多药耐药细胞系K562/A02裸小鼠皮下移植瘤模型的建立与鉴定

目的建立一种耐药特性稳定的裸小鼠移植瘤动物模型,为进行体内肿瘤耐药机制研究和逆转药物的筛选提供实验模型和实验基础.方法将具有典型MDR表型的K562/A02耐药细胞接种于裸小鼠右侧背侧皮下,而亲本K562细胞接种裸小鼠左侧背侧皮下,接种细胞数为1×107细胞/只,观察肿瘤成瘤情况及生长特性,并比较裸小鼠移植瘤细胞和体外培养细胞对化疗药物的敏感性,同时利用RT-PCR和免疫组织化学染色对裸小鼠K562/A02移植瘤基因表型进行鉴定.结果1)K562/A02裸小鼠移植瘤的成瘤率为100%,大约于第6～9天成瘤(体积＞100mm3),敏感肿瘤生长速度与耐药肿瘤无明显差别;2)裸小鼠K562/A02移植瘤肿瘤细胞和体外培养原代细胞对阿霉素的IC50分别为185.24μg/ml和235.25μg/ml,而K562/A02移植瘤肿瘤的IC50分别为3.07μg/ml和3.26μg/ml;阿霉素治疗组能够明显减慢K562敏感移植瘤的生长,而对K562/A02耐药移植瘤的生长几乎无作用;3)K562/A02裸小鼠移植瘤肿瘤细胞具有mdr1/Pgp表达,而K562裸小鼠移植瘤肿瘤细胞则无mdr1/Pgp表达.结论以K562/A02细胞建立的裸小鼠耐药移植瘤模型,仍保持近似体外的耐药活性和基因表型,为耐药机制和逆转研究提供了良好的体内模型.     

通过活体动物光学成像系统建立乳腺癌动物模型

目的: 通过活体动物光学成像系统建立稳定表达荧光素酶的乳腺癌裸鼠移植瘤模型.方法: 用脂质体包裹带有neo抗性基因及荧光素酶报告基因的真核表达质粒pGL4.17[luc2/neo],体外转染人乳腺癌细胞株MCF-7,将获得稳定表达荧光素酶的乳腺癌细胞株(MCF-7-luc)进行皮下接种BALB/c裸鼠及尾静脉接种SCID小鼠,建立BALB/c裸鼠皮下移植瘤模型和SCID小鼠尾静脉移植瘤模型,通过活体成像系统监测体内肿瘤的生长及转移情况.结果: 经过筛选得到1个能够用于建立裸鼠皮下移植瘤模型及SCID小鼠尾静脉移植瘤模型的稳定表达荧光素酶的乳腺癌细胞株,并成功建立了乳腺癌裸鼠皮下移植瘤模型及SCID小鼠尾静脉移植瘤模型.结论: 通过活体动物光学成像系统成功获得的稳定表达荧光素酶的乳腺癌皮下移植瘤和尾静脉移植瘤模型,为人乳腺癌的相关研究提供了一个直观、方便、灵敏和可靠的平台.     

自杀基因作为一种“安全性开关”控制CAR-T细胞毒性的临床前研究

目的：探讨小分子化学诱导药AP1903能否在体内外终止过表达iCasp9自杀基因的CD19靶向嵌合抗原受体修饰T（CD19CAR-T）细胞毒性功能。方法：构建过表达iCasp9的CD19CAR-T（iCasp9-CD19CAR-T）细胞并和AP1903共孵育，采用流式细胞术检测细胞表型及凋亡的方法，分别在K562和T细胞上验证iCasp9/CID自杀基因系统，在体内（观察荷Raji细胞移植瘤NCG小鼠的生存率）和体外（流式细胞术检测细胞的杀伤功能）检测AP1903给药情况下iCasp9-CD19CAR-T细胞的杀伤功能。结果：和 CD19CAR-T 细胞相比，iCasp9-CD19CAR-T 细胞的增殖能力、表型及体内外杀伤功能均无显著差异（均 P>0.05）。AP1903给药2 h后双表达iCasp9和CD19CAR的K562和T细胞分别有（33.8±0.9）%和（27.95±0.35）%的细胞出现凋亡，AP1903给药 24 h 后双表达 iCasp9 和 CD19CAR 的 K562 和 T 细胞均已经全部死亡。检测 AP1903 给药和未给药两种条件下的 iCasp9-CD19CAR-T细胞体外杀伤效率，前者明显低于后者（P<0.01）；iCasp9-CD19CAR-T细胞治疗荷Raji细胞移植瘤NCG小鼠，其60 d 生存率同样是AP1903给药的明显低于未给药的（P<0.01）。结论：小分子化学诱导药物AP1903能在体内外有效终止iCasp9-CD19CAR-T细胞毒性功能。     

双自杀基因CD和TK对K562细胞体内外杀伤作用的实验研究

目的:探讨双自杀基因CD和TK对K562细胞的体内外抑制作用及前体药物对肿瘤的杀伤作用。方法:将目的基因转染入K562细胞,MTT法观察细胞在体内外的增殖状况及5-FC/GCV对转染细胞的杀伤作用,电子显微镜观察其超微结构变化。将K562/CDgly TK和K562细胞接种于裸鼠皮下,观察各种肿瘤细胞在体内的成瘤情况及对前体药物治疗的敏感性。结果:单独使用GCV或5-FC对K562及K562/CDgly TK细胞产生明显的杀伤作用,联合应用该2种药物对肿瘤细胞的杀伤作用更强。将K562细胞和K562/CDgly TK细胞接种于小鼠皮下后小鼠成瘤率为100%,GCV或5-FC可明显抑制裸鼠体内的肿瘤形成,联合应用GCV和5-FC治疗K562/CDglyTK细胞在小鼠体内形成的肿瘤,较单独应用GCV和5-FC及对照组小鼠形成的肿瘤体积明显缩小,生存期也明显延长。结论:双自杀基因在体内外对K562细胞均有明显的抑制作用,可增加前体药物GCV和5-FC对瘤细胞的杀伤率。     

增强子结合蛋白C/EBPα对白血病BALB/c裸鼠的肿瘤抑制作用

  目的　探讨增强子结合蛋白C／EBPα在白血病裸鼠体内的肿瘤抑制作用。方法　将30只BALB／c裸鼠随机分转染组（10只）、空载组（10只）、对照组（10只）,建立皮下瘤模型,并将30只BALB／c裸鼠如上分组建立白血病模型,将C／EBPα稳定表达细胞株pEGFP-C／EBPα-K562、空载细胞株pEGFP-K562及白血病细胞株K562分别经皮下和尾静脉注射到相应组裸鼠体内,形成皮下瘤和血液病模型。观测皮下肿瘤的变化,应用TUNEL检测细胞的凋亡,瑞特-吉姆萨染色观察血液病模型裸鼠外周血和骨髓中白血病细胞增殖能力,RT-PCR检测增殖相关基因的表达。结果　皮下瘤模型中pEGFP-C／EBPα-K562组肿瘤质量及最大直径为（2.4±0.1）g和（11±2）mm,空载组和对照组分别为（5.1±0.3）g、（19±3）mm和（5.7±0.4）g、（23±3）mm（均P＜0.05）,TUNEL检测发现pEGFP-C／EBPα-K562组肿瘤细胞凋亡明显增加（P＜0.05）;血液病模型鼠外周血可见白血病细胞,pEGFP-C／EBPα-K562组白血病细胞增殖能力明显低于空载组和对照组,可见明显细胞分化现象,RT-PCR检测发现p53基因上调和c-myc基因下调。结论　增强子结合蛋白C／EBPα在白血病小鼠体内具有促进肿瘤细胞凋亡和抑制白血病细胞增殖的能力,并能促进白血病细胞分化。C／EBPα的白血病抑制作用可能是通过对相应基因的调控来实现。     

表达增强型绿色荧光蛋白的小鼠骨肉瘤体外和体内荷瘤模型的建立

背景与目的：小鼠骨肉瘤模型在骨恶性肿瘤的研究中应用较为广泛，良好的活细胞标记方法将有利于研究骨肉瘤的侵袭和转移生物学行为，本研究拟建立稳定表达增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）基因的小鼠骨肉瘤S180细胞株，同时对EGFP-S180细胞进行体内和体外的初步研究。方法：采用电穿孔转染法将EGFP基因导入小鼠骨肉瘤S180细胞，通过G418筛选,建立稳定表达EGFP蛋白的小鼠骨肉瘤EGFP-S180细胞株；激光共聚焦显微镜检测转染前后癌基因c-Myc的表达；建立相应的皮下接种和腹腔接种荷瘤小鼠模型。结果：建立了稳定表达EGFP的EGFP-S180细胞株，转染与未转染细胞的c-Myc表达差异无统计学意义（P〉0.05），小鼠皮下接种和腹腔接种成瘤的时间无显著性差异（P〉0.05）。结论：稳定表达EGFP的S180细胞株没有显著改变肿瘤细胞S180的生物学行为，建立稳定表达EGFP的S180细胞株在体内外研究的方法是可行的，为骨肉瘤体内外的示踪研究提供了实验基础。     

转染4-1BBL基因的胃癌细胞在小鼠体内的抗肿瘤及免疫调节作用

目的:观察转染4-1BBL基因的小鼠胃癌细胞株MFC的体内致瘤性及其对小鼠免疫功能的影响。方法:通过脂质体介导以重组质粒pMKITneo和pMKITneo/4-1BBL转染小鼠胃癌细胞株MFC。用RT-PCR法检测目的基因的表达。计数法绘制体外培养细胞生长曲线。将转染前后肿瘤细胞,分别接种于615小鼠背部皮下观察其致瘤性。流式细胞仪测定荷瘤小鼠外周血NK、CD4 T和CD8 T细胞数,并测定荷瘤小鼠肿瘤结节内细胞凋亡率。结果:MFC/4-1BBL细胞中可检测到4-1BBLmRNA表达。转染前后肿瘤细胞的体外生长速度无明显变化。MFC/4-1BBL细胞在小鼠体内的致瘤性较MFC/pMKITneo细胞和野生型MFC细胞明显降低。接种MFC/4-1BBL细胞的小鼠外周血NK细胞及CD8 T细胞数明显增多;其肿瘤结节内细胞凋亡率亦明显增高。结论:转染4-1BBL基因后并未影响MFC细胞的体外生长,但其在小鼠体内的致瘤性明显降低。MFC/4-1BBL细胞可诱导荷瘤小鼠体内CD8 T和NK细胞增殖,而且能促进肿瘤细胞的凋亡。     

GRIM-19表达对子宫颈癌HeLa细胞增殖的影响

目的:通过体内和体外实验探讨GRIM-19表达对子宫颈癌HeLa细胞增殖的影响.方法:采用脂质体转染法将GRIM-19重组质粒转染HeLa细胞,建立稳定表达GRIM-19的细胞株;采用RT-PCR和Western印迹法检测HeLa细胞转染GRIM19重组质粒前后GRIM-19 mRNA和蛋白水平的表达情况;应用MTT法检测各组细胞增殖水平,并将HeLa/con、HeLa/GRIM-19细胞接种到裸鼠皮下以检测荷瘤情况;同时对移植瘤检测其GRIM-19 mRNA及其下游STAT3 mRNA的表达水平.结果:建立稳定表达GRIM-19基因的HeLa/GRIM-19细胞株.RT-PCR结果显示GRIM-19基因在HeLa/GRIM-19细胞中mRHA表达明显增加,Western 印迹法检测同样发现GRIM-19蛋白的表达显著升高,而STAT3的表达明显受到抑制.MTT检测结果显示HeLa/ GRIM-19细胞增殖较HeLa/con明显减慢(P<0.05),其接种后肿瘤生长速度亦明显减慢(P<0.01).移植瘤的RT-PCR结果表明GRIM-19表达升高、STAT3表达降低.结论:GRIM-19的表达可以抑制子宫颈癌细胞的增殖,可能作为子宫颈癌治疗的一个新靶点.     

重组腺病毒Ad5F35-SH2-DED对K562细胞裸鼠致瘤能力的影响

目的:研究融合蛋白SH2-DED(SD)对白血病K562细胞裸鼠皮下移植瘤的影响.方法:建立K562细胞裸鼠皮下移植瘤的重组腺病毒预防和治疗模型.预防模型采用皮下注射经重组腺病毒Ad5F35-SD或Ad5F35-SmD预处理的K562细胞;治疗模型采用皮下注射K562细胞建立皮下移植瘤后,进行Ad5F35-SD或Ad5F35-SmD的瘤体内多点注射.观察裸鼠移植瘤的生长情况以及移植瘤肿瘤细胞的形态学变化.应用TUNEL法和电子显微镜观察移植瘤肿瘤细胞的凋亡情况.结果:治疗模型组中,Ad5F35-SD处理组移植瘤体积明显缩小,肿瘤细胞核浓缩,细胞质染色加深.TUNEL法和电子显微镜检测结果提示,肿瘤细胞发生凋亡,且凋亡相关蛋白caspase-3和caspase-8表达上调.Ad5F35-SD在预防模型组中同样具有抑制移植瘤生长的作用.结论:重组腺病毒Ad5F35-SD能够抑制K562细胞在裸鼠体内的致瘤能力以及裸鼠皮下移植瘤的生长,并促进肿瘤细胞凋亡.     

CCAAT/增强子结合蛋白α对K562细胞分化和凋亡的影响及其机制

目的:研究CCAAT/增强子结合蛋白α(CCAAT/enhancer-binding protein-alpha,C/EBPα)对K562细胞株分化和凋亡的影响及对相关基因的调控,为慢性粒细胞白血病的治疗提供新的治疗靶点.方法:将C/EBPα表达质粒pEGFP-C/EBPα及空载体对照质粒pEGFP分别经阳离子脂质体2000介导转染K562细胞,用G418筛选出C/EBPα稳定表达细胞株.Wright-Giemsa染色观察细胞形态学变化,FCM分析细胞表面分化抗原CD11b的表达、细胞周期及细胞凋亡,电子显微镜观察细胞凋亡,RT-PCR和Western印迹法检测细胞中相关基因Per 2、cyclin B1和C-myc的表达.结果:筛选得到稳定表达C/EBPα的细胞株pEGFP-C/EBPα-K562.与空载体转染组及对照组细胞相比,转染组K562细胞分化明显,同时粒系细胞表面分化抗原CD11b表达增加;细胞周期分析发现,转染组细胞中G2期细胞增多,与空载体组和对照组相比,差异有统计学意义(P＜0.05),同时出现细胞凋亡峰.细胞凋亡检测结果显示,转染组细胞凋亡明显增加(21.1%),与空载体组(6.0%)和对照组(4.2%)比较,差异有统计学意义(P＜0.05);电子显微镜观察发现,转染组细胞中出现染色质浓集、断裂和核固缩等现象,并见凋亡小体;RT-PCR和Western印迹法检测发现,C/EBPα明显上调Per 2 mRNA和蛋白表达,抑制cyclinB1、C-myc mRNA和蛋白的表达.结论:C/EBPα能促进K562细胞分化,并诱导细胞凋亡,其机制可能是通过对细胞周期相关基因的调控来实现的.     

pcDNA3.1-CD74-ROS1重组质粒构建与表达对肺癌细胞增殖及迁移的影响

目的 构建C-ros致癌基因1（ROS1）融合基因CD74-ROS1重组质粒,并转染A549细胞株,检测其对肺癌细胞迁移能力的影响。方法 通过PCR技术获得CD74和ROS1基因片段,利用融合PCR法构建CD74-ROS1融合基因,构建pcDNA3.1-CD74-ROS1重组质粒,通过脂质体转染技术转入A549细胞中,利用Real-time PCR和Western blot技术检测CD74-ROS1的表达。MTT法观察细胞增殖情况,结晶紫染色法观察细胞形态变化,划痕实验检测CD74-ROS1对肺癌细胞迁移能力的影响。结果 酶切及测序结果表明CD74-ROS1融合基因构建成功,阳性克隆的CD74-ROS1蛋白表达显著增加。CD74-ROS1过表达导致细胞增殖能力增强,细胞形态上发生上皮-间质样转变且细胞迁移能力增强。结论 pcDNA3.1-CD74-ROS1重组质粒构建成功,并在A549细胞中稳定表达,CD74-ROS1过表达能够促进肺癌细胞增殖、形态转变且增强其迁移能力。     

Adenovirus-mediated human topoisomerase IIalpha gene transfer increases the sensitivity of etoposide-resistant human and mouse breast cancer cells

Cellular resistance to chemotherapeutic agents is attributable to several mechanisms, including alteration of topoisomerase IIa gene expression. Our previous studies have shown that transient transfection with a vector containing either Drosophila or human topoisomerase IIalpha gene into drug-resistant tumor cells enhanced their drug sensitivity. Furthermore, we constructed a recombinant adenovirus, Ad-hTopoIIalpha, containing the human topoisomerase IIa gene that was able to selectively increase etoposide sensitivity in drug-resistant tumor cells. We also examined Ad-hTopoIIalpha for therapeutic efficacy in vitro using additional etoposide-resistant cell lines, including a mouse breast cancer cell line and a human leukemia cell line. The etoposide-resistant mouse breast cancer cell line FvP, which is derived from FM3A, and etoposide-resistant human breast cancer cell line, MDA-VP, which derived from MDA-P cells showed increased sensitivity to etoposide as well as increased expression of human Topoisomerase IIa mRNA, but this was not seen in FM3A and MDA-P cells. On the other hand, the etoposide-resistant human leukemia cell line K562/MX2 and the parental cell line K562/P did not show enhanced sensitivity against etoposide or an increase in human Topoisomerase IIa mRNA. Using a recombinant adenovirus containing beta-galactosidase gene (Ad-beta-gal), K562 cells were not transducted by the recombinant adenovirus, while both etoposide-sensitive FM3A cells and etoposide resistant FvP cells were transducted by recombinant adenovirus. Ad-hTOP2alpha and etopside treatment showed reduced inoculated tumor weight in the mice. We concluded that a recombinant adenovirus containing the human Topoisomerase IIalpha gene might be a powerful tool for overcoming drug resistance in breast cancer cells, but not in leukemia cells.     

人CD34分化抗原转录调控序列的克隆与调控表达

目的：克隆有特异调控活性的人ＣＤ３４基因５’端转录调控序列。方法：根据已知的ＣＤ３４抗原基因５’－端启动子调控序列,自行设计２对引物,用巢式－ＰＣＲ方法从染色体ＤＮＡ中成功的克隆６６１ｂｐ长度的ＣＤ３４抗原转录调控序列（ＣＤ３４ＴＲＳ,ＣＤ３４ＴＲＳ片段插入启动子报告质粒ｐＥＧＦＰ－１,观察重组质粒ｐＣＤ３４ＥＧＦＰ在造血系细胞和非造血系细胞中的调控表达作用。结果：经限制性内切酶酶切鉴定及ＤＮＡ序列分析证实,克隆的ＣＤ３４启动子序列与文献报道的顺序基本一致,能特异调探ＥＧＦＰ基因在造血细胞系细胞Ｋ５６２中表达。结论：所克隆的人ＣＤ３４分化抗原转录调控序列有特异调控真核基因表达作用,本研究为构建造血干细胞特异性表达载体奠定了基础。     

不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究

背景与目的:信号肽(signal peptide,SP)是一段存在于前体蛋白N-端的短肽链,能够调节前体蛋白的折叠和转移,在蛋白质的分泌过程中扮演着极其重要的角色.近年来,靶向CD19的嵌合抗原受体(chimeric antigen receptor,CAR)T细胞在白血病治疗中取得了重大突破,关于CAR结构的胞内域改...     

Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted.     

CD19 expression and growth inhibition of tumours in human multiple myeloma

Multiple myeloma (MM) is a proliferative disorder of monoclonal plasma cells which accumulate in human bone marrow (BM). CD19 is a hallmark differentiation antigen of the B cell lineage and positively regulates antigen receptor signal transduction in mature B cells. We have previously shown that malignant plasma cells (myeloma cells) isolated from the MM patients lack the CD19 expression, while non-malignant plasma cells isolated from the healthy donors do express the CD19 antigens. It is also intriguing that there exists both CD19- and CD19+ plasma cells in some cases in pre-myeloma states including monoclonal gammopathy of undetermined significance (MGUS). It indicates that MGUS is usually composed of phenotypically non-malignant (CD19+) and malignant (CD19-) plasma cells. Furthermore, we recently demonstrate that, expression of the CD19 gene markedly inhibits the proliferation of human myeloma cell lines in vitro, and exhibits the reduced tumorigenicity in vivo and no anchorage-independent growth in vitro of a tumorigenic myeloma cell line. This inhibitory effect might result from the CD19-mediated intracellular signals because it is not observed in cells expressing the mutant CD19, which lacks the cytoplasmic domain. In this review, we suggest that loss of CD19 in MM could contribute to the proliferative advantage of the malignant plasma cell clones in this disease. Furthermore, we propose the usefulness of the phenotypic analysis of plasma cells in human plasma cell dyscrasia as a new diagnostic tool, and the CD19 gene as a potential target for the gene therapy in MM.     

hTERT启动子调控的SEA-CD80基因共表达重组腺病毒载体的构建及在不同肿瘤细胞中的表达鉴定

目的：构建肿瘤靶向性葡萄球菌肠毒素 A 和 CD80基因共表达重组腺病毒载体。方法：采用 PCR 技术从质粒 pShuttle - AFP - CD80扩增人 CD80全长 cDNA 片段,克隆至已构建载体 pMD18- T - BIS,取代小鼠CD80 cDNA 片段。重组质粒命名为 pMD18- T - hBIS。经酶切,将 CWV 增强子修饰的人端粒酶反转录酶启动子从已构建载体 pGL3- CWVE - hTP,亚克隆至穿梭质粒 pShuttle2,然后经酶切将片段 hCD80- IRES - SEA从 pMD18- T - hBIS 质粒亚克隆至 CWV 增强子修饰的人端粒酶反转录酶启动子下游,构建质粒 pShuttle2-CE - hTP - hBIS。再与腺病毒骨架质粒 pAdEasy -1共转化 E. coli BJ5183,以获得的重组子（命名为 Ad - CE- hTP - BIS）转染 Ad293细胞制备病毒并纯化。将病毒分别感染人肝癌细胞 H7721、宫颈癌细胞株 Hela 细胞和原代培养的人牙龈成纤维细胞,经免疫荧光染色后,激光共聚焦显微镜观察 SEA 和 CD80在细胞膜的表达情况。结果：重组腺病毒能够使 CD80和 SEA 在人肝癌细胞 H7721、宫颈癌细胞株 Hela 细胞膜上共表达,而在人牙龈成纤维细胞中不表达。结论：成功地构建了多肿瘤靶向性 SEA 和 CD80基因共表达重组腺病毒载体。为应用 SEA 和 CD80基因对人多种肿瘤进行靶向基因治疗奠定了基础。