局部重复应用控释BCNU聚乳酸微球治疗U251胶质瘤的实验研究

 目的研究局部重复应用聚乳酸[poly(D,L-lactic acid),PLA]控释卡莫司汀[1,3-bis(2-chlorethyl)-1-nitrosourea,BCNU]微球(BCNU-PLA)对U251人脑胶质瘤的生长抑制作用。方法应用沉淀方法制备包载BCNU的PLA纳米微球。应用扫描电子显微镜观察载药微球形态,分别应用Bratton-Marshall比色法和紫外分光光度法测定微球载药率与药物释放曲线。应用MTT方法检测PBS、BCNU、PLA微球和BCNU-PLA等不同处理方法对U251胶质瘤细胞增殖活性的影响。应用U251裸鼠皮下移植瘤模型为研究对象,连续3次进行PBS、BCNU、PLA微球和BCNU-PLA的治疗,观察实验动物的生存期与皮下肿瘤生长情况。结果扫描电子显微镜观察载药纳米微球呈球形,具有良好的单分散性;BCNU-PLA平均粒径为23.5μm。BCNU-PLA平均载药率为11.5%;以相对分子质量30×10³的PLA制备BCNU-PLA微球的体外药物释放分析显示:BCNU在释放开始的10d表现出较大的突释,释放量达到载药量的40%;随后进入控释期,至4周左右释放了所载药物的50%。与BCNU原药治疗组比较,BCNU-PLA控释微球治疗在体外持续抑制U251胶质瘤细胞的生长;在体内明显抑制裸鼠皮下荷U251胶质瘤的生长,并显著改善荷瘤鼠的全身状况。结论BCNU-PLA载药微球可有效抑制皮下荷U251胶质瘤的生长,是局部化疗治疗胶质瘤的一种新策略。     

Akt1和Akt2基因转染对人胃黏膜上皮细胞GES-1生长的影响

目的观察Akt1和Akt2基因转染人胃黏膜上皮GES-1细胞后对其生长的影响。方法采用脂质体法将分别含Akt1和Akt2基因全长cDNA序列的p-LXSN-Akt1(Akt1组)和p-LXSN-Akt2(Akt2组)质粒转染人胃黏膜上皮GES-1细胞,以转染p-LXSN空质粒的细胞作为空载组,正常未转染细胞作为对照组,并经抗生素G418筛选抗性克隆。提取各组细胞的总蛋白,蛋白质印迹法检测转染后细胞Akt1、Akt2蛋白及细胞周期蛋白D1(CyclinD1)的表达水平;采用MTT法检测转染后细胞的增殖活性;应用流式细胞仪检测Akt1和Akt2基因转染对细胞周期的影响。结果 Akt1和Akt2组均获得了稳定表达Akt1和Akt2基因的GES-1细胞模型。Akt1组:①Akt1蛋白相对表达量(0.96±0.05)分别是空载组(0.47±0.02)和对照组(0.64±0.03)的2.03±0.22和1.48±0.19倍(均P0.01),CyclinD1蛋白相对表达量(0.74±0.01)分别是空载组(0.29±0.01)和对照组(0.22±0.01)的2.57±0.05和3.44±0.04倍(均P0.01);②吸光度(A490)值于第1、2天分别与空载组和对照组比较差异无统计学意义,第3～6天差异均有统计学意义(均P0.01);③S期细胞数与空载组和对照组比较分别增多约20.00%和21.10%,G2期细胞数减少约20.80%和15.48%(均P0.01)。Akt2组:①Akt2蛋白相对表达量(0.95±0.01)分别是空载组(0.62±0.02)和对照组(0.56±0.01)的1.53±0.04和1.69±0.01倍(均P0.01),CyclinD1蛋白相对表达量(0.31±0.02)与空载组(0.29±0.01)和对照组(0.22±0.01)比较差异均无统计学意义;②A490值于第1～6天分别与空载组和对照组比较差别均无统计学意义;③S期细胞数与空载组和对照组比较差异均无统计学意义。结论 Akt1基因可使GES-1细胞S期细胞数增多,并提高细胞的增殖能力;而Akt2基因对GES-1细胞的细胞周期和增殖能力的影响不明显。     

尿道下裂合并阴茎阴囊转位的外科治疗

作者１９７８年至１９９２年采用分期手术尿道下裂合并阴茎阴囊转位３４例。其中阴茎阴囊型尿道下裂２３例，会阴型尿道下裂１１例；不完全型阴茎阴囊转位２４例，完全型阴茎阴囊转位１０例。３４例中３２例阴茎阴囊转位复位满意。本后并发尿道瘘３例，尿道口狭窄２例。文中对阴茎阴囊转位的分型、手术适应证及其手术方法进行了讨论。     

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.     

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.     

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

目的 构建靶向性蛋白激酶B1(PKB1/Akt1)和环氧合酶-2(COX-2)的短发夹RNA(shRNA)腺病毒载体,观察其在人胃癌细胞株SGC-7901中的表达.方法 利用同源重组技术构建重组腺病毒载体pGSadeno-Akt1+COX-2(pGSadeno-A+C),经酶切及测序鉴定后转染人胚肾细胞HEK293包装成为重组rAd5-A+C腺病毒,并测定病毒的滴度.体外转染人胃癌细胞株SGC-7901后,Real-time PCR和蛋白印迹分别检测Akt1和COX-2 mRNA和蛋白质的表达.结果 成功构建rAd5-A+C重组腺病毒,测定包装的病毒滴度为1.0×1010pfu/ml.转染组Akt1和COX-2 mRNA表达明显下调,其△Ct值分别是(12.26±0.05)和(5.41±0.09),比rAd5-HK空载组(10.63±0.02)、(3.75±0.08)和对照组(10.57±0.02)、(3.73±0.08)增高(P<0.01),而空载组和对照组比较ΔCt值无明显变化(P>0.05).同样转染组Akt1和COX-2蛋白表达量与空载组和对照组比较分别下调70.5%和63.7%(P<0.01),空载组和对照组比较Akt1和COX-2蛋白表达差异无统计学意义(P>0.05).结论 靶向性Akt1和COX-2的shRNA腺病毒载体可以特异性抑制Akt1和COX-2的表达,可能成为胃癌靶向性Akt1和COX-2基因治疗的新策略.     

腺病毒介导的靶向蛋白激酶B1和环氧合酶2短发夹RNA抑制人胃腺癌细胞的侵袭和转移

目的 构建靶向性蛋白激酶B1(Aktl)和环氧合酶2(COX-2)短发夹RNA(shRNA)腺病毒载体,研究其对SGC-7901人胃腺癌细胞侵袭和转移的抑制效果.方法 构建腺病毒载体pGSadeno-Aktl+COX-2(rAd5-A+C)并体外转染SGC-7901人胃癌细胞株后,用实时定量PCR和蛋白印迹分别检测对照组SGC-7901、空载组rAd5-HK和治疗组rAd5-A+C中Aktl、COX-2 mRNA和蛋白质的表达,其中以对照组SGC-7901、空载组rAd5-HK作为阴性对照.用ELISA法分别检测它们的MMP-2和MMP-9含量;用Transwell法分析它们的侵袭和转移能力.结果 构建的rAd5-A+C重组腺病毒载体在转染SGC-7901细胞48 h后可显著抑制Aktl和COX-2 mRNA的表达,而治疗组rAd5-A+C的Aktl和COX-2蛋白表达量与对照组和空载组相比分别下调68.4%和60.2%(均P<0.01);rAd5-A+C组中MMP-2和MMP-9含量分别为(39.7±1.7)ng/ml(31.3±3.6)ng/ml,明显低于对照组SGC-7901(278.4±15.5)ng/ml(225.4±15.1)ng/ml和卒载组rAd5-HK(275.5±2.1)ng/ml、(226.0±23.3)ng/ml(P=0.01、P=0.021).结论 腺病毒介导的靶向性Aktl和COX-2shRNA可抑制人胃腺癌细胞的侵袭和转移.     

Wnt2信号通路重要成员在胶质瘤中的表达及其意义

近来发现Wnt信号通路对个体发育和肿瘤生成具有重要作用[1-2].为进一步探讨Wnt通路在胶质瘤中是否存在表达异常及其在胶质瘤生成中的作用,我们采用RT-PcR、组织芯片免疫组织化学染色以及蛋白印迹法,对人脑胶质瘤Wnt2通路相关因子的表达变化及其意义进行了分析.     

骨髓基质干细胞培养、鉴定及标记的初步研究

目的：对骨髓基质干细胞的培养方法、鉴定及标记技术进行初步研究。方法：采用贴壁法培养大鼠骨髓细胞，经反复传代细胞逐渐纯化，以流式细胞术检测细胞表面抗原。以BrdU标记骨髓基质干细胞，将骨髓基质干细胞注入大鼠脑内，2周后取脑作石蜡切片，进行免疫荧光染色。结果：通过贴壁法培养大鼠骨髓细胞可获得较纯化的细胞，并能在体外长期培养，流式细胞术检测显示CD34、CD31、CD45阴性，CD90、CD44、CDT1阳性。BrdU可掺入DNA合成期的骨髓基质干细胞。结论：贴壁法可获得高纯度的骨髓基质干细胞，BrdU可作为骨髓基质干细胞体内示踪的理想标记物。     

反义表皮生长因子受体与p53基因联合治疗脑胶质瘤

基于前期研究我们发现不论是表皮生长因子受体(EGFR)扩增/过表达或p53基因突变率均随星形细胞瘤的恶性程度增高而增高[1],体外实验也表明联合应用反义EGFR RNA及p53基因转染胶质母细胞瘤细胞株对抑制细胞增殖和诱导凋亡的作用显著,较单独应用其中之一为强[2],为此,我们进行了体内实验.