Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
Objective To observe the differences in protein contents of three transforming growth factorbeta(TGF-β) isoforms, β1, β2, β3 andtheir receptor(I) in hypertrophic scar and normal skin and to explore their influence on scar formation. Methods Eight cases of hypertrophic scar and their corresponding normal skin were detected to compare the expression and distribution of TGF-β1, β2, β3 and receptor(I) with immunohistochemistry and common pathological methods. Results Positive signals of TGF-β1, β2, and β3 could all be deteted in normal skin, mainly in the cytoplasm and extracellular matrix of epidermal cells; in addition, those factors could also be found in interfollicular keratinocytes and sweat gland cells; and the positive particles of TGF-β R(I) were mostly located in the membrane of keratinocytes and some fibroblasts. In hypertrophic scar, TGF-β1 and β3 could be detected in epidermal basal cells; TGFβ2 chiefly distributed in epidermal cells and some fibroblast cells; the protein contents of TGF-β1 and β3 were significantly lower than that of normal skin, while the change of TGF-β2 content was undistinguished when compared withnormalskin. In two kinds of tissues, the distribution and the content of TGF-β R(I) hadno obviously difference. ConclusionThe different expression and distribution of TGF-β1, β2 andβ3 between hypertrophic scar and normal skin may beassociated with the mechanism controlling scar formation, in which the role of the TGF-βR (I) and downstream signal factors need to be further studied.
OBJECTIVE: To localize the distribution of basic fibroblast growth factor (bFGF) and transforming growth factor-beta(TGF-beta) in tissues from dermal chronic ulcer and hypertrophic scar and to explore their effects on tissue repair. METHODS: Twenty-one cases were detected to localize the distribution of bFGF and TGF-beta, among them, there were 8 cases with dermal chronic ulcers, 8 cases with hypertrophic scars, and 5 cases of normal skin. RESULTS: Positive signal of bFGF and TGF-beta could be found in normal skin, mainly in the keratinocytes. In dermal chronic ulcers, positive signal of bFGF and TGF-beta could be found in granulation tissues. bFGF was localized mainly in fibroblasts cells and endothelial cells and TGF-beta mainly in inflammatory cells. In hypertrophic scar, the localization and signal density of bFGF was similar with those in granulation tissues, but the staining of TGF-beta was negative. CONCLUSION: The different distribution of bFGF and TGF-beta in dermal chronic ulcer and hypertrophic scar may be the reason of different results of tissue repair. The pathogenesis of wound healing delay in a condition of high concentration of growth factors may come from the binding disorder of growth factors and their receptors. bFGF may be involved in all process of formation of hypertrophic scar, but TGF-beta may only play roles in the early stage.
Objective To explore the expression characteristics of chaperone interacting protein (CHIP) in normal, scar and chronic ulcer tissues and its relationship with wound healing. Methods Twenty biopsies including scar tissues(n=8), chronic ulcer tissues(n=4) and normal tissues(n=8)were used in this study. The immunohistochemical staining (power visionTMtwo-step histostaining reagent) was used to explore the amount and expression characteristics of such protein.Results The positive expression of CHIP was observed in fibroblasts, endothelial cells and epidermal cells in dermis and epidermis. It was not seen ininflammatory cells. The expression amount of CHIP in scar tissues, chronic ulcer tissues and normal tissues was 89%, 83% and 17% respectively. Conclusion Although the function of CHIP is not fully understood at present, the fact that this protein is expressed only at the mitogenic cells indicates that it may be involved in mitogenic regulation during wound healing.
Objective:To detect collagen I synthesis activity in the vitreous of PVR induced by macrophages in rabbits. Methods:PC Ⅲ (Procollagen Ⅲ ) concentrations were measured by radioim- munoassay in the vitreous samples of 14 rabbit eyes with experimental PVR and 14 control eyes. Results:The mean PC Ⅲ concentration on the 7th day after macrophage injection as 257.58mu;g/L(range,236.04~266.88mu;g/L,n= 4)and significantly increased on the 14th day later. On the 28th day the mean concentration of PC Ⅲ as 912.23mu;g/L (range, 881.36~943.10mu;g/L ;n= 2). There was a significant difference between the 7th and the 14th, 21st of 28th day statistically(P＜0.05). PC Ⅲ was not detected in control eyes. Conclusion:The PC Ⅲ level in the vitreous of rabbit eyes with experimental PVR increased significantly from the 7th to the 28th day after macrophages injection and is well consistent with the time course of scarring and the development of traction retinal detachment in the PVR model. （Chin J Ocul Fundus Dis,1996,12: 43-44）
【摘要】 目的 原肌球蛋白是肌球蛋白主要相关蛋白之一，在细胞骨架与运动中起着重要的作用。探讨原肌球蛋白在增生性瘢痕中的作用，有助于揭示瘢痕挛缩的产生机制。 方法 收集2006年3月-2008年7月48例患者不同时期增生与非增生瘢痕组织标本，利用基因芯片筛选出的瘢痕相关基因——原肌球蛋白基因特异片段，制备成寡核苷酸探针与瘢痕组织切片进行原位杂交。同时，将各标本进行原代细胞培养，制备成纤维细胞爬片，进行原位杂交。 结果 原肌球蛋白基因在3、6个月的增生性瘢痕中表达均明显强于9、12个月增生性瘢痕及非增生性瘢痕，阳性细胞比例也高于9、12个月增生性瘢痕及非增生性瘢痕。 结论 瘢痕增生挛缩与细胞骨架运动的相关基因存在密切关系，而原肌球蛋白可能在其中起着重要作用。【Abstract】 Objective Tropomyosin is one of the proteins of cytoskeleton and cell movement. The aim of this study is to investigate the effect of gene expression of fibroblast tropomyosin on the formation and contraction of hypertrophic scar. Methods According to the results of differently expressed genes, in hypertrophic scar by gene microarray, topomyosin, one of the most important genes, was selected and made into oligonucleotide probe. Twenty-four hypertrophic scars and 24 non-hypertrophic scars and 12 normal skins were used and these scar were taken on 3, 6, 9, and 12 months after burned between March 2006 and July 2008. Frozen section and cultured fibroblasts were made to detect the expression of the gene by in situ hybridization. Results Expression of tropomyosin was defected in scar tissue, but those in the hypertrophic scar on 3 and 6 months after burn were significantly ber than those in the hypertrophic scar on 9 and 12 months after burn and non-hypertrophic scar. Conclusion Overexpression of cytoskeletal relative genes causes the contraction of scar and tropomyosin acts leading and key functions.
Objective To determine whether local delivery of c-myc shRNA could inhibit hyperplasia and lithogenic potentiality in a rat model of chronic proliferative cholangitis (CPC) via specific blockade of the c-myc expression. Methods The CPC animal model (CPC group) was established via retrograde insertion of a 5-0 nylon thread into the common bile duct through Vater’s papilla. Three kinds of c-myc shRNAs were then respectively injected in c-myc shRNA group, which were included shRNA-1, shRNA-2, and shRNA-3, respectively. Negative control group and sham operation group were established for comparison. Subsequently, histopathological changes of bile duct wall were observed by HE, Massion, and PAS/AB staining; c-myc protein was detected by immunohistochemistry method; 5-bromodeoxyuridine (BrdU) protein was tested by immumofluorescence method; c-myc, Mucin 3, and Procollagen Ⅰ mRNAs were detected by real time PCR; Ki-67 protein was determined by Western blot; Activity of β-glucuronidase was measured by modified Fisherman method. Results ①Compared with the CPC and negative control groups, biliary tract mucosa epithelium (HE staining), submucosal acid mucinous gland (mid-blue staining, PAS/AB staining), and degree of over-hyperplasia of collagen fiber in bile duct wall (blue staining, Massion staining) were weaker in the c-myc shRNA group. ②The expressions of c-myc mRNA, Mucin 3 mRNA, Procollagen Ⅰ mRNA, Ki-67 protein, and β-G activity in the c-myc shRNA group were lower than those of the CPC and negative control groups (Plt;0.05), but higher than those of the sham operation group (Plt;0.05). Conclusion c-myc shRNA treatment could effectively inhibit the hyperplastic behavior and lithogenic potential of CPC, which might help to prevent the biliary restenosis and stone recurrence.