Manishen W J, Sivananthan K, Orr F W
Am J Pathol. 1986 Apr;123(1):39-45.
Demineralized extracts of bone matrix and conditioned media from cultured fetal rat calvaria have been reported to contain growth stimulatory activity for bone cells. To investigate the potential role of these local bone growth factors in the development of bone metastases, we chose the Walker 256 carcinosarcoma, a rat mammary tumor which causes osteolytic bone metastases and hypercalcemia. 45Ca-labeled, 19-day fetal Sprague-Dawley rat calvaria were cultured for 96 hours in BGJb medium. Walker cells from ascites tumors or cultures were grown in unconditioned media or in conditioned media harvested from the bone cultures, in the presence of 10% fetal calf serum. Media were changed every 2 days, cells were counted daily for 5 days, and 3H-thymidine uptake into acid insoluble residues was measured. The growth of tumor cells was 5-6-fold greater in conditioned media than in unconditioned media and the effect was dose dependent. Cells cultured in conditioned media demonstrated a approximately 3-fold enhancement of 3H-thymidine incorporation. Generation of growth stimulatory activity correlated with the extent of bone resorption, measured by release of 45Ca from the fetal parietal bones (r = 0.85; P less than 0.001). Conditioned media from bones cultured with 10(-7) M prostaglandin E2 (PGE2) contained greater amounts of growth stimulatory activity than untreated conditioned media, but PGE2 itself did not stimulate tumor cell growth. Addition of 3.5 mM PO4 to bone cultures blocked bone resorption and the generation of growth factors. Growth stimulatory activity was stable to heat (56 C for 30 minutes) and trypsin digestion, with an apparent molecular weight of less than 17,000 daltons by high-performance liquid chromatography. Conditioned medium also stimulated the growth of 13762 rat mammary adenocarcinoma cells, MB-MDA-231 human breast carcinoma cells, TE-85 osteosarcoma cells, a murine fibrosarcoma and rat embryonic fibroblasts, with the most potent effects noted for Walker tumor cells, the TE-85 osteosarcoma, and human breast carcinoma lines. These results suggest a mechanism by which bone resorption could promote the development of skeletal metastasis.
据报道，骨基质的脱矿质提取物和培养的胎鼠颅骨的条件培养基含有对骨细胞的生长刺激活性。为了研究这些局部骨生长因子在骨转移发展中的潜在作用，我们选择了Walker 256癌肉瘤，一种导致溶骨性骨转移和高钙血症的大鼠乳腺肿瘤。将45Ca标记的19天龄胎鼠Sprague-Dawley大鼠颅骨在BGJb培养基中培养96小时。腹水肿瘤或培养物中的Walker细胞在未条件培养基中或在从骨培养物收获的条件培养基中生长，培养基中含有10%胎牛血清。每2天更换一次培养基，连续5天每天计数细胞，并测量3H-胸腺嘧啶核苷掺入酸不溶性残渣的量。肿瘤细胞在条件培养基中的生长比在未条件培养基中高5-6倍，且该效应呈剂量依赖性。在条件培养基中培养的细胞显示3H-胸腺嘧啶核苷掺入增加约3倍。生长刺激活性的产生与骨吸收程度相关，骨吸收程度通过胎鼠顶骨中45Ca的释放来测量（r = 0.85；P < 0.001）。与未处理的条件培养基相比，用10(-7) M前列腺素E2（PGE2）培养的骨的条件培养基含有更多的生长刺激活性，但PGE2本身不刺激肿瘤细胞生长。向骨培养物中添加3.5 mM PO4可阻断骨吸收和生长因子的产生。生长刺激活性对热（56℃30分钟）和胰蛋白酶消化稳定，通过高效液相色谱法测定其表观分子量小于17,000道尔顿。条件培养基还刺激13762大鼠乳腺腺癌细胞、MB-MDA-231人乳腺癌细胞、TE-85骨肉瘤细胞、小鼠纤维肉瘤和大鼠胚胎成纤维细胞的生长，对Walker肿瘤细胞、TE-85骨肉瘤和人乳腺癌细胞系的作用最为显著。这些结果提示了一种骨吸收促进骨骼转移发展的机制。
