CD自杀基因系统对胶质瘤细胞作用的实验研究

将含大肠杆菌胞嘧啶脱氨酶(EC-CD)基因的重组逆转录病毒感染大鼠神经胶质瘤细胞C6,获得稳定表达CD的克隆细胞C6/CD。CD可以将无毒性的原药5-氟胞嘧啶(5-FC)转化成高度细胞毒性物质5-氟尿嘧啶(5-FU)。生长抑制试验表明C6/CD细胞对5-FC高度敏感,IC_(50)为3μmol/L;5-FC对C6细胞基本无毒性,IC_(50)近6000μmol/L;而C6/CD和C 6细胞均对低浓度5-FU敏感,IC_(50)小于1μmol/L。混合细胞试验表明C6/CD细胞在5-FC存在下对C6细胞有旁杀伤效应。本工作在细胞水平建立了一个治疗神经胶质瘤的自杀基因系统,过去无人报道过。     

CD/5-FC对KSHV肿瘤转化基因K12诱导NIH3T3转化细胞的杀伤作用

采用分子克隆技术构建含卡波济肉瘤相关疱疹病毒(Kaposi’s sarcoma—associated herpesvirus,KSHV)转化基因K12和含胞嘧啶脱氨酶(CD)基因重组逆转录病毒表达质粒,分别命名为LZRS-K12和pLXSN—Fcy∷Fur。将LZRS-K12质粒转染NIH3T3细胞系,诱导细胞转化获得具有肿瘤细胞生物学特性的N／K12细胞,进一步将pLXSN—Fcy∷Fur质粒转染N／K12细胞,获得含CD基因的N／K12／FF细胞。加入5-氟胞嘧啶(5-FC)后,采用MTT法、流式细胞仪和免疫组化(IHC)等方法,分别检测细胞存活率、凋亡率及凋亡相关蛋白的表达。结果显示,N／K12／FF细胞在使用5-FC后存活率明显下降。流式细胞仪检测结果显示,5-FC作用后的N／K12／FF细胞的凋亡率与作用前相比增加了4．6％。与此同时,经5-FC作用后的N／K12和N／K12／FF细胞的生长周期发生了明显变化,均表现为G0-G1期细胞比率降低,S和G2-M期细胞比率增高。5-FC使得N／K12细胞的增殖主要阻滞在G2-M期,而N／K12／FF细胞的增殖主要阻滞在S期。IHC检测结果表明,N／K12细胞在5-FC使用前后Fas和Fas-L表达改变均不明显;而N／K12／FF细胞在5-FC使用后的Fas和Fas-L表达水平均明显高于5-FC使用前的水平。提示,CD／5-FC对KSHV肿瘤转化基因K12诱导NIH3T3转化细胞具有杀伤作用;凋亡有可能介导了部分杀伤过程;Fas和Fas-L有可能部分参与凋亡的诱导。     

健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的实验研究

目的:观察健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的作用。方法:脂质体lipofectamine将含有双自杀基因的腺病毒载体pAd-CD/TK导人293细胞,收集病毒上清转染人肝癌细胞BEL7402,MTT法测定BEL7402细胞存活率。裸鼠人肝癌模型转染CD/TK双自杀基因后,给予5-FC500mg/kg,GCV 100mg/kg腹腔注射,同时予健脾化瘀中药960复方灌胃。观察肿瘤生长情况。结果:给予前体药物5-FC和GCV后,CD/TK转染细胞被杀死。并表现出较强的旁观者效应。转染细胞比例达到10%即表现出较强的杀伤作用(P<0.01)。健脾化瘀中药960复方具有提高旁观者效应作用,1.67ml/kg和2.5ml/kg960复方含药血清组细胞存活率显著低于对照组(P<0.01)。转染基因组应用5-FC和GCV治疗后,裸鼠肝癌的生长明显受到抑制(P<0.05),抑瘤率39.42%,单用中药组抑瘤率18.04%,中药与CD/TK 5-FC/GCV联合运用组,较单纯CD/5-FC/HSV-tk/GCV对裸鼠肿瘤模型的生长抑制作用更加明显(P<0.05),抑瘤率55.10%。结伦:腺病毒介导CD/TK自杀基因可有效地杀死人肝癌BEL7402细胞,健脾化瘀中药960复方具有显著提高CD/TK双自杀基因对人肝癌细胞的抑杀作用。     

Wistar大鼠C6胶质瘤模型中的肿瘤消退现象研究

目的:建立Wistar大鼠C6胶质瘤模型,研究其生物学特性(病理特性,新生血管特性,模型最佳应用时间段,肿瘤自发消退现象)并对其进行MRI动态扫描观察,了解肿瘤的自然生长规律,与人脑胶质瘤的自然生长规律相比较.方法:Wistar大鼠40只,分成2组,一组30只,二组10只,分别将C6胶质瘤细胞悬液和DMEM培养基立体定向接种于大鼠的右侧尾状核,分别于接种后7,14,21,28,35,50天进行增强MRI扫描,了解肿瘤生长特性,脑组织水肿情况,肿瘤消退现象等.于荷瘤鼠死亡当日取脑组织,液氮速冻后行脏染色,抗CD31免疫组化检查.结果:大部分肿瘤模型于接种后7天可在MRI上见到肿瘤生长,14-28天为快速增长期,并有大量新生血管形成,脑屏障严重破坏,大部分荷瘤鼠死于28天内,有3只大鼠20天以后出现肿瘤自发消退现象,病理切片可见大量炎细胞浸润,肿瘤内部出现软化灶.结论:立体定向建立大鼠C6胶质瘤模型与人脑胶质母细胞瘤具有相似性,部分荷瘤鼠在5周左右有肿瘤自发消退,肿瘤新生血管于7天开始出现并增多,4周后基本趋于稳定.因此利用此模型进行试验研究的最佳时期在14-28天.     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34⁺富集细胞在SCF+IL-3+IL-6+FL+Tpo细胞因子组合下的体外扩增特性,发现：CD34⁺富集细胞具有很高的扩增潜力,在本实验条件下其总细胞持续扩增了8周,扩增倍数达31270.9±8640.5倍;而MNC在培养至第4周扩增就已呈现下降趋势,最大仅扩增了53.3±6.2倍。对比集落和CD34⁺细胞的扩增发现,MNC的集落密度和CD34⁺细胞含量由第0天至第7天有一个上升的过程,而CD34⁺富集细胞在培养过程中,集落密度和CD34⁺细胞含量却始终呈下降趋势。在体外培养过程中,CD34⁺富集细胞的CFU-GM和CD34⁺细胞最大分别扩增了185.7±14.1和191.7±188.8倍,明显高于MNC的12.4±3.2和50.6±33.2倍;而CD34⁺富集细胞和MNC的BFU-E则只实现了少量扩增,分别为7.2±5.2和10.1±3.4倍。结果显示,从CD34⁺富集细胞出发扩增造血干/祖细胞,可以得到更多的CD34⁺细胞和CFU-GM集落形成细胞。      

欧前胡素调节Gas6/Axl轴对膀胱癌荷瘤小鼠免疫功能的影响

摘要 目的：探讨欧前胡素（IMP）调节生长停滞特异蛋白6（Gas6）/Axl轴对膀胱癌荷瘤小鼠免疫功能的影响。方法：从60只SPF级雄性C57BL/6小鼠中随机选取12只小鼠作为对照组（CON组）,其余小鼠平均分为Model组、IMP组、Gas6组、IMP+Gas6组,每组12只,往其左侧前肢皮下注射0.2 mL浓度为1×10⁷个/mL的鼠源性膀胱癌肿瘤细胞系T739细胞溶液（Model组和IMP组注射对照载体的T739稳转株细胞,Gas6组和IMP+Gas6组注射过表达Gas6的T739稳转细胞株）造模。造模成功后,IMP组及IMP+Gas6组通过腹腔注射5 mg/kg的IMP;CON组、Model组及Gas6组通过腹腔注射等量的生理盐水,2天1次,持续5次。测量小鼠肿瘤体积和重量;测定胸腺指数和脾脏指数;酶联免疫吸附测定（ELISA）法检测血清白介素2（IL-2）、γ干扰素（IFN-γ）、血管内皮生长因子（VEGF）水平;苏木精-伊红（HE）染色检查肿瘤组织病理变化;流式细胞术检测T细胞亚群;TUNEL染色检测细胞凋亡;蛋白质印迹法（Western blot）检测Gas6/Axl通路相关蛋白表达。结果：Model组肿瘤细胞整体上排列松散,出现不同程度的核固缩、核碎裂现象;IMP则可以逆转该现象,使肿瘤细胞排列重新变得紧密,细胞核固缩程度提升,核碎裂现象降低;Model组较CON组肿瘤体积、肿瘤质量、CD8⁺细胞百分比、Gas6、Axl蛋白水平、VEGF水平显著增加（P<0.05）,胸腺指数和脾脏指数、IL-2、IFN-γ、CD4⁺细胞百分比、CD4⁺/CD8⁺、肿瘤细胞凋亡率显著下降（P<0.05）;与Model组相比,IMP组胸腺指数和脾脏指数、IL-2、IFN-γ、CD4⁺细胞百分比、CD4⁺/CD8⁺、肿瘤细胞凋亡率显著升高（P<0.05）,肿瘤体积、肿瘤质量、CD8⁺细胞百分比、Gas6、Axl蛋白水平、VEGF水平显著下降（P<0.05）;而Gas6组结果与IMP组趋势相反;而IMP+Gas6组的结果显示：IMP能逆转Gas6过表达后对膀胱癌荷瘤小鼠的促瘤作用。结论：IMP可能通过下调Gas6/Axl信号通路改善膀胱癌荷瘤小鼠免疫功能,从而产生抑瘤效果。     

CD自杀基因系统对T淋巴细胞作用的实验研究

去除供者骨髓中的T淋巴细胞可有效防止移植物抗宿主病(GVHD)的发生.用含大肠杆菌胞嘧啶脱氨酶（EC-CD）基因的高滴度逆转录病毒(1.5×10⁵ CFU/ml)感染小鼠T淋巴细胞,G418筛选,获得稳定表达CD基因的T/ pCD₂细胞.经PCR和RT-PCR方法检测证明CD基因已成功地导入T淋巴细胞中并有效表达.分别用不同浓度的5-FCyt作用于T/ pCD₂及T淋巴细胞,光镜下观察不同时间细胞数目变化及MTT法检测细胞活性.结果表明,5-FCyt浓度大于1 μmol/L时,即对T/ pCD₂细胞有显著的杀伤作用,而对正常T淋巴细胞基本无毒性,且T/ pCD₂细胞在加入药物后生存时间（3～5 d）明显短于未转染的T淋巴细胞（大于14 d）.     

人核糖核酸抑制因子基因在人脐血干细胞中的转染表达及对小鼠黑色素瘤基因治疗的研究

探讨人核糖核酸抑制因子 (hRI)基因在人脐血干细胞中的转染及表达情况 ,及转染后对小鼠B16黑色素瘤生长的影响。用免疫磁珠分离系统 (MACS)分离纯化人脐血CD34⁺ 细胞后 ,用制备的含hRI基因的逆转录病毒上清转染脐血CD34⁺ 细胞 ,采用克隆形成法和PCR法检测转染效率 ,Western blot和免疫荧光法检测基因表达 ,同时观察RI对荷瘤C57BL小鼠B16黑色素瘤生长的影响。应用MACS能高度纯化人脐血CD34⁺ 细胞 ,使分选后的脐血CD34⁺ 细胞纯度平均达96.15%。hRI基因能够转染到脐血CD34⁺ 细胞上 ,转染效率达 35% ,Western blot和免疫荧光检测转染后CD34⁺ 细胞hRI基因有阳性表达。经转hRICD34⁺ 细胞治疗 ,使小鼠B16黑色素瘤的生长速度减慢 ,成瘤率和瘤重降低 ,成瘤潜伏期延长。     

Ⅰ-Ak基因克隆转导及对肿瘤细胞成瘤的影响

采用RT-PCR方法合成小鼠MHCⅡ类分子Ⅰ-A^k基因α和β链cDNA,插入逆转录病毒载体pLSXN,构建Ⅰ-A^k α和Ⅰ-A^k β表达载体,采用脂质体介导的重组质粒转移方法将Ⅰ-A^k α和Ⅰ-A^k β基因导入EL4小鼠淋巴瘤细胞和P815小鼠肥大细胞瘤细胞,经流式细胞仪检测在细胞表面有Ⅰ-A^k表达.将以上两种细胞注射到同源小鼠C57BL/6(H-2^d)皮下,观察到肿瘤产生后又消退,证明在肿瘤细胞中单独导入同种异型MHCⅡ类分子基因也能激活肿瘤的细胞免疫,为进一步开展肿瘤的基因治疗奠定了基础.     

肝癌肿瘤干细胞的分离鉴定及差异性小分子RNA筛选

旨在分离并鉴定肝癌肿瘤干细胞,并对其异常表达的microRNA进行了初步筛选。首先利用流式细胞术及免疫荧光技术分别在肝癌细胞系及肝癌组织标本中确认了CD90⁺细胞的存在;随后利用免疫磁珠分选技术从肝癌细胞系MHCC97-H中分离出了CD90⁺细胞,化疗药物耐药性实验表明,CD90⁺细胞具有明显的化疗药物耐受能力;两次裸鼠皮下成瘤性实验也表明CD90⁺细胞具有较强的成瘤能力,而CD90^-细胞不具备成瘤能力。上述实验充分说明:CD90⁺细胞具有肝癌肿瘤干细胞特性。因此利用该细胞模型进行了肝癌肿瘤干细胞CD90⁺细胞和非肝癌肿瘤干细胞CD90^-细胞之间差异表达的microRNA的检测,结果表明,CD90⁺细胞与CD90^-细胞之间共有92个microRNAs的表达存在差异性,其中在CD90⁺细胞中高表达的microRNAs有52个;在CD90^-细胞中高表达的microRNAs有40个。     

Therapeutic effect of genetically modified human neural stem cells encoding cytosine deaminase on experimental glioma

The aim of this study was to determine the efficacy of neural stem cell-based suicidal gene therapy in rats bearing human glioma. F3 human neural stem cells (NSCs) were transduced to encode cytosine deaminase (CD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Intratumoral or intravenous transplantation of F3.CD human NSCs led to marked reduction in tumor burden and significantly prolonged the survival of brain tumor-bearing rats. The systemic administration of 5-FC with direct intratumoral/intravenous transplantation of F3.CD cells had remarkable therapeutic effect in rats with human glioma cells as compared with transplantation of parental F3 cells. There was 74% reduction in tumor volume in rats receiving direct transplantation of F3.CD cells into tumor site, and 67% reduction in tumor volume in rats receiving intravenous injection of F3.CD cells as compared to control animals transplanted with human glioma U373 cells alone. The combination of F3.CD and 5-FC was a highly effective in the glioma rat model. Our observations suggest that genetically engineered NSCs encoding suicide gene CD could provide clinical application of suicide gene therapy for patients with glioma.     

Combined radiation and enzyme/prodrug treatment for head and neck cancer in an orthotopic animal model.

In an effort to improve the therapeutic outcome for squamous cell cancer of the head and neck, we have used the enzyme cytosine deaminase (CD) and the prodrug 5-fluorocytosine (5-FC) as a means to deliver the chemotherapeutic agent 5-fluorouracil (5-FU) in a tumor-specific manner and have evaluated the use of this treatment in combination with external-beam radiation. Infection of SCCVII cells in culture with a CD-expressing retrovirus and treatment with 5-FC was cytotoxic depending on the time of treatment and dose of 5-FC. An orthotopic model of squamous cell cancer of the head and neck was used in vivo to study the CD/5-FC system both alone and with concurrent radiation due to the radiosensitizing properties that 5-FU generates in situ. Treated mice were imaged using magnetic resonance imaging (MRI), and their survival was evaluated. Neither 5-FU nor radiation either alone or combined provided a survival advantage. In contrast, 5-FC treatment prolonged survival and decreased tumor burden compared to control animals, but the tumors recurred after the treatment ceased. Finally, combined treatment with concurrent administration of 5-FC and radiation resulted in a synergistic decrease in tumor growth and enhanced survival over treatment with 5-FC or radiation alone.     

Implication of functional activity for determining therapeutic efficacy of suicide genes in vitro

Gene-Directed Enzyme Prodrug Therapy (GDEPT), commonly known as suicide gene therapy, provides a selective approach to eradicate tumor cells that is currently considered as an alternate approach to conventional therapy for cancers due to its high efficacy. Herein, we have demonstrated functional activity of the cytosine deaminase (CD) and the hybrid cytosine deaminase-uracil phosphoribosyltransferase (CD-UPRT) suicide genes in transfected cell lines. We have monitored retention profiles of various metabolites that were formed during enzymatic conversion of the prodrug 5-flurocytosine (5-FC) using reverse phase HPLC method. Therapeutic effect of suicide genes was established by cell viability and toxicity assay, whereas apoptotic induction was confirmed by DNA fragmentation ELISA. Our results demonstrated that 5-FC/CD-UPRT-mediated apoptotic cell death was more than 5-FC/CD, which could be further potentiated with anticancer compound curcumin. Such results corroborated 5-FC/CD-UPRT in combination with curcumin as a better chemosensitization method.     

An Increase in CD3+CD4+CD25+ Regulatory T Cells after Administration of Umbilical Cord-Derived Mesenchymal Stem Cells during Sepsis

Sepsis remains an important cause of death worldwide, and vigorous immune responses during sepsis could be beneficial for bacterial clearance but at the price of collateral damage to self tissues. Mesenchymal stem cells (MSCs) have been found to modulate the immune system and attenuate sepsis. In the present study, MSCs derived from bone marrow and umbilical cord were used and compared. With a cecal ligation and puncture (CLP) model, the mechanisms of MSC-mediated immunoregulation during sepsis were studied by determining the changes of circulating inflammation-associated cytokine profiles and peripheral blood mononuclear cells 18 hours after CLP-induced sepsis. In vitro, bone marrow-derived MSCs (BMMSCs) and umbilical cord-derived MSCs (UCMSCs) showed a similar morphology and surface marker expression. UCMSCs had stronger potential for osteogenesis but lower for adipogenesis than BMMSCs. Compared with rats receiving PBS only after CLP, the percentage of circulating CD3+CD4+CD25+ regulatory T (Treg) cells and the ratio of Treg cells/T cells were elevated significantly in rats receiving MSCs. Further experiment regarding Treg cell function demonstrated that the immunosuppressive capacity of Treg cells from rats with CLP-induced sepsis was decreased, but could be restored by administration of MSCs. Compared with rats receiving PBS only after CLP, serum levels of interleukin-6 and tumor necrosis factor-α were significantly lower in rats receiving MSCs after CLP. There were no differences between BMMSCs and UCMSCs. In summary, this work provides the first in vivo evidence that administering BMMSCs or UCMSCs to rats with CLP-induced sepsis could increase circulating CD3+CD4+CD25+ Treg cells and Treg cells/T cells ratio, enhance Treg cell suppressive function, and decrease serum levels of interleukin-6 and tumor necrosis factor-α, suggesting the immunomodulatory association of Treg cells and MSCs during sepsis.     

布美他尼与光动力疗法联合应用对大鼠C6胶质瘤的治疗作用

目的：探讨布美他尼对PDT诱导加剧的大鼠C6胶质瘤瘤周水肿的治疗作用。方法：培养C6胶质瘤细胞,建立C6大鼠胶质瘤模型,分成：A：空白对照组,B：光动力治疗组,C：布美他尼治疗组,D：光动力和布美他尼联合治疗组。荷瘤21天后取材测量瘤重,瘤周水含量,微血管密度,NKCC-1和ZO-1的表达。另外MRI监测肿瘤生长,记录大鼠生存时间。结果：PDT能够抑制胶质瘤细胞生长增殖,杀伤肿瘤细胞,促使肿瘤细胞凋亡,下调紧密连接相关蛋白的表达来打开紧密连接,从而延长生存期,同时诱使NKCC-1过表达引起瘤周水肿加剧。结论：应用布美他尼联合治疗能抑制PDT单独应用引起的NKCC-1过表达,减轻PDT加重的瘤周水肿,增强PDT的杀肿瘤作用,而不减少ZO-1的表达。     

The growth of K562 human leukemia cells was inhibited by therapeutic neural stem cells in cellular and xenograft mouse models

To confirm the anti-tumor effect of engineered neural stem cells (NSCs) expressing cytosine deaminase (CD) and interferon-β (IFN-β) with prodrug 5-fluorocytosine (FC), K562 chronic myeloid leukemia (CML) cells were co-cultured with the neural stem cell lines HB1.F3.CD and HB1.F3.CD.IFN-β in 5-FC containing media. A significant decrease in the viability of K562 cells was observed by the treatment of the NSC lines, HB1.F3.CD and HB1.F3.CD.IFN-β, compared with the control. A modified trans-well assay showed that engineered human NSCs significantly migrated toward K562 CML cells more than human normal lung cells. In addition, the important chemoattractant factors involved in the specific migration ability of stem cells were found to be expressed in K562 CML cells. In a xenograft mouse model, NSC treatments via subcutaneous and intravenous injections resulted in significant inhibitions of tumor mass growth and extended survival dates of the mice. Taken together, these results suggest that gene therapy using genetically engineered stem cells expressing CD and IFN-β may be effective for treating CML in these mouse models.     

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.     

In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-α and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2

This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, Hep-2/TIC group; Hep-2/CD group; Hep-2/TNF-α group; Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.