应用微阵列观察弥漫性星形细胞瘤基因表达谱

目的　探讨弥漫性星形细胞瘤 (WHOⅡ级 )的肿瘤相关基因表达情况。方法　术中收集新鲜弥漫性星形细胞瘤组织 9例及 1例正常脑组织 ,提取总RNA ,逆转录成3 2 P标记的cDNA探针 ,与Atlas人肿瘤微阵列杂交 ,通过放射自显影获得微阵列杂交图 ,应用AtlasImageTM1.0 1a综合分析其相互之间差异。结果　与正常脑组织相比 ,弥漫性星形细胞瘤的差异表达基因数平均为63 .0± 49.9,其中至少在一半以上标本中具有相同表达趋势的基因为 3 3个 ,这些基因的功能多样 ,甚至完全相反。结论　弥漫性星形细胞瘤是一种多基因病变 ,应用微阵列有助于发现该肿瘤的基因变化规律以及研究肿瘤内基因与基因的相互作用关系。     

miR-27b影响β-catenin／Tcf-4通路调控U87胶质瘤细胞生物学行为

目的研究miR-27b在胶质瘤细胞系U87中的表达、功能及作用机制。方法在多个胶质瘤细胞系及胶质瘤标本中检测miR-27b的表达;利用反义miR-27b转染U87,下调rniR-27b的表达,应用流式细胞术和MTT法,评价细胞生长、增殖、凋亡及周期变化：采用荧光素酶法及Western印迹法检测miR-27b作用的初步机制及通路蛋白的变化。结果miR-27b在胶质瘤细胞系及胶质瘤标本中均呈高表达;转染反义miR-27b后,表达降低86％,MTT法显示转染反义miR-27b后细胞生长受到抑制且凋亡细胞增加13％;荧光素酶实验结果显示：miR-27b发挥作用可能与B—catenin／Tcf-4通路相关,Western印迹法显示通路相关蛋白STAT3、c-myc和CyclinD1在转染反义miR-27b后表达下降。结论miR-27b可促进人脑胶质瘤细胞生长、增殖,具有原癌基因的作用,其发挥作用可能与β-catenin／Tcf-4通路密切相关。     

反义miR-21抑制异种移植U87人脑胶质瘤生长的体内研究

目的 探讨敲低miR-21表达抑制裸鼠皮下荷U87人脑胶质瘤生长的疗效和机制.方法 原位注射miR-21反义寡聚核苷酸(AS-miR-21)治疗裸鼠皮下荷U87人脑胶质瘤.定时测量肿瘤大小评估原位注射AS-miR-21的治疗效果.使用RT-PCR和原位杂交方法 鉴定治疗后miR-21表达水平,采用HE染色和免疫组织化学染色(增殖细胞核抗原、细胞周期抑制因子-21、基质金属蛋白酶-9和隔蛋白-7)评价治疗后肿瘤生物学性状的变化,TUNEL法检测肿瘤细胞凋亡. 结果 肿瘤生长曲线显示AS-miR-21治疗组肿瘤生长速度及体积明显小于对照组与无义序列治疗组,差异有统计学意义(F=6.056,P=0.007);RT-PCR检测显示AS-miR-21治疗组miR-21表达下调为对照组的(0.031±0.008)%;原位杂交显示AS-miR-21治疗组miR-21表达水平较对照组与无义序列治疗组下调:组织病理学检测表明AS-miR-21治疗后肿瘤恶性度降低;TUNEL法检测可见AS-miR-21治疗组细胞凋亡数明显高于对照组与无义序列治疗组,差异有统计学意义(F=141.021,P=0.000). 结论 以miR-21作为靶点治疗异种移植U87人脑胶质瘤效果令人满意,miR-21可作为人脑胶质瘤基因治疗的侯选靶点.     

RNA干扰技术靶向P13K/AKT信号通路抵制胃腺癌SGC7901细胞侵袭转移的研究

Objective To observe the inhibitory effects on the invasion of gastric adenocarcinoma SGC7901 cells by small hairpin RNA targeting PIK3R1 and AKT1 in vitro. Methods The recombinant adenovirus vector plasmid expression vector which contained PIK3R1 and AKT1 shRNA was transfected into SGC7901 cells. Real time PCR and Western blot were used to detect the expression of P1K3R1 and AKT1. ELESA was used to measure the change in the MMP-2 and MMP-9 expression. The invasion ability of the tumor ceils was examined by Scarification, Transwell and 3-dimensional matrigel matrix tests. Re-sults rAdS-A-P mediated shRNA targeting PIK3R1 ,and AKT1 dramatically down-regulated their expres-sion in SGC7901 cells. MMP-2 and MMP-9 were downregulated,and TIMP-2 was upregulated. The extra-cellular levels of MMP-2 and MMP-9 were decreased. Scarification test indicated that the invision ability was decreased obviously. Transwell showed that the number of cells invading through the matrigel in con-trol. nonsense sequence, and rAd5-A-P transfection groups was 105.0±4.0,102.5±6.4, and 67.0±3.9 respectively. In the rAdS-A-P transfection group, cells formed only small aggregates as compared with the control and nonsense sequence groups in 3-dimensional matrigel matrix growth test. Conclusion ShRNA targeting PIK3R1 ,and AKTI downregulated significantly their expression in a sequence-specific manner, and inhibited the invasion of gastric adenocarcinoma SGC7901 cells in vitro.     

"十四五"胶质瘤基础与转化研究目标与共识

国家"十四五"发展规划和2035年远景目标纲要提出,把保障人民健康放在优先发展的战略位置,全面推进健康中国建设.目前,我国神经系统肿瘤的发病人数和死亡人数均居世界首位,严重威胁我国人民身体健康.在此大背景下,中国医师协会脑胶质瘤专业委员会基础研究与转化学组整理总结目前胶质瘤研究进展,着眼于全新诊断治疗的策略突破、发病和...     

紫杉醇联合miR-200a对胃腺癌SGC-7901细胞增殖侵袭能力的影响

目的观察紫杉醇联合应用 miR-200 a 对胃癌 SGC-7901细胞增殖侵袭能力的影响。方法体外应用50 nmol/L紫杉醇单独处理胃癌SGC-7901细胞或联合应用脂质体转染miR-200 a模拟物（miR-200a mimics）,通过流式细胞仪检测细胞凋亡水平和周期分布,克隆形成试验观察细胞的增殖能力,Tr-answell实验和细胞划痕试验观察细胞的侵袭迁移能力。通过以上实验比较单独应用紫杉醇与联合应用miR-200 a对胃癌细胞增值侵袭能力影响的差异,并探讨可能的作用机制。结果流式细胞凋亡检测表明50 nmol/L紫杉醇联合应用 miR-200a 细胞早期凋亡率高于单独用药组[（22.79±0.43）％ vs．（11.76±0.64）％,P＜0.05],联合组阻滞于G2/M期细胞比例（63.74±1.76）％高于单独应用紫杉醇组的（51.75±2.01）％,结果具有统计学差异（ P＜0.01）;细胞克隆形成实验显示联合用药组克隆形成率与紫杉醇组相比[（4.03±0.25）％vs．（7.80±0.30）％]显著降低（P＜0.01）;Transwell侵袭实验表明联合用药组穿过小室的细胞数目少于紫杉醇组（22.00±2.00 vs．56.33±5.13,P＜0.01）;细胞划痕试验表明在划痕后48 h,联合组细胞迁移水平与单独用药组迁移率已有显著降低[21.43±2.34）％ vs．（54.26±2.49）％, P ＜0.01]。结论紫杉醇联合应用miR-200 a能增加紫杉醇对胃癌SGC-7901细胞增殖侵袭能力的抑制作用,为今后胃癌的化疗及基因靶向治疗提供新的思路。     

靶向性蛋白激酶B1和环氧合酶-2 shRNA腺病毒载体的构建和在人胃癌细胞的表达

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting protein kinase BI (PKB1/Akt1) and cyclooxygenase-2 (COX-2) and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Akt1 and COX-2 shRNA expression frames were sub-cloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl + COX-2 ( pGSadeno-A + C) vector. Furthermore after screening and amplification,recombinant ade-novirus vector was digested with Pacl and transfected into HEK293 cells. The replication adenovirus rAd5-A + C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A + C,Akt1 and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A + C,SGC-7901 and gen-eral rAd5-HK were selected as the negative controls. Results The recombinant adenovirus rAd5-A + C was constructed successfully and its titer reached 1.0 ×1010 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly, and their ACt values ( 12.26±0.05 and 5.41±0.09 respectively ) were higher than rAd5-HK group (10.63±0.02 and 3.75 +0.08 respectively) and control group (10.57± 0.02 and 3.73±0.08 respectively) (P <0.01 ). There was no significant difference between rAd5-HK and control groups (P >0.05). Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively ( P < 0.01 ) in rAd5-HK group as compared with control group ( P > 0.05 ). Conclu-sion The shRNA aclenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression,and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.     

LY294002抑制PI3K/AKT信号通路对胃腺癌SGC-7901细胞的影响

目的：观察应用PI3K抑制剂2- （4-吗啉基） -8-苯基-4氢-1-苯并吡喃-4酮 （LY294002） 对胃腺癌细胞株SGC-7901中磷脂酰肌酸3-激酶 （PI3K） /丝氨酸苏氨酸激酶 （AKT） 信号通路的变化以及由此产生的细胞生长、 侵袭及凋亡等方面的影响。方法： 应用LY294002作用于胃腺癌细胞株SGC-7901, 通过Western blot检测PI3K/AKT信号通路中相关蛋白质P-AKT、 基质金属蛋白酶-2 （MMP-2）、 基质金属蛋白酶-9 （MMP-9）、 B细胞淋巴瘤/白血病-2 （Bcl-2）、 细胞周期素 （CyclinD1） 的表达情况。噻唑蓝比色法 （MTT） 和流式细胞法及Transwell等实验评价胃腺癌细胞增殖、 侵袭、 凋亡等变化。结果： 与空白对照组和DMSO组相比,LY294002组能有效抑制P-AKT、 MMP-2、 MMP-9、 Bcl-2及CyclinD1蛋白的表达。LY294002组自第2天起生存率明显下降 （P<0.05）。Transwell穿过细胞数结果分别为空白对照组 （81.0±2.65）、 DMSO组 （81.3±1.52）、 LY294002组 （44.6±2.52）, 3组相比较差异具有统计学意义 （F=255.1, P=0.000）。LY294002组细胞周期被阻滞在G0/G1期, S期减（P<0.05）, 早期凋亡的细胞数明显增多（F=149.66, P=0.000）。结论： LY294002是一种对PI3K/AKT信号传导通路具有抑制作用的抑制剂, 靶向PI3K的LY294002可以有效抑制胃腺癌SGC-7901细胞的PI3K/AKT信号通路中相关蛋白质的表达, 在体外显著抑制细胞的增殖、 侵袭, 并促进细胞的凋亡, 因此为临床肿瘤防治提供了新思路, 针对信号传导通路的靶向治疗有望成为肿瘤治疗的新途径。     

立体定位注射靶向功能载药纳米粒子治疗恶性脑胶质瘤

以转铁蛋白溶液为外水相，纳米沉淀技术制备了表面含转铁蛋白的包载卡莫司汀的聚乳酸纳米微粒，药物释放时间达一周以上。体外细胞实验验证纳米微粒表面转铁蛋白保持了活性，纳米粒子与胶质瘤细胞具有较强的结合能力。立体定位注射纳米微粒至荷瘤鼠瘤腔，SPECT考察载药纳米微粒的体内分布，表明纳米微粒在颅内滞留。与对照组相比，注射载药纳米微粒的大鼠生存期显著延长，核磁共振图象显示其中2只大鼠颅内肿瘤消退，表明载药纳米粒子对恶性胶质瘤具有体内抑制作用。     

人脑胶质瘤细胞系miRNA表达谱初步研究

目的 确定部分人脑胶质瘤细胞系miRNA表达谱的初步特征.方法 提取人脑胶质母细胞瘤细胞系U251、TJ861、TJ905、TJ899和A172以及星形细胞瘤细胞系H4细胞的miRNA,miRNA微阵列芯片杂交,扫描后分析差异表达的miRNA.选择差异表达的miR-21设计反义寡核苷酸处理U251细胞后原位杂交和Western blot检查SEPT 7的表达.结果 在胶质瘤细胞系中hsa-miR-21等8种miRNA一致表达上调;hsa-miR-1等18种miRNA一致表达下调.miR-21锁核酸修饰反义寡核苷酸处理U251细胞72h后,原位杂交发现阳性信号集中在细胞核的miR-21表达显著下降,Western blot发现U251细胞中SEPT 7的表达明显上调.结论 miRNA的差异表达可能是胶质瘤的重要分子生物学标签,并在基因表达调控中具有潜在的研究价值.