抗HBsAg-碱性磷酸酶双功能抗体分子的构建

为构建带有碱性磷酸酶活性的双功能基因工程抗体, 用PCR方法克隆大肠杆菌碱性磷酸酶基因, 通过酶切分析和DNA序列测定核实后,将其重组到抗乙肝表面抗原(HBsAg) Fab段的Fd羧基端,构建重组融合蛋白表达载体pHBFAP, 转化大肠杆菌XL1-Blue, 经异丙基硫代-β-D-半乳糖苷诱导表达后, 采用ELISA法检测到培养上清中存在与HBsAg的结合活性和碱性磷酸酶的催化活性, 显示抗HBsAg-碱性磷酸酶双功能抗体分子在大肠杆菌中获得了表达.     

珍珠质自然涂层钛种植体表面对MC3T3E1成骨样细胞的作用

为了研究珍珠质自然涂层钛种植体表面的体外生物相容性,将珍珠质自然涂层的钛片与MC3T3E1成骨样细胞复合培养以观察细胞的生长、增殖和分化.分别以羟基磷灰石涂层钛片和没有涂层的纯钛片作为对照组,以MC3T3E1细胞单纯培养作为空白组,分别培养3天,5天和7天,通过倒置相差显微镜和扫描电镜观察细胞生长情况,流式细胞技术检测细胞增殖活性,金氏比色法检测碱性磷酸酶(ALP)活性以及蛋白质印迹(Western blotting)法测定转化生长因子-β1(TGF-β1)表达水平.结果发现,细胞在珍珠质周围能形成良好附着,在其表面生长丰满.细胞培养第3天,第5天和第7天时,珍珠质表面的细胞增殖指数分别为(35.9±2.5)%、(69.7±3.3)%和(58.2±2.6)%,ALP活性分别为(6.123±2.917)U/g、(17.486±1.986)U/g和(23.987±1.372)U/g.第5天和7天时,实验组的细胞增殖指数、ALP活性和TGF-β1表达水平显著高于对照组和空白组(P<0.05).珍珠质自然涂层钛表面有利于MC3T3E1细胞的生长、增殖和分化,表明了珍珠质涂层能提高种植体表面的生物相容性,有可能会促进种植后的骨整合.     

Endo180半胱氨酸富集区与I型胶原蛋白C末端分子的体外相互作用

I型胶原蛋白(type I collagen)是由2条α1链和1条α2链构成的纤维状异源三聚体.它是构成细胞外基质最主要的成分. I型胶原蛋白和内吞胶原蛋白受体Endo180(Endocytic 180)之间的相互作用在细胞基质的粘附和降解过程中起着关键性作用. 但内吞胶原蛋白受体Endo180与胶原蛋白的结合区域尚未有相关的报道.本研究在体外通过酶联免疫吸附、蛋白免疫沉淀和Western印迹等多种定量分析实验发现,用碱性磷酸酶（alkaline phosphatase,AP）标记I型胶原蛋白C末端（氨基酸1151～1464）后形成的分泌型三聚体融合蛋白AP-Coll-S, 可与用抗体恒定区（antibody constant fragment, Fc）标记的Endo180的半胱氨酸富集区（CysR) 结合. 从而表明,AP-Coll-S和CysR-Fc之间存在相互作用. 本研究结果为进一步研究胶原蛋白和其受体Endo180的分子间相互作用提供新依据.     

Effects of Ulmus davidiana planch on mineralization, bone morphogenetic protein-2, alkaline phosphatase, type I collagen, and collagenase-1 in bone cells

Summary Ulmus davidiana Planch (Ulmaceae) (UD) long has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation, and bone among other functions. The herbal medicine also is being used in Oriental medicine to treat osteoporosis. In a preliminary study, treatment of osteoclasts containing long bone cells with the water extract of UD bark prevented the intracellular maturation of cathepsin K (cat K), and thus, it was considered that UD is a pro-drug of a potent bone-resorption inhibitor. To further clarify the role of UD in ossification, we investigated the effects of UD on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, we assessed the effects of UD on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. UD enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the UD was observed at relatively low doses (significant at 5–50 μg/ml and maximal at 50 μg/ml). Northern blot analysis showed that UD (100 μg/ml) increases in bone morphogenic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. UD slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that UD has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that is could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Crystal structure of alkaline phosphatase from the Antarctic bacterium TAB5

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.