亚砷酸对裸鼠宫颈癌移植瘤模型的干预研究

目的建立宫颈癌移植瘤模型,研究亚砷酸的体内干预效果及机制。方法将Hela细胞注射于裸鼠皮下接种,成瘤后腹腔分别连续注射亚砷酸、卡铂及生理盐水14d,观察肿瘤大小和裸鼠精神状态,用流式细胞术（FCM）检测细胞凋亡和细胞周期。结果亚砷酸组小鼠精神状态良好。与对照组比较,亚砷酸组治疗7d后肿瘤生长速度减慢;治疗14d肿瘤重量显著性降低（P〈0．05）,抑瘤率达到35．8％,高于卡铂组的27．9％;治疗14d肿瘤细胞的凋亡率显著升高（P〈0．01）,增殖指数（PI）显著降低（P〈0．01）,处于G0／G1周期的细胞明显增多（P〈O．01）,处于G2/M周期的细胞明显减少（P〈0．05）。结论亚砷酸具有抑制宫颈癌移植瘤生长的作用,且未见明显毒副作用,其抑瘤的机理可能与诱导肿瘤细胞凋亡、干扰肿瘤细胞生长周期和抑制肿瘤细胞增殖有关。     

靶向诱骗受体3的小干扰RNA抑制人结肠癌裸鼠皮下移植瘤的实验研究

目的：以脂质体为载体,探讨靶向诱骗受体3（DcR3）的小干扰RNA（siRNA）对人结肠癌SW480细胞裸鼠皮下移植瘤生长的抑制作用。方法：裸鼠背部皮下种植结肠癌SW480细胞,建立结肠癌SW480细胞裸鼠皮下移植瘤模型;针对靶标DcR3基因,利用Dharmacon公司的软件设计合成siRNA序列,通过瘤体局部多点注射脂质体与DcR3siRNA混合物,将DcR3siRNA转染到裸鼠背部皮下移植瘤内,同时设立阴性对照组和空白对照组;观察肿瘤治疗前后体积变化,评价DcR3siRNA对肿瘤的抑制作用;比较肿瘤治疗前后的细胞形态学改变;免疫组织化学及RT-PCR检测DcR3基因表达;原位细胞凋亡检测肿瘤的凋亡。结果：治疗组肿瘤体积明显小于对照组,肿瘤组织内坏死面积增大,细胞凋亡率显著增高,DcR3蛋白表达水平降低;治疗过程中裸鼠生长良好,无明显毒性反应。结论：脂质体介导的DcR3siRNA对裸鼠皮下结肠癌SW480细胞移植瘤有明显的抑制、杀伤作用,细胞凋亡是DcR3siRNA致肿瘤细胞死亡的重要形式。     

体内致敏的树突状细胞诱导特异性抗肿瘤免疫的基础研究

目的证实树突状细胞（dendritic cells,DC）可在体内通过吞噬凋亡肿瘤细胞获取抗原物质,探讨其在肿瘤免疫治疗中的意义.方法以615小鼠的前胃癌细胞株造模,在体外用rmGM-CSF和rmIL-4从荷瘤小鼠骨髓细胞分化、诱导未成熟树突状细胞.分为4组：小剂量化疗组、树突状细胞组、小剂量化疗＋树突状细胞组和对照组,以BAX试剂盒检测肿瘤细胞凋亡.在瘤体内注射树突状细胞,观察给药侧瘤体及对侧瘤体体积,生存期,和特异性细胞毒性T淋巴细胞（CTLs）对肿瘤细胞的特异性杀伤作用.结果小剂量化疗能诱导肿瘤细胞凋亡.小剂量化疗后瘤内应用树突状细胞,给药侧瘤体及对侧瘤体体积明显缩小（P＜0.05）,小鼠的生存率提高,体内凋亡肿瘤细胞致敏的DC诱导的CTL对MFC有显著的杀伤作用,在效靶比为40：1、20：1、10：1和5：1时72 h的杀伤率分别为87.64%、70.32%、34.63%和13.87%.并能特异性杀伤小鼠前胃癌细胞MFC（P＜0.01）.结论体外诱导分化的未成熟DC,能于体内捕获小剂量化疗诱导的凋亡肿瘤细胞所携带的肿瘤抗原,诱导机体特异性抗肿瘤免疫反应.     

特异性人端粒酶催化亚单位基因小干扰RNA对HeLa细胞生长的抑制作用

通过设计并化学合成人端粒酶催化亚单位(hTERT)特异性siRNA,观察其对hTERT表达水平及肿瘤细胞生长的影响。将hTERT-siRNA以脂质体法转染入HeLa细胞,应用RT-PCR、实时定量TRAP、Western印迹、软琼脂克隆形成实验、荷瘤裸鼠肿瘤内注射等方法检测细胞内hTERTmRNA、蛋白质表达水平及对肿瘤细胞生长的影响。RT-PCR、实时定量TRAP和Western印迹的结果显示hTERT-siRNA明显降低了HeLa细胞内hTERT的mRNA及蛋白质表达水平并伴随有端粒酶活性的下降。克隆形成实验表明hTERT-siRNA组的体外肿瘤形成能力受到抑制。荷瘤裸鼠肿瘤内注射hTERT-siRNA使肿瘤平均体积显著小于对照组。TUNEL凋亡检测表明hTERT-siRNA转染组的凋亡率明显高于对照组。研究表明hTERT特异性siRNA可以明显抑制HeLa细胞内hTERT的表达水平,对其生长有明显抑制作用,是一种有前途的肿瘤治疗新方法。     

AS2O3抑制人结肠癌生长的体内外实验研究

目的：通过研究三氧化二砷（AS2O3）对人结肠癌细胞株LS-174T在体内外实验中增殖情况的影响,探讨As2O3治疗结肠癌的机制。方法：1、试验分组：体外细胞培养及体内动物实验均分三组：对照组,As2O3低剂量组,As2O3高剂量组。2、MTT法测定细胞增殖数的情况。3、丫啶橙/溴乙啶（AO/EB）双荧光染色检测细胞凋亡率。4、免疫组织化学法测定结肠癌细胞和裸鼠实体肿瘤中HIF-1α和VEGF的表达。5、测量各组裸鼠实体肿瘤的重量及体积。结果：体外试验中,由MTT法可以看出,As2O3治疗组细胞生存率明显低于对照组（P〈0.01）,其中AS2O3高剂量组明显低于低剂量组。双荧光染色结果As2O3高剂量组细胞凋亡百分率明显高于低剂量组和对照组（P〈0.01）。免疫组化结果显示结肠癌细胞HIF-1α和VEGF在对照组的阳性表达率明显高于As2O3治疗组（P〈0.01）,低剂量组高于高剂量组（P〈0.05）。体内实验中,治疗组平均瘤重和瘤体积均较对照组明显减低,（P〈0.01）。HIF-1α及VEGF在裸鼠肿瘤组织中均有阳性表达,其中对照组较AS2O3治疗组高（P〈0.01）。结论：As2O3能够抑制体内外人结肠癌细胞的生长,并能明显抑制HIF-1α和VEGF的表达,从而抑制肿瘤的生长。     

RNAi沉默SDF-1基因对人胃癌裸鼠原位移植瘤生物学行为的影响及机制

目的:探讨RNAi技术沉默基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)对人胃癌裸鼠原位移植瘤生物学行为的影响及可能机制。方法:利用RNAi-SDF-1细胞及对照组细胞建立人胃癌裸鼠皮下移植瘤,Western Blot检测裸鼠皮下移植瘤SDF-1蛋白表达情况。利用裸鼠皮下移植瘤建立其原位移植瘤模型,8周后处死裸鼠解剖尸体,检测原位移植瘤生长、凋亡及远处脏器转移情况。Western Blot检测SDF-1沉默对通路蛋白Akt、p-Akt、NF-κB、p-NF-κB以及侵袭转移相关基因E-cadherin、MMP-7表达的影响。结果:成功建立RNAi-SDF-1裸鼠原位移植瘤模型。与空白及阴性对照组比较,RNAi-SDF-1组裸鼠原位移植瘤生长缓慢,体积与质量均明显降低,差异有统计学意义(P0.01)。流式细胞术结果显示,RNAi-SDF-1组肿瘤细胞凋亡率明显高于空白及阴性对照组(P0.01)。尸体解剖结果显示,RNAi-SDF-1组肿瘤腹腔淋巴结及肝脏转移率明显低于空白及阴性对照组(P0.05)。Western Blot结果显示,与空白及阴性对照组比较,RNAi-SDF-1组肿瘤组织中Akt、p-Akt、NF-κB、p-NF-κB、MMP-7表达水平明显降低,E-cadherin表达水平明显升高(P0.01)。结论:RNAi-SDF-1能够有效抑制人胃癌SGC7901细胞裸鼠原位移植瘤的生长,诱导肿瘤细胞凋亡,抑制其腹腔淋巴结、肝脏转移,其机制可能与抑制PI3K-Akt、NF-κB信号通路及MMP-7的表达并上调E-cadherin的表达相关。     

紫杉醇抑制前列腺癌增殖的体内实验研究

目的探讨紫杉醇对人前列腺癌细胞PC-3增殖的体内抑制作用。方法建立体内绿色荧光蛋白（GFP）标记的人雄激素非依赖性前列腺癌细胞PC-3裸鼠原位移植瘤模型,观察紫杉醇对裸鼠前列腺癌原位移植瘤的体积、重量的影响。结果裸鼠模型体内实验显示,与对照组（100μL生理盐水）相比,紫杉醇处理组（0.5 mg/kg）在给药第18天后能显著抑制前列腺肿瘤的体积（P〈0.05）;紫杉醇处理组在抑制前列腺肿瘤重量方面与对照组相比亦有明显抑制作用（P〈0.05）。与对照组相比G31P处理组VEGF（P〈0.05）的表达差异具有统计学意义（免疫组化法）。结论紫杉醇在体内实验中能明显抑制人雄激素非依赖性前列腺癌细胞系PC-3的增殖。     

CDA基因沉默对人慢性髓系白血病细胞增殖和凋亡的影响

目的:探讨胞苷脱氨酶(CDA)基因沉默在治疗人慢性髓系白血病(CML)中的潜在价值。方法:通过RT-PCR和Western blot检测CML患者和造血干细胞移植供体的骨髓单个核细胞中的CDA表达。对CML KCL-22细胞系转染shRNA和过表达CDA的p BS/U6-Neo质粒来诱导CDA基因沉默或过表达。通过细胞计数试剂盒8(CCK-8)测定和细胞集落形成实验评价细胞增殖,通过流式细胞仪检测细胞凋亡。此外,将0.2 m L不同处理的细胞悬浮液(106个细胞/m L)注射到裸鼠中建立裸鼠肿瘤异种移植模型。结果:与造血干细胞移植供体相比,CML患者的骨髓单个核细胞中的CDA m RNA和蛋白表达显著升高(P 0.05)。转染shRNA-CDA显著降低了KCL-22细胞的细胞活力和细胞集落数(P0.05)。与对照组(4.32%)相比,shRNA-CDA组(13.45%)的细胞凋亡率显著升高(P0.05)。与对照组相比,shRNA-CDA组的BCL-2蛋白表达水平显著降低,而cleaved caspase-3显著升高(P0.05)。与对照组相比,shRNA-CDA组的PI3K蛋白表达水平和Akt磷酸化水平显著降低(P0.05)。接种30 d后,与对照组相比,shRNA-CDA组裸鼠的肿瘤重量和肿瘤体积均显著降低(P0.05)。结论:CDA在人慢性髓系白血病中高表达,CDA基因沉默可在体内和体外抑制肿瘤细胞的生长。其机制与抑制PI3K/Akt信号通路的激活有关。     

慢病毒靶向携带Survivin siRNA抗肺腺癌的体内研究

目的:探讨慢病毒载体介导的靶向survivin基因小干涉RNA(survivin-siRNA)对裸鼠移植人肺腺癌的体内抑瘤活性。方法:构建表达survivin-siRNA的慢病毒载体和移植人肺腺癌裸鼠模型,肿瘤组织局部注射survivin-siRNA慢病毒载体,观察肿瘤体积及随时间生长变化;PI染色检测细胞凋亡;流式细胞术检测肿瘤细胞周期变化。结果:慢病毒载体介导survivin-siRNA对裸鼠肺腺癌的抑瘤率为46.07%;30～35%的肿瘤细胞凋亡;G1期细胞比例明显增加,S期细胞比例则明显减少。结论:靶向survivin的RNAi能有效抑制裸鼠移植人肺腺癌的生长,诱导肿瘤细胞凋亡和细胞周期改变。     

Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type.

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.     

利用Gaussia萤光素酶建立乳腺癌实时定量检测动物模型

目的：建立-种基于分泌型萤光素酶的实时定量检测实验动物体内肿瘤大小的方法。方法：以分泌型Gaussia萤光素酶（Gluc）为报告基因,以嘌呤霉素为筛选基因,将两者用T2A元件连接后克隆到慢病毒载体,包装慢病毒后感染乳腺癌MCF-7细胞,经嘌呤霉素筛选得到稳定转染细胞MCF-7-Gluc,并检测细胞上清中Gluc活性随时问和细胞数目的变化;将MCF-7-Gluc扩大培养后经皮下注射到雌性BALB／c裸鼠前肢腋下,待肿瘤形成后,检测外周血液中Gluc活性与肿瘤体积的相关性。结果：体外实验显示稳定转染细胞MCF-7-Gluc分泌到细胞上清的Gluc活性与时间和细胞数量在-定范围内均呈现良好的线性关系,体内实验显示裸鼠血液中的Gluc活性与肿瘤体积呈正相关。结论：Gluc技术可作为-种灵活、方便、实时定量检测活体动物体内肿瘤大小的有效工具。     

Synthesis and biological evaluation of cinnamyl compounds as potent antitumor agents

A series of cinnamyl compounds related to 2'-hydroxycinnamaldehyde were synthesized and their antitumor effects against human cancer cells evaluated. Hydroxylamine derivative 6 inhibited the growth of human cancer cells and human colon tumor xenograft in nude mice. Its antitumor effects belong to the induction of apoptosis and arresting cell cycle at G(2)/M phase, which is confirmed by detection of apoptosis markers and cell cycle analysis.     

纳米氧化铁双歧杆菌LTA对实验性胃癌抑制效果的影响

目的探讨纳米氧化铁（Fe3O4）双歧杆菌脂磷壁酸（Lipoteichoic acid,LTA）对实验性胃癌体内抑制效果的影响及其抑瘤途径。方法用人胃癌BGC823细胞建立裸鼠移植瘤模型,用硝酸还原酶法检测各剂量组裸鼠腹腔巨噬细胞分泌NO和iNOS的含量,用免疫组化法检测移植瘤内VEGF、Survivin和TAMs的表达。结果纳米Fe3O4-LTA各组与阴性对照组相比,NO生成量增加、iNOS活性增高,差异有非常显著性（P〈0.01）。移植瘤内VEGF、Survivin和TAMs蛋白表达显著低于阴性对照组（P〈0.01）。结论纳米Fe3O4-LTA对人胃癌裸鼠移植瘤的生长具有明显的抑制作用,其抑瘤机制可能与激活巨噬细胞,使其分泌多种具有杀瘤作用的活性因子以及下调胃癌内VEGF、Survivin和TAMs的表达,进而抑制其血管形成有关。     

干扰OVA66基因表达对移植瘤生物学功能的影响

目的探讨干扰OVA66基因表达对移植瘤细胞生物学功能的影响。方法采用脂质体法,以重组的pSUPER-shRNA-OVA66和空载体转染HeLa细胞获得稳定表达细胞株,经RT-PCR和Western blot鉴定OVA66基因的表达。将两组细胞接种于BALB／cnu／nu裸鼠的前肢腋下,连续观察干扰OVA66表达对肿瘤的生长影响。并于接种4周后,应用RT-PCR,免疫组化、免疫荧光等方法检测移植瘤细胞中OVA66的蛋白表达。组织病理分析肿瘤细胞生长和转移的特征。结果经检测pSUPER-shRNA-OVA66能有效抑制目的基因的表达。比较两组荷瘤小鼠中肿瘤的生长曲线发现,OVA66实验组的肿瘤生长速度明显低于对照组,并且肿瘤细胞体内转移和浸润能力显著降低。结论干扰OVA66基因和蛋白表达,可以抑制肿瘤细胞的生长,浸润和侵袭能力。     

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis

KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model. Received: 10 July 1999 / Accepted: 21 January 2000     

Adeno-associated virus sensitizes HeLa cell tumors to gamma rays.

Infection with the helper virus-dependent human parvovirus adeno-associated virus (AAV) is known to interfere with cellular transformation in vitro and oncogenesis in vivo. Here we report on sensitization to gamma irradiation by AAV infection of cells in culture and of tumors established from HeLa cells grafted into immunodeficient (nude) mice: infection of HeLa cells with AAV type 2 enhanced cell killing and reduced plating efficiency after irradiation compared with uninfected cells. Similarly, HeLa cell tumors in nude mice displayed a reduced growth rate and were more sensitive to gamma irradiation when the animals were infected with AAV type 2 prior to or after tumor cell inoculation. Since no pathogenicity is known for AAV, the ability of this virus to render radiotherapy of human tumor cells more efficient may up open novel approaches in cancer treatment.     

苦杏仁苷介导人肝癌细胞HuH-7凋亡的实验研究

摘要 目的：通过体内体外实验探讨苦杏仁苷在肝癌中的抗肿瘤作用。方法：MTT 法检测不同浓度的苦杏仁苷对肝癌 HuH-7细胞存活率的影响;DAPI染色法观察苦杏仁苷介导HuH-7细胞的凋亡形态学变化;流式细胞术检测苦杏仁苷干预后HuH-7细胞凋亡率变化;Western Blot法检测细胞凋亡相关蛋白Bax、Bcl-2的表达,计算Bax/Bcl-2的比值。建立裸鼠HuH-7细胞移植瘤模型,观察苦杏仁苷对荷瘤裸鼠移植瘤体积的影响。结果：体外实验结果证实苦杏仁苷能够诱导人肝癌HuH-7细胞凋亡的发生（P<0.05）。随着苦杏仁苷浓度的增加,HuH-7细胞的存活率降低,凋亡率升高,干预后Bax/Bcl-2比值明显升高（P<0.05）。体内实验结果表明,裸鼠HuH-7细胞移植瘤的体积增长速度减慢（P<0.05）。结论：苦杏仁苷能够诱导人肝癌HuH-7细胞和裸鼠HuH-7细胞移植瘤细胞发生凋亡,减缓肿瘤生长,从而发挥抗肿瘤作用。     

Obovatol inhibits colorectal cancer growth by inhibiting tumor cell proliferation and inducing apoptosis

Neolignans such as obovatol, honokiol, and magnolol have been previously reported to show various biological activities including anti-inflammation and antitumor effects. This is the first demonstration on the in vivo antitumor effect of obovatol on human colorectal carcinoma SW620 cells. Nude mice were implanted with SW620 cells and fed with vehicle or 5mg/kg/d dose of obovatol for 20 days. Obovatol inhibited tumor growth that accounted for 50% decrease in tumor volume and 44.6% decrease in tumor weight at the end of the experiment without any adverse health effect. In nude mice bearing SW620-incubated tumor, obovatol exhibited more potent antitumor activity than honolkiol. In addition, DNA flow cytometric analysis shows that obovatol progresses to apoptosis as detected by flow cytometry after double staining with annexin V and propidium iodide. Thus, we suggest that obovatol is a potent inducer of cell apoptosis in SW620 cells, and a potent antitumor agent.