血管紧张素Ⅱ受体拮抗剂对肝硬化大鼠TGFβ1基因表达影响的实验研究

目的研究血管紧张素Ⅱ(AngiotensinⅡ)Ⅰ型受体(AT1)拮抗剂Losartan对肝硬化大鼠TGF β1(transforming growth factor β1,转化生长因子β1)基因表达的影响.方法 41只雄性SD大鼠分为4组对照组(10只)、模型组(11只)、及Losartan治疗组(20只)[早期(10)、中期(10)],除对照组外所有大鼠均给予50% CCL4灌胃,3*!mg*kg-1*5d-1,共9周.治疗组早期组同时给予血管紧张素受体拮抗剂Losartan灌胃,中期组于造模中期(5周)开始给药,用量10*!mg*kg-1*d-1至处死前.实验结束后,处死取肝脏液氮冻存及福尔马林固定标本,分别行免疫组织化学及RT-PCR检测,TGF β1蛋白及基因表达情况.结果 与正常组相比,模型组TGF β1表达明显增强,由0.93±0.05升高到1.94±0.13(P<0.01),而两治疗组与模型组相比,losartan明显降低了TGF β1 mRNA的表达,分别降至1.17±0.16、1.02±0.11(P<0.05),而早、中期两组差异无显著意义(P>0.05).结论 Losartan对肝硬化时TGF β1表达有明显抑制作用.     

Lefty蛋白在人胚胎皮肤、成人正常皮肤及增生性瘢痕中的表达

目的 了解Lefty蛋白在人胚胎皮肤、成人正常皮肤及增生性瘢痕(HS)组织中的表达,探讨其在HS形成中的作用及与胚胎无瘢痕愈合的关系. 方法 留取人胚胎皮肤、成人正常皮肤及HS标本,制成冰冻切片,行免疫荧光染色.激光共聚焦显微镜下观察各标本中成纤维细胞形态及Lefty蛋白的表达,用阳性细胞率作为表达量. 结果 人胚胎皮肤成纤维细胞呈梭形,胞核呈椭圆形或梭形,排列规则;成人正常皮肤及HS组织成纤维细胞呈长梭形,胞核呈梭形或星形,前者排列较规则,而后者排列不规则.HS组织中Lefty蛋白阳性细胞率为15.38%,低于成人正常皮肤(67.92%)和人胚胎皮肤(81.67%,P＜0.01);而成人正常皮肤阳性细胞率也低于人胚胎皮肤(P＜0.05). 结论 Lefty蛋白可能对瘢痕的形成有抑制作用,其高表达可能与胚胎无瘢痕愈合相关.     

Evidence for a role of transforming growth factor (TGF)-beta1 in the induction of postglomerular albuminuria in diabetic nephropathy: amelioration by soluble TGF-beta type II receptor

Transforming growth factor-beta (TGF-beta) has previously been implicated in the progression of diabetic nephropathy, including the onset of fibrosis and albuminuria. Here we report for the first time the use of a high-affinity TGF-beta1 binding molecule, the soluble human TGF-beta type II receptor (sTbetaRII.Fc), in the treatment of diabetic nephropathy in 12-week streptozotocin-induced diabetic Sprague-Dawley rats. In vitro studies using immortalized rat proximal tubule cells revealed that 50 pmol/l TGF-beta1 disrupted albumin uptake (P < 0.001 vs. control), an inhibition significantly reversed by the use of the sTbetaRII.Fc (1,200 pmol/l). In vivo studies demonstrated that treatment with sTbetaRII.Fc reduced urinary albumin excretion by 36% at 4 weeks, 59% at 8 weeks (P < 0.001), and 45% at 12 weeks (P < 0.01 for diabetic vs. treated). This was correlated with an increase in megalin expression (P < 0.05 for diabetic vs. treated) and a reduction in collagen IV expression following sTbetaRII.Fc treatment (P < 0.001 for diabetic vs. treated). These changes occurred independently of changes in blood glucose levels. This study demonstrates that the sTbetaRII.Fc is a potential new agent for the treatment of fibrosis and albuminuria in diabetic nephropathy and may reduce albuminuria by reducing TGF-beta1-induced disruptions of renal proximal tubule cell uptake of albumin.     

核心蛋白多糖及其mRNA在正常皮肤和增生性瘢痕组织中的表达

目的　检测不同时期增生性瘢痕组织中核心蛋白多糖的含量及其mRNA的表达水平 ,探讨核心蛋白多糖含量的变化与其合成的关系。　方法　取 10例手术剩余的正常皮肤、包皮和 2 2例手术切除的瘢痕组织作标本 ,采用免疫组织化学和蛋白免疫印迹法检测核心蛋白多糖在标本组织中的含量及其分布 ,用原位杂交技术检测其mRNA表达水平。　结果　正常皮肤的真皮中核心蛋白多糖含量丰富 ,mRNA呈低表达。增生性瘢痕组织中核心蛋白多糖含量在 6个月内 (184 0 3± 7193)极少 ,7～ 12个月 (4 2 937± 96 6 2 )逐渐增加 ,13～ 36个月 (86 2 31± 16 2 85 )持续增加 ,36个月以后 (13094 3± 174 5 8)其含量与正常皮肤比较 ,差异无显著性意义 (P >0.0 5);其mRNA水平在 6个月内低于正常 ,7～ 12个月呈高表达 ,13～ 36个月呈持续高表达 ,36个月后逐渐下降至正常皮肤表达水平。　结论　增生性瘢痕组织中核心蛋白多糖减少的主要原因是其合成减少 ,核心蛋白多糖的增加与瘢痕组织稳定的时间相一致 ,提示核心蛋白多糖的延迟出现可能与增生性瘢痕的形成有关。     

Recombinant human decorin inhibits cell proliferation and downregulates TGF-beta1 production in hypertrophic scar fibroblasts

Hypertrophic scarring remains a major problem for patients who have suffered deep burns. The pathophysiology underlying hypertrophic scar formation may be driven by the biological activity of transforming growth factor beta1 (TGF-beta(1)). Decorin is a human proteoglycan that inactivates the effect of TGF-beta(1) and therefore displays a beneficial effect of antifibrosis in various tissues. Hypertrophic scarring is a fibroproliferative disorder of the dermis that occurs following wounding. This study investigated the effects of decorin on cell proliferation, TGF-beta(1) production, and collagen synthesis in hypertrophic scar fibroblasts. The cell proliferation rates, cell cycle distribution, low-molecular-weight apoptotic DNA and TGF-beta(1) levels, and contents of type I and type III collagen amino-terminal propeptide (PINP, PIIINP) in supernatants were assessed. Fibroblast proliferation was significantly (P<0.05) inhibited by decorin, and this effect was dose-dependent. The fibroblast population became stationary at decorin concentrations of 100 and 200 nM. Decorin inhibited fibroblast proliferation by inducing cell growth arrest but not apoptosis. TGF-beta(1) and PINP levels were significantly (P<0.05) lower in fibroblasts treated with 10, 50, 100, 200 nM of decorin compared with fibroblasts without decorin addition. However, there was no significant difference in PIIINP concentration between the decorin-treated group and the control group. These results suggest that decorin has a down-regulatory effect on cell proliferation, TGF-beta(1) production, and collagen synthesis in hypertrophic scar fibroblasts. Improved understanding of such a regulatory mechanisms may eventually be of therapeutic significance in the control of hypertrophic scarring.     

转化生长因子β1在增生性瘢痕中的表达及临床意义

目的探讨转化生长因子β1在增生性瘢痕中的表达及临床意义.方法采用免疫组化技术检测了67例增生性瘢痕和11例正常皮肤对照组中转化生长因子β1的表达.结果转化生长因子β1在增生性瘢痕中表达的阳性率为100%,且高表达为54例,低表达为13例.11例正常皮肤仅有3例为低表达,阳性率27.27%.统计学分析两组间有显著差异(P＜0.01).结论转化生长因子β1在增生性瘢痕中的表达增强,提示转化生长因子β1可能参与了增生性瘢痕的形成并起着重要的作用,故在预防和治疗烧伤后增生性瘢痕中具有一定的临床意义.     

TGF-beta 1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2)

BACKGROUND: The prognosis of primary renal disease is often dependent on the degree of tubulointerstitial scarring. Scarring is caused by proliferation and excessive matrix production of renal fibroblasts and possibly other cellular elements. Transforming growth factor-beta (TGF-beta) is the most important cytokine for the induction of matrix synthesis in the kidney. However, its effects on renal fibroblast proliferation have not been determined. We have recently demonstrated that the expression of basic fibroblast growth factor (FGF-2) is robustly up-regulated in human kidneys with tubulointerstitial fibrosis and that FGF-2 is a potent inducer of fibroblast proliferation. The present study examined the interaction between TGF-beta 1 and FGF-2 in human renal fibroblasts. METHODS: Experiments were performed on a transformed medullary fibroblast line and on primary cortical kidney and skin fibroblasts isolated from human biopsies. mRNA levels of FGF-2 and TGF-beta 1 were analyzed by Northern blot analyses. Changes in protein expression were examined by immunoblots and enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine incorporation assays and cell counts were used to analyze cell proliferation. The expression of cell cycle-regulatory proteins cyclin-dependent kinase (cdk) 2 and the cdk inhibitor p27(kip1) were determined by immunoblots. RESULTS: Stimulation of renal fibroblasts with FGF-2 resulted in no change of TGF-beta 1 mRNA expression, whereas incubation of the cells with TGF-beta 1 induced FGF-2 mRNA up to 3.51 +/- 0.21-fold after six hours. This increase could be blocked almost completely by the addition of cyclohexamide, indicating that the process is in large part dependent on protein synthesis. The up-regulation in FGF-2 mRNA expression was paralleled by de novo detection of FGF-2 protein in the supernatant, peaking after 12 to 24 hours, as determined by Western blot and ELISA, whereas cellular protein was only increased up to 2.1-fold. Interestingly, both methods detected release of FGF-2 protein to the supernatant already at three hours, indicating a role for TGF-beta1 in directly releasing preformed FGF-2. Since TGF-beta 1 induced FGF-2, which results in fibroblast proliferation, we hypothesized that TGF-beta1 may cause fibroblast proliferation mediated by FGF-2. This hypothesis was verified by cell proliferation assays demonstrating that stimulation of renal fibroblasts with TGF-beta1 resulted in an up to 3.21 +/- 0.28-fold increase in bromodeoxyuridine incorporation and a 1.95 +/- 0.16-fold increase in cell number after 72 hours. This mitogenic effect of TGF-beta1 could be blocked completely by the addition of a neutralizing antibody to FGF-2 or the tyrosine kinase inhibitor tyrphostin AG1296, which blocks FGF receptor (FGFR) tyrosine kinase activity. Conversely, a neutralizing antibody to epidermal growth factor (EGF) or the tyrphostin B42, which inhibits EGF receptor signal transduction, had no effect. Interestingly, a neutralizing antibody to PDGF had only minor effects in primary kidney fibroblasts but reduced TGF-beta 1-induced proliferation considerably in primary skin fibroblasts. Finally, TGF-beta1-induced proliferation in kidney fibroblasts was paralleled by a robust increase in cdk 2 protein expression up to 72 hours, whereas p27(kip1), whose activity is maintained by TGF-beta in epithelial cells, was down-regulated up to 48 hours. CONCLUSIONS: Our studies demonstrate, to our knowledge for the first time, that TGF-beta1 induces proliferation in human renal fibroblasts and that this process is mediated largely by FGF-2. The induction of proliferation by TGF-beta 1 via induction of FGF-2 may play an important role in the autonomy of renal fibroblast growth and thus in the pathogenesis of human fibrogenesis.     

肝细胞生长因子对正常皮肤和增生性瘢痕成纤维细胞的影响

目的:采用携带肝细胞生长因子(HGF)腺病毒转染正常皮肤成纤维细胞(NFB)和增生性瘢痕成纤维细胞(HFB)后,观察HGF对这两种细胞作用的差异,为HGF基因治疗病理性瘢痕提供理论基础。方法:分离培养HFB和NFB,以不同感染复数(multiplicity of infection,MOI)携带绿色荧光蛋白基因的腺病毒(Ad-GFP)转染细胞,用流式细胞仪检测转染效率;以MOI为100携带HGF基因的腺病毒(Ad-HGF)转染HFB和NFB48h后,酶联免疫吸附法检测细胞上清中HGF、基质金属蛋白酶-1(MMP-1)和转化生长因子-β1(TGF-β1)的表达;应用免疫组织化学方法检测正常皮肤和增生性瘢痕组织中MMP-1和TGF-β1的分布。结果:NFB和HFB在腺病毒转染效率和HGF表达方面差异无显著性,但HGF能明显促进HFB分泌MMP-1,抑制HFB表达TGF-β1。结论:促进HFB分泌MMP-1和抑制HFB表达TGF-β1可能是HGF治疗病理性瘢痕的细胞和分子生物学基础。     

Distribution of intracellular and extracellular expression of transforming growth factor—β1（TGF—β1） in human testis and their association with spermatogenesis

AIM: Spermatogenic dysfunction may result from thickening of seminiferous tubular basement membrane (BM) with tubular sclerosis. Transforming growth factor beta1 (TGF-beta1) plays an important role in fibrogenesis. The intracellular and extracellular expression of TGF-beta1 in the testis were immunohistochemically determined, using LC antibody (LC) for intracellular TGF-beta1 and CC antibody (CC) for extracellular TGF-beta1. METHODS: Twenty-three testicular biopsy specimens were obtained from varicocele and five from Sertoli-cell-only (SCO) patients, and five from normal volunteers. The relative area involved by the expression of TGF-beta1 for CC or LC (TGF-beta1 index for CC or LC) was examined, and semen parameters and serum hormonal levels and TGF-beta1 were analyzed. The Johnson score (JS), the BM thickness, and the tubular diameter were also determined. RESULTS: Immunoreactivity for CC was hardly detected. That for LC was detected in the Sertoli and germ cells. The TGF-beta1 index for LC was significantly higher in the varicoceles than in the normal testes. Interestingly, that for LC was significantly higher in the varicoceles than in the SCO. The level of serum TGF-beta1 was significantly higher in varicoceles than in the normal testes. CONCLUSION: The distribution of the intracellular and extracellular expression of TGF-beta1 in human testis was demonstrated. It suggests that TGF-beta1 is related to fibrosis of seminiferous tubules and may lead to spermatogenic disruption.     

Bone matrix insulin-like growth factor (IGF)-I, IGF-II and transforming growth factor (TGF)-beta1 levels in men and postmenopausal women with osteoporosis: lack of association with circulating growth factors and bone mineral density

Previous clinical studies have suggested a positive correlation between serum insulin-like growth factor components and bone mass in both men and women with or without osteoporosis. The aim of the present study was to analyze the relationship between the skeletal levels of insulin-like growth factors and transforming growth factor-b1 and bone mineral density in a group of men and postmenopausal women in whom osteoporosis was diagnosed previously. Bone matrix extraction was achieved by passive dialysis against tetrasodium EDTA-guanidine-HCL. IGF's were quantified by radioimmunoassay. TGF-b1 was assessed by a specific enzyme-linked immunoassay. No correlation between BMD and the concentration of IGF-I, IGF-II and TGF-b1 in bone matrix was detected in either men or postmenopausal women with osteoporosis. In addition, circulating growth factors levels failed to be associated with the concentration of IGF-I, IGF-II and TGF-b1 in the skeleton. Thus, our study provides no evidence for a major role of bone matrix IGF's or TGF-b1 as determinants of bone mass in men or postmenopausal women with osteoporosis.     

移植人皮肤建立的增生性瘢痕裸鼠模型瘢痕中TGF-β1变化的实验研究

目的 研究移植人皮肤建立的增生性瘢痕裸鼠模型瘢痕中不同时期TGF-β1的变化,探讨TGF-β1的表达与瘢痕增生的关系及对比与人的增生性瘢痕的变化规律,证明用该裸鼠模型进行实验研究的可行性.方法 将人的全厚皮片移植于裸鼠背部,待皮片脱落后局部产生瘢痕增生,切取不同时期的瘢痕组织标本,用免疫组织化学染色的方法对TGF-β1进行染色,并对染色结果进行图像分析.结果 移植术后TGF-β1在瘢痕组织中的表达为,1个月和3个月时高表达,6个月时表达明显减少,而在正常皮肤中很少有阳性表达.结论 TGF-β1在该裸鼠模型中的表达与人的增生性瘢痕的变化规律相似,TGF-β1的表达与瘢痕增生有密切关系,该模型是一种较为理想的增生性瘢痕的动物实验模型.