Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.
【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 investigate the results of human amniotic membrane(HAM) which are loaded with marrow mesenchymal stem cells(MSCs) and epidermis cells in treating fullthickness skin defect combined with radiation injury. Methods Eight minipigs were used in this study. Three round fullthickness wounds（Ф3.67cm）, which combined with radiation injury, were created on the dorsum of each side close to the vertebral column in each animal. Among 48 wounds, 24 left side wounds were treated with HAM loaded with MSCs and epidermis cells as experimental group (group A), 16 right side wounds with simple HAM (HAM group, group B) and 8 right side wounds with oil gauze as control (group C). The granulation tissue, reepithelization and wound area were observed after 1,2 and 3 weeks. Immunohistochemistry was performed using vWF as a marker for blood vessels.Image analysis was employed to test new area of wound at different interval time and healing rate of wound.Results The healing time of group A was 6 to 7 days faster than that of group C and 5 to 6 days faster than that of group B. After 15-17 days of graft, there were significant differences in new area of wound and healing rate between group A and groups B,C(Plt;001). New epidermis fully covered whole wound surface in group A, and their granulation tissue, which contained a lot of vWF, fibroblasts, capillaries and collagen, grew well. Many inflammatory cells still were seen in groups B and C, and their contents of vWF, fibroblasts, capillaries and collagen in granulation tissue were smaller than that in group A.Conclusion The graft of HAM loaded with MSCs and epidermis cells played an effective role in promoting healing of wound combined radiation injury with high quality.
Objective To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20％ fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group（20％O2, DMEM-LG, Group A）, the hypoxic group（1％O2, DMEM-LG,Group B）, the normoxic osteoblast induction group（20％O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group（1％O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed bythe cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarinred staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (Plt;0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A(Plt;0.01). After the induction, hMSCs in the Group B had a decreasednumber of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increasedactivity of ALP, which was significantly lower than that in the Group C(Plt;0.01). The RT-PCR examination revealed that ALP,osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group Csignificantly increased （P＜0.01). By comparisonamong the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.ConclusionHypoxia can promote the proliferation rate of hMSCs, enhance the colonyforming ability and inhibit the differentiation of the osteoblasts.
Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor β3 (TGF-β3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P lt; 0.05); group D was significantly larger than group C in size (P lt; 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P lt; 0.05). Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-β3-induced chondrogenic differentiation in vitro.
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.
Objective To explore effects of zinc on the contents of cycl in D2, cycl in-dependent kinase 4 (CDK4), and their DNA and total cellular protein in human umbil ical cord blood-drived mesenchymal stem cells (hUCBMSCs). Methods hUCBMSCs were isolated and cultured by density gradient centrifugation adherence method in vitro. At the serial subcultivation, the hUCBMSCs were randomly divided into 7 groups. In control group, hUCBMSCs were cultured with DMEM medium (containing 15%FBS). In treatment groups, hUCBMSCs were cultured with DMEM medium (containing 15%FBS plusZnSO4•7H2O). The final concentrations of zinc were 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 mg/L, respectively. The cellular surface antigens of CD29, CD34, CD44, and CD45 at the 3rd generation of hUCBMSCs were detected by flow cytometry. MTT assay was used to detect cell activity of the 3rd generation of hUCBMSCs. The optimum concentration of zinc was selected by the results of MTT as experimental group. The cell growth curves of experimental group and control group were drown by counting cell. The cell surface antigen, reproductive cycle, and DNA content were detected by flow cytometry motheds. The contents of cycl in D2 and CDK4 were detected by Western blot method. Results The positive expression rates of CD29 and CD44 were more than 70% in hUCBMSCs. The cell activity of 2.5 mg/L treatment group was superior to other treatment groups, as experimental group. At 7, 14, and 28 days, the contents of DNA, total cellular protein, cycl in D2, and CDK4 of hUCBMSCs were significantly higher in experimental group than those in control group (P lt; 0.01). The percentage of hUCBMSCs at S stage and prol iferation index in experimental group were also significantly higher than those in control group (P lt; 0.01). Conclusion Zinc (0.5-4.5 mg/L) has the promoting effect on the hUCBMSCs activity, and 2.5 mg/L is the optimal concentration. Zinc (2.5 mg/L) can accelerate the prol iferation and DNA reproduction of hUCBMSCs and increase the contents of cycl in D2 , CDK4, and cellular total protein.
Objective To investigate the feasibility of differentiation of the marrow mesenchymal stem cells (MSCs) into the cells of the skin appendages andthe mechanism of their involvement in the wound healing. Methods The bone marrow was collected from Wistar rats by the flushing of the femurs, MSCs were isolated and purified by the density gradient centrifugation. Then, the MSCs were amplified and labelled with 5-bromo-2′-deoxyuridine (BrdU). The full-thickness skin wounds with an area of 1 cm×1 cm were made on the midback of the homogeneous male Wistar rats. At the same time, 1×106/ml BrdU-labelled MSCs were infused from thepenile vein. The specimens were harvested from the wound tissues on the 3rd dayand the 7th day after operation and were immunohistochemically stained by either BrdU or BrdU and pan-keratin. Results The BrdU positive cells appeared in thehypodermia, the sebaceous glands, and the hair follicles of the wounds, as wellas the medullary canal of the femurs. The double-staining showed the BrdU positive cells in the sebaceous glands and the hair follicles of the wounds expressedpan-keratin simultaneously. Conclusion During the course of the wound healing, MSCs are involved in the wound repair and can differentiate into the cells ofthe skin appendages under the microenvironment of the wound.
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.