High Five昆虫细胞系的致瘤性研究

目的检测High Five昆虫细胞系对BALB/c裸鼠致瘤性情况,以确保其作为疫苗生产细胞株的安全性。方法将裸鼠随机分为5组,分别为High Five基础细胞库细胞试验组、High Five最高限制代次细胞试验组、HEp-2细胞阳性对照组、CEF细胞阴性对照组和不作任何处理的空白对照组,用细胞悬液背部皮下接种裸鼠。3周和12周后对裸鼠解剖及病理组织学检查,其是否有肿瘤形成。结果阳性对照HEp-2细胞组裸鼠接种后3周注射部位形成结节,病理组织学检查为鳞状细胞癌;阴性对照CEF细胞组和High Five细胞组均无结节形成,接种后12周经解剖用病理组织学检查,均无肿瘤形成。结论基础代次和最高限制代次High Five细胞均不具有致瘤性,可安全地应用于疫苗生产。     

人脐带间充质干细胞体内移植的安全性研究

目的:探讨人脐带间充质干细胞(hUCMSC)的成瘤性及其对荷瘤鼠肿瘤生长的影响。方法:分离培养hUCMSC,取第6代细胞裸鼠皮下移植,观察其成瘤性;对荷瘤鼠尾静脉注射移植hUCMSC,观察其对肿瘤生长的影响;体外共培养hUCMSC和MCF-7肿瘤细胞,观察hUCMSC对MCF-7细胞克隆形成率的影响。结果:hUCMSC裸鼠皮下移植30 d,未观察到有肿瘤形成;尾静脉注射移植hUCMSC对荷瘤鼠肿瘤的生长无明显影响;体外共培养结果表明,hUCMSC对MCF-7肿瘤细胞的克隆形成无明显影响。结论:hUCMSC体内移植无成瘤性;静脉移植后对肿瘤生长无显著影响。     

亲骨转移乳腺癌细胞MDA-MB-231BO成瘤性的初步研究

目的通过比较亲骨转移乳腺癌细胞（MDA-MB-231BO）和亲代乳腺癌细胞（MDA-MB-231）的生长曲线和致瘤性,初步探讨MDA-MB-231BO细胞的生物学特性。方法MTT法测定两种细胞的生长曲线,并将两种乳腺癌细胞接种于裸鼠腋窝处皮下,建立乳腺癌细胞异种移植瘤动物模型,30 d后处死裸鼠,肿瘤组织及相关脏器官做病理检查。结果MTT法测得MDA-MB-231BO细胞生长速率高于MDA-MB-231细胞。接种两种乳腺癌细胞的裸鼠均长出肿瘤,成瘤率为100%。病理检查符合人乳腺癌细胞特征,MDA-MB-231BO组瘤体体积明显大于MDA-MB-231组（P〈0.05）。结论MDA-MB-231BO细胞生长速率高于MDA-MB-231细胞,而且MDA-MB-231BO在裸鼠体内的致瘤性强于MDA-MB-231。     

慢病毒介导荧光素酶表达的人鼻咽癌细胞小鼠模型的建立

目的：建立荧光素酶标记的人鼻咽癌细胞裸鼠模型,活体成像系统监测肿瘤的生长并与肿瘤的体积进行对比。方法：构建表达荧光素酶基因2（1uc2）的慢病毒载体,与辅助质粒共转染293T细胞以制备慢病毒,感染人鼻咽癌SUNEl细胞后经嘌呤霉素筛选获得表达luc2的细胞株。活体成像设备体外检测不同数量细胞的发光强度,最后以5×10 6个细胞皮下接种BALB／cnu／nu裸鼠,活体成像系统动态记录接种后肿瘤的信号并与肿瘤的体积对比。结果：成功构建慢病毒表达质粒pLenti．1uc2并包装出慢病毒颗粒,病毒感染后嘌呤霉素筛选6天得到鼻咽癌细胞株SUNEl一luc2。细胞株传代后有稳定的发光强度,且经活体检测的每秒光子数与细胞数成正相关（R2=0．96）;活体成像观察发现裸鼠接种第2天接种部位的发光强度就达到3-2×10^8,而且成瘤过程中发光强度的变化与肿瘤大小一致。结论：成功构建适用于活体成像的人鼻咽癌SUNEl细胞的裸鼠成瘤模型,该模型从细胞接种开始即可有效动态监测鼻咽癌皮下瘤的生长及转移,从而为鼻咽癌的成瘤机制及药物干预研究提供一个新的手段。     

乙型肝炎病毒和黄曲霉素协同作用诱发人肝细胞癌细胞株的建立

为了研究人乙型肝炎病毒（HBV）和黄曲霉素（AFB1）在肝癌发生过程中的作用,我们用HBV感染的人胚胎肝细胞移植至裸鼠背部皮下,以后每周皮下注射AFB1,在裸鼠体内成功地诱发了人肝细胞癌。选3只裸鼠所形成的肿瘤组织,分别再接种裸鼠传代。在裸鼠体内传至5代后,将瘤组织在体外培养、传代,建立了3个肝癌细胞株,分别为CBH－1a、CBH－1b和CBH－2。对3个细胞株进行生物学特性分析发现,细胞生长迅速,接触抑制消失,细胞增殖核主数Ki67阳性细胞占38．2％,用EMA单抗检测证实为人来源细胞,核酸原位杂交显示,细胞中HBV－X和HBV－S基因阳性,PCR可扩增出X基因,证明HBV基因已到瘤细胞中,3个细胞株细胞再接种裸鼠皮下,可再生成肿瘤,此实验证明了HBV协同AFB1在个肝细胞癌发生过程中的病因作用。同时,为进一步研究HBV和AFB1在肝癌发生过程中的分子机制提供了细胞水平的模型。     

过表达PTPα促进K562细胞体外增殖能力和体内致瘤性

探讨蛋白质酪氨酸磷酸酶α（PTPα）在血液肿瘤细胞中的特异性表达,研究过量表达PTPα对人红白血病细胞K562生物学行为的影响及在裸鼠体内致瘤能力的改变.首先应用RT-PCR和Western 印迹检测3种不同类型造血系肿瘤细胞（K562、NB4、Jurkat T）中PTPα的表达水平.根据检测结果,选择K562细胞作为研究对象,利用脂质体将 PTPα 真核表达载体转染K562细胞,通过G418筛选获得阳性克隆,RT-PCR和Western 印迹验证过表达情况;经MTT法检测细胞增值能力的改变;用流式细胞仪检测细胞周期分布和细胞分化状态;并将阳性克隆细胞皮下接种裸鼠,观察 PTPα 基因转染前后细胞系在裸鼠体内的致瘤能力及瘤体的组织化学变化.上述实验结果表明,通过G418压力筛选获得了 PTPα 高表达多克隆细胞系K562-PTPα;经体外增殖实验分析,实验组K562-PTPα细胞与未转染组细胞K562和转染空载体组细胞K562-splice相比,细胞生长速度增快,G₂/M期细胞比例增加( P <0.05),而细胞分化状态无明显变化;裸鼠体内致瘤实验显示,K562组、K562-splice组和K562 PTPα组平均瘤重分别为(1.1±0.3)g、(1.3±0.2)g和(2.5±0.5)g;病理切片显示,K562-PTPα组瘤体组织分化程度较对照组低、恶性程度高,细胞的致瘤能力增强( P <0.01).综上所述,过表达PTPα使K562细胞具有更强的体外增殖能力和体内致瘤性,表明PTPα可能在造血系统恶性肿瘤的发生发展中发挥促进作用.     

人卵巢浆液性囊腺癌永生化细胞系BUPH∶OVCA-3的建立及其生物学特性

介绍人卵巢浆液性囊腺癌永生化细胞系的建立 ,研究其生物学特性 .以卵巢浆液性乳头状囊腺癌的腹水细胞为材料 ,进行体外培养 .将永生化基因———SV4 0T抗原基因转染第 2代细胞 ,得到永生化细胞系 .通过光学显微镜、生长曲线测定、染色体分析、双层软琼脂培养、裸鼠接种、免疫组化等 ,研究其生物学特性 ,并与其来源细胞的生物学特性进行比较 .建立了一株人卵巢浆液性囊腺癌永生化细胞系 ,命名为BUPH∶OVCA 3,现已传至 6 0余代 .其生物学特性为 ,细胞生长旺盛 ;具有人体恶性细胞的核型特征 ;细胞恶性度较低 ,不具有集落形成能力及裸鼠接种致瘤性 ;除较未永生化细胞生长速率增快 ,饱和密度增加外 ,仍保留上皮细胞的分化表型 .结果表明 ,BUPH∶OVCA 3为一株恶性度较低的人卵巢浆液性囊腺癌永生化细胞系 ,保留其来源细胞的生物学特性 ,可作为研究恶性度较低的卵巢上皮癌的体外模型     

全反式维甲酸对U87-MG细胞裸鼠皮下移植瘤模型中血管生成拟态的影响

目的:明确全反式维甲酸对人恶性脑胶质瘤U87-MG细胞体内血管拟态形成的影响。方法:体外培养U87-MG细胞,并建立裸鼠皮下移植瘤模型,利用全反式维甲酸进行干预,检测荷瘤裸鼠肿瘤生长情况;利用HE染色对肿瘤组织进行细胞形态学观察;利用CD34-PAS双染法观察肿瘤血管拟态的形成情况。结果:U87-MG细胞在裸鼠体内成功荷瘤并出现血管拟态;全反式维甲酸显著抑制了U87-MG细胞实体瘤的生长和血管拟态的形成。结论:全反式维甲酸具有显著的抗肿瘤能力,其作用机制与血管拟态形成相关。     

中脑神经前体细胞的体外分离、培养和特性

为了解中脑神经前体细胞的体外培养特性和建立中脑神经前体细胞的体外分化调控机制提供细胞模型。本实验采用含有丝分裂源表皮生长因子(EGF)的无血清培养基培养来源于大鼠胚胎E14.5天的中脑神经前体细胞,应用免疫细胞化学方法了解其前体细胞特性。结果发现中脑神经前体细胞呈神经前体细胞特征性标记Nestin免疫染色阳性,无分化细胞标记;细胞克隆实验证实中脑神经前体细 胞有自我更新能力;在EGF刺激下增殖迅速;当撤去EGF后置于含胎牛血清的培养基和被覆多聚赖氨酸(PLL)的培养皿内,中脑神经前体细胞可分化成神经元和星形胶质细胞。本试验证明我们培养的中脑神经前体细胞具有增殖、自我更新能力和多向分化潜能特性。     

人脐带间充质干细胞对裸鼠的促瘤和致瘤性研究

目的研究人脐带间充质干细胞(hUCMSCs)对动物是否具有促瘤性,以及不同代次是否有致瘤性,为其临床应用提供安全性依据。方法分别用人淋巴瘤细胞Raji接种于CB-17 SCID小鼠皮下、用人结肠癌细胞WiDr接种于BALB/c裸鼠皮下,建立皮下移植瘤模型,WiDr成模动物用完全随机法分为4组(n=8):模型对照组、hUCMSCs低、中、高剂量组;Raji成模动物用完全随机法分为5组(n=7):空白对照组、模型对照组、hUCMSCs低、中、高剂量组。hUCMSCs均静脉注射给药,观察hUCMSCs对皮下移植瘤的增殖是否有影响。另外,观察不同代次的hUCMSCs是否有致瘤性,NOD/SCID小鼠分别皮下接种hUCMSCs的第2、6、10代的细胞,注射后3周,16周对动物剖检,观察hUCMSCs皮下结节的形成及是否有肿瘤转化的倾向。对照组和hUCMSCs组的瘤体积及瘤重采用成组t检验进行统计分析,并计算hUCMSCs组相比对照组的相对肿瘤抑制率T/C%。结果 WiDr肿瘤模型中,第43天hUCMSCs中、高剂量组的瘤体积均显著低于模型对照组,分别为(586.7±274.4)mm~3、(689.5±114.8)mm~3 vs(945.9±234.0)mm~3,P0.05,但T/C%值40%;第51天hUCMSCs低、中高剂量组瘤体积均显著低于模型对照组,分别为(777.8±346.7)mm~3、(793.1±358.6)mm~3、(800.7±116.5)mm~3 vs(1300.0±356.8)mm~3,P均0.05,但T/C%值均40%;表明hUCMSCs在3个剂量下均未对WiDr肿瘤的生长有影响,无显著促进或抑制作用。Raji肿瘤模型中,hUCMSCs治疗组瘤体积与对照组相比在所有时间点差异均无统计学意义(P0.05),显示hUCMSCs在本实验条件下对肿瘤生长没有明显的抑制和促进作用。两个模型的病理检测均未见对肿瘤增殖有明显影响。3个不同代次的hUCMSCs注射到NOD/SCID后16周对动物剖检,也没有观察到肿瘤转化的倾向。结论 hUCMSCs对裸鼠WiDr和Raji移植瘤模型的肿瘤增殖无促进作用,不同代次的hUCMSCs在NOD/SCID裸鼠体内移植也不具有致瘤性。     

Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells

In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17). After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem cell-like features and expressed neural stem cell markers nestin and CD133. The T1 cells were involved in abnormal karyotype, genomic instability and fast proliferation. Importantly, after long-term in vitro culture, the T1 cells did not result in subcutaneous xenografts, but induced intracranial tumor formation, indicating that they adjusted themselves to the intracranial microenvironment. We further found that the T1 cells exhibited an overexpressed level of EGFR, and the CD133 positive T1 cells showed a truncation mutation in the exons 2-7 of the EGFR (EGFRvIII) gene. These results suggest that continuous expansion of neural stem cells in culture may lead to malignant spontaneous transformation. This phenomenon may be functionally related to EGFR by EGFRvIII gene mutation.     

人乳头状瘤病毒协同60钴照射促进食管上皮细胞恶性转化

为探明人乳头状瘤病毒(HPV)和促癌剂对食管上皮致癌作用,人胚食管上皮细胞转染HPV协同60钴(60Co)放射观察其恶性转化.用HPV18E6E7AAV转染的人胚食管上皮(SHEE),培养至13代,分为4组,实验组分别用60Co2、4、8Gy照射,每周1次共4周;SHEE未经照射为对照组.细胞形态用相差显微镜观察;细胞DNA合成和定量用3H-TdR掺入和用流式细胞仪分析;染色体众数用常规方法分析;致瘤性用软琼脂培养和裸小鼠接种;HPVDNA用PCR检测.经60Co照射后细胞呈凋亡和坏死(危象期).8周后SHEE 4Gy组细胞增殖,增殖指数(34%)和3H-TdR摄入增高,软琼脂培养和裸鼠接种出现致瘤性.对照组SHEE组细胞增殖指数24%,伴有少数3H-TdR掺入,裸鼠未成瘤.染色体众数:对照组,58～62;4Gy组,63～65;两组HPV18E6E7 PCR呈阳性条带.此结果表明,用HPV18E6E7协同60Coγ射线可以使人胚食管上皮恶性转化,60Co γ射线有加速食管上皮细胞恶性转化作用.     

Induction of transformed phenotypes in sheep fibroblasts by culture fluids from cells persistently infected with bovine leukemia virus

FLK cells are fetal lamb kidney cells persistently infected with bovine leukemia virus (BLV). 3178 cells, originating from calf-form bovine lymphosarcoma, also showed persistent production of BLV and alteration of cell morphology, after treatment with 5'-iodo-2'-deoxyuridine. In the present paper, the first in vitro transformation of sheep fibroblasts by inoculation with BLV materials from these two cell lines is described. In a few passages after inoculation with these viral materials, morphological alteration occurred. The morphologically altered cells were grown as stable cultures and showed such transformed phenotypes as growth in soft agar medium, increased uptake of 2-deoxy-D-glucose and tumorigenicity in athymic nude mice. This result, together with our previous observation of simultaneous induction of BLV expression and morphological alteration of 3178 cells, suggests the presence of some transforming capacity in these BLV materials similar to that in, for example, murine or avian acute leukemia viruses. The possible acquisition of such capacity during the prolonged passage is discussed.     

Decreased tumorigenicity correlates with expression of altered cell surface carbohydrates in Lec9 CHO cells.

To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lec1) that expressed a Lec1 glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in gamma-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice.     

恶性横纹肌样瘤（MRT）的起源研究——高变异率HeLa细胞致裸鼠产生MRT的实验研究

HeLa细胞KB株、X株、NM20／X株、H株的染色体众数依次为60±3（超二倍体）、62±3（超二倍体）、68±3（超二倍体和亚四倍体）和78±2（亚四倍体）,所占比率分别为72%～76%,69%,52%和40%。在纯化3代的肿瘤阴性对照二倍体猫肾（染色体众数38所占比率80%）和犬肾原代细胞皮下接种裸鼠的致癌／致瘤率分别为0%（0／22）和0%（0／10）,X株HeLa细胞冻融裂解物皮下接种裸鼠产生进行性缩小肿瘤的比率为20%（1／5）的前提下,HeLa细胞KB株、X株、NM20／X株皮下接种裸鼠产生进行性生长恶性肿瘤的比率分别为100%（10／ 10）,100%（25／25）和100%（5／5）,H株细胞皮下接种裸鼠产生恶性肿瘤的比率为50%（5／10）。其中,只有HeLa细胞KB株10～11代（染色体结构畸变率高达20%,出现18%双着丝点和2%断片）以超高数量接种的1组4只裸鼠（0.17ml12.75×10     

Phellinus Linteus Extract Sensitizes Advanced Prostate Cancer Cells to Apoptosis in Athymic Nude Mice

Phellinus linteus (PL) mushroom possesses anti-tumor property. We previously reported that the treatment with PL caused cultured human prostate cancer cells to undergo apoptosis. To further studying the mechanisms of PL-mediated apoptosis, we performed xenograft assay, together with in vitro assays, to evaluate the effect of PL on the genesis and progression of the tumors formed from the inoculation of prostate cancer PC3 or DU145 cells. After the inoculation, nude mice were injected with PL every two days for 12 days. Although PL treatment did not prevent the formation of the inoculated tumors, the growth rate of the tumors after PL treatment was dramatically attenuated. We then tested the effect of PL on the tumors 12 days after the inoculation. After inoculated tumors reached a certain size, PL was administrated to the mice by subcutaneous injection. The histochemistry or immunochemistry analysis showed that apoptosis occurred with the activation of caspase 3 in the tumors formed by inoculating prostate cancer DU145 or PC3 cells. The data was in a good agreement with that from cultured cells. Thus, our in vivo study suggests that PL not only is able to attenuate tumor growth, but also to cause tumor regression by inducing apoptosis.     

Growth of tumor cells with different sensitivities for murine natural killer cells in young and adult athymic nude mice

Of four tumor cell lines, the murine YAC lymphoma, the human K562 lymphoma, and the human prostatic carcinomas PC3 and PC93, the susceptibility to murine natural killer (NK) cells as well as the tumorigenicity in young (3.5-4 weeks old) and in adult (8-10 weeks old) nude mice were studied. In young nude mice, which exhibited a lower level of NK cell activity than adult nude mice, the formation of solid tumors after inoculation of tumor cell suspensions occurred more frequently and with a shorter time lag than in adult animals. These effects were observed not only with the NK-sensitive YAC cells, but also with the relatively NK-insensitive PC3 and PC93 cells, indicating that also factors other than NK cell susceptibility may influence the growth of tumor cells in nude mice. Therefore, the use of young nude mice may enhance the rate of success of heterotransplantation of human tumors, regardless of the NK cell susceptibility of the tumor cells.     

Tumorigenicity of Vero cells

One of the current criteria for evaluating the acceptability of cell lines for use in vaccine production is lack of tumorigenicity. Vero cells represent an example of a class of cells known as continuous cell lines. They were derived from African green monkey kidney, and their growth properties and culture characteristics have many advantages over other cell substrates for use in vaccine production. We have tested Vero cells for tumorigenicity in nude mice and in a human muscle organ culture system, and found a significant increase in their tumorigenic potential with increasing passage numbers. Cells at passage 232 and higher produced nodules in all nude mice inoculated. Histologically the nodules were well defined, anaplastic tumors, which exhibited some of the characteristics of renal adenocarcinomas. In about 6 to 8 days all of the nodules began to regress. Data were obtained that suggested an immune mechanism was the basis for the regression phenomenon.