Stimulation of DNA synthesis by epidermal growth factor in osteoblast-like cells

Summary Normal and malignant osteoblast-like cells in culture have been shown to possess specific, high affinity receptors for epidermal growth factor (EGF). In this study, the mitogenic response to EGF was examined in a clonal line of a rat osteogenic sarcoma (UMR 106) and in osteoblast-rich newborn rat calvarial cells. Twenty-four hour treatment of UMR 106 cells with EGF in doses ranging from 10⁻¹² m to 2 × 10⁻⁸ m stimulated the incorporation of [³H]thymidine and DNA synthesis in a dose-dependent manner. This short-term stimulatory effect was sustained in long-term culture with a dose-dependent increase in cell proliferation by calvarial cells. A lag period of 8 h occurred before significant stimulation of [³H]thymidine incorporation was observed. Commitment to increased incorporation of [³H]thymidine required a minimum of 6 h continuous incubation with EGF. These results establish the osteoblast as a target cell for EGF action on bone.     

Stimulation of DNA synthesis by epidermal growth factor in osteoblast-like cells

Normal and malignant osteoblast-like cells in culture have been shown to possess specific, high affinity receptors for epidermal growth factor (EGF). In this study, the mitogenic response to EGF was examined in a clonal line of a rat osteogenic sarcoma (UMR 106) and in osteoblast-rich newborn rat calvarial cells. Twenty-four hour treatment of UMR 106 cells with EGF in doses ranging from 10(-12) M to 2 X 10(-8) M stimulated the incorporation of [3H]thymidine and DNA synthesis in a dose-dependent manner. This short-term stimulatory effect was sustained in long-term culture with a dose-dependent increase in cell proliferation by calvarial cells. A lag period of 8 h occurred before significant stimulation of [3H]thymidine incorporation was observed. Commitment to increased incorporation of [3H]thymidine required a minimum of 6 h continuous incubation with EGF. These results establish the osteoblast as a target cell for EGF action on bone.     

尼古丁对兔骨髓基质干细胞增殖及向软骨细胞方向分化的影响

目的：探讨不同浓度尼古丁对单层培养的骨髓基质干细胞及向软骨方向分化的影响。方法：获取兔骨髓基质干细胞,倒置显微镜下观察细胞形态。取第3代细胞,加入不同浓度（0、1×10。、l×10。、1X10。M）的尼古丁,分别于1、4、7、14d用MTT法检测其对骨髓基质干细胞增殖的影响。取第3代细胞进行诱导分化,用逆转录聚合酶链式反应（RT—PCR）法检测Ⅱ型胶原和蛋白聚糖的表达。结果：镜下见细胞形态由圆形向梭形转化;1x100、1×10M尼古丁浓度能够促进骨髓基质干细胞的增殖,1×10。M尼古丁则会抑制骨髓基质干细胞的增殖活性。1×10^-7、1×10^-6M（特别是1×10。M）的尼古丁能够增加诱导后Ⅱ型胶原的表达,并随时间延长而增加。而1×10。M尼古丁在第7天时能促进诱导后蛋白聚糖的表达,1X10。M尼古丁则能抑制Ⅱ型胶原及蛋白聚糖mRNA表达。结论：低浓度的尼古丁能够促进骨髓基质干细胞的增殖及向软骨细胞方向分化,高浓度的尼古丁则对其有抑制作用,由此推测在软骨组织工程中,局部话时i舌号柏府厍I．f芦．士丁暑一种能白骞俚{井骨唇莆篡席干细胎．向款骨细日白．方向奔让．的有前导曲治疗方法．     

Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production.

Pulsed electromagnetic field stimulation has been used to promote the healing of chronic nonunions and fractures with delayed healing, but relatively little is known about its effects on osteogenic cells or the mechanisms involved. The purpose of this study was to examine the response of osteoblast-like cells to a pulsed electromagnetic field signal used clinically and to determine if the signal modulates the production of autocrine factors associated with differentiation. Confluent cultures of MG63 human osteoblast-like cells were placed between Helmholtz coils and exposed to a pulsed electromagnetic signal consisting of a burst of 20 pulses repeating at 15 Hz for 8 hours per day for 1, 2, or 4 days. Controls were cultured under identical conditions, but no signal was applied. Treated and control cultures were alternated between two comparable incubators and, therefore, between active coils; measurement of the temperature of the incubators and the culture medium indicated that application of the signal did not generate heat above the level found in the control incubator or culture medium. The pulsed electromagnetic signal caused a reduction in cell proliferation on the basis of cell number and [3H]thymidine incorporation. Cellular alkaline phosphatase-specific activity increased in the cultures exposed to the signal, with maximum effects at day 1. In contrast, enzyme activity in the cell-layer lysates, which included alkaline phosphatase-enriched extracellular matrix vesicles, continued to increase with the time of exposure to the signal. After 1 and 2 days of exposure, collagen synthesis and osteocalcin production were greater than in the control cultures. Prostaglandin E2 in the treated cultures was significantly reduced at 1 and 2 days, whereas transforming growth factor-beta1 was increased; at 4 days of treatment, however, the levels of both local factors were similar to those in the controls. The results indicate enhanced differentiation as the net effect of pulsed electromagnetic fields on osteoblasts, as evidenced by decreased proliferation and increased alkaline phosphatase-specific activity, osteocalcin synthesis, and collagen production. Pulsed electromagnetic field stimulation appears to promote the production of matrix vesicles on the basis of higher levels of alkaline phosphatase at 4 days in the cell layers than in the isolated cells, commensurate with osteogenic differentiation in response to transforming growth factor-beta1. The results indicate that osteoblasts are sensitive to pulsed electromagnetic field stimulation, which alters cell activity through changes in local factor production.     

人类白细胞抗原-G阳性的脐带间充质干细胞 体外诱导调节性T细胞的实验研究

目的  探讨人类白细胞抗原（HLA）-G阳性的脐带间充质干细胞在体外诱导调节性T细胞（Treg）产生的效果。方法  从新生儿脐带中分离脐带间充质干细胞,采用脂质体转染的方式将PEGFP-N1-HLA-G质粒转染到脐带间充质干细胞中,设为PEGFP-N1-HLA-G组; 转染空载体PEGFP-N1质粒的脐带间充质干细胞设为PEGFP-N1组; 相同条件下,未加入空载体的脐带间充质干细胞设为空白对照组。采用流式细胞仪检测脐带间充质干细胞标志物; 采用蛋白质免疫印迹法鉴定各组细胞HLA-G蛋白的表达; 各组细胞与健康人外周血中CD4⁺T细胞混合培养24 h和48 h后,采用流式细胞仪检测CD4⁺ CD25⁺ Foxp3⁺Treg占全部T细胞的比例。结果  脐带间充质干细胞CD45、CD34和HLA-DR呈阴性表达,CD29、CD44和CD105呈阳性表达; PEGFP-N1-HLA-G组可以表达HLA-G蛋白,与空白对照组和PEGFP-N1组比较差异均有统计学意义（均为P < 0.01）。PEGFP-N1-HLA-G组细胞在与CD4⁺T细胞混合培养24 h后,CD4⁺ CD25⁺ Foxp3⁺Treg占全部T细胞的（15.3±1.9）%,在培养48 h后,CD4⁺ CD25⁺ Foxp3⁺Treg占全部T细胞的（14.3±2.1）%,与空白对照组和PEGFP-N1组比较,差异均有统计学意义（均为P < 0.05）。结论  HLA-G基因修饰后脐带间充质干细胞能够有效地在体外诱导CD4⁺ CD25⁺ Foxp3⁺Treg的产生。     

In vitro and in vivo studies of pressurization of femoral cement in total hip arthroplasty

Improvements in cementing techniques in the absence of pressurization of the cement have led to major increases in the long-term success rate of fixation of the femoral components of cemented total hip arthroplasty (THA). The strength of the cement-bone interface is strongly related to cement intrusion into the bone. The depth of cement intrusion, in turn, is correlated with the cement-intrusion pressure. Thus, adding cement pressurization to those current techniques that have already been validated may further increase the long-term durability of fixation of the femoral component of cemented THA. To assess cement pressurization in the proximal femur for THA, the authors compared in vitro the efficacy of three existing pressurization systems (the Johnson and Johnson system [New Brunswick, NJ], the Miller system [Zimmer, Warsaw, IN], and the Zimmer system [Zimmer]) in cadaver femurs using pressure transducers and evaluated their ease and optimization for clinical use. The authors then selected one (the Zimmer system) for use in studies in vivo to quantify the actual pressures achieved in the medullary canal in vivo under surgical conditions using pressure transducers placed throughout the femoral cortex. Each of the three commercially available femoral cement pressurization systems has its own advantages and disadvantages. All three systems were shown to produce average peak cement-intrusion pressures in vitro of over 21 N/cm² (30 psi) throughout the cement mantle including, importantly, in the proximal portion of the femur. Under laboratory conditions all three systems produced adequate pressurization for optimal cement penetration into cancellous bone. Because the repeated application of pressure is more valuable than the single thrust provided by the Johnson and Johnson cement compactor, the Miller and Zimmer systems seemed preferable. Because the Miller system does not pressurize the most proximal portion of the canal (the volume occupied by the silicone conical seal) and because the proximal portion of the cement mantle is important to longterm fixation of the femoral component, the Zimmer system was selected for the in vivo study. The cement-intrusion pressures in the proximal portion of the femurs obtained in vivo in the operating room during the 10 primary and 5 revision THAs had a mean value from all 15 patients of 19 ± 8 N/cm² (27 ± 11 psi). The primary cases had a significantly higher intrusion pressure (22 ± 7 N/cm² [32 ± 10 psi]) than the revision cases (13 ± 6 N/cm² [19 ± 9 psi]). In a prior study from the authors' laboratory using bovine cancellous bone, which is more dense than human cancellous bone, cement pressures of 14 N/cm² (20 psi) were shown to produce a cement-intrusion depth of 5 mm. An intrusion depth of 3–4 mm has been shown to be optimal for the strength of the cement-bone interface. Therefore, 10 N/cm² (15 psi) is likely to be adequate to achieve optimal cement-intrusion depth in humans. The results of this study show that a pressure of 10 N/cm² (15 psi) was obtained in 9 of 10 primary cases and 3 of 5 revision cases.     

尿毒症毒素硫酸吲哚酚在体外对人肾小球足细胞骨架的损伤及其机理研究

目的硫酸吲哚酚(IS)是一种公认的结合蛋白质的肠源性尿毒症毒素,在慢性肾脏病患者体内蓄积可导致和促进慢性肾脏病的发生和发展。本实验探索了IS是否能引起人肾小球足细胞骨架的损伤及其可能的机理。 方法以体外培养的人肾小球足细胞系作为研究对象。采用台盼蓝拒染法、MTT法检测足细胞活力;采用免疫荧光法检测足细胞骨架纤维状肌动蛋白(F－actin)、骨架蛋白Synaptopodin改变;Western印迹法分析骨架蛋白Synaptopodin蛋白水平;RT－qPCR法检测Synaptopodin mRNA表达;琼脂糖凝胶电泳法分析足细胞内蛋白激酶A(PKA)活性。 结果IS使足细胞F－actin、Synaptopodin蛋白荧光减弱;IS还下调Synaptopodin的mRNA (P<0.01)。IS刺激使PKA磷酸化增多;PKA通路阻断剂H89可以上调Synaptopodin的蛋白及mRNA表达(P＝0.002)。 结论IS可下调足细胞骨架F－actin、Synaptopodin表达,PKA信号通路可能部分参与了Synaptopodin表达的调节,本研究结果为防治和干预CKD发生发展提供了新的思路和潜在的治疗靶点。     

Comparison of the type-2 insulin-like growth factor receptor in normal osteoblasts and osteosarcoma-derived osteoblast-like cells

Insulin-like growth factor-II is known to stimulate the proliferation and differentiation of osteoblasts in part through activation of the type-2 insulin-like growth factor receptor. The present study examined the type-2 insulin-like growth factor receptors of three normal osteoblast-like cells and three osteosarcoma-derived osteoblast-like cells (OGA, SU, and IMAI) from humans. [¹²⁵I]insulin-like growth factor-II was used for the binding studies. All of the cell types had high affinity binding sites for insulin-like growth factor-II (dissociation constants [Kd] ? 1 nM). The concentration of these sites was 10 to 24-fold higher in normal osteoblasts than in the osteosarcoma cells studied. Unlabeled insulin-like growth factor-II inhibited the binding of [¹²⁵I]insulin-like growth factor-II to the cells in a dose-dependent manner; however, unlabeled insulin-like growth factor-I and insulin were less effective. Covalent crosslinking of insulin-like growth factor-II binding sites gave molecular mass estimates of M_r 250,000 in human osteoblast cells, 250,000 and 130,000 in OGA cells. 240,000 in SU cells, and 250,000 and 130,000 in IMAI cells. Unlabeled insulin-like growth factor-II inhibited all affinity labeling. In Northern blot analysis, the type-2 insulin-like growth factor receptor mRNA of normal osteoblasts was seen in greater abundance than it was in osteosarcoma cells. These results indicate that the numbers of type-2 insulin-like growth factor receptors differ between normal and transformed osteoblasts and that the differential expression of the receptor may be due to the differentiation of osteoblasts.