PPARγ agonist pioglitazone attenuates cortical neuron lesion and gliosis in rat brain of post-traumatic injury

AIM: To investigate the neuroprotective effect of pioglitazone (Pio), a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ), on the traumatic brain injury (TBI) in rats. METHODS: SD rats were randomly divided into 4 groups: sham group, vehicle+TBI group, Pio+TBI group and Pio+T0070907+TBI group. TBI was induced by the method of controlled cortical impact (CCI) injury. Neutral red staining technique was used to determine the cortical lesion volume. NeuN, GFAP and OX-42 were measured by immunohistochemical technique to evaluate the morphology of neurons, activation and infiltration of astrocytes and microglia at the edge of cortical lesion. RESULTS: CCI injury in rat elicited activation and proliferation of the astrocytes and microglia. The glial scar wall formation at the edge of cortical lesion, which was accompanied by the loss of neurons, was observed. Pio significantly reduced the cortical lesion volume, the activation and infiltration of the astrocytes and microglia, and the loss of pyramidal neurons at the edge of cortical lesion. T0070907, an antagonist of PPARγ, reversed the effects of Pio. CONCLUSION: Pioglitazone exerts a neuroprotective efficacy, attenuates the loss of neurons and cortical lesion volume following CCI injury by inhibiting the activation and infiltration of astrocytes and microglia, especially glial scar formation.     

Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells

AIM: To investigate the function of aged bone marrow mesenchymal stem cells (BMSCs) fused with young BMSCs in mice. METHODS: The cell fusion model, which was made by C57BL/6 mouse BMSCs labeled with PKH26 membrane red fluorescence (young cells, age of 2-3 months, Y) and (old cells, age of 18-24 months, O), and young and old BMSCs of green fluorescent protein (GFP) transgenic C57BL/6 mouse, was established by the induction of polyethylene glycol 1500 (PEG 1500). The cell fusion rate and cell surface markers were detected by flow cytometry. The morphology and nuclear characteristics of the fused cells were observed under fluorescence microscopy. In this study, the age dependent changes in BMSCs proliferation and differentiation potential in Y group, O group, and another three fusion groups (Y-Y group, Y-O group, O-O group) were examined. The proliferation potentials in 5 groups were compared by counting cell numbers at days 2, 4, 6, and 8. The osteogenic and adipogenic differentiation potentials of the cells in 5 groups were determined by using standard differentiation procedures. RESULTS: The fusion rate of 30.45%±4.13% was obtained by PEG 1500 induction. No significant difference of the fusion rates in Y-Y, Y-O and O-O groups was observed. Fused BMSCs coincided with the common BMSCs were reactive to the BMSCs lineage-specific CD44, Sca-1 surface markers and negative for the hematopoietic stem cells (HSCs) lineage-specific surface markers such as CD34, CD117, CD31, and CD45. The percentage of increasing cell numbers in Y-O group was significantly higher than that in O-O group at days 2, 4, 6, and 8. The positive rate of the area stained with Alizarin red, which represents osteogenic differentiation potential of BMSCs, was significantly higher in Y-O group than that in O-O group ［(25.46%±1.52%) vs (13.85%±1.69%), P<0.01］. In Y-O group, the higher rate of the positive area stained with oil red O, which represents adipogenic differentiation potential of BMSCs, was observed as compared to that in O-O group ［(12.99%±2.61%) vs (6.03%±1.71%), P<0.05］. CONCLUSION: Aged bone marrow stem cells can be rejuvenated by cell fusion with young bone marrow stem cells, particularly the proliferation and differentiation potentials.     

Morphology and growth capacity of bone marrow stromal cells in vitro from APBSCT patients post pretreatment

AIM: To investigate the morphology and capacity of bone marrow colony forming unity-fibroblast (CFU-F) from APBSCT patients before and after pretreatment. METHODS: 21 case peripheral blood stem cell transplantation (PBSCT) patients were treated with pretreatment. The changes of morphology of the bone marrow stromal cells were assayed by light microscope and electron microscope, respectively. The numbers of CFU-F were assayed by Dexter type. RESULTS: The bone marrow stromal cells occured different types of morphology from PBSCT patients treated with chemotherapy or chemotherapy-TBI pretreatment, respectively, compared with controls. The transmission electron microscope showed that the endoplasmic reticula was dilated, the matrix of mitochondria appeared pale and the cristae of mitochondria became shorter in stromal cells from chemotherapy-TBI patients compared with those of controls. The structure of mitochondria from combined chemotherapy-TBI pretreatment appeared severe degeneration and disorder. The numbers of CFU-F from combined radiation-chemotherapy injury were significantly decreased compared with that before pretreatment and the chemotherapy injury (P<0.01), respectively. CONCLUSION: The change of cell morphology and capacity of CFU-F for bone marrow stromal cells is one of impairment injury mechanism of bone marrow hematopoietic inductive microenvironment from PBSCT patients post pretreatment.     

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.     

Astrocyte activity and autophagy after radiation-induced brain injury in rats

AIM: To investigate the activity of astrocytes and autophagy-related changes after radiation-induced brain injury (RBI) in rats.METHODS: A total of 36 Sprague-Dawley rats, weighing 180~200 g, were trained for 4 d in the Morris water maze. They were randomly divided into sham group, model group and 3-methyladenine (3-MA) group. The rats in model group and 3-MA group were given single whole-brain X-ray irradiation at a dose of 20 Gy after intraperitoneal anesthesia. After the irradiation was completed, the rats in model group was given 5 μL of NaCl into the lateral ventricle, and the rats in 3-MA group was injected with 3-MA at 600 nmol into the lateral ventricle. After 8 weeks of feeding, Morris water maze was used for measuring the learning and memory abilities. The brain tissues were taken and HE staining was used to observe the pathological changes of the hippocampus. The protein level of GFAP was determined by immunohistochemistry and Western blot for evaluating astrocyte activity. Dual fluorescence staining of GFAP and LC3 was performed for evaluating the changes of autophagy in the astrocytes. The protein level of cleaved caspase-3 detected by Western blot and TUNEL staining in the ipsilateral hippocampus were used to evaluate the apoptosis. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were examined by ELISA to assess the inflammatory response in the hippocampus.RESULTS: Radiation inhibited astrocyte activity, activated autophagy in astrocytes, and aggravated brain damage. 3-MA promoted the activation of astrocytes and promoted the repair of brain tissue damage.CONCLUSION: The injury of rat hippocampus after radiation is obvious, and the number of astrocytes is significantly reduced. 3-MA significantly attenuates the damage. This finding may provide a new approach for the treatment of radiation-induced brain injury.     

Relationship between changes of bone mineral density and bone marrow angiogenesis in ovariectomized rats

AIM: To explore the relationship between the changes of bone mineral density (BMD) and bone marrow angiogenesis in ovariectomized rats. METHODS: Thirty female Sprague-Dawley rats (3 month-age) were randomly divided into ovariectomized (OVX) groups and sham operated (sham) groups, and executed after 4 weeks, 8 weeks and 12 weeks respectively. The bone mineral density (BMD) of left femora was measured. The right distal femoral epiphysis was fixed in formalin, decalcificated by EDTA-Na2, dehydrated and embedded in paraffin. Four-μm-thick slices were obtained from the paraffin section and stained with haematoxylin-eosin (HE) for bone marrow pathological examination. The number of bone marrow microvessels was examined by means of immunohistochemical staining for CD34 to stain the endothelial cells of blood vessels to definite the bone marrow microvascular density (MVD). Plasma levels of vascular endothelial growth factor (VEGF) were examined by the method of ELISA. RESULTS: The BMD of femoral in 8-week OVX group were decreased significantly compared to the 8-week sham group (P<0.05), suggesting the establishment of osteoporosis model. Meanwhile, the area of hematopoietic tissue decreased and the area of adipose tissue increased. These changes became obviously in 12-week OVX group, and the area of trabecular bone and the bone marrow MVD significantly decreased compared to the 12-week sham group. A positive correlation between MVD and BMD, area of hematopoietic or trabecular bone as well as a negative correlation between MVD and area of adipose tissue were observed. The plasma levels of VEGF in OVX group were not significantly different from that in sham group, and had no correlation relationship with the indexes of bone marrow pathology. CONCLUSION: There has an increase in MVD companied with the bone mass loss and hematopoietic tissue decreased in ovarietomized rats, which provide experiment proof to treat osteoporosis with the means of promoting of MVD in bone marrow.     

Influence of normal conditioned medium of astrocytes on the damaged neurons of cerebral hypoxia and reoxygenation in vitro

AIM: To study the nutritional role of the normal conditioned medium of astrocytes (NCMA) on the injured cerebral cortex neurons induced by cerebral hypoxia and reoxygenation in vitro. METHODS: The normal and damaged neurons induced by hypoxia and reoxygenation were cultured with normal conditioned medium of astrocytes (NCMA) after the cerebral cortical astrocytes and neurons of rats were isolated. The viability and survival rate of cultured neurons were investigated by MTT method. RESULTS: NCMA increased the viability and survival rate of cultured neurons. CONCLUSION: NCMA has nutritional and supporting roles on cultured neurons.     

Astrocyte proliferation in the rat hippocampus after middle cerebral artery occlusion

AIM: To study rat astrocyte proliferation in ipsilateral hippocampus following focal cerebral ischemia. METHODS: Ischemia was induced by temporary middle cerebral artery occlusion (MCAO). In hippocampus of rats at 3, 7 and 30 days after MCAO, the numbers and anatomic distribution of glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The protein expression of GFAP and proliferating cell nuclear antigen (PCNA) in the ipsilateral hippocampus were analyzed by Western blot analysis. RESULTS: Astrocytes appeared hypertrophic, with increased process thickness and numbers at 7 days after MCAO, and the highest density of astrocytes were seen at 30 days in the CA1, CA2 regions of the ipsilateral hippocampus. Western blot analysis revealed that GFAP levels were normal at 3 days, but increased by 7 days and remained elevation at 30 days. Western blot analysis of PCNA protein also revealed identified upregulation PCNA at 3 days after MCAO and the expression peaked at 7 days. CONCLUSION: This study demonstrates that focal cerebral ischemia in the rat results in a rapid response, a process often referred to as reactive astrogliosis or glial scarring, from resident astrocytes of the ipsilateral hippocampus to the side of ischemia.     

Changes of Ski expression levels in rat activated astrocytes

AIM: To explore the time-dependent change of Ski protein expression in normal and activated astrocytes in rats.METHODS: The astrocytes were obtained from rat cerebral cortex and cultured in vitro. The astrocytes were treated with LPS and scratch injury for activation. Western blot analysis was used to determine glial fibrillary acidic protein (GFAP) and Ski protein levels in activated astrocytes at a series of time points. The indirect immunofluorescence staining method was performed to detect the location of Ski protein in the astrocytes.RESULTS: The protein of GFAP was naturally expressed in the astrocytes, beginning to increase after treated with LPS and scratch injury. Little protein expression of Ski in the normal astrocytes was observed. The Ski protein expression began to increase after treated with 1 mg/L LPS, peaked at 4 d (P<0.05) and then deceased, but was stills higher than that in the normal cells. The protein expression level of Ski after scratch injury was highly consistent with above mentioned. Ski was mainly observed in the nucleus of the normal cells and the cells treated with LPS for 6 d, while it was observed in the cytoplasm 2 and 4 d after treated with LPS.CONCLUSION: The protein of Ski is expressed in the astrocytes, and the expression level is increased in activated astrocytes, mainly located in the nucelus. Ski may plays an essential roles in the processes of activation and proliferation of astrocytes.     

Effect of bone marrow mononuclear cells on necrotic and apoptotic cell death induced by renal ischemia-reperfusion in rats

AIM: To investigate the effect of bone marrow mononuclear cells (BMMNCs) on necrotic and apoptotic cell death and to test whether BMMNCs can restore the renal tissues with ischemia-reperfusion injury to tubular structural integrity in rats. METHODS: BMMNCs were isolated from the femurs and tibiae of male SD rats with Percoll density gradient centrifugation and labeled with DAPI in vitro. All model rats were subjected to clamping bilateral renal pedicles for 45 min, followed by injection of BMMNCs or saline into the inferior vena cava, and were sacrificed to collect kidneys at 1, 2, 7, 14 d after operation, respectively. Fluorescence microscope was performed to detect the DAPI labels in the renal paraffin slices. All kidney samples were examined by HE staining under light microscope. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Immunohistochemical staining was used to examine the expression of proliferating cell nuclear antigen (PCNA). RESULTS: There were lots of cells with blue fluorescence in the injured kidney samples under fluorescence microscope, some of which lay in the renal tubular epithelium. Tubular cell necrosis and degeneration were observed in kidneys of control group, no necrosis and significant tubular cell degeneration was observed in kidneys of transplantation group. Decreased numbers of apoptotic cells were detected in kidney samples of transplantation group as compared with control group. The numbers of PCNA-positive cells in kidney samples of transplantation group were higher than those in control group. CONCLUSION: Transplanted BMMNCs migrate into the injured kidney, some of which might directly reconstitute the tubular epithelium. Transplantation of these cells attenuates necrotic and apoptotic cell death and accelerates the proliferation of the renal tubular epithelium after ischemia-reperfusion injury. It is suggested that these cells might contribute to restore injured kidney to the tubular structural integrity.     

Effect of E-selectin pretreatment on cerebral ischemia-reperfusion injury in rats

AIM:To study the effect of nasal mucosal tolerance to E-selectin on cerebral ischemia-reperfusion injury.METHODS:Two different doses (single and booster) of E-selectin or PBS were dropped into membrana mucosa nasi of rats. The middle cerebral artery occlusion (MCAO) model referring to Zea Longa method with modifications was performed 48 h after the last dose of E-selectin or PBS. After 2 h ischemia and 22 h reperfusion, the numbers of CD3+CD4+T-lymphocyte and CD3+CD8+T lymphocyte subgroup in the blood were examined with flow cytometry. Rats were killed, then part of the animals was used to measure the cerebral infarction volume by TTC staining. mRNA expressions of E-selectin, ICAM-1 and lymphocyte function-associated antigen-1(LFA-1) were determined by RT-PCR and activity of SOD was determined by xanthinoxidanse method in ischemic cortex of the other part of animals. RESULTS:The ratio of the numbers of CD3+CD4+T-lymphocytes and CD3+CD8+T-lymphocytes increased in E-selectin single pretreatment group (P<0.05). Compared to other groups, E-selectin booster pretreatment group showed decreased CD3+CD8+T-lymphocytes (P<0.05), increased ratio of the numbers of CD3+CD4+T-lymphocytes and CD3+CD8+T-lymphocytes (P<0.05), reduced cerebral infarction volume by 40.87% (P<0.05), heightened activity of SOD (P<0.05), lowed E-selectin mRNA and ICAM-1 mRNA expression (P<0.05), and less tendency of LFA-1 mRNA expression.CONCLUSION:E-selectin induces cerebral ischemic tolerance and relieves cerebral ischemia-reperfusion injury. The mechanisms are related to the changes in the ratio of CD4+T-lymphocyte and CD8+T-lymphocyte. The heightened activity of SOD, the lowed mRNA expressions of E-selectin and ICAM-1, as well as the less tendency of LFA-1 mRNA expression are also involved.     

Isolation of exosomes and effect of stellate ganglion block on the number of exosomes in post-hemorrhagic shock mesenteric lymph of rats

AIM To isolate the exosomes in mesenteric lymph, verify the source of exosomes, and observe the effect of stellate ganglion block （SGB） on the number of exosomes in post-hemorrhagic shock mesenteric lymph （PHSML） of rats. METHODS Twenty-four male rats were randomly divided into sham, sham+SGB, shock, and shock+SGB groups. SGB was performed before the establishment of hemorrhagic shock model using the routine methods in our lab. The PHSML was drained for exosomes isolation. The exosomes were identified through particle size analysis and CD63 protein expression. The expression of epithelial cell adhesion molecule （EpCAM） was detected to identify whether the exosomes were derived from epithelial cell. The number of exosomes in various mesenteric lymphs was measured using the flow cytometry. RESULTS The diameter of granular material extracted from mesenteric lymph was about 100 nm. The positive expression of exosomes pecific protein CD63 indicated the successful isolation of exosomes, and the EpCAM expression verified the exosomes were derived from intestinal epithelial cells. The number of exosomes in mesenteric lymph isolated from the rats of Shock group was obviously increased compared to that from the Sham group （P<0.05）, while the exosomes from the Shock+SGB group was markedly decreased when compared to Shock group （P<0.05）. CONCLUSION The current study establishes the isolation technique of exosomes in mesenteric lymph, and proved the exosomes were derived from the intestinal epithelial cells. SGB treatment reduces the number of exosomes in PHSML.     

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.     

Effect of inhibiting release of exosomes on biological characteristics of bone marrow mesenchymal stem cells

AIM:To investigate the effect of inhibiting the release of exosomes on the biological characteristics of bone marrow mesenchymal stem cells (BM-MSCs) and the underlying mechanisms. METHODS:The exosome releasing-deficient mouse model was constructed by knockout of Rab27a using TALEN technique. The BM-MSCs were isolated and cultured. The exosomes were extracted from the culture medium using total exosome isolation kit and quantified by nanoparticle tracking analysis (NTA). The size and morphology of the exosomes were observed under transmission electron microscope. To evaluate the proliferation ability of BM-MSCs, the BM-MSCs were labeled with 5-ethynyl-2'-deoxyuridine (EdU) and the expression level of proliferating cell nuclear antigen (PCNA) was determined by Western blot. Moreover, hypoxia tolerance of BM-MSCs in vitro was evaluated via TUNEL staining and MTS assay. RESULTS:The count of exosomes released by BM-MSCs isolated from Rab27a knockout mice was significantly reduced. Inhibition of exosome release resulted in decreases in the viability of the BM-MSCs and their resistance to hypoxia. CONCLUSION:Inhibition of exosome release from the BM-MSCs results in significantly decreased proliferation ability and resistance to hypoxia.     

Role of caveolin-1 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of caveolin-1 in bone marrow mesenchymal stem cells (MSCs) differentiation into neurons. METHODS: The MSCs used in the experiment were divided into non-transfected group, transfected group (transfected with Rn-caveolin-1 siRNA), positive control group (transfected with Rn-MAPK-1 control siRNA) and negative control group (transfected with negative control siRNA). The MSCs were induced by β-ME to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The mRNA expression of caveolin-1 and MAPK-1 was detected by RT-PCR. NSE, NF-M and GFAP, the neural cell specific markers, were detected by immunocytochemistry staining. The survival ratio of MSCs was detected by MTT method. RESULTS: The fluorescence of MSCs was mostly displayed at 72th h after transfection and the efficiency of transfection was up to 81.5%±2.8%. Meanwhile, the mRNA expression of caveolin-1 in transfected MSCs was decreased (P<0.05). No significant difference of the survival MSCs ratio between transfected group and other groups was observed by MTT method. β-ME induced MSCs into neural cells. The differentiation efficiency of MSCs transfected with Rn-caveolin-1 siRNA was the highest and the expression of NSE and NF-M was increased significantly compared to the other groups (P<0.01). The expression of caveolin-1 was increased persistently with time and the highest expression was observed 6 d after induction. Furthermore, there was significant difference between before induction and 6 d after induction. CONCLUSION: Lipid raft labeled with careolin as marker protein has important role in regulating MSCs differentiation into neurons.     

Protective effects of auricularia auricular polysaccharide on chronic cerebral ischemia injury in rats

AIM: To investigate the effects of auricularia auricular polysaccharide (AAP) on chronic cerebral ischemia injury in rats. METHODS: The chronic cerebral ischemia mode1 was made by permanent middle cerebral artery occlusion (MCAO) on the right side. AAP at different doses (50 mg/kg and 100 mg/kg) was intragastrically administered at the onset of ischemia and in the following days after operation, once a day for 4 weeks. After 4 weeks of MCAO, Morris water maze test was introduced to examine the learning and memory functions. Nissl staining was performed to detect the survival neurons in hippocampal slices. Level of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in brain tissue were measured. RESULTS: Rats treated with AAP showed a shorter escaping latency in spacial navigation test because the AAP treated rats spent less time to find the platform in spatial probe test. More survival neurons in hippocampal slices were observed from AAP treated rats. Also, the MDA level in brain tissue was reduced and SOD activity in brain tissue was increased in the AAP treated rats with MCAO. CONCLUSION: AAP protects rats from chronic brain ischemic injury, in which its anti-oxidative effect might be involved.     

Colquhounia root tablet inhibits the expression of adhesion molecule in acute lung injury of rats

AIM: To study the effects of colquhounia root tablet on the expression of adhesion molecule in acute lung injury of rats.METHODS: The rats were divided into 3 groups: ALI group,colquhounia root tablet＋ALI group and control group .ALI animal model was performed by treatment with oleic acid.The positive expression rates of CD11a,CD11b and CD18 in polymorphonuclear neutrophils and monocytes were analyzed by flow cytometry.ICAM-1 expression in lung tissue was determined by immunohistochemistry,histopathological examination and biological markers were measured from lung specimens.RESULTS: Colquhounia root tablet decreased the expression of CD11a,CD11b and CD18 in polymorphonuclear neutrophils and monocytes,and ICAM-1 in lung tissue (P<0.01),pathologic changes and the biological markers of ALI significantly ameliorated.CONCLUSION: The increase in the expression of CD11a/CD18 and CD11b/CD18 in polymorphonuclear neutrophils and monocytes,and expression of ICAM-1 in lung tissues of ALI may be involved in the formation of ALI.Colquhounia root tablet effectively ameliorates the lung damage,mechanism of which may be related to inhibition of adhesion molecule expression.     

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.     

Neuroprotective effect of wortmannin on epileptic rats by inhibiting autophagy

AIM: To investigate the role of autophagy in hippocampus injury induced by seizures and to observe the neuroprotective effects of autophagy inhibitor wortmannin(WM) on epileptic rats.METHODS: The Wistar rats were randomly divided into control group, model groups at 2 h, 8 h, 16 h, 24 h or 72 h after seizure induction by pilocarpine, and WM pretreatment group. The methods of HE and Nissl staining were used to evaluate the hippocampus injury. The expression of microtubule-associated protein 1 light chain 3(LC3) was detected by Western blotting. The ratio of LC3II to LC3I was calculated and used to represent the activity of autophagy. RESULTS: The significant increase in the ratio of LC3II to LC3I began at 2 h, peaked at 24 h, and maintained at high level at least to 72 h after seizure induction. Obvious neural injury and neuron depletion were observed in hippocampus CA1 area at 24 h after seizure induction. The number of surviving neurons at 24 h was sharply decreased in rats with seizures(75.50±5.92) as compared to the controls(110.67±18.56, P＜0.01). WM significantly decreased the neuron depletion induced by seizures(100.88±18.73, P＜0.05). Moreover, WM significantly decreased the ratio of LC3II to LC3I in rats with seizures at 24 h(P＜0.05). CONCLUSION: Autophagy was activated in hippocampus injury induced by seizures. WM reduces the transformation of LC3II to LC3I to inhibit the autophagy activated by seizures. WM has neuroprotective effect on epileptic rats by increasing the surviving neurons in hippocampus CA1 area.     

CCL3 promotes hBMSC proliferation and inhibits exosome secretion

AIM: To explore the regulatory effect of chemokine CCL3 on exosome secretion from human bone marrow mesenchymal stem cells (hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL3 at different concentrations in vitro. The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting. Exosome secretion from hBMSCs was qualitatively analyzed by transmission electron microscope (TEM) and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis (NTA).RESULTS: Compared with control group, the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3 (P<0.05). The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL3 group in a dose-dependent manner (P<0.05). The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related specific receptors, CCR1, CCR5 and CCR9. Compared with control group, the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced. However, no significant difference of fluorescence intensity for CCR5 and CCR1 was observed between the 2 groups. The results of NTA demonstrated that the secretion capacity of CCL3-induced hBMSCs was far less than that in control group (P<0.05). However, the microvesicles larger than 100 nm in CCL3 groups were increased (P<0.05). The above results indicated that the higher concentration of CCL3 induced the lower secretion of exosomes. In addition, the results of flow cytometry demonstrated that CCL3-induced hBMSCs showed lower quantity of CD9⁺ exosomes than those in control group (P<0.01).CONCLUSION: CCL3 promotes the proliferation of hBMSCs but depresses the secretion of exosomes in a dose-dependent manner. CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size. CCL3 induces the expression of CCR9 in hBMSCs.