Experimental treatment of the model mice of Duchenne muscular dystrophy by bone marrow transplantation

AIM: To detect dystrophin expression in skeletal muscles of mdx mice after bone marrow transplantation (BMT), and to evaluate the effect of BMT on Duchenne muscular dystrophy (DMD). METHODS: Bone marrow cells were cultured for three days, and then transplanted into mdx mice irradiated lethally through tail veins. After 4 and 6 months, dystrophin expression on myocytes membranes in mdx mice was detected by fluorescent immunohistochemical staining. The centrally nucleated fibers (CNF) were calculated by HE staining, and the physiologic parameters measured and the motor function detected by traction test, rotating rods test and rotating wheels test were also observed. RESULTS: Until 4 and 6 months after BMT, dystrophin was expressed partly on myocytes membranes in mdx mice, and the ratio of CNF decreased, physiologic functions improved, the motor ability reinforced in treated group. CONCLUSION: After BMT, marrow stem cells settled in injured skeletal muscles and bone marrow, then differentiated into myocytes with dystrophin expression and caused the improvement of pathology, physiology and motor function in treated group finally. These results give a powerful proof for the treatment of DMD with BMT.     

Distribution of mesenchymal stem cells in mdx mice after transplantation

AIM: To investigate the distribution of mesenchymal stem cells in mdx mice after transplantation. METHODS: P5 mesenchymal stem cells (MSCs) of SD rats labeled with ［3H］－TdR were injected intravenously into the mdx mice preconditioned with 7 Gy γ ray. The mice were killed at 24 h, 48 h, 2 weeks, 1 month, 2 months, 4 months after transplantation of MSCs. Blood, lung, liver, bone marrow, heart, and skeletal muscle were collected, then the irradiated quantity was detected to calculate tissue specific localization account using scintillascope. RESULTS: Specific localization account in lung was the highest at 24 h. At 48 h liver was the highest. After transplantation the account of bone marrow increased and at 2 weeks reached the highest, then decreased as time going but was still higher than that of other organs. The account of skeletal muscle and heart also increased. CONCLUSION: At early time after transplantation, the MSCs labeled by ［3H］－TdR mainly distribute in lung and liver, then homing to bone marrow increasingly and the account is the highest at 2 weeks. MSCs migrate to injured organs, such as skeletal muscle and heart. The migration suggests that MSCs can settle down in muscles and provide evidence for MSCs to differentiate into myocytes.     

Transplantation of bone marrow mesenchymal stem cells improves motor function in a DMD mouse model

AIM: To observe amelioration of motor function in a Duchenne muscular dystrophy (DMD) mouse model (dko mice) after transplantation of bone marrow mesenchymal stem cells (MSCs). METHODS: Passage fifth MSCs cultured in vitro were transplanted into dko mice by tail vein, motor functions of experimental mice and matched control mice, including traction, rotating rods, rotated wheel, upside down, turning over and walking (all were recorded by Sony digital camera) were tested 15 weeks after transplantation. The fluorescent expression of dystrophin and utrophin in gastrocnemius muscle tissue of dko mice was detected by SABC-Cy3, and average optical density of positive fibers was calculated. RESULTS: MSCs grew in colony over passage third, and there was low immunologic reaction by vein transplantation. There was dystrophin and utrophin fluorescent expression in sarcolemma of dko mice 15 weeks after transplantation, but no any fluorescent expression in controls. There was significant difference in fluorescent average optical density of positive fibers between two groups (P＜0.05). Amelioration of motor functions in dko mice was found 15 weeks after MSCs transplantation compared with the control mice (P＜0.05). CONCLUSION: Transplantation of MSCs ameliorates the positive and passive motor functions of dko mice.     

Myoblast transplantation in mdx mice prevents muscle damage by exercise

AIM: To observe skeletal muscle damage of mdx mice after overload exercise, and protection to muscle damage induced by exercise due to myoblast transplantation (MTT). METHODS: Muscle samples of C₅₇ mice were minced and digested with trypsin, and myoblasts were cultured ex vivo, purified and detected by immunohistochemistry stains. The myoblasts were injected into muscle of left limb of mdx mice, whereas the right limb was injected with DMEM liquid as control. Mice were submitted to exercise for 3 days starting 1 month after MTT, and then Evans blue was injected intravenously through the tail vein. The muscle cryostat sections of mdx mice were made, and then detected the immunofluorescence of dystrophin. Under a fluorescence microscope, the number of fiber stained with Evans blue and dystrophin was counted, analyzed quantitatively with image software. RESULTS: Under a fluorescence microscope, only 10.37%±2.87% muscle fibers in the myoblast grafted muscles were stained with Evans blue In contrast, 26.82%±14.85% muscle fibers in right control muscles were stained. Significant differences between these two groups were showed (P＜0.05). A average of 48.32%±6.54% of muscle fibers were dystrophin positive in grafted muscles, whereas few dystrophin positive muscle fibers in control side. None of the fibers expression dystrophin was stained with Evans blue. CONCLUSION: MTT in mdx mice has a protective effect against muscle damage by exercise.     

Distribution and identification of immunocytes in humanized SCID mouse model

AIM: Humanized-NOD/SCID(hu-NOD/SCID) mouse model was established and the level of immune reconstitution was assessed in this model. METHODS: Mononuclear cells (MNC) and CD34⁺ cells were isolated or sorted from cord blood(CB). Human CD45, CD19, CD3 markers on cells from NOD/SCID murine peripheral blood(PB), bone marrow(BM), thymus were detected by FCM from 4 to 10 weeks after hematopoietic stem cell transplantation. After 10 weeks, the gene expressions of the human β₂M and RAG2 were detected by RT-PCR in PB or bone marrow of mice model. RESULTS: Human CD45, CD19, CD3 cells populations in PB and BM were found by flow cytometry in mice model transplanted with CD34+ cells or CB MNC from 4 to 10 weeks. The highest positivity of human lymphocytes was at 8 week after transplantation. The levels of human cell engraftment in mice transplanted with CD34⁺ cells were higher than those in mice transplanted with CB MNC. The mRNA of human β₂M and RAG2 were found by RT-PCR in BM.CONCLUSION: The higher level of human lymphocyte engraftment is established in NOD/SCID mouse model transplanted with CD34⁺ compared with CB MNC. The maturation of T lymphocytes could be happened in bone marrow of mice model.     

Effect of bone marrow mesenchymal stem cells on engraftment of hematopoietic stem/progenitor cells in sensitized mice

AIM: To evaluate the effects of bone marrow-derived mesenchymal stem cells (MSCs) on engraftment of hematopoietic stem/progenitor cells in sensitized mice. METHODS: Mouse bone marrow-derived MSCs were cultured by adherent culture method. MSCs combined with or without hematopoietic stem/progenitor cells were implanted into the sensitized mouse model, which was established by allogeneic splenocyte transfusion, and were divided into 6 groups: MSC intervention groups, including sensitized mice with MSCs on day 11, sensitized mice with MSCs on day 0 and sensitized-mice with MSCs both on day 11 and day 0; control groups, including sensitized mice without MSC intervention, non-sensitized mice without MSC intervention and non-sensitized mice without MSCs or transplantation of hematopoietic stem/progenitor cells. The survivors were assessed after transplantation and hematopoietic recovery was monitored weekly including hematological change, immune function reconstruction, bone marrow cell recovery, chimera analysis and graft-versus-host disease development. RESULTS: Compared with different control groups, MSC intervention did not prolong the survival rates of the sensitized model mice after lethal irradiation. CONCLUSION: Under the experimental conditions, MSC combined with C57BL/6 bone marrow hematopoietic stem/progenitor cells fail to promote the growth of engraftment in C57BL/6 allogeneic splenocyte-sensitized BALB/c mice in vivo.     

Comparison between mobilization and transplantation of bone marrow stem cells for the therapy of myocardial infarction in rabbits

AIM: To compare bone marrow stem cell mobilization with bone marrow-derived mononuclear cells (BMCs) transplantation for the therapy of myocardial infarction (MI) in rabbits, and to explore more effective and practical stem cell therapeutic strategy for MI. METHODS: In mobilization group (M, n=10), granulocyte-colony stimulating factor (G-CSF) (30 μg·kg-1·d-1) was injected subcutaneously 3 hours after MI and every 24 hours for 5 days. On the 5th day, the BMCs from 10 mL peripheral blood were labeled with bromodeoxyuridine (BrdU) for 24-48 hours, then reinjected intravenously. In transplantation group (T, n=10), BMCs transplantation was performed 5-7 days after MI. After being obtained from bone marrow (3-5 mL) of iliac crest and labeled with BrdU for 24-48 hours, BMCs were transplanted into infracted myocardium through intramyocardial injection. Control animals (C, n=10) did not receive any treatment after MI. Echocardiography were performed for the evaluation of cardiac function 1 week and 5 weeks after MI. Hemodynamic studies and histological study were performed 5 weeks after MI. RESULTS: LV ejection fraction increased significantly in group M, had no change in group T, and decreased 1 week and 5 weeks after MI in group C. Group M and group T had higher LV max +dp/dt and max -dp/dt, lower LV end-diastolic pressure compared with group C 5 weeks after MI. Histological studies revealed that there were BrdU positive cells in the infarcted area in group M and group T. The vascular density of group M and group T in the infarcted area was significantly greater in comparison with group C. No regeneration of smooth muscle cells and cardiomyocytes were found in the infarcted area. CONCLUSION: Bone marrow stem cell mobilization with G-CSF and transplantation of BMCs both significantly improve the cardiac function for the therapy of MI through vascular genesis in the infarcted area. Bone marrow stem cell mobilization may offer a new and non-invasive therapeutic strategy for MI.     

Generation of insulin-producing cells from PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells

AIM: To differentiate bone marrow mesenchymal stem cells (BMSCs) into functional insulin-producing cells to produce sufficient pancreatic islet cells for transplantation. METHODS: Recombinant adenovirus vectors carrying PDX1 and NKX6.1 genes were constructed and the bone marrow mesenchymal stem cells were infected by the recombinant adenovirus combined with several cytokines for differentiation. The expressions of PDX1, NKX6.1 and insulin and C-peptide in the differentiated bone marrow mesenchymal stem cells were detected by RT-PCR and Western blotting. After the differentiated bone marrow mesenchymal stem cells were transplanted into subrenal capsule of diabetic mice, cell morphology of the grafts as well as their secretion of insulin and C-peptide were detected. Besides, regulating capacities of grafts on the blood glucose level of the diabetic mice were also detected. RESULTS: BMSCs induced by recombinant adenovirus (pAdxsi-CMV-PDX1/CMV-NKX6.1) and several cytokines showed positive dithizone staining and the expressions of β-cell related molecule such as insulin and glucose transporter 2 were detected by RT-PCR, which showed a sustaining and stable expression. The similar results were showed by Western blotting, immunohistochemical staining and indirect immunofluorescence. The insulin secretions in the cells stimulated with glucose at concentrations of 5.5 and 25 mmol/L in the experimental group were (1 240.4±109.3) mU/L and (3 539.8±245.1) mU/L, respectively, and were significantly higher than those in control group. Moreover, transplantation of the cells to STZ mice in treatment group made serum glucose recover to normal level. CONCLUSION: PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells differentiate into insulin-producing cells in vitro. When these cells transplanted into STZ induced diabetic mice, their serum glucose could return to the normal level and they could live well. Thus this is a promising method for diabetes treatment.     

Therapeutic action of transplantation of marrow mesenchymal stem cells into infarction area after cerebral middle artery occlusion in rats

AIM: To explore the differentiation and the functional behavior of marrow mesenchymal stem cells (MSC) transplanted into the cerebral infarction area after cerebral middle artery ischemia in rats. METHODS: MSC were isolated from human rib marrow and cultured in L DMEM medium in vitro. The model of rat cerebral infarction by cerebra middle artery occlusion was established, and the identified MSC were transplanted intracerebrally 10 days later. Immunohistochemistry technique was used to identify the cell survivor and its differentiation to the neurogenesis in the transplantation site, and at 2 weeks and 6 weeks after transplantation, the functional tests were comparatively studied. RESULTS: The results showed that the survivor of transplanted MSC was differentiated to neural phenotype cells, and the functional behavior of the injury rats was recovered significantly after MSC transplantation (P<0.05). CONCLUSION: Our data suggest that transplantation of MSC may be a powerful autoplastic therapy for the stroke.     

Effect of mesenchymal stem cells infusion on hematopoietic recostitution after peripheral blood stem cell transplantation in mice

AIM: To study the effect of mesenchymal stem cells (MSCs) infusion on hematopoietic recovery after peripheral blood stem cell transplantation in mice. METHODS: BALB/c mice conditioned by high dose chemotherapy/radiotherapy were infused with10⁶ peripheral blood mononuclear cells (PBMC) mobilized by granulocyte colony-stimulating factor (PBSCT group),10⁴ MSCs culture-expanded in vitro and10⁶ PBMC(experimental group1),10⁶ MSCs and10⁶ PBMC(experimental gruop 2). Survival rate within 4 weeks, white blood cell count, bone marrow nucleated cells (BMNC), granulocyte-macrophage colony forming unit(GM-CFU) and fibroblast colony forming unit (F-CFU) were examined. RESULTS: Survival rate, BMNC, GM-CFU, F-CFU were significantly higher in experimental group 2 than that in PBSCT group (P<0.05), WBC recovery was faster (P<0.01) and F-CFU level was higher in experimental group1than that in PBSCT group (P<0.05). CONCLUSION: Mesenchymal stem cells infusion enhanced hematopoietic reconstitution after peripheral blood stem cell transplantation.     

Remaining residual bone marrow cells through bone marrow transplantation from female to male in mice

AIM:To investigate the remaining residual bone marrow cells after bone marrow transplantation (BMT) from female to male in mice by detecting the male Y chromosome from the blood cells. METHODS:Bone marrow cells from either male or female C57BL/6 mice were injected via tail vein to the corresponding male or female mice at 1×107 per animal 6 h after irradiation exposure to different doses of ［137Cs］. The survival rate of BMT was calculated after 14 d. The numbers of leukocytes in peripheral blood were measured and Y chromosome levels were also assayed in the male recipent mice. RESULTS:With radiation doses of 1 000 rad and 950 rad, the hematopoietic function of the female recipient mice quickly restored, but the male recipient mice had only 48% survival rate. With the radiation dose of 900 rad, the male recipient mice all survived and their hematopoietic function quickly restored. The peripheral leukocyte counts returned to normal 13 d after BMT. The Y chromosome genes in the peripheral blood cells were detected in 5 weeks after BMT in the male recipient mice, suggesting that the bone marrow cells in the male mice were completely destroyed by radiation, and the bone marrow cells from female mice completely replaced those in the male mice. CONCLUSION: After irradiation at a dose of 900 rad, the male mice can be used as BMT recipients without endogenous bone marrow cells. This study warrants male recipient mouse model in BMT for further investigation on the function of bone marrow cell-specific genes after global gene manipulations of the animals.     

Differentiation of bone marrow mesenchymal stem cells in dilated cardiomyopathy

AIM: To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy (DCM). METHODS: 20 rabbits were randomly divided into two groups. MSCs were isolated from bone marrow and cultured. Adriamycin was applied to the two groups to create rabbit DCM models. At 3 weeks after the creation of DCM models, the experiment group animals received intramyocardial injection of autologous MSCs. 4 weeks after transplantation, the implanted sites were examined to identify the labelled cells and to investigate its differentiation through immunofluorescence. RESULTS: At 4 weeks after the MSCs transplantation, the implanted cells were found in the experiment group and some differentiated into cardiomyocytes and vascular endothelial cells, which was not founded in the control group. CONCLUSION: Autologous bone marrow mesenchymal stem cells can differentiate into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy.     

Transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells improves motor function in a MCAO rat model

AIM:To observe the amelioration of motor function in a media cerebral artery occlusion (MCAO) rat model after transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells. METHODS:Transplant neuron-like cells derived from rat bone marrow mesenchymal stem cells, which were isolated, cultivated, predicated and induced in vitro, were introduced into infracted cerebral cortex. L-DMEM were injected in control group. Screen test, beam test, prehensile traction test and Morris maze test were conducted at 2 and 8 weeks alternately after transplantation. Rat brain tissues were stained with TTC and percentage of infracted volume was analyzed. RESULTS:There was significant difference between control group and test group in the time and length for rat finding aim and in the grade and time of screen test, beam test, Prehensile traction test (P<0.05). CONCLUSION:Transplantation of neuron-like cells induced from rat bone marrow mesenchymal stem cells improves the motor function of MCAO rats.     

Skeletal muscles of mdx mice were damaged after overload exercise

AIM:To observe the effects of overload exercise on skeletal muscles in X-linked muscular dystrophy(mdx) mice.METHODS:Mdx mice and C57 mice were carried out swimming and hanging tail movement tests (mdx mice as control did not exercise). It lasted for 13 minutes each time per day, and lasted 3 days. Evans blue was injected into tail vain. The mice were killed the next day, and the hind limbs were taken photographs after skins were flayed. The gastrocnemius muscles and diaphragms cryostat sections were made. Under a fluorescence microscope, Evans blue staining was seen. Then the sections were tested by routine HE staining, the histological change of muscles was analyzed under a light microscope.RESULTS:Many blue colored longitudinal lines were observed in skeletal muscles of mdx mice, whereas they were hardly seen in control mdx and C57 mice. Under a fluorescence microscope, some muscle fibers of mdx mice were stained with Evans blue, few muscle fibers of control mdx mice were stained, and C57 mice were not. Under a light microscope, HE staining of muscles showed some degenerated muscle fibers became round in shape and the myonuclei became condensed, or necrotic fibers had amorphous structures, most of them in the degenerated and necrotic fibers of diaphragms C57 mice did not have these changes.CONCLUSION:Overload exercise did harm to skeletal muscles of mdx mice; Vital staining with Evans blue is useful not only for distinguishing degenerating muscle fibers, but also for studying the degeneration process in dystrophin-deficient muscle.     

Effect of human β-NGF gene-modified bone marrow-derived mesenchymal stem cells on rotational behavior of rats with Parkinson disease

AIM：To investigate the effect of human β-nerve growth factor (β-NGF) gene-modified bone marrow-derived mesenchymal stem cells (MSCs) transplantation on the rotational behavior improvement in a rat model of Parkinson disease (PD). METHODS：The rat model of PD was established successfully and the animals were divided into 4 groups:β-NGF-MSCs group (transplanted with 5×105 β-NGF-engineered MSCs), MSCs group (transplanted with 5×105 MSCs), DMEM/F12 group (5 μL transplantation medium was injected in the right striatum of the rats) and PD model group (without transplantation). The rotational scores were assessed 2 weeks, 4 weeks and 6 weeks after transplantation. At different time points after transplantation, the rats were tested for apomorphine (APO)-induced rotational behavior and the brains of the PD model rats were examined by fluorescence microscopy and immunohistochemical staining. RESULTS：Transplantation of human β-NGF gene-modified MSCs effectively improved the behavioral performance in the rats. At the 2nd, 4th and 6th weeks after cell transplantation, the rotational frequencies after injection of APO decreased significantly in β-NGF-MSCs group compared with MSCs group and PD group (P<0.05). Both β-NGF gene-modified MSCs and MSCs survived in the brains of PD model rats, had good compatibility with the host cells, and showed no signs of destroying the host and the glial cicatrisation. The β-NGF gene-modified MSCs expressed β-NGF stablely in the brains of PD model rats, and showed obvious improvement of the rotational behavior in the PD model rats induced by APO compared with MSCs group. CONCLUSION:The behavior of the rats with PD is significantly improved by transplanting β-NGF-modified MSCs in right striatum, and β-NGF gene therapy has potential clinical value．     

Orthotopic transplant fetal rat pancreatic stem cells for treating diabetes mellitus

AIM: To observe the possibility of differentiation of fetal rat pancreatic stem cells into islet-like cell cluster by transplantation of fetal rat pancreatic stem cells into pancreatic parenchyma in diabetic SD rats. METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16. SRY DNA was examined to discriminate gender by fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult SD rats were divided into three groups including 10 pancreatic parenchymal orthotopic transplantation, 10 experimental controls and 10 normal controls. In orthotopic transplantation group, 1×10⁶ male fetal pancreatic stem cells per rat were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume of PBS was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodically. 8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assessed by Western blotting. RESULTS: 5 of 12 fetal rats were male according to FISH. After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry with 74.1% of PSCs expressed nestin. The orthotopic transplantation of PSCs led to stable reduction in hyperglycemia and increase in insulin level in serum (3 weeks after transplantation), culminating (5 weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipients pancreata 8 weeks post-transplantation. The expression levels of insulin mRNA and protein in the orthotopic transplanted recipients pancreata were higher than those in experimental control (P<0.05), and the expressions of PDX-1 mRNA and protein were also higher than those in normal control (P<0.05). CONCLUSION: When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat differentiates into islet-like cell cluster, gains comparable function with normal islets and reverses experimental diabetes.     

水杨酸和高温锻炼与葡萄抗热性及抗氧化的关系

 对‘京秀’葡萄(Vitis vinifera CV．Jingxiu)高温锻炼和外施水杨酸(salicylic acid，SA)都能显著提高其幼苗的抗热性。高温锻炼1 h，叶片内自由态sA含量急剧升高，其后又迅速下降。在抗热性诱导过程中(高温锻炼12 h或水杨酸喷施后6 h)，抗氧化酶系统中的过氧化物酶(POD)、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)、抗坏血酸过氧化物酶(APX)的活性都明显升高，但过氧化氢酶(CAT)活性下降；高温锻炼或外施SA 1 h，过氧化氢(H2o2)含量急剧升高，之后又迅速下降，可能起着一种信号分子的作用。以上结果说明内源sA可能通过提高抗氧化酶活性参与了高温锻炼过程，外施sA和高温锻炼有相似的提高抗热性机制。     

Origin of smooth muscle cells in vascular neointima from recipient bone marrow cells in rat aortic allograft

AIM: To study the expression of Sry gene in neointimal smooth muscle cells (NI-SMCs),and to investigate the origin of NI-SMCs in rat aortic allograft.METHODS: Sex-mismatched bone marrow transplantation was performed from male Wistar rat to female Wistar rat.Four weeks after transplantation,the aortic transplant model was constituted by means of micro-surgery in rat.The recipients were divided into four groups: female-female aortic isografts,female-female aortic allografts,male-male aortic allografts,female-chimera aortic allografts.Eight weeks after transplantation,aortic grafts were removed and processed for histological evaluation and immunohistochemistry assay.The Sry gene-specific PCR was performed on the genome of NI-SMCs to analyze its origin involved in aortic allograft.RESULTS: Excessive accumulation of α-SMA-positive SMCs resulted in significant neointima formation in rat aortic allografts.The neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all aortic allograft groups compared with those in aortic isograft group (P<0.01).PCR assay indicated that a distinct DNA band with 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively,but not in the female-female aortic allograft group,was observed.CONCLUSION: As the origin of NI-SMCs,recipient bone marrow cells contribute to the pathological neointimal hyperplasia of aortic allograft.     

The prevention of acute graft versus host disease by Rhodamine 123-mediated photodynamic therapy

AIM: To evaluate the effect and safety of Rhodamine 123 (Rh123)-mediated photodynamic treatment (PDT) on acute graft versus host disease. METHODS: An acute graft versus host disease (aGVHD) mice model was established using C57B/6 mice as donors and BALB/c mice as recipients. Mixed lymphocytic cells were cultured with Rh123 (50 nmol/L) and irradiated by argon laser 30 mW/cm2 for 3 min, then transplanted to BALB/c recipient mice mixed with donor bone marrow. Hepatopoietic recovery, aGVHD occurrence, survival time after transplantation and pathological changes were observed. In addition, CD3+CD69+ positive rates of MLC were examined by flowcytometry. RESULTS: Occurrence of aGVHD decreased, degree of pathological manifestation became milder, survival rates were higher than non PDT groups. CD3+CD69+ rates of MLC cells treated with photodynamic therapy (PDT) and cultured for 24 h significantly decreased. CONCLUSION: Rh123-mediated PDT can effectively prevent aGVHD of allogeneic bone marrow transplantation in mice.     

Effectiveness and pathologic changes of transplantation of human mesenchymal stem cells in SOD1-G93A mouse model of familial amyotrophic lateral sclerosis

AIM: To study intravenous transplantation of human mesenchymal stem cells (hMSCs) on the life span and pathological change of SOD1-G93A amyotrophic lateral sclerosis (ALS) mice. METHODS: hMSCs were cultured and expanded from heparinized bone marrow cells from healthy donors and the purity and features were identified with FCM. hMSCs (3×106) resuspended in 0.3 mL DMEM or 0.3 mL DMEM only were injected into the tail vein of genotyped SOD1-G93A ALS mice. The mice were evaluated for signs of motor deficit with 4-point scoring system according to Weydt and the onset and life span were assessed. The pathological change was observed with Nissl staining and number of motor neuron was counted. RESULTS: The onset symptoms in untreated SOD1-G93A ALS mice appeared at (156.6±3.6) d of age and the average life span was (188.3±3.5) d. hMSCs transplantation delayed the onset of ALS type symptoms about 14 d and prolonged the life span about 18 d compared to the untreated SOD1-G93A littermates. The loss of motor neurons in untreated mice was much faster and severer than that in hMSCs transplanted mice. At 16 th week and 20 th week, motor neurons of untreated mice were significantly fewer than those of transplanted mice. β-globin gene in brain was detected in transplanted ALS mice. CONCLUSION: hMSCs migrate to central nervous system after intravenous transplantation, prolong the life span and delay the onset and motor neuron loss in SOD1-G93A ALS mice.