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).     

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.     

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.     

COA:细胞外基质来源的双层支架的研制及其修复犬膝关节负重区骨软骨缺损的实验研究

背景与目的 骨软骨损伤常见且修复困难,本研究探讨采用软骨细胞外基质（CECM)与脱细胞骨基质（ACBM)为材料制作新型组织工程骨软骨双层支架的可行性,并检测其联合骨髓基质干细胞（bone marrow mesenchymal stem cells,BMSCs）修复犬膝关节负重区骨软骨联合缺损的疗效。 方法 ⑴新型组织工程骨软骨双层支架的骨部分以犬松质骨制备的ACBM为原料,软骨部分以人CECM为材料：将天然软骨粉碎成微丝并脱细胞处理后配成30 g/L的乳状悬液,将ACBM浸于盛CECM悬液的模具中,采用冷冻冻干法制备CECM/ACBM双层支架并交联。进行组织学、扫描电镜、Micro-CT观察,测定支架孔隙率,采用MTT法分析支架浸提液毒性。⑵ 将成软骨诱导的BMSCs种植到双相支架上体外构建组织工程骨软骨复合体,并以此复合体修复犬膝关节股骨髁负重区骨软骨缺损,分为细胞-双相支架组(实验组)和单纯支架组（对照组）,分别在3和6个月时取材,根据大体、组织学、生物力学、生物化学、Micro-CT等检测结果进行半定量或定量评估。 结果 支架评估：组织学显示双层支架去细胞彻底,无细胞碎片残留;CECM部分番红O及Ⅱ型胶原免疫荧光染色呈阳性。扫描电镜及Micro-CT观察显示支架内孔洞相互贯通呈海绵状,CECM部分孔径(155±34）μm,孔隙率为91.3%±2%;ACBM部分具有天然骨的孔径和空隙率,骨软骨部分结合良好。MTT法显示,不同浓度支架浸提液与对照培养液吸光度值比较,差异无统计学意义（P>0.05）。在修复犬膝关节骨软骨缺损的实验中,结果显示两组以纤维软骨或透明软骨对缺损有不同的修复,大体以及组织学评分表明实验组明显优于对照组,生物力学测试表明6月实验组修复软骨的刚度为正常膝关节软骨的70.1%,与生物化学分析结果一致;软骨下骨的刚度达到正常膝关节的74.96%。Micro-CT结果表明实验组与对照组软骨下骨重建不具备明显差异。结论 CECM /ACBM骨软骨双层支架保留了骨、软骨的细胞外基质成分,具备良好的孔径和孔隙率,骨-软骨两层间结合良好,无毒,生物相容性良好,可作为支架载体用于组织工程骨软骨复合体的构建。骨软骨双相支架复合成软骨诱导的BMSCs能成功修复犬膝关节负重区的骨软骨缺损,     

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.     

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     

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...     

The Expression of the Plasmid DNA Encoding TGF—β1 in Endothelium after Injection into the Anterior Chamber

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-β1 gene transfer to inhibit corneal graft rejection.Two days after direct injection of pMAM TGF-β1 mediated by liposome into the anterior chamber of rabbits,one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia.By means of immunohistochemical technique,the plasmid pMAM TGF-β1 expression product TGF-β1 in the endothelia was detected.Specific TGF-β1 expression was positive in the endothelia on both the paraffin slide and the single layer slide.The results showed that by direct injection into the anterior chamber,foreign plasmid DNA could be transferred into the endothelia and its expression was obtained.This may provide a foundation for further study on TGF-β1 participating in local induction of corneal immune tolerance.     

Fabrication of a novel hybrid scaffold for tissue engineered heart valve

The aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells （MSCs） onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly（3-hydroxybutyrate-co-4-hydroxybutyrate） using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically （hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy）, biochemically （DNA and 4-hydroxyproline） and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline （P〉0.05）. However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls （P〈0.05）. This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.     

三种形貌静电纺丝膜片的制备及其与牙周膜细胞的相容性

目的探讨三种形貌静电纺丝膜片的制备方法,并比较其对牙周膜细胞（periodontal ligament cells,PDLCs）的细胞相容性。方法通过控制纺丝溶液成分、空气湿度、流速,制成3种静电纺丝膜片,利用扫描电镜观察其形貌;将PDLCs与三种膜片共培养,DAPI染色观察PDLCs的形貌、分布;MTT法检测PDCFs的增殖;活-死细胞染色法比较PDLCs的活力。结果研究显示,制得纤维状,液滴状,圆盘状三种形貌的膜片;在共培养第7、11天,PDLCs在圆盘状膜片上的细胞增殖和活性明显优于另两种膜片（P〈0.01）。结论通过调控静电纺丝的溶液成分、空气湿度、温度及流速,可以改变纺丝膜片的微观结构和形貌,从而影响材料的细胞相容性。圆盘状膜片具有更好的细胞相容性。     

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…     

Role of protein kinase C in advanced glycation end products-induced epithelial-mesenchymal transition in renal proximal tubular epithelial cells

The role of protein kinase C （PKC） activation in advanced glycation end products （AGEs）-induced epithelial-mesenchymal transition in renal proximal tubular epithelial cells was investigated. HKC cells were divided into three groups： normal group, AGE-BSA group （100 mg/L AGE-BSA） and AGE-BSA＋PKC inhibitor （10 μmol/L chelerythrine chloride） group. PKC activity was measured by PKC assay kit. The expression of Vimentin, and phosphorylated β-catenin was detected by using Western blotting, and the content of TGF-β1 was examined by ELISA method. The intracellular disposition of Vimentin was observed by fluorescence microscopy. As compared with normal group, PKC activity was increased significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was enhanced significantly in AGE-BSA group. The expression of Vimentin, phosphorylated β-catenin, and TGF-β1 was significantly blocked by chelerythrine chloride. High expression of Vimentin, phosphorylated β-catenin, and TGF-β1 induced by AGE-BSA may be mediated via the activation of PKC signal transduction pathway.     

Dysregulation of the TGF-β Postreceptor＇Signaling Pathway in Cell Lines Derived from Primary or Metastatic Ovarian Cancer

Summary Transforming growth factor-beta (TGF-β) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF-β. Mechanisms of resistance to TGF-β caused by modulation of cell cycle regulators and/or inactivation of components of the TGF-β signaling transduction pathway such as C-myc and Smad4 are not well understood. To investigate the potential association between loss of sensitivity to TGF-β and expression status of transforming growth factor receptor II (TβRII), Smad4, CDC25A and C-myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi-quantitative RT-PCR. Normal ovarian surface tissues were used as controls. The expression of TβR II was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88% (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20% (1/5) of non-tumorigenic cell lines (P<0.05). C-myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF-β of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF-β induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF-β is not associated with a lack of TβRII. XI Ling, female, born in 1972, M.D., Ph.D., Doctor in Charge This project was supported by grants from the National Crackajack Youth Foundation (30025017) and the National Emphasis Basis Research Project of China (2002CB513100).     

Changes of CD4^＋CD25^＋ Regulatory T Cells in Patients with Acute Coronary Syndrome and the Effects of Atorvastatin

The function of CD4＋CD25＋ regulatory T lymphocytes （Treg） in patients with acute coronary syndrome （ACS） and the effects of atorvastatin were investigated. Forty-eight patients with ACS were randomly divided into two groups： group C receiving conventional therapy （n=24）, and group C＋A receiving conventional therapy＋atorvastatin （10 mg/day, n=24）. T lymphocytes from ACS patients （before and 2 weeks after the treatment） or 18 healthy subjects were separated and the flow cytometry was used to measure the percentage of Treg. The inhibitory ability of Treg on effector T cells was determined by mixed lymphocyte reaction （MLR）. ELISA was used to measure the serum levels of cytokines （IL-10, TGF-β1 and IFN-γ） before and after treatment. The results showed that as compared with normal control group, Treg percentage was decreased significantly （P〈0.01）, the inhibitory ability of Treg on the T lymphocytes proliferation was reduced （P〈0.01）, IFN-γ levels were increased and IL-10 and TGF-β1 levels were lowered in ACS patients. After treatment with atorvastatin, Treg percentage and the inhibitory ability of Treg on T lymphocytes proliferation were significantly increased in ACS patients. Serum IFN-γ was decreased significantly, while IL-10 and TGF-β1 were elevated significantly as compared with the non-atorvastatin group. The number of Treg was positively correlated with serum TGF-β1, but negatively with serum IFN-γ and CRP. It was concluded that ACS was associated with decreased number and defected function of Treg, which may play an important role in initiating immune-inflammatory response in ACS. The inhibitory effects of atorvastatin on inflammation in ACS may be due to its beneficial effects on Treg and restoration of immune homeostasis.     

Effects of heparin on transforming growth factor-β1 and extracellular matrix components in the glomeruli of diabetic rats

Summary The effects of heparin on the expression of transforming growth factor-β₁ (TGF-β₁) and two extracellular matrix components laminin (LN) and fibronectin (FN) in diabetic rat glomeruli were investigated. Twenty-six rats were randomly divided into control group (C,n=8), diabetic group (D,n=9), and diabetes+heparin group (DH,n=9). After 8-week therapy of heparin (200 U once daily by abdominal injection), TGF-β₁, LN and FN expression in glomeruli was detected by immunohistochemical method. The results showed that the expression levels of TGF-β₁, LN and FN were higher in group D than in group C. It was found that heparin could reduce 24-h urinary albumin excretion and inhibit overexpression of TGF-β₁, LN and FN in glomeruli of diabetic rats. It suggested that the inhibitory effect of heparin on diabetic glomerular sclerosis was at least partly related with the inhibition of TGF-β₁ expression. LI Yuanhong, female, born in 1973, Resident