裸小鼠原位移植筛选高转移性人骨肉瘤细胞及其生物 …

目的 在裸小鼠体内筛选人骨肉瘤高肺转移性细胞亚群,为实验研究提供模型。方法 利用原位移植方法,将人骨肉瘤细胞系ＳＯＳＰ－９６０７皮下移植瘤接种于裸鼠胫骨骨髓腔。筛选到第２代时,有１例发生肺转移,以皮下扩增转移灶,再次原位移植,第３代肺转移率达８３％（５／６）。用组织块法培养收集。比较其与纱在形态结构、细胞核型,ｖｉｍｅｎｔｉｎ,ａｃｔｉｎ,ＮＳＥ抗原表达及体积倍时间待贩差别。结果 同母系相比较,该     

高转移性人成骨肉瘤细胞系在裸小鼠体内的筛选和维持

目的：筛选人成骨肉瘤高转移细胞并探讨维持其高转移性状的方法。方法：采用低转移性成骨肉瘤细胞系在裸小鼠腹腔内连续传代和体外培养肺转移灶筛选高转移亚系,通过体内、外交替传代维持其高转移性状,并用流式细胞仪、染色体分析等方法,研究该高转移亚系的细胞周期、形态、增殖等生物学特性。结果：筛选所得腹水型高转移亚系自发性肺转移率达100％。在细胞形态、增殖速度、染色体数目等方面与母系有较大差别,经体内、外交替传代高转移性状得以维持,且出现较高比例的脑、骨、肌肉转移。结论：腹腔连续传代培养转移灶和体内、外交替传代是筛选和维持高转移细胞的可靠方法。     

胃腺癌细胞侵袭亚系的筛选及母、亚两系生物学特性的比较

应用人羊膜成功地筛选了侵袭性较强的胃腺癌细胞亚系，并比较了母、亚两系某些生物学特性。实验证明亚系细胞体外移动性和体内移植瘤的生长率及转移率均明显高于母系细胞。结论：（１）羊膜基底膜可作为筛选某些肿瘤细胞亚系的有效侵袭滤膜；（２）胃腺癌细胞系具有肿瘤异质性；（３）筛选的亚系细胞较母系细胞具有明显的高侵袭力和高转移力。本文对肿瘤异质性与侵袭机理进行了探讨。     

胃腺癌细胞侵袭亚系的筛选及母,亚两系生物学特征的比较

应用人羊膜成功地筛选了侵袭性较强的胃腺癌细胞亚系，并比较了母、亚两系某些生物学特性。实验证明亚系细胞体外移动性和体内移植瘤的生长率及转称率均明显高于母系细胞。结论：（１）羊膜基底膜可作为筛选某些肿瘤细胞亚系的有效侵袭滤膜；（２）胃腺癌细胞系具有肿瘤异质性；（３）筛选的亚系细胞较母系细胞具有明显的高侵袭力和高转移力。本文对肿瘤异质性与侵袭机理进行了探讨。     

胃癌单克隆细胞球和CD44＋及CD133＋亚群成瘤能力的比较

目的研究胃癌干细胞亚群的筛选方法,探讨建立胃癌干细胞稳定亚群的可行性。方法利用流式细胞仪从MKN45、MKN25、SGC7901细胞株和人胃癌组织中分选出不同CD44和CD133表型的亚群,比较不同亚群和无血清培养基培养的单克隆细胞球细胞在裸鼠移植瘤模型中的成瘤能力。结果不同细胞的CD44＋和CD133＋亚群在低数量级时几乎不具有裸鼠皮下成瘤能力,在高数量级接种时成瘤能力与母系细胞差异无统计学意义;单克隆细胞球细胞在各数量级下的成瘤能力、瘤体积和生瘤速度均显著高于母系细胞。结论与CD44和CD133等细胞表面标志物筛选法相比,单克隆细胞球细胞可更好的作为胃癌干细胞研究的细胞学基础。     

骨肉瘤动物模型的建立

背景：为进一步研究骨肉瘤的发病机制,国内外学者通过不同方法建立了多种骨肉瘤动物模型。 目的：对骨肉瘤模型的建立方法以及这些模型的特点进行综述。 方法：应用计算机检索国内外数据库中关于骨肉瘤动物模型的文章,最终选择17篇文献进行综述。 结果与结论：骨肉瘤模型主要分为实验性动物模型和自发性动物模型,目前大多骨肉瘤模型多属于实验性动物模型,其建立方法主要包括放射性核素诱导、化学物质诱导以及生物致瘤因素诱导、异体移植等。对肺高转移细胞进行筛选并建立相应模型的方法主要有3种,体内筛选、体外筛选、体内外筛选共同建立模型。不论是建立骨肉瘤细胞系还是动物模型,一般都需要体外重复传代培养,体内多次筛选等处理,不同学者虽然建立方法大致相同,但处理细节过程不同,这样建立的动物模型稳定性到底如何,目前没有统一的认识和标准。     

小鼠肺腺癌细胞系四株克隆化亚系的转移潜能研究

本文对小鼠肺腺癌细胞系（ＬＡ－７９５）进行单细胞克隆化培养，分离；经筛选获得具有裸小鼠体内移植自发肺转移力相异的４株克隆化亚系，说明母系确实在存在转移潜在异质性，体外实验比较此四株亚系的结果显示：癌细胞的转移潜能不仅与其细胞增殖，分裂及胞浆骨架分布的不均紊乱程度有关（Ｐ〈０．０１），而且与其胞浆内架聚集于胞核－侧及内源性非特异性酯酶的活性表达有关（Ｐ〈０．０１）；同时，高转移潜能亚系比转移潜能亚系     

人鼻咽癌CNE－2Z细胞的不同转移潜能克隆株在严重联合免疫缺陷小鼠体内的检验和比较研究

将人类鼻咽癌不同克隆株的细胞悬液移植在严重联合免疫缺陷（ＳＣＩＤ）小鼠和ＢＡＬＢ／ｃ（ｕｎ／ｕｎ）裸小鼠的颈背侧皮下，５６ｄ后处死全部动物进行观察。结果发现，在ＳＣＩＤ小鼠体内移植后ＣＮＥ２Ｌ２为高转移克隆株，其淋巴结转移率为１００％，肺转移率为７１％；而ＣＮＥ２Ｌ４为低转移克隆株，其肺转移率为１３％，淋巴结未见癌转移。这是从１个细胞母系中新筛选出的１个高转移和１个低转移的癌细胞克隆株。实验结果还显示，同ＢＡＬＢ／ｃ（ｕｎ／ｕｎ）裸小鼠相比，ＳＣＩＤ小鼠的恶性表型的表达能力高。另外，皮下移植时肿瘤细胞的数量可能与转移表型的表达有相关性，移植的瘤细胞数越多，转移率越高，反之亦然。     

人鼻咽癌CNE—2Z细胞的不同转移潜能克隆株在严重联合免疫缺…

将人类鼻咽癌不同克隆株的细胞悬液植在严重联合免疫缺陷（ＳＣＩＤ）小鼠和ＢＡＬＢ／ｃ（ｎｕ／ｎｕ）裸小鼠的颈背侧皮下，５６ｄ后处死全部动物进行观察，结果发现，在ＳＣＩＤ小鼠体内移植后ＣＮＥ２Ｌ２为高转移克隆株，其淋巴结转移率为１００％，肺转移率为７１５；而ＣＮＥ２Ｌ４为低转移克隆株，其肺转移率为１３％，淋巴结未见癌转移。这是从１个细胞母系中新筛选出的１个高转移和１个低转移的癌细胞克隆株。实验结果还显     

鞘糖脂微结构域1相关磷酸化蛋白基因的筛选及其对转移性前列腺癌细胞生物学行为的影响

目的 筛选肿瘤转移相关新基因,探讨鞘精脂微结构域1相关磷酸化蛋白基因(PAG1)转染对人前列腺癌细胞系PC-3M的高转移亚系PC-3M-1E8体外生物学行为的影响.方法 利用PC-3M高转移亚系PC-3M-1E8、低转移亚系PC-3M-284,人肺巨细胞痛细胞系(PG)高转移亚系PG-BE1和低转移亚系PG-LH7 cDNA制作4张基因芯片,筛选出PC-3M和PG高、低转移亚系共同差异表达基因.对在两个转移亚系共同表达下调的PAG1基因做进一步研究,采用即时定量PCR及Western blot验证PAG1在PC-3M细胞系中的表达.构建pcDNA3.0-PAG1真核表达载体,稳定转染PC-3M-1E8细胞.MTT比色实验及软琼脂集落形成实验检测肿瘤细胞体外增殖能力;流式细胞术检测肿瘤细胞周期及凋亡;Matrigel穿膜实验检测肿瘤细胞体外侵袭能力.结果 基因芯片初步筛选出PC-3M高、低转移亚系差异表达基因共327个,上调基因123个,下调基因204个.PG高、低转移亚系差异表达基因共281个,上调基因167个,下调基因114个.PC-3M与PG高转移亚系共同表达下调基因9个、上调基因8个.即时定量PCR及Western blot证实PAG1在PC-3M高转移亚系中表达低于低转移亚系.MTT比色及软琼脂集落形成实验显示转染pcDNA3.0-PAG1组细胞增殖速度明显低于转染空载体组和未转染组(均P<0.05).细胞周期检测转染pcDNA3.0-PAG1组比转染空载体组和未转染组处于G_0～G_1期的细胞百分数明显增加(P<0.05).转染pcDNA3.0-PAG1组与转染空载体组和未转染组相比凋亡细胞百分率无显著差异(P>0.05).体外穿膜侵袭实验结果表明转染pcDNA3.0-PAG1组比转染空载体组和未转染组穿膜细胞数目明显减少,分别为(35.1±4.9)、(127.6±6.6)和(135.0±5.0)个(P<0.05).结论 利用同一母系来源的高、低转移亚系制作基因芯片可以摒除转移无关基因的干扰,筛选出差异表达的肿瘤转移相关基因.PAG1基因稳定转染能抑制人前列腺癌高转移亚系PC-3M-1E8细胞的体外增殖能力和侵袭能力,PAG1基因可能是一个潜在的肿瘤增殖、侵袭和转移的抑制基因.     

Establishment and characterization of a new highly metastatic human osteosarcoma cell line

Osteosarcoma is the most common primary malignancy of bone in children and young adults. There is a paucity of tumorigenic and highly metastatic human osteosarcoma cell lines that have not been further transformed by exogenous means. Here we establish and characterize a highly metastatic human osteosarcoma cell line that is derived from a poorly metastatic MG63 line through serial passage in nude mice via intratibial injections. The occasional pulmonary metastases developed from MG63 were harvested and repassaged in mice until a highly metastatic subline (MG63.2) was established. The parental MG63 and highly metastatic MG63.2 cells were further characterized in vitro and in vivo. MG63.2 cells demonstrated increased cell migration and invasion compared to the parental MG63 cells. Conversely, cell adhesion was significantly greater in MG63 cells when compared to the MG63.2 cells. MG63.2 cells grew at a slightly slower rate than that of the parental cells. When injected into nude mice, MG63.2 cells had a greater than 200-fold increase in developing pulmonary metastases compared to the parental MG63 cells. MG63.2 cells also formed larger primary tumors when compared to the parental MG63 cells. Further analysis revealed that ezrin expression was up-regulated in the metastatic MG63.2 cells. Interestingly, expressions of MMP-2 and MMP-9 were down-regulated, and expression of TIMP-2 was up-regulated in the MG63.2 cells. Taken together, we have established and characterized a highly metastatic human osteosarcoma cell line that should serve as a valuable tool for future investigations on the pathogenesis, metastasis, and potential treatments of human osteosarcoma.     

A novel spontaneous metastasis model of human osteosarcoma developed using orthotopic transplantation of intact tumor tissue into tibia of nude mice

Evaluation of potential new treatment strategies requires adequate experimental tumor models which resemble the clinical situation as closely as possible. The purpose of the present study was to establish a new human osteosarcoma spontaneous metastasis model using orthotopic transplantation of histologically intact tumor tissue into the tibia of nude mice. Intact tumor pieces, obtained from the 32nd serial passage of subcutaneously growing human osteosarcoma xenografts, were implanted into the proximal tibia in 31 nude mice. Animals were sacrificed and autopsied 2, 4, 6, and 8 weeks after transplantation and examined macroscopically and microscopically for local tumor growth and metastases. All mice developed local intratibial bone tumors that were radiographically and histologically similar to primary human osteosarcoma. Lung metastases were observed in all mice, local and distant lymph node metastases in 15 (48%), and liver metastases in 6 (19%) mice. The microscopic appearance of the metastases was similar to that observed in the donor patientÕs tumor, corresponding subcutaneous xenografts and orthotopically transplanted intratibial tumors. This spontaneous metastasis model of human osteosarcoma in nude mice may resemble a clinical situation and could thus be useful for studies on local tumor growth, metastasis formation and therapy.     

Establishment of human osteosarcoma cell lines with high metastatic potential to lungs and their utilities for therapeutic studies on metastatic osteosarcoma

Relevant animal models for metastasis of osteosarcoma is needed to understand the biology and to develop the treatment modality of metastasis of human osteosarcoma. Therefore, we screened six human osteosarcoma cell lines for metastatic ability in nude mice. The HuO9 cell line was identified as being metastatic to the lung after intravenous injection. We established two sublines, HuO9-M112 and HuO9-M132, with high metastatic potential to the lung from the parental HuO9 cells by in vivo selection. There were no differences between these two sublines and the parental cells in the growth rate in vitro and the tumorigenicity after subcutaneous injection in nude mice, however, mice injected with the metastatic sublines became moribund earlier than mice injected with the parental HuO9 cells did. Thus, adriamycin (ADR) and recombinant interleukin-12 (IL-12) were administered to mice injected with the HuO9-M112 subline to suppress experimental lung metastases. Production of lung colonies was significantly suppressed and the prognoses of mice were significantly improved by both ADR and IL-12 treatments. These results indicate that both ADR and IL-12 are effective agents against pulmonary metastatic osteosarcoma, and that these sublines are useful for studies on the biological behavior and treatment of pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

An Orthotopic Model of Human Osteosarcoma Growth and Spontaneous Pulmonary Metastasis

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. In order to investigate the pathogenesis of human osteosarcoma, there is a great need to develop a clinically relevant animal model. Here we report the development of an osteosarcoma animal model using three related human osteosarcoma lines, the parental TE-85 and two derivative lines MNNG/HOS and 143B. In vitro characterization demonstrated that the 143B line had the greatest cell migration and the least cell adhesion activities among the three lines. The 143B line also exhibited the greatest ability for anchorage independent growth. When GFP-tagged osteosarcoma cells were injected into the proximal tibia of athymic mice, we found that 143B cells were highly tumorigenic and metastatic, and MNNG/HOS cells were tumorigenic but significantly less metastatic. TE85 cells were neither tumorigenic nor metastatic. The number of pulmonary metastases was found 50-fold higher in 143B injected animals than that in MNNG/HOS injected mice. No pulmonary metastases were detected in TE85 injected animals for up to 8 weeks. Primary tumors formed by MNNG/HOS and 143B cells could be visualized by whole body fluorescence imaging, while the pulmonary metastases were visualized on the necropsied samples. The GFP tagged 143B cells (and to a lesser extent, MNNG/HOS cells) were readily recovered from lung metastases. This clinically relevant model of human osteosarcoma provides varying degrees of tumor growth at the primary site and metastatic potential. Thus, this orthotopic model should be a valuable tool to investigate factors that promote or inhibit osteosarcoma growth and/or metastasis.     

人绒毛膜癌裸鼠原位移植模型的建立

目的建立人绒毛膜癌裸鼠原位移植瘤模型。方法培养人绒毛膜癌细胞系JAR,制备JAR单细胞悬液,给5只8周龄BALB/c裸鼠经皮下注射建立皮下移植瘤模型。待裸鼠皮下成瘤后,无菌条件下取瘤组织并切成1 mm^3组织块,通过手术方式植入10只10周龄BALB/c裸鼠子宫腔内,裸鼠濒死状时4%水合氯醛(10 g/kg)腹腔注射麻醉处死,观察子宫成瘤及腹腔转移情况。解剖取子宫原位移植瘤、腹腔内转移瘤、腹腔淋巴结及其他脏器组织标本,通过组织病理学检查进行鉴定。结果10只BALB/c裸鼠中共有7只裸鼠子宫内可见移植瘤肿块形成,其中2只可同时观察到子宫移植瘤和腹膜转移瘤。在病理学形态和结构上,皮下移植瘤模型、原位移植模型和腹膜转移瘤的瘤细胞与人绒毛膜癌细胞系JAR一致。结论成功建立人绒毛膜癌JAR细胞的BALB/c裸鼠原位移植瘤模型。     

High collagenolytic activity in spontaneously highly metastatic variants derived from a human pancreatic cancer cell line (SUIT-2) in nude mice

Cell lines with high metastatic capacity to the lung were established by sequential passage of a human pancreatic cancer cell line (SUIT-2) through the lung of a nude mouse, via the lateral tail vein and from a subcutaneous inoculum. Cells of the parental SUIT-2 and sublines S2-VPx (x-cycle selection from SUIT-2 cells, by Vein-Pulmonary metastasis-culture) and S2-CPx (x-cycle selection, by Cutis-Pulmonary metastasis-culture) were injected intravenously or subcutaneously into nude mice to produce experimental or spontaneous lung metastasis. The S2-VP10 cell line produced pulmonary metastases in 100% of the nude mice, when injected intravenously. It failed, however, to produce more lung colonies than its parent cell line, when injected subcutaneously. The S2-CP8 cell line produced extensive pulmonary metastases in 100% of the nude mice, when injected either intravenously or subcutaneously. This study indicates that the nude mouse provided a good model for in vivo selection of metastatic cells from SUIT-2 cells both experimentally and spontaneously, and that the SUIT-2, S2-VPx, and S2-CPx cell lines will be valuable in the study of human cancer metastasis. We previously reported high levels of ezrin expression in the S2-VP10 and S2-CP8 cell lines. Here we show that these cell lines exhibit a greater capacity to invade or attach to various extracellular matrix components than the parent SUIT-2 cells. The S2-CP8 cell lines also exhibit greater level of type-I and type-IV collagen-degrading activity than the parent SUIT-2 cell line and the S2-VP10 cell line, which shows similar collagen-degrading activity to the parent SUIT-2 cells. In RT-PCR studies, SUIT-2, S2-CP8 and S2-VP10 cell lines constitutively expressed many matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP7, MMP-9, MMP-10 and MMP-14). These results suggest that some parameters that enhance adhesion and invasion are important to both experimental and spontaneous metastasis and the collagen degrading enzymes are predicted to play a key-role during spontaneous metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

A nude mouse model of human osteosarcoma lung metastases for evaluating new therapeutic strategies

The purpose of these studies was to develop a metastatic osteosarcoma nude mouse model to evaluate the in vivo efficacy of new therapeutic compounds. Human SAOS-2 osteosarcoma cells (10⁶ cells) were injected i.v. into nude mice. Cells isolated from a rare pulmonary metastases 6 months later were established (SAOS-LM1) in culture and re-injected. This procedure was repeated 5 additional times to produce the SAOS-LM6 cell line. Visible pulmonary nodules were present 8 weeks following i.v. injection of 10⁶ SAOS-LM6 cells as compared to 17 weeks using SAOS-LM2 cells. Microscopic SAOS-LM6 pulmonary metastases were demonstrated at 6 weeks. Administration of adriamycin on week 9 resulted in regression of macroscopic SAOS-LM6 lung tumors. The ability of the model to be used to evaluate the effectiveness of a biologic agent against microscopic disease was also verified. It was concluded that this model can assess therapeutic efficacy and therefore, may have a role in investigating the potential of novel approaches aimed at eliminating pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

ESTABLISHMENT OF A NEW HUMAN CHONDROSARCOMATOUS CELL LINE

A new cultured cell line (HuOS) was established from tumor cells obtained from the pulmonary metastatic foci of a patient diagnosed as chondroblastic osteosarcoma. The tumor cell line was maintained for over 19 months, and morphological and biological characteristics were studied. These cells retained their malignant properties and produced nodules when transferred intramuscularly to nude mice. Morphologically, these nodules revealed a chondromatous pattern.     

Establishment of a new human chondrosarcomatous cell line.

A new cultured cell line (HuOS) was established from tumor cells obtained from the pulmonary metastatic foci of a patient diagnosed as chondroblastic osteosarcoma. The tumor cell line was maintained for over 19 months, and morphological and biological characteristics were studied. These cells retained their malignant properties and produced nodules when transferred intramuscularly to nude mice. Morphologically, these nodules revealed a chondromatous pattern.