Growth-associated protein 43 and neural cell adhesion molecule expression following bone marrow-derived mesenchymal stem cell transplantation in a rat model of ischemic brain injury

BACKGROUND： Transplantation of bone marrow-derived mesenchymal stem cells （BMSCs） improves motor functional recovery, but the mechanisms remain unclear. OBJECTIVE： To investigate expression of growth-associated protein 43 （GAP-43） and neural cell adhesion molecule following BMSC transplantation to the lateral ventricle in rats with acute focal cerebral ischemic brain damage. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment using immunohistochemistry was performed at the laboratories of Department of Neurology, Renmin Hospital of Wuhan University and Doctoral Scientific Research Work Station of C-BONS PHARMA, Hubei Province, China, from January 2007 to December 2008. MATERIALS： Monoclonal mouse anti-rat 5-bromo-2-deoxyuridine and neural cell adhesion molecule antibodies were purchased from Sigma, USA; monoclonal mouse anti-rat GAP-43 antibody was purchased from Wuhan Boster, China. METHODS： Rat models of right middle cerebral artery occlusion were established using the thread method. At 1 day after middle cerebral artery occlusion, 20μL culture solution, containing 5×10^5 BMSCs, was transplanted to the left lateral ventricle using micro-injection. MAIN OUTCOME MEASURES： Scores of neurological impairment were measured to assess neural function. Expression of GAP-43 and neural cell adhesion molecule at the lesion areas was examined by immunohistochemistry. RESULTS： GAP-43 and neural cell adhesion molecule expression was low in brain tissues of the sham-operated group, but expression increased at the ischemic boundary （P 〈 0.05）. Transplantation of BMSCs further enhanced expression of GAP-43 and neural cell adhesion molecule （P 〈 0.05） and remarkably improved neurological impairment of ischemic rats （P 〈 0.05）. CONCLUSION： BMSC transplantation promoted neurological recovery in rats by upregulating expression of GAP-43 and neural cell adhesion molecule.

Biochemical properties of norepinephrine as a kind of neurotransmitter secreted by bone marrow-derived neural stem cells induced and differentiated in vitro

BACKGROUND： It has been proved by many experimental studies from the aspects of morphology and immunocytochemistry in recent years that bone marrow stromal cells （BMSCs） can in vitro induce and differentiate into the cells possessing the properties of nerve cells. But the functions of BMSCs-derived neural stem cells（NSCs） and the differentiated neuron-like cells are still unclear. OBJECTIVE： To observe whether bone marrow-derived NSCs can secrete norepinephrine （NE） under the condition of in vitro culture, induce and differentiation, and analyze the biochemical properties of BMSCs-derived NSCs. DESIGN： A non-randomized and controlled experimental observation SETTING ： Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University MATERIALS： This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5- month-old healthy New Zealand white rabbits. METHODS： This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5 month-old healthy New Zealand white rabbits. BMSCs of rabbits were isolated and performed in vitro culture, induce and differentiation with culture medium of NSCs and differentiation-inducing factor, then identified with immunocytochemical method. Experimental grouping： ①Negative control group： L-02 hepatic cell and RPMI1640 culture medium were used. ② Background culture group： Only culture medium of NSCs as culture solution was added into BMSCs to perform culture, and 0.1 volume fraction of imported fetal bovine serum was supplemented 72 hours later. ③Differentiation inducing factor group： After culture for 72 hours, retinoic acid and glial cell line-derived neurotrophic factors were added in the culture medium of BMSCs and NSCs as corresponding inducing factors. The level of NE in each group was detected on the day of culture and 5, 7, 14 and 20 days after culture with high performance liquid chromatography （HPLC）. The procedure was conducted 3 times in each group.Standard working curve was made according to the corresponding relationship of NE concentration and peak area. The concentration of NE every 1×10^7 cells was calculated according to standard curve and cell counting. MAIN OUTCOME MEASURES ： The level of NE of cultured cells was detected with HPLC; immunocytochemistrical identification of Nestin and neuron specific nuclear protein was performed. RESULTS： ① On the 14^th day after cell culture, BMSCs turned into magnus and round cells which presented Nestin-positive antigen, then changed into neuron-like cells with long processus and presented neuron specific nuclear protein -positive antigen at the 20^th day following culture. ② The ratio of NE concentration and peak area has good linear relationship, and regression equation was Y=1.168 36＋0.000 272 8X,r=-0.998 4. Coefficient variation （CV） was 〈 5% and the recovery rate was 92.39%（ Y referred to concentration and X was peak area）.③NE was well detached within 10 minutes under the condition of this experiment. ④ NE was detected in NSCs and their culture mediums, which were cultured for 7, 14 and 20 days respectively, but no NE in BMSCs, NSCs-free culture medium and L-02 hepatic cell which were as negative control under the HPLC examination. Analysis of variance showed that the level of NE gradually increased following the elongation of culture time （P 〈 0.01 ）. No significant difference in the level of NE existed at the same time between differentiation inducing factor group and basic culture group（P 〉 0.05）. CONCLUSION ： BMSCs of rabbits can proliferate in vitro and express Nestin antigen; They can differentiate into neuron-like cells, express specific neucleoprotein of mature neurons, synthesize and secrete NE as a kind of neurotransmitter.

In vitro differentiation of adipose-derived stem cells and bone marrow-derived stromal stem cells into neuronal-like cells

Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.

Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model

Sodium nitrite（Na NO2） is an inorganic salt used broadly in chemical industry. Na NO2 is highly reactive with hemoglobin causing hypoxia. Mesenchymal stem cells（MSCs） are capable of differentiating into a variety of tissue specific cells and MSC therapy is a potential method for improving brain functions. This work aims to investigate the possible therapeutic role of bone marrow-derived MSCs against Na NO2 induced hypoxic brain injury. Rats were divided into control group（treated for 3 or 6 weeks）, hypoxic（HP） group（subcutaneous injection of 35 mg/kg Na NO2 for 3 weeks to induce hypoxic brain injury）, HP recovery groups N-2 w R and N-3 w R（treated with the same dose of Na NO2 for 2 and 3 weeks respectively, followed by 4-week or 3-week self-recovery respectively）, and MSCs treated groups N-2 w SC and N-3 w SC（treated with the same dose of Na NO2 for 2 and 3 weeks respectively, followed by one injection of 2 × 106 MSCs via the tail vein in combination with 4 week self-recovery or intravenous injection of Na NO2 for 1 week in combination with 3 week self-recovery）. The levels of neurotransmitters（norepinephrine, dopamine, serotonin）, energy substances（adenosine monophosphate, adenosine diphosphate, adenosine triphosphate）, and oxidative stress markers（malondialdehyde, nitric oxide, 8-hydroxy-2′-deoxyguanosine, glutathione reduced form, and oxidized glutathione） in the frontal cortex and midbrain were measured using high performance liquid chromatography. At the same time, hematoxylin-eosin staining was performed to observe the pathological change of the injured brain tissue. Compared with HP group, pathological change of brain tissue was milder, the levels of malondialdehyde, nitric oxide, oxidized glutathione, 8-hydroxy-2′-deoxyguanosine, norepinephrine, serotonin, glutathione reduced form, and adenosine triphosphate in the frontal cortex and midbrain were significantly decreased, and glutathione reduced form/oxidized glutathione and adenosine monophosphate/adenosine triphosphate ratio were significantly increased in the MSCs treated groups. These findings suggest that bone marrow-derived MSCs exhibit neuroprotective effects against Na NO2-induced hypoxic brain injury through exerting anti-oxidative effects and providing energy to the brain.

Effects of lateral ventricular transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene on cognition in a rat model of Alzheimer's disease

In the present study, transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene into the lateral ventricle of a rat model of Alzheimer＇s disease, resulted in significant attenuation of nerve cell damage in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor and tyrosine kinase B mRNA and protein levels were significantly increased, and learning and memory were significantly improved. Results indicate that transplantation of bone marrow-derived mesenchymal stem cells modified with brain-derived neurotrophic factor gene can significantly improve cognitive function in a rat model of Alzheimer＇s disease, possibly by increasing the levels of brain-derived neurotrophic factor and tyrosine kinase B in the hippocampus.

MicroRNA changes of bone marrow-derived mesenchymal stem cells differentiated into neuronal-like cells by Schwann cell-conditioned medium

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow- derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesencaymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis iden:ified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathv/ays were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury

Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury.

Bone marrow-derived mesenchymal stem cells increase dopamine synthesis in the injured striatum

Previous studies showed that tyrosine hydroxylase or neurturin gene-modified cells transplanted into rats with Parkinson＇s disease significantly improved behavior and increased striatal dopamine content. In the present study, we transplanted tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells into the damaged striatum of Parkinson＇s disease model rats. Several weeks after cell transplantation, in addition to an improvement of motor function tyrosine hydroxylase and neurturin proteins were up-regulated in the injured striatum, and importantly, levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid increased significantly. Furthermore, the density of the D2 dopamine receptor in the postsynaptic membranes of dopaminergic neurons was decreased. These results indicate that transplantation of tyrosine hydroxylase and neurturin gene-modified bone marrow-derived mesenchymal stem cells increases dopamine synthesis and significantly improves the behavior of rats with Parkinson＇s disease.

Neuronal-like differentiation of bone marrow-derived mesenchymal stem cells induced by striatal extracts from a rat model of Parkinson's disease

A rat model of Parkinson＇s disease was established by 6-hydroxydopamine injection into the medial forebrain bundle. Bone marrow-derived mesenchymal stem cells （BMSCs） were isolated from the femur and tibia, and were co-cultured with 10% and 60% lesioned or intact striatal extracts. The results showed that when exposed to lesioned striatal extracts, BMSCs developed bipolar or multi-polar morphologies, and there was an increase in the percentage of cells that expressed glial fibrillary acidic protein （GFAP）, nestin and neuron-specific enolase （NSE）. Moreover, the percentage of NSE-positive cells increased with increasing concentrations of lesioned striatal extracts. However, intact striatal extracts only increased the percentage of GFAP-positive cells. The findings suggest that striatal extracts from Parkinson＇s disease rats induce BMSCs to differentiate into neuronal-like cells in vitro.

Cell viability and dopamine secretion of 6-hydroxydopamine-treated PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells

In the present study, PC12 cells induced by 6-hydroxydopamine as a model of Parkinson＇s Disease, were used to investigate the protective effects of bone marrow-derived mesenchymal stem cells bone marrow-derived mesenchymal stem cells against 6-hydroxydopamine-induced neurotoxicity and to verify whether the mechanism of action relates to abnormal a-synuclein accumulation in cells Results showed that co-culture with bone marrow-derived mesenchymal stem cells enhanced PC12 cell viability and dopamine secretion in a cell dose-dependent manner. MitoLight staining was used to confirm that PC12 cells co-cultured with bone marrow-derived mesenchymal stem cells demonstrate reduced levels of cell apoptosis. Immunocytochemistry and western blot analysis found the quantity of α-synuclein accumulation was significantly reduced in PC12 cell and bone marrow-derived mesenchymal stem cell co-cultures. These results indicate that bone marrow-derived mesenchymal stem cells can attenuate 6-hydroxydopamine-induced cytotoxicity by reducing abnormal α-synuclein accumulation in PC12 cells.

Distribution and differentiation of bone marrow-derived mesenchymal stem cells in vivo after intraperitoneal and tail vein injection into rats in the recovery phase of stroke: Which path is better?

BACKGROUND： Stereotactic injection （striatum or lateral ventricle） and vascular injection （ tail vein or carotid artery） are now often used in cellular therapy for cerebral infarction. Stereotactic injection can accurately deliver cells to the infarct area, but requires a stereotactic device and causes secondary trauma; vascular injection is easy and better for host neurological deficit recovery, but can cause thrombosis. OBJECTIVE： To compare the therapeutic potential of adult bone marrow-derived mesenchymal stem cells （BMSCs） transplantation by intraperitoneal versus intravenous administration to cerebral ischemic rats. DESIGN, TIME AND SE＇B＇ING： A randomized controlled animal experiment was performed at the Cell Room and Pathology Laboratory, Brain Hospital Affiliated to Nanjing Medical University from November 2007 to September 2008. MATERIALS： BMSCs were derived from 20 healthy Sprague-Dawley rats aged 4-6 weeks. METHODS： Forty-five adult middle cerebral artery occlusion （MCAO） rats were randomly divided into control, intravenous and intraperitoneal injection groups, with 15 rats in each group. At 21 days after modeling, rats in the control group received 1 mL of 0.01 mol/L phosphate buffered saline via tail vein injection and each experimental rat received 4 x 106 BMSCs labeled by bromodeoxyuridine （BrdU） via intravenous or intraperitoneal injection. MAIN OUTCOME MEASURES： Angiogenin expression and survival of transplanted cells were measured by immunohistochemical staining of brain tissue in infarction hemisphere at 7, 14 or 21 days after BMSC transplantation. Co-expression of BrdU/microtubule-associated protein 2 or BrdU/glial fibrillary acidic protein was observed by double-labeled immunofluorescence of cerebral cortex. Evaluation of nerve function adhesion-removal test was performed on the 14 or 21 days after BMSCs treatment. using the neurological injury severity score and the 1st and 21st day before and after MCAO, and at 3, 7 RESULTS： Angiogenin-positive new vessels were distributed in the bilateral striatum, hippocampus and cerebral cortex of each group of rats at each time point, most markedly in the intravenous injection group. There were significantly more BrdU-positive cells in the intravenous injection group than in the intraperitoneal injection group （P 〈 0.01）. Co-expression of BrdU/ microtubule-associated protein 2 or BrdU/glial fibrillary acidic protein were almost only seen in the intravenous group by fluorescence microscopy. After transplantation, BMSCs significantly restored nerve function in rats, particularly in the intravenous injection group. CONCLUSION： BMSCs were able to enter brain tissue via the tail vein or peritoneal injection and improve neurological function by promoting the regeneration of nerves and blood vessels in vivo, more effectively after intravenous than intraperitoneal injection.

Ultra-early treatment of bone marrow-derived mesenchymal stem cells for focal cerebral ischemia/ reperfusion injury

The time point at which bone marrow-derived mesenchymal stem cells（BMSCs）can be used in transplantation for the treatment of ischemic brain injury remains unclear.In the present study,BMSCs were transplanted to the ischemic site 90 minutes post-ischemia.The results demonstrated that the transplanted BMSCs improved neurological function,reduced infarct volume,increased survivin expression,decreased caspase-3 expression and reduced apoptosis.This suggests that BMSCs transplanted at an ultra-early stage ameliorated brain ischemia by increasing survivin expression,decreasing caspase-3 expression and reducing apoptosis at the ischemia/reperfusion injury site.

Pre-degenerated peripheral nerves co-cultured with bone marrow-derived cells: a new technique for harvesting high-purity Schwann cells

Schwann cells play an important role in the peripheral nervous system, especially in nerve repair following injury, so artificial nerve regen- eration requires an effective technique for obtaining purified Schwann cells. In vivo and in vitro pre-degeneration of peripheral nerves have been shown to obtain high-purity Schwann cells. We believed that in vitro pre-degeneration was simple and controllable, and available for the clinic. Thus, we co-cultured the crushed sciatic nerves with bone marrow-derived cells in vitro. Results demonstrated that, 3 hours after injury, a large number of mononuclear cells moved to the crushed nerves and a large number of bone marrow-derived cells infiltrated the nerve segments. These changes promoted the degradation of the nerve segments, and the dedifferentiation and proliferation of Schwann cells. Neural cell adhesion molecule and glial fibrillary acidic protein expression were detected in the crushed nerves. Schwann cell yield was 9.08 ± 2.01 ×104/mg. The purity of primary cultured Schwann cells was 88.4 ± 5.79%. These indicate a successful new method for ob- taining Schwann cells of high purity and yield from adult crushed sciatic nerve using bone marrow-derived cells.

Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

大鼠骨髓间充质干细胞体外培养及其向视网膜神经样细胞诱导分化的实验研究

目的研究大鼠骨髓间充质干细胞(rMSCs)体外培养和扩增及其向视网膜神经细胞诱导分化的可能性。方法取SD大鼠双侧股骨和胫骨骨髓细胞进行体外培养。培养视网膜神经细胞,收集视网膜细胞培养上清液,配制成条件分化液。将培养的rMSCs先经神经选择诱导后再换用条件分化液诱导,并对诱导的细胞进行免疫荧光鉴定。结果体外培养的rMSCs生长较好并扩增迅速,经2种分化液诱导后可先分化为神经先祖细胞,继而分化为视网膜神经样细胞。分别应用nestin、NeuN、GFAP、Thy1.1行免疫荧光检测,细胞呈阳性反应。结论rMSCs能于体外培养存活和扩增,并且在一定条件下可被诱导分化为视网膜神经样细胞,体外自制分化条件及模拟眼内环境行骨髓间充质干细胞的诱导,将为进一步的眼内移植提供理论依据。

骨髓基质细胞分泌蛋白的蛋白质质谱分析

目的通过Shotgun蛋白质组学分析,初步检测骨髓基质细胞(BMSCs)条件液中可能含有的蛋白质。方法将BMSCs条件液混匀后经超滤浓缩分为大于5kD和小于5kD两部分,并培养神经干细胞(NSCs),观察其后代中神经元、星形胶质细胞和少突胶质细胞的比例,鉴定出能调节NSCs分化的部分,并进行Shotgun蛋白质组学分析。结果BMSCs条件液分子量大于5kD的部分可以调节NSCs向神经元和少突胶质细胞方向分化,这部分条件液先经过聚丙烯酰胺凝胶电泳(SDS-PAGE)发现大部分蛋白集中在14kD以上,将蛋白条带酶解以后,经Shotgun分析共鉴定到456种蛋白质,其中154个相似蛋白质,17个假想蛋白质,56个未知蛋白质,剩余的229个蛋白质中大多为细胞骨架蛋白、分泌蛋白、信号转导蛋白、酶类和转运蛋白等。结论BMSCs分泌的多种蛋白质对NSCs的分化起调节作用,明确了BMSCs条件液中可能含有的成分。

骨髓基质细胞条件培养液诱导神经干细胞分化的信号转导机制

研究MAPK信号转导通路在骨髓基质细胞条件培养液诱导神经干细胞分化中的作用及机制。用骨髓基质细胞条件培养液培养神经干细胞,并在培养体系中加入MAPK信号转导通路抑制剂PD98059(ERK1/2通路抑制剂)、SB203580(p38通路抑制剂)和genistin(JNK通路抑制剂),7天后经免疫荧光染色鉴定其后代中神经元和星形胶质细胞的比例,并将骨髓基质细胞条件培养液进行蛋白质微阵列检测。结果发现与对照组相比,PD98059和SB203580组神经干细胞分化为神经元比例降低,而星形胶质细胞比例增高,蛋白质微阵列测检发现骨髓基质细胞条件培养液中含有IL-4、IL-6、LIX和TNF-α4种细胞因子。研究结果显示骨髓基质细胞分泌的IL-4、IL-6、LIX和TNF-α4种细胞因子可能通过ERK1/2和p38信号转导通路调节神经干细胞的分化。

脑脊液诱导的大鼠BMSC—Ns移植对脊髓损伤大鼠神经营养因子分泌的影响

目的观察侧脑室移植脑脊液诱导的骨髓源性神经样细胞（BMSC—Ns）对脊髓损伤大鼠BBB评分及神经营养因子分泌的影响。方法全骨髓贴壁法分离培养大鼠骨髓间充质干细胞（BMSCs），人脑脊液诱导得到BMSC—Ns；钳夹法制作sD大鼠脊髓损伤模型，将造模后脊髓损伤大鼠随机分为A、B、C组，每组18只。采用侧脑室注射途径将细胞移植入大鼠体内，A组移植BMSC—Ns（1×10^6／20／μl），B组移植BMSCs（1×10^6／20μl），C组侧脑室注射等体积生理盐水。于细胞移植前和细胞移植后第1、2、3、4、5、6周对脊髓损伤大鼠进行BBB评分；于细胞移植后第1、4周通过ELISA法测定脑脊液中脑源性神经营养因子（BDNF）、神经生长因子（NGF）、神经营养因子－3（NT－3）的浓度。结果在移植后的各个时间点，A组脊髓损伤大鼠的BBB评分显著高于B组和C组，B组的BBB评分又显著高于c组，差异有统计学意义（P〈0．05）。不同时间点亚组间比较显示：细胞移植后前3个时间点A组和B组BBB评分呈现逐渐上升的趋势，亚组间比较差异有统计学意义（P〈0．05）；第3周后上升趋势平缓，各时间点BBB评分差异无统计学意义（P〉0．05）。细胞移植后A组、B组大鼠脑脊液中BDNF、NGF、NT－3含量明显高于C组；A组又高于B组，差异均有统计学意义（P〈0．05）。结论侧脑室移植BMSC—Ns和BMSCs可明显改善脊髓损伤大鼠神经功能，并且可以促进脑脊液中BDNF、NGF、NT－3的分泌，BMSC—Ns效果优于BMSCs．

BDNF对大鼠骨髓源神经干细胞诱导分化的影响

目的观察脑源性神经生长因子(Brain Derived Neurotrophic Factor,BDNF)对体外培养的Wistar大鼠骨髓源神经干细胞诱导分化的影响。方法运用无血清培养基成功培养Wistar大鼠骨髓源神经干细胞,并对分离获得的悬浮生长的神经细胞球,运用免疫组织化学法检测CD133和Nestin表达情况。采用10%胎牛血清诱导其分化,并添加10 ng/m L BDNF,分化7 d后,采用免疫荧光细胞化学染色方法检测分化后GFAP、Map2、β-tubulin III、Galc的表达情况。结果大鼠骨髓基质细胞在无血清培养基中,呈悬浮状态生长,形成细胞球,经免疫荧光检测,细胞球表达CD133和Nestin。将细胞球转入含血清培养基分化7 d后,表达GFAP的阳性细胞占61.78%±3.54%,Map2阳性细胞占6.28%±0.80%,β-tubulin III阳性细胞占7.43%±1.09%,Galc阳性细胞占2.79%±0.62%。添加10 ng/m L BDNF后,表达GFAP的阳性细胞占62.76%±2.94%,Map2阳性细胞占14.29%±2.45%,β-tubulin III阳性细胞占13.13%±2.42%,Galc阳性细胞占2.97%±0.82%。分别经两独立样本t检测,BDNF组分化为Map2及β-tubulin III阳性细胞,显著高于血清组(P<0.05),但BDNF组分化为GFAP及Galc阳性细胞与血清组相比,无显著性差异(P>0.05)。结论 BDNF可显著提高Wistar大鼠骨髓源神经干细胞分化为神经元的比例。

Stem Cell Ophthalmology Treatment Study(SCOTS) for retinal and optic nerve diseases: a case report of improvement in relapsing auto-immune optic neuropathy

We present the results from a patient with relapsing optic neuropathy treated within the Stem Cell Ophthalmology Treatment Study （SCOTS）. SCOTS is an Institutional Review Board ap- proved clinical trial and has become the largest ophthalmology stem cell study registered at the National Institutes of Health to date （www.clinicaltrials.gov Identifier NCT 01920867）. SCOTS utilizes autologous bone marrow-derived stem cells （BMSCs） for treatment of retinal and optic nerve diseases. Pre-treatment and post-treatment comprehensive eye exams of a 54 year old female patient were performed both at the Florida Study Center, USA and at The Eye Center of Columbus, USA. As a consequence of a relapsing optic neuritis, the patient＇s previously normal visual acuity decreased to between 20/350 and 20/400 in the right eye and to 20/70 in the left eye. Significant visual field loss developed bilaterally. The patient underwent a right eye vitrectomy with injection of BMSCs into the optic nerve of the right eyeand retrobulbar, subtenon and in- travitreal injection of BMSCs in the left eye. At 15 months after SCOTS treatment, the patient＇s visual acuity had improved to 20/150 in the right eye and 20/20 in the left eye. Bilateral visual fields improved markedly. Both macular thickness and fast retinal nerve fiber layer thickness were maximally improved at 3 and 6 months after SCOTS treatment. The patient also reduced her mycophenylate dose from 1,500 mg per day to 500 mg per day and required no steroid pulse therapy during the 15-month follow up.