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 observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse musclederived stem cells（MDSCs） producing the connective tissue growth factor（CTGF）at different time points in vitro. Methods MDSCs from the primarycultureof the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culturein vitro with TGF-β1 (10 ng/ml) for 24 hours. Then, MDSCs were randomlydivided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ng/ml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivityrate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate gt;99%. The Western blot test showed that the ratios of CTGF to the average absorbance of βactin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups AD，the absorbance increased gradually, with a significant difference between the abovementioned groups (Plt;0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P＞0.05). The RT-PCR test showed that the △Ct values in GroupsA-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263,2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (Plt;0.05). But in Groups E and F, the promotive tendency became less significant（Pgt;0.05）. Conclusion TGF-β1 can promote the production of CTGF inthe mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.
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 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.
Objective To explore an experimental method of transfecting the marrow stromal stem cells (MSCs) with the reconstructed PGL3-t ransforming growth factor-β1 (TGF-β1) gene and to evaluate the feasibility of selfinduction of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for a further “gene enhanced tissue engineering” research. Methods The rabbit MSCs was transfected with the reconstructed PGL3-TGF-β1gene by the Liposo mesMethod, the growth of the cells were observed, and the growth curve was drawn. The living activity of the transfected cells in the experimental group was evalua ted by MTT, and the result was significantly different when compared with that in the control group. By the immunohistochemistry method (SABC), the antigens of TGF-β1 and collagen Ⅱ were examined at 2 and 7 days of the cell culture afte r transfe ction with PGL3-TGF-β1gene. The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysis was perfor med with the software of the SPSS 11.0, compared with the control group and the blank group. Results Transfection of the cultured rabbit MSCs in vitro with the reconstructed PGL3-TGF-β1gene by the Liposomes Method achie ved a success, with a detection of the Luceraferase activity. The result was significantly different from that in the control group (Plt;0.01). Tested by MTT, the living acti vity of the transfected cells was proved to be significantly decreased (Plt;0.01 vs. the control group). By the immunohistochemistry method (SABC) to study TGF-β1 positive particles were detected in the experimental group，but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the ttest (Plt;0.01). By the immunohistochemistry me thod (SABC) to study collagen Ⅱ, there were more positive particles in the transfected cells in t he experimental group than in the control and the blank groups, and there was a significant difference between the experimental group and the two other groups based on the t-test (Plt;0.01). Conclusion Transfection of the rabbit MSCs with the reconstructed PGL3-TGF-β1 gene by the Liposomes Method is successful. There may be some damage to the cells when transfection is performed. The transfecte d BMS cells with PGL3-TGF-β1 gene can express and excrete TGF-β1when cultured in vitro. The transfected MSCs that secret TGF-β1 can be self-induced into the chondrocytes after being infected for 7 days when cultured in vitro.
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 investigate the effects of basic fibroblast growth factor (bFGF) on the promoter activities of human alpha 1(I) procollagen gene and the interaction between bFGF and transforming growth factor-beta 1 (TGF-beta 1). METHODS: Fibroblasts of the hypertrophic scar and normal skin from a 3-year-old patient were primarily cultured and subcultured in vitro. Both of the fibroblasts were transient transfected with phCOL 2.5, containing -2.5 kb of 5’f lank sequence of human alpha 1(I) procollagen gene and CAT reporter gene by FuGENE transfection reagent; and treated thereafter by 16 ng/ml bFGF, 2 ng/ml TGF-beta 1 and 16 ng/ml bFGF + 2 ng/ml TGF beta 1 for 24 hours. The relative CAT expression values were determined by CAT-ELISA. RESULTS: TGF-beta 1 bly induced the CAT expression level, however, bFGF not only inhibited the basal CAT expression but also reduced the CAT expression up-regulated by TGF-beta 1 in normal skin and hypertrophic scar fibroblasts (P lt; 0.05). CONCLUSION: bFGF can reduce the promoter activities of human alpha 1(I) procollagen gene and antagonize the role of TGF-beta 1 in up-regulating the promoter activities of human alpha 1(I) procollagen gene in normal skin and hyertrophic scar fibroblasts.
Objective To access the protective effects of glucosamine hydrochloride capsules (OTL) on articular cartilage in osteoarthritis of rabbit. Methods Thirty-six New Zealand white rabbits were divided randomly into three groups (n=12): sham group (group A), anterior cruciate l igament transection (ACLT)/normal sal ine group (group B), and ACLT/ OTL group (group C). Rabbits in groups B, C received ACLT on the right knee. Rabbits in group A were not given ACLT ascontrol. Group C received a daily administration of OTL at a dose of 150 mg/kg of body weight for 12 weeks; in contrast, group B received normal sal ine at the same dose. All rabbits were sacrificed after 12 weeks. The right femoral condyle were removed and observed at pathologic changes with HE staining and graded by Mankin’s scale, the expression level of transforming growth factor β1 (TGF-β1) and interleukin 1β (IL-1β) were detected by immunohistochemical staining. Results All rabbits survived at the end of experiment and incision healed well. The gross observation showed that joint synovia increased and articular surface was smooth and integrity in group A; that ulcer was observed on the articular surface of group B; and that articular surface was smooth and integrity in group C. There were sigificant differences in articular cartilage scores between 3 groups (P lt; 0.05). The histological observation showed that the articular cartilage had normal structure and the cells arranged regularly in group A; that the articular cartilage became thin and the cells arranged irregularly in group B; and that the cells arranged with a clear layer and had regular shape in group C. The Mankin scores were 1.04 ± 0.13, 7.97 ± 0.12, and 2.81 ± 0.36 in groups A, B, and C, respectively; showing significant difference between 3 groups (P lt; 0.05). The result of immunohistochemistry showed that the expressions of TGF-β1 were 50.62 ± 1.51, 24.81 ± 1.28, and 41.57 ± 1.69 and the expressions of IL-1β were 13.12 ± 1.21, 62.53 ±2.37, and 30.67 ± 1.28; showing significant differences between 3 groups (P lt; 0.05). Conclusion A daily administration ofOTL at a dose of 150 mg/kg for 12 weeks can partially decrease the expression levels of IL-1β and increase the expression levels of TGF-β1, which delays the development of osteoarthritis.
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.