壳聚糖季铵盐包裹甲状旁腺激素相关肽制备纳米缓释颗粒

摘要 背景：小范围研究显示,低剂量、间歇性应用甲状旁腺激素相关肽能有效治疗绝经后妇女骨质疏松症。但其存在着易变性、半衰期短、价格昂贵等缺陷,因此研制应用缓释系统控制甲状旁腺激素相关肽的释放速度,提高其生物利用效率极为必要。 目的：制备一种新型纳米载药颗粒,探讨其对甲状旁腺激素相关肽的包封及体外释放特性。 方法：采用离子交联法制备壳聚糖季铵盐纳米载药颗粒,用傅里叶红外光谱、透射电镜等进行表征,检测纳米颗粒的包封及体外释放特性。 结果与结论：在常温磁力搅拌条件下,当壳聚糖季铵盐与三聚磷酸钠投药量为5︰1~2︰1时可形成纳米颗粒,粒径100~ 180 nm,为规则球形,甲状旁腺激素相关肽投药质量浓度越高时包封率增大但载药量有所减少,体外PBS溶液中纳米载药颗粒表现出缓慢释放特性。 关键词：壳聚糖;壳聚糖季铵盐;纳米粒子;甲状旁腺激素相关肽;体外释放 doi:10.3969/j.issn.1673-8225.2011.03.019     

Differential Regulation of RGS-2 by Constant and Oscillating PTH Concentrations

PTH has diverse effects on bone metabolism: anabolic when given intermittently, catabolic when given continuously. The cellular mechanisms underlying the varying target cell response are not clear yet. PTH induces RGS-2, a member of the Regulator of G-protein Signaling protein family, via cAMP/PKA, and inactivates PKC-mediated signaling. To investigate intracellular signaling pathways with different PTH concentration-time patterns, we treated UMR 106-01 osteoblast-like cells in a perfusion system. PTH was administered intermittently (4 min/h, 10⁻⁷ M) or continuously at an equivalent cumulative dose (6.6 × 10⁻⁹ M). cAMP was measured using radioimmunoassay, mRNA levels using real-time rtPCR and ribonuclease protection assay, and protein levels using Western immunoblotting. A single PTH pulse transiently increased cAMP levels by 2000% ± 1200%. In contrast to continuous PTH exposure, cAMP induction remained unchanged with intermittent PTH, ruling out desensitization of the PTH receptor. In continuously perfused cells, RGS-2 abundance was three to five times higher than in cells intermittently exposed to PTH for up to 12 h. MKP-1 and -3 were significantly less induced with pulsatile PTH; exposure-mode-dependent differences in MMP-13 and IGFBP-5 were small. Pulsatile but not continuous PTH administration prevents PTHrP receptor desensitization and accumulation of RGS-2 in osteoblasts, which should preserve PKC-dependent signaling.     

Vitamin D Insufficiency in Greenlanders on a Westernized Fare: Ethnic Differences in Calcitropic Hormones Between Greenlanders and Danes

We studied the influence of age, gender, latitude, season, diet and ethnicity on plasma 25-hydroxyvitamin D 25 OHD, PTH, 1,25-dihydroxyvitamin D, vitamin D-binding protein, bone-specific alkaline phosphatase, and osteocalcin levels in 46 Greenlanders living in Nuuk (64°N) on a traditional fare (group A), 45 Greenlanders living in Nuuk on a westernized fare (group B), 54 Greenlanders (group C), and 43 Danes (Group D) living in Denmark (55°N) on a westernized fare. Blood specimens were drawn both summer and winter. Vitamin D insufficiency (plasma 25 OHD <40 nmol/l) was common in all four study groups during summer (23–74%) and winter (42–81%). Compared to groups A and D, vitamin D insufficiency was significantly more frequent in groups B and C. In all groups, summer levels of 25 OHD were above winter levels. Multiple regression analysis revealed a significant effect of ethnicity. Compared to Danes, Greenlanders had higher 1,25-dihydroxyvitamin D levels, but lower 25 OHD and PTH levels despite relatively low plasma calcium concentrations. In addition to ethnicity, 25(OH)D levels were influenced by age, season (summer > winter), and diet (a traditional Inuit diet>westernized diet). Ethnical differences exist between Greenlanders and Danes. Our results suggest that Greenlanders may have an inherent lower set-point for calcium-regulated PTH release or an enhanced renal 1,25(OH)₂D production. In addition to ethnicity, age, season, and diet were important determinants of vitamin D status. Changes from a traditional to a westernized fare are associated with a reduced vitamin D status in Greenlanders. Vitamin D supplementation should be considered.     

In vitro and in vivo activities of C-terminally truncated PTH peptides reveal a disconnect between cAMP signaling and functional activity

There is considerable evidence implicating the cAMP-signaling pathway in the anabolic action of PTH; and to date, all PTH and PTHrp peptides that stimulate cyclic AMP are active in animal models of osteogenesis. We have tested two C-terminally truncated peptides, PTH(1–29) and a modified PTH(1–21) (MPTH(1–21)), in in vitro and in vivo assays of PTH action. Each of the C-terminally truncated peptides was of low nanomolar potency in assays of receptor binding and cAMP stimulation. However, when we tested these peptides for functional response in Saos-2 cells stably transfected with a cyclic AMP response element (CRE) reporter, the C-terminally truncated peptides were two to four times less potent than would be expected from their binding and cAMP-stimulating properties. Furthermore, PTH(1–29), although active, was approximately 20-fold less potent than PTH(1–34) in a rat model of osteogenesis while MPTH(1–21) was inactive. The relative lack of activity of these peptides in vivo suggests that while activation of the cAMP pathway may be important for the anabolic effect of PTH fragments, it is not, of itself, predictive of their in vivo activity.     

Calcium-sensing receptors and parathyroid hormone-related protein in the caudal neurosecretory system of the flounder (Platichthys flesus)

The caudal neurosecretory system of the flounder (Platichthys flesus) has been examined by immunocytochemistry and in situ hybridization for the expression of parathyroid hormone-related protein (PTHrP) and calcium-sensing receptors (CaSR). The N-terminus nucleotide and deduced amino acid sequences of flounder PTHrP were determined and used to prepare oligonucleotide probes and homologous antiserum. The Dahlgren cells of the posterior spinal cord and their axons contained PTHrP protein which was also detected around the capillaries of the urophysis. PTHrP gene expression was abundant in the Dahlgren perikarya and axons in the spinal cord, but it was absent from nerve endings in the urophysis. Calcium-sensing receptor protein was present in the Dahlgren perikarya and axons, also with abundant gene expression, but there was neither protein nor mRNA in the urophysis. There were no apparent differences between freshwater- and seawater-adapted fish in either CaSR or PTHrP expression in the caudal neurosecretory system. These observations suggest that Dahlgren cells produce PTHrP which may be released from axons abutting capillaries in the urophysis. However, the sensing of ionic calcium appears to be confined to the perikarya of the Dahlgren cells in the spinal cord neuropil, suggesting that they are responsive to calcium in the central nervous system rather than the general circulation.     

Biosynthesis of proteoglycan in bone and cartilage of parathyroid hormone-related protein knockout mice

Proteoglycans are suggested to regulate cell adhesion, differentiation and mineralization of hard tissues. In vitro studies have shown that many humoral and local factors regulate proteoglycan synthesis. Among them, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have potent stimulating effects on proteoglycan synthesis. However, the exact role of PTHrP on the biosynthesis and metabolism of proteoglycans during skeletal development is not clear. To clarify this point, we examined bony and cartilaginous explants of newborn mice with disrupted PTHrP alleles. Ribs of homozygous PTHrP-knockout mice and wild-type littermates were dissected into bony and cartilaginous regions and metabolically labeled with [³⁵S]sulfate in culture. Radiolabeled proteoglycans were analyzed by column chromatography. The elution profiles of [³⁵S]-labeled proteoglycan from cartilaginous explants did not differ between homozygous PTHrP-knockout mice and wild-type littermates. However, the amount of labeled proteoglycan in homozygous PTHrP-knockout mice was only 4%–5% that of wild-type littermates. In contrast with cartilaginous explants, the amount of labeled proteoglycans in bony explants did not differ between the two genotypes. Interestingly, besides the common major peak (K_d = 0.10–0.16) observed in the bony explants of both genotypes, a minor peak (K_d = 0.42) was specifically present in homozygous PTHrP-knockout mice. This minor peak was earlier than that of free glycosaminoglycan (GAG) chains, suggesting that the core protein, but not GAG chain, was cleaved in the bony explants of homozygous PTHrP. These findings demonstrate a crucial and nonredundant role of PTHrP in the regulation of proteoglycan synthesis and metabolism during skeletal development. Received: February 21, 2000 / Accepted: June 16, 2000     

Parathyroid hormone-related protein expression and secretion in a skin organotypic culture system

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the epidermis, where it is thought to play a role in the regulation of keratinocyte growth and differentiation. Most in vitro studies of normal keratinocytes use monolayer cell cultures, which have limitations, including the inability to reproduce the stratified structure of the epidermis. The objective of this study was to investigate PTHrP production and secretion, and mRNA expression in skin organotypic cultures. The cultures consisted of an artificial dermis with differentiating keratinocytes grown at the air-liquid interface. Immunohistochemical assessment of cytokeratins 14 and 10/13, involucrin, and proliferative cell nuclear antigen (PCNA) demonstrated that keratinocytes differentiated in a manner similar to keratinocytes in normal epidermis. PTHrP expression was demonstrated in all viable layers of the epidermis, as well as in some fibroblasts of the collagen lattice by immunohistochemistry and in situ hybridization. Since most fibroblasts expressed α-smooth muscle actin, these cells were interpreted to be consistent with myofibroblasts. PTHrP expression by myofibroblasts suggests a possible role for PTHrP in the regulation of contractibility of these cells. PTHrP was also detected in conditioned media for 50 days. In conclusion, because of its superior tissue morphology and ability to induce organized keratinocyte differentiation, this culture system will be an excellent model to study the role of PTHrP in pathologic and physiologic processes involving the epidermis in vitro.     

Renal Cell Carcinoma Presenting with Paraneoplastic Hypercalcemic Coma: A Case Report and Review of the Literature

We report a case of a 62-year-old woman with renal cell carcinoma (RCC) presenting with a hypercalcemia-induced coma. A laboratory evaluation indicated nonparathyroid-mediated hypercalcemia with an initial serum calcium level of 18.6 mg/dL. Our patient’s parathyroid hormone (PTH)-related peptide level was undetectable. Initial imaging was negative, but PET scan identified a mass in the upper pole of the left kidney. Our patient underwent partial nephrectomy, and the mass was identified as RCC on final pathology. After surgery, her hypercalcemia resolved and PTH returned to normal limits. This case report describes a patient with RCC with the unusual presentation of hypercalcemic coma. We review the differential diagnosis of malignant hypercalcemia and the evaluation of hypercalcemia occurring with RCC. This case illustrates the need to carefully review and interpret all available data, especially when conventional testing in the work-up of hypercalcemia is unrevealing. Supported by the Atlanta Research and Education Foundation, Atlanta Veterans Administration Medical Center, and UV Foundation