慢病毒转染自杀基因后联合更昔洛韦治疗卵巢上皮性癌的实验研究

目的：探讨以抗Ⅰ型粘蛋白单链抗体（anti-MUC1)为靶向的慢病毒高效转染自杀基因后联合更昔洛韦（GCV）对Ⅰ型粘蛋白阳性（MUC1^ ）卵 巢上皮性癌细胞的靶向性基因治疗。方法：包装以anti-MUC1为靶向的慢病毒,介导单纯疱疹病毒胸苷激酶（HSV－tk)基因转染卵巢上皮性癌细胞株SKOV3（MUC1^ ）及正常戊纤维细胞GF（MUC1^-）,荧光显微镜下观察转染效果,聚合酶链反应（PCR）、逆转录聚合酶链反应（RT－PCR）技术鉴定HSF－tk基因在靶细胞（SKOV3及GF）中的整合转录结果。学显微镜观察GCV作用后细胞形态变化,用四甲基偶氮唑蓝（MTT）法测定GCV的体外杀伤效应。结果：荧光显微镜下观察到以anti-MUC1为靶向的慢病毒可有效转染SKOV3细胞,PCR、RT－PCR均证明HSF－tk基因已有靶细胞SKOV3中整合且转录表达;而在GF细胞中未见HSF－tk基因的整合及转录表达,光学显微镜下观察到HSF－tk基因康后的细胞在GCV作用后出现明显形态变化;MTT法显示GCV对转染HSF－tk基因后的SKOV3细胞有显著杀伤的作用,其半数抑制浓度为0．10mg/L.结论：以anti-MUC1为靶向的慢病毒可将HSF－tk基因高效、靶向性转染MUC1^ 卵巢上皮性癌细胞,联合应用GVC可高效、靶向性杀伤MUC1^ 卵巢上皮性癌细胞。     

单链抗体导向卵巢上皮癌进行靶向基因转移的实验研究

目的 :探讨靶向性基因转移载体—含抗MUC 1单链抗体的慢病毒对MUC 1+卵巢上皮癌细胞系SKOV 3细胞进行基因转移的特异性 ,为卵巢上皮癌的靶向性基因治疗提供实验基础。方法 :将针对SKOV 3细胞上抗原MUC 1的单链抗体基因构建到慢病毒包膜结构质粒 (pM .TC .G) ,以报告基因绿色荧光蛋白 (GFP)基因作为目的基因。用磷酸钙沉淀法进行Anti MUC 1(ScFv)慢病毒包装 ,感染共培养的卵巢癌细胞SKOV 3(MUC 1+)和正常人成纤维细胞GF(MUC 1- ) ,荧光显微镜下观察感染效果。竞争抑制实验即病毒感染前加入Anti MUC 1单抗是否抑制病毒感染。结果 :荧光显微镜下观察到Anti MUC 1(ScFv)介导的慢病毒对共培养的SKOV 3(MUC 1+)和GF(MUC 1- )细胞的感染有明显差别 ,非Anti MUC 1(ScFv)介导的慢病毒对共培养的SKOV 3(MUC 1+)和GF(MUC 1- )的感染未见显著性差别 ,证明Anti MUC 1(ScFv)介导的慢病毒可对MUC 1+的卵巢细胞靶向性转染。病毒感染前加入Anti MUC 1单抗则可抑制病毒靶向性感染。此竟争抑制实验表明 ,病毒感染是由其抗体组成部分介导 ,故有抗体靶向性。结论 :Ani MUC 1(ScFv)介导的慢病毒可对MUC - 1+卵巢上皮癌细胞靶向性基因转移。     

转座子piggyBac介导的HSV-tk基因在妇科恶性肿瘤细胞中长期稳定表达的研究

目的 研究piggyBac(PB)转座子介导转移外源基因HSV-tk在多种妇科恶性肿瘤细胞中的表达情况,探讨其作为一种可整合的非病毒载体在肿瘤基因治疗中的应用价值.方法 通过PCR技术扩增HSV-tk基因的编码区,定向克隆至PB表达载体PB[Act-RFP]DS,经酶切、测序鉴定为重组载体PB[Act-RFP,HSV-tk]DS(pPB/TK).联合3种小同的转染试剂FuGENE HD、jetPEI及lipofectamine 2000,分别将pPB/TK转染入4种常见的妇科恶性肿瘤细胞株HeLa、JEG-3、SKOV3和HEC-1B.转染后以mRFP1为报告基因,经荧光显微镜观察和流式细胞仪分析转染效率;逆转录(RT)-PCR技术检测HSV-tk和mRFP1基因的表达;四甲基偶氮唑蓝实验测定不同浓度的更昔洛韦(GCV)对转染后细胞的杀伤作用.转染后细胞经流式细胞仪分选,极限稀释单克隆培养建立稳定转染株,并以反向PCR技术确定转座子插入基因组的位点.结果 (1)成功构建重组载体pPB/TK.(2)在3种转染试剂的辅助下,pPB/TK均可有效转染入HeLa、HEC-1B、SKOV3和JEG-3细胞;不同转染试剂辅助下pPB/TK在同种细胞中的转染效率各不相同,在HeLa细胞中,FuGENE HD的转染效率[(78.7±9.2)%]高于lipofectamine 2000[(54.1±11.4)%]和jetPEI][(46.5±7.4)%],分别比较,差异均有统计学意义(P<0.05);同一转染试剂辅助下pPB/TK在不同细胞中的转染效率也不相同,以FuGENE HD为转染试剂,pPB/TK在HeLa、JEG-3和SKOV3细胞中的转染效率分别为(78.7 ±9.2)%、(74.4±8.9)%和(83.2±9.7)%,高于HEC-1B细胞的(39.5±8.7)%,分别比较,差异均有统计学意义(P<0.05).(3)RT-PCR结果显示,4种细胞均有HSV-tk和mRFP1基因mRNA的表达.(4)GCV对转染有pPB/TK的HeLa、JEG-3、SKOV3和HEC-1B细胞的半数抑制浓度分别为1.29、3.35、0.09和13.28 μg/ml.10μg/ml GCV对转染有pPB/TK的SKOV3细胞抑制率高于单独转染pORF-HSVtk者,细胞抑制率分别为(86±9)%和(52±12)%,两者比较,差异有统计学意义(P<0.05).(5)稳定转染株在转染后3个月仍可观察到mRFP1表达,并可榆测纠基因组中有TTAA位点的插入整合.结论 PB转座子可介导外源基因在多种妇科恶性肿瘤细胞中的长期稳定表达,有望为肿瘤基因治疗提供更为安全有效的转基因技术平台.     

内皮前体细胞转染TRAIL基因后治疗卵巢上皮性癌的实验研究

目的探讨内皮前体细胞(EPC)转染TRAIL基因后对卵巢上皮性癌细胞的体外杀伤作用。方法采用细胞表面特异性标志物CD133磁珠分离法,从脐血中分离EPC,并进行体外培养、扩增和鉴定。构建带有TRAIL基因的质粒(即TRAIL质粒),用脂质体将TRAIL质粒转染入EPC,酶标法测定培养上清液中EPC分泌的TRAIL蛋白的表达水平,流式细胞仪分析其对卵巢上皮性癌细胞的促凋亡作用。结果采用磁珠分离法可从脐血中分离出EPC,EPC占分离后细胞总数的(84±3)%。成功构建TRAIL质粒,转染TRAIL质粒后EPC分泌的TRAIL蛋白水平增加,转染72 h时可达532.8pg/m l;而仅转染脂质体及转染绿色荧光蛋白者为12.4及9.2 pg/m l。将转染TRAIL质粒后的EPC培养上清液与卵巢上皮性癌细胞共同孵育24 h后,卵巢上皮性癌细胞凋亡率最高可达24.1%。结论TRAIL基因转染EPC后,可使EPC分泌的TRAIL蛋白水平增加,并诱导卵巢上皮性癌细胞凋亡。提示,EPC可作为基因治疗的靶向性载体,有望应用于卵巢上皮性癌的治疗。     

白细胞介素7基因转染卵巢癌细胞的体外研究

目的　观察白细胞介素 (IL) 7基因体外转染卵巢癌细胞后 ,细胞的增殖特性 ,表面抗原表达和细胞因子分泌的变化 ,及对淋巴因子激活的杀伤细胞 (LAK细胞 )杀伤敏感性的影响。方法　对卵巢上皮性癌细胞株SKOV3进行体外培养 ,将重组质粒pBK CMVIL 7导入SKOV3建立SKOV3 IL 7,将双相表达载体pBK CMV导入SKOV3建立SKOV3 Neo ,应用倒置相差显微镜、四甲基偶氮唑蓝比色试验及流式细胞术 ,观察IL 7基因转染前后 ,SKOV3的增殖特性变化 ;间接标记异硫氰酸荧光素 流式细胞术 ,测定细胞表面抗原表达 ;酶联免疫吸附试验测定细胞因子分泌 ;应用乳酸脱氢酶释放法观察细胞对LAK细胞杀伤敏感性的影响。结果　IL 7基因转染前后 ,SKOV3细胞基本形态、增殖特性及细胞周期分布无明显变化 ;细胞表面人类白细胞抗原ABC(HLA ABC)表达阳性率为 91 8%～99 9% ;人类白细胞抗原DR(HLA DR)未能检测到 ;IL 7基因转染后SKOV3细胞间粘附分子I(ICAM I)表达显著提高 ,SKOV3 IL 7为 (4 0 6± 3 7) % ,SKOV3 Neo为 (2 3 2± 2 7) % ,SKOV3为 (18 9± 7 2 ) % ;转化生长因子 β1 (TGF β1 )表达显著降低 ,SKOV3 IL 7为每 2 4h、10 6 细胞中 (下同 ) 15 8ng L ,SKOV3 Neo为 72 6 μg L ,SKOV3为 396ng L ;IL 7基因转染后 ,SKOV3细胞对LAK细胞     

肿瘤相关粘蛋白MUC1及其同种型MUC1/Y与卵巢上皮性肿瘤关系的研究

目的探讨肿瘤相关粘蛋白MUC1及其同种型 MUC1/Y粘蛋白在卵巢上皮性肿瘤的变化特征.方法采用免疫组化ABC法,对72例卵巢上皮性、浆液性和粘液性肿瘤组织和8例正常卵巢组织 MUC1和MUC1/Y粘蛋白进行检测.结果 MUC1在卵巢癌中的表达率(80%)高于良性上皮性肿瘤(45%)和正常卵巢组织(37.5%), P<0.005.MUC1/Y仅在卵巢癌中表达,二者高表达,与卵巢癌的恶性程度高、组织分化差、癌瘤播散及淋巴结转移有关.结论 MUC1、MUC1/Y作为一种新型的肿瘤标志物,与卵巢癌的发生、发展密切相关.     

上皮性卵巢癌中Wnt/β-catenin信号通路对间皮素表达的影响

目的:探讨上皮性卵巢癌(EOC)中Wnt/β-catenin信号通路对间皮素基因表达的调控作用。方法:免疫组化法检测EOC组织中Wnt-1、β-catenin及间皮素蛋白表达,并分析其共定位表达的相关性。卵巢细胞系SKOV3中,分别以Li Cl、Wnt-3a模拟激活,β-catenin靶向RNAi慢病毒阻断Wnt/β-catenin通路,Real-time PCR及Western blot法检测间皮素mRNA及蛋白表达。结果:EOC组织中Wnt-1蛋白表达与良性卵巢上皮性肿瘤和正常卵巢上皮比较,差异无统计学意义(P0.05)。β-catenin异位表达率为76.56%,间皮素阳性表达率为79.69%,均高于良性卵巢上皮性肿瘤和正常卵巢上皮,差异均有统计学意义(P均0.01)。EOC组织中间皮素蛋白表达与β-catenin异位呈正相关(r=0.564,P0.05)。以Li Cl和Wnt-3a模拟激活SKOV3细胞Wnt/β-catenin通路后,间皮素mRNA表达明显高于对照组(F=27.576,P0.01),间皮素蛋白表达明显上调。构建β-catenin靶向的RNAi慢病毒,转染SKOV3细胞成功后,间皮素表达无明显下调。结论:Wnt/β-catenin信号通路可能参与了EOC中间皮素基因的表达调控。     

肿瘤相关粘蛋白MUC1及其mRNA与卵巢上皮性肿瘤关系的研究

目的 :初步探讨肿瘤相关粘蛋白MUC1基因及其蛋白与卵巢上皮性肿瘤的关系。方法 :采用原位杂交法和免疫组化ABC法 ,检测 6 5例卵巢上皮性肿瘤组织和 8例正常卵巢组织MUC1mRNA及其蛋白的表达。结果 :MUC1mRNA和MUC1阳性表达皆位于胞浆和胞膜 ,多呈灶状分布。MUC1mRNA和MUC1在卵巢癌中的检出率分别为 75 .6 % ,80 .0 % ,显著高于良性上皮性肿瘤的 4 0 % ,4 5 %和正常卵巢组织的 2 5 .0 % ,37.5 % (P <0 .0 0 5 )。二者高表达与卵巢癌的期别晚、组织分化差及淋巴结转移有关。结论 :MUC1作为新型肿瘤标志物 ,与卵巢癌的发生、发展密切相关     

ERCC1基因与卵巢上皮癌细胞铂类耐药相关性探讨

目的 检测切除修复交叉互补1（ERCC1）基因在人卵巢上皮癌顺铂(DDP)耐药细胞株SKOV3／DDP及其亲本细胞株SKOV3中的表达,探讨ERCC1基因在卵巢上皮癌顺铂耐药中的意义以及有效沉默ERCC1基因能否有效逆转SKOV3／DDP细胞对DDP耐药。方法 于2013-2014年在南方医科大学采用实时荧光定量PCR(RT-PCR)和Western印迹法检测 ERCC1 mRNA和蛋白在SKOV3／DDP及SKOV3细胞中的表达。人工构建3个靶向沉默ERCC1基因的短发夹状RNA(shRNA)质粒表达载体,慢病毒载体法转染至SKOV3／DDP细胞,RT-PCR和Western印迹法检测ERCC1基因水平并挑选沉默效果最佳的表达载体。RT-PCR和Western印迹法检测稳定转染后SKOV3／DDP细胞中ERCC1基因的表达,CCK-8法检测转染细胞对DDP的耐药性。结果 SKOV3／DDP细胞中ERCC1 mRNA和蛋白的表达量明显高于SKOV3,差异有统计学意义(P<0.01)。稳定转染ERCC1 shRNA后的SKOV3／DDP细胞,ERCC1的mRNA及蛋白的表达量明显下降,差异有统计学意义(P<0.01)。细胞活性检测提示特异性沉默ERCC1基因能增加SKOV3/DDP 细胞对顺铂的敏感性(P<0.05)。结论 ERCC1基因在SKOV3／DDP细胞中的表达明显升高,有效沉默ERCC1 基因能有效逆转SKOV3/DDP细胞对DDP耐药,增加卵巢上皮癌DDP耐药细胞对DDP的敏感性,为卵巢上皮癌耐药的治疗提供新靶点。     

Ad.Ela对SKOV3细胞的转染效率及体外杀伤作用

目的：研究腺病毒Ela质粒对HER-2/neu高表达卵巢癌细胞系SKOV3的转染效率和体外治疗作用。方法：脂质体介导腺病毒载体重组,腺病毒扩增、纯化、滴度测定,不同滴度病毒转染卵巢癌细胞的效率分析,以20：1的Ad.Ela抑制SKOV3细胞生长。结果：体外20：1 AdRSVlacZ感染SKOV3细胞1次,基因转染效率732%。此后增加病毒量,转染效率曲线接近平台。103*"SKOV3细胞分别以2×104 PFU的Ad.Ela+、Ad.Ela-和PBS感染1次,结果显示,Ad.Ela+治疗后卵巢癌细胞生长明显抑制,FACS检测转染后的SKOV3细胞,P185蛋白的表达明显下降。结论：Ad.Ela对HER-2/neu高表达细胞SKOV3的生长有显著抑制作用。     

Association of placental inflammation with fetomaternal hemorrhage and loss of placental mucin-1

Background  

Fetomaternal hemorrhage (FMH) poses an immediate risk to the fetus and, in case of Rhesus-immunization, to future pregnancies. Given that altered endothelial permeability is part of the pathophysiology of inflammation, in this study we investigated whether placental inflammatory processes like chorioamnionitis (ChoA) or preeclampsia (PE) lead to increased rates of FMH compared to the established risk factor of placenta previa (PP). Putative accompanying markers of trophoblastic damage were also explored.     

Glutathione S-transferases P1-1 and A1-1 in ovarian cyst fluids

PURPOSE: The purpose of the present study was to determine the gluthathione S-transferases (GST) P1-1 and A1-1 levels in cyst fluid from malignant, borderline, and benign ovarian tumors. The clinical relevance of these enzymes in cyst fluid was investigated, including the possible relation with resistance to chemotherapy. METHODS: A total of 90 ovarian cysts were punctured for cyst fluid collection. GSTP1-1 and GSTA1-1 concentrations were determined by ELISA in cyst fluid from 23 malignant, 9 borderline, and 51 benign primary ovarian tumors, and levels were correlated with histopathological data. RESULTS: Significantly higher GSTP1-I concentrations were found in cyst fluid from malignant (median: 477 ng/ml), compared with benign (median: 52 ng/ml) ovarian cysts (p < 0.0001), as well as in fluid from borderline (median: 366 ng/ml) compared with benign cysts (p < 0.0001). No significant differences were found in cyst fluid GSTA1-1 concentrations between the histologic subgroups. In cyst fluid from malignant tumors higher GSTPI-1 and lower GSTAI-1 concentrations were found in patients with worse prognostic factors: FIGO II-III-IV, grade 2-3, residual tumor > 2 cm, presence of ascites, patients with recurrent disease, and survival, but differences were not significant. In the subgroup of patients that received cisplatin-based chemotherapy (n = 14) significantly higher GSTP1-1 (p = 0.01) concentrations were found in patients with recurrence compared with patients without recurrence. Considering only FIGO stage I patients, a differentiation could be made between patients with or without recurrence based on cyst fluid GSTP I - I concentrations. CONCLUSIONS: Determination of glutathione S-transferases P 1-1 in cyst fluid samples from ovarian tumors can be of additiona] value in the differentiation between histologic subgroups. In case of possible low malignant potential cysts where sampling of the most representative tissue can be an issue, determination of GSTP- I concentrations in cyst fluid may optimise histopathologic classification. Cyst fluid GSTP1-1 seems to be a good marker for aggressiveness of the ovarian tumor, and it may predict response to chemotherapy.     

Analysis of GSTM1, GSTT1, and CYP1A1 in idiopathic male infertility

Enzymes belonging to the glutathione-S-transferase (GST) and cytochrome P450 (CYP) families are involved in a 2-stage detoxification process of a wide range of environmental toxins and carcinogens. In order to investigate whether there is a genetic association of the biotransformation enzymes and idiopathic male fertility, we studied GSTT1, GSTM1, and CYP1A1*2A polymorphisms in 150 infertile men and 200 healthy men as controls from Northern Iran. Genotyping of the GSTT1 and GSTM1 genes were performed using the multiplex polymerase chain reaction (PCR). However, the CYP1A1 polymorphism was determined using PCR-restriction fragment length polymorphism (RFLP). The GSTM1 and GSTT1 null genotypes were present at frequencies of 0.61 and 0.34 in infertile cases, whereas in controls the frequencies were 0.33 and 0.17, respectively. Double-null genotype was found to be elevated among infertile men (odds ratio [OR] = 3.75, 95% confidence interval [CI] = 2.42-6.45; P < .0001). The frequency of TT, TC, and CC genotypes of CYP1A1 polymorphism in the controls were 42.5%, 45.5%, and 12%, respectively, while those in the infertile men were 38.7%, 48%, and 13.3%. The CYP1A1*2A did not display any association with male infertility. We observed an association between male infertility and the GSTM1 and GSTT1 null deletion, but not with the CYP1A1 polymorphism in North Iranian men with idiopathic infertility.     

CYP1A1 MspI多态性及其合并GSTM1基因缺失与子宫内膜异位症的关系

目的:探讨新疆维吾尔族、汉族人群中解毒酶细胞色素P4501A1(CYP1A1)基因MspI多态性、CYP1A1/MspI合并GSTM1基因缺失基因型与子宫内膜异位症(内异症)的关系。方法:用聚合酶链反应限制性片段长度多态性技术,检测维吾尔族107例正常妇女与41例内异症患者、汉族105例正常妇女与80例内异症患者CYP1A1基因限制性内切酶MspI位点的3种基因型的分布频率。结果:CYP1A1基因MspI位点基因型在维吾尔族正常对照组的分布频率为TT(48.6%)、TC(42.9%)、CC(8.5%),等位基因分布频率为T(70.1%)、C(29.9%),内异症组的基因型分布频率为TT(39.1%)、TC(46.3%)、CC(14.6%),等位基因分布频率为T(62.2%)、C(37.8%),差异无显著性(P>0.05);在汉族正常对照组基因型分布频率为TT(41.9%)、TC(46.7%)、CC(11.4%),等位基因分布频率为T(65.2%)、C(34.8%),内异症组基因型分布频率为TT(42.5%)、TC(51.2%)、CC(6.3%),等位基因分布频率为T(68.1%)、C(31.9%),差异无显著性。两个民族对照组之间与内异症组之间基因型频率与等位基因频率比较差异无显著性。在CYP1A1/MspI合并GSTM1(/)基因型的人群中,维吾尔族对照组与内异症组的基因型频率与等位基因频率分布比较均有显著差异(P<0.05),其TC+CC与TT比较,OR值为3.556(P<0.05)。汉族对照组与内异症组的比较无统计学差异。结论:解毒酶CYP1A1基因MspI多态性本身可能与维吾尔族及汉族内异症发病无关,而CYP1A1/MspI合并GSTM1(/)基因型可能与维吾尔族内异症发病有关。     

CYP1A1 and CYP1B1 genetic polymorphisms and uterine leiomyoma risk in Chinese women

Purpose  The aim of the study was to evaluate the association of CYP1A1 and CYP1B1 polymorphisms with uterine leiomyoma in Chinese women. Methods  We investigated 100 women with clinically diagnosed uterine leiomyoma and 110 healthy normal subjects from Chinese women. The genetic distribution of two CYP1A1 polymorphisms at MspI, Ile462Val and four CYP1B1 polymorphisms at Arg48Gly, Ala119Ser, Leu432Val, Asp449Asp were analyzed by polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing method. Results  All the SNPs showed polymorphisms in Chinese women. The genotype A/G and the allele G on Ile462Val was significantly different between uterine leiomyoma patients and controls (P < 0.05). Conclusion  These results suggest that the genotype of CYP1A1 Ile462Val was associated with the increased risk of uterine leiomyomas in Chinese women. Capsule This is the first report that demonstrates the polymorphism at Ile462Val of CYP1A1 to be associated with uterine leiomyoma in Chinese women.