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.
ObjectiveTo explore the effect and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on healing quality of acetic acid-induced gastric ulcer.
MethodsForty-eight clean grade male Wistar rats were used to establish the model of gastric ulcer with acetic acid and were randomly divided into 3 groups after 3 days of modeling, 16 rats each group. After the abdominal cavity was open and stomach was pulled out, no treatment was given in group A, 150 μL phosphate buffered saline (PBS) and 150 μL BMSCs at passage 4+PBS (1×108 cells/100 μL) were injected into the gastric wall surrounding the ulcer at 5 different points in groups B and C respectively. After 10 days, the ulcer area was measured, the mucosal thickness and the number of dilated glands were tested in the regenerative mucosa by histological method. And the expression of vascular endothelial growth factor (VEGF) was detected at ulcerative margin by immunohistochemical method.
ResultsThe ulcer area in group C was significantly smaller than that of groups A and B (P<0.01), but no significant difference was found between groups A and B (P>0.05). HE staining showed that group C had thicker regenerative gastric mucosa, less dilated glands, and more regular mucosal structure than groups A and B, showing significant differences in regenerative gastric mucosa thickness and dilated glands number (P<0.01), but no significant difference between groups A and B (P>0.05). Immunohistochemical staining showed that the positive expression of VEGF in the ulcer margin mucosa of group C was significantly higher than that of groups A and B. The integral absorbance (IA) value of VEGF expression in group C was significantly higher than that in groups A and B (P<0.01), but no significant difference between groups A and B (P>0.05).
ConclusionBMSCs can accelerate ulcer healing by the secretion of VEGF, and improve the quality of ulcer healing.
Objective To review the biochemical characteristics, appl ication progress, and prospects of the adiposederived stem cells (ADSCs). Methods The recent original experimental and cl inical l iterature about ADSCs was extensively reviewed and analyzed. Results ADSCs can be readily harvested in large numbers from adipose tissue with properties of stable prol iferation and potential differentiation in vitro. Significant progress of ADSCs is made in the animal experimentand the cl inical appl ication. It has been widely used in the cl inical treatment of cardiovascular disease, metabol ic disease, encephalopathy, and tissue engineering repair. Conclusion ADSCs have gradually replaced bone marrow mesenchymal stem cells and become the focused hot spot of regenerative medicine and stem cells.
ObjectiveTo investigate the effect of echinococcus granulosus protoscolices on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into fibroblasts.MethodsFemur bone marrow of 4-week-old C57BL/6 mice was taken and BMSCs were isolated and cultured by adherent culture. Echinococcus granulosus protoscolices was extracted from the liver of sheep infected with echinococcus granulosus. The experiment was divided into two groups. The experimental group was co-cultured with the 3rd generation BMSCs and the echinococcus granulosus protoscolices, and the control group was the 3rd generation BMSCs. Before and after co-culture, the morphology of BMSCs and the activity of echinococcus granulosus protoscolices were observed by inverted microscope. After cultured for 1, 3, 5, and 7 days, the mRNA expressions of transforming growth factor β1 (TGF-β1), collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescent quantitative PCR, the protein expressions of TGF-β1, collagen type Ⅰ, collagen type Ⅲ, Smad7, and phosphorylated Smad2/3 were detected by Western blot, and the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the two groups were detected by ELISA.ResultsAfter 7 days of co-culture, the morphology of BMSCs changed into fusiform and irregular triangle, which was closer to the mouse fibroblasts. The relative mRNA expressions of TGF-β1, collagen type Ⅰ, and collagen type Ⅲ in the experimental group were significantly higher than those in the control group; the relative protein expressions of TGF-β1, collagen type Ⅰ, collagen type Ⅲ, and phosphorylated Smad2/3 in the experimental group were significantly higher than those in the control group, and the relative protein expression of Smad7 in the experimental group was significantly lower than that in the control group; the contents of collagen type Ⅰ and collagen type Ⅲ in the supernatant of the experimental group were significantly higher than those in the control group. The differences between the two groups were significant (P<0.05).ConclusionEchinococcus granulosus protoscolices may promote the secretion of collagen type Ⅰ, collagen type Ⅲ, and TGF-β1 by TGF-β1/Smad signal pathway, which can promote the fibrosis of BMSCs that related to the formation of fibrocystic wall by echinococcosis.
Objective
To explore the paracrine effect of bone marrow mesenchymal stem cells (BMSCs) on dexamethasone-induced inhibition of osteoblast function in vitro.
Methods
The serum free conditioned medium of mouse BMSCs cultured for 24 hours was prepared for spare use. The 3rd passage of MC3T3-E1 cells were divided into 4 groups: the control group (group A), dexamethasone group (group B), dexamethasone+BMSCs conditioned medium (1:1) group (group C), and BMSCs conditioned medium group (group D). After 24 hours of culture, the alkaline phosphatase (ALP) content was determined; the protein expressions of RUNX2 and Osteocalcin were detected by Western blot; and the gene expressions of collagen type I-α 1 (COL1A1), RUNX2, ALP, and Osteocalcin were detected by real-time fluorescence quantitative PCR (RT-qPCR); alizarin red staining was used to observe calcium nodules formation at 21 days.
Results
After cultured for 24 hours, ALP content was significantly lower in groups B, C, and D than group A, and in group B than groups C and D (P < 0.05), but no significant difference was found between groups C and D (P > 0.05). The relative protein expression of RUNX2 of group B was significantly lower than that of groups A, C, and D (P < 0.05), but difference was not significant between groups A, C, and D (P > 0.05). The relative protein expression of Osteocalcin was significantly lower in group B than groups A, C, and D, in groups A and C than group D (P < 0.05), but difference had no significance between groups A and C (P > 0.05). The relative gene expressions of RUNX2, Osteocalcin, COL1A1, and ALP of groups B, C, and D were significantly lower than those of group A (P < 0.05); the relative gene expressions of RUNX2, Osteocalcin, and ALP were significantly higher in group D than groups B and C, in group C than group B (P < 0.05). The gene expression of COL1A1 was significantly higher in group D than group B (P < 0.05), but difference was not significant between groups B and C, and between groups C and D (P > 0.05). The cells of group A all died at 6 days after culture; at 21 days, the calcium no dule staining was positive by alizarin red in groups B, C and D, and the degree of the staining gradually increased from groups B to D.
Conclusion
BMSCs conditioned medium can alleviate the inhibitory effect of dexamethasone on osteoblasts function.
ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) derived exosomes (MSCs-exosomes) in tissue repair.
MethodsThe literature about MSCs-exosomes in tissue repair was reviewed and analyzed.
ResultsExosomes are biologically active microvesicles released from MSCs which are loaded with functional proteins, RNA, and microRNA. Exosomes can inhibit apoptosis, stimulate proliferation, alter cell phenotype in tissue repair of several diseases through cell-to-cell communication.
ConclusionMSCs-exosomes is a novel source for the treatment of tissue repair. Further research of MSCs-exosomes biofunction, paracellular transport, and treatment mechanism will help the transform to clinical application.
Objective To investigate the effects of the misshapen auricular chondrocytes from microtia in inducing chondrogenesis of human adipose derived stem cells (ADSCs) in vitro. Methods Human ADSCs at passage 3 and misshapen auricular chondrocytes at passage 2 were harvested and mixed at a ratio of 7 ∶ 3 as experimental group (group A, 1.0 × 106 mixed cells). Misshapen auricular chondrocytes or ADSCs at the same cell number served as control groups (groups B and C, respectively). All samples were incubated in the centrifuge tubes. At 28 days after incubation, the morphological examination was done and the wet weight was measured; the content of glycosaminoglycan (GAG) was detected by Alcian blue colorimetry; the expressions of collagen type II and Aggrecan were determined with RT-PCR; and HE staining, toluidine blue staining, Safranin O staining of GAG, and collagen type II immunohistochemical staining were used for histological and immunohistochemical observations. Results At 28 days after incubation, all specimens formed disc tissue that was translucent and white with smooth surface and good elasticity in groups A and B; the specimens shrank into yellow spherical tissue without elasticity in group C. The wet weight and GAG content of specimens in groups A and B were significantly higher than those in group C (P lt; 0.05), but no significant difference was found between groups A and B in the wet weight (t=1.820 3, P=0.068 7) and in GAG content (t=1.861 4, P=0.062 7). In groups A and B, obvious expressions of collagen type II and Aggrecan mRNA could be detected by RT-PCR, but no obvious expressions were observed in group C; the expressions in groups A and B were significantly higher than those in group C (P lt; 0.05), but no significant difference was found between groups A and B in collagen type II mRNA expression (t=1.457 6, P=0.144 9) and Aggrecan mRNA expression (t=1.519 5, P=0.128 6). Mature cartilage lacunas and different degrees of dyeing for the extracellular matrix could be observed in groups A and B; no mature cartilage lacunas or collagen type II could be observed in group C. The expression of collagen type II around cartilage lacuna was observed in groups A and B, but no expression in group C; the gray values of groups A and B were significantly lower than that of group C (P lt; 0.01), but no significant difference was found between groups A and B (t=1.661 5, P=0.09 7 0). Conclusion Misshapen auricular chondrocytes from microtia can induce chondrogenic differentiation of human ADSCs in vitro.
Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.
ObjectiveTo investigate the multi-directional differentiation potential and other biological characteristics of chicken umbilical cord mesenchymal stem cells (UMSC), as well as their reparative effects on bleomycin (BLM)-induced lung injury in mice. MethodsAn acute lung injury model in mice was established by injecting BLM into the bronchus. UMSC were then transplanted via the tail vein. The reparative effects of UMSC on lung injury were evaluated through pathological section observation, survival and differentiation of transplanted cells in mice, and detection of hydroxyproline (HYP) content, among other indicators. ResultsThe UMSC successfully isolated in this study positively expressed specific surface markers CD29, CD44, CD90, and CD166, while the expression of CD34 and CD45 was negative. Induced UMSC could differentiate into adipocytes, osteocytes, chondrocytes, and alveolar epithelial cells. Animal experiments revealed that BLM-treated mice exhibited damaged alveolar structures, significant inflammatory cell infiltration, abnormal collagen deposition, and pulmonary fibrosis. However, after UMSC transplantation, the extent and severity of lung damage were reduced, and the HYP content in lung tissue decreased but remained higher than that of the control group. ConclusionUMSC can continuously proliferate and maintain their biological characteristics under in vitro culture conditions. They possess the ability to migrate to damaged sites and undergo directional differentiation, demonstrating a certain reparative effect on BLM-induced acute lung injury in mice.
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment.
MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide.
ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups.
ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.
