肿瘤表遗传学（六）

第六讲 染色质重塑 真核生物染色质是一切遗传学过程的物质基础，是多层次、高度可塑性的核蛋白多聚体。染色质构型局部和整体的动态改变，是基因功能调控的关键因素。染色质重塑（chromatin remodeling）是以染色质构型改变为基础的表遗传学机制，     

SRS－淋巴瘤小鼠淋巴细胞染色质理化性质的研究

本实验对正常及ＳＲＳ－淋巴瘤小鼠淋巴细胞染色质进行了化学组分、ＤＮａｓｅⅠ有限度消化、染色质附着蛋白释放，热变性的对比分析。结果表明：（１）ＳＲＳ－淋巴瘤细胞染色质的非组蛋白（ｎｏｎ－ｈｉｓｔｏｎｅｃｈｒｏｍｏｓｏｍａｌｐｒｏｔｅｉｎｓ，ＮＨＣＰ）和ＲＮＡ高于正常细胞，提示随着肿瘤的发生染色质转录活性和与转录相关的ＮＨＣＰ呈升高趋势；（２）ＳＲＳ－淋巴瘤细胞染色质的ＤＮａｓｅⅠ消化率明显高于正常淋巴细胞，提示肿瘤细胞含有较多优先被核酸酶消化的活性染色质；（３）ＳＲＳ－淋巴瘤细胞较正常淋巴细胞蛋白释放量明显升高，提示参与活性染色质结合的ＮＨＣＰ与染色质活性位点及转录活性有密切相关；（４）ＳＲＳ－淋巴瘤细胞染色质比正常淋巴细胞增色效应明显，提示肿瘤细胞比正常细胞染色质结构疏松。     

SRS－淋巴瘤小鼠淋巴细胞活性染色质抗原特异性研究

本文用ＳＤＳ－ＰＡＧＥ分离正常及ＳＲＳ－淋巴瘤小鼠淋巴细胞染色质ＮＨＣＰ，发现ＳＲＳ－淋巴瘤细胞染色质存在１３０Ｋｄ，１２０Ｋｄ，１１０Ｋｄ，９４Ｋｄ，５７Ｋｄ肿瘤相关性ＮＨＣＰ两种染色质经ＤＮａｓｅⅠ有限度消化，优先被消化的活性染色质部位释放的蛋白不同。ＳＲＳ－淋巴瘤细胞消化后释放的ＮＨＣＰ多于正常淋巴细胞，并具时相性差异。经多种免疫学方法证实，ＳＲＳ－淋巴瘤ＮＨＣＰ具有肿瘤相关性，它的ＦＬＩＳＡ结果远高于正常淋巴细胞，ＳＲＳ－淋巴瘤染色质有限度消化时ＮＨＣＰ释放的时相性变化，在ＥＬＩＳＡ中得到证实。免疫印迹结果表明：经正常染色质重吸收后的抗ＳＲＳ－淋巴瘤染色质ＮＨＣＰ的抗血清与正常淋巴细胞染色质不显带，而与ＳＲＳ－淋巴瘤细胞染色质显出多条阳性带，这些肿瘤相关性ＮＨＣＰ存在于不同处理的样品中。以上结果提示：ＳＲＳ－淋巴瘤相关性ＮＨＣＰ位于活性染色质部位，它们可能是参与肿瘤基因表达的调控蛋白一反式作用因子。     

关于X染色质

Ｘ染色质是一呈异固缩状态、失去转录活性的Ｘ染色体，新近的研究发现Ｘ染色质上的基因部分失活而部分仍有活性；认为Ｘ染色质上有非赖昂化片段，并引起不完全剂量补偿作用，在生殖细胞中失活的Ｘ染色体将要复活，对Ｘ染色质的研究发现其本身有一失活中心，并且这一失活中心由另一失活诱导基因诱发引起失活，进而弥散地引起其它基因失活，这就是Ｘ染色质产生的机理，也有人认为失活信号不是染色体本身而来自外部。另外Ｘ染色质上的一     

SRS—淋巴瘤小鼠淋巴细胞染色质理化性质的研究

本实验对正常及ＳＲＳ－淋巴瘤小鼠淋巴细胞染色质进行了化学组分，ＤＮａｓｅ Ｉ有限度消化，染色质附着蛋白释放，热变性的对比分析。结果表明：（１）ＳＲＳ－淋巴瘤细胞染色质的非组蛋白和ＲＮＡ高于正常细胞，提示随着肿瘤的发生染色质转录活性和与转录相关的ＮＨＣＰ呈升高趋势；（２）ＳＲＳ－淋巴瘤细胞染色质的ＤＮａｓｅ Ｉ消化率明显高于正常淋巴细胞，提示肿瘤细胞含有较多优先被核酸酶消化的活性染色质（３）ＳＲＳ－     

9号染色体异染色质区的变异分析

目的分析疑有染色体异常个体的9号染色体异染色质区的变异。方法采集疑有染色体异常的3075名个体静脉血，其中男性1653例，女性1422例，以1515名正常人作对照，培养其淋巴细胞进行染色体核型分析。结果疑有染色体异常个体的9号染色体异染色质区的变异率为5．56％，而正常对照组9号染色体异染色质区的变异率仅为1．32％，9号染色体异染色质区的变异包括9qh＋、9qh-及inv（9）。结论疑有染色体异常个体的9号染色体异染色质区的变异率高于正常人4．2倍（P＜0．01），这说明9号染色体异染色质区的变异可能参与一些染色体病的发生。     

9号染色体异染色质区变异的遗传效应分析

人类染色体正常情况有46条，其区带排列有序。在配子结合时，同源染色体相应位点一一对应。在异常情况时，染色体可发生易位、倒位、重复、插入、异染色质区变异等结构异常和数目异常，由于剂量效应和位置效应，导致染色体病发生。在异染色质区变异的染色体中，9号染色体异染色质区的变异是最常见的。现就我院遗传中心5008例遗传咨询者外周血染色体检查发现的99例9号染色体异染色质区改变的遗传效应报道如下。     

SRS－淋巴瘤细胞染色质非组蛋白的研究

制备ＳＬＮＩＬＣ和ＮＭＬＣ染色质，用６％～１２％梯度胶的ＳＤＳ－ＰＡＧＥ分离ＳＬＭＬＣ和ＮｈＬＣ染色质ＮＨＣＰ。结果表明：ＳＬＭＬＣ染色质含有１３０ＫＤ、１２０ＫＤ、１１０ＫＤ、９４ＫＤ、５７ＫＤ、３７ＫＤ相关的ＮＨＣＰ，它们分别位于不同处理的ＳＬＮＩＬＣ染色质样品中；免疫化学分析显示：ＮＭＬＣ与重吸收后的抗ＳＬＭＬＣ抗血清不产生阳性带，而经不同处理的ＳＬＭＬＣ染色质样品与这种抗血清仍显阳性条带。这些结果表明：ＳＬＭＬＣ和ＮＭＩＬＣ染色质ＮＩＩＣＰ存在差别，这些相关的ＮＨＣＰ可能与ＳＲＳ－淋巴瘤的发生有关。     

染色体C显带检测的方法改进

染色体C显带技术是使结构异染色质或高度重复的DNA区着色。在人类染色体中，这些区域位于着丝粒区和Y染色体的长臂上。在优良的C带标本上，结构异染色质应当是着色很深的，而常染色质只呈现出染色体的一般外貌，我们在刘权章主编的《人类染色体方法学》介绍的方法基础上，做了几点改进，取得了比较满意的效果，现介绍如下。     

SRS—淋巴瘤小鼠淋巴细胞活性染色质抗原特异性研究

本文用ＳＤＳ－ＰＡＧＥ分离正常及ＳＲＳ－淋巴瘤小鼠淋巴细胞染色质ＮＨＣＰ，发现ＳＲＳ－淋巴瘤细胞染色质存在１３０Ｋｄ，１２０Ｋｄ，１１０Ｋｄ，９４Ｋｄ，５７Ｋｄ肿瘤相关性ＮＨＣＰ。两种染色质经ＤＮａｓｅ Ｉ有限度消化，优先被消化的活性染色质部位释放的蛋白不同。ＳＲＳ－淋巴瘤细胞消化后释放的ＮＨＣＰ多于正常淋巴细胞，并具时相性差异。经多种免疫学方法证实，ＳＲＳ－淋巴瘤ＮＨＣＰ具有肿瘤相关性，它的ＥＬ     

PTHrP and PTH/PTHrP receptor 1 expression in odontogenic cells of normal and HHM model rat incisors

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). We examined PTHrP and its receptor (PTHR1) expression patterns in odontogenic cells in normal and HHM model rat incisors. Nontreated nude rats serving as the normal control and HHM model rats produced by implantation of PTHrP-expressing tumor (LC-6) cells were prepared. HHM rats fractured its incisor, and histopathologically, restrict population of odontoblasts showed findings classified as "shortening of high columnar odontoblasts" and "dentin niche." The incisors were immunostained against PTHrP and PTHR1. In normal rats, PTHrP and PTHR1 colocalized in ameloblasts, cementoblasts, and odontoblastic cells from mesenchymal cells to columnar odontoblasts. In high columnar odontoblasts, PTHrP solely expressed. In the HHM animals, although the expression patterns were identical to those of the normal rats in normal area, the shortened high columnar odontoblasts maintained PTHR1 expression and dentin niche comprising odontoblastic cells expressed both proteins. In the HHM model, the protein expression patterns changed in the odontoblastic cells with histological anomalies, and thus direct relations between the anomalies and PTHrP/PTHR1 axis are suggested.     

Col1a1-GFP transgene expression in developing incisors

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of alpha 1(I) collagen in various tissues including odontoblasts.     

Ultrastructural and immunocytochemical analyses of osteopontin in reactionary and reparative dentine formed after extrusion of upper rat incisors

Reactionary dentine and reparative dentine are two strategies used by the dentine-pulp complex to respond to injury. The reactionary dentine is secreted by original odontoblasts, while the reparative dentine is formed by odontoblast-like cells. Osteopontin (OPN) is a non-collagenous protein usually present in the repair of mineralized tissues. It is likely to be present in newly formed dentine but there are no studies attempting to detect it in reactionary and reparative dentine. The aim of the present study was to examine the ultrastructural characteristics, as well as the presence and distribution of OPN in reactionary and reparative dentine by provoking extrusion of the rat incisor. The right upper incisors of 3-month-old male rats were extruded 3 mm and then repositioned into their original sockets. At 3, 7, 10, 15, 20, 30 and 60 days after surgery, the incisors were fixed in glutaraldehyde-formaldehyde and then processed for scanning and transmission electron microscopy and for immunocytochemistry for OPN. After extrusive trauma, the dentine-pulp interface showed the presence of reactionary and reparative dentine, which varied in aspect, thickness and related cells. OPN was not detected in the physiological and reactionary dentine, while it was strongly immunoreactive in the matrix that surrounded the entrapped cells of reparative dentine. In addition, original odontoblasts subjacent to the physiological dentine contained OPN in their Golgi region. The present findings showed that reparative dentine shares some structural characteristics with primary bone, especially in relation to its OPN content. The odontoblast-like cells resemble osteoblasts rather than odontoblasts.     

Col1a1-GFP Transgene Expression in Developing Incisors

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of &#102 1(I) collagen in various tissues including odontoblasts.     

Microtubules, intermediate filaments, and actin filaments in the odontoblast of rat incisor

Actin filaments, intermediate filaments, and microtubules in the odontoblasts of rat incisors were investigated electron microscopically using heavy meromyosin and taxol. Actin filaments were abundant at the periphery of the odontoblast process in the form of a network or in bundles. In a branch of the odontoblast process, longitudinally oriented actin filament bundles were found. Most actin filaments were associated with the plasma membrane via electron-dense material which stained with tannic acid. The intermediate filaments had a diameter of 11 to 13 nm. They were distributed throughout the cytoplasm of odontoblasts. They ran lengthwise in the core of the odontoblast process, which showed a different distribution compared with that of actin filaments. Microtubules, which were disrupted after Triton X-100 but preserved by addition of taxol, tended to be associated with intermediate filaments. Such a relation was also seen in conventional preparations. Coated vesicles, which were abundant at the periphery of the odontoblast process, were often associated with actin filaments. Therefore, it is suggested that actin filaments, in the odontoblast process at least, play a role associated with the coated vesicles at the periphery of the process, and may be involved in coated vesicle transport.     

Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.     

Ca-binding domains in the odontoblast layer of rat molars and incisors under normal and pathological conditions

We recently reported the presence of high concentrations of a Ca-binding matrix in the circumpulpal dentin of rat incisors which had been prevented from mineralization by a systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP), a type of bisphosphonates, thus suggesting the role of the putative Ca-binding matrix in the appositional mineralization of circumpulpal dentin (TAKANO et al., 1998, 2000; OHMA et al., 2000). In this study, we examined the distribution of Ca-binding domains in the pulp tissue of normal rat teeth and its changes under the influence of HEBP, in order to identify and clarify the role of the Ca-binding matrix in the physiological process of dentin mineralization. Observation of the normal rat tooth pulp showed occasional, tiny extracellular deposits of Ca-enriched material in the odontoblast layer, associated primarily with pericapillary regions. Such deposits were immunopositive for dentin sialoprotein (DSP), displayed high levels of X-ray peaks for calcium and phosphorus, and showed a drastic increase in amount by daily injections of HEBP. A brief vascular perfusion of high Ca-containing solution in normal animals caused the extensive deposition of Ca-P complexes along the basolateral membranes of odontoblasts but not in the other regions of the pulp tissue. These data suggest the existence of DSP-enriched extracellular Ca-binding domains in the odontoblast layer and also indicate a novel Ca-binding property of the basolateral membranes of odontoblasts. Since DSP is primarily synthesized as dentin sialophosphoprotein (DSPP) and later cleaved into dentin phosphophoryn (DPP) and DSP in odontoblasts, and since DSP has no notable affinity for Ca, the sites of DSP-immunopositive Ca-P deposits in the odontoblast layer may also contain DPP, a highly phosphorylated acidic protein having a strong binding property for calcium. Characteristic Ca-binding properties seen in the odontoblast layer appear to be related to the regulation of the appositional mineralization of circumpulpal dentin.     

Prenatal expression of growth hormone receptor/binding protein and insulin-like growth factor-I (IGF-I) in the enamel organ

Immunohistochemistry was used to study the ontogeny of GH receptor/binding protein (GHR/BP) and IGF-I from the 13-day-old embryo (E13) to the E19 rat fetus in the developing incisor and molar. Analysis of serial sections revealed diffuse staining of GHR/BP and IGF-I at the bud and early cap stages within both the mesenchyme of the dental papilla and the ectodermal-erived enamel organ. Just before transition to the cap stage, immunoreactivity of GHR/BP and IGF-I increased in the epithelial bud and extended to the condensed dental mesenchyme. At the cap stage, the dental epithelium showed an intense expression of GHR/BP and IGF-I, whereas the dental mesenchymal cells showed very weak staining. The inner enamel epithelium and the outer enamel epithelium were positive for both GHR/BP and IGF-I in the bell stage. Differentiating ameloblasts, odontoblasts and the secretory ameloblasts and odontoblasts continued to express GHR/BP and IGF-I in incisors. These findings support the premise that growth hormone and IGF-I may play a role in embryonic tooth development by regulating the epithelial-mesenchymal interactions that influence events in growth and cytodifferentiation.     

Nuclear uptake of 1,25-dihydroxyvitamin D3 in developing rodent teeth: an autoradiographic study

Target cells for 1,25(OH)2 vitamin D3 metabolites are identified in developing rodent teeth by the use of thaw-mount autoradiography. Following the injection of [26, 27-3H]-1,25(OH)2 vitamin D3 into 18-day- and 20-day-old fetal rats and neonatal mice, nuclear concentration of radioactivity is found in different cell types. In incisors of both animal groups, strong nuclear labeling is present predominantly in pulp cells, while relatively weakly labeled cells are found in the layers of odontoblasts, ameloblasts, and stratum intermedium. In molars, nuclear labeling is absent in fetal rats, but is present in 2-day-old neonates in pulp cells and cells in the layers of stratum intermedium of the first molars, but not in the second molars. The absence of labeled pulp cells in the progenitor regions of incisors and in molars of 20-day-old fetal rats, and differential ontogenic appearance of labeled pulp cells in molars, indicates that there is a critical period of receptor emergence. The finding that labeled pulp cells exist in the regions of incisors and molars where secretory odontoblasts are present suggests that nuclear uptake of 1,25(OH)2 vitamin D3 is related to cell maturation and differentiation, and topographically related to the formation of dentin. The results further suggest that, in contrast to bone, the predominant effect of 1,25(OH)2 vitamin D3 is not on tooth cells which are directly involved in the formation of calcified tissue, i.e., ameloblasts and odontoblasts, but rather on supporting tissues such as pulp cells and stratum intermedium.