Objective To research the gene expression of transforming growth factor β1 (TGF-β1) in zone Ⅱ flexor tendon wound healing of rabbit. Methods Sixty New Zealand white rabbits forepaws(left side) underwent complete transection and the middle digit flexor digitorum profundus tendon in zone Ⅱ were repairedby Kessler methods as the experimental group. The normal right forepaws served as the control group. The tendons and tendon sheaths were harvested at 1, 7, 14, 21, 28and 56 days after repair(n=10). The expression patterns ofTGF-β1 wereanalyzed by in situ hybridization and immunohistochemistry staining methods. Results The in situ hybridization examination revealed thatTGF-β1 mRNA expression upregulated at 1 day, reached the peak levels at 1421 days and remained high levels up to 56 days in the experimental group. The expression ofTGF-β1 mRNA in control group was lowerthan that in the experimental group, showing statistically significant difference (Plt;0.05). The results of immunohistochemical staining was similar to that of in situ hybridization. Conclusion The normal tendon and tendon sheath cells are capable ofTGF-β1 production. The cytokine is activated in tendon wound condition. The upregulation of this cytokine in both tendon and tendon sheath cells are coincidence with both extrinsic and intrinsic mechanisms for tendonrepair.
Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
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 It is reported that transforming growth factor β1 (TGF-β1) has the protective effects on the articular cartilage in osteoarthritis (OA). To investigate the significance of the expressions of matrix metalloproteinase 9 (MMP-9), TGF-β1 mRNA and corresponding proteins in OA. Methods The specimens of articular cartilage and synovium were collected from voluntary donators, including 60 cases of OA (experimental group) and 20 cases of traumatic amputation,cruciate l igament rupture, discoid cartilage injury, and menisci injury (normal control group). The pathological changes were observed by HE staining. MMP-9 and TGF-β1 protein expressions were detected by immunohistochemical technique, and the mRNA expressions of MMP-9 and TGF-β1 were detected through in situ hybridization technique; and their correlation was analysed. Results HE staining showed: shrinkage, necrosis, and irregular arrange of the articular chondrocytes, extracellular matrix fracture, hypertrophy and hyperplasia synovium, infiltration of lymphoid and mononuclear cells and prol iferation of many small blood vessels in the experimental group; regular arrangement of the articular chondrocytes, the homogeneously staining matrix, and synovial tissue without chronic inflammation and significant prol iferation in the normal control group. The mRNA and protein expressions of MMP-9 and TGF-β1 were positive in 2 groups. The positive-stained cells included chondrocytes, synovial l ining cells, and vascular endothel ial cells, fibroblasts, and inflammatory infiltrated cells in subsynovial layer. The expressions of mRNA and corresponding protein of MMP-9 and TGF-β1 in the experimental group were significantly higher than those in the normal control group (P lt; 0.01). There was a positive correlation between MMP-9 mRNA and protein expression (r=0.924, P=0.000), and between TGF-β1 mRNA and protein expression (r=0.941, P=0.000) in the experimental group. There was a negative correlation between the expression of MMP-9 protein and TGF-β1 protein (r= — 0.762, P=0.000), and between the expression of MMP-9 mRNA and TGF-β1 mRNA (r= — 0.681, P=0.000) in the experimental group. Conclusion The higher expression of TGF-β1 can protect articular cartilage by down-regulating the expression of MMP-9 of chondrocytes and synoviocytes in OA, which may delay the biological behavior of OA such as occurrence and progress, etc.
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 To observe the effect of angiotensin Ⅱ (Ang Ⅱ) or/and transforming growth factor β(TGF-β) on human skin fibroblast proliferation, and to explore the possible signaling mechanism involved in their actions. Methods Cultured human skin fibroblasts were treated with different concentrations of Ang Ⅱ (1×10-10 , 1×10-9，1×10-8 and 1×10-7 mol/L) , TGF-β(0.1, 1.0 and 10.0 ng/ml), and 1×10 -10 mol/L Ang Ⅱ＋0.1 ng/ml TGF-β, respectively. The cell proliferation was determined by3Hthymidine (3H-TdR) incorporation. The phosphorylation of extracellular signalregulated kinases (ERK) was detected by Western blot. Results Ang Ⅱ at 1×10-9，1×10-8，1× 10-7 mol/L or TGF-β at 1.0, 10.0 ng/ml increased 3H-TdR incorporation into cultured skin fibroblasts dose-dependently. Ang Ⅱ and TGF-β at lower doses (1×10-10 mol/L and 0.1 ng/ml, respectively) did not affect 3H-TdR incorporation into fibroblasts (Pgt;0.05), whereas co-administration of both Ang Ⅱ and TGF-β at these doses significantly increased 3H-TdR incorporation intofibroblasts(Plt;0.05). Ang Ⅱ at 1×10-7 mol/L or TGF-β at 10.0 ng/ml significantly increased ERK phosphorylation of fibroblasts after stimulation (Plt;0.01). Smaller doses of Ang Ⅱ (1×10-10 mol/L) or TGF-β (0.1 ng/ml) did not influence ERKphosphorylation of fibroblasts, whereas co-administration of Ang II and TGF-β at these doses significantly enhanced ERK phosphorylation (Plt;0.05). Total protein levels of ERK did not differ at different doses. Conclusion These results indicate that Ang Ⅱ and TGF-β synergistically increase skin fibroblast proliferation, which is at least partly via enhancement of ERK activity.
ObjectiveTo investigate the effect of hamstring tendon transfected with adenovirus-mediated transforming growth factor β1 (AdTGF-β1) genes on the histomorphology of tendon-bone interface healing after anterior cruciate ligament (ACL) reconstruction in rabbits. MethodsAdTGF-β1 and AdGFP were diluted to 5×108 PFU/mL with DMEM. Forty-eight New Zealand white rabbits were divided into 3 groups randomly (n=16), weighing 1.6-2.5 kg for ACL reconstruction with hamstring tendon autograft. Hamstring tendon was cultured and transfected with AdTGF-β1 (group A) and AdGFP (group B) for 12 hours before ACL reconstruction, and was cultured with DMEM in group C. After 12 hours of transfection, the expression of green fluorescence was observed in groups A and B under fluorescence microscopy; TGF-β1 protein level was detected by ELISA in group A. At 2, 4, 8, and 12 weeks after operation, the specimens were harvested for HE and Masson staining; the number of fibroblasts was counted, and the Buark grading was used to evaluate tendon-bone interface healing. ResultsGreen fluorescence was observed after 12 hours of transfection in groups A and B. TGF-β1 protein level reached (221.0±12.2) ng/mL at 12 hours in group A. The histological observation showed that few fibroblasts and collagen fibers were found, and Sharpey fibers appeared in group A; regular Sharpey fibers were seen in the interface, and integrity interface in some areas at 12 weeks. But fibroblasts of groups B and C were less than those of group A, with loose tendon-bone interface; no integrity interface was observed at 12 weeks. The number of fibroblasts and Buark grading of group A were significantly higher than those of groups B and C (P<0.05), but no significant difference was found between groups B and C (P>0.05). ConclusionHamstring tendon transfected with AdTGF-β1 gene can promote the healing of tendon-bone interface after ACL reconstruction.
Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
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 study the relation between expressions of transforming growth factor β1 （TGF-β1）, transforming growth factor receptor type Ⅰ (TβRⅠ) and cell proliferation, cell cycle in gallbladder carcinomas, to disclose the mechanism of TGF-β1 and TβRⅠin the gallbladder carcinogenesis，and to evaluate their values in the prognosis of gallbladder carcinomas. Methods Thirty five gallbladder carcinomas 〔age （57.94± 4.61） years, 14 male cases and 21 female cases〕 comprised 32 adenocarcinomas, 2 adenosquamous carcinoma and 1 squamous cell carcinomas. Formalin fixed, paraffin embedded sections from gallbladder carcinomas were immunostained with TGF-β1, TβRⅠ, PCNA, cyclin E antibodies by immunochemical assays. Gallbladder adenoma and chronic cholecystitis were collected as non-malignant controls. Patients of gallbladder carcinomas were followed up. Results Positive immunostaining rate of TGF-β1 was 57.14% in gallbladder carcinomas, which was significantly higher than that in gallbladder adenomas and chronic cholecystitis (P＜0.01, respectively). Expression of TGF-β1 was associated with Nevin stage, lymph nodes and distant metastasis (P＜0.05, P＜0.01, respectively). Expression of TGF-β1 was positively correlated with expression of PCNA LI and cyclin E (r=0.523 2, P=0.001 3; r=0.406 5, P=0.015 4), and 34.29% of gallbladder carcinomas were immunostained positively for TβRⅠ. Expression of TβRⅠwas significantly lower in gallbladder carcinomas than that in gallbladder adenomas and cholecystitis (P＜0.05, respectively). It was significantly lower in gallbladder carcinomas patients with lymph nodes and distant metastases than in those without (P＜0.05). Expression of TβRⅠwas negatively correlated with PCNA LI (r=-0.402 4, P=0.016 6). Patients with negative expression of TGF-β1 and/or positive expression of TβRⅠ had significant longer survival rates than those with positive expression of TGF-β1 and/or negative expression of TβRⅠ(P＜0.01, P＜0.05, respectively). Expressions of TGF-β1 and TβRⅠ correlated with prognosis of gallbladder carcinomas closely. Conclusion TGF-β1 and TβRⅠ have close correlation with cell proliferation, cell cycle of gallbladder carcinomas and are important biological markers of carcinogenesis and progress of gallbladder carcinomas. The escape of growth inhibition of TGF-β1 due to low expression of TβRⅠand carcinogenesis of TGF-β1 may play an important role in gallbladder carcinogenesis. TGF-β1 and TβRⅠare valuable indices for judging the prognosis of gallbladder carcinoma.