骨肉瘤高转移细胞SOSP-M_1生物学稳定性及组织学起源的鉴定

目的　检测人骨肉瘤细胞高转移亚系SOSP M1 在裸小鼠体内传代过程中的生物学稳定性并鉴定其组织学特性。　方法　利用原位移植将细胞系在 33只裸鼠体内连续传代 ,组织块培养收集各代肺转移灶的肿瘤细胞 ,观察各代肿瘤细胞致瘤率、转移率、形态结构、骨形成蛋白、波形蛋白、肌动蛋白、神经元特异性烯醇化酶的表达情况以及遗传学方面的变化。　结果　各代肿瘤细胞致瘤率均为 10 0 % ,体内增殖稳定 ,肺转移率在 80 %以上。其显微及超微结构形态、抗原表达、染色体数目及结构变化均符合人骨肉瘤的特征。　结论　该细胞亚系在裸鼠体内传代生物学特性相对稳定 ,是人骨肉瘤实验研究的良好模型。     

表达增强型绿色荧光蛋白的骨肉瘤细胞亚株建立及生物学特性

目的 建立经绿色荧光蛋白基因转染的骨肉瘤细胞亚株并研究其生物特性。方法 利用脂质体转染增强型绿色荧光蛋白真核表达质粒（pEGFP-N1）人人骨肉瘤MG63细胞系，通过有限稀释法和细胞电泳，获得两株细胞克隆M6和M8，经体外细胞增殖、软琼脂形成、生长曲线、裸鼠成瘤试验综合分析其生物学行为改变。应用组织形态学观察、染色体分析、软琼脂克隆形成法研究癌细胞的生物学特性。结果 M6和M8两株在细胞电泳率和侵袭性上差异有统计学意义（P〈0．05），其中M6的群体倍增时间为38．4h，软琼脂形成率为18．7％，M8群体倍增时间为23．0h，软琼脂形成率为29．3％。裸小鼠背部皮下接种M6和M8，发现M8成瘤时间短，细胞增殖快，但在4周内两者均不发生转移。结论 骨肉瘤细胞亚系有不同的转移特性，GFP的整合及表达未对MG63细胞的生长状态造成明显影响，可作为报告基因进一步了解骨肉瘤细胞转移的差异性分析。     

人成骨肉瘤高转移性细胞亚群的筛选及生物学特性的观察

目的 从低转移性人成骨肉瘤细胞系ＳＯＳＰ－９６０７中筛选出高转移亚群,并且建立人成骨肉瘤裸鼠转移模型。方法 采用裸鼠腹腔传代的方法,获得腹水型自发性肺转移细胞,经体外培养和体内再次接种后获得高转移亚群。应用细胞计数法、染色体显示法、流式细胞术及裸鼠体内实验法研究细胞的生物学特性。结果 筛选出的高转移亚群ＳＯＳＰ－Ｍ在转移分析实验中达到１００％肺转移率,除肺转移外还出现其他器官的转移。其染色体为５０     

肿瘤转移相关基因1在人骨肉瘤细胞的表达与转移侵袭的关系

目的比较肿瘤转移相关基因(MTA1)在人骨肉瘤细胞高低转移株的表达水平,探讨MTA1表达水平与骨肉瘤细胞转移潜能的相关性。方法采用半定量逆转录-聚合酶链反应 (RT-PCR)检测MG-63骨肉瘤细胞高低转移株MTA1的表达情况,用Boyden小室体外侵袭实验检测两株MG-63细胞的体外侵袭力;用脂质体介导的MTA1基因转染MG-63低转移株细胞,通过 RT-PCR检测MTA1的表达;Boyden小室体外侵袭实验检测转染前后细胞侵袭力的变化。结果 RT-PCR结果显示MTA1在MG-63低转移细胞株中表达水平低(1．32),在高转移细胞株中表达水平高(6．27,P<0．05);Boyden小室体外侵袭实验显示MG-63高转移株细胞体外侵袭力强,其穿膜细胞相对百分率为(46．3±2．4)％,低转移株细胞体外侵袭力较弱,其穿膜细胞相对百分率(12．6± 1．1)％,两者差异有统计学意义(P<0．05);转染MTA1基因后,低转移细胞株转移潜能较未转染细胞明显增高。结论 MTA1与人骨肉瘤细胞转移潜能有密切关系。     

乳腺癌转移抑制蛋白1下调PI3K/AKT1通路抑制骨肉瘤细胞转移

目的 探讨乳腺癌转移抑制蛋白1(BRMS1)在骨肉瘤转移中的作用和机制。方法免疫组化检测骨肉瘤组织中BRMS1蛋白表达;荧光定量聚合酶链反应(Q-PCR)法检测8例骨肉瘤组织和8个骨肉瘤细胞系BRMS1 mRNA表达的变化;western blot检测正常成骨细胞系(HOSB)及人骨肉瘤细胞系(OS187、COL、LM7、SJSA、MG63、HOS、SAOS-2、CCH-D)和BRMS1蛋白和AKT1蛋白磷酸化的水平;侵袭小室(Transwell)法检测过表达BRMS1的HOS细胞转移能力的变化。结果 骨肉瘤组织中BRMS1蛋白表达较癌旁正常骨组织明显减少。骨肉瘤细胞OS187、COL、LM7、SJSA、MG63、HOS、SAOS-2和CCH-D BRMS1 mRNA和蛋白的表达水平较正常成骨细胞HOSB明显降低,而AKT1蛋白磷酸化水平明显升高。HOS细胞过表达BRMS1后,HOS细胞转移能力和AKT1蛋白磷酸化水平明显降低。运用AKT1蛋白磷酸化抑制剂LY294002明显抑制HOS细胞转移能力。结论 BRMS1可以抑制骨肉瘤细胞HOS细胞转移,其机制可能与抑制AKT1蛋白磷酸化有关。     

人原发性胃恶性淋巴瘤裸鼠原位移植高转移模型的建立

目的 建立人原发性胃恶性淋巴瘤裸小鼠原位移植高转移模型.方法 采用人原发性胃恶性淋巴瘤术中新鲜瘤组织块植入裸小鼠胃壁黏膜下层,观察原位移植的成瘤率和移植瘤的侵袭、转移,并进行形态学（光镜、电镜、免疫组织化学）、染色体核型和流式细胞分析.结果在裸小鼠体内建成了一株人原发性胃恶性淋巴瘤原位移植高转移模型（HGBL-0305）.移植瘤的组织病理学为原发性胃弥漫性大B细胞淋巴瘤.免疫组织化学显示,CD19、CD20、CD22、CD79α阳性,CD3、CD7阴性.染色体众数范围56～69条;移植瘤细胞DNA指数为1.47±0.12,均为异倍体.目前该瘤株在裸鼠体内生长4年,已经传至45代,共移植裸鼠156只;肿瘤移植生长率和液氮冻存复苏成活率均为100%.人胃恶性淋巴瘤在裸鼠胃内自主侵袭性生长,浸润破坏胃壁各层组织结构.HGBL-0305模型的肝转移率为69.5%,脾转移率为55.6%,淋巴结转移率为45.7%,腹腔种植转移率为30.5%.结论 HGBL-0305模型是成功的人原发性胃恶性淋巴瘤裸鼠原位移植自发性高转移模型,完整地模拟了人原发性胃恶性淋巴瘤患者的自然临床病理过程,为研究原发性胃恶性淋巴瘤发病机制、转移生物学和抗转移治疗提供了理想的动物模型.     

稳定表达TSLC1基因骨肉瘤MG63细胞系的建立及鉴定

目的 建立稳定表达人肺癌抑癌基因-1（TSLC1）骨肉瘤细胞系并对其进行鉴定.方法 脂质体转染的方法 将真核表达载体pCI-TSLC1稳定转染至MG63细胞系,经克隆化培养以及G418筛选,获得了1株稳定高表达TSLC1蛋白的细胞系.对其表达的蛋白性质进行了鉴定,对其分泌动态、细胞纯度、遗传稳定性、外源基因整合等生物学特性做了鉴定.结果 获得高表达TSLC1的稳定细胞系,命名为M8T.生物学性状研究结果 表明该转基因细胞系纯度好,遗传性状稳定,抽提细胞RNA进RT-PCR,扩增到与预期结果 大小一致的DNA片段,说明TSLC1基因成功整合到细胞基因组中.细胞系无细菌和霉菌污染.结论 成功建立了稳定表达TSLC1的骨肉瘤细胞系M8T.该细胞系遗传性质稳定,为进一步研究抑癌基因TSLC1在骨肉瘤中的作用奠定了基础.     

凝血酶敏感蛋-1、血管内皮生长因子及微血管密度对骨肉瘤复发转移的判断价值

目的探讨凝血酶敏感蛋-1（TSP-1）、血管肉皮细胞生长因子（VEGF）、微血管密度（MVD）在判断骨肉瘤患者复发转移中的价值。方法采用免疫组织化学法检测65例骨肉瘤手术切除标本的VEGF、TSP-1蛋白的表达情况并计数MVD，对上述指标与术后复发转移率之间的关系进行回顾性分析。结果全组病例TSP-1、VEGF蛋白表达阳性率分别为30．77％（20／65）和61．54％（40／65）；MVD平均值为（23．68±9．42）／200倍视野；VEGF阳性组的术后复发转移率高于VEGF阴性组（P〈0．05）；TSP-1阳性组的术后复发转移率低于TSP-1阴性组（P〈0．05）；多因素分析表明，MVD和淋巴结状况为判断骨肉瘤术后复发转移的独立因素（P〈0．05）。结论MVD是判断骨肉瘤术后复发转移的独立因素，具有判断术后复发转移的风险及预后的价值。     

人原发性胃恶性淋巴瘤裸鼠原位移植高转移模型的建立

Objective To construct a mouse model of highly metastatic gastric lymphoma with orthotopic transplantation of human primary gastric lymphoma specimen. Methods A fresh surgical specimen of primary gastric lymphoma was obtained intraoperatively and implanted into the submucosa of stomach in nude mice. Tumor formation, invasion, metastasis, morphological characteristics under light microscopy and electron microscopy, immunohistochemistry,and the karyotype of orthotopically transplanted tumor cells were studied. Results An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice (HGBL-0305) was successfully established. Histopathology of transplanted tumors showed primary gastric diffuse large B cell lymphoma. CD19, CD20, CD22 and CD79α were positive, while CD3 and CD7 were negative. The number of chromosome ranged from 56 to 69. DNA index(DI) was 1.47±0.12(i.e. heteroploid). Until now, HGBL-0305 model has been maintained for 45 generations by orthotopic passage for almost 4 years in nude mice. A total of 156 nude mice were used for transplantation. The growth rate and resuscitation rate of liquid nitrogen cryopreservation of transplanted tumor cells were both 100%. The autonomic growth of the transplanted tumor cells invaded and destructed all the layers of the nude mice stomach. The metastasis rates of liver, spleen, lymph node, and peritoneal seeding were 69.5%, 55.6%, 45.7%, and 30.5%, respectively. Conclusions An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice is successfully established. HGBL-0305 model may simulate the natural course of primary gastric lymphoma in human and provides an ideal animal model for studies on pathogenesis, metastasis biology and anti-metastatic therapies of primary gastric lymphoma.     

人原发性胃恶性淋巴瘤裸鼠原位移植高转移模型的建立

Objective To construct a mouse model of highly metastatic gastric lymphoma with orthotopic transplantation of human primary gastric lymphoma specimen. Methods A fresh surgical specimen of primary gastric lymphoma was obtained intraoperatively and implanted into the submucosa of stomach in nude mice. Tumor formation, invasion, metastasis, morphological characteristics under light microscopy and electron microscopy, immunohistochemistry,and the karyotype of orthotopically transplanted tumor cells were studied. Results An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice (HGBL-0305) was successfully established. Histopathology of transplanted tumors showed primary gastric diffuse large B cell lymphoma. CD19, CD20, CD22 and CD79α were positive, while CD3 and CD7 were negative. The number of chromosome ranged from 56 to 69. DNA index(DI) was 1.47±0.12(i.e. heteroploid). Until now, HGBL-0305 model has been maintained for 45 generations by orthotopic passage for almost 4 years in nude mice. A total of 156 nude mice were used for transplantation. The growth rate and resuscitation rate of liquid nitrogen cryopreservation of transplanted tumor cells were both 100%. The autonomic growth of the transplanted tumor cells invaded and destructed all the layers of the nude mice stomach. The metastasis rates of liver, spleen, lymph node, and peritoneal seeding were 69.5%, 55.6%, 45.7%, and 30.5%, respectively. Conclusions An orthotopic highly metastatic model of human primary gastric lymphoma in nude mice is successfully established. HGBL-0305 model may simulate the natural course of primary gastric lymphoma in human and provides an ideal animal model for studies on pathogenesis, metastasis biology and anti-metastatic therapies of primary gastric lymphoma.     

高低不同转移特性骨肉瘤亚克隆细胞系的分离与鉴定

目的　建立高低不同转移特性人成骨肉瘤MG 63亚克隆细胞系 ,为研究成骨肉瘤转移机制提供较好的实验模型。方法　通过体外培养和裸鼠体内移植 ,初步分离并建立了 2个亚克隆细胞系A1和A2 ,并利用细胞电泳、细胞增殖、琼脂克隆形成、体外侵袭实验、裸鼠体内异位和原位移植对两者的生物学特性进行比较、分析和鉴定。结果　A1和A2的电泳率、琼脂克隆形成能力、体外侵袭能力、自发性肺转移率分别为 (1.0 8± 0 .12 ) μm ,(0 .64± 0 .13 ) μm ;2 1.0± 2 .3 ,9.5±2 .9;186.0± 16.7,84.0± 12 .6;10 0 .0 0 % ,6.67% ,A1均明显高于A2 ,两者的差异有统计学意义(P <0 .0 1)。结论　高低不同转移特性的人成骨肉瘤MG 63亚克隆细胞系的建立 ,能为成骨肉瘤转移机制的研究提供较理想的实验模型。     

Characterization of a liver metastatic cell line derived from a human gastric cancer cell line

This study was carried out to investigate whether there is any difference of biological characteristics between a gastric cancer cell line (KATOIII) and another cell line derived from liver metastasis of the same cell line (KATOIII-H2). The liver metastasis was produced by intrasplenic injection of the fluid containing of KATOIII in nude mouse and new cell line was established using the cells of metastatic site. The results are as follows. 1) Inoculation of KATOIII-H2 into the spleen produced liver metastases in all of the experimental animals, whereas the same procedure with KATOIII produced metastasis only in 30% of the animals. 2) KATOIII-H2 exhibited more prominent platelet-aggregating activity than KATOIII. 3) There is no difference between two cell lines on doubling time, histological findings of the xenografts and chromosomal number. 4) DNA index of KATOIII-H2 is lower than KATOIII and the trisomy in NO. 20 chromosome of KATOIII-H2 was noted. The results indicate that metastatic potential is different between two cell lines and this fact is probably in a part because of the different platelet-aggregating activity of each cell line.     

Establishment of a cell line producing bone morphogenetic protein from a human osteosarcoma

A human osteosarcoma cell line was established from a biopsy specimen from a 13-year-old girl. The osteosarcoma tissue was maintained in athymic nude mice (Balb C nu/nu) by serial transplantation for three years. The tumor was excised from a host mouse and digested with collagenase. The isolated cells were cultured by 98 passages in 14 months, and clones of osteosarcoma cells were obtained by limiting dilution. A clone named human osteosarcoma cell 6 (H-OS-6) that showed the osteoblastic phenotypes of productions of bone morphogenetic protein (BMP) and alkaline phosphatase and a response to human parathyroid hormone (h-PTH 1-34) was selected. The morphology of its chromosomes indicated its human origin. This human osteosarcoma cell line is unique in producing BMP under in vitro conditions.     

组织蛋白酶L反义基因转染对高转移人骨肉瘤细胞生物学行为的影响

目的构建人组织蛋白酶L（CATL）基因的正、反义真核表达载体。观察反义CATL核酸转染后对高转移人骨肉瘤细胞体内、外侵袭特性的抑制效应。方法采用RT-PCR的方法,从人骨肉瘤组织中扩增出1001bp的CATL全长cDNA片段,以BamHⅠ及XbaⅠ双酶切,定向克隆到真核表达载体pcDNA 3.0中。对重组质粒进行限制性内切酶酶切分析及DNA序列测定。应用脂质体介导的基因转染技术将重组质粒导入高转移人骨肉瘤细胞中,观察转染后细胞的生长、体外侵袭能力及裸鼠体内成瘤及自发转移能力等指标的变化。结果正、反义真核表达载体成功构建并转染入高转移人骨肉瘤细胞中。基因转染对细胞的体外生长无明显影响。反义载体转染后细胞的体外侵袭能力和裸鼠体内成瘤性及自发转移能力均受到一定程度抑制。结论反义CATL基因可显著抑制骨肉瘤细胞的体内、外侵袭力。     

人成骨肉瘤细胞系SOSP-9607的建立及其生物学特性的观察

目的 建立人成骨肉瘤细胞系。方法 采用原代组织培养法对成骨肉瘤手术标本进行培养,对存活细胞进行形态学观察、组织化学染色、乳酸脱氢酶（ＬＤＨ）同工酶谱、细胞周期检查、核型分析、异种移植。结果 建立细胞系ＳＯＳＰ－９６０７,其形态学表现、组织化学染色、ＬＤＨ同工酶谱等均符合成骨肉瘤的特征。经１年体外培养,已连续传代１２０次,细胞倍增时间２９．８ｈ,细胞周期测定Ｇ１期为４８．２％、Ｇ２期为２０．９％、Ｓ     

1,25-Dihydroxyvitamin D3 metabolism in a human osteosarcoma cell line and human bone cells

The metabolism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by a human osteoblastic sarcoma cell line, U-2 OS, and by primary cultures of human bone-derived cells was examined at physiologic (5 x 10(-11) M) and pharmacologic (3.5 x 10(-7) M) substrate concentrations. For metabolite identification purposes, cells nearing confluency were incubated for 18 h with 3.5 x 10(-7) M 1,25-(OH)2D3 in serum-free medium. The putative vitamin D metabolites produced during this incubation were isolated from a total lipid extract of cells and medium. Identification of the metabolites was achieved by comigration with authentic standards on three high-performance liquid chromatography systems, UV spectral analysis, mass spectrometry, and chemical modification by sodium borohydride and sodium metaperiodate. The identified metabolites produced from 1,25-(OH)2D3 by the human osteosarcoma cells include 1,24,25-trihydroxyvitamin D3; 24-oxo-1,25-dihydroxyvitamin D3; 24-oxo-1,23,25-trihydroxyvitamin D3; and 24,25,26,27-tetranor-1,23-dihydroxyvitamin D3. Evidence is presented that (1) 1,25-(OH)2D3 metabolism occurs constitutively in U-2 OS osteosarcoma cells at a physiologic substrate concentration (5 x 11(-11) M), (2) the pathway can be further induced by pharmacologic 1,25-(OH)2D3 concentrations (10(-7) M), and (3) this pathway is present in primary cultures of normal human bone-derived cells.     

Recognition by cellular and humoral autologous immunity in a human osteosarcoma cell line

Because of the difficulty of developing pairs of osteosarcoma cell lines and cytotoxic T lymphocytes (CTLs), no osteosarcoma tumor antigens that are useful for antiosteosarcoma immunotherapy have yet been identified. In parallel with continuous attempts to develop such pairs from osteosarcoma, we employed serological identification using a recombinant expression cloning (SEREX) method to identify B cell-defined antigens. Consequently, a human osteosarcoma cell line, OS2000, was established from a primary osteosarcoma of a patient cured of hereditary retinoblastoma. Repetitious in vitro stimulations by OS2000 cells to the autologous peripheral T cells induced cytotoxic activity in the autologous osteosarcoma cells but not in the nontumor cells. The cytotoxicity was inhibited by anti-HLA class I monoclonal antibody. SEREX analysis revealed that autologous humoral immunity reacted to two proteins expressed in OS2000. One was the self HLA-Cw*0102 molecule, and the other was wild-type smooth muscle myosin light chain (SMMLC). However, no antigenicity of these proteins was seen versus the sera of the other patients. In conclusion, our results demonstrated the presence of host cellular and humoral immune responses to autologous osteosarcoma cells. This offered the opportunity to identify osteosarcoma antigens recognized by autologous immunity.     

同源性骨肉瘤新细胞系Zos和Zos-M的生物学比较研究

目的 对来源于同一患者原发灶和跳跃转移灶的两株新建骨肉瘤细胞系-Zos和Zos-M的特性和生物学行为进行鉴定和比较研究.方法 用组织培养块法从1例骨肉瘤患者原发灶和跳跃转移灶骨肉瘤组织,分离并分别建立两株新的骨肉瘤细胞系-Zos和Zos-M;利用形态学观察、核型和细胞周期分析、生长曲线和倍增时间检测、成骨性标志检测和基质胶侵袭试验对两株细胞系进行体外鉴定.通过皮下、原位移植和实验性转移检测细胞系的体内成瘤和转移能力.应用MTT法检测Zos和Zos-M对传统化疗药物的敏感性;RT-PCR方法检测并比较二者转移相关基因的表达.结果 Zos和Zos-M体外连续培养100代后仍保持稳定,形态学检测、RT-PCR检测碱性磷酸酶、骨钙蛋白和骨桥蛋白等成骨系标志提示均符合骨肉瘤特征.Zos和Zos-M倍增时间分别为33.65 h和31.58 h.核型分析表现为非整倍体和多种染色体结构异常.Zos、Zos-M皮下和原位成瘤率均为100%,Zos-M实验性转移率为37.5%(3/8),Zos无实验性转移(0/8).转移相关基因表达检测表明cadherin-11在Zos-M的低表达可能与其高转移能力相关.并且Zos和Zos-M与传统骨肉瘤细胞系相比,对常用化疗药物的敏感性差.结论 人骨肉瘤细胞系Zos和Zos-M及相关动物模型的建立为治疗骨肉瘤的药物筛选提供新的模型,其具有相同的遗传背景和不同的侵袭转移能力,为骨肉瘤转移的研究提供良好的模型.

Abstract：

Objective To characterize and compare the different biological behaviors of two novel human osteosarcoma cell lines, Zos and Zos-M, established respectively from the primary site and the skip metastasis of an osteosarcoma patient. Methods Two novel human osteosarcoma cell lines, Zos and Zos-M,were developed using tissue plant culture method. The vitro examinations included observations of morphology, analysis of karyotype and cell cycle, calculation of doubling time and growth curve, detection of osteoblastic markers and matrigel invasion assay. Subcutaneous, intratibial and intravenous inoculations into nude mice were performed to study the in vivo tumorigenicity and metastatic potentials of both cell lines.MTT were used to detect sensitivity of the cell lines to chemotherapeutic drugs. RT-PCR was performed to assess the expression of and some metastasis-related genes. Results Both cell lines proliferated actively and remained stable for more than 100 passages in vitro without interruption. The morphology and expression of osteoblastic markers of Zos and Zos-M were conformed to the characteristic of osteosarcoma. The karyotype analysis displayed aneuploidy and various structural abnormalities. The population doubling time of Zos and Zos-M were 33.65 h and 31.58 h respectively. Both cell lines were less sensitive to the current chemotherapy protocols compared to U-2OS. Zos and Zos-M were 100% tumorigenic by subcutaneous and othotopic injection. 37.5% of nude mice injected Zos-M and none of nude mice injected Zos developed lung metastasis after intravenous injection. The comparison of the expression patterns of some metastasis-related genes revealed that the decreased expression of cadherin-11 in Zos-M may correlate with a high potential of metastases. Conclusion The two novel established human osteosarcoma cell lines, Zos and Zos-M and related animal models could serve as models for the study of drug resistance and screening of new therapeutics for osteosarcoma. In addition, the study also provide tools for the study of metastasis because the same genetic background and different potential of metastasis of Zos and Zos-M.     

A newly established human osteosarcoma cell line with osteoblastic properties

A human osteosarcoma cell line, HuO9, was established from a tumor that was heterotransplanted into athymic nude mice. Antiserum against nude mouse spleen cells was added to the early passage cultures to eliminate the host fibroblastic cells. The cell line retained a high activity of liver/bone/kidney-type alkaline phosphatase (ALP) and secreted osteocalcin, i.e., bone gamma-carboxyglutamic acid-containing protein (BGP), into the medium. The addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) increased the ALP activity as well as the level of BGP secreted into the medium. The ALP of 1,25(OH)2D3-treated cells has the same inhibition characteristics to heat and amino acids as that of untreated cells. Synthetic human parathyroid hormone stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) approximately 100-fold within five minutes. However, the stimulation was not observed with a synthetic human thyrocalcitonin. When HuO9 cells were transplanted into the back of a nude mouse, a tumor with an abundant osteoid formation and mineralization was produced. The results indicate that the HuO9 cell line expresses well-differentiated osteoblastic phenotypes. HuO9 is the first established human cell line to produce BGP, and it provides a useful model for the studies of osteoblasts and the regulatory mechanisms of BGP production.