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.
目的 通过观察卵巢早衰 POF 患者外周血CD4+CD25+调节性T细胞 Treg 及干扰素-γ（IFN-γ）、转化生长因子-β(TGF-β)的变化，探讨POF的免疫学发病机制。 方法 收集2011年12月－2012年9月就诊的POF患者17例，卵巢储备功能减退 DOR 患者11例，以及生殖中心健康育龄女性16例，流式细胞仪定量检测外周血Treg数量，Elisa方法检测血清IFN-γ、TGF-β的水平，并以FSH/LH评价卵巢储备功能，进行相关性分析。 结果 与对照组相比，POF组和DOR组IFN-γ水平增高 P＜0.01 、TGF-β水平降低 P＜0.01 ，POF患者及DOR患者Treg比例降低 P＜0.01 ，IFN-γ的增高与卵巢储备功能的下降呈显著正相关 r=0.70，P＜0.01 。 结论 Treg 和IFN-γ、TGF-β水平与卵巢早衰密切相关，IFN-γ对评估卵巢储备功能、预测卵巢早衰具有参考价值。
Objective To investigate the effects of exogenous bone morphogenetic protein(BMP) and transforming growth factor-β(TGF-β) on biomechanical property for ulna of fracture healing.Methods Thirty-six adult rabbits were made the model of right ulnar fracture and treated locally with TGF-β/PLA, BMP/PLA，TGF-β+BMP/PLA or PLA(as control group). Fracture healing was evaluated by measurement of the mechanical parameters and geometric parameters.Results As compared with control group, the geometric parameters, the bending broken load, the ultimatebending strength, the bending elastic modulus, the ultimate flexural strength, the flexural elastic modulus, the ultimate compressing strength, the compressingelastic modulus, and the ultimate tensile strength for ulna of fracture healingincreased significantly in the treatment groups(P＜0.01). These parameters were higher in TGF-β+BMP/PLA group than in TGF-β/PLA group or in BMP/PLA group andin TGF-β/PLA group than in BMP/PLA group(P＜0.05). There was no significant difference in bone density between the treatment groups and control group. Conclusion Local application of exogenous TGF-β and BMP canincrease the callus formation and enhance biomechanical strength of bone after fracture healing. A combination of TGF-β and BMP has synergetic effect in enhancing fracture healing.
For observation of the change of transforming growth factor-beta 1 (TGF-beta 1) gene expression in the process of skin wound healing, the following experiments were performed. Sixteen Wistar rats were chosen. At each side of the rat’s back, a 1 cm x 1.5 cm middle-thick skin wound was made. After 3, 6, 9 and 12 days, the specimens were taken from the wounds. For each specimen, half of it was used for RNA extraction, and underwent dot blotting; and the other half was frozen immediately and underwent in situ hybridization. The probes were dig-labeled PDGF-BB cDNA probe and TGF-beta 1 probe. The results showed that TGF-beta 1 gene was expressed mainly in fibroblast, epithelial cell and capillary endothelial cell. The peak of TGF-beta 1 mRNA content was in the 6th day postoperatively. After that, the content of TGF-beta 1 decreased to normal. It was suggested that TGF-beta 1 gene expression was in close relation with healing process. TGF-beta 1 may play an important regulatory role in the skin wound healing.
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 investigate the role of IFN-γ in suppressing bleomycin-induced pulmonary fibrosis in rats.Methods Seventy-five SD rats were randomly divided into five groups (15 rats in each group)，ie.a normal group，a bleomycin-induced pulmonary fibrosis model group，a dexamethasone-treated group，a high-dose IFN-γ-treated group (150 000 U/kg) and a low-dose IFN-γ-treated group (50 000 U/kg).Five rats in each group were randomly killed in 7th day，14th day and 28th day after relative treatment respectively，and lung tissue samples were harvested for histopathology study.HE and Masson staining were used to determine the extent of alveolus inflammation and pulmonary fibrosis respectively.Histoimmunochemical method were adapted to determine protein levels of TGF-β1，CTGF，type Ⅰcollagen and type Ⅲ collagen in pulmonary tissues.Results Histopathological study showed that treatment with either dexamethasone or IFN-γ (both high dose and low dose) remarkably meliorated the extent of alveolus inflammation and suppressed pulmonary fibrosis (compared with model group，all Plt;0.05).Histoimmunochemical study suggested that both dexamethasone and IFN-γ could inhibit the expression of TGF-β1，CTGF，type Ⅰand type Ⅲ collagen (compared with model group，all Plt;0.05)，and the suppression of TGF-β1，type Ⅰand type Ⅲ collagen expression was more obvious in high-dose IFN-γ-treated group than those in low-dose group (Plt;0.05).Conclusions INF-γ possesses apparent anti-fibrosis effect that is similar to dexamethasone but with less side effect.Such effect may resulted from reduced production of type Ⅰand type Ⅲ collagen through expression inhibition of cytokines such as TGF-β1 and CTGF.
ObjectiveTo investigate the early diagnostic value of transforming growth factor-β1(TGF-β1) on acute rejection after liver transplantation in rhesus by detecting the expression of TGF-β1 in the liver tissue. MethodsLiver transplantation models in rhesus were constructed by the improved vascular dual cuff, supporting tube of biliary tract, and artery anastomosis method.The successful models were randomly divided into experimental group (no immunosuppressant treatment in perioperative period) and control group (treated by immunosuppressant in perioperative period).Then the blood samples and liver tissues were collected at 6, 12, 24, and 72 hours after surgery.Allograft rejections of liver tissue after liver transplantation were monitored by liver function test, hematoxylin-eosin staining and Banff score.Finally, the expression level of TGF-β1 was detected by Western blot analysis or immunohistochemistry technique. Results①The acute rejection happened in all the rhesus at 12 h, 24 h and 72 h after liver transplantation, especially at 72 h after liver transplantation in the experimental group, the Banff grade levels of acute rejection in the liver tissue was more severe than that in the control group (P < 0.05).②The levels of ALT, AST, and TBIL after liver transplantation was gradually increased, which were similar at 6 h and 12 h after transplantation between the two groups, but which at 24 h and 72 h after transplantation in the experimental group were significantly higher than those in the control group (P < 0.05).③The results of TGF-β1 protein expression using immunohistochemical detection:The percentage of positive area of TGF-β1 of liver tissue at 12 h in the experimental group was significantly higher than that in the control group (P < 0.05).With the extension of time, it was gradually increased and significantly higher than that in the control group at 24 h or 72 h (P < 0.05).④The semi-quantitative results of TGF-β1 protein expression using Western blot detection:The TGF-β1 protein expressions began to increase at 6 h after liver transplantation in the experimental group and the control group, and the magnitude of increase was more obvious in the experimental group.The TGF-β1 protein expressions at different time (6 h, 12 h, 24 h, and 72 h) in the experimental group were significantly higher than those in the control group (P value was 0.003, 0.001, 0.001, and 0.001, respectively). ConclusionsThe elevated level of TGF-β1 of liver tissue after liver transplantation might suggest the enhanced cellular immune function, it might have certain significance for early diagnosis of acute rejection after liver transplantation.
Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) gene transfer on the biological characteristics of osteoblasts. Methods The expression of TGF-β1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-β1 activity in the supernatant (minklung epithelium cell growth -inhibition test). The effects of gene transfer andsupernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3 H-TdR and MTT. Results The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-β1 gene could express TGF-β1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1∶1, 1∶2, 1∶4, could inhibit growth of Mv-1-Lu evidently and the ratios ofinhibition were 16.3%, 22.7%, 28.2% respectively. TGF-β1 gene transfer hadno effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALPactivity of osteoblasts. Conclusion TGF-β1 gene transfer promotes the expression of TGF-β1 and the biological characteristics of trasfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.
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.