【Abstract】 Objective To explore the new therapy for pulmonary fibrosis by observing the effects of insulin-like growth factor 1 ( IGF-1) treated mesenchymal stemcells ( MSCs) in rats with bleomycin-induced pulmonary fibrosis. Methods Bone marrowmesenchymal stemcells ( BMSCs) were harvested from6-week old male SD rats and cultured in vitro for the experiment. 48 SD rats were randomly divided into 4 groups, ie.a negative control group ( N) , a positive control group/bleomycin group ( B) , a MSCs grafting group ( M) ,and an IGF-1 treated MSCs grafting group ( I) . The rats in group B, M and I were intratracheally injected with bleomycin ( 1 mL,5 mg/kg) to induce pulmonary fibrosis. Group N were given saline as control. Group M/ I were injected the suspension of the CM-Dil labled-MSCs ( with no treatment/pre-incubated with IGF-1 for 48 hours) ( 0. 5mL,2 ×106 ) via the tail vein 2 days after injected bleomycin, and group B were injected with saline ( 0. 5 mL) simultaneously. The rats were sacrificed at 7,14,28 days after modeling. The histological changes of lung tissue were studied by HE and Masson’s trichrome staining. Hydroxyproline level in lung tissue was measured by digestion method. Frozen sections were made to observe the distribution of BMSCs in lung tissue, and the mRNA expression of hepatocyte growth factor ( HGF) was assayed by RTPCR.Results It was found that the red fluorescence of BMSCs existed in group M and I under the microscope and the integrated of optical density ( IOD) of group I was higher than that of group M at any time point. But the fluorescence was attenuated both in group M and group I until day 28. In the earlier period, the alveolitis in group B was more severe than that in the two cells-grafting groups in which group I was obviously milder. But there was no significant difference among group I, M and group N on day 28.Pulmonary fibrosis in group B, Mand I was significantly more severe than that in group N on day 14, but itwas milder in group M and I than that in group B on day 28. Otherwise, no difference existed between the two cells-grafting groups all the time. The content of hydroxyproline in group B was significantly higher than that in the other three groups all through the experiment, while there was on significant difference betweengroup I and group N fromthe beginning to the end. The value of group M was higher than those of group I and group N in the earlier period but decreased to the level of negative control group on day 28. Content of HGF mRNA in group Nand group I was maintained at a low level during the whole experiment process. The expression of HGF mRNA in group I was comparable to group M on day 7 and exceeded on day 14, the difference of which was more remarkable on day 28. Conclusions IGF-1 can enhance the migratory capacity of MSCs which may be a more effective treatment of lung disease. The mechanismmight be relatedto the increasing expression of HGF in MSCs.
Objective To construct chemically extracted acellular nerve allograft (CEANA) with Schwann cells (SCs) from different tissues and to compare the effect of repairing peripheral nerve defect. Methods Bone marrow mesenchymal stem cells (BMSCs) and adi pose-derived stem cells (ADSCs) were isolated and cultured from 3 4-week-old SD mice with weighing 80-120 g. BMSCs and ADSCs were induced to differentiated MSC (dMSC) and differentiated ADSC (dADSC) in vitro.dMSC and dADSC were identified by p75 protein and gl ial fibrillary acidic protein (GFAP). SCs were isolated and culturedfrom 10 3-day-old SD mice with weighing 6-8 g. CEANA were made from bilateral sciatic nerves of 20 adult Wistar mice with weighing 200-250 g. Forty adult SD mice were made the model of left sciatic nerve defect (15 mm) and divided into 5 groups (n=8 per group) according to CEANA with different sources of SCs: autografting (group A), acellular grafting with SCs (5 × 105) (group B), acellular grafting with dMSCs (5 × 105) (group C), acellular grafting with dADSCs (5 × 105) (group D), and acellular grafting alone (group E). Motor and sensory nerve recovery was assessed by Von Frey and tension of the triceps surae muscle testing 12 weeks after operation. Then wet weight recovery ratio of triceps surae muscles was measured and histomorphometric assessment of nerve grafts was evaluated. Results BMSCs and ADSCs did not express antigens CD34 and CD45, and expressed antigen CD90. BMSCs and ADSC were differentiated into similar morphous of SCs and confirmed by the detection of SCs-specific cellsurface markers. The mean 50% withdrawal threshold in groups A, B, C, D, and E was (13.8 ± 2.3), (15.4 ± 6.5), (16.9 ± 5.3), (16.3 ± 3.5), and (20.0 ± 5.3) g, showing significant difference between group A and group E (P lt; 0.01). The recovery of tension of the triceps surae muscle in groups A, B, C, D, and E was 87.0% ± 9.7%, 70.0% ± 6.6%, 69.0% ± 6.7%, 65.0% ± 9.8%, and 45.0%± 12.1%, showing significant differences between groups A, B, C, D, and group E (P lt; 0.05). No inflammatory reactionexisted around nerve graft. The histological observation indicated that the number of myel inated nerve fiber and the myel in sheath thickness in group E were significantly smaller than that in groups B, C, and D (P lt; 0.01). The fiber diameter of group B was significantly bigger than that of groups C and D (P lt; 0.05) Conclusion CEANA supplementing with dADSC has similar repair effect in peripheral nerve defect to supplementing with dMSC or SCs. dADSC, as an ideal seeding cell in nerve tissue engineering, can be benefit for treatment of peripheral nerve injuries.
ObjectiveTo investigate the effect of transforming growth factorβ1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, prol iferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured. MethodsBMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and prol iferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types I andⅢ, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR. ResultsA single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion ( > 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05); the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05). The ratios of collagen type Ⅰ to type Ⅲ were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type Ⅰ and type Ⅲ and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05). ConclusionDirectly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.
Objective To study the effect of recombinant lentiviral vector mediated human hepatocyte growth factor (hHGF) gene-modified bone marrow mesenchymal stem cells (BMSCs) on the immunological rejection after allograft l iver transplantation in rats, and to reveal the mechanism of immune tolerance. Methods Eight male Sprague Dawley (SD)rats of clean grade (aged 3 to 4 weeks, weighing 75-85 g) were selected for the isolation and culture of BMSCs; 64 adult male SD rats of clean grade (weighing 200-250 g) were used as donors; and 64 adult male Wistar rats of clean grade (weighing 230-280 g) were used as receptors. After establ ishing a stable model of rat allogeneic l iver transplantation, 1 mL sal ine, 2 ×106/mL of BMSCs 1 mL, 2 × 106/mL of BMSCs/green fluorescent protein 1 mL, and 2 × 106/mL of BMSCs/hHGF 1 mL were injected via the portal vein in groups A, B, C, and D respectively. Then the survival time of the rats was observed. The hepatic function was determined and the histological observation of the l iver was performed. The hHGF mRNA expression was detected by RT-PCR, the level of cytokine including hHGF, interleukin 2 (IL-2), IL-4, IL-10, and interferon γ (IFN-γ) by ELISA assay, the level of apoptosis by TUNEL method, and the expression level of prol iferating cell nuclear antigen (PCNA) by immunohistochemical method. Results The survival time of group D was significantly higher than that of groups A, B, and C (P lt; 0.01); the survival time of groups B and C was significantly higher than that of group A (P lt; 0.01), but there was no significant difference between group B and group C (P gt; 0.05). RT-PCR demonstrated the transcription of hHGF mRNA in the grafts of group D; the serum cytokine hHGF reached to (6.2 ± 1.0) ng/mL. Compared with groups B and C, group D exhibited significant inhibitory effect, significantly improved l iver function, and showed mild acute rejection. In addition, the levels of cytokine IL-2 and IFN-γ decreased; the levels of cytokine IL-4 and IL-10 increased; the level of apoptosis reduced; and the expression level of PCNA increased. Except for the expression of IL-4 (P gt; 0.05), there were significant differences in the other indexes between group D and groups B, C (P lt; 0.05). Conclusion BMSCs/hHGF implanting to rat l iver allograft via portal vein can induce immune tolerance. Compared with injection of BMSCs alone, BMSCs/hHGF treatment can alleviate acute rejection and prolong the survival time significantly. The immunosuppressive effect of BMSCs/hHGF is correlated with Th2 shifts up of Th1/Th2 shift, reduced apoptosis, promoted l iver regeneration.
Objective To construct lentiviral vector carrying the human hepatocyte growth factor (hHGF) gene, and then to get hHGF gene/modified bone marrow mesenchymal stem cells (BMSCs) by infecting the BMSCs. Methods The hHGF gene was obtained with PCR from pcDNA-hHGF plasmid. The recombination lentiviral vector plasmid hHGF was constructed with Age I digestion and gene recombinant, then was identified with PCR and sequencing. Mediated by Lipofectamine2000, the three plasmids system of lentiviral vector including pGC-E1-hHGF, pHelper 1.0, and pHelper 2.0 was co-transfected to 293T cells to produce hHGF gene. The supernatant was collected and concentrated by ultracentrifugation and the titer of lentivirus was measured by real-time quantitative PCR. The BMSCs were infected by the constructed lentivirus and the multipl icities of infection (MOI) was identified with fluorescent microscope, the efficiency of infection with flow cytometry (FCM) analysis, the hHGF level with ELISA analysis, and the expression of hHGF gene with RT-PCR. Results Lentiviral vector carrying hHGF gene was constructed successfully. The titer of lentivirus was 1 × 108 TU/mL. The infection efficiency of BMSCs by hHGF lentiviral was high and reached 98% by FCM, and the best MOI was 10. A great mount of green fluorescence was observed with the fluorescent microscope at 28 days after infection. Peak concentration of hHGF secreted by BMSCs/hHGF reached 40.5 ng/mL at 5 days. The concentration could maintain a high level until 28 days after infection. RT-PCR showed that BMSCs/hHGF could express hHGF gene. Conclusion By lentiviral vector, hHGF gene was integrated into BMSCs genome, and it can express stably.
【Abstract】 Objective To investigate the effect of salidroside on rat bone marrow mesenchymal stem cells (BMSCs)differentiation into the chol inergic nerve cells, so as to provide the theory basis of the combination of salidroside and stem cellsfor cl inical therapy of nervous system diseases. Methods BMSCs were isolated from 2 Wistar rats (aged 4-6 weeks, weighing 120 g), which were identified by CD34, CD45, CD90, and CD106 with flow cytometry. According to inducing method, BMSCs at passage 2 were divided into 3 groups: In groups A and B, BMSCs were induced by salidroside (20 μg/mL) and retinoic acid (5 μmol/mL) respectively for 1, 3, 6, and 9 days, in group C, BMSCs were cultured with serum-free DMEM/F12 medium as control. MTT assay was used to detect the cellular prol iferation activity. The immunofluorescence chemical technology was used to detect the expressions of nerver growth factor (NGF) and relevant marker molecule of nerve cells, including neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), β-Tubulin III, gl ial fibrillary acidic protein (GFAP), and the marker of cholinergic neuron, such as Acetylcholine (Ach) and NGF. RT-PCR was used to detect mRNA expressions of NSE, β-Tubulin III, GFAP, brain derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA). ELISA was used to detect the levels of BDNF and NGF, and the expression level of NGF protein was analyzed by Western blot. Results The results of the flow cytometry showed that the cultured cells were CD90 and CD106 positive, and CD34 and CD45 negative, which indicated that the cells were BMSCs. The cellular proliferation activity in groups A and B were significantly higher than that in group C at 6 days and 9 days (P lt; 0.05). RT-PCR results showed that the expression level of NSE, BDNF, β-Tubulin III, GFAP mRNA were increased in groupA at 6 days; In group B, that expression level of NSE mRNA was up-regulated at 6 days, that expression level of BDNF mRNA increased at 1 days and reached the peak at 6 days, and that expression level of β-Tubulin III mRNA was up-regulated at 3 days, which was significantly higher than that at the other time points, and than that in group C (P lt; 0.01). But no GABA mRNA expression was detected in each group. Immunofluorescence chemical technology staining showed that the positive rates of NSE, MAP2, β-Tubulin III, and GFAP were significantly higher in group A than those in group C at 3 days; the positive rates of Ach were significantly higher at 3, 6, and 9 days than those at 1 day in groups A and B, and in groups A and B than in group C (P lt; 0.01); the positive rates of NGF in groups A and B were significantly higher than those in group C (P lt; 0.01). The levels of BDNF and NGF in groups A and B were significantly higher than those in group C at 1, 3, 6, and 9 days (P lt; 0.01), but no significant difference of BDNF was found between groups A and B (P gt; 0.05). The expression level of NGF protein in groups A and B were significantly higher than that in group C (P lt; 0.01). The NGF expression reached the peak at 6 days in group A and at 3 days in group B. Conclusion Sal idroside could induce rat BMSCs differentiate into chol inergic nerve cells in vitro.
ObjectiveTo study the potential protective effects of bone marrow mesenchymal stem cells (BMSCs) on chondrocytes injured by interleukin 1β (IL-1β), and the resistant capacity of chondrocytes when co-cultured indirectly with BMSCs against IL-1β. MethodsSix Sprague Dawley (SD) rats were randomly divided into experimental group (articular cartilage defects) and control group. The content and gene expression of IL-1β were detected at 6 hours after surgical intervention by quantitative real time RCR (qRT-PCR) and ELISA. BMSCs repairing function test: the 18-holes cultured chondrocytes were randomly divided into 3 groups (n=6): cells of blank group were not treated;cells of injured group and co-cultured group were intervened by IL-1β, and Transwell chamber was used to establish co-culture system of BMSCs with chondrocyte in co-cultured group. The mRNA relative expressions of cysteinyl aspartate specific proteinase 3 (Caspase 3), a disintegrin and metalloprotease with Thrombospondin motifs 4 (ADAMTS-4), and ADAMTS-5 were measured via qRT-PCR in chondrocytes, meanwhile Caspase-3 content was detected via ELISA, and the cell apoptosis rate was detected via flow cytometry. BMSCs protecting function test: the 12-holes cultured chondrocytes were randomly divided into 2 groups (n=6), Transwell chamber was used to establish co-culture system of BMSCs with chondrocyte in co-cultured group before the 2 groups were both intervened by IL-1β, then the same detected indexes were taken as the BMSCs repairing function test. ResultsAnimal in vivo studies showed that relative expression of IL-1β mRNA and IL-1β contents were significantly higher in experimental group than control group (P<0.05). BMSCs repair tests showed that mRNA relative expressions of Caspase-3, ADAMTS-4, and ADAMTS-5, Caspase-3 content, and cell apoptosis rate were significantly higher in injured group and co-cultured group than blank group, and in injured group than co-cultured group (P<0.05). BMSCs protect tests showed that mRNA relative expressions of Caspase-3, ADAMTS-4, and ADAMTS-5, Caspase-3 content, and cell apoptosis rate in co-cultured group were significantly lower than those in control group (P<0.05). ConclusionBMSCs, as seed cells for tissue engineering, have potential for applications to anti-inflammation and anti-apoptosis.
Objective To summarize the recent advance in the research of tissue engineered nerve grafts. Methods The cl inical and experimental research papers about tissue engineered nerve grafts were extensively reviewed and analyzed. Results The porosity, mechanical property and surface topography of a nerve scaffold, which was either made up of natural biodegradable polymers or synthetic polyesters, were pivotal factors that influenced the capacity of the scaffold in supporting nerve regeneration. Of various candidate supporting cells for nerve tissue engineering, the bone marrowmesenchymal stem cells had been paid more attention because of their advantages. Several model designs of drug del ivery systems for controlled release of growth factors had been attempted. In cl inical settings, short nerve gaps were demonstrated to be treatable with several nerve conduits which were commercially available, with functional recovery approximating tonerve autografting. Conclusion The field of nerve tissue engineering has witnessed a rapid development not only in experimental research but also in cl inical appl ication.
Abstract： Objective To investigate the effects of hepatocyte growth factor（HGF）gene transfected bone marrow mesenchymal stem cells （MSCs）transplantation in pigs with chronic ischemic heart disease. Methods MSCs were isolated from pig bone marrow by density gradient centrifugation and adherent cell culture, purified, and determined by cellsurface antigens（CD34, CD44, CD71, Ⅷ factor and desmin）. MSCs were transfected by adenovirus expressing hepatocyte growth factor（AdHGF）, and the influence of HGF on the biological characteristics of MSCs was tested. The pig model of chronic myocardial ischemia was established by placing Ameroid ring inside the left circumflex coronary artery via leftthoracotomy. A total of 40 pigs were randomly divided into 5 groups （n=8） and were injected 5×106/ml MSCs＋ 4×109 pfu 200 μl AdHGF （MSCs＋ AdHGF group）, 4×109 pfu 200 μl AdHGF （AdHGF group）, 5×106/ml MSCs 200 μl（MSCs group），4×109 pfu 200 μl AdNull （AdNull group）and 1 ml saline（control group） into the ischemic myocardiumrespectively. Echocardiogram, digital subtraction angiography （DSA） of coronary artery, single photon emission computed tomography（SPECT） myocardial perfusion imaging and cardiomyocyte apoptosis were examined after 4 weeks. Results Positive CD44 and CD71 and negative CD34, Ⅷ factorand desmin were detected in MSCs by flow cytometer. HGF had a b influence on stimulating the proliferation and differentiation of MSCs. Echocardiogram examination showed that left ventricular end-diastolic volume（LVEDV），left ventricular ejection fraction（LVEF），fractional shortening（FS）of MSCs＋ AdHGF group were significantly increased after treatment （P＜ 0.05）. DSA detection showed that ischemic neovascularization of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group （P＜ 0.05）. SPECT showed that the left ventricular myocardium of MSCs＋ AdHGF group appeared thickened,myocardial perfusion was significantly improved and the myocardial motion was significantly increased （P＜ 0.05）. Vascular density of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group by HE stain of myocardium ［（39.4±1.2）/ HPF vs. （36.5±1.4）/ HPF and（34.5±1.7）/ HPF，P＜ 0.05］. Cardiomyocyte apoptosis rate of MSCs＋ AdHGF group was significantly lower than those of AdHGF group and MSCs group by TUNEL stain （P＜ 0.05）. Conclusion Combination transplantation can promote the angiogenesis of chronic ischemic myocardium, inhibit cardiomyocyte apoptosis and improve heart function in pigs with chronic ischemic heart disease. The effect of HGF gene transfected MSCs transplantation is better than that of MSCs or HGF transplantation alone.
Objective To review the basic research, the cl inical progress, and the mechanism of bone marrow mesenchymal stem cells (BMSCs) in acute lung injury (ALI). Methods The l iterature concerning the basic and cl inical researches of BMSCs in ALI was reviewed. Results BMSCs can take the initiative to “homing” the site of lung injury and take partici pate in repair by means of differentiation, meanwhile BMSCs could regulate and balance local and systematic inflammatory response and immune disorders in ALI. Currently, the mechanism of BMSCs on anti-inflammation and immune regulation in ALI is not clear. Conclusion BMSCs have comprehensive biological effect on ALI, providing a potential cl inical treatment and also laying the foundation for gene therapy and stem cell therapy of ALI in the future.