS: Compared the mean numbers of regenerating RGCs between experiment group and control group at 3, 4, 5 weeks, the difference was significant (P
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AIM: To investigate the mechanism by which human amnion induced mouse embryonic stem (ES) cells to differentiate into epidermal like cells. METHODS: ES-BALB/c cells were cocultured with human amnion in transwells for 4-5 days, and those cultured alone without amnion were taken as control group. The morphological differentiation were observed. The committed differentiation of ES cells into epidermal like cells were detected by integrin-?_1, CK19, CK15 and involucrin immunohistochemistry, respectively. RESULTS: After 4-5 days of coculture, ES cells differentiated into single layer of epidermal like cells, fitted tightly, with polyhedral in shape. The immunohistochemical staining results showed that, most of the cells were integin-?_1 positive, only a few cells were CK19 and CK15 positively stained. Most of the cells in control group died, the survived ones were different in morphological shapes, and no integrin-?_1, CK19 and CK15 positive cells were found. CONCLUSION: Soluble substances secreted by human amnion may play an important role in inducing the differentiation of mouse ES cells into epidermal like cells. [
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AIM: To explore the effects of neurotrophin-3 (NT-3)-genetically modified Schwann cells (NT-3-SCs) on differentiation of neural stem cells (NSCs) into the neuron-like cells. METHODS: The NSCs were co-cultured with NT-3-SCs. Report gene LacZ genetically modified Schwann cells (LacZ-SCs) and normal SCs respectively in vitro. 7 d later, the differentiation of NSCs was studied by immunohistochemistry, and the percentage of neuron-like cells was calculated. RESULTS: NSCs differentiated to the GFAP-positive cells (glial-like cells) and NF-positive cells (neuron-like cells) in vitro. Compared to the normal SCs, NT-3-SCs more efficiently promoted NSCs to differentiate into the neuron-like cells. The effect of LacZ-SCs was as the same to the normal SCs. CONCLUSION: NT-3-SCs promote NSCs to differentiate into the neuron-like cells. [
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AIM: To investigate whether berberine can ind uce rat mesenchymal stem cells (MSCs) to differentiate into neuron -like cells in vitro. METHODS: MSCs were separated from young rat femurs marrow and expanded in culture medium. MSCs were induced to di fferentiate by berberine. The morphological changes of MSCs were evaluated by li g ht microscope.Neuron-spcific enolase (NSE), neurofilament (NF), glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. RESULTS: Induced by be rberine for 8 hours,MSCs exhibited neurotype . The expression of NSE and NF in the neuron-like cells was positive, but the glial astrocyte marker GFAP didn't express. CONCLUSION: Berberine may induce adult rat MSCs to differe ntiate into neuron-like cells in vitro.
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Immunohistochemistry was utilized to investigate the light and electron micros-copic localization of neurotensin-like (NT) immunoreactive amacrine cells in thechicken retina.The results showed that the NT-immunoreactive cell bodies wereoval and situated in either the second or third row of cells from the inner borderof the inner nuclear layer.The processes of such cells extended into the innerplexiform layer where they ramified as a fine plexus in sublamina 1 and as a denseplexus in sublamina 3 and 4.At the ultrastructural level,NT positive soma exhibited a rather dense and evenlydistributed immune reaction product throughout their cytoplasm.The nucleus ofNT-amacrine cells possessed a round,unindented nuclear membrane.NT positiveprocesses of such cells receive synaptic input from processes of unlabeled amacrineand bipolar cells.They formed synaptic output onto processes of nonimmuno-reactive amacrine cells and bipolar cells.Moreover,each of the above synapticrelationships were identified in each of sublamina 1 and 3 to 4 of the inner plexiform layer.In addition,NT positive processes fromed synaptic output to processesdevoid of synaptic vesicles,which may originated from ganglion cells.They alsoformed synaptic output to somas situated in the innermost cell row of the innernuclear layer.Identification of synaptic elements and retinal circuitry were also discussed.
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Objective To investigate the effects of Colera Toxin(CTx) on the regeneration of retinal ganglion cells (RGCs) after distal axotomy in adult hamsters. Method After transecting the optic nerve(ON) intracranically,an autologus sciatic nerve (attached graft,AG) was removed and connected to the proximal stump of the ON.CTx was injected and/or a 2mm segment of sciatic nerve(SN) was inserted intravitreally.Animals were divideded into six groups:control group 1(AG group) and control group 2(solution group);AG+SN group;AG+CTx group;AG+SN+CTx group;effect and dosage group.Animals in the former five groups were allowed to survive for 4-6 weeks respectively.Granular blue fluorescent retrograde labeling method was used to measure the quantity of regenerating RGCs of control and experiment animals. Results The mean number of regenerating RGCs in AG+CTx groups were increased and significantly higher than those in control group 1 and control group 2 at each time point(P
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Objective To provide a new way for treatment of full thickness skin defects by embryonic stem(ES) cell-derived epidermal-like stem cells. Methods Epidermal like stem cells,labeled by Hoechst 33342 and carried by a layer of biomembrane,were transplanted into the defective skin of mice.The differentiation tissue of donor cells was sampled each week.The sections were observed with HE staining,immunohistochemical and di-labeled immunofluorescence methods to test the expression of ?1 integrin,CK15,CK19,CK10,CEA. Results The full thickness skin defects were healed in 2 weeks.The newborn skin was thicker than the normal skin.The basal layer cells proliferated.There were more bulky cellular poles towards dermis.The cells labeled by Hoechst 33342,located in the newborn epidermis and tubular or follicular structures in dermis,expressed ?1 integrin and CK15 positive respectively in the first 3 weeks.There were sweat gland-like,sebaceous gland-like and hair follicle-like structures in the newborn dermis after 4 weeks.Basal cells of keratinized stratified squamous epithelium expressed CK19 and CK10 positive respectively and sweat gland-like structure expressed CEA positive.Conclusion ES cell-derived epidermal stem cells can restore mice full thickness skin defects.There are epidermis,sweat gland-like,sebaceous gland-like and hair follicle-like structures in the newborn skin.
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Objective The present study was aimed at investigating the effects of cholera toxin(CTx) on promoting the regeneration of retinal ganglion cells(RGCs) in hamster retina. Methods After optic nerve (ON) transection, an autologus sciatic nerve (attached graft, AG) was removed and sutured to the proximal stump of the ON. CT X was injected or (and) a small segment of sciatic nerve (SN) inserted intravitrously. Animals were separated into 5 groups:regenerating control group(AG groups and solution groups); AG+CT X groups; AG+SN groups; AG+CT X+SN groups; Effect and Dosage groups; Animals in the former 4 groups were allowed to survive for 2-6 weeks respectively. The regenerated RGCs were labeled retrogradely by granular blue, and the changes in number of regenerating RGCs in each retina were observed under fluorescent microscope. Results The mean numbers of regenerating RGCs in AG+CT X groups were increased and significantly higher than those in AG groups and solution groups at each time point( P
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The immunohistochemical single and double label techniques were used to study the localization and coexistence of immunoreaetive enkephalin (ENK) and somatostatin (SOM) in amacrine cells of the chicken retina. The single label experiments showed that certain SOM-immunoreactive amacrine cells are similar in morphology, location in the inner nuclear layer (INL) and the manner of processes ramified in the inner plexiform layer (IPL) to some ENK-immunoreactive amacrine cells, although the plexus of SOM-immunoreactive processes in sublamina 3 and 4 were not as dense as ENK-immunoreactive plexus and SOM-immunoreactive processes in sublamina 5 were not observed. The double label studies indicated that some amacrine cells showed both SOM and ENK positive immunoreactivity and some other amacrine cells showed only SOM or ENK positive immunoreactivity.The coexistence of two neuropeptides or a neuropeptide and a classical neurotransrnitter in the retinal amacrine cells were also discussed.
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Objective To investigate the effects of Cholera Toxin(CTx)on the cAMP level and the survival as well as the apoptosis of retinal ganglion cells (RGCs) after distal axotomy of the optic nerve.Method After transecting the optic nerve intracranially, CTx was injected intravitreally. Fluorescent retrograde tracing method and TUNEL(TdT-mediated dUTP nick end labeling)technique were used to show the surviving RGCs and the apoptotic cells in the ganglion cell layer. The Brown's radioimmunoassay method was used to measure the cAMP level of the retina. Result The cAMP level of the normal retina was 6.22?2.02pmol/g/retina. The mean density of RGCs in the normal retina was 2 192?66/mm 2 and it decreased to 1 520?116/mm 2、736?39/mm 2 and 466?53/mm 2 at 1W、2W and 3W respectively after distal axotomy. The densities of RGCs in the distal axotomy groups treated with CTx and killed at 1W、2W and 3W were 1 642?122/mm 2、1 091?107/mm 2、 748?35/mm 2 respectively and were significantly higher than those of distal axotomy group without CTx treatment. Conclusions The results show that CTx can elevate cAMP level of the retina and promote the survival of RGCs and inhibit the apoptosis of RGCs after distal axotomy of the optic nerve in adult hamsters.
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Objective To investigate the differentiation potenitial of ES cell derived epidermal like stem cells,to lay a base for the study of their differentiation mechanism,as well as seek new source to provide seed cells for skin engineering. Methods Mouse ES cells labeled or unlabeled by Hoechst 33342 were cocultrued with human amnion for 4 days.The epidermal like stem cell clones formed on the surface of amnion were digested with trypsin and transplanted into hypodermis of nude mice for 10,20 and 45 days,then the differentiation pattern of the donor cells were observed and estimated with morphological and immunohistochemical method. Results The grafts may differentiate into tubular or follicular like structures lined with simple or stratified epithelial like cells which expressed ? 1 integin,CK19,CK15,PK involucrin and CEA respectively after 10 to 30 days of transplantation.Keratinized stratified squamous epithelium,sebaceous gland like,sweat gland like and hair follicle like structures were observed after 45 days ofter transplantation.Conclusion ES cell derived epidermal like cells might have differentation potential to diffreentiate into keratinized stratified squamous epithelium,sebaceous like,sweat gland like and hair follicle like structures.
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Objective To investigate the condition which can induce mouse ES cells to differentiate into epidermal-like stem cells for the clinical application of ES cells-derived epidermal-like stem cells and the research on the mechanism of committed differentiation of ES cells. Methods Coculture mouse ES cells with human amnion for 3-4 days, and the committed differentiation were detected by flow cytometry and immunohistochemistry. In experimental group 1, amnion was pasted covering the whole bottom of the wells, with the epithelial surface upward, and in group 2 covering the half bottom. No amnion was used in control. Results After 3-4 days of co-culture, epidermal-like stem cell clones were formed on the epithelial surface of amnion in group 1 and group 2, and expressed high levels of integrin-? 1, CK19 and CK15. The percentages of integrin-? 1, CK19 and CK15 positive cells counted in group 1 by flow cytometry were 89.2%, 86.8% and 71.2% respectively, versus the control group of 8.4%,9.6% and 11.8%, the differences were significant in all the three indices (Z tests P