Exosomes from hypoxic pretreated BMSCs attenuate primary Sjögren's syndrome-induced skin injury via PPARγ-mediated M2-like macrophage activation.

Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cells (BMSC) overexpressing basic fibroblast growth factor (bFGF) on osteoporotic fracture in rats. Methods 80 female SD rats were randomly divided into control group, model group, BMSC group and BMSC+ bFGF group. The osteoporotic fracture model was established by resection of ovariectomized in model group, BMSC group and BMSC+ bFGF group. In group BMSC and group BMSC+ bFGF, BMSC and bFGF overexpressing BMSC were transplanted at 1, 14 and 21 d after operation respectively, while rats in the control and model groups were given the same volume of saline injection. The protein level were analyzed by Western blotting in BMSC group and BMSC+ bFGF group. Determination of bone density of rats in four group were detected by bone densitometry and fracture healing was evaluated by X-ray. The changes of bone biomechanical parameters were analyzed using three point bending test. alkaline phosphatase (ALP), runt related transcription factor-2 (Runx2) and bone morphogenetic protein-2 (BMP-2) mRNA level in four groups were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results Western blot showed that the expression of bFGF factor was up-regulated in BMSC+ bFGF group. Compared with the model group [2.02±0.37, 0.051±0.002], the X-ray scores and bone mineral density significantly increased in the BMSC [3.12±0.46, 0.800±0.004] and BMSC+ bFGF groups [4.43±0.51, 0.128±0.006], and the biological parameters (maximum load, elastic load, stiffness and maximum deflection) significantly improved (P=0.032, 0.013; P=0.000, 0.000). Compared with the BMSC group, the X-ray score and bone mineral density in the BMSC+ bFGF group significantly increased (P=0.010, 0.010) and the biological parameters (maximum load, elastic load, stiffness and maximum deflection) significantly improved (P=0.002, 0.000; P=0.000, 0.000). Compared with the model group (1.41±0.19, 1.31±0.20, 1.43±0.24), the levels of ALP, Runx2 and BMP-2 mRNA in callus tissue of BMSC group (2.16±0.33, 2.48±0.31, 3.17±0.33) and BMSC+ bFGF group (4.48±0.48, 5.13±0.41, 4.89±0.49) significantly increased (P=0.000). Compared with group BMSC, the levels of ALP, Runx2 and BMP-2 mRNA in callus tissue of BMSC+ bFGF group more significantly increased (P=0.000). Conclusion Overexpression of bFGF in BMSC can increase the expression of bone morphogenetic protein, increase bone mineral density, promote the healing of osteoporotic fracture, and enhance the biological stress of fracture. Key words: Bone marrow mesenchymal stem cells; Basic fibroblast growth factor; Osteoporotic fractures; Bone mineral density; Bone healing; Biology

Objective To investigate the therapeutic effect and possible mechanism of adenosine A2A receptor agonist (CGS21680) combined with bone marrow mesenchymal stem cells (BMMSC) transplantation in acute liver failure (ALF). Methods Fifty male C57BL/6 mice, 6—8 weeks old, were fed with standard diet for 1 week and randomly divided into 5 groups according to random number table: healthy control group (n=6), model group (n=11), BMMSC group (n=11), CGS21680/BMMSC group (n=11) and CGS21680 group (n=11). Except healthy control group, the other mice were injected with D-GalN and lipopolysaccharide (LPS) to establish ALF model. Ten hours later, CGS21680/BMMSC group and CGS21680 group were injected intraperitoneally with adenosine A2A receptor agonist CGS21680 (2.1 mg/kg). In addition, the BMMSC group and CGS21680/BMMSC group were injected BMMSC (1×106) through tail vein. After 24 hours, pathological changes of liver tissue was observed by hematoxylin and eosin staining. The change of proportion of mouse splenic Treg among CD4+ T lymphocytes was detected by flow cytometry. Toll-like receptor (TLR)4 and nuclear factor (NF)-κB expression levels in liver tissue were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blot. One-way analysis of variance (one-way ANOVA) and SNK-q test was conducted for data analysis. Results Serum IL-6 levels were (23.67±2.97) pg/mL in healthy control group, (151.47±6.03) pg/mL in model control group, (72.10±3.74) pg/mL in BMMSC group, (53.35±2.50) pg/mL in CGS21680/BMMSC group and (84.85±3.25) pg/mL in CGS21680 group. The differences between healthy control group and the other 4 groups were all statistically significant (t=46.02, 25.51, 19.58 and 34.03, respectively, all P<0.01). Serum TNF-ɑ levels were (24.62±3.19) pg/mL in healthy control group, (102.25±2.10) pg/mL in model control group, (54.71±2.23) pg/mL in BMMSC group, (42.20±4.72) pg/mL in CGS21680/BMMSC group and (81.76±3.50) pg/mL in CGS21680 group. The differences between healthy control group and the other 4 groups were all statistically significant (t=46.49, 19.97, 7.72 and 29.57, respectively, all P<0.01). The differences of spleen Treg proportion in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=51.67, 12.22, 5.91 and 18.21, respectively, all P<0.01). The differences of TLR4 mRNA levels of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=26.31, 21.33, 13.24 and 27.14, respectively, all P<0.05). The differences of NF-κB mRNA level of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=16.56, 16.34, 7.83 and 13.11, respectively, all P<0.05). The differences of TLR4 protein level in liver tissue of healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=35.60, 10.38, 6.05 and 18.02, respectively, all P<0.05). The differences of liver NF-κB protein level in the healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=10.80, 7.30, 4.61 and 13.24, respectively, all P<0.05). Conclusions Adenosine A2A receptor agonist combined with BMMSC can significantly up-regulate the proportion of Treg cells in acute liver failure mice and inhibit the TLR4/NF-κB pathway activation, with both coordinated regulation, and further inhibit the liver inflammation. Key words: Mesenchymal stem cells; Liver failure, acute; Receptor, adenosine A2A; TLR4/ NF-κB signal pathway

To investigate the feasibility of construction of tissue engineered cartilage by co-culture of bone marrow mesenchymal stem cells (BMSCs) and costal chondrocytes (CCs), and to provide theoretical basis and experimental basis for clinical repair of articular cartilage defects by Wuzhishan miniature pig knee cartilage defects with co-cultured cells. Methods: Density gradient centrifugation method was used to isolate BMSCs from Wuzhishan miniature pig. The double enzyme digestion method was used to isolate CCs. The passage 3 generation of BMSCs and passage 2 generation of CCs were randomly divided into 3 groups: a co-culture group of BMSCs:CCs for 1:2 (Group A), a simple CCs (Group B), and a simple BMSCs (Group C). The cell growth curve was drawn, and the content of glycosaminoglycan (GAG) of external separation in chondrocytes was determined. The 12 Wuzhishan miniature pigs were randomly divided into a co-culture cells/collagen membrane experimental group, a collagen membrane control group and the blank group. In the co-culture cells/collagen membrane experimental group, the co-cultured cells/collagen membrane were implanted into the cartilage defects of the mandibular condyle; in the collagen membrane control group, only collagen membrane was implanted; while in the blank group, nothing was implanted. Six animals were sacrificed at 8 and 16 weeks after surgery respectively (2 animals in each group). General observation, cartilage histological score and histopathological examination were carried out. Results: The BMSCs and co-culture cells grew well. The biological activity of CCs was good. After 16 weeks of operation, the repair tissues in the co-cultured cells/collagen membrane experimental group showed hyaline cartilage features: smooth, flat, and integrated well with the surrounding cartilage and subchondral bone. The collagen membrane in the collagen membrane control group was fibrously repaired. Repair tissue gross score in the co-culture cells/collagen membrane experimental group was significantly better than that in the collagen membrane control group and the blank group (both P<0.05), but there was no significant difference between the collagen membrane control group and the blank group (P>0.05). Conclusion: BMSCs, CCs and co-cultured cells can function as the seed cells for cartilage tissue engineering, and the co-culture cells (BMSCs:CCs=1:2) possess more advantages; the short-term effect of co-culture cells with collagen membrane on repairing cartilage defects is satisfied.

Epac1 activation optimizes cellular functions of BMSCs and promotes wound healing via Erk/ACLY/PGC-1α signaling pathway

Objective To investigate the effects of bone marrow mesenchymal stromal cells (BMSCs) transplantation after mannitol pretreatment on behavioral performance and synaptophysin expression in the CA3region in hippocampus of vascular dementia (VD) rats. Methods The BMSCs of rats were isolated and purified by the whole bone marrow adherence method.The rats were subjected for permanent ligation of bilateral common carotid arteries at an interval of 3 days for each carotid artery.At the same time，Sham group was set in parallel.Four weeks after modeling，the VD rats were divided randomly into five groups: (1) VD control group; (2) culture media group; (3) mannitol group; (4) BMSCs group; (5) mannitol with BMSCs group.Morris water maze performance and synaptophysin expression in the CA3region in hippocampus were observed at 4 weeks after transplantation. Results The morris water maze performance significantly improved in mannitol with BMSCs group when compared with BMSCs group，VD control group，culture media group，mannitol group.Moreover，the escape latency of fifth day decreased significantly ((9.3±2.9)，(14.1±3.5)，(23.5±4.4)，(22.8±4.4)，(23.2±2.8) s，F=43.900，P=0.000))，and the platform quadrant residence time increased significantly ((40.8±6.3)，(34.9±5.8)，(26.4±4.8)，(27.4±7.0)，(28.5±6.2) s，F=13.000，P=0.000)).The synaptophysin expressions of the hippocampal CA3region were significantly increased in the mannitol with BMSCs group (39 624±7798) when compared with BMSCs group，VD control group，culture media group，mannitol group (27 060±4668，18 294±6446，19 956±4244，18 946±4953，F=39.206，P=0.000). Conclusions Intravenous BMSCs transplantation after mannitol pretreatment improves the behavioral performance of VD rats and facilitates the synaptophysin expression of hippocampal CA3region in VD rats than BMSCs transplantation alone.Mannitol pretreatment can amplify the therapeutic effect of intravenous BMSCs transplantation in VD rats. Key words: Dementia，vascular; Mannitol; Mesenchymal stromal cells; Bone marrow cells; Mesenchymal stem cell transplantation; Synaptophysin; Hippocampus

Bone marrow mesenchymal stem cells (BMSCs) has been considered as a therapeutic strategy for acute liver injury (ALI). However, the insufficient homing of BMSCs in vivo limited their applications. Ultrasound targeted microbubble destruction (UTMD) has shown the potential in promoting the homing of stem cells into ischemic myocardium. We therefore explored whether UTMD could promote BMSCs' homing in ALI rat models and its corresponding therapeutic effect. Methods: Bone marrow mesenchymal stem cells (BMSCs) were obtained from the femurs and tibias of Sprague-Dawley (SD) rats. The characteristics of BMSCs including the proliferative viability, diversified differentiation ability and specific cell surface markers' expression were verified by flow cytometry. In order to find the appropriate ultrasound parameters, the normal rat liver was exposed to different intensity of ultrasound (1.0 W/cm2, 1.5 W/cm2, 2.0 W/cm2) with 300µl microbubbles, and the expression of tumor necrosis factor $a$ (TNF-a) and stromal cell-derived factor 1 (SDF-1) were detected at 24h post-treatment. The ALI models were established via a single intraperitoneal injection of D-galactose on 32 SD rats, which then were randomly assigned into four groups: Control, BMSCs, UTMD, and UTMD+BMSCs. After the BMSCs were stably transfected with a lentivirus for expressing green fluorescent protein (GFP), these cells were then injected through tail vein in a concentration of 2×106/ml. Forty-eight hours after the treatment, the protein expression levels of SDF-1, intercellular cell adhesion molecule1(ICAM-1), vascular cell adhesion molecule 1(VCAM-1 and monocyte chemotactic protein 1 (MCP-1) of exposed liver were analyzed, and the number of GFP-transfected BMSCs were observed as well. Serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities were tested after 48 h/72 h/168 h of treatment. The histology of liver was evaluated at 168h. Results: The isolated rat BMSCs demonstrated good proliferation, osteogenic and adipogenic differentiation and the expression of CD29+, CD90+, CD45-, CD11b/c-. After the application of UTMD, the expression level of SDF-1 and TNF-a on exposed liver was markedly higher in 2.0 W/cm2 group than in the 1.5 W/cm2 and 1.0 W/cm2 group ( $p$ $p$ <0.05), as well as the protein expression of SDF-1, ICAM-1, VCAM-1 and MCP-1 (p<0.01). The ALT, AST and ALP activities were much lower in UTMD+BMSCs group. The pathological features of liver injury were alleviated to a large extent in UTMD+BMSCs group. Conclusion: Through upregulation of adhesion molecular and cytokines, UTMD could enhance the homing of BMSCs in rat models of ALI and improve the function of acute injured liver.

The implantation of BMSCs (bone marrow mesenchymal stem cells) has emerged as a potential method of treating tissue damage, but the in vivo differentiation of BMSCs in an injured pancreas and its therapeutic effects have not been determined. Our aim has been to investigate the potential of BMSCs to contribute to the parenchyma and mesenchymal components of the pancreas during rapid regeneration, with preliminary exploration of the molecular mechanisms of this process. GFP(+) (green fluorescent protein(+) ) BMSCs were intravenously infused into the tail veins of mice that had received a 65-70% partial pancreatectomy, while mice that had only received a partial pancreatectomy and mice that had only been injected with BMSCs served as controls. Four weeks later, the injected GFP(+) BMSCs were diffusely engrafted in the pancreatic parenchyma and mesenchyma of the recipient mice with pancreatic injuries and had differentiated into pancreatic ductal epithelial cells (accounting for 1.7±0.3%), vascular endothelial cells (3.2±0.6%) and PSCs (pancreatic stellate cells) (5.2±1.6%), but no β or neural cells. Significantly, more engrafted and differentiated GFP(+) BMSCs were observed in the regenerating pancreas than in the normal pancreas. For the mice that received a partial pancreatectomy, the pancreatic weight/body weight of the mice with BMSC treatment was greater than mice without BMSC treatment (P<0.05). In addition, real-time RT-PCR (reverse transcription-PCR) showed that the expression levels of miR-9 (microRNA 9) and miR-204 in the engrafted BMSCs (5.2- and 2.6-fold, P<0.05, respectively) were increased compared with wild-type BMSCs. We also observed a significant reduction in the expression of miR-375 (0.71-fold, P<0.05) in engrafted GFP(+) BMSCs compared with wild-type BMSCs. BMSCs can therefore be a potential cell bank for treating pancreatic injuries by contributing to a variety of cell types. This process might be related to the expression of miR-9, miR-204 and miR-375.

BackgroundAutologous stem cell therapy is a promising strategy for cardiovascular diseases including diabetic cardiomyopathy (DCM), but conclusions from clinical trials were compromised. We assumed that diabetes might induce the dysfunction of stem cells and thus limit its therapeutic effect. This study aimed to compare the effect of diabetes and nondiabetes-derived bone marrow mesenchymal stem cells (BMSCs) transplantation on DCM and explored the potential mechanism.MethodsRats with diabetes were induced using high-fat diets and streptozotocin (STZ) injection. BMSCs harvested from diabetic and nondiabetic rats were infused into DCM rats, and the effects on the heart were identified by echocardiography and histopathology. The inhibition or overexpression of SAHH in nondiabetic and diabetic BMSCs was used to confirm its key role in stem cell activity and cardiac therapy.ResultsCompared with normal BMSCs, the therapeutic effects of diabetic rat-derived stem cells on improving cardiac function and adverse remodeling were significantly attenuated. In vitro, diabetic BMSCs had lower cell viability and paracrine function than nondiabetic BMSCs. It was further found that diabetic BMSCs had obvious mitochondrial oxidative stress damage and S-adenosylhomocysteine (SAH) accumulation due to S-adenosylhomocysteine hydrolase (SAHH) deficiency. SAHH inhibition by adenosine dialdehyde (ADA) or shSAHH plasmid in normal BMSCs significantly reduced the favorable effects on endothelial cell proliferation and tube-forming capacity. In contrast, SAHH overexpression in diabetic BMSCs significantly improved cellular activity and paracrine function. Transplantation of BMSCs with SAHH overexpression improved cardiac adverse remodeling and angiogenesis. Activation of the Nrf2 signaling pathway may be one of the key mechanisms of SAHH-mediated improvement of stem cell viability and cardiac repair.ConclusionsDiabetes leads to compromised bioactivity and repair capacity of BMSCs. Our study suggests that SAHH activation may improve the cardioprotective effect of autologous transplantation of diabetes-derived BMSCs on patients with DCM.Graphical abstractDiabetes induced the inhibition of S-adenosylhomocysteine (SAH) expression and aging phenotype in BMSCs and thus decreased the cell viability and paracrine function. Compared with normal BMSCs, the therapeutic effects of diabetic rat-derived BMSCs on improving cardiac function and adverse remodeling were significantly attenuated. SAHH overexpression in diabetic BMSCs significantly rescued cellular function partly via activating Nrf2/HO-1 signal. Transplantation of diabetic BMSCs with SAHH overexpression improved angiogenesis and cardiac adverse remodeling in rats.

Background Limbal stem cell deficiency usually leads to blindness, and traditional therapy is limited. Recent research demonstrated that bone mesenchymal stem cells ( BMSCs ) and human amniotic epithelial cells(AECs) could differentiate into many kinds of cells including corneal epithelial cells, but the outcome and effect of these cells on corneal stem cell deficiency are still unclear. Objective This study aimed to observe and compare the effects of rabbit BMSCs and human AECs transplantation for rabbit limbal stem cell deficiency. Methods Eighteen clean New Zealand rabbits were randomly divided into the amniotic stroma(AS) group, rabbit BMSCs group and human AECs group with 6 rabbits for each group. Limbal stem cell deficiency models were established by putting a piece of filter paper that had been soaked in a NaOH solution at the corneal center. Rabbit BMSCs were isolated and purified by density gradient centrifugation combined with the attachment culture method, and human AECs were collected by a sequential trypsin digestion technique,and the third generation rabbit BMSCs and the first generation human AECs were identified with RT-PCR. After that,cells were inoculated onto the denuded AS and grafted to the corneal surface of the experimental animals. Twenty-eight days after cell transplantation, the therapeutic effects were evaluated based on the corneal neovascularization and opacity scores. Corneal histopathological examination and immunohistochemistry were performed to evaluate and compare the effectiveness among AS,rabbit BMSCs and human AECs on corneal stem cell deficiency. The procedure complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The third generation of rabbit BMSCs grew well after 12 hours, and the first generation of human AECs formed a membrane-like monolayer after 48 hours of incubation on AS. Immunohistochemistry staining showed that, 28 days after transplantation, the surface cells of the cornea were positive for cytokeratin 3 in both the rabbit BMSCs group and human AECs group.Compared with the AS group,the corneal neovascularization and opacity grades were significantly decreased in the rabbit BMSCs group( Z=-2. 983, P =0. 003 ; Z =-2. 844, P =0. 004 ) and human AECs group ( Z =-2. 817, P =0. 005 ; Z =-2.041, P =0. 041 ). Histopathological analysis exhibited that stratified corneal epithelial-like cells formed on the corneal stroms 28 days after grafting and no signs of goblet cells and neovascularization were found. Less inflammatory cells and regular collagen fiber could be seen in the rabbit BMSCs group and human AECs group. In addition,clinical observation also revealed that the corneas were much clearer in the rabbit BMSCs group than the human A ECs group( Z =-2. 091 , P=0. 037 ), but the corneal neovascularization score was similar between them (Z = -0. 267,P=-0. 789). Conclusions Rabbit BMSCs and human AECs can differentiate into corneal epithelial-like cells on the damaged corneal surface and further demonstrate remarkable inhibitory effects on corneal neovascularization and inflammatory cells. The more dominant and prominent effect is the role played by rabbit BMSCs in the improvement of corneal transparency. Key words: Bone mesenchymal stem cell; Amniotic epithelial cell; Limbal stem cell; Corneal neovascularization; Stem cell transplantation

Effects of a PDGF-stem cell-hydrogel compound on skin wound healing in mice.

Mesenchymal stem cells (MSCs) treatment has emerged as a promising approach for treating Sjögren's syndrome (SS). Impaired immunoregulatory activities of bone marrow mesenchymal stem cells (BMMSCs) are found in both SS patients and animal models, and the underlying mechanism is poorly understood. Increased expression of BMP6 is reported to be related to SS. The aim herein was to determine the effects of BMP6 on BMMSCs function. BMMSCs were isolated from SS patients and NOD mice and showed a high level of BMP6 expression. The effects of BMP6 on BMMSCs function were investigated using in vitro BMMSCs differentiation and in vitro and in vivo T cell proliferation and polarization assays. BMP6 increased osteogenic differentiation of BMMSCs and inhibited the immunomodulatory properties of BMMSCs. BMP6 enhanced T cell proliferation and Th1/Th17 differentiation in a T cell-BMMSC coculture system. Mechanistically, BMP6 downregulated PGE2 and upregulated IFN-gamma via Id1 (inhibitor of DNA-binding protein 1). Neutralizing BMP6 and knockdown of Id1 could restore the BMMSCs immunosuppressive function both in vitro and in vivo. The present results suggest a novel role of Id1 in BMP-mediated MSCs function, which may contribute to a better understanding of the mechanism of action of MSCs in treating autoimmune diseases.

Study on the function of Huazhuo Jiedu Decoction in promoting the homing of bone marrow mesenchymal stem cells and contributing to the treatment of ulcerative colitis

Background and ObjectivesSarco/endoplasmic reticulum Ca2＋-ATPase (SERCA) inhibition was proved in streptozotocin (STZ)-diabetic rats. The present study aimed at investigating and comparing the therapeutic effect of bone marrow mesenchymal stem cells (BMMSCs), BMMSCs combined with ascorbic acid (AA) and SERCA1a gene transfected BMMSCs in induced type I diabetic myopathy of male albino rat.Methods and Results54 rats were divided into donor group of 6 rats for isolation, propagation and characterization of BMMSCs and SERCA1a transfected BMMSCs, groups I∼V 48 rats. Group I of 8 control rats, group II (Diabetic) of 10 rats given STZ 50 mg/kg intraperitoneal, group III (BMMSCs) of 10 rats given STZ and BMMSCs intravenous (IV), group IV (BMMSCs and AA) of 10 rats given STZ, BMMSCs IV and AA 500 mg/kg and group V (SERCA 1a transfected BMMSCs) of 10 rats given STZ and SERCA1a transfected BMMSCs IV. The rats were sacrificed after 8 weeks. Gastrocnemius specimens were subjected to biochemical, histological, morphometric and statistical studies. Diabetic rats revealed inflammatory and degenerative muscle changes, a significant increase in blood glucose level, mean DNA fragmentation and mean MDA values and a significant decrease in mean GSH and catalase values, area of pale nuclei, area% of CD105 and CD34 ＋ve cells, SERCA1a protein and gene values. The morphological changes regressed by therapy. In group III significant decrease in DNA fragmentation and MDA, significant increase in GSH and catalase, significant increase in the mean area of pale nuclei, area % of CD105 and CD34 ＋ve cells versus diabetic group. In group IV, same findings as group III versus diabetic and BMMSCs groups. In group V, same findings as group IV versus diabetic and treated groups. Western blot and PCR proved a mean value of SERCA1a protein and gene comparable to the control group. Mean calcium concentration values revealed a significant increase in the diabetic group, in BMMSCs and AA group versus control and SERCA1a group.ConclusionsSERCA1a transfected BMMSCs proved a definite therapeutic effect, more remarkable than BMMSCs combined with AA. This effect was evidenced histologically and confirmed by significant changes in the biochemical tests indicating oxidative stress, muscle calcium concentration, morphometric parameters and PCR values of SERCA1a.

Objective To observe the in vivo gene expression profile of the recombinant adenoassociated-2 virus mediated human GM-CSF, mouse GM-CSF (rAAV-2-hGM-CSF, rAAV-2-mGM-CSE )vector modified bone marrow mesenchymal stem cell (BMSC). Methods We transduced the BMSC by rAAV-2-hGM-CSF, rAAV-2-mGM-CSF at the condition which have acquired before respectively, then transfused the in vitro gene modified BMSC after 12 days proliferation in vitro to 6 weeks old nude mice through tail vein,while the BMSC transfused in control group hadn' t been gene modified. 2, 4, 6, 8 weeks after transfusion, count the total white blood cells and detect the hGM-CSF, mGM-CSF concentration in nude mice serum at that time point. Results Nude mice serum hGM-CSF levels were 23.77, 25.32, 19.77, 15.25 ng/L at 2, 4, 6, 8 weeks after transfusion compare to 36.25 ng/L, the in vitro level before transfusion; mGM-CSF levels were 34.96, 34.84, 35.50, 32.93 ng/L at 2, 4, 6, 8 weeks after transfusion compare to 25.14 ng/L, the in vitro level before transfusion; at the same time point the nude mice serum mGM-CSF levels were 17.34,17.44, 14.68, 16.85 ng/L in control group, rAAV-2-mGM-CSF transduced BMSC made the nude mice white blood cell count increased, but no changes in nude mice white blood cell count at rAAV-2-hGM-CSFtransduced BMSC and control group. Conclusion BMSC as a gene therapy vehicle, it can be gene modified in vitro, then the gene modified BMSC could let the therapeutic gene to have therapeutic effects in vivo. Key words: Bone marrow; Mesenchymal stem cell; rAAV-2; Gene expression

Transplantation of bone marrow mesenchymal stem cells is a promising new strategy for the repair of infarcted cardiac tissue. However, the majority of transplanted bone marrow mesenchymal stem cells (BMSCs) die soon after transplantation, due in part to oxidative stress in the ischemic region. Oxidative stress is known to induce apoptosis through the activation of caspase-3. The aim of this study is to determine whether small interfering RNA targeting caspase-3 can inhibit the apoptosis of rat BMSCs in vitro. Caspase-3 siRNA expression vectors were prepared and transfected into rat BMSCs in the presence of liposomes. Western blot assay and real-time polymerase chain reaction (RT-PCR) were performed to detect caspase-3 expression. A retrovirus packaging system was employed to package 293FT cells producing caspase-3 siRNA virus, which were transfected into rat BMSCs. Those stably expressing caspase-3 siRNA were screened by Western blot assay and RT-PCR to determine caspase-3 expression levels. Stable transfection of caspase-3 siRNA significantly decreased caspase-3 protein (0.26 ± 0.001 vs. 0.42 ± 0.004, P < 0.05) and mRNA expression (0.19 ± 0.002 vs. 1, P < 0.05) in BMSCs compared to non-transfected BMSCs. Cells were incubated in H2O2 to induce apoptosis, which was detected by TUNEL staining, and BMSC morphology was not altered by either transient or stable transfection of caspase-3 siRNA. H2O2-induced apoptosis of BMSCs stably transfected with caspase-3 siRNA was dramatically reduced compared to that of normal BMSCs (11.0 ± 3.2 vs. 25.8 ± 4.2, P < 0.05). Caspase-3 knockdown BMSCs are thus more resistant to apoptosis than normal BMSCs, potentially increasing their survival rates under conditions that cause oxidative stress.