Objective To investigate the role of transforming growth factorβ3 (TGF-β3) on the amylase secretion of rat submandibular gland cells(RSGCs).Methods The RSGCs were cultured and identified. The expressions of CK 8.13, S100 and Vimentin in the RSGCs were examined by immunohistochemical staining. The experimental group was divided into 5 groups according to differentconcentrations of TGF-β3 (0.5, 1.0, 5.0, 10.0 and 25.0 ng/ml) and no TGF-β3 culture was used as control group. The effects ofTGF-β3 on the cell proliferation and amylase secretion were examined at the24th, the 48th, the 72nd and the 96th hour. MTT colorimetric method was used to estimate vital force of culture cells. Amylase protein was assayed by autobiochemistry equipment and Western blotting.Results The RSGCs were stained positively for CK 8.13 and S-100, but negatively for Vimentin. There were no significant differences in absorbency between the experimental groups and the control group(Pgt;0.05). Compared with the control group,TGF-β3 at concentrations of 0.5-10.0 ng/ml significantly stimulated the amylase secretion of RSGCs after 72 and 96 hours(Plt;0.01). But high concentration of TGF-β3 (25.0ng/ml) showed no stimulation. Western blotting demonstrated that the cultured RSGCs and submandibular gland had the same band of amylase electrophoresis.Conclusion TGF-β3 can stimulate RSGCs to differentiate and to secrete amylase, but TGF-β3 has no effect on proliferation ofRSGCs.
Objective Platelet-rich plasma (PRP) secretes many growth factors, including transforming growth factor β1 (TGF-β1), platelet derived growth factor, vascular endothl ial growth factor, insul in-l ike growth factor 1, and so on, which can promote cell prol iferation, chemotaxis, and collagen synthesis in wound heal ing. To investigate the effects of PRPon the tendon heal ing, and to explore the mechanism of action so as to provide the experimental basis for the tissue engineered tendons. Methods Forty healthy New Zealand white rabbits, weighing 2.5-3.0 kg and male or female, were randomly divided into the experimental group (n=20) and the control group (n=20). PRP was prepared from arterial blood of rabbit’s ears through twice centrifugation method of Landesberg. The platelet concentrations of whole blood and PRP were determined. The right achilles tendons of the rabbits were transected to make rupture models. In experimental group, the tendon was sutured after PRP (0.5 mL) was immediately appl ied at repair site. In control group, the tendon was sutured directly after transection. At 1, 2, 4, and 6 weeks after operation, the tendons of 5 rabbits in each group were harvested for morphological, histological, and immunohistochemical observations; the fibroblast counting, the content of collagen fibers, and the expression of TGF-β1 were detected. Results The concentration of platelet of PRP was 4.03 times of whole blood. All the animals survived till the end of the experiment, and the incision healed well. No death, infection, and other compl ications occurred. With time, the tendons almost healed in 2 groups, and the fibrous tissue at anastomosis site was more remarkable in control group than in experimental group. The histological observation showed significant differences in fibroblast counting at 1, 2, and 4 weeks after operation between 2 groups (P lt; 0.05), while no significant difference at 6 weeks (P gt; 0.05). The contents of collagen fibers in the parenchyma at repair site in experimental group were significantly higher than those in control group at each time point (P lt; 0.05). Immunohistochemistry staining showed the expression of TGF-β1 in experimental group was upregulated at 1 week and 2 weeks and reached the peak at the 2nd week, and subsequently downregulated at 4 and 6 weeks in comparison with the control group, showing signficant differences between 2 groups at each time point (P lt; 0.05). Conclusion PRP can facil itate rabbit’ s tendons heal ing and significantly improve the heal ing qual ity, which may be associated with its advancing the peak time of the TGF-β1 expression in tendon.
Objective To determine whether the transforminggrowth factor β1 (TGF-β1) is a key regulatory molecule required for an increase or a balance of extracellular matrix (ECM) and DNA synthesis in the goat passaged nucleus pulposus (NP) cells. Methods The NP cells isolated from the goat intervertebral discs were cultured in vitro for a serial of passages and transfected with the replicationincompetent adenoviral vectors carrying the human TGF-β1 (hTGF-β1) or lacZ genes. Then, they were cultured in monolayer or alginate bead 3dimensional (3-D) systems for 10 days.The changes in the production and the molecular components of ECM that occurredin the NP cells transfected with Ad/hTGF-β1 or the controls were evaluated by Westernblot and absorbance of glycosaminoglycan (GAG)-Alcian Blue complexes. Differences of DNA synthesis in the variant cells and culture systems were assessed by fluorometric analysis of the DNA content. ResultsA quantitation in the variant culture systems indicated that in monolayers the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher cell viability and more DNA synthesis(P＜0.05); however, in the alginate 3-D culture system, the NP cells transfected with Ad/hTGF-β1 did not have any significant difference from the controls(P＞0.05). The Western blotting analysis ofthe protein sample isolated from the variant cells for TGF-β1, type Ⅱ collagen, and Aggrecan expression indicated that in the monolayers and alginate 3-D culture systems the NP cells at Passage 3 transfected with Ad/hTGF-β1 revealed much higher protein levels than the controls(P＜0.05)； whereas the type Ⅰcollagen content was much lower than the controls (P＜0.05), but a significatly increased ratio of type Ⅱ/type Ⅰ collagen was found in both of the cell culture systems(P＜0.05). The GAG quantification also showed a positive result in both the cell culture systems and the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher GAG content than the controls(P＜0.05). Conclusion To a greaterextent, hTGF-β1 can play a key role in maintaining the phenotype of the NP cells and can still have an effect of the phenotypic modulation after a serial of the cell passages. The NP cells that are genetically manipulated to express hTGF-β1 have a promising effect on the restoration of the intervertebral disc defects. The NP cells transfected with Ad/hTGF-β1 cultured in the 3-D alginate bead systems can show a nearly native phenotype.
OBJECTIVE To study the relationship between the changes of mRNA expression in wound tissues of diabetic ulcers and tissue repair. METHODS The mRNA expression of TGF-beta 1 and IL-6 in eight bioptic samples of diabetic ulcers were detected by RT-PCR and pathologic methods, and the surrounding normal skins from the same patients were measured as control group. RESULTS The mRNA expression levels of TGF-beta 1 were markedly decreased in the diabetic ulcers compared with control group, while the mRNA expression levels of IL-6 were increased at the same reaction conditions. CONCLUSION The different changes of mRNA expression level of TGF-beta 1 and IL-6 in wound tissue result in low production and decreased activity of TGF-beta 1 and IL-6, which lower the reparative ability of wound tissue.
ObjectiveTo study the effect of transforming growth factor β3 (TGF-β3), bone morphogenetic protein 2 (BMP-2), and dexamethasone (DEX) on the chondrogenic differentiation of rabbit synovial mesenchymal stem cells (SMSCs). MethodsSMSCs were isolated from the knee joints of 5 rabbits (weighing, 1.8-2.5 kg), and were identified by morphogenetic observation, flow cytometry detection for cell surface antigen, and adipogenic and osteogenic differentiations. The SMSCs were cultured in the PELLET system for chondrogenic differentiation. The cell pellets were divided into 8 groups: TGF-β3 was added in group A, BMP-2 in group B, DEX in group C, TGF-β3+BMP-2 in group C, TGF-β3+DEX in group E, BMP-2+DEX in group F, and TGF-β3+BMP-2+DEX in group G; group H served as control group. The diameter, weight, collagen type II (immuohistochemistry staining), proteoglycan (toluidine blue staining), and expression of cartilage related genes [real time quantitative PCR (RT-qPCR) technique] were compared to evaluate the effect of cytokines on the chondrogenic differentiation of SMSCs. Meanwhile, the DNA content of cell pellets was tested to assess the relationship between the increase weight of cell pellets and the cell proliferation. ResultsSMSCs were isolated from the knee joints of rabbits successfully and the findings indicated that the rabbit synovium-derived cells had characteristics of mesenchymal stem cells. The diameter, weight, collagen type II, proteoglycan, and expression of cartilage related genes of pellets in groups A-F were significantly lower than those of group G (P<0.05). RT-qPCR detection results showed that the relative expressions of cartilage related genes (SOX-9, Aggrecan, collagen type II, collagen type X, and BMP receptor II) in group G were significantly higher than those in the other groups (P<0.01). Meanwhile, with the increase of the volume of pellet, the DNA content reduced about 70% at 7 days, about 80% at 14 days, and about 88% at 21 days. ConclusionThe combination of TGF-β3, BMP-2, and DEX can make the capacity of chondrogenesis of SMSCs maximized. The increase of the pellet volume is caused by the extracellular matrix rather than by cell proliferation.
ObjectiveTo investigate the effect of transforming growth factor β3 (TGF-β3) at different concentrations on the differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated with TGF-β3 at the concentrations of 5.0, 2.5, 1.0, and 0 ng/mL. At 1, 3, and 5 days, the mRNA expressions of tendogenic differentiation related genes[collagen type Ⅰ,tenascin C (TNC), tenomodulin (TNMD), scleraxis (Scx)], osteogenic differentiation related genes[Runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP)], chondrogenic differentiation genes (Sox9 and collagen type Ⅱ),and adipogenic differentiation genes[AP2 and peroxisome proliferator-activated receptor γ (PPARγ)] were measured by real-time quantitative PCR (qRT-PCR). ResultsTSCs could differentiated in different directions after treated with TGF-β3 at different concentrations at different time points. TGF-β3 was able to induce TSCs differentiated into tenocytes, which was related to the concentration and time of duration, and the two factors have interaction. Stimulation of TGF-β3 at low concentration and for short time could inhibit non-tendogenic differentiation of TSCs, but at high concentration and for long time, TGF-β3 enhanced TSCs differented into osteocytes or chondrocytes. ConclusionEffects of TGF-β3 on TSCs differentiation are complicated and depend on the concentration and time of duration, which may be a key factor between tendogenic and non-tendogenic differentiations of TSCs.
Objective To study biological effect of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP-2) on theSchwann cell(SC) in vitro. Methods Cultured SC from newbornSDrats were implanted at 5×103/well in 96-well-plate (36 wells in each group, altogether 3 groups):TGF-β group (group A) treated with 50 ng/ml TGF-β; rhBMP-2 group (group B) treated with 50 ng/ml rhBMP-2 and control group (group C). SC proliferation activity was assessed by MTT and flow cytometry (FCM) methods, and nerve growth factor (NGF) synthesis in SC culture media was detected by ELISA method. Results MTT observation indicated that there was significant difference in the growth curve among 3 groups until the 8th and 9th day. Group A had more obvious rising tendency than group B and group C. FCM observation indicated that the proliferation index of group A and group B was higher than that of group C（Plt;0.05）. ELISA observation indicated that there was significant difference in the NGF concentration of the culture medium among the 3 groups(P＜0.05). Group A had the highest NGF concentration. Conclusion Exogenous TGF-β and rhBMP-2 can promote SC’s ability to proliferate NGF, but TGF-β is more effective than rhBMP-2.
OBJECTIVE: To study the effect of overexpression of truncated type II TGF-beta receptor on transforming growth factor-beta 1(TGF-beta 1) autoproduction in normal dermal fibroblasts. METHODS: In vitro cultured dermal fibroblasts were treated with recombinant human TGF-beta 1(rhTGF-beta 1) (5 ng/ml) or recombinant adenovirus containing truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-beta 1 were observed with Northern blotting. RESULTS: rhTGF-beta 1 up-regulated the gene expression of TGF-beta 1 and type I procollagen. Overexpression of truncated receptor II down-regulated the gene expression of TGF-beta 1. CONCLUSION: Overexpression of the truncated TGF-beta receptor II decreases TGF-beta 1 autoproduction via blocking TGF-beta receptor signal. The results may provided a new strategy for scar gene therapy.
Objective To investigate the effects of the insulin-like growth factor 1 (IGF-1), the transforming growth factor β1(TGFβ1), and the basic fibroblast growth factor (bFGF) on proliferation and cell phenotype of the human fetal meniscal cells, and to find out the best combination and concentration of the growth factors for the meniscus tissue engineering. Methods The fetus came from the healthy woman accidental abortion and the procedure had got her approval.The human fetal meniscal fibrochondrocytes were cultured in vitro. The cell phenotype was identifiedby the collagen type Ⅱ immunohistochemistry and Aggrecan immunofluorescence. Inthe growth factor groups, the 3rd passage meniscal cells synchronized by the serum starvation method and were mixed with IGF-1 (1, 10, 50, 100 μg/L), TGF-β1 (0.1, 1.0, 5.0, 10.0, 50.0 μg/L), and bFGF (5, 10, 50, 100, 200 μg/L), respectively, and in the combination groups, the combinations of bFGF and TGF-β1, bFGF and IGF-1, TGF-β1 and IGF-1 were established at their optimal effect concentrations. The control group was also established for comparison. The dose-response relationship was studied at 48 h and 72 h bythe MTT colorimetric method. Results The 3rd passage meniscalcells could express collagen type Ⅱ and Aggrecan before and after the addition of the three growth factors. The proliferating effects of the growth factors (IGF-1 50 μg/L，TGF-β1 5 μg/L，bFGF 50 μg/L) on the 3rd passage cells at 48 h and 72 h were significantly better in the growth factor groups than in the control group (Plt;0.05)，and the combination groups of bFGF 50 μg/L and IGF-1 50 μg/L, IGF-1 50 μg/L and TGF-β1 5 μg/L showed a significantly higher proliferatingeffect than that in the single growth factor group (Plt;0.05). bFGF 50 μg/L and TGF-β1 5 μg/L had no synergetic effect (Pgt;0.05). Conclusion IGF-1, TGF-β1 and bFGF can promote the proliferation of the human fetal meniscal cells, respectively, and the combinations of bFGF and IGF-1, IGF-1 and TGF-β1 at their optimal concentrations can have better proliferating effects than the single growth factor. They can be used for the in vitro amplification of the meniscal seed cells.
Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.