X射线照射诱导鼻咽癌细胞hMSH2的表达

目的研究X射线照射对鼻咽癌CNE-1细胞错配修复基因hMSH2表达的影响,探讨放射损伤后肿瘤细胞的DNA修复机制。方法应用逆转录-PCR(RT-PCR)、免疫细胞化学及蛋白免疫印迹(Western blot)方法,检测X射线照射后对照组和实验组(照射总剂量分别为0Gy和10Gy)细胞中hM-SH2基因mRNA及蛋白表达。结果实验组细胞hMSH2基因mRNA及蛋白表达在照射终止后逐渐上调,其表达较对照组显著增强(P<0.01)。结论X射线照射可诱导鼻咽癌细胞hMSH2的表达,有助于放射损伤后肿瘤细胞DNA修复,这可能是肿瘤放疗敏感性降低的原因之一。     

错配修复基因hMLH1和hMSH2的表达与人卵巢癌顺铂耐药的关系

目的:探讨DNA错配修复基因hMLH1和hMSH2的表达在人卵巢癌细胞顺铂耐药现象中的作用.方法:MTT法检测卵巢癌细胞对顺铂的敏感性,Hoechst免疫荧光染色检测顺铂作用下敏感及耐药细胞的凋亡情况,并使用RT-PCR技术及West-ern blotting技术分别检测顺铂敏感性人卵巢癌细胞及耐药顺铂人卵巢癌细胞中错配修复基因hMLH1及hMSH2的转录与翻译表达变化.结果:同一顺铂浓度作用下,耐药顺铂人卵巢癌细胞的凋亡率明显低于顺铂敏感性人卵巢癌细胞,且后者的错配修复基因hMLH1和hMSH2的mRNA及蛋白表达水平均显著高于前者.结论:顺铂可诱导卵巢癌细胞凋亡.在耐药顺铂人卵巢癌细胞中,错配修复基因hMLH1和hMSH2低表达可能是导致顺铂耐药发生的重要原因.     

化疗对肺癌人类错配修复基因hMLH1 和hMSH2表达的影响

0引言DNA错配修复系统是生物在进化过程中形成的一套纠正体内DNA损伤的体系,可以及时有效地纠正因各种体内外因素导致的基因错配.研究发现DNA错配修复基因中的hMLH1和hMSH2介入了一些肿瘤的发生与发展[1].已有研究发现hMLH1和hMSH2在非小细胞肺癌中的表达降低,提示两者很可能在肺癌的发病机制中起重要作用[2-3].由于相当一部分患者术前往往先接受化疗,而化疗对肺癌中hMLH1和hMSH2表达有何影响尚不明确.     

大肠腺瘤及癌变组织hMLH1和hMSH2表达与细胞凋亡的研究

目的:探讨hMLH1和hMSH2基因在大肠腺瘤及其癌变中的作用,及其表达对细胞凋亡的影响.方法:采用免疫组织化学染色检测63例大肠腺瘤、20例腺瘤癌变和20例大肠癌组织hMLH1和hMSH2表达;同时采用TUNEL法检测其细胞凋亡指数(AI).结果:大肠腺瘤、腺瘤癌变和大肠癌组织错配修复基因hMLH1、hMSH2的表达率逐渐降低,与正常大肠相比相差显著,随腺瘤不典型增生程度增加其阳性率逐渐降低;大肠腺瘤、腺瘤癌变和大肠癌中hMLH1表达缺失者细胞凋亡指数较显著高于其阳性者,且大肠腺瘤不典型增生Ⅰ、Ⅱ、Ⅲ级hMLH1-与hMLH1+组存在差异显著;而hMSH2-与hMSH2-间AI仅大肠腺瘤组有显著性差异,不典型增生Ⅰ、Ⅱ级组hMSH2-与hMSH2+间AI差异显著,而不典型增生Ⅲ级组hMSH2蛋白的表达阴性与阳性间AI无统计学差异.结论:DNA错配修复基因突变或功能缺失与大肠癌的发生有关,可能系大肠癌发生过程中的早期事件,且可能与大肠肿瘤细胞凋亡活性增加相关.     

表没食子儿茶素没食子酸酯对人成淋巴细胞株hMLH1 和hMSH2 mRNA表达的影响

目的： 探讨表没食子儿茶素没食子酸酯[(-)epigallocatechin gallate ，EGCG]对人成淋巴细胞株错配修复基因hMLH1和hMSH2 mRNA表达水平的影响。方法：将正常人成淋巴细胞株GM12593和乳腺癌患者成淋巴细胞株GM13705分别置于含有0、5、10、20 μmol/L EGCG的RPMI-1640中进行6 d干预培养后，采用实时荧光定量PCR (FQ-PCR)技术检测干预前后错配修复基因hMLH1和hMSH2 mRNA表达水平的变化。结果：经20 μmol/L EGCG干预培养6 d后，GM12593细胞hMLH1与hMSH2 mRNA表达水平均显著高于其他浓度组 (P均<0.05)，且显著高于同等浓度时GM13705细胞中上述2个基因的表达水平(P<0.05)；EGCG对GM13705 目标基因的表达无显著影响 (P>0.05)。结论：EGCG具有上调正常人成淋巴细胞株hMLH1与hMSH2 mRNA表达水平的潜力，可能通过增加错配修复起始复合物的数量，来帮助错配修复机制的启动，维护基因组的稳定性。     

大肠腺瘤及癌变组织hMLH1和hMSH2表达与细胞凋亡的研究

目的：探讨hMLH1和hMSH2基因在大肠腺瘤及其癌变中的作用,及其表达对细胞凋亡的影响。方法：采用免疫组织化学染色检测63例大肠腺瘤、20例腺瘤癌变和20例大肠癌组织hMLH1和hMSH2表达;同时采用TUNEL法检测其细胞凋亡指数（AI）。结果：大肠腺瘤、腺瘤癌变和大肠癌组织错配修复基因hMLH1、hMSH2的表达率逐渐降低,与正常大肠相比相差显著,随腺瘤不典型增生程度增加其阳性率逐渐降低;大肠腺瘤、腺瘤癌变和大肠癌中hMLH1表达缺失者细胞凋亡指数较显著高于其阳性者,且大肠腺瘤不典型增生Ⅰ、Ⅱ、Ⅲ级hMLH1-与hMLH1＋组存在差异显著;而hMSH2-与hMSH2-间AI仅大肠腺瘤组有显著性差异,不典型增生Ⅰ、Ⅱ级组hMSH2-与hMSH2＋间AI差异显著,而不典型增生Ⅲ级组hMSH2蛋白的表达阴性与阳性间AI无统计学差异。结论：DNA错配修复基因突变或功能缺失与大肠癌的发生有关,可能系大肠癌发生过程中的早期事件,且可能与大肠肿瘤细胞凋亡活性增加相关。     

散发性胆囊肿瘤错配修复基因hMLH1、hMSH2和nm23H1基因蛋白表达的研究

目的探讨hMLH1和hMSH22种错配修复基因和抑癌基因nm23H1蛋白在胆囊肿瘤组织中的表达,评估其在胆囊肿瘤发生、发展和预后判断中的临床意义,为揭示肿瘤发生的病理机制提供实验依据。方法采用Envision免疫组织化学方法,检测70例胆囊肿瘤组织和10例胆囊炎症组织hMLH1、hMSH2和nm23H1蛋白表达变化。结果①在胆囊癌、胆囊良性肿瘤和炎症组织中,hMLH1、hMSH2和nm23H1蛋白表达阳性率差异显著(P<0.05);②在胆囊癌组织中,hMLH1、hMSH2蛋白表达阳性率分别为51.06%、42.55%;有淋巴结转移者hMLH1蛋白表达阳性率(25.00%)和NevinⅣ Ⅴ期表达阳性率(29.17%)分别低于无淋巴结转移者(70.37%)和NevinⅠ Ⅱ Ⅲ期(73.91%),P<0.01,伴肝脏浸润者(27.78%)低于无肝脏浸润者(65.51%),P<0.05;但hMSH2蛋白表达阳性率无显著性差异;③在胆囊癌组织中,nm23H1蛋白表达阳性率为46.81%,有淋巴结转移者(25.00%)低于无淋巴结转移者(62.96%),P<0.01;NevinⅣ Ⅴ期(29.17%)低于Ⅰ Ⅱ Ⅲ期(65.22%),P<0.05;④在胆囊癌组织中,hMSH2蛋白表达阳性者中nm23H1蛋白表达阳性率(65.00%)显著高于hMSH2蛋白表达阴性者(33.33%),P<0.05。结论实验结果提示,DNA错配修复基因hMLH1、hMSH2和nm23H1基因相互协同,参与了胆囊肿瘤发生、发展和转移的过程,可能是胆囊肿瘤发生的1个重要分子机制。     

表没食子儿茶素没食子酸酯（EGCG）对鼻咽癌细胞CNE1、CNE2放射增敏作用的初步研究

目的:研究表没食子儿茶素没食子酸酯（EGCG）对鼻咽癌细胞CNE1、CEN2的放射增敏作用及其可能机制。方法:体外培养CNE1、CNE2细胞,CCK-8实验得到EGCG IC20值50μg/ml,即为实验浓度。实验分为4组,对照组:含有和其他组相等浓度的DMSO溶解剂;药物组:DMSO溶解的EGCG;照射组:X线照射组;实验组:EGCG联合X线照射组。体外培养CNE1、CNE2细胞至对数生长期,药物组及实验组分别给予药物即EGCG处理,培养24h后进行相应剂量的X线照射后收集细胞。采用克隆形成实验检测不同射线剂量（0、2、4、6、8、10Gy）处理后各组的克隆形成率、细胞存活率及放射增敏比（SER）,流式细胞分析法检测各组细胞的凋亡情况及细胞周期,Western bolt检测Akt蛋白的表达,RT-PCR检测Akt mRNA的表达。结果:平板克隆实验:照射组及实验组随着X线剂量的逐步增加,克隆形成率随之下降,细胞存活分数下降,呈现剂量依赖效应（P＜0.05）;相同剂量下,实验组比照射组克隆形成率低,实验组Do、Dq明显低于单纯照射组（P＜0.05）,CNE1细胞株SER为1.4962,CNE2细胞株SER为1.1846,说明EGCG具有放射增敏作用（P＜0.05）。流式细胞分析:各实验组与对照组相比,G2/M期百分比升高（P＜0.05）,表明存在G2/M期阻滞,单纯射线组比单纯药物组对G2/M期的阻滞作用更明显,二者联合的实验组表现为协同作用。实时荧光定量实验:各实验组与对照组相比,Akt mRNA表达下降（P＜0.05）,实验组比单纯照射组及单纯药物组更明显。Western blot实验:各实验组与对照组相比,Akt蛋白表达均有下降（P＜0.05）,实验组比单纯照射组及单纯药物组更明显。结论:EGCG对鼻咽癌细胞CNE1、CNE2具有放射增敏作用,其机制可能是通过抑制细胞增殖、促进细胞凋亡、抑制细胞周期、抑制射线诱导的Akt活化而产生。     

急性白血病组蛋白乙酰化修饰及其对错配修复基因表达的调控作用

 目的　探讨急性白血病患者组蛋白乙酰化修饰规律,并探索组蛋白乙酰化对错配修复基因hMSH2 和hMLH1差异表达的调控作用。方法　用反转录-聚合酶链反应（RT-PCR）方法检测56例急性白血病患者和30名健康志愿者单个核细胞（MNC）的错配修复基因hMSH2 和hMLH1 mRNA的表达,用Western blot法检测组蛋白H3、H4、去乙酰化酶（HDAC1）、hMSH2 和hMLH1基因的蛋白表达情况。用组蛋白去乙酰转移酶抑制剂（TSA）诱导30例白血病患者MNC乙酰化,并检测处理后MNC的组蛋白H3、H4、HDAC1、hMSH2 和hMLH1的表达状态变化。结果　急性白血病组的hMSH2 和hMLH1、组蛋白H3、H4的蛋白表达量分别为0.4610±0.1211、0.4013±0.1143、0.4103±0.1241和0.4251±0.1081,均明显低于健康志愿者组的蛋白表达量（0.9461±0.1841、0.9960±0.2021、0.8971±0.1194、0.9513±0.1953）,差异均有统计学意义（t值分别为3.341、3.935、2.843、3.575,P＜0.05）;而急性白血病患者组的HDAC1表达（0.8841±0.2018）高于健康志愿者组的表达量（0.5142±0.1340）,差异有统计学意义（t＝2.634,P＜0.05）;TSA作用于白血病单个核细胞后,组蛋白H3、H4、hMSH2 和hMLH1的表达上调,分别比阴性对照组表达上调2.9倍、3.4倍、1.5倍和1.6倍,而HDAC1的蛋白表达出现明显的抑制,表达下调为阴性对照组的40 %。结论　急性白血病患者的组蛋白乙酰化呈低表达现象,组蛋白乙酰化在急性白血病患者中对错配修复基因差异表达具有调控作用。     

沉默CDKN1A基因对鼻咽癌放射抗拒性CNE-2R细胞放射敏感性的影响

目的：探讨CDKN1A基因的表达与鼻咽癌放射敏感性的关系。方法构建慢病毒表达载体LV-CDKN1A-RNAi并转染鼻咽癌放射抗拒性CNE-2R细胞,设转染LV-CDKN1A-RNAi慢病毒的CNE-2R细胞为实验组,转染阴性对照慢病毒的CNE-2R细胞为阴性对照组,未转染的CNE-2R细胞为空白对照组。用CCK-8法、细胞克隆形成实验及流式细胞术分别检测各组细胞增殖、放射敏感性及细胞周期的变化。结果成功构建了CDKN1A基因沉默的CNE-2R细胞,CCK-8法检测显示实验组CNE-2R细胞在照射6 Gy后生长受到抑制,且随时间延长其抑制作用更为明显。细胞克隆形成实验显示实验组CNE-2R细胞放射敏感性增强（放射增敏比为SER=1.24）。流式细胞术检测显示实验组与对照组细胞相比, G0/G1期和G2/M期细胞分布在X射线照射6 Gy前后明显改变（P约0.05）。结论 CDKN1A基因沉默能增强鼻咽癌放射抗拒性CNE-2R细胞的放射敏感性,CDKN1A基因的表达可能与鼻咽癌放射敏感性相关,有望成为鼻咽癌治疗的新靶点。     

Analysis of MLH1 and MSH2 expression in ovarian cancer before and after platinum drug-based chemotherapy.

Preclinical studies have demonstrated a relationship between DNA mismatch repair (MMR) status and sensitivity to cisplatin and carboplatin. MMR-deficient cells are resistant to both drugs, and selection for cisplatin resistance in vitro is sometimes accompanied by loss of MMR protein expression. We used immunohistochemical staining techniques to investigate hMLH1 and hMSH2 expression in paired ovarian tumor sections from 54 ovarian cancer patients before and after platinum-based therapy. We sought associations between hMLH1 and hMSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. hMLH1 and hMSH2 staining decreased significantly after platinum-based therapy. The percent of malignant cells that stained positive correlated with the intensity of nuclear staining for both proteins; staining for hMLH1 correlated well with staining for hMSH2. Unexpectedly, expression of nuclear hMLH1 correlated negatively with response to treatment. Expression of nuclear hMLH1 and hMSH2 was positively correlated with pretreatment CA125 level, and expression of nuclear hMSH2 was positively correlated with change in CA125 level after treatment. Tumor stage was associated with expression of nuclear hMSH2, and tumor histological subtype was associated with both hMLH1 and hMSH2 staining. No association was found between expression of either protein and overall survival. These results indicate that the tumor is biologically altered after chemotherapy consistent with treatment-induced selection for cells expressing lower hMLH1 and hMSH2 levels. However, immunohistochemical staining for either hMLH1 or hMSH2 was not highly predictive of drug sensitivity as measured by response or survival.     

Changes of hMSH2 and hMLH1 Expression in Nasopharyngeal Carcinoma Cells after X-Radiation

OBJECTIVE To study the effect of X-radiation on expression of hMSH2 and hMLH1 in nasopharyngeal carcinoma （NPC） CNE-1 cells, and explore the mechanism of DNA mismatch repair after radiation. METHODS The cells were divided into experimental and control groups. Experimental cells were exposed to 10 Gy radiation administered as 2 Gy per fraction. Control cells were not radiated. The distribution of cells in the cell cycle was analyzed using flow cytometry. The expression of hMSH2 and hMLH1 was examined by RT-PCR and Western blots. RESULTS The expression of hMSH2 mRNA in experimental cells was significantly greater compared to control cells at 0-3rd weeks and decreased at the 4th week following radiation （P〈0.01）. The expression of hMSH2 protein in experimental cells was up-regulated and significantly greater compared to control cells at the 2nd-4th weeks after radiation （P〈0.01）. There were no significant differences in hMLH1 mRNA and protein expression between experimental and control cells （P 〉0.05）. CONCLUSION Radiation induces hMSH2 expression; hMSH2 has a role in the process of DNA repair, which maybe responsible for reduction of radiosensitivity after radiation.     

X射线对人肺癌A549细胞Bmi-1表达的影响

目的：研究X射线照射对人肺癌A549细胞株中Bmi-1 mRNA和蛋白表达的影响。方法：用不同剂量（0、2、4、6Gy）的X射线照射体外培养的人肺癌A549细胞株,分别采用实时荧光定量PCR和Western blot技术检测照射0、6、12、24、48和72 h后Bmi-1 mRNA和蛋白的表达水平。结果：与对照组比较,2、4、6 Gy X射线照射A549细胞后48 h内Bmi-1 mRNA表达升高,差异均有统计学意义（P〈0.05）;（2 Gy 48 h组除外）,2 Gy组照射后6 h、4 Gy组12 h、6 Gy组24 h升高最显著（P〈0.05）。照射后48 h内蛋白表达升高（4Gy除外）,48 h后蛋白表达逐渐下降,至72 h时接近未照射组水平。各时间点（除4 Gy 48 h和72 h外）的蛋白表达与对照组相比差异均有统计学意义（P〈0.05）。结论：在本实验条件下,2～6 Gy剂量X射线照射48 h内可使人肺癌A549细胞Bmi-1表达升高,之后表达逐渐降低。     

Reduced expression of hMSH2 protein is correlated to poor survival for soft tissue sarcoma patients

BACKGROUND: Deregulation of DNA mismatch repair is a common mechanism for the development of hereditary nonpolyposis colon carcinoma and related familiar cancers, but it also plays a role in the tumorigenesis of sporadic cancers. Although the protein expression of the two main components of DNA mismatch repair, hMSH2 and hMLH1, has been described in soft tissue sarcoma (STS) patients, its prognostic impact is yet to be determined. METHODS: The authors investigated the expression of the DNA repair proteins hMSH2 and hMLH1 by Western blot analysis in tumor samples of 57 STS patients. The correlation between the expression of hMSH2/hMLH1 and survival was studied in a Cox proportional hazards regression model, which was adjusted for the prognostic effects of staging, tumor entity, and radicality of tumor resection. RESULTS: Nine of 57 STS (16%) showed reduced expression of hMSH2 and reduced expression of hMLH1 was detected in seven STS patients (12%). In a Kaplan-Meier analysis, the median survival for patients with reduced expression of the hMSH2 protein was 18 months, whereas the patients with a normal expression of hMSH2 survived for an average of 68 months. A multivariate Cox proportional hazards regression model revealed a significant correlation between the reduced expression of the hMSH2 protein and poor survival (relative risk = 4.7; 95% confidence interval [CI]: 1.3-17.2; P = 0.019). CONCLUSIONS: Reduced expression of the hMSH2 protein is an independent negative prognostic factor for STS patients.     

Expression of DNA mismatch repair proteins hMSH2 and hMLH1 and the cyclin G1 inhibitor, p21(waf1/cip1) in pediatric tumors: correlation with response to therapy

The expression of the DNA mismatch repair proteins hMSH2 and hMLH1 and p21(waf1) the cyclin G1 inhibitor, may determine response of adult cancers to anti-cancer drugs, that include alkylating agents and platinum-based drugs. The role of DNA mismatch repair proteins (hMSH2 and hMLH1) and p21(waf1) in pediatric tumor responses to chemotherapy and irradiation is described in the present study of 23 pediatric solid cancers (4 wilms' tumors, 9 neuroblastomas, 3 hepatoblastomas, 3 lymphomas and 4 sarcomas) using immunohistochemical methods. Immunostaining was scored for intensity (0-3) and extent (0-3). Most tumors stained strongly for hMSH2 and weakly or negative for hMLH1. All the hMLH1 negative tumors (1 wilms', 1 hepatoblastoma, 1 sarcoma, 2 lymphomas and 2 neuroblastomas) achieved complete response. p21(waf1) negative and positive tumors achieved relatively similar treatment response. The results suggest that the expression of DNA mismatch repair proteins hMLH1 and hMSH2, and p21(waf1) do not influence individual cancer responses to treatment and the results may reflect the use of multiple drugs and irradiation that cause many different types of DNA damage.     

错配修复基因的异常表达与淮安食管癌发病的关系

背景与目的： 研究错配修复相关基因hMLH1和hMSH2的mRNA表达水平与食管癌发病的关系。 材料与方法： 收集112例淮安地区食管癌新发病例的食管癌组织及远端癌旁组织,采用实时荧光定量PCR方法定量分析食管癌组织与癌旁组织中hMLH1和hMSH2基因的表达水平,应用t检验和logistic回归分析评价基因的表达水平与食管癌的关系。 结果： hMSH1基因的mRNA表达水平在食管癌与癌旁组织间的差异不明显,而hMSH2基因的mRNA表达水平在食管癌组织显著低于癌旁组织(下调了51.2％),条件logistic回归分析表明hMSH2的表达下调与食管组织癌变有统计学关联(OR=1.311,95％CI：1.075~1.599)。 结论： 错配修复相关基因hMSH2的表达水平下降提示食管癌组织错配修复能力异常,可能是淮安地区食管癌发病的危险因素之一。     

子宫颈鳞癌HPV16感染对错配修复基因表达的影响

目的:探讨子宫颈癌发病机制中HPV感染对错配修复(mismatchrepair,MMR)基因表达影响的意义。方法:77例子宫颈鳞癌标本采用免疫组织化学SP法检测HPV16、hMSH2与hMLH1的表达。并分析这些表达与临床病理参数的关系。结果:HPV感染与非感染组子宫颈癌hMLH1的阳性表达率分别为69.0%(29/42)和40.8%(12/28),rs=0.260,P=0.029;hMSH2的阳性表达率分别为64.2%(27/42)和46.4%(13/28),rs=0.177,P=0.143。HPV感染、hMLH1及hMSH2阳性表达率与分化程度、临床分期及淋巴结转移无显著相关。结论:HPV感染组细胞hMSH2与hMLH1蛋白表达上调的现象表明HPV16感染增加子宫颈上皮细胞DNA复制时的碱基错配。因此,这可能是HPV16的致癌机制之一。