突变的大肠癌线粒体DNA转染NIH3T3及LST细胞的研究

目的了解大肠癌细胞株（SW480，LOVO，HT29）线粒体DNA的突变，克隆突变的大肠癌线粒体DNA（mtDNA）基因，构建peDNA3．1（＋）-mtDNA真核表达重组体，并导入NIH3T3及LST细胞，以探讨线粒体基因突变与肿瘤发生的关系。方法提取大肠癌细胞株（SW480，LOVO，HT29）mtDNA，扩增D—LOOP区，产物用DNA自动测序法进行序列分析。利用DNA重组技术将其定向插入真核表达质粒peDNA3．1（＋），并用脂质体法导人NIH3T3及LST细胞。用MitoCa．ptureMitochondrialApoptosisDetectionKit试剂盒染色后用流式细胞仪及荧光显微镜检测转染细胞的凋亡情况。扩增并测序分析转染细胞的D—LOOP区突变特点。结果检测出大肠癌细胞株SW480，LOVO，HT29细胞mtDNAD—LOOP分别有10，9，8个突变位点。转染前后，各组间细胞凋亡无明显变化。转染细胞的核基因组可扩增出目的基因及Neo基因。4株NIH3T3转染细胞mtDNAD-环区分别检测到9，11，8，4个突变点，并相应有3，4，3，2个多态性变化。结论（1）转染突变的大肠癌细胞mtDNA后转染细胞的mtDNA均可发生多处的突变位点。（2）通过转染后突变的外源性的mtDNA可以整合到核基因组内。（3）突变的mtDNA转染LST细胞及NIH3T3细胞后，不影响转染细胞的凋亡改变。（4）mtDNA的突变可能通过影响体细胞mtDNA的突变和通过外源性mtDNA在核内的整合从而影响癌基因或抑癌基因的表达异常，从而参与肿瘤的发生发展。     

结直肠癌组织中线粒体DNA D-环区突变的研究

背景：近年线粒体DNA与肿瘤之间的相关性已成为新的研究热点，目前已在多种恶性肿瘤或癌前病变中检测到线粒体突变。目的：研究线粒体DNAD-环区在结直肠癌组织中的突变情况，探讨其在结直肠癌发生、发展中的作用。方法：以聚合酶链反应（PCR）扩增40例结直肠癌患者肿瘤组织和癌旁组织的线粒体DNAD-环区，扩增产物直接测序。结果：40例结直肠癌患者中，共发现50个D-环区多态性，其中2个为BLAST数据库中未记录的新多态性。14例患者存在线粒体DNAD-环区突变，突变率为35％，突变位点12个，其中9个点突变，2个微卫星不稳定，1个缺失。结论：结直肠癌组织线粒体DNAD-环区具有高度多态性和高突变率，可能与结直肠癌的发生、发展有关。     

线粒体DNA变异在大肠癌发病中的作用与机制研究

目前认为肿瘤的生物学特征不仅取决于核内遗传物质，而且与核外线粒体DNA的改变也可能有重要的关系。为研究线粒体DNA的变异在大肠癌发病中的作用与机制，本文对4株大肠癌细胞系和1株原代培养的正常肠上皮细胞的线粒体DNA的D-环区进行了对比研究。     

大肠侧向发育型肿瘤线粒体DNA D-环区的突变

目的:了解大肠侧向发育型肿瘤(laterally spreading tumor, LST)线粒体DNA的突变,探讨其与肿瘤发生的关系. 方法:提取LST线粒体DNA(mtDNA),扩增D-环区,产物用DNA自动测序法进行序列分析. 结果:共检测到4个碱基突变.第16223位C突变为T,第16298位C突变为T,第16362位T突变为C,第16519 位T突变为C. 结论:线粒体DNA D-环区是—个具有高度多态性和突变性的区域,在LST中突变率较高.     

Tiam1与大肠癌细胞EMT的关系

目的:探讨Tiaml和EMT在不同转移能力的大肠癌细胞中的关系.方法:用Real time-RT PCR法分析Tiaml mRNA,Ecadherin mRNA和Vimentin mRNA在6种大肠癌细胞系中的表达:通过免疫组织化学检测Tiaml,Ecadherin和Vimentin在LoVo和HT29中的表达.通过考马斯亮蓝染色观察LoVo和HT29的细胞骨架.结果:在SW620,SW480/M5,HT29,LoVo和LS174T中,Tiam1 mRNA的表达水平分别是SW480的0,51,7,67,0,00,0,36,0,06倍,差异具有统计学意义(P<0.05);Ecadherin mRNA的表达水平分别是SW480的3.18,2.27,5.92,0.00,0.61倍,差异具有统计学意义(P<0.05);Vimentin mRNA的表达水平分别是SW480的6.08,0.02,0.35,11.72,0.00倍,差异具有统计学意义(P<0.05).细胞免疫组织化学显示LoVo细胞的Tiaml表达呈阳性(++),Vimentin表达呈强阳性(+++),Ecadherin表达阴性(-);HT29细胞的Ecadaerin表达呈阳性(++),Tiaml和Vimentin表达阴性(-).LoVo细胞质中丝网状的骨架蛋白结构及点状肌动蛋白小体比HT29多.结论:Tiam促进大肠癌转移的机制可能与EMT发生有关.     

线粒体DNA ND1基因nt3394 T→C突变与老年人2型糖尿病

目的：了解线粒体烟酰胺腺嘌呤脱氧核苷酸（NADH）脱氢酶第一亚单位（ND1）基因中339位点T→C突变与我国老年人2型糖尿病的关系。方法：收集无血缘关系的208例老年2型糖尿病患者，同时以180例无糖尿病家族史的老年糖耐量正常者作为对照，用聚合酶链反应扩增。限糖尿病患者，同时以180例无糖尿病家族史的老年糖耐量正常者作为对照，用聚合酶链反应扩增、限制性内切酶HaeⅢ消化进行点突变筛选。结果：发现9例患者存在线粒体DNA ND1基因3394位点T→C突变（4．3％），非糖尿病者中仅有1例突变（0．6％），两组比较差异有显著性（P＜0．05）。结论：线粒体DNA ND1基因中3394位点T→C突变可能与我国老年人2型糖尿病的发病有关。     

不同转移潜能大肠癌细胞株基因表达谱的差异分析

目的从基因表达谱轮廓分析不同转移潜能大肠癌细胞株的特点,探讨大肠癌亲器官转移的分子机制。方法应用Affymetrix HG-U133 Plus 2.0原位合成寡核苷酸芯片检测同一亲本分别具有不同转移潜能和转移器官亲和性的大肠癌细胞株SW620、SW480和SW480肝转移细胞株基因表达谱差,以SW480为对照细胞株,筛查"大肠癌转移相关基因"和"亲器官转移相关基因",应用GOTM软件对基因功能进行分析。结果筛查出SW620、SW480肝转移细胞株两组共同表达的"大肠癌转移相关基因"共422个,筛查出的"亲器官转移相关基因或ESTs"3 054个。"转移相关基因&ESTs"功能主要涉及细胞代谢、细胞生理进程调节、信号传导、大分子代谢、基本代谢和代谢调节等。"亲器官转移相关基因或ESTs"的GO分析结果表明SW620、SW480肝转移细胞株在基因表达谱功能分类差异主要表现在细胞黏附、信号传导、细胞运动、调节酶活性、核酸代谢和金属离子结合等方面。结论不同转移潜能大肠癌细胞株基因表达谱存在差异,通过对基因表达谱轮廓分析,可进一步了解大肠癌转移器官亲和性的分子机制和筛查转移相关基因。     

雌激素受体β过表达对大肠癌细胞增殖凋亡的影响

目的观察雌激素受体β（ERβ）过表达对大肠癌细胞增殖和凋亡的影响,并探讨其机制。方法以脂质体介导的ERβ基因转染SW480细胞,G418筛选阳性克隆（SW480-C1-ERβ）。RT—PCR及WesternBlot鉴定ERβ的过表达。以正常SW480细胞和转染了空质粒的SW480细胞（SW480-pEGFP—C1）作为对照,在无雌激素或有雌激素作用的条件下,采用MTT法检测细胞增殖,流式细胞术检测细胞凋亡率,实时荧光定量RT—PCR方法检测凋亡相关基因survivin和Bax表达。结果SW480和SW480-pEGFP—C1细胞中ERβ mRNA和蛋白表达较低,而稳定转染的SW480-C1-ERβ细胞中ERβmRNA和蛋白表达明显增加。在有或无雌激素作用的条件下均可发现,与SW480细胞或SW480-pEGFP—C1细胞比较,SW480-C1-ERβ细胞增殖速度减慢,凋亡率明显增高,survivin表达减低,Bax表达增高;在SW480-C1-ERβ细胞中,有雌激素作用者较无雌激素作用者以上变化更明显。结论ERβ过表达可以同时以配体非依赖性和配体依赖性的方式抑制细胞增殖和增加细胞凋亡,可能与调控凋亡相关基因survivin和Bax表达有关。     

广西地区妊娠期糖尿病患者所生新生儿线粒体tRNALeu（UUR）基因3243位点突变的研究

目的探讨线粒体tRNALeu(UUR)基因3243位点A→G突变在广西地区妊娠期糖尿病患者所分娩的新生儿中发生的频率。方法采用DNA测序技术,对广西地区50例妊娠期糖尿病产妇所分娩的新生儿和50名正常健康对照者分娩的新生儿进行线粒体tRNALeu(UUR)基因3243位点检测。结果妊娠期糖尿病患者分娩的新生儿及正常对照者分娩的新生儿脐血中均未检测到线粒体tRNALeu(UUR)基因3243位点A→G突变。结论线粒体tRNALeu(UUR)基因3243位点A→G突变尚不能作为该地区孕妇妊娠期糖尿病产前筛查的参考指标。     

奥沙利铂不同浓度和时间对诱导大肠癌细胞系SW480耐药效果的影响

目的采取两种方式体外诱导大肠癌耐奥沙利铂耐药细胞系,比较其耐药机制。方法分别采用低浓度长时间诱导法和高浓度短时间诱导法诱导大肠癌细胞系SW480,建立大肠癌奥沙利铂(Oxaliplatin,OXP)耐药细胞系SW480/OXP1和SW480/OXP2。CCK8法检测SW480、SW480/OXP1和SW480/OXP2耐药指数,绘制细胞生长曲线,检测细胞周期分布;罗丹明123检测细胞膜泵功能,Western blotting检测P-gp和E-cadherin水平。结果 SW480/OXP1对OXP、5-氟尿、顺铂耐药指数分别为10.34±0.35、3.32±0.52、2.76±0.26,SW480/OXP2对OXP、5-氟尿、顺铂耐药指数分别为7.89±0.62、2.78±0.37、2.1±0.23;SW480、SW480/OXP1和SW480/OXP2的群体倍增时间分别为30.50 h、36.64 h和35.87 h。SW480/OXP1和SW480/OXP2的G0/G1比率较亲本细胞上升,而S期比率较亲本细胞下降;正常SW480的罗丹明123排出明显缓于SW480/OXP1和SW480/OXP2。SW480/OXP1和SW480/OXP2的P-gp表达较SW480升高,且SW480/OXP1高于SW480/OXP2。SW480/OXP1和SW480/OXP2的E-cadherin表达均低于SW480,且SW480/OXP2低于SW480/OXP1。结论 OXP低浓度长时间诱导法和高浓度短时间诱导法都可使SW480产生多药耐药性,但是它们的特性仍有部分差异,应按照需要选择合适的诱导方式。     

Reduced expression of β-catenin inhibitor Chibby in colon carcinoma cell lines

AIM: To analyse the Chibby expression and its function in colon carcinoma cell lines and colorectal carcinoma (CRC).METHODS: Chibby expression levels were investigated by quantitative RT-PCR in a panel of seven different colon carcinoma cell lines. By sequencing, we analysed mutational status of Chibby. To test whether Chibby exhibited effects on β-catenin signalling in colon carcinoma cells, we transfected SW480 cells with Chibby expression plasmid and, subsequently, analysed activity of β-catenin and tested for alterations in cellular phenotype. In addition, we examined Chibby mRNA levels in samples of colorectal carcinomas and adjacent normal tissues by using quantitative RT-PCR and hybridised gene chips with samples from CRC and normal tissues.RESULTS: Chibby mRNA expression was strongly downregulated in colon carcinoma cell lines in comparison to normal colon epithelial cells and no mutation in any of the examined colon carcinoma cell lines was found. Further, we could show that Chibby inhibited β-catenin activity in TOPflash assays when over-expressed in SW480 cells. Proliferation and invasion assays with Chibby transfected SW480 cells did not reveal profound differences compared to control cells. In contrast to these in vitro data, quantitative RT-PCR analyses of Chibby mRNA levels in CRC tumor samples did not show significant differences to specimens in adjacent non-cancerous tissue.Consistent with these findings, gene chips analysing tissue samples of tumors and corresponding normal tissue did not show altered Chibby expressionCONCLUSION: Altered Chibby expression might beobserved in vitro in different colon carcinoma cell lines.However, this finding could not be confirmed in vitro in CRC tumors, indicating that Chibby is not likely to promote CRC tumor development or progression. As Chibby is an important inhibitor of β-catenin signalling, our data implicate that the usability of colon carcinoma cell lines for in vitro studies analysing the Wnt/β-catenin pathway in colorectal carcinoma needs extensive verification.     

Reduced expression of beta-catenin inhibitor Chibby in colon carcinoma cell lines

AIM: To analyse the Chibby expression and its function in colon carcinoma cell lines and colorectal carcinoma (CRC). METHODS: Chibby expression levels were investigated by quantitative RT-PCR in a panel of seven different colon carcinoma cell lines. By sequencing, we analysed mutational status of Chibby. To test whether Chibby exhibited effects on beta-catenin signalling in colon carcinoma cells, we transfected SW480 cells with Chibby expression plasmid and, subsequently, analysed activity of beta-catenin and tested for alterations in cellular phenotype. In addition, we examined Chibby mRNA levels in samples of colorectal carcinomas and adjacent normal tissues by using quantitative RT-PCR and hybridised gene chips with samples from CRC and normal tissues. RESULTS: Chibby mRNA expression was strongly down-regulated in colon carcinoma cell lines in comparison to normal colon epithelial cells and no mutation in any of the examined colon carcinoma cell lines was found. Further, we could show that Chibby inhibited beta-catenin activity in TOPflash assays when over-expressed in SW480 cells. Proliferation and invasion assays with Chibby transfected SW480 cells did not reveal profound differences compared to control cells. In contrast to these in vitro data, quantitative RT-PCR analyses of Chibby mRNA levels in CRC tumor samples did not show significant differences to specimens in adjacent non-cancerous tissue. Consistent with these findings, gene chips analysing tissue samples of tumors and corresponding normal tissue did not show altered Chibby expression. CONCLUSION: Altered Chibby expression might be observed in vitro in different colon carcinoma cell lines. However, this finding could not be confirmed in vitro in CRC tumors, indicating that Chibby is not likely to promote CRC tumor development or progression. As Chibby is an important inhibitor of beta-catenin signalling, our data implicate that the usability of colon carcinoma cell lines for in vitro studies analysing the Wnt/beta-catenin pathway in colorectal carcinoma needs extensive verification.     

Study on the mutations in the D-loop region of mitochondrial DNA in cervical carcinoma

Background  Study on genesis and development of tumor is mainly concentrated on gene mutation in nucleus. But in recent years, the role of mitochondrial DNA (mtDNA) mutation in tumor genesis has been given more attention, which is the only extra-nucleus DNA in cells of higher animals. Carcinoma of the uterine cervix is a common tumor in gynecology. There are few reports of mtDNA mutation in this area. The focus of this study was to investigate the mitochondrial DNA mutation in tumor tissues of cervical carcinomas patients and their relationship to tumorigenesis and tumor development. Methods  The D-loop region of 24 cervical carcinomas together with the adjacent normal tissues were amplified by PCR and sequenced. Results  Among the 24 cervical carcinomas, 30 mutations were identified with the mutations rate of 37.5% (9/24).There were eight microsatellite instabilities among the mutations and 13 new polymorphisms which were not reported previously in the GeneBank. Conclusions  The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutations rate is relatively high in patients with cervical carcinomas. Daozhen Chen and Huiying Zhan contributed equally to this work.     

Mutation in D-loop region of mitochondrial DNA in gastric cancer and its significance

AIM:To investigate the mutation in D-loop region of mitochondrial DNA in gastric cancer and its influence on the changes of reactive oxygen species (ROS) and cell cycle. METHODS: The D-loop region was amplified by PCR and sequenced.Reactive oxygen species and cell cycle were detected by flow cytometry in 20 specimens from gastric cancer and adjacent normal tissues.According to the sequence results,gastric cancer tissue was divided into mutation group and control group.Reactive oxygen species,apoptosis and proliferation in the two groups were compared. RESULTS:Among the 20 gastric cancer specimens, 18 mutations were identified in 7 patients,the mutation rate being 35%.There were four microsatellite instabilities in the mutations. No mutation was found in the adjacent tissues. Reactive oxygen species,apoptosis,and proliferation in the mutation group were all significantly higher than those in control group. CONCLUSION: Mutation in D-loop region plays a role in the genesis and development of gastric cancer.     

Mutation of Mitochondrial DNA in Livers From Patients With Alcoholic Hepatitis and Nonalcoholic Steatohepatitis

Background: In alcoholic hepatitis (Al-Hep) and nonalcoholic steatohepatitis (NASH), triglycerides accumulate in hepatocytes. We examined the hypothesis that mutations in mitochondrial DNA may take place by mitochondrial overwork, resulting in dysfunction of mitochondria.
Subjects and Methods: Subjects of this research were 8 cases each of Al-Hep, NASH, and fatty liver (FL). Total DNA was extracted from the biopsied liver samples. DNA fragments were amplified by PCR and DNA sequences determined in the control and coding regions of mitochondrion.
Results: When the numbers of mutations per 1,000 bases of mitochondrial DNA were compared between each group, no significant differences were found among D-loop, HV1, and HV2 mitochondrial DNA regions. However, there were significantly more mutations in ND1 and COII of Al-Hep and NASH than in FL, and mutations were comparatively at random. Neither a region in which mutations were focused nor differences among the groups were recognized. When details of the base mutation in a control region were investigated by group, the transition type of mutation between T:A≪–≫C:G occurred in at least 70%. Also, a transition-type mutation was found mostly in a coding region, which was similar to the mutation pattern in the control region, except for the ND1 and COII regions where there were hardly any mutations.
Conclusions: As gene mutations of mitochondrial DNA appeared frequently in Al-Hep, and also in NASH, mitochondrial dysfunction caused by mutation in mitochondrial DNA may be involved in the pathogenesis of both diseases.     

急性髓系白血病患者线粒体DNA的D-loop区存在突变

目的研究线粒体DNA的D-loop区突变与白血病发生、发展的关系。方法应用盐析法对8例初治的成人急性髓系白血病患者及其生母和正常无关对照者的外周血基因组DNA提取、线粒体基因D-loop区PCR扩增、序列测定,对比分析白血病患者的线粒体基因D-loop区序列与其生母和健康无关对照者的线粒体基因D-loop区序列与标准剑桥线粒体基因D-loop区序列的差异。结果8例白血病患者中6例存在突变(突变率75%),共查出突变位点11个,突变类型均为D-loop区的T-C、A-C、G-A碱基替代突变。结论线粒体DNA的D-loop区在白血病患者中的突变率较高,D-loop区的突变在白血病发生发展过程中可能起作用。