Specific inhibitory protein Dkk-1 blocking Wnt/β-catenin signaling pathway improve protectives effect on the extracellular matrix

The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells.The model of nucleus pul...     

Modulation of Matrix Metalloproteinase and TIMP-1 Expression by TGF-β1 in Cultured Human RPE Cells

Matrix metalloproteinases(MMPs)are a familyof zinc binding,calcium-dependent endopeptidases thatfunction by degrading extracellular matrix(ECM)components.The functional effects of these enzymesare in part controlled byinteractions withtissueinhibi-tors of metalloproteinases(TI MPs)acting as naturalMMPinhibitors.Aprecise balance between MMPandTI MPactivities may be i mportant for the integrity ofECMcomponents[1].It shows that the expression andactivity of MMPs could be regulated by va…     

Expression of transforming growth factor β<Subscript>1</Subscript> in mesenchymal stem cells: Potential utility in molecular tissue engineering for osteochondral repair

Summary The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-β₁ genes in bone marrow-derived mesenchymal stem cells (MSCs)in vitro. The full-length rat TGF-β₁ cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418, a synthetic neomycin analog. The transient and stable expression of TGF-β₁ by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently transfected MSCsin vitro with the TGF-β₁ gene causing a marked up-regulation in TGF-β₁ expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were susceptible toin vitro lipofectamine mediated TGF-β₁ gene transfer and that transgene expression persisted for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, i.e. molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineering, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness articular cartilage defects that occur as a result of injury and osteoarthritis. GUO Xiaodong, male, born in 1970, Doctor in Charge     

Osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β1 genein vitro

Summary To study the osteogenic potential of cultured bone marrow stromal cells (BMSCs) transfected with transforming growth factor β₁ (TGF-β₁) genein vitro, cultured BMSCs were transfected with the complexes of pcDNA₃-TGF-β₁ and Lipofectamine Reagentin vitro. The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type I propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type I expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF-β₁ gene could promote the osteogenic potential of cultured BMSCs.     

In vitro chondrogenic phenotype differentiation of bone marrow-derived mesenchymal stem cells

Summary In order to study the chondrogenic phenotype differentiation of adult sheep bone marrow-derived mesenchymal stem cells (MSCs) in a defined medium as potential seed cells for cartilage tissue engineering, MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. The third passage of MSCs were induced with H-DMEM containing TGF-β3. IGF I. Dexamethasone and VitC. The shape and ultrastructure of cells were observed, toluidine blue stain for GAG and immunohistochemistry for type II collagen were applied for chondrogenic phenotype identification. After 14 days of induction, MSCs changed from a spindle-like appearance to a polynal shape, a large amount of endoplasmic reticulum. Golgi complex and mitochondria were observed, and the differentiation of MSCs chondrogenic phenotype was verified by positive staining of toluidine blue and immunohistochemistry. MSCs derived from bone marrow can differentiate to chondrogenic phenotype when inducedin vitro and can be used as optimal seed cells for cartilage tissue engineering. ZHANG Yufu, male, born in 1976, M. D., Ph. D. This project was supported by a grant from the National Basic Science Research and Development Foundation (2001AA216031).     

Ginsenoside Rb1, a panoxadiol saponin against oxidative damage and renal interstitial fibrosis in rats with unilateral ureteral obstruction

Objective:To investigate the possible protective effect and mechanism of ginsenoside Rb1 against oxidative damage and renal interstitial fibrosis on rats with unilateral ureteral obstruction(UUO). Methods:In total,80 male rats were randomly divided into 4 groups,20 in each group:the sham operated group (SOR),UUO group,UUO with ginsenoside Rb1 treatment group(treated with intraperitoneal injection of 50 mg/ kg daily) and UUO with Losartan treatment group(as the positive control,treated with 20 mg/kg by ga...     

Role of transforming growth factor-β1 in the process of fibrosis of denervated skeletal muscle

In order to investigate the biological function of transforming growth factor-β1(TGF-β1) during fibrosis in denervated skeletal muscle,we recruited sciatic nerve injury model of SD rats in which denervated gastrocnemius was isolated for analysis.At different time points after operation,denervated muscle was examined by several methods.Masson trichrome staining showed morphological changes of denervated skeletal muscle.Quantitative RT-PCR detected the rapid increase of TGF-β1 expression at mRNA level after nerve injury.It was found that a peak of TGF-β1 mRNA expression appeared one week post-operation.The expression of collagen Ⅰ(COL Ⅰ) mRNA was up-regulated in the nerve injury model as well,and reached highest level two weeks post-injury.Immunoblot revealed similar expression pattern of TGF-β1 and COL Ⅰ in denervated muscles at protein level.In addition,we found that the area of the gastrocnemius muscle fiber was decreased gradually along with increased interstitital fibrosis.Interestingly,this pathological change could be prevented,at least partly,by local injection of TGF-β1 antibodies,which could be contributed to the reduced production of COL Ⅰ by inhibiting function of TGF-β1.Taken together,in this study,we demonstrated that the expression of TGF-β1 was increased significantly in denervated skeletal muscle,which might play a crucial role during muscle fibrosis after nerve transection.     

The role of TGF-β1 in mice hepatic fibrosis bySchistosomiasis Japonica

Summary To investigate the role of transforming growth factor-β₁ (TGF-β₁) in mice with hepatic fibrosis caused bySchistosomiasis Japonica, ELISA, VG staining and multimedia color hieroglyph quantitative analysis were used to study the change of the serum TGF-β₁, liver collagen fiber and reticular fiber in mice. The level of serum TGF-β₁ in experimental group was significantly higher than that in control group (P < 0. 01 orP < 0. 05) 8, 10, 12 weeks after infected by schistosomiasis. After infection, the level of liver collagen fiber and reticular fiber, and that of TGF -β₁ increased over time (P < 0. 01 orP < 0. 05). In mice infected bySchistosomiasis Japonica, the level of TGF-β₁ increased with prolongation of infection time, and with the increase of liver collagen fiber and reticular fiber. TGFβ₁ plays an important role of immunomodulation in hepatic fibrosis formation caused bySchistosomiasis Japonica.     

Molecular tissue engineering: Applications for modulation of mesenchymal stem cells proliferation by transforming growth factor β1 gene transfer

Summary The effect of transforming growth factor β₁ (TGF-β₁) gene transfection on the proliferation of bone marrow-derived mesenchymal stem cells (MSCs) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF-β₁ gene at different doses was transduced into the rat bone marrow-derived MSCs to examine the effects of TGF-β₁ gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectaminemediated 1 μg TGF-β₁ gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine= 1μg/3μl) flow cytometry and immunohistochemical analyses revealed a significant increase in the³H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF-β₁ could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases. This project was supported by a grant from National Natural Science Foundation of China (No. 30170270).     

Effect of TGF-β1 on the expression of IL-12, IL-15, IL-18, IL-4 and IL-10 in heart transplantation rejection in rats

Summary To investigate the effect of TGF-β1 on the expressions of IL-12, IL-15, IL-18, IL-4 and IL-10 in heart transplantation rejection in rats, a model of rat cervical heterotopic heart transplantation was set up and the model rats were randomly divided into three groups: control group, transplant group and TGF-β1 group. The mRNA expression levels of IL-12, IL-15, IL-18, IL-4 and IL-10 were determined by RT-PCR at the 5th day after the transplantation. The mRNA expression levels of IL-12, IL-15, IL-18 were increased obviously and those of IL-4, IL-10 were significantly decreased in the transplant group as compared with the control group (P<0.01). In the TGF-β1 group, the mRNA expression levels of IL-12, IL-15, IL-18 were significantly decreased and those of IL-4, IL-10 were significantly increased as compared with the transplant group (P<0.01). The immunosuppressive effect of TGF-β1 on heart transplantation rejection was related to its inhibition of the expressions of Th1-type cytokines (IL-12, IL-15, IL-18 etc) and its promotion of the expressions of Th2-tpye cytokines (IL-4, IL-10).     

Effect of Calcium Dobesilate on Nephropathy in Type 2 Diabetic Rats

In order to investigate the effects and mechanisms of calcium dobesilate on renal lesions in experimental type 2 diabetic rats, dibetic rats were randomly divided into control group (group C) and experimental group (group D) treated with calcium dobesitate. The serum creatinine (Scr),protein kinase C (PKC), creatinine clearance (Ccr), transforming growth factor-beta, (TGF-β1),type Ⅳ collagen were compared among the groups after 24 weeks. The renal tissues were observed under light microscopy and electron microscopy. The results showed that after 24 weeks, Scr,PKC, TGF-β1 in group D were significantly lower than in group C, meanwhile, renal pathologic changes in group D were improved. Ccr had no difference between group C and group D. It was concluded that calcium dobesilate could ameliorate renal lesions in diabetic rats through inhibiting PKC and TGF-β1.     

Expression of TGF-β in region of bone defect repaired by collagen nano-beta-tricalcium phosphate composite artificial bone

Summary The distribution and function of transforming growth factor-beta (TGF-β) in the region of bone defect repaired by collagen/nano-beta-tricalcium phosphate composite artificial bone (Co/N-TCP) and the ability of Co/N-TCP recruiting osteoblasts to precipitate the repair of bone defect were investigated. Twenty-four domestic rabbits were operated on bilateral cranial bone to create an experimental bone defect of 8.0 mm in diameter through the whole bone. On the left, Co/N-TCP was implanted as experimental group, but on the right, Co/TCP was implanted as control group. At 2nd, 4th, 8th, 12th week after operation, all animals were sacrificed and the implanted materials with surrounding bone were taken out. Immunohistochemical staining was performed for TGF-β assay by avidin-biotin complex method (SABC). Simultaneously, TGF-β was quantitatively analyzed by HPIAS-1000 imaging analysis system. The immunohistochemical staining for TGF-β revealed that osteoblasts and immature osteocytes highly expressed TGF-β. Diffused TGF-β positive staining particles appeared in the mesenchymal and fibrous-tissue. There was no significant difference in the TGF-β positive staining between two groups in the medial region to original osseous beds at different time points (P>0.05). However, in distal original osseous bed of the defected region, the positive expression of TGF-β in the Co/N-TCP group was significantly stronger than in the control group (P<0.05 or 0.01). The Co/N-TCP has good bioactivities and ability of stimulating and conducting TGF-β to aggregate and precipitate the healing of bone defect. LING Xiang, male, born in 1967, Doctor in Charge     

The Dynamic Change of TGF-β1 in the Myocardial Remodeling of Rat after Myocardial Infarction

Chronic heart failure is the leading cause of mortality and morbidity in most countries. Ventricular remodeling was the important pathophysiological process of heart failure. Mechanical overload, neurohormones and system nerve adrenal gland system can evoke remodeling. There are plentiful evidence indicating that inflammation plays an important role in the ischemic cardiac disease[1—3]. In this study we used the MI rat to observe the morpho- logical change of the ventricle, expression of the…     

Implication of EMT Induced by TGF-β1 in Pancreatic Cancer

This study examined the implication of EMT induced by TGF-β1 in pancreatic cancer invasion. TGF-β1 expression was determined in 29 cases of human pancreatic carcinoma （PC） by immunohistochemistry and the results were compared with those of pathological examination. Moreover, the effects of TGF-β1 on the phenotype and invasion of pancreatic cancer cell line Panc-1 were also investigated. TGF-β1 was detected in 12 cases （41.4 %） of PC. Significant correlation was found between the expression of TGF-β1 and lymph node involvement （P=0.047） and the depth of invasion （P=0.035）. TGF-β1 obviously promoted EMT of Panc-1 cell lines and their invasion ability was substantially enhanced. TGF-β1 may promote the malignancy of pancreatic cancer by triggering EMT.     

Effect of Coiquhoumia Root on the Expression of Transforming Growth Factor-β in Mesangiai Proliferation Giomerulonephritis Model

Summary： To study the efficacy and the mechanism of Colquhoumia root （ Tripterygium hypoglaucure （Le,vL） Hutch） in the treatment of mesangial proliferation glomerulonephritis （MsPGN）, SD rats were injected with anti-thymoeyte serum （ATS） to make MsPGN model （anti-Thyl model）. The rats were then divided into 3 groups： normal control group, anti-Thyl model group and treatment group. Histopathologieal （HE, PAS）, immunohistoehemieal, RT-PCR technique and computer imaging analysis system were used to evaluate mesangial matrix production, the expression of TGF-β protein and mRNA in the tissues of kidney. Our result showed that proteinuria and the ratio of extraeellular matrix/glomerular capillaries area （ECM/CA） were increased significantly in model group. The expression of both TGF-β protein and mRNA in glomeruli was much higher in model group than in control group （P〈0.01）. After the treatment with Colquhoumia root, proteinuria, ECM/CA and the expression of both TGF-β1 protein and mRNA in glomeruli were significantly decreased in treatment group as compared with those in model group. It is concluded that Colquhoumia root is effective in reducing proteinuria and mesangial matrix proliferation in MsPGN and it may achieve these effects by inhibiting the expressions of TGF-β1 protein and mRNA of mesangial cells.     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-β<Subscript>2</Subscript><Emphasis Type="Italic">in vitro</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Summary Whether transforming growth factor-β₂ (TGF-β₂) induces apoptosis of human trabecular meshwork cells was investigatedin vitro. Cultured 3–5 passage human trabecular meshwork cells were treated with 0 (control). 0. 32. 1, 3. 2 ng/ml TGF-β₂ for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy. TUNEL technique and flow cytometry. The results showed character istic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshowork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44)%. (4.43±1.17)% and (9.60±2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-β₂ with the difference being significant between experimental group and control group [(1.41±0.34)%]. It was concluded that TGF-β₂ can induce apoptosis of human trabecular meshwork, cellsin vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people. CAO Yang, male, born in 1972, M. D., Ph. D., Associate Professor This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

Influence of High Level TGF-β1 on Scleral Thickness

In order to explore the role of TGF-β1 in scleral remodeling and the possible mechanism,the influence of high level TGF-β1 on scleral thickness and the expression of MMP-2 and TIMP-2 was investigated in a TGF-β1 transgenic mouse model.Alb/TGF-β1(Cys223,225Ser) TGF-β1 transgenic mice were used as experimental subjects and non-transgenic littermates as controls.Plasma levels of TGF-β1 were determined by ELISA.TGF-β1,MMP-2 and TIMP-2 levels in sclera were detected by using Western blot.The thickness of posterior sclera was measured by computerized image analysis of a midsagittal section.Mean difference was analyzed with independent t-test.The results showed plasma levels of TGF-β1 in transgenic mice were 1.68 times as much as that in the controls(P<0.01).TGF-β1 levels in the sclera of transgenic mice were 2.68 times of the controls(P<0.01).Posterior scleral thickness in transgenic mice were significantly thicker than in the controls.There was no significant difference in the MMP-2 levels between transgenic mice and controls,but the TIMP-2 levels were increased significantly in transsgenic mice as compared with those in the controls.It was suggested that high levels of TGF-β1 in transgenic mice could result in the increased scleral thickness by inducing the expression of TIMP-2 to suppress the activity of MMP-2,finally inhibiting the degradation of collagen.     

Experimental study on effect of folium ginkgo biloba in treating pulmonary interstitial fibrosis in rats

Experimental Study on Effect of Folium Ginkgo Bilobain Treating Pulmonary Interstitial Fibrosis in Rats@陈建 @何冰 @刘新民 @王海斌 @章巍 @张英     

Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. In-tracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhib-ited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein syn-thesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.