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 explore the autocrine regulation for the transforming growth factor-beta 1(TGF-β1) by the fibroblasts in vitro. METHODS: Fibroblasts were cultured in vitro which isolated from the normal prepuce by circumcision. TGF-β1 concentration were determined with ELISA for the different passages and the different time at the same passage. RESULTS: The TGF-β1 concentration was different for the distinct passage of fibroblasts and achieved the peak (450 ng/L) at the sixth passage. Among the same fibroblasts, the TGF-β1 concentration was different in the various days and passage of the summit (680 ng/L) on the fifth day, which was 2.5 times as high as that of the first day. CONCLUSION: The fibroblasts possess the ability of autocrine of the TGF-β1 and have some regularity. It will provide the theory basis for the research about TGF-β1 regulation mechanism and the medical application about salvia miltiorrhiza.
Objective To investigate the effects of adenovirus-mediated melanoma differentiation-associated gene-7 (mda-7)/IL-24 and/or adriamycin (ADM) on transplanted human hepatoma in nude mice and to explore a new way for hepatoma gene therapy combined with chemotherapy. Methods The recombinant adenovirus vector carrying Ad.mda-7 was constructed; Ad.mda-7 and/or ADM were injected into the tumor-bearing mice. Their effects on the growth of the tumor and the survival time of the mice were observed. The expressions of VEGF and TGF-β1 were detected by an immunohistochemistry method. Results Ad.mda-7 was constructed and expressed in vivo successfully. Compared with other three groups 〔control group (43.4±1.67) d, ADM group (64.2±4.14) d, Ad.mda-7 group (61.4±1.67) d〕, the mice treated with Ad.mda-7 combined with ADM had longer average survival time 〔(83.8±4.82) d, P＜0.01〕; the average size of tumor treated with Ad.mda-7 combined with ADM diminished significantly compared with that treated with ADM or Ad.mda-7 separately (P＜0.01). VEGF and TGF-β1 expressions of Ad.mda-7 group were (56.2±7.7)%, (35.2±4.5)%, respectively, and were lower than those in ADM group (VEGF: P＜0.05; TGF-β1: P＜0.01). VEGF expression of Ad.mda-7+ADM group was (37.3±5.0)%, and was significantly lower than that in other three groups (P＜0.01). TGF-β1 expression of Ad.mda-7+ADM group was (31.2±3.1)% and significantly lower than that in control group and ADM group (P＜0.01), however, there was no significant difference compared with Ad.mda-7 group (Pgt;0.05). Conclusion Ad.mda-7 combined with ADM has b antitumor potency and synergistic effects and suppresses the growth of human HCC xenograft in nude mice, possibly by inducing the apoptosis of hepatoma cell lines and suppressing tumor angiogenesis.
Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
Objective
To observe whether transforming growth factor-beta;2（TGF-beta;2）could promote the differentiation of retinal stem cells in rats cultured in vitro.
Methods
The retinal stem cells were separated from the embryonic ratsprime; eyes under the dissecting microscope, cultured, and subcultured. The cells were identified by nestin and Chx-10 immunofluorescence. The sixth generation of cells were induced and differentiated, immunofluorescent stained with anti-glial fibrillary acidic protein，anti-opsin, anti-b-tubulin, and anti-protein kinase C, and identified the final cells.
Results
The cultured cells after induced by TGF-beta;2 differentiated to the mature cells. The results of immunofluorescence showed that the differentiated cells induced by TGF-beta;2 were more than which induced by the embryonic bovine blood serum.
Conclusion
TGF-beta;2 may induce the retinal stem cell differentiating into retinal cells. The inductive and differentiating effect of TGF-beta;2 is ber than which of the blood serum.
(Chin J Ocul Fundus Dis, 2007, 23: 104-107)
【Abstract】Objective To investigate the apoptosis induced by TGF-β1 in human hepatic carcinoma cell lines and its relationship with p53 gene and Smad. Methods Three human hepatic carcinoma cell lines which involving in various status of the p53 gene were used in this study. TGF-β1-induced apoptosis in hepatic carcinoma cell lines was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. To study the mechanism of TGF-β1-induced apoptosis, these cell lines were transfected with a TGF-β1-inducible luciferase reporter plasmid containing Smad 4 binding elements (SBE) and luciferase gene using Lipofectamine 2000, then treated with TGF-β1, relative luciferase activity was assayed. Results Of three cell lines studied with TUNEL assay, TGF-β1 induced apoptosis was observed in HepG2 cells (wild type p53). Huh-7 (mutant p53) and Hep3B (deleted p53) cell lines showed less apoptosis. Luciferase activity assay indicated that the response to TGF-β1 induction in HepG2 cells was increased dramatically but was not significant in Huh-7 and Hep3B cell lines. Conclusion HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh7 cell lines. Smad 4 is a central mediator of the TGF-β1 signal transduction pathway.
Objective To validate the mechanism of effect of hepatic artery ischemia on biliary fibrosis after liver transplantation and the prevention method. Methods Eighteen male dogs were established into the concise auto orthotopic liver transplantation models and assigned into three groups randomly: hepatic artery ischemia (HAI) group, TBB group (transferred the blood by a bridge duct ) and control group, each group contained 6 dogs. After opening portal vein, the samples were cut from liver in each group at the time of 6 h, 3 d and 14 d. The pathological modifications of intrahepatic bile ducts were observed and expression of transforming growth factor-β1 (TGF-β1) were detected in the three times. Expressions of Smad3 and phosphate-Smad3 as well as mRNA of α-smooth muscle actin (α-SMA) in intrahepatic bile ducts were detected 14 d after opening portal vein.Results Compared with control group, the collagen deposition and lumens stenosis in biliary vessel wall were more obviously in HAI group. In TBB group, the pathological modifications were slighter compared with HAI group. The positive cell index of TGF-β1 reached peak on day 3 after opening portal vein, then decreased in TBB group, and which in HAI group kept increase and was significantly higher than that in TBB group （Plt;0.05）. The expression level of phosphate-Smad3 and transcriptional level of α-SMA mRNA were 1.04±0.13 and 1.12±0.55 in TBB group on day 14 after opening portal vein, which were significantly higher than those in control group (0.59±0.09 and 0.46±0.18) and lower than those in HAI group (1.82±0.18 and 1.86±0.73), the diversities among three groups were significant (Plt;0.05). There was not significant difference of expression of Smads among three groups (Pgt;0.05). Conclusions Hepatic artery ischemia could increase the deposition of collagen fibers and the transdifferentiation of myofibroblast in bile duct and result in the biliary fibrosis by activating the TGF-β1/Smads signaling pathway. The bridging bypass device could lessen the biliary fibrosis caused by hepatic artery ischemia by inhibiting the activation of TGF-β1/Smads signal transduction passageway.
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.
ObjectiveTo study the serum transforming growth factorβ1 (TGF-β1) and interleukin-23 (IL-23) expression in the patients with chronic gastric ulcer or gastric cancer, and to investigate the clinical value of TGF-β1 and IL-23 on the prevention and treatment of gastric cancer.
MethodsThe serum levels of TGF-β1 and IL-23 in cancer group (83 cases), gastric ulcer group (184 cases), and control group (58 cases) were detected by using ELISA assay method. The difference of serum TGF-β1 and IL-23 levels in patients with gastric cancer with different pathological parameters were compared.
ResultsThe serum levels of TGF-β1〔(15.96±3.92) ng/mL〕and IL-23〔(645.25±234.18) ng/mL〕in gastric cancer group were higher than those of the gastric ulcer group〔(10.10±3.58) ng/mL, (496.10±108.32) ng/mL〕and normal control group〔(9.87±2.86) ng/mL, (372.75±89.27) ng/mL〕, the difference were statistically significant (P < 0.05). The levels of serum TGF-β1 in gastric cancer patients of stageⅠ-Ⅱ, ⅢandⅣwere successively increased, and the differences were statistically significant (P < 0.05). The levels of serum TGF-β1 in poorly differentiated gastric cancer or with lymph node metastasis patients were higher than those in high-middle differentiation or without lymph node metastasis patients, the difference were statistically significant (P < 0.05). There were no significant difference in the levels of serum TGF-β1 between different tumor diameter and different location (P > 0.05). The level of serum IL-23 in patients with stageⅠ-Ⅱwas higher than that in stageⅢandⅣ, the difference was statistically significant (P < 0.05). Ther were no significant difference in serum IL-23 levels between the different degree of differentiation, lymph node metastasis or not, different tumor diameter and different location of the tumor (P > 0.05).
ConclusionTGF-β1 and IL-23 have important reference value in judging the stage and malignancy degree of gastric cancer.
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.
