肌腱细胞类胰岛素生长因子-1受体密度分析

为了了解类胰岛素生长因子１（ＩＧＦ１）受体在不同代次体外培养的肌腱细胞的分布，同时观察同一肌腱细胞周期中不同亚时相的ＩＧＦ１受体数目的差别，采用免疫组化和流式细胞仪分析法，对体外培养的原代、第６代和第１３代肌腱细胞的ＩＧＦ１受体密度进行了分析，并以第６代肌腱细胞为对象，进行了同一细胞周期不同亚时相的ＩＧＦ１受体数目差别分析。结果表明，ＩＧＦ１受体在体外培养的原代、第６代和第１３代肌腱细胞的密度大体相同，而在同一细胞周期中分裂前期及分裂期（Ｇ２Ｍ期）的受体数目比ＤＮＡ合成前期（Ｇ１期）的受体数目多（Ｐ＜０．０１）。提示，在肌腱细胞培养的传代过程中和在同一细胞周期的不同亚时相，肌腱细胞均维持一个相对稳定的ＩＧＦ１受体密度。为组织工程人工肌腱的构建和肌腱细胞生长的调控提供了理论基础     

类胰岛素生长因子—1及其受体mRNA在培养肌腱细胞内的表达

为了进一步探讨类胰岛素生长因子－１（ＩＧＦ－１）受体系统在培养肌腱细胞群体生命活动中的变化，采用地高辛标记的ＩＧＦ－１及其受体基因的寡核苷酸探针，对原代、第６代及第１３代肌腱细胞ＲＮＡ进行杂交，探测ＩＧＦ－１及其受体在上述细胞中的ｍＲＮＡ表达。结果发现，上述三种细胞均表达ＩＧＦ－１受体ｍＲＮＡ；只有原代和第６代细胞表达ＩＧＦ－１ｍＲＮＡ，而第１３代细胞不表达ＩＧＦ－１ｍＲＮＡ。提示，ＩＧＦ－１ｍＲＮＡ不表达可能是导致第１３代肌腱细胞增殖能力下降的原因之一。     

类胰岛素生长因子—1作用下肌腱细胞的周期改变

为了明确类胰岛素生长因子－１（ＩＧＦ－１）促进肌腱细胞生长的作用机制，以便进一步探索调控肌腱细胞生长的手段，采用体外培养的第６代肌腱细胞，加入ＩＧＦ－１共同培养后，与对照组一起，用流式细胞技术进行细胞周期亚时相的定量分析。结果表明，实验组的ＤＮＡ合成前期（Ｇ１期）时间、ＤＮＡ合成期（Ｓ期）时间和分裂前期及分裂期（Ｇ２Ｍ期）时间分别为１１．８，２１．４和６．８小时；而对照组的时间分别为２５．６，２２．６和２１．８小时。ＩＧＦ－１使肌腱细胞的Ｇ１期和Ｇ２Ｍ期所需要的时间大大缩短。说明ＩＧＦ－１对肌腱细胞生长的促进作用是通过加快Ｇ１期和Ｇ２Ｍ期的进程实现的     

TGFβ1及其I、II型受体在肾癌中的表达

目的探讨肾癌中转化生长因子β１（ＴＧＦβ１）及其Ｉ、Ｉ型受体（ＴＧＦβＲＩ、ＴＧＦβＲＩ）表达及其意义。方法采用免疫组化技术（ＳＰ法）对４６例肾癌,１１例正常肾组织ＴＧＦβ１、ＴＧＦβＲＩ、ＲＩ进行检测,结合临床资料进行分析。结果肾癌组ＴＧＦβ１表达量较正常肾组高（Ｐ＜０．０５）;ＴＧＦβＲＩ在肾癌组及正常肾组均表达;ＴＧＦβＲＩ在肾癌中阳性率较正常肾阳性率低（Ｐ＜０．０１）,ＴＧＦβＲＩ在高分期,高分级肾癌中表达阳性率较低分期、低分级肾癌低（Ｐ＜０．０５）;ＴＧＦβ１表达量低及ＴＧＦβＲＩ表达阴性肾癌者预后差。结论ＴＧＦβ１对肾癌是一种重要负性调节因子,可抑制肾癌进展,ＴＧＦβＲＩ缺失使ＴＧＦβ系统完整性受到破坏,ＴＧＦβ１失去负性调节作用。ＴＧＦβ１及ＴＧＦβＲＩ表达可做为判断肾癌预后的指标之一     

IGF-Ⅰ受体反义寡核苷酸对胰腺癌生长的抑制作用

目的　观察 Ｉ Ｇ ＦⅠ受体反义寡核苷酸对胰腺癌细胞生长的抑制作用,为反义核酸疗法在胰腺癌外科综合治疗提供依据。 方法　以免疫组织化学 Ｓ Ｐ法检测 Ｉ Ｇ Ｆ１ 受体在体外培养的胰腺癌细胞系 Ｓ Ｗ１９９０ 的表达;用脂质体包裹合成的 Ｉ Ｇ ＦⅠ受体反义寡核苷酸（ Ａ Ｓ Ｏ Ｎ） 与 Ｓ Ｗ１９９０ 细胞共培养,并接种于裸鼠体内,观察细胞增殖情况。 结果　 Ｓ Ｗ１９９０ 细胞高表达 Ｉ Ｇ ＦⅠ受体,经上述处理细胞增殖抑制率达到８００ ％ ; Ｉ Ｇ ＦⅠ受体表达抑制率为８３９ ％ 。对照组裸鼠体内全部出瘤（１０／１０ ,１００ ％ ） ,处理组出瘤率为２０ ％ （２／１０） ,二者差异有显著意义（ Ｐ＜ ００１） 。 结论　 Ｉ Ｇ ＦⅠ受体反义寡核苷酸可显著抑制胰腺癌细胞系 Ｓ Ｗ１９９０ 在体外及体内的生长,其作用是经抑制受体表达所介导。     

胰岛素样生长因子Ⅰ、Ⅱ与其受体mRNA在前列腺增生组织中表达

采用地高辛标记的ｃＲＮＡ探针行细胞原位杂交技术，对良性前列腺增生（ＢＰＨ）组织中胰岛素样生长因子及受体的基因表达情况进行了研究，结果显示：１４／１６例ＢＰＨ组织中ＩＧＦⅠｍＲＮＡ阳性表达，定位在上皮和基质组织；１１／１２例ＢＰＨ组织中，ＩＧＦⅡｍＲＮＡ阳性表达，定位基质；ＩＧＦⅠ受体ｍＲＮＡ在所检测的８例标本中均有表达，主要定位于上皮，而ＩＧＦⅡ受体ｍＲＮＡ无表达。研究证实ＩＧＦⅠ、ＩＧＦⅡｍＲＮＡ在ＢＰＨ组织中都有表达，提示与ＢＰＨ的发病有关。ＢＰＨ组织中ＩＧＦⅠ受体是ＩＧＦ发挥作用的唯一受体，ＩＧＦ通过自分泌和旁分泌方式对前列腺细胞起作用。     

增殖性肾炎患者肾小球细胞胰岛素样生长因子—1的表达及其意义

目的　探讨人类增殖性肾炎（ＰＧＮ） 肾小球中胰岛素样生长因子１（ＩＧＦ１） 的表达及其意义。方法　采用原位杂交、免疫组织化学方法检测肾活检组织中ＩＧＦ１ ｍＲＮＡ及其蛋白质表达，并分析其与患者肾小球内细胞外基质（ＥＣＭ） 增生的关系。结果　增殖性肾炎组中ＩＧＦ１ 的表达水平显著高于非增殖性肾炎组和正常组（ Ｐ＜０－０１），ＩＧＦ１ 表达水平与ＥＣＭ 增生程度呈正相关。结论　肾小球局部ＩＧＦ１ 基因异常表达参与了ＰＧＮ的发病机制。     

类胰岛素生长因子1促腱细胞生长的实验研究

探索促进肌腱细胞增殖的方法。方法：将原代人死胎肌腱细胞和第６代，第１３代冻存后复苏的人胎儿肌腱细胞进行体外培养，并在培养基中加入不同浓度的类胰岛素生长因子１。通过绘制生长曲线％３Ｈ－ＴｄＲ渗入等方法，观察ＩＧＦ－１对肌腱细胞的作用。结果：ＩＧＦ－１能使培养的肌腱细胞增殖加快，提前进入平顶期。     

Fas/APO-1 在泌尿系肿瘤细胞中的表达

为了探讨Ｆａｓ／ＡＰＯ１在泌尿系恶性肿瘤发生发展中的作用，采用免疫细胞化学方法对６种泌尿系肿瘤细胞，膀胱癌（Ｔ２４，ＥＪ，ＢＩＵ８７），肾癌（ＧＲＣ１，ＲＣＣ９４９），前列腺癌（ＰＣ３Ｍ）和一种原代培养正常肾间质成纤维细胞Ｆａｓ／ＡＰＯ１分子的表达进行了检测。结果显示原代培养正常肾间质成纤维细胞和Ｔ２４细胞系Ｆａｓ／ＡＰＯ１分子表达较强，ＰＣ３Ｍ属中等强度表达，ＲＣＣ９４９与ＢＩＵ８７属微弱表达，而ＥＪ及ＧＲＣ１则不表达。Ｆａｓ／ＡＰＯ１在泌尿系恶性肿瘤培养细胞中的表达强度偏低或完全不表达，可能参与了泌尿系恶性肿瘤的发生和发展。     

白介素1β对肾小球系膜细胞表达纤维蛋白溶解酶原激活物抑制物-1和纤维连接蛋白的影响

目的探讨白介素１β（ＩＬ１β）对人肾小球系膜细胞表达纤维蛋白溶解酶原（纤溶酶原）激活物抑制物１（ＰＡＩ１）和纤维连接蛋白（ＦＮ）的影响。方法应用Ｎｏｒｔｈｅｒｎ杂交检测体外培养的肾小球系膜细胞在ＩＬ１β刺激后ＰＡＩ１和ＦＮ的ｍＲＮＡ表达,采用纤维蛋白平板法检测系膜细胞培养上清中ＰＡＩ１的活性,利用ＥＬＩＳＡ法检测ＦＮ的水平。结果ＩＬ１β刺激组系膜细胞ＰＡＩ１和ＦＮｍＲＮＡ的表达显著增高（与对照组比,Ｐ＜００５）,细胞培养上清中ＰＡＩ１的活性和ＦＮ的水平亦明显增加（与对照组比,Ｐ＜００１）。结论ＩＬ１β可以上调系膜细胞ＰＡＩ１和ＦＮ蛋白质及ｍＲＮＡ的表达,提示ＩＬ１β可能通过抑制细胞外基质降解和增加细胞外基质合成而导致细胞外基质的积聚。     

Insulin-like growth factor-1 sustains stem cell mediated renal repair

In mice with cisplatin-induced acute kidney injury, administration of bone marrow-derived mesenchymal stem cells (MSC) restores renal tubular structure and improves renal function, but the underlying mechanism is unclear. Here, we examined the process of kidney cell repair in co-culture experiments with MSC and cisplatin-injured proximal tubular epithelial cells (PTEC). Exposure of PTEC to cisplatin markedly reduced cell viability at 4 days, but co-culture with MSC provided a protective effect by promoting tubular cell proliferation. This effect was mediated by insulin-like growth factor-1 (IGF-1), highly expressed by MSC as mRNA and protein, since blocking the growth factor's function with a specific antibody attenuated cell proliferation of PTEC. Confirming this, knocking down IGF-1 expression in MSC by small interfering-RNA also resulted in a significant decrease in PTEC proliferation and increased apoptosis. Furthermore, in the murine model of cisplatin-induced kidney injury, administering IGF-1 gene-silenced MSC limited their protective effect on renal function and tubular structure. These findings indicate that MSC exert beneficial effects on tubular cell repair in acute kidney injury by producing the mitogenic and pro-survival factor IGF-1.     

Insulin-like growth factor-1 receptors mediate infragenicular vascular smooth muscle cell proliferation in response to glucose and insulin not by insulin receptors.

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation is an early event in the pathogenesis of atherosclerosis. Insulin and glucose are known to stimulate the growth of VSMC. Cell membrane receptors play an important role in the proliferation of VSMC in response to growth factors. Insulin and insulin-like growth factor-1 (IGF-1) have demonstrated a cross reactivity for receptor binding and function. By using monoclonal antibodies directed against insulin (IRA) and IGF-1 (IGF-1RA) receptors, we attempt to further delineate the mechanism for the proliferation of VSMC in response to insulin and glucose. METHODS: Human infragenicular VSMC isolated from diabetic patients undergoing below-knee amputations were used. Cells from passages 3 to 6 were grown in serum-free media with a glucose concentrations of 0.1% or 0.2%, both with and without insulin (100 ng/mL). The baseline cell density was 4,635 +/- 329 cells/mL. IRA or IGF-1RA was added to the media, with the control group receiving neither antibody. Cells were grown in 5% CO2 at 37 degrees C for 6 days. Analysis of variance was used for statistical analysis, with P <0.05 considered significant. In addition, DNA synthesis was measured using thymidine incorporation assays in the same groups of cells receiving IRA, IGF-1RA, and no antibody. RESULTS: IGF-1RA prevented the proliferation of VSMC in response to insulin and glucose, while IRA had no effect on cell growth. There was no significant growth when IGF-1RA was added to the media, while the control group and the group receiving IRA demonstrated significant growth compared with the baseline concentration of 4,635 +/- 329 cells/mL at all concentrations of insulin and glucose. [3H]thymidine incorporation assays confirmed the cell count results. CONCLUSIONS: These results suggest that the mitogenic effects of insulin and glucose on infragenicular VSMC are due to stimulation of the IGF-1 receptor. VSMC antiproliferative strategies employing receptor blockade should be directed against the IGF-1 receptor, not the insulin receptor.     

Renal tubulointerstitial injury from ureteral obstruction in the neonatal rat is attenuated by IGF-1

BACKGROUND: The administration of insulin-like growth factor-1 (IGF-1) has been shown to ameliorate the renal injury resulting from ischemic acute renal failure. As there are a number of similarities between acute renal failure and obstructive nephropathy, we examined the effects of IGF-1 on the renal cellular response to unilateral ureteral obstruction (UUO) in the neonatal rat. METHODS: Forty-five rats were subjected to UUO or sham operation within the first 48 hours of life and received IGF-1 (2 mg/kg/day) or saline for the following three or seven days, after which kidneys were removed for study by morphometry and immunohistochemistry. To determine the effects of UUO on endogenous expression of IGF-1 and its receptor, six additional rats were subjected to UUO or sham operation, and mRNA was measured by solution hybridization. RESULTS: There was no effect of seven days of UUO on the renal expression of endogenous IGF-1 or its receptor. Moreover, seven days of exogenous IGF-1 did not improve the suppression of nephrogenesis, the delay in glomerular maturation, or the reduction in tubular proliferation induced by ipsilateral UUO. However, in the obstructed kidney, IGF-1 reduced tubular expression of vimentin, apoptosis, and tubular atrophy by 38 to 50% (P < 0.05). In addition, IGF-1 also decreased renal interstitial collagen deposition in the obstructed kidney by 44% (P < 0.05). Following three days of UUO, the administration of IGF-1 also reduced tubular apoptosis (P < 0.05), but did not alter tubular proliferation. CONCLUSIONS: IGF-1 has a profound salutary effect on the tubular and interstitial response to UUO in early development, without affecting glomerular injury or development. These results suggest that IGF-1 may have therapeutic potential in the management of congenital obstructive nephropathy.     

Inhibitory effects of nordihydroguaiaretic acid (NDGA) on the IGF-1 receptor and androgen dependent growth of LAPC-4 prostate cancer cells

BACKGROUND: Nordihydroguaiaretic acid (NDGA) is an inhibitor of the IGF-1 receptor (IGF-1R) in breast and other cancers, and concomitantly inhibits tumor growth both in cultured cells and animals. The current study evaluates the effect of NDGA on the androgen-stimulated growth of human prostate cancer cells. METHODS: LAPC-4 prostate cancer cells in tissue culture were androgen starved for 3 days, 1 nM dihydrotestosterone (DHT) and other androgens were then added for up to 7 days, and cell proliferation measured. IGF-1R protein expression was measured by Western blot, and IGF-1R mRNA expression by quantitative PCR. IGF-1R receptor kinase activation was measured by ELISA. RESULTS: After 7 days, LAPC-4 growth was doubled by 1 nM DHT. NDGA had a rapid effect to inhibit IGF-1R autophosphorylation induced by IGF-1. DHT increased the expression of IGF-1R protein and mRNA levels. Maximal IGF-1R protein levels were observed 3 days after the addition of androgen. In addition, NDGA, at 10 microM or less, inhibited DHT-induced proliferation in both cells grown in plates and cells grown in soft agar. Androgen receptor (AR) studies by FRET revealed that NDGA had no conformational effects on the AR in response to ligand. CONCLUSIONS: NDGA blocks the DHT-induced growth of LAPC-4 prostate cancer cells by several mechanisms including rapid inhibition of the IGF-1R kinase, and a dose-dependent inhibition of androgen stimulation of IGF-1R expression. Clinical studies of this agent will determine its efficacy in the setting of androgen-dependent prostate cancer.     

胰岛素样生长因子Ⅰ型受体单克隆抗体对结肠癌细胞生长的影响

目的探讨胰岛素样生长因子Ⅰ型受体（insulin-like growth factorⅠ receptor,IGF-ⅠR）在人结肠癌细胞株HT-29上的表达,以及两种胰岛素样生长因子Ⅰ型受体单克隆抗体（IGF-ⅠRMcAb）对HT-29细胞增殖的影响.方法免疫组织化学法检测HT-29的IGF-ⅠR表达,MTT法检测两种IGF-ⅠR McAb对HT-29的抑制增殖作用及诱导凋亡情况.结果人结肠癌细胞株HT-29细胞膜高表达IGF-ⅠR.IGF-ⅠR McAb能抑制HT-29细胞增殖,McAb对HT-29细胞的抑制作用随抗体浓度增大而增大.IGF-ⅠR McAb诱导HT-29凋亡,与对照组相比差异有统计学意义（P＜0.01）.结论IGF-ⅠR McAb通过阻断IGF-ⅠR抑制结肠癌细胞增殖、诱导细胞凋亡.     

胰岛素样生长因子-I对人脐带间充质干细胞增殖影响的量效关系

目的：探讨不同剂量胰岛素样生长因子-I对人脐带间充质干细胞（hUCMSCs）增殖的影响及其量效关系。方法：取第2代hUCMSCs接种于96孔板,分为7组（G1,G2…,G7）。前5组分别加入含不同剂量IGF-I（40,60,80,100,120μg/L）的体积分数为0.02的胎牛血清培养液;第6组加入体积分数为0.05的胎牛血清培养液做为阳性对照,第7组加入体积分数为0.02的胎牛血清培养液做为阴性对照,采用MTT法观察不同剂量的IGF-I对细胞增殖的影响。结果：①第3天、第6天,G1组、G2组和G7组之间差异无显著性（P〉0.05）;G3组、G4组和G5组之间差异无显著性（P〉0.05）;②第3天,G6组增殖高于G1组与G2组,低于G3组、G4组和G5组,差异有显著性（P〈0.000或〈0.006）;第4天,G6组增殖高于其余各组,差异有显著性（P〈0.000或〈0.006）。③第3天、第6天,G1组、G2组和G6组低于G3组,G4组,G5组,差异有显著性（P〈0.000）;G6组增殖高于G7组,统计分析差异有显著性（P〈0.000）。结论：外源性IGF-I对hUCMSCs增殖的促进作用,随IGF-I浓度增高而有增强趋势,100μg/L为其较适合浓度,为实现hUCMSCs体外快速扩增提供适宜的条件。     

Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro

OBJECTIVE: Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model. METHODS: Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization. RESULTS: IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks. CONCLUSION: The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.