裸鼠脊髓转移灶粘液表皮样癌细胞系Ms的建立及生物学特性

目的：建立Mc3细胞的脏器转移细胞系。方法：采用瘤细胞尾静脉注射法、组织块细胞培养法．从裸鼠体内获得Mc3细胞系脊髓转移细胞亚系。用染色体最示、细胞形态及细胞致瘤性观察证明其性质；用细胞生长曲线、流式细胞仪测定增值速度。结果：从50只受试课鼠中获得一只截瘫的裸鼠．取其脊髓组织行原代及传代培养获得细胞并建立细胞系．传代50次以上．生长稳定．倍增时间为13 h．s期细胞占22．7％．染色体众数为61，保持人类染色体形态、细胞形态与其母细胞相似．相差显微镜观察活细胞晕”铺路石”样外观。裸鼠肺内转移肿瘤组织切片显示为粘液表皮样癌．将该细胞命名为Ms。结论：Ms细胞系是Mc3细胞系在裸鼠体内的脊髓转移细胞亚系．其生物学性质与Me3细胞小不同     

Docetaxel对涎腺粘液表皮样癌高转移细胞增殖及转移力的抑制作用

目的 :研究Docetaxel对涎腺粘液表皮样癌高转移细胞M3 SP4 增殖及转移力的抑制作用。方法 :用细胞计数法、克隆形成法、流式细胞术、癌细胞裸鼠尾静脉注射法、癌细胞裸鼠颌下腺原位接种法研究Docetaxel对M3 SP4 细胞增殖及转移力的抑制作用。结果 :Docetaxel对M3 SP4 细胞具有浓度及时间依赖性生长抑制作用 ,作用 72h后 ,IC3 0 和IC50 分别为 0 .34nmol/L和 0 .6 3nmol/L ;IC3 0 浓度的药物作用于M3 SP4 细胞 ,对照组及处理组细胞群体倍增时间分别为 32 .7h和 43h ;对照组及药物作用浓度分别为 0 .0 5nmol/L及 0 .1nmol/L时 ,克隆形成率为 ( 2 9.2± 1.4) %和 ( 2 0 .2± 0 .8) % ,( 2 .8± 0 .4) % ;在裸鼠体内实验中对照组及治疗组〔30mg/(kg·周 )〕肺表面的转移结节数为 11± 3.4和 0 ;裸鼠颌下腺重量 ( g)为 1.2 0± 0 .2 3和 0 .31±0 .0 5。结论 :Docetaxel可明显抑制M3 SP4 细胞的增殖及转移力。     

涎腺腺样囊性癌裸鼠肺转移灶细胞时相分析及定量形态学研究

本研究利用流式细胞仪及Ｖｉｄａｓ自动图象分析系统，检测了涎腺腺样囊性裸鼠肺转移灶细胞周期时相分布及其定量形态学参数，结果表明：肺转移灶细胞Ｓ期明显低于皮下移植癌细胞（Ｐ＜０．０１），而Ｇ０／Ｇ１期高于皮下移植癌；肺转移灶细胞之核浆比、核面积、核周长、等效径、形态因子等定量形态学参数提示肺转移灶细胞趋向高分化。本研究结果对于解释涎腺腺样囊性癌临床生物学行为及制定临床治疗计划均有重要意义。     

外源性PTEN基因诱导黏液表皮样癌细胞系凋亡的研究

目的：探讨转染外源性PTEN抑癌基因对黏液表皮样癌细胞系M3SP2的诱导凋亡作用。方法：用含野生型PTEN抑癌基因逆转录病毒载体转染高转移性涎腺黏液表皮样癌细胞系M3SP2,转染空载体亲本细胞为对照细胞。体外细胞凋亡采用苏木精-伊红染色、电镜、TUNEL染色和流式细胞仪检测,裸鼠移植瘤细胞凋亡应用苏木精-伊红染色形态学判定。Bc l-2、P53和H-ras蛋白表达用免疫组化染色检测。结果：试验细胞M3SP2-PTEN与对照细胞M3SP2-pBp比较,出现较多的典型的凋亡细胞特征。试验细胞和对照细胞凋亡率分别为7.5%-13.6%和1.2%-2.3%;裸鼠移植瘤M3SP2-PTEN组和M3SP2-pBp组的凋亡指数分别为17.4%和3.5%。免疫组化染色显示M3SP2-PTEN细胞与对照细胞比较,P53蛋白表达增强,Bc l-2和H-ras蛋白表达减弱。结论：外源性PTEN抑癌基因能显著诱导高转移性涎腺黏液表皮样癌细胞凋亡,并且与P53、Bc l-2和H-ras蛋白表达有一定关系。     

涎腺腺样囊性癌裸鼠转移灶细胞时相分析及定量形态学研究

本研究利用流式细胞仪及Ｖｉｄａｓ自动图象分析系统，检测了涎腺样囊性裸鼠肺转移灶细胞周期时相分布及其定量形态学参数，结果表明：肺转移灶细胞Ｓ期明显低于皮下移植癌细胞，而Ｇ０／Ｇ１期高于皮下移植癌，肺转移灶细胞之核浆比，核面积，核周长，等效径，形态因子等定量形态学参数提示肺移灶细胞趋向高分化。本研究结果对于解释涎腺腺样囊性癌临床生物学行为及制定临床治疗计划均有重要意义。     

PTEN基因对粘液表皮样癌细胞裸鼠移植瘤生长的抑制作用

目的研究外源野生型PTEN抑癌基因对人高转移性粘液表皮样癌细胞裸鼠移植瘤生长的抑制作用。方法取PTEN抑癌基因转染粘液表皮样癌细胞的M3SP2-PTEN及逆转录病毒空载体转染的对照细胞M3SP2-pBp,接种于裸鼠背部皮下,测定移植瘤生长曲线,并对移植瘤进行组织学观察。结果对照组移植瘤生长迅速,肿瘤体积倍增时间为32.68h。HE染色切片显示,细胞数量多、排列紧密,核分裂相较多见;而实验组M3SP2-PTEN细胞移植瘤生长比较缓慢,肿瘤体积倍增时间延长为36.58h,肿瘤结节体积明显小于对照组(P<0.01),抑瘤率达63.82%。HE染色切片显示,细胞排列比较松散,较多的细胞出现变性和坏死,部分细胞染色体边集并出现凋亡小体。结论外源野生型PTEN抑癌基因能够显著抑制人高转移性粘液表皮样癌细胞在裸鼠体内的生长,其作用机制与PTEN基因诱导癌细胞变性和凋亡有关。     

雌激素受体在人粘液表皮样癌细胞系中的表达

目的:测定雌激素受体在人涎腺粘液表皮样癌细胞系Mec-1以及高转移株Mc-3上的表达,为口腔肿瘤临床内分泌治疗提供依据.方法:用免疫细胞化学方法和RT-PCR方法检测雌激素受体在人涎腺粘液表皮样癌细胞上的表达,并对其表达量进行相对定量分析.结果:经免疫细胞化学和RT-PCR方法可检测到ER在人涎腺粘液表皮样癌细胞上有表达,并且Mc3细胞系ER表达强于MEC-1;两种细胞系ER-α表达均略强于ER-β.结论:雌激素有可能通过它的受体对人粘液表皮样癌细胞产生一定的影响.ERα/ERβ比率升高在粘液表皮样癌发生过程中可能起一定作用, ER的表达升高可能与肿瘤转移力有关.     

涎腺疾病

水通道蛋白-1，5在干燥综合征小鼠下颌下腺中的表达;：17β-雌二醇对涎腺黏液表皮样癌高转移细胞Mc3黏附、侵袭和移动力的影响;治疗角结膜干燥症自体颌下腺移植的血管处理;雌二醇对人涎腺腺样囊性癌细胞增殖和转移作用的研究;异丙肾上腺素对SD大鼠原代颌下腺细胞增殖的影响     

17β-雌二醇对涎腺黏液表皮样癌高转移细胞M3SP4的作用

目的:研究17β雌二醇(E2)对涎腺黏液表皮样癌高转移细胞系M3SP4细胞的作用。方法:采用细胞记数法、MTT、软琼脂克隆形成法及相关癌基因免疫组织化学的方法研究17β雌二醇对M3SP4细胞的增殖和对VEGF、cerbB2、Ki67、P16的影响。结果:雌激素在10-10～10-5mol/L时对M3SP4有促进作用,其中在10-7mmol/L时对M3SP4的促增殖作用最显著,体外实验表明细胞群体倍增时间减少了10.8%,克隆形成率增加了225%,免疫组化显示VEGF、cerbB2、Ki67的表达增强,而P16的表达明显降低。体内实验,实验组裸鼠在每天给予0.0052mg/d的E2时,肿瘤细胞M3SP4的生长增加65%,转移增加了609%。VEGF、Ki67和cerbB2有较强表达,明显强于对照组;相反抑癌基因P16的表达较弱。结论:雌激素可刺激涎腺黏液表皮样癌M3SP4细胞的增殖和转移能力。     

PTEN抑癌基因对黏液表皮样癌实验性裸鼠肺转移的抑制作用

目的　探讨外源野生型PTEN抑癌基因对人高转移性黏液表皮样癌M3SP2细胞系实验性裸鼠肺转移能力的影响。方法　用脂质体介导方法将PTEN抑癌基因导入M3SP2细胞系 (M3SP2 -PTEN细胞 ) ,转染空载体的细胞系作为对照M3SP2 -pBp细胞 ) ,应用裸鼠肺转移实验测定癌细胞的体内转移能力 ,并进行组织学观察。结果　对照组裸鼠肺转移结节数为 (2 4 0± 1.2 )个 ,组织学观察瘤细胞转移灶体积大 ,细胞生长活跃 ,可见较多核分裂。实验组裸鼠肺转移结节数量减少为 (8 5± 3 4 )个 ,转移抑制率为 6 5 6 % ;组织学观察可见转移灶数量少、体积小 ,并有较多的坏死和凋亡细胞。结论　转染野生型PTEN抑癌基因能降低人高转移性黏液表皮样癌细胞转移能力。     

唾液腺黏液表皮样癌干细胞的分离、鉴定及生物学特征分析

目的: 筛选出唾液腺黏液表皮样癌细胞的特异基因,进一步了解肿瘤干细胞的成瘤机制。方法: 通过有限稀释法对肿瘤细胞行单细胞培养,观察肿瘤干细胞单克隆生长的情况。应用免疫磁珠分离技术,确定唾液腺黏液表皮样癌干细胞的相对特异性表面标志。采用SPSS 11.5软件包对数据进行统计学分析。结果: CD133在唾液腺黏液表皮样癌细胞株M3SP2单克隆细胞株中表达较高,具有显著差异（P<0.05）。结论: CD133可能是唾液腺黏液表皮样癌细胞株M3SP2单克隆细胞株的表面特异性抗原标记物或组合之一。     

TGF‐β1 regulates the invasive and metastatic potential of mucoepidermoid carcinoma cells

J Oral Pathol Med (2011) 40 : 762–768 Background: Patients with mucoepidermoid carcinoma exhibit poor long‐term prognosis because of the lack of therapeutic strategies that effectively block tumor progression. We have previously characterized the Ms cells as a highly metastatic mucoepidermoid carcinoma cell line that expresses high levels of transforming growth factor β1 (TGF‐β1). Here, we studied the effect of suppressing TGF‐β1 by RNA silencing on the invasive and metastatic potential of mucoepidermoid carcinoma. Methods: Cell motility, substratum adhesion, and transmembrane invasion were estimated by migration, matrigel adhesion, and matrigel invasion assay. Matrix metalloproteinase (MMP)‐2 and MMP‐9 activity were determined using gelatin gel zymography. Balb/c nu/nu nude mice lung metastatic model was used to test the metastatic ability of the Ms cells. Lung metastatic tumors were experimentally induced by mice tail vein inoculation of cancer cells. Results: TGF‐β1 silencing inhibits cell motility, substratum adhesion, and transmembrane invasion. In vivo, a significant decrease in lung metastasis was observed when mice received tail vein injections of TGF‐β1‐silenced mucoepidermoid carcinoma cells, as compared to controls. Conclusion: These results unveil a critical role for TGF‐β1 in the progression of mucoepidermoid carcinomas and suggest that patients with this malignancy may benefit from therapeutic inhibition of the effectors of the TGF‐β1 pathway.     

Variant sublines with different metastatic potentials selected in nude mice from human oral squamous cell carcinomas

Variant sublines LMF3, LMF4 and LMF5 with high metastatic potential were established from a human oral squamous carcinoma cell line HSC-3. These sublines metastasized to the draining lymph nodes after subcutaneous inoculation into nude mice. They were obtained by sequential selection in vivo from the parent HSC-3. At each step, the cells which metastasized to lymph nodes were cultured and reinoculated into nude mice. Two other cell lines HSC-2 and HSC-4 were also established from other patients, that had neither invasive nor metastatic potential. Biologic properties were compared among high metastatic, low metastatic and non-metastatic cells. Metastatic cells grew rapidly and invaded into surrounding tissues at the inoculated site. The incidence of pulmonary colonization after intravenous injection of tumor cells was high in selected variants. Metastatic cells formed diffuse colonies in type I collagen matrix and had a higher tendency to adhere to type IV collagen network.     

PTEN抑癌基因对高转移性黏液表皮样癌细胞系生物学行为的影响

目的　探讨外源野生型PTEN抑癌基因对人高转移性黏液表皮样癌细胞系M3SP2体外黏附、迁移和侵袭特性的影响。方法　用脂质体介导方法将PTEN抑癌基因导入M3SP2细胞系 (M3SP2 -PTEN细胞 ) ,转染空载体的细胞系作为对照(M3SP2 -pBp细胞 ) ,分别用四甲基偶氮唑蓝 (MTT)比色法、细胞迁移试验及细胞侵袭试验测定M3SP2 -PTEN细胞和M3SP2 -pBp细胞体外黏附、迁移和侵袭能力。结果　M3SP2 -PTEN细胞在Metrigel和Fn基质上黏附数量减少 ,与对照组比较差异有显著性 (P <0 0 1) ,黏附抑制率分别为 34 3%和 4 9 4 %。M3SP2 -PTEN细胞在Matrigel基质上迁移距离小 ,与对照组比较差异有显著性 (P <0 0 5 ) ,迁移抑制率为 2 6 0 %。M3SP2 -PTEN细胞体外侵袭细胞数量减少 ,与对照组比较差异有显著性 (P <0 0 1) ,侵袭抑制率为 31 4 %。结论　外源性PTEN抑癌基因对高转移性黏液表皮样癌细胞系M3SP2体外黏附、迁移和侵袭具有抑制作用。     

外源性PTEN抑癌基因对高转移性黏液表皮样癌细胞系形态的影响

目的 :观察外源野生型PTEN基因对高转移性黏液表皮样癌细胞系形态的影响。方法 :应用倒置显微镜观察法、HE染色观察、扫描电镜及透射电镜观察法 ,比较野生型PTEN抑癌基因转染的黏液表皮样癌细胞M 3SP2 PTEN与对照的黏液表皮样癌细胞M 3SP2 pBp的形态学差异。 结果 :M 3SP2 pBp细胞体外生长活跃 ,细胞数量多 ,核分裂相多 ,细胞表面微绒毛丰富 ,细胞核切迹明显 ,细胞质中线粒体丰富 ;M 3SP2 PTEN细胞生长缓慢 ,分裂相很少 ,细胞数量少 ,部分细胞空泡变性 ,染色质凝集 ,细胞膜彭出 ,线粒体数量少并发生肿胀 ,较多的溶酶体融合。结论 :外源野生型PTEN基因的转染能导致体外培养的高转移性黏液表皮样癌细胞变性、坏死或凋亡。     

A cervical lymph node metastatic model of human tongue carcinoma: Serial and orthotopic transplantation of histologically intact patient specimens in nude mice.

PURPOSE: A lymph node metastatic model of human tongue carcinoma using orthotopic and serial transplantation was established in nude mice to study the invasive and metastatic properties of human tongue cancer. Materials and Methods: Lymph node metastatic specimens of human tongue carcinoma were transplanted into nude mice orthotopically. Tumors dissected from the metastatic lymph nodes of the nude mice were serially transplanted into tongues of disease-free nude mice at 4-week intervals. RESULTS: All mice developed aggressive and diffuse well-differentiated squamous cell carcinoma at tongue recipient sites. Tumor cells invaded to lymphatic vessels. In addition, increased cervical lymph node metastasis was noted in the first (3 of 14, or 21%), second (4 of 11, or 36%), third (6 of 10, or 60%), or fourth (11 of 14, or 79%) transplantation. In mice, 2 of 14 lung metastases were found in the fourth round of transplantation. CONCLUSION: After surgical specimens of the lymph node metastasis for human tongue cancer were transplanted into the tongue of nude mice, the clinical characteristics of human tongue carcinoma, especially invasion and metastasis, were observed. This metastatic model involving orthotopic and serial transplantation should be useful for studies on the mechanisms, treatment, and prevention of human carcinoma of tongue.     

Establishment and characterization of metastasizing cell lines from a heterotransplanted human adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is a rare malignant tumor of the head and neck occurring in the salivary glands. We established a human ACC line which is serially transplantable in nude mice and designated it as KOA-1. The KOA-1 tumor doubled in 9.3 days and retained the histologic characteristics of a solid pattern of ACC even after 22 serial passages. The KOA-1 metastasized to the lung 2 months after subcutaneous transplantation into nude mice. We further established a subline of KOA-1 by an in vivo selection method and named it KOA-1L3. The KOA-1L3 doubled in 1.3 days and metastasized to the lung within 1 week after subcutaneous transplantation. These tumor lines may serve as a useful model for exploration of the biological behavior and treatment of human ACC.