胰岛素样生长因子结合蛋白相关蛋白1在活化肝星状细胞中的表达及意义

目的 研究胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding protein-related protein-1,IGFBP-rP1)在转化生长因子β1(tronstorming growth factor β1,TGFβ1)诱导的肝星状细胞中表达的变化及抗IGFBP-rP1抗体对TGFβ1诱导的肝星状细胞产生Ⅰ型胶原的影响.方法 大鼠肝星状细胞株(HSC-T6)体外培养,分别设空白对照组(加入等量PBS)、不同浓度的TGFβ1.处理组、不同浓度的抗IGFBP-rP1抗体处理组,处理因素作用24h,采用免疫组织化学染色、Western blot检测IGFBP-rP1在HSC-T6中的表达,ELISA检测Ⅰ型胶原的含量并进行IGFBP-rP1与Ⅰ型胶原的相关性分析.结果 TGFβ1各处理组IGFBP-rP1的表达均较空白对照组显著增强.抗IGFBP-rP1抗体可以拮抗TGFβ1诱导的HSC-T6产生的过多Ⅰ型胶原.IGFBP-rP1与Ⅰ型胶原呈正相关(r=0.833,P＜0.01).结论 IGFBP-rP1参与了肝纤维化的形成,且可能在肝纤维化的发生、发展中占有重要地位.     

小鼠肝组织中胰岛素样生长因子结合蛋白相关蛋白1在肝纤维化发生及发展中的作用

目的 探讨小鼠肝组织中胰岛素样生长因子结合蛋白相关蛋白1(IGFBPrP1)在肝纤维化发生及发展过程中的作用.方法 采用腹腔注射硫代乙酰胺制备小鼠肝纤维化模型,按时间将小鼠分为模型4、5、6周组(各10只),并分别设立正常对照组(各6只),采用Masson染色检测肝组织胶原沉积,免疫组织化学检测肝组织中IGFBPrP1、α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(Collagen Ⅰ)、纤维连接蛋白(FN)、TGF-β1和Smad3蛋白表达和分布,同时以Western blot检测IGFBPrP1、α-SMA和Smad3蛋白表达.采用单因素方差分析、Pearson等级相关检验进行分析,以P<0.05为差异有统计学意义.结果 免疫组织化学检测结果发现,模型组小鼠肝组织中IGFBPrP1由0.21±0.03上升到5.03±0.09,α-SMA由0.11±0.04上升到10.09±0.18,Collagen Ⅰ由0.22±0.01上升到11.01±±0.16,FN由0.31±0.09上升到19.81±1.62,TGF-β1由0.49±0.02上升到5.97±0.19,Smad3由0.22±0.03上升到2.03±0.07,这些检测因子在模型组的表达与正常对照组比较,随时间的增加而明显增强(F=783.141,998.200,886.715,935.242,931.241,697.118,P<0.05).在肝纤维化形成过程中,IGFBPrP1的表达与α-SMA、Collagen Ⅰ、FN、TGF-β1和Smad3的表达均呈正相关(r=0.906,0.927,0.988,0.947,0.977,P<0.05).Western blot检测结果发现,模型组小鼠肝组织的IGFBPrP1蛋白表达量由0.23±0.01上升到0.92±0.07,α-SMA蛋白表达量由0.36±0.02上升到1.39±0.03,FN蛋白表达量由0.03±0.00上升到0.12±0.02,Smad3蛋白表达量由0.09±0.01上升到0.56±0.04,模型组小鼠的蛋白表达量均较正常对照组明显升高(F=57.316,201.214,103.871,72.966,P<0.05).在肝纤维化形成过程中,IGFBPrP1的表达与α-SMA、FN和Smad3的表达均呈正相关(r=0.982,0.924,0.965,P<0.05).结论 IGFBPrP1随着肝纤维化程度的加重,其表达水平逐渐上调,IGFBPrP1的促肝纤维化作用可能与促进肝星状细胞激活、使细胞外基质的重要组成成分Collagen Ⅰ和FN的合成与分泌增加及影响TGF-β1/Smad3信号通路有关.     

胰岛素样生长因子和胰岛素样生长因子结合蛋白在软骨细胞生长和发育过程中的调控作用研究进展

胰岛素样生长因子（Insulin-like growth factors,IGFs）,是机体生理情况下调控生长和发育的重要生长因子。IGFs在长骨发育,即软骨内化骨的调控中发挥重要作用。IGF-I参与调节了间充质干细胞的成软骨过程并进而参与了软骨组织稳态的保持。胰岛素样生长因子结合蛋白（Insulin-like growth factor-binding proteins,IGFBPs）代表了一个能与IGF-I和IGF-2相结合的进化上保守的蛋白质家族,包括6个独立的家族成员IGFBP1、2、3、4、5、6,它们具有调控和储存转运IGFs的作用以及独立于IGFs的作用,在机体生长和发育中发挥重要的功能,在软骨组织的发育中同样扮演着重要的角色。本文综述了IGFs和IGFBPs对软骨细胞生长和发育的生理调节功能,从而为IGFs在软骨组织工程领域的应用提供参考。     

小鼠肝组织中胰岛素样生长因子结合蛋白相关蛋白1在肝纤维化发生及发展中的作用

Objective To investigate the effect of insulin-like growth factor binding protein-related protein 1 ( IGFBPrP1 ) in the formation and development of hepatic fibrosis. Methods Hepatic fibrosis model of mice was made by intraperitoneal injecting with thioacetamide, then the mice were sacrificed four, five and six weeks later (10 mice were sacrificed at each time point, model groups). Mice in the control groups were treated by normal saline (6 mice were sacrificed at each time point). Collagen accumulation in liver tissues was detected by Masson stain. Distribution and dynamic expressions of IGFBPrP1, alpha-smooth muscle actin ( α-SMA ),Collagen Ⅰ , fibronectin ( FN), TGF-β1 and Smad3 in different groups were detected by immunohistochemistry.The expressions of IGFBPrP1, α-SMA and Smad3 were detected by Western blot. All data were analyzed using the analysis of variance (ANOVA), Pearson rank correlation coefficient. Results The expressions of IGFBPrP1 in the liver tissues were increased from 0.21 ±0.03 to 5.03 ±0.09, α-SMA from 0. 11 ±0.04 to 10.09 ±0. 18,Collagen Ⅰ from 0.22 ±0.01 to 11.01 ±0. 16, FN from 0.31 ±0.09 to 19.81 ±1.62, TGF-β1 from 0.49 ±0.02 to 5.97 ± 0. 19, and Smad3 from 0.22 ± 0.03 to 2.03 ± 0.07. Compared with the control groups, the expressions of IGFBPrP1, α-SMA, Collagen Ⅰ , FN, TGF-β1 and Smad3 in the model groups were significantly increased as time passed by ( F = 783. 141,998. 200,886. 715,935. 242, 931. 241,697. 118, P < 0. 05 ). During the formation of hepatic fibrosis, the expression of IGFBPrP1 was positively correlated with the expressions of α-SMA,Collagen Ⅰ , FN, TGF-β1 and Smad3 ( r = 0. 906, 0. 927, 0. 988, 0. 947, 0. 977, P < 0.05 ). The results of Western blot showed that the protein expression of IGFBPrP1 was increased from 0. 23 ± 0.01 to 0.92 ± 0.07,α-SMA from 0.36 ± 0. 02 to 1.39 ± 0.03, FN from 0.03 ± 0.00 to 0.12 ± 0.02, and Smad3 from 0.09 ± 0. 01 to 0.56 ±0.04. The protein expressions of IGFBPrP1, α-SMA, FN and Smad3 were significantly increased in the model groups when compared with the control groups (F =57. 316, 201. 214, 103. 871, 72. 966, P <0.05).During the formation of hepatic fibrosis. IGFBPrP1 was positively correlated with the expressions of α-SMA, FN and Smad3 (r = 0. 982, 0. 924, 0. 965, P < 0.05 ). Conclusions The expression of IGFBPrP1 increases as the aggravation of the fibrosis. IGFBPrP1 promotes the formation and development of hepatic fibrosis by activating hepatic stellate cells, accelerating the synthesis and secretion of Collagen Ⅰ and FN which are the principal components of extracellular matrix, and affecting the TGF-β1/Smad3 pathway.     

血清胰岛素样生长因子结合蛋白7的水平与多囊卵巢综合征及其胰岛素抵抗的关系

目的 探讨血清胰岛素样生长因子结合蛋白7(IGFBP7)水平与多囊卵巢综合征(PCOS)及其胰岛素抵抗(IR)的关系。方法 选择2021年10月至2022年12月于徐州市中心医院生殖中心就诊的PCOS患者为PCOS组(91例),收集同时期同年龄段因输卵管因素或男方因素行辅助生殖的女性为对照组(85例)。收集所有研究对象的一般资料,包括年龄、体质量指数(BMI)、腰围等;检测研究对象血清中部分生化指标如血糖、脂代谢相关指标、生殖激素水平等。采用二元Logistic回归分析评价血清IGFBP7水平与PCOS及其IR发生的关系;采用多因素线性回归分析筛查与PCOS患者IGFBP7水平升高有关的因素。结果 PCOS组患者的BMI、腰围、抗苗勒管激素(AMH)、黄体生成素(LH)、总睾酮(TT)、游离睾酮(FT)、游离雄激素指数(FAI)、空腹血糖(FPG)、空腹胰岛素(FIN)、胰岛素抵抗的稳态模型指数(HOMA-IR)、总胆固醇(CHOL)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-c)、IGFBP7水平均显著高于对照组(P<0.05),而年龄、高密度脂蛋白胆固醇(HDL-c)、性...     

胰岛素和胰岛素样生长因子及其结合蛋白与结直肠癌发生的关系

目的探讨血清胰岛素、胰岛素样生长因子（IGF-1）、IGF结合蛋白（IGFBPs）体质量指数（BMI）、腰臀围比（WHR）的变化及与结直肠癌发生、发展的关系。方法检测对象为2006年6月至2007年10月间住院收治和门诊复查的结直肠癌患者615例（术前检测244例,术后371例）和健康对照者150例。采用酶联免疫吸附法检测血清胰岛素、IGF-1和IGFBPS水平。结果结直肠癌患者术前血清胰岛素、IGF-1水平和IGF-I／IGFBP-3比值与健康对照组、术后患者比较,均明显升高,IGFBP-3水平明显降低,差异均有统计学意义（P〈0.05,P〈0.01）。结直肠癌术后未发生转移者与有肝或腹腔远处转移者胰岛素、IGF-1、IGFBP-1、IGFBP-3、IGF-1／IGFBP-3比较,差异均无统计学意义（P〉0.05）。结直肠癌患者WHR明显高于健康对照组（P〈0.01和P〈0.05）;而BMI与健康对照组比较,差异无统计学意义（P〉0.05）。结肠癌患者WHR、BMI与胰岛素水平、IGF-1／IGFBP-3比值呈正相关（P〈0.01,P〈0.05）,与IGFBP-3呈负相关（P〈0.01,P〈0.05）;直肠癌患者WHR与血清瘦素、胰岛素水平及BMI与血清IGFBP-1水平均呈正相关（P〈0.05）,与其他无相关性（P〉0.05）。结论胰岛素、IGF-1水平和IGF-I／IGFBP-3比值升高及IGFBP-3水平降低,可能与结直肠癌的发生有关,但与肿瘤转移与否无关,中心性肥胖是结肠癌发生的危险因素之一。     

Smad3沉默对IGFBPrP1诱导肝星状细胞分泌细胞外基质作用的影响

目的 明确胰岛素样生长因子结合蛋白相关蛋白1(insulin-like growth factor binding protein related protein1,IGFBPrP1)是否通过Smad3信号通路影响肝星状细胞分泌细胞外基质.方法 (1)化学合成2对针对Smad3基因的siRNAs(siRNA1,siRNA2),转染肝星状细胞株(HSC-T6).采用实时定量PCR和Western blot法筛选抑制效率较高的siRNA用于干扰实验;(2)将肝星状细胞株(HSC-T6)分为4组:阴性对照组、siRNA-Smad3转染组、siRNA-Smad3+IGFBPrP1组和IGFBPrP1组.将筛选的抑制效率较高的siRNA转染HSC-T6细胞株,田Western b1ot检测各组Smad3、纤维连接蛋白及Ⅰ型胶原的表达.结果 (1)siRNA2-Smad3对Smad3的抑制效率较高;(2)与阴性对照组相比,siRNA-Smad3转染组Smad3蛋白的表达显著下降(P＜0.01).与IGFBPrP1组相比,siRNA-Smad3+IGFBPrP1组纤维连接蛋白和Ⅰ型胶原蛋白的表达均显著降低(P＜0.01).结论 IGFBPrP1影响肝星状细胞分泌细胞外基质的机制之一是通过Smad3信号通路来实现的.

Abstract：

Objective To identify the effect of IGFBPrP1 on the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway. Methods (1)Two pairs of chemically synthesized siRNAs (siRNA1, siRNA2) targeting Smad3 were transfected into HSC-T6 cells,real-time PCR and Western blot were used to evaluate the silence efficiency, and the better siRNA was used. (2)HSC-T6 cells were divided into four groups: Negative control group, siRNA-Smad3 transfection group, siRNA-Smad3+IGFBPrP1 group and IGFBPrP1 group. The better siRNA was chosen to transfect into HSC-T6 cells. The protein expressions of Smad3, fibronectin and Collagen Ⅰ were evaluated by Western blot. Results (1)siRNA2-Smad3 inhibited Smad3 gene expression stronger than another siRNA. (2)After transfection of siRNA2-Smad3, the protein expression of Smad3 was significantly decreased compared to the negative control group(P＜0.01). The protein expression of fibronectin and Collagen Ⅰ in IGFBPrP1 stimulating HSCs treated with siRNA2-Smad3 were significantly decreased compared to that in IGFBPrP1 stimulating HSC without siRNA2-Smad3 (P ＜0. 01 ).Conclusion IGFBPrP1 induces the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway.     

胰岛素和胰岛素样生长因子及其结合蛋白与结直肠癌发生的关系

目的 探讨血清胰岛素、胰岛素样生长因子(IGF-1)、IGF结合蛋白(IGFBPs)及体质量指数(BMI)、腰臀围比(WHR)的变化及与结直肠癌发生、发展的关系.方法 检测对象为2006年6月至2007年10月间住院收治和门诊复查的结直肠癌患者615例(术前检测244例,术后371例)和健康对照者150例.采用酶联免疫吸附法检测血清胰岛素、IGF-1和IGFBPS水平.结果 结直肠癌患者术前血清胰岛素、IGF-1水平和IGF-Ⅰ/IGFBP-3比值与健康对照组、术后患者比较,均明显升高,IGFBP-3水平明显降低,差异均有统计学意义(P<0.05,P<0.01).结直肠癌术后未发生转移者与有肝或腹腔远处转移者胰岛素、IGF-1、IGFBP-1、IGFBP-3、IGF-1/IGFBP-3比较.差异均无统计学意义(P>0.05).结直肠癌患者WHR明显高于健康对照组(P<0.01和P<0.05):而BMI与健康对照组比较,差异无统计学意义(P>0.05).结肠癌患者WHR、BMI与胰岛素水平、IGF-1/IGFBP-3比值呈正相关(P<0.01,P<0.05),与IGFBP-3呈负相关(P<0.01,P<0.05);直肠癌患者WHR与血清瘦素、胰岛素水平及BMI与血清IGFBP-1水平均呈正相关(P<0.05),与其他无相关性(P>0.05).结论 胰岛素、IGF-1水平和IGF-Ⅰ/IGFBP-3比值升高及IGFBP-3水平降低,可能与结直肠癌的发生有关,但与肿瘤转移与否无关,中心性肥胖是结肠癌发生的危险因素之一.     

胰岛素和胰岛素样生长因子及其结合蛋白与结直肠癌发生的关系

目的 探讨血清胰岛素、胰岛素样生长因子(IGF-1)、IGF结合蛋白(IGFBPs)及体质量指数(BMI)、腰臀围比(WHR)的变化及与结直肠癌发生、发展的关系.方法 检测对象为2006年6月至2007年10月间住院收治和门诊复查的结直肠癌患者615例(术前检测244例,术后371例)和健康对照者150例.采用酶联免疫吸附法检测血清胰岛素、IGF-1和IGFBPS水平.结果 结直肠癌患者术前血清胰岛素、IGF-1水平和IGF-Ⅰ/IGFBP-3比值与健康对照组、术后患者比较,均明显升高,IGFBP-3水平明显降低,差异均有统计学意义(P<0.05,P<0.01).结直肠癌术后未发生转移者与有肝或腹腔远处转移者胰岛素、IGF-1、IGFBP-1、IGFBP-3、IGF-1/IGFBP-3比较.差异均无统计学意义(P>0.05).结直肠癌患者WHR明显高于健康对照组(P<0.01和P<0.05):而BMI与健康对照组比较,差异无统计学意义(P>0.05).结肠癌患者WHR、BMI与胰岛素水平、IGF-1/IGFBP-3比值呈正相关(P<0.01,P<0.05),与IGFBP-3呈负相关(P<0.01,P<0.05);直肠癌患者WHR与血清瘦素、胰岛素水平及BMI与血清IGFBP-1水平均呈正相关(P<0.05),与其他无相关性(P>0.05).结论 胰岛素、IGF-1水平和IGF-Ⅰ/IGFBP-3比值升高及IGFBP-3水平降低,可能与结直肠癌的发生有关,但与肿瘤转移与否无关,中心性肥胖是结肠癌发生的危险因素之一.     

胰岛素和胰岛素样生长因子及其结合蛋白与结直肠癌发生的关系

目的 探讨血清胰岛素、胰岛素样生长因子(IGF-1)、IGF结合蛋白(IGFBPs)及体质量指数(BMI)、腰臀围比(WHR)的变化及与结直肠癌发生、发展的关系.方法 检测对象为2006年6月至2007年10月间住院收治和门诊复查的结直肠癌患者615例(术前检测244例,术后371例)和健康对照者150例.采用酶联免疫吸附法检测血清胰岛素、IGF-1和IGFBPS水平.结果 结直肠癌患者术前血清胰岛素、IGF-1水平和IGF-Ⅰ/IGFBP-3比值与健康对照组、术后患者比较,均明显升高,IGFBP-3水平明显降低,差异均有统计学意义(P<0.05,P<0.01).结直肠癌术后未发生转移者与有肝或腹腔远处转移者胰岛素、IGF-1、IGFBP-1、IGFBP-3、IGF-1/IGFBP-3比较.差异均无统计学意义(P>0.05).结直肠癌患者WHR明显高于健康对照组(P<0.01和P<0.05):而BMI与健康对照组比较,差异无统计学意义(P>0.05).结肠癌患者WHR、BMI与胰岛素水平、IGF-1/IGFBP-3比值呈正相关(P<0.01,P<0.05),与IGFBP-3呈负相关(P<0.01,P<0.05);直肠癌患者WHR与血清瘦素、胰岛素水平及BMI与血清IGFBP-1水平均呈正相关(P<0.05),与其他无相关性(P>0.05).结论 胰岛素、IGF-1水平和IGF-Ⅰ/IGFBP-3比值升高及IGFBP-3水平降低,可能与结直肠癌的发生有关,但与肿瘤转移与否无关,中心性肥胖是结肠癌发生的危险因素之一.     

Prognostic value of insulin-like growth factor 1 and insulin-like growth factor binding protein 3 blood levels in breast cancer

High circulating insulin-like growth factor 1 (IGF-1) levels are firmly established as a risk factor for developing breast cancer, especially estrogen positive tumors. The effect of circulating IGF-1 on prognosis once a tumor is established is unknown. The authors explored the effect of IGF-1 blood levels and of it's main binding protein, IGFBP-3, on overall survival and occurrence of second primary breast tumors in breast cancer patients, as well as reproductive and lifestyle factors that could modify this risk. Patients were accrued from six hospitals in the Netherlands between 1998 and 2003. Total IGF-1 and IGFBP-3 were measured in 582 plasma samples.No significant association between IGF-1 and IGFBP-3 plasma levels and overall survival was found. However, in a multivariate Cox regression model including standard prognostic variables high IGF-1 levels were related to worse overall survival in patients receiving endocrine therapy (HR = 1.37, 95% CI: 1.11, 1.69, P 0.004). These data at least indicate that higher IGF-1 levels, and as a consequence most likely IGF-1-induced signaling, are related to a less favorable overall survival in breast cancer patients treated with endocrine therapy. Interventions aimed at reducing circulating levels of IGF-1 in hormone receptor positive breast cancer may improve survival.     

Circulating levels of insulin-like growth factor binding protein 3 (IGFBP-3) and insulin-like growth factor I (IGF-I) in perimenopausal women

The study investigated possible menopause-related changes in circulating insulin-like growth factor binding protein 3 (IGFBP-3) levels and their relationship with insulin-like growth factor I (IGF-I) plasma levels. Forty-three healthy women, aged 45–55 years, were studied (22 premenopausal and 21 postmeno-pausal, matched for age and body mass index); in all subjects plasma IGF-I and IGFBP-3 levels were measured by radioimmunoassay. No difference was found between mean IGFBP-3 plasma levels in the two groups studied (premenopausal 3.42±0.49 v postmenopausal 3.46±0.58 mg/l), while mean IGF-I levels were significantly lower in postmenopausal as compared with premenopausal women (136.7±37.86 v 175.7±51.91 ng/ml,p<0.02). Multiple regression analysis showed no significant effect of age, body mass index and years since menopause on IGFBP-3 levels; however, considering the IGF-I/IGFBP-3 ratio as a possible parameter of circulating free somatomedin C, an inverse correlation was found with years since menopause (n=43,r=–0.499,p<0.001). We conclude that lack of oestrogen induces different effects on circulating IGF-I and IGFBP-3, possibly reflecting a real decrease in IGF-I activity.     

肿瘤坏死因子-α对肝星状细胞形态及表达结缔组织生长因子的影响

目的 通过观察肿瘤坏死因子（TNF）-α对肝星状细胞（HSC）形态和结缔组织生长因子（CTGF）表达的影响，探讨TNF—α在肝纤维化中的作用机制。方法 采用体外培养大鼠肝星状细胞，加入TNF-α和TGF-β1，透射电镜观察肝星状细胞的形态学改变并应用逆转录聚合酶链反应（RT-PCR）技术检测不同处理组HSC中CTGF的表达。结果 TNF-α、TGF—β1均诱导HSC中CT-GF表达；10μg／L浓度的TNF-α作用6、24和48h后，检测到CTGFmRNA表达，而TGF-β1在1μg/L浓度作用4h后，即可诱导HSC中CTGFmRNA表达。结论 TNF-α诱导HSC中CTGF的表达可能参与早期肝纤维化的形成。     

The multi-functional role of insulin-like growth factor binding proteins in bone

The insulin-like growth factor (IGF) system is an important regulator of bone formation. The IGFs (IGF-I and IGF-II) are the most abundant growth factors produced by bone, and are regulated by their six high affinity binding proteins (IGFBPs). The IGFBPs are produced by osteoblasts and are responsible for transporting the IGFs and extending their half-lives. In general, IGFBP-1, -2, -4, and -6 inhibit and IGFBP-3 and –5 stimulate osteoblast function. IGFBP-4 and -5 are the most abundant IGFBPs produced by osteoblasts, and therefore they are the primary focus of this review. IGFBP-5 is an important stimulator of bone formation and may also function independently of IGFs. IGFBP-4 inhibits osteoblast function by sequestering IGF and preventing it from binding to its receptor. This review focuses on the specific IGF-dependent and IGF-independent roles of the IGFBPs in bone formation, as well as their potential mechanisms of action. In addition, discussion of the regulation of the IGFBPs by post-translational modification (i.e., proteolysis) has been included. Studies on the regulation of production and actions of IGFBPs suggest that the IGFBP system in bone is pleiotropic and capable of serving multiple effector inputs from systemic and local sources.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

Increased apoptosis and decreased proliferation of colorectal cancer cells using insulin-like growth factor binding protein-4 gene delivered locally by gene transfer

OBJECTIVE: Insulin-like growth factor (IGF)-I induces proliferation of transformed cells. Its binding proteins (IGFBP) are involved in local regulation of IGF. This study assessed the effects of overexpression of IGFBP-4 on the development of cancer in vivo. METHOD: Nude mice were subcutaneously inoculated with HT-29 colorectal cancer cells (3 x 10(6)). When the tumour became visible (1 week after inoculation), animals received either 150 microg of mammalian expression vector containing IGFBP-4 cDNA or vector alone (n = 6 each) by peritumoural injection. Tumour size was measured during the growth. After 3 weeks of IGFBP-4 induction, animals were killed and tumour tissue samples were collected for examining the level of IGFBP-4 expression. Tumour mitotic activities were determined by counting numbers of mitotic cells on the tissue section. Apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUDP nick end labelling assay. RESULTS: Following IGFBP-4 treatment, tumour showed large necrotic areas, significantly increased numbers of apoptotic cells (36.67 +/- 7.36 vs 7.07 +/- 1.91, P < 0.01 vs control), decreased cells undergoing mitosis (2.31 +/- 0.32 vs 3.61 +/- 0.27, P < 0.01 vs control) and higher expression of IGFBP-4 (P < 0.05 vs control). CONCLUSION: IGFBP-4 gene transfer increased apoptosis and decreased mitosis, but tumour volume was not significantly altered possibly due to cellular debris filling the centre of tumours.     

Insulin-like growth factor binding protein-3 protease activity in the urine of children with chronic renal failure

The insulin-like growth factors, IGF-1 and IGF-II, are polypeptides that potentiate cellular growth. In addition to binding to specific cell surface receptors, the IGFs bind with high affinity to a family of proteins, the insulin-like growth factor binding proteins (IGFBPs). Serum and urine IGFBP patterns are altered in individuals with chronic renal failure (CRF). We recently reported that the urinary IGFBP pattern of CRF patients is unique for increased insulin-like growth factor binding protein-1 (U-IGFBP-1) levels. In this study, we used western ligand blotting (WLB), western immunoblotting (WIB), and radioimmunoassay (RIA) to further evaluate serum and urine IGFBP profiles of children with CRF (n=14). Five patients with CRF displayed decreased serum IGFBP-3 profiles by WLB. Serum IGFBP-3 WIB profiles were remarkable for 30- and 20-kDa fragments of IGFBP-3 not seen in control serum. Serum IGFBP-3 levels, as determined by RIA, were slightly elevated. Serum levels of IGFBP-2 also were increased, although not at a level reaching statistical significance. WLB of CRF urine revealed a large increase in U-IGFBP-1 and a complete absence of urinary IGFBP-3. Recent studies of serum from pregnant women and seminal plasma have demonstrated a similar absence of intact IGFBP-3, due to the presence of a specific IGFBP-3 protease. To evaluate whether an IGFBP-3 protease accounts for the absence of intact U-IGFBP-3 in children with CRF, urine and serum samples from individuals with CRF and controls were tested. An IGFBP-3 protease assay using concentrated urine revealed the presence of two distinct proteases found only in the CRF urines. Serum IGFBP-3 protease activity was no greater in patients with CRF than in controls. We conclude that the decrease in intact urinary IGFBP-3 is due to proteolysis by an IGFBP-3 protease.     

特异性siRNA抑制肝星状细胞Smad2表达

目的构建Smad2特异性siRNA表达克隆,探讨其在肝星状细胞(HSCs)中是否能够有效地抑制Smad2的表达。方法利用生物软件进行Smad2mRNA二级结构模拟,选择合适的靶位点,使用pBSKU6载体表达Smad2特异性siRNA,在体外转染HSC鄄T6细胞株,应用RT鄄PCR和Western鄄blot技术检测Smad2mRNA和蛋白的表达情况。结果成功构建siRNA表达克隆,转染后Smad2在基因和蛋白的表达水平均受到了明显抑制。在构建成功的4个克隆中,CR4抑制mRNA表达最为显著,达90%以上;抑制蛋白表达率也达到80%左右。同时CR1和CR4能有效地下调HSC分泌Ⅲ型胶原。结论在体外实验中,载体表达的特异性siRNA可有效地抑制HSCs中Smad2的表达,并可对激活的HSCs分泌细胞外基质产生有益的影响。