Effect of bone marrow stem cell transplantation on mdx mice at different ages

AIM: To study the effect of bone marrow stem cell transplantation on mdx mice at different ages. METHODS: The bone marrow stem cells of C57BL/6 mice (4-to-weeks age) were cultured in vitro for 3 days, then injected intravenously into the 6-week and 8-week aged mdx, which were preconditioned with 7 Gy γ ray. 12 weeks after being transplanted, the mdx mice were studied for the dystrophin protein expression on the skeletal muscle membrane. RESULTS: Three months after transplanted with bone marrow stem cells, about 16% and 7% muscles cells in 6-week and 8-week mdx mice expressed dystrophin protein， respectively. CONCLUSION: 12 weeks after transplantation with bone marrow stem cells of homologous series mice, different amounts of dystrophin protein expressed on the membrane of skeletal muscle cells were observed in different aged mdx mice. Bone marrow stem cell transplantation show more benefic effect for younger mdx mice.     

Effect of mesenchymal stem cell transplantation on papain and ［60Co］ irradiation-induced rat pulmonary emphysema

AIM: To investigate the effect of bone marrow mesenchymal stem cells(MSCs) transplantation on papain and ［⁶⁰Co］ irradiation-induced rat pulmonary emphysema and the underlying mechanisms.METHODS: Female rats were randomly divided into three groups: control group, emphysema group, emphysema+MSCs transplantation group. Rats were exposed to ［⁶⁰Co］ irradiation and instilled with papain intratracheally. Bone marrow MSCs from male rats were infused through tail veins. Morphologic analysis of the lung tissue was performed. The engraftment of male bone marrow MSCs in female recipient lung was determined by PCR of Sry gene and Y chromosome fluorescence in situ hybridization (Y-FISH). Sry gene was amplified by PCR using the genomic DNA from the lungs as template. Surfactant protein C (SP-C) immunofluorescent staining and Y-FISH were performed on the same lung section to determine whether the engrafted bone marrow MSCs differentiated into type II pneumocytes. RESULTS: Destruction of alveolar walls was observed in rat lungs from emphysema group and emphysema+MSCs transplantation group. MSCs transplantation significantly ameliorated the emphysematous changes. Significant differences in the mean linear interval (MLI), the mean alveoli number (MAN) and mean alveoli area (MAA) between emphysema group and emphysema+MSCs transplantation group were observed. DNA fragment of Sry gene was amplified in the genomic DNA from the rat lungs in emphysema+MSCs transplantation group. Y chromosome positive cells were observed in the lungs tissue from emphysema+MSCs transplantation group. Some of the Y chromosome positive cells also expressed SP-C, the marker of type II pneumocytes. CONCLUSION: Bone marrow MSCs transplantation improves papain and irradiation-induced pulmonary emphysema. The underlying mechanisms may include the engraftment of bone marrow MSCs in the lungs and differentiation of MSCs into type II pneumocytes.     

Effect of irradiation on the engraftment of rat bone marrow mesenchymal stem cells in different organs of recipient rats

AIM: To investigate the effect of irradiation on the engraftment of rat bone marrow mesenchymal stem cells in different organs of recipient rats and possible mechanisms. METHODS: The apoptosis in the irradiated rats heart, kidney, liver and lung was assessed by terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) at day 14 after irradiation. MSCs cultured from male rats were delivered systemically into irradiated ［60COγ］ and nonirradiated syngeneic female rats sacrificed at day 28 after MSCs transplantation. Tracking of MSCs was determined by PCR and Y chromosome fluorescent in situ hybridization (Y-FISH). RESULTS: The numbers of apoptotic cells in the heart, kidney, liver and lung were significantly greater in the irradiated rats compared with those in the nonirradiated controls. Sry gene was amplified in the irradiated recipient heart, kidney, liver and lung. Moreover, Y chromosome positive cells in these organs of the irradiated recipient rats were observed. However, no Sry gene sequence and cells with Y chromosome were found in the nonirradiated recipients. CONCLUSION: Whole body irradiation induces apoptosis and results in engraftment of MSCs in the recipients heart, kidney, liver and lung.     

Biological characteristics of bone marrow mesenchymal stem cells from GFP transgenic mice transfected with human β-nerve growth factor gene

AIM:To investigate the changes of biological characteristics of GFP transgenic mouse bone marrow mesenchymal stem cells (MSCs), which were transfected with human β-nerve growth factor (β-NGF) gene in a recombinant eukaryotic expression vector. METHODS:MSCs obtained from GFP transgenic mice were isolated, cultured and purified by the whole bone marrow adherence methods. Human β-NGF gene was transfected into the MSCs by a recombinant eukaryotic expression vector. The β-NGF expression in the MSCs was detected by the method of immunocytochemistry. Hippocampal neurons from neonatal mice were cultured with culture supernatant of the MSCs transfected with pcDNA3-β-NGF and the biological characteristics of the MSCs were investigated 3 d after culture under inverted phase-contrast microscope. RESULTS:The β-NGF positive rate of MSCs in pcDNA3-β-NGF transfection group ［(37.12±2.14)%］ was significantly higher than that in MSCs control group ［(2.36±0.62)%］ and blank control group ［(1.43±0.76)%］.The neurite length of neonatal mouse hippocampal neurons cultured with culture supernatant from pcDNA3-β-NGF-transfected MSCs ［(31±3)μm］ was significantly longer than that in negative control group ［(23±4)μm］, suggesting that MSCs transfected with β-NGF gene maintained better biological characteristics. CONCLUSION: The constructed recombinant eukaryotic expression vector of human β-NGF gene can be transfected into MSCs efficiently and NGF can be effectively expressed in MSCs. MSCs transfected with β-NGF gene are capable of stable expression and secretion of β-NGF, and maintenance of better biological characteristics.     

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.     

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.     

Adrenomedullin gene transfection enhances the therapeutic effects of transplantation of bone marrow mesenchymal stem cells on cardiac function and ventricle remodeling in acute myocardial infarction rats

AIM: To investigate adrenomedullin gene transfection enhances the therapeutic effects of homogeneous transplantation of bone marrow mesenchymal stem cells (MSCs) on cardiac function and ventricle remodeling in acute myocardial infarction rats. METHODS: MSCs were isolated and expanded using the preplating method. The infection efficiency of adenovirus vector to MSCs was tested by X-gal staining. Ad-ADM expression in MSCs and its secretion in culture medium were measured by ELISA. The left anterior descending branch of rats was ligated to establish a myocardial infarction model. The MSCs were labeled by DAPI, and were directly implanted into the acute infarct site via focal injection. Four weeks later, cardiac function was evaluated using physiological recorder. Hearts were harvested and sliced to be analyzed by immunohistochemistry (factor Ⅷ and ADM) and the DAPI-labeled cells were identified. Sirius red staining was used to identify interstitial collagen on slides. Analysis of collagen type I and III was performed using a polarized filter on sections stained for collagen with Sirius red, and the ratio of collagen type I and III were detected. RESULTS: With X-gal staining, MSCs were effectively transfected by adenovirus in vitro. The transfection efficiency showed the dose-effect relationship with multiplicities of infection (MOI). When MOI was 150, the infection efficiency was 95.4%. The expression of ADM was traced in culture medium and expressed in the time-dependent manner. A maximum production of ADM was observed at 7 d after infection ［(26.53±1.42) ng/L vs (1.34±0.08) ng/L, P<0.05］, and ADM secretion reduced to normal level at 15 d ［(2.20±1.44) ng/L vs (1.52±0.33) ng/L, P>0.05］. DAPI-labeled MSCs transplantation was found in the hearts of the recipients. Immunohistochemical studies demonstrated that intense immunostaining for ADM was higher in Ad-ADM plus MSCs group, compared to other groups. Compared with control, MSCs transplantation significantly increased capillary density in infarct area (P<0.01). A combination of Ad-ADM trensfection and MSCs transplantation demonstrated a further increase in capillary density compared with Ad-ADM or MSCs alone. MSCs transplantation decreased the ratio of collagen type I and III, obviously improved the left ventricular functions. Furthermore the combination treatment resulted in further decrease in the ratio of collagen type I and III, and significantly improved the left ventricular functions. CONCLUSION: Ad-ADM transfection enhances the angiogenic potency of MSCs transplantation and decreases the ratio of collagen type I and III through increasing ADM expression in infarct area, thus contributes to reverse the ventricular remodeling and improves the cardiac function.     

Transfusion of mesenchymal stem cells improve the hematopoiesis in mice with aplastic anemia by regulating immune cells

AIM: To study the possible immunologic mechanism of mesenchymal stem cells (MSCs) by which the hematopoiesis of mice with aplastic anemia (AA) is improved. METHODS: The mice model of immuno-mediated aplastic anemia was established. Thirty BALB/c mice were divided into 3 groups: radiation group, AA model group and MSCs group. The mice in radiation group only had processing of 5 Gy ［60Co］γ radiation. The mice in AA model group were intravenously injected with the cell suspension of thymocyte and lymph-node cell mixture (prepared from DBA/2 mice, 1×106 cells) after 5 Gy ［60Co］γ radiation. The animals in MSCs group received the same treatment as the mice in AA model group did, and afterwards was injected with 1×106 MSCs intravenously at 3 days. All the mice in the 3 groups were observed and analyzed the following parameters: peripheral blood cells, pathological features of bone marrow, the relation between the changes of peripheral CD4+, CD8+, CD4+/CD8+, NK cells and the hematopoiesis. RESULTS: After 5 Gy ［60Co］γ radiation, the peripheral blood cells in all groups decreased at 7 days, while at 21 days those in MSCs group and radiation group restored to normal but those in AA model group was still at lower level. At 7 days, the cells of CD4+, CD8+ and CD4+/CD8+ in each group were similar without statistical difference, but the number of NK cells in MSCs group was quite lower than that in the other 2 groups (P<0.05). At 14 days, the CD4+ cells in all groups were obviously decreased, while the CD8+ cells in MSCs group and AA model group were significantly higher than those in radiation group. At 21 days, the CD8+ and NK cells in MSCs group were quite the same as those in radiation group while those in AA model group were obviously higher compared to the other 2 groups. The number of adipose cells observed in the pathological slice of femoral bone in MSCs group and radiation group was no difference while that in AA model group was much higher. CONCLUSION: MSCs transfusion improves the hematopoiesis in mice with aplastic anemia by regulating the functions of immune cells.     

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.     

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.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

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.     

Effect of mesenchymal stem cells transfected with human heme oxygenase-1(HO-1)gene on myocardial apoptosis and angiogenesis

AIM: To investigate the effects of mesenchymal stem cells(MSCs)transfected with human heme oxygenase-1(HO-1)gene on myocardial apoptosis and angiogenesis. METHODS: MSCs were acquired from the bone marrow of adult rats. The cells were isolated, purified, cultured, and transfected with Adv-HO-1 in vitro before transplantation. At 1 h after left coronary artery ligation, Adv-HO-1-MSCs or MSCs were directly injected into the border of cardiac infarction in rats. Western blotting analysis was used to measure HO-1, and Bax protein expression in the border of cardiac infarction. ELISA was used to measure the expressions of VEGF and bFGF in the border of cardiac infarction. At 4 weeks after transplantation, the heart functions in survival rats were examined by the Buxco system. The rats were killed, then the myocardial infarct size was measured with Masson’s trichrome, and the expression of CD34 in myocardial infarction area was detected by immunohistochemical method. RESULTS: HO-1-MSCs exhibited increased HO-1 expression. The expression of HO-1, VEGF and bFGF in the border of cardiac infarction in the rats treated with HO-1-MSCs were higher than those in the rats treated with MSCs and PBS(P<0.01). However, the expression of apoptotic protein Bax was significantly lower than that in the rats treated with MSCs and PBS(P<0.01). The number of capillary vessels in the border of cardiac infarction in the rats treated with HO-1-MSCs was significantly higher than that in the rats treated with MSCs and PBS. The cardiac function in the rats treated with HO-1-MSCs was better than that in the rats treated with MSCs and PBS(P<0.01). CONCLUSION: The favorable effect on heart function appears to be a combined outcome of HO-1 and paracrine factors released by MSCs.     

Preliminary investigation on combining autologous marrow stromal cells transplantation with granulocyte colony stimulating factor for improvement of rabbit cardiac performance after actue myocardiac infarction

AIM: To test the hypothesis that autologous marrow stromal cells (MSCs) transplantation combined with granulocyte colony stimulating factor (G-CSF) can enhance cardiac function of ischemic hearts in vivo.METHODS: In order to achieve a safe and persistent effect,we explored the potential of autologous MSCs transplantation.Acute myocardial infarction induced by occlusion of left anterior descending artery,autologous MSCs labeled with BrdU bromodeoxyuridine in vitro were administered intramyocardially into the infarct area of the same donor rabbits and G-CSF was administrated by subcutaneous injection.Four weeks later,the transplanted labeled MSCs were detected by laser scanning confocal microscopy and the cardiac functions were examined by echocardiogram and multichannel physiologic recorder.Myocardial infarct size was measured from mid-transverse sections stained with Massons trichrome.RESULTS: After 4 weeks,transplanted MSCs were demonstrated myogenic differentiation with the expression of α-sarcomeric actin and connexin 43 located in intercalated disk.MSCs combined with G-CSF transplantation improved the left ventricular contractility and reduced myocardial infarct size markedly compared to that without G-CSF tratment.CONCLUSION: Our finding indicates that autologous MSCs combined with G-CSF transplantation may represent a promising therapeutic strategy on ischemic heart disease.     

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.     

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.     

南京不同类型梅花品种香气成分的比较研究

1 材料与方法 采用活体植株动态顶空套袋采集法和TCT／GC／MS（热脱附／气相色谱／质谱联用）技术分析南京梅花山真梅种系直枝梅类宫粉型‘玉露宫粉’、玉蝶型‘宇治里’、黄香型‘黄金鹤’、朱砂型‘姬千鸟’、绿萼型‘变绿萼’、洒金型‘复瓣晚跳’及垂枝梅类白碧垂枝型‘双碧垂枝’的香气组成。2002年3月513采样，将采样后的吸附管套上聚四氟乙烯套，放在干燥器中低温保存。样品分析时间为2002年4月27日。同时采集和分析空气作对照。采用Xcalibur 1．2版本软件及NIST98谱图库进行梅花香气成分的检索，兼顾挥发物出峰的保留时间鉴定。     

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.     

Influences of coculture with bone marrow mesenchymal stem cells on dopaminergic neurons in brain slice treated with 1-methyl-4-phenylpytidinium

AIM: To study the protective effects of bone marrow-derived mesenchymal stem cells on damaged dopaminergic neurons induced by 1-methyl-4-phenylpytidinium(MPP+).METHODS: The parkinson disease(PD) models were established in newborn rats. Bone marrow mesenchymal stem cells(MSCs) obtained from adult bone marrow were cultured, isolated and purified. MSCs were co-cultured with brain slice and the immunohistochemical technique, electron microscopy, propidium iodide staining were used to observe the changes of neurons. RESULTS: In the MPP+ treatment group, the neurites grew slowly and sparsely, dead cells were found in all regions. In the co-culture group, the neuritis grew densely, only a few cells were dead, the number of tyrosine hydroxylase(TH)-stained neurons increased and the structure of organellae was normal. CONCLUSION: MSCs may protect dopaminergic neurons against damage induced by MPP+. These results provide some data for cell transplantation therapy to Parkinsons disease.