Role of Oct3/4 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of Oct3/4 in inducing differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons in vitro. METHODS: Lentivirus (LV) vector containing Oct3/4 gene was constructed and transfected into rat bone marrow MSCs. The MSCs were divided into non-transfection group, transfection group (transfected with Oct3/4 -LV) and negative control group (transfected with FU-PCG-NC-LV). β-mercaptoethanol (β-ME) was used to induce differentiation of MSCs into neurons. Morphological changes and the fluorescence in transfected MSCs were observed under inverted fluorescence microscope. The expression of Oct3/4 and microtubulin-associated protein 2(MAP-2) at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of Oct3/4 and the neural cell specific markers neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP) were determined by immunocytochemical method. The viability of the MSCs was analyzed by MTT assay. RESULTS: The results of PCR confirmed that the Oct3/4 -LV was successfully constructed and the virus titer was 2×10¹¹ TU/L. The best transfection efficiency and survival rate appeared when multiply of infection(MOI) was 10 and at 48 h, and the fluorescence of MSCs was mostly displayed. The efficiency of transfection was up to 83.4%±2.2%. The shape of the MSCs was changed in transfection group, and the survival rate of the MSCs in transfection group was significant lower than that in other groups (P<0.05). MSCs were induced by β-ME to differentiate into neurons and the best efficiency of induction was observed in transfection group. The typical neuronal morphology was observed in transfection group after induction and the expression levels of NSE and MAP-2 were higher than those in other groups (P<0.05). Compared with other groups, the expression of Oct3/4 in transfection group was significantly increased (P<0.01). Furthermore, the expression of Oct3/4 was time-dependently decreased and there was significant difference between before induction and 5 h after induction (P<0.05). CONCLUSION: Oct3/4 may have an important role in regulating the differentiation of rat MSCs into neurons.     

Transplantation of exogenous mesenchymal stem cells treats post-infarction ventricular remodeling in rats

AIM: This study was performed to investigate the feasibility and efficiency of exogenous mesenchymal stem cells (MSCs) transplantation on post-infarction ventricular remodeling and heart function in rats and compare the effects between adult rat MSCs and neonate rat MSCs transplantation. METHODS: 1-2 hours after left coronary artery ligation, MSCs cultured in ex vivo, marked with BrdU, were injected directly into the border of infarcts in exogenous rats. 6 weeks after transplantation, rat heart function, ventricular remodeling and pathological results were measured. RESULTS: MSCs transplantation decreased LV end-diastolic diameter and end-systolic diameter, limited LV chamber dilatation and reduced collagen content significantly. The numbers of blood vessels and cardiomyocytes were increased. BrdU-labelled MSCs with oval nucleus were widely distributed. There were no significant difference between adult rat MSCs and neonate rat MSCs transplanted groups. CONCLUSION: MSCs can survive and home in exogenous host infarct hearts without addition of any immunosuppressant. MSCs transplantation has benificial effects on remodeling processes and contributes to improvement of cardiac function, which may be related with the reduction of the amount of the collagen, promotion of myogenesis and angiogenesis.     

Effects of Ginkgo biloba extract on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats

AIM:To study the effects of Ginkgo biloba extract (EGB) on myocardial TGF-β1 and collagen expression and interstitial fibrosis in type I diabetic cardiomyopathy rats. METHODS:Thirty male SD rats were randomly divided into normal control group (CON), diabetes mellitus group (DM) and EGB treatment group (EGB). Streptozocin was intraperitoneally injected into the animals in the latter 2 groups to induce type I diabetic rat model. The rats in EGB group were intraperitoneally injected with EGB. At the end of the 12th week, the body weight of each rat and its left ventri-cular weight, blood glucose, glycosylated hemoglobin and serum insulin concentration were measured. The left ventricular end-diastolic volume (LVEDV), the left ventricular end-systolic volume (LVESV), the left ventricular ejection fraction (LVEF) and the stroke volume (SV) were determined by echocardiography. The content of collagen in left ventricular myocardium, and the expression of transforming growth factor β1 (TGF-β1), procollagen type I and collagen type III were assayed by Sirius red staining, immunohistochemical staining and RT-PCR, respectively. Left ventricular myocardial cells of the neonatal SD rats were isolated and cultured in vitro with low-glucose culture medium (LG group), high-glucose culture medium (HG group) or high-glucose culture medium plus EGB (HG+EGB group). The mRNA levels of TGF-β1, procollagen type I and collagen type III were detected by RT-PCR. RESULTS:Compared with CON group, blood glucose, glycosylated hemoglobin, left ventricular weight index, the content of collagen, and the expression of TGF-β1, procollagen type I and collagen type III in left ventricular myocardial tissues of DM group were significantly increased, while the levels of blood insulin, LVEDV and SV were significantly decreased. However, compared with DM group, blood glucose, glycosylated hemoglobin, left ventricule weight index, the content of collagen, and the expression levels of TGF-β1, procollagen type I and collagen type III in the left ventricular myocardial tissues of EGB-treated rats were significantly decreased, while the levels of blood insulin, LVEDV and SV were significantly increased. Compared with LG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly increased. However, compared with HG group, the mRNA expression levels of TGF-β1, procollagen type I and collagen type III were significantly decreased after treated with EGB. CONCLUSION: EGB retards the process of myocardial fibrosis and improves the cardiac functions in type I diabetic cardiomyopathy rats by down-regulating the expression of TGF-β1, reducing the synthesis and deposition of collagen type I and collagen type III.     

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 lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cell(MSCs) differentiation into neurons.METHODS: The lentiviral vector of microRNA-9-1(microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group(transfected with microRNA-9-1-LV) and negative control group(transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol(β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtublin-associated protein 2(MAP-2) was detected by RT-PCR. The expression of neuron-specific markers,neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed successful construction of mouse microRNA-9-1-LV. The virus titer was 1×10¹² TU/L(TU, transduction unit). The best transfection efficiency(up to 91.3%±4.2%) and survival rate appeared when multiply of infection(MOI)was 20 and on 4th day. β-ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other group(P<0.05).CONCLUSION: MicroRNA-9-1-LV has high transfection efficiency in mouse MSCs. Higher differentiation rate from mouse MSCs to neurons is induced by β-ME after the cells are transfected with microRNA-9-1-LV.     

Autologus marrow stromal cell transplantation improves rabbit cardiac performance after myocardiac infarction

AIM: We tested the hypothesis that marrow stromal cells (MSCs), when implanted into self-myocardium, can undergo milieu-dependent differentiation, express cardiomyogenic phenotypes and enhance angiogenesis and 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. One week after myocardial infarction induced by occlusion of left anterior descending artery, autologous MSCs labeled with BrdU (bromodeoxyuridine) in vitro was administered intramyocardially into the infarct area of the same donor rabbits. RESULTS: By 1 months, transplanted MSCs demonstrated to be myogenic differentiation with the expression of α-sarcomeric actin (5C5). MSCs implantation significantly increased vascular density in the infarct zone and resulted in markedly improved the left ventricular contractility. CONCLUSION: The finding indicates that autologous MSCs transplantation may represent a promising therapeutic strategy with free of ethical concerns and immune rejection.     

Apoptosis of colorectal cancer cells transfected with human mutant p27

AIM: To study the inducing effect of human mutant p27 gene on apoptosis of the colorectal cancer cells. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the colorectal cancer cell SW480. The inducing effect of Ad-p27mt on apoptosis in colorectal cancer cells was measured by flow cytometry, DNA fragment analysis and TUNEL method. RESULTS: Ad-p27mt was successfully constructed. When the multiplicity of infection (MOI) was ≥50, the infection efficiency reached 100%. After 24 h of infection, there was an apoptotic hypodiploid peak observed by flow cytometry before G1 and there were apoptotic characteristic bands in the DNA electrophoresis. The apoptotic index detected by TUNEL method was 82.6±3.2 (Ad-p27mt group) and 5.0±3.5 (control group), respectively, the difference of which was significant (P<0.01). CONCLUSION: Human mutant p27 gene transfection effectively induces apoptosis in the colorectal cancer cells.     

Bone marrow mesenchymal stem cells modified by adenoviral vector containing CTLA4Ig inhibit immune response ex vivo

AIM: To investigate the feasibility and infection efficiency of MSCs as the target cells of gene delivery mediated by adenoviral vector carrying CTLA4Ig gene, and to study the mechanism of transgenic MSC to inhibit immune response ex vivo. METHODS: The recombinant adenovirus containing CTLA4Ig gene was constructed, by which rat MSCs with various multiplicity of infection (MOI) were conducted. The infection efficiency was analyzed with FACS and fluorescence microscope. The expression of CTLA4Ig protein in transgenic MSCs was detected by FACS and western blot. Co-culturing the transgenic MSCs with mixed lymphocytes, the inhibitory effect of transgenic MSCs on lymphocyte proliferation was also observed. RESULTS: The adenoviral vector delivered CTLA4Ig gene with high efficiency to MSCs. The expression of CTLA4Ig protein was detected in transgenic MSCs. The gene modified MSCs inhibited the proliferation of mixed lymphocytes and maximal inhibition rate was observed on day 4 of MLR. The inhibition induced by CTLA4Ig was donor-specific. CONCLUSION: MSCs is a promising target cell for gene delivery. The expressed CTLA4Ig specifically inhibited the lymphocyte proliferation ex vivo.     

Sedum sarmentosum Bunge extract inhibits epithelial-mesenchymal transition and collagen accumulation in aristolochic acid-treated rat renal tubular epithelial cells

AIM:To investigate the effect of Sedum sarmentosum Bunge (SSB) extract on epithelial-mesenchymal transition (EMT) and collagen accumulation induced by aristolochic acid (AA) in renal tubular epithelial cells. METHODS:Rat renal tubular epithelial NRK-52E cells were randomly divided into 3 groups, including control group (only treated with solvent), AA group (treated with AA at concentrations ranging from 1 to 100 mg/L) and SSB group (treated with AA at a concentration of 10 mg/L plus SSB extract at concentrations ranging from 10 to 2 000 mg/L). After cultured for 24 h, the morphology of the NRK-52E cells was observed under inverted phase-contrast microscope. The level of transforming growth factor β1 (TGF-β1) in the culture supernatant was measured by ELISA. Immunofluorescent analysis was performed to detect the expression of epithelial marker α-smooth muscle actin (α-SMA), mesenchymal marker E-cadherin, and extracellular cell matrix component type III collagen. The mRNA expression of E-cadherin, α-SMA, bone morphogenetic protein 7 (BMP-7) and type I collagen was also quantified by real-time PCR. RESULTS: Fibrosis-like reaction observed under microscope was obviously increased in AA-treated NRK-52E cells, and aggravated as the increase in the concentration of AA. AA at concentrations of 1 and 10 mg/L increased the expression of α-SMA, type I and type III collagens, and decreased the expression of E-cadherin. With SSB extract treatment, fibrosis in NRK-52E cells was alleviated, accompanied with the decreasing expression of α-SMA, type I and type III collagen, and the enhancing expression of E-cadherin and BMP-7.Moreover, SSB extract down-regulated TGF-β1 level in a concentration-dependent manner. CONCLUSION: AA-induced fibrosis-like reaction in renal tubular epithelial cells is reduced by the treatment with SSB extract. The possible mechanism is that SSB extract decreases TGF-β1 level, and inhibits renal EMT and collagen accumulation induced by AA.［KEY WORDS］Sedum sarmentosum Bunge|Aristolochic acid|Transforming growth factor β1|Epithelial-mesenchymal transition|Collagen     

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.     

Transfection efficiency and cytotoxicity in human T lymphocytes infected with Ad5 adenoviral vector

AIM:To determine the transfection efficiencies and to evaluate the cytotoxicity of infection with Ad5 adenoviral vector in human T lymphocytes. METHODS:The T-lymphoma Jurkat cell line, normal CD3+ T cells and pre-stimulated CD3+ T cells were transfected with Ad5 adenovirus at multiplicities of infection (MOI) ranging from 20 to 400. GFP expression was analyzed by flow cytometry 48 h after transfection. The cells were harvested 24 h, 48 h and 72 h after transfection. The cell cycle was analyzed using propidium iodide staining and the apoptosis of T lymphocytes was detected by annexin V/7-AAD staining. Trypan blue exclusion assay was used to determine the survival cell numbers after 48 h or 72 h of transfection. RESULTS:The transfection of primary human T cells by the Ad5 virus was less efficient than that of a T-lymphoma cell line. Similar transfection efficiency was observed in both CD4+ T lymphocytes and CD8+ T lymphocytes. The activation of T lymphocytes resulted in a decrease in Ad5 transfection efficiency in CD8+ T cells. Following transfection (24 h, 48 h and 72 h), the percentages of G 0/G 1-phase cells, S-phase cells, G 2/M-phase cells and apoptotic cells at all MOI did not change significantly. Therefore, transfection by the Ad5 adenoviral vector did not influence the cell cycle and survival cell numbers. CONCLUSION:The Ad5 adenoviral vector, which shows little cytotoxicity to T lymphocytes, may be a valuable tool for T cell receptor gene therapy.     

Inhibitory effects of FRNK on collagen synthesis in hepatic stellate cells

AIM:To investigate the effect of focal adhesion kinase-related non-kinase (FRNK) on collagen synthesis in hepatic stellate cells (HSCs).METHODS:HSCs were transfected with FRNK by cationic liposome method. The expressions of FRNK at the protein level in HSCs were detected by Western blotting analysis. HSCs collagen synthesis capability was examined by［3H］-Pro incorporation assay.RESULTS:The exposure of HSCs to FRNK led to the up-regulated expression of FRNK protein, and it was at 48 h after transfection that the FRNK protein content was the highest. Moreover, after FRNK was transfected successfully in HSCs, the total collagen synthesis and type I collagen synthesis were inhibited.CONCLUSION:After FRNK is transfected successfully in HSCs using Lipofectamine, the synthesis of total and type I collagen in HSCs is inhibited.     

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.     

Effects of rhTGF-β1 on proliferation and osteogenic differentiation of MSCs in SD rats

AIM：To investigate the effects of recombinant human transforming growth factor β1 (rhTGF-β1) on the ability of proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), as well as its effects on the expression of bone morphogenetic protein 2 (BMP-2), Smad4 and core binding factor α1 (Cbfa1). METHODS：SD rat MSCs were isolated and purified by the differential time adherent method. MTT assay was used to confirm the optimal concentration of rhTGF-β1 for the proliferation of MSCs. The optimal concentration for differentiation of MSCs into osteoblast was also determined by observing the activity and positive staining of alkaline phosphatase. According to the different induction conditions, MSCs were divided into 4 groups:control group, classic group, rhTGF-β1 group, and rhTGF-β1+classic group. Alkaline phosphatase, type I collagen, bone Gla protein and calcium nodes were detected to evaluate the osteogenic differentiation. BMP-2 was detected by ELISA and the mRNA expression of Smad4 and Cbfa1 was analyzed by real-time quantitative PCR. RESULTS：The optimal concentrations of rhTGF-β1 for the proliferation of MSCs and for the osteogenic differentiation of MSCs were 10 and 5 μg/L, respectively. The MSCs in classical group and rhTGF-β1 group were promoted to osteogenic differentiation, and the mRNA expression of BMP-2, Smad4 and Cbfa1 was increased. rhTGF-β1 induced osteogenic differentiation of MSCs in the early and middle terms. However, in rhTGF-β1+classic group, the osteogenic differentiation of MSCs was more obvious in the late term. CONCLUSION:The induction conditions of classical group, rhTGF-β1 group and rhTGF-β1+ classical group promote the differentiation of MSCs by increasing BMP-2 secretion and starting the TGF-β superfamily/Smads signaling pathway to regulate the differentiation of MSCs.     

Recombinant MIF induces synthesis of collagen type III in lung fibroblasts via Rho-kinase

AIM: To investigate the effects of recombinant macrophage migration inhibitory factor (rMIF) on the synthesis of collagen type III in fibroblasts.METHODS: The MRC-5 fibroblasts were divided into 2 groups.The cells in treatment group were exposed to rMIF at the concentrations of 25-100 μg/L for 48 h. The cells without rMIF treatment served as control group. Half an hour before challenging with rMIF (100 μg/L), Y27632 (a Rho-kinase inhibitor) was added to the cells in both 2 groups. After challenged for 48 h, total RNA and protein in the cells were extracted. The expression of collagen type III at mRNA and protein levels was analyzed by RT-PCR and Western blotting, respectively.RESULTS: Different concentrations of rMIF significantly increased the expression of collagen type III at mRNA and protein levels in a dose-dependent manner as compared with the control cells (r=0.862 and r=0.914; all P<0.01). These increases were abolished by Y27632 pretreatment(P<0.01).CONCLUSION: rMIF increases the synthesis of collagen type III in MRC-5 cells and Rho-kinase regulates the MIF-induced synthesis of collagen, suggesting that MIF may play important roles in the pathogenesis of airway remodeling.     

Effects of bone mesenchymal stem cell transplantation on silicosis fibrosis in rats

AIM: To study the effects of exogenous bone mesenchymal stem cell (BMSC) transplantation on silicosis fibrosis in rats, and to explore the dose-effect relationship. METHODS: BMSCs were isolated and cultured from male 5-week-old SD rats in vitro. Fifty healthy female SD rats were randomly divided into 5 groups: control group, silicosis model group, BMSCs treatment A group (1×10⁹ cells/L), BMSCs treatment B group (3×10⁹ cells/L) and BMSCs treatment C group (5×10⁹ cells/L). The silicosis model was made by one-time infusion of silica dust suspension using the non-exposed tracheal intubation, and different doses of BMSCs were given for intervention therapy. All the rats were sacrificed on the 21st day after the model was established. The morphological changes of the lung tissues were observed by HE staining and Masson staining. The localization and distribution of tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) were determined by the method of immunohistochemistry. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III were detected by Western blotting. The sex-determining region (SRY) protein was searched by an immunofluorescence method to confirm the homing of BMSCs. RESULTS: Compared with control group, the silicosis model group had significant alveolitis changes, silicon nodule formation, collagen deposition and other pathological characteristics. Compared with silicosis model group, the pathological changes in BMSCs treatment A group were improved. The conditions of BMSCs treatment B group were also improved significantly. However,the pathological changes in BMSCs treatment C group were increased obviously. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III in the lung tissues ranked as follows: BMSCs treatment C group > silicosis model group > BMSCs treatment A group > BMSCs treatment B group > control group. The difference between BMSCs treatment C group and silicosis model group was not statistically significant, and the differences between the other groups were statistically significant. The SRY-positive cells were observed in BMSCs treatment B group, but no significant expression in the heart, liver, spleen and kidney tissues was observed. CONCLUSION: The exogenous BMSC transplantation antagonizes the development of silicosis fibrosis in rats, which has dose-effect relationship.     

Effect of livin-modified BM-MSCs transplantation on cardiac function following acute myocardial infarction in a rat model

AIM: To study the effect of livin gene-modified bone marrow mesenchymal stem cells(BM-MSCs) transplantation on the cardiac function following acute myocardial infarction in a rat model and the expression of livin, caspase-3, caspase-7 and caspase-9 in the livin gene-modified BM-MSCs. METHODS: The MSCs were obtained by the whole bone marrow culture method, and the apoptosis of the MSCs after infection with adenovirus vector carrying enhanced green fluorescent protein(EGFP) gene and livin recombinant vector(rAd-livin) were detected by flow cytometry. The expression of livin, caspase-3, caspase-7 and caspase-9 was detected by Western blot. After permanent left anterior descending artery occlusion, the rats were randomized to receive intramyocardial injection of DMEM without cells(vehicle group), or containing MSCs(MSCs group), MSCs(EGFP)(rAd-control/MSCs group) or MSCs(livin)(rAd-livin/MSCs group). Left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), the maximum increased rate of left ventricular pressure(-dp/dt_max) and the maximum decline rate of left ventricular pressure(+dp/dt_max) were recorded for evaluating the cardiac functions. RESULTS: The apoptosis of rAd-livin/MSCs was significantly decreased as compared with MSCs and rAd-control/MSCs(P<0.05). Meanwhile, the expression of caspase-3, caspase-7 and caspase-9 was significantly downregulated as compared with the other 2 groups(P<0.05). The cardiac function in rAd-livin/MSCs group was significantly improved as compared with DMEM group, and those in the other 2 groups got the similar results, but the function in rAd-livin/MSCs group was better improved. Meanwhile, the number of surviving cells in rAd-livin/MSCs group was significantly improved as compared with the other 2 groups. CONCLUSION: The apoptosis of MSCs is decreased after rAd-livin transfection, and the expression of caspase-3, caspase-7 and caspase-9 is also significantly downregulated while the expression of livin is significantly upregulated. Transplantation of livin-modified BM-MSCs by lentiviral vector results in better prognosis for treating myocardial infarction by enhancing cell survival.     

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.