Localization of osteonectin expression in human fetal skeletal tissues byin situ hybridization

Summary The expression of osteonectin gene was studied in developing human fetuses by Northern analysis andin situ hybridization. The highest levels of osteonectin mRNA were detected in RNA extracted from calvarial bones, growth plates, and skin. Low mRNA levels were present in several parenchymal tissues.In situ hybridization of developing long bones revealed three cell types with high osteonectin mRNA levels: osteoblasts, cells of the periosteum, and hypertrophic chondrocytes. Weaker signals were detected in osteocytes, fibroblasts of tendons, ligaments and skin, and in cells of the epidermis. Apart from the hypertrophic chondrocytes, only low osteonectin mRNA levels were seen in cartilage. The localization of osteonectin mRNA in fetal growth plates is consistent with the hypothesis that the protein plays a role in the mineralization of bone and cartilage matrices.     

原位杂交检测骨肉瘤中MDM2和P~(53)基因表达及其临床意义

目的 :从基因水平研究 MDM2和 P5 3 基因在骨肉瘤中的表达 ,观察其在骨肉瘤发病中的作用及与临床病理和预后间的关系。方法 :用地高辛标记原位杂交技术研究 40例骨肉瘤 MDM2和 P5 3表达 ,并分析两种基因表达间的相互关系。结果 :MDM2及 P5 3表达阳性率分别为 77.5 % (31/ 40 )、6 7.5 % (2 7/ 40 )。 MDM2与 P5 3基因表达呈显著正相关(P<0 .0 5 )。表达阳性率及表达强度与肿瘤分化程度、转移与否及生存率差异显著 (P<0 .0 5 )。结论 :MDM2和 P5 3基因改变是骨肉瘤的常见现象 ,参与骨肉瘤的发生和发展。其阳性检出率有助于判断骨肉瘤的恶性程度及预测肿瘤的转移及预后。     

Insulinlike growth factor gene expression in human fracture callus

Summary The effects of insulinlike growth factors on bone and cartilage-derived cells in culture have been extensively investigated, but there is little information on their rolein vivo in bone, especially in fracture healing. This study investigated insulinlike growth factor (IGF) I and II mRNA expression in normally healing human fractures byin situ hybridization. Endothelial and mesenchymal cells at the granulation tissue stage expressed IGF-II mRNA. At the stage of bone and cartilage formation, osteoblasts and nonhypertrophic chondrocytes expressed mRNA for both IGF-I and II. Some osteoclasts were positive for IGF-II mRNA at the stage of bone remodeling. The greater time span of IGF-II expression relative to IGF-I reflects the predominance of IGF-II in human bone matrix. Taken together with the known effects of IGFs on bone and cartilage cellsin vitro, these findings support a role for IGFs in local cellular regulation in human fracture healing.     

Cultured embryonic bone shafts show osteogenic responses to mechanical loading

Summary Pairs of 17-day embryonic chick tibiotarsi were removed and maintained in organ culture. One of each pair was subjected to a single 20-minute period of intermittent loading at 0.4 Hz, producing peak longitudinal compressive strains of 650 microstrain (). In the 18-hour culture period following loading, alkaline phosphatase levels in the osteoblasts of the loaded tibiotarsi were maintained whereas in controls they declined. In situ hybridization using a collagen type I cRNA riboprobe showed a substantial increase in expression of mRNA for collagen type I in the periosteal tissue of bones that were cultured for 18 hours after loading compared with that in similarly cultured controls and bones cullured for 4 hours. These results demonstrate that appropriate loading of embryonic chick bones in organ culture elicits adaptive regulation of matrix synthesis as evidenced by increased expression of the gene for type I collagen and alkaline phosphatase activity. This model may be useful as it must contain all the obligatory steps between strain change in the matrix and modified osteogenic activity.     

Osteoclast development in the coculture system of periostless metatarsal bones and hemopoietic cells studied by in situ hybridization with a probe for Y chromosomes

In the coculture system of periostless metatarsal bones of 17-day-old fetal mice and osteoclast progenitors, osteoclasts will develop. Our goal in the present report was to provide further evidence that in the coculture system of fetal metatarsal bone rudiments with hemopoietic cells, the osteoclasts developing inside the bone rudiments are exclusively derived from the cells suspended in the plasma clot and not from endogenous precursor cells of the bone explants themselves, by using the technique of in situ hybridization with a probe for the mouse Y chromosome. Osteoclast formation in unstripped male metatarsal rudiments, occurring after 3–4 days of culture, was compared with osteoclast formation in cocultures of female metatarsal rudiments and male bone marrow cells, occurring after 5–6 days of culture. Osteoclasts were recognized by their tartrate-resistant acid phosphatase activity. In paraffin sections of cultured male metatarsals, the mean percentage of microscopically identifiable osteoclast nuclei, in which the Y chromosome could be detected, was 43.1±4.2% (n=12). For cocultures of female metatarsal bones and male bone marrow cells this mean percentage was 40.9±5.7% (n=17). Statistical comparison by means of the two sample t-test indicated no significant difference in the percentages of osteoclast nuclei containing the Y chromosome for both groups. We concluded that the osteoclasts do derive from cocultured cells and not from precursor cells in the bone explant itself. Therefore, the coculture system is a reliable in vitro system for studying osteoclast formation from progenitor/precursor cells.     

腰椎间盘纤维性主胶原基因表达的观察

目的探讨腰间盘组织主要胶原成分－Ｉ型Ⅱ型胶原在细胞中基因表达的规律，作为今后的基因调控工作的基础。方法 利用核酸分子的原位杂交技术，分别以Ⅰ型、Ⅱ型胶原基因的ｃＤＮＡ探针对胎儿，成人和病理椎间盘组织切片中椎间盘细胞的胶原ｍＲＮＡ进行原位杂交，观察不同阶段胶原基因表达的水平。以胶原的特异性天狼星红染色进行不同阶段的胶原蛋白水平的观察。结果 Ｉ型胶原ｍＲＮＡ的阳性杂交信号集中于纤维环的外层，Ⅱ型胶原基     

人类胰腺癌Ki—67基因表达的研究

目的 应用原位杂交结合免疫组化分析胰腺癌Ｋｉ－６７基因的ｍＲＮＡ转录和蛋白翻译,研究该基因结构和功能表达的关系。方法 胰腺癌４０例,正常胰腺及良性病变９例;扁桃体组织及Ｈｅｌａ细胞作为阳性对照。通过地高辛标记的Ｋｉ－６７ｃＲＮＡ探针原位杂交和Ｋｉ－６７等效单克隆抗体的免疫组化分析该基因的ｍＲＮＡ转录和蛋白翻译。结果正常胰腺及良性病变Ｋｉ－６７ 指数均小于２０％;胰腺主分化腺癌、低分化腺癌各２０例,     

Localization of type X collagen in canine growth plate and adult canine articular cartilage

Type X collagen was extracted from ends of canine growth plates by pepsin digestion after 4 M guanidine hydrochloride extraction, purified by stepwise salt precipitation (2.0 M NaCl in 0.5 M acetic acid), and chromatographed on a Bio-Gel A1.5 M column in 1.0 M CaCl2. Without reduction on sodium dodecyl sulfate (SDS) polyacrylamide gels, the preparation yielded a single, high-molecular-weight (mol wt) band; after reduction, a single band of relative mol wt 5.0 x 10(4) was found. Polyclonal sera were raised against the purified collagen and used in the immunolocalization of canine type X collagen. As expected, indirect immunoperoxidase (IP) or indirect immunofluorescent staining with the polyclonal sera demonstrated that most of the immunoreactivity was localized in the zone of provisional calcification of the growth plate and in cartilage remnants in the metaphyseal region of the physis. A progressive decrease in staining toward the diaphysis of the fetal canine long bone was apparent as the trabecular structures were remodeled to bone. Unexpectedly, type X collagen was also detected in the zone of calcified, mature articular cartilage. It was concentrated in the pericellular matrix of the chondrocytes, appeared at or just above the tidemark, and was expressed immediately before mineralization. Identification of type X collagen in both the canine growth plate and the zone of calcified articular cartilage suggests that cells in the deep layer of cartilage and in the zone of calcified cartilage in the adult animal retain some characteristics of a growth plate and may be involved in regulation of mineralization at this critical interface. The expression of growth plate-like properties would allow the deep chondrocytes of mature articular cartilage to play a role in remodeling of the joint with age and in the pathogenesis of osteoarthritis.     

Gene expression of collagen types IX and X in the lumbar disc

To study gene expression of collagen typesIX and X in human lumbar intervertebral discs duringaing and degeneration and to explore the role of collagentypes IX and X in disc degeneration.     

Expression of type I,type II,and type X collagen genes during altered endochondral ossification in the femoral epiphysis of osteosclerotic (oc/oc) mice

The osteosclerotic (oc/oc) mouse, a genetically distinct murine mutation that has a functional defect in its osteoclasts, also has rickets and shows an altered endochondral ossification in the epiphyseal growth plate. The disorder is morphologically characterized by an abnormal extension of hypertrophic cartilage at 10 days after birth, which is later (21 days after birth) incorporated into the metaphyseal woven bone without breakdown of the cartilage matrix following vascular invasion of chondrocyte lacunae. In situ hybridization revealed that the extending hypertrophic chondrocytes expressed type I and type II collagen mRNA, as well as that of type X collagen and that the osteoblasts in the metaphysis expressed type II and type X collagen mRNA, in addition to type I collagen mRNA. The topographic distribution of the signals suggests a possible co-expression of each collagen gene in the individual cells. Immunohistochemically, an overlapping deposition of type I, type II, and type X collagen was observed in both the extending cartilage and metaphyseal bony trabeculae. Such aberrant gene expression and synthesis of collagen indicate that pathologic ossification takes place in the epiphyseal/metaphyseal junction of oc/oc mouse femur in different way than in normal endochondral ossification. This abnormality is probably not due to a developmental disorder in the epiphyseal plate but to the failure in conversion of cartilage into bone, since the epiphyseal plate otherwise appeared normal, showing orderly stratified zones with a proper expression of cartilage-specific genes.     

Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages.     

转移抑制基因Kiss-1和KAI-1在胃癌组织中表达的原位杂交研究

目的研究胃癌及其癌旁组织Kiss-1和KAI-1转移抑制基因信使核糖核酸（Kiss-1mRNA和KAI-1 mRNA）的表达水平及其临床意义。方法将49例胃癌组织和20例癌旁组织常规制作石蜡包埋切片．用Kiss-1 mRNA和KAI-1 mRNA染色方法作为原位杂交染色法。结果胃癌组织中Kiss-1 mRNA和KAI-1 mRNA表达阳性率明显低于癌旁组织（P〈0．01）；正常至轻度不典型增生癌旁组织两者Kiss-1 mRNA和KAI-1 mRNA表达阳性率及其评分明显高于中至重度不典型增生病例（P〈0．05，P〈0．01）。侵袭深度T1～T2、区域淋巴结无转移、第1站淋巴结转移及无远处转移的胃癌Kiss-1 mRNA和KAI-1 mRNA表达阳性率及其评分明显高于侵袭深度T3～T4、区域淋巴结有转移、第2站或第3站淋巴结转移及有远处转移的病例（P〈0．05，P〈0．01）；但两者的表达与胃癌其他临床病理特征无明显关系。胃癌组织中Kiss-1 mRNA表达评分与KAI-1 mRNA表达评分呈密切正相关（r=0．53，P〈0．01）。结论Kiss-1 mRNA和KAI-1 mRNA表达可能是反映胃癌侵袭和转移潜力及预后的重要生物学标记物，对胃良性病变者检测Kiss-1 mRNA和／或KAI-1 mRNA可能对早期发现及预防胃癌发生有重要临床意义。     

Patterns and Localization of Gene Expression During Intramembranous Bone Regeneration in the Rat Femoral Marrow Ablation Model

Tissue formation and repair are dependent upon cascades of biological events, but the signals involved and the possible gene coexpression patterns during intramembranous bone repair are only poorly understood. We sought to place this mode of regeneration in context by profiling quantitative gene expression for a panel of 39 genes between days 1 and 14 following rat femoral marrow ablation. In situ hybridization was employed to localize a subset of genes. Additionally, principal components analysis was conducted to identify underlying factors suggestive of coexpression patterns. During inflammation (days 1–5), several genes, including cyclooxygenase-1 and -2, showed downregulation. Other proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β, exhibited increasing levels around day 5. During repair (days 3–10), growth factors, receptors, and inhibitor genes for transforming growth factor- β; basic fibroblast growth factor; bone morphogenetic proteins 2, 4, and 7; vascular endothelial growth factor; and insulin-like growth factor-I were upregulated. In addition, the gene for core binding factor-α1 and markers of osteoblast function such as alkaline phosphatase, collagen type I, osteonectin, osteopontin, and osteocalcin had peak expression at day 5 or 7. The remodeling phase (days 10–14) was characterized by peaks for cytokines associated with osteoclastic activity including receptor activator of nuclear factor-κB, receptor activator of nuclear factor-κB ligand (RANKL), cathepsin K, tumor necrosis factor-α, interleukin-6, and cyclooxygenase-2. In situ hybridization showed that the most common sites of increased signal were within osteoblastic cells on trabecular and endosteal surfaces. Principal components analysis identified eight underlying factors that together explained over 80% of the variance in the data. Shinji Kuroda and Amarjit S. Virdi, contributed equally to this report.     

猪TGF-β1基因重组慢病毒载体的构建及其在BMSCs中的表达

目的构建猪TGF-β1重组慢病毒表达载体,并转染BMSCs,为构建组织工程骨软骨提供TGF-β1修饰的BMSCs,作为持续、高效的种子细胞。方法将已获取的目的基因TGF-β1cDNA包装至慢病毒载体中,通过PCR及基因测序对阳性克隆进行鉴定,并测定病毒滴度。取2月龄巴马香猪(体重约15 kg)骨髓制备BMSCs,取第2～3代用于实验。用TGF-β1重组慢病毒载体以感染复数(multiplicity of infection,MOI)为10、50、70、100、150分别转染BMSCs,通过激光共聚焦显微镜观察,并以Western blot检测不同MOI值的转染效果,确定最佳MOI值。用TGF-β1重组慢病毒载体以最佳MOI值感染BMSCs作为实验组,以空载体转染的BMSCs(空载体组)及未转染的BMSCs(空白组)作为对照,通过RT-PCR、免疫细胞化学染色、ELISA等方法检测TGF-β1基因及蛋白在BMSCs中的表达情况,并检测Ⅱ型胶原表达情况。结果经PCR及基因测序鉴定TGF-β1重组慢病毒表达载体构建成功,并成功转染BMSCs,激光共聚焦显微镜下可观察到强绿色荧光;Western blot示MOI为70时转染效果最佳;RT-PCR示实验组TGF-β1基因的表达量明显高于空载体组及空白组,差异有统计学意义(P<0.05);免疫细胞化学染色示实验组TGF-β1蛋白及Ⅱ型胶原呈阳性表达,而空载体组及空白组呈弱阳性或阴性表达;ELISA示实验组TGF-β1蛋白至转染后21 d仍有较高表达。结论 TGF-β1重组慢病毒表达载体可成功转染BMSCs,TGF-β1蛋白可长期、稳定表达,促使BMSCs向成软骨细胞方向分化。     

Increased expression of tumor-associated trypsin inhibitor, TATI, in prostate cancer and in androgen-independent 22Rv1 cells

OBJECTIVES: Tumor-associated-trypsin inhibitor (TATI) is frequently coexpressed with trypsinogen in tumors. Recently, we found expression of trypsinogens in prostate cancer. We have now studied whether TATI is also expressed in prostate cancer and if TATI expression is associated with Gleason grade, proliferation, and neuroendocrine differentiation. METHODS: Expression of TATI and prostate-specific antigen (PSA) was studied by immunohistochemistry and in situ hybridization, and that of chromogranin A (CgA) and Ki-67 by immunohistochemistry. Immunofluorometric assays were used to quantify TATI and PSA in serum from prostate cancer patients and in medium of 22Rv1 prostate cancer cells. RESULTS: TATI expression was weak in benign prostatic epithelium and moderate to strong in prostate cancer and high-grade prostatic intraepithelial neoplasia. There was no correlation between TATI and Ki-67 immunostaining in a tissue microarray of 115 prostate cancer cores, but strong expression of TATI was associated with higher Gleason grade (p=0.002) and CgA immunostaining intensity (p=0.012). Serum TATI was elevated in 44% (29 of 66) of patients with prostate cancer, and the levels correlated with serum PSA (p<0.0001, r=0.306). DU145, PC-3, LNCaP, and 22Rv1 cells contained TATI mRNA as determined by RT-PCR, but only 22Rv1 cells produced detectable TATI protein. The synthetic androgen R1881 decreased secretion of TATI from 22Rv1 cells. CONCLUSIONS: We demonstrate for the first time that TATI is expressed in the benign and malignant prostate. Increased TATI protein expression is found in high-grade tumors and in 22Rv1 cells in which it is regulated by androgens.     

<Emphasis Type="Italic">In Vivo</Emphasis> Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes

Mechanical stimulation is essential for maintaining skeletal integrity. Mechanosensitive osteocytes are important during the osteogenic response. The growth hormone-insulin-like growth factor (GH-IGF) axis plays a key role during regulation of bone formation and remodeling. Insulin-like growth factor binding proteins (IGFBPs) are able to modulate IGF activity. The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae. Female Wistar rats were assigned to three groups (n = 5): load, sham, and control. The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft. The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections. Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts, some trabecular and subendocortical osteocytes, intracortical endothelial cells of blood vessels, and periosteum. Megakaryocytes, macrophages, and myeloid cells also expressed IGFBP-2 mRNA. Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts, bone marrow cells, and chondrocytes. An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded (1.68-fold) and sham-loaded (1.35-fold) endocortical tibial shaft. In conclusion, 6 hours after a single loading session, the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae. Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes. We suggest that IGFBP-2 plays a role in the lamellar bone formation process.     

T(2) relaxation time mapping reveals age- and species-related diversity of collagen network architecture in articular cartilage

OBJECTIVE: The magnetic resonance imaging (MRI) parameter T(2) relaxation time has been shown to be sensitive to the collagen network architecture of articular cartilage. The aim of the study was to investigate the agreement of T(2) relaxation time mapping and polarized light microscopy (PLM) for the determination of histological properties (i.e., zone and fibril organization) of articular cartilage. METHODS: T(2) relaxation time was determined at 9.4 T field strength in healthy adult human, juvenile bovine and juvenile porcine patellar cartilage, and related to collagen anisotropy and fibril angle as measured by quantitative PLM. RESULTS: Both T(2) and PLM revealed a mutually consistent but varying number of collagen-associated laminae (3, 3-5 or 3-7 laminae in human, porcine and bovine cartilage, respectively). Up to 44% of the depth-wise variation in T(2) was accounted for by the changing anisotropy of collagen fibrils, confirming that T(2) contrast of articular cartilage is strongly affected by the collagen fibril anisotropy. A good correspondence was observed between the thickness of T(2)-laminae and collagenous zones as determined from PLM anisotropy measurements (r=0.91, r=0.95 and r=0.91 for human, bovine and porcine specimens, respectively). CONCLUSIONS: According to the present results, T(2) mapping is capable of detecting histological differences in cartilage collagen architecture among species, likely to be strongly related to the differences in maturation of the tissue. This diversity in the MRI appearance of healthy articular cartilage should also be recognized when using juvenile animal tissue as a model for mature human cartilage in experimental studies.     

Anin situ assay system to measure ornithine decarboxylase activity in primary cultures of chicken osteoblasts: Effects of bone-seeking hormones

Summary We present a rapid and uncomplicatedin situ assay for measuring ornithine decarboxylase activity in small cell quantities. This method is more economic than thein situ methods described by others. In addition, our system is faster and less complicated since it avoids manipulation of the CO₂-trapping paper. Applying this method we demonstrate that parathyroid hormone, PGE₁, and other inducers of intracellular cAMP levels, like IBMX and forskolin can induce ODC activity in primary cultures of chicken osteoblasts. Salmon calcitonin does not induce ODC activity, and 1,25 (OH)₂D₃ at higher concentrations can even give an inhibition of ODC activity. We confirm the recent findings that ODC activity is also dependent on calcium.