Ribozyme对癌基因Ki-rasG12V mRNA的剪切及其特异性

目的：设计切割ｋｉ－ｒａｓ＾Ｇ１２Ｖ ｍＲＮＡ的特异性ｒｉｂｏｚｙｍｅ（Ｒｚ２１７）,明确其对癌基因ｋｉ－ｒａｓ＾Ｇ１２ＶｍＲＮＡ的细胞内外切割活性,为以ｋｉ－ｒａｓ＾Ｇ１２ＶｍＲＮＡ为特异性靶分子的基因治疗及癌基因ｋｉ－ｒａｓ的功能研究提供一种新的途径。方法：依Ｓｙｍｏｎｓ总结的“锤头结构”原理,设计一种能特异性切割ｋｉ－ｒａｓ＾Ｇ１２ＶｍＲＮＡ的ｒｉｂｏｚｙｍｅ,利用ＤＮＡ重组技术构建ｋｉ－ｒａｓ＾Ｇ１２Ｖ外显子１ｍＲＮＡ,在含Ｍｇ＾２＋溶液中ｒｉｂｏｚｙｍｅ Ｒｚ２１７对其靶ＲＮＡ分子进行切割。以ＲＴ－ＰＣＲ对感染ｒｉｂｏｚｙｍｅ Ｒｚ２１７真核表达质粒的细胞ｋｉｒａｓ＾Ｇ１２Ｖ ｍＲＮＡ进行半定量分析。结果：ｋｉ－ｒａｓ＾Ｇ１２Ｖ外显子１体外转录ｍＲＮＡ分子,能被ｒｉｂｏｚｙｍｅ Ｒｚ２１７定点切割而野     

鼻咽癌组织中CD44V6 mRNA及其蛋白表达的临床意义

目的：探讨鼻咽癌（NPC）组织中CD44V6 mRNA及其蛋白表达的临床意义。方法：应用催化信号放大原位杂交和免疫组织化学方法，检测65例NPC组织中CD44V6mRNA及其蛋白的表达情况，结合临床病理因素及随访资料进行分析。结果：在NPC组织中CD44V6 mRNA及其蛋白表达阳性率分别为47．7％（31／65）和52．3％（34／65），CD44V6mRNA及其蛋白阳性表达与NPC原发病灶的分期和淋巴结转移均正相关，CD44V6mRNA及其蛋白阳性表达者5年内复发转移率高（P＜0．01），5年以上生存率低（P＜0．05），结论：CD44V6基因表达可作为预测NPC转移，复发和评估预后的重要生物学指标。     

Cyclin D1(CCNDl)A870G 基因多态性与保定地区结肠癌的关系

为了探讨cyclin D1(CCNDl)A870G 基因多态性与保定地区结肠癌患者的年龄、性别、高胆固醇血症、家族史、 病理分型、病理分期的关系,我们从200 例保定地区结肠癌患者和200 例保定地区健康体检者的血中提取DNA,用聚合 酶链反应和限制性酶切片段长度多态体分析对提取的 DNA 进行多肽性分析。结果显示,病例组和对照组 AA\ AG\GG 基因 频率无明显差异。高胆固醇及重度吸烟的结肠癌患者GG 基因型频率明显高于其他基因型,有明显差异（P<0.05）,重度 吸烟及高胆固醇血症在健康体检人群中GG 基因型无明显差异（P>0.05）。由此推断高胆固醇血症及吸烟可作为在cyclin D1(CCNDl)A870G 基因多态性中GG 基因型中结肠癌发生的危险因子,遗传因素的结肠癌患者AA 基因型频率明显高于其 他基因型,有明显差异（P<0.05）。由此推断遗传因素可作为在cyclin D1(CCNDl)A870G 基因多态性中AA 基因型中结肠 癌发生的危险因子,这将为进一步研究结肠癌分子生物学的发生机制提供依据。     

切割MDR1 RNA的核酶(Ribozyme)对肝癌多药耐药细胞株BEL-7402/DOX化疗耐药性的逆转作用

为逆转肿瘤多药耐药基因(MDR1)产物P-gp蛋白所介导的肿瘤细胞对多种化疗药物的耐受性,设计合成了一种能切割MDR1 mRNA第196密码子GUC序列的锤头状核酶(Ribozlyme)并定向克隆于转录病毒载体pDOR-neo的BamH Ⅰ位点.经病毒包装细胞PA317包装后感染人肝癌多药耐药细胞株BEL-7402/DOX细胞,经G418筛选得到稳定的转化细胞株.Northem Blot杂交证实包装细胞PA317及转化的BEL-7402/DOX细胞中均有病毒的高表达,RT-PCR证实转化细胞中MDR1 mRNA与未转化细胞相比明显减少甚至不能扩增出来,流式细胞技术检测转化细胞P-gp的表达与非转化细胞的93.4～97.5%相比下降至8.2～14.6%.MTT法检测证实转化细胞对多种化疗药物重新产生较高的敏感性.结果表明,表达Ribozyme的逆转录病毒载体转化肝癌多药耐药细胞BEL-7402/DOX后能有效抑制MDR1的表达和翻译,使已产生耐药的肿瘤细胞的多药耐药表型发生逆转.     

Camptothecin(CPT)和Cytosine arabinoside(Ara-C)联合应用对MOLT-4细胞株周期特异性凋亡的影响

目的以急性T淋巴细胞白血病细胞株MOLT-4为模型,探讨作用于同一细胞周期的化疗药物 Camptothecin(CPT)和Cytosine arabinoside(Ara-C)联合应用时是否具有抗癌增效作用、引起细胞凋亡的周期时相性是否发生改变及对细胞周期检测点(ckeckpoint)的影响.方法喜树碱(CPT)(0.25 μmol/L),阿糖胞苷(Ara-C)(1.00 μmol/L),及喜树碱(CPT)(0.25 μmol/L) 阿糖胞苷(Ara-C)(1.00 μmol/L) 二药共同诱导急性早幼粒白血病细胞株MOLT-4细胞4 h,分别采用激光共聚焦显微镜(Confocal)观察其细胞的形态变化,采用Sub-G1法,API法及cyclins/DNA双参数法,运用流式细胞术分析凋亡、凋亡细胞的周期时相性及CyclinE的表达规律.以Western blot印迹法检测Bcl-2蛋白的表达.结果喜树碱和阿糖胞苷联合应用时,凋亡细胞的数目明显增加,二者具有协同效应.发生凋亡的细胞大多数仍然是S期细胞.CyclinE的表达升高,bcl-2蛋白的表达降低.结论作用于同一细胞周期的化疗药物CPT和Ara-C联合应用时,具有协同效应但并不改变细胞周期的作用点(Checkpoint).CyclinE和Bcl-2蛋白似乎在药物作用时起着关键性的调节作用,并对检测点的敏感性产生重要影响.     

PEI-RGD/125I(αV)ASODN 的制备及其对肝癌HepG2细胞侵袭力的抑制

目的：探讨以PEIRGD(polyethyleneimineArgGlyAsp)为转染载体的125I(αV)ASODN投递在体外对HepG2肝癌细胞侵袭力的影响。方法:125I标记整合素αV亚基的ASODN,以聚乙烯亚胺衍生物PEIRGD为载体制备PEIRGD/125I(αV)ASODN复合物,通过受体介导方式转染进入HepG2细胞,利用Boyden小室侵袭模型检测复合物对HepG2细胞侵袭力的影响。结果:(1)125I(αV)ASODN的标记率为（73.78±4.09）%,放化纯度为（96.68±1.38）%,37 ℃放置48 h后的放化纯度仍>90%,表明其稳定性良好;(2) HepG2细胞对PEIRGD/ 125I(αV)ASODN的摄取于4 μl/2 μg时达到峰值\[（12.77±085）%\],之后明显降低,故选择2 μl/1 μg作为PEIRGD/ 125I(αV)ASODN对HepG2细胞的作用剂量;(3)相对于其他实验组和对照组,PEIRGD/ 125I(αV)ASODN组显著降低了HepG2细胞的侵袭能力（P<0.01）。结论:以PEIRGD为载体投递 125I(αV)ASODN能有效抑制HepG2细胞的侵袭力。     

~(131)Ⅰ与~(132)Ⅰ对大鼠甲状腺损伤的形态计量学比较研究

应用图像分析技术和形态计量学方法，测量了１３１Ⅰ、１３２Ⅰ注入后２０月大鼠甲状腺组织损伤体视学参数的变化。结果表明，各剂量组胶体和滤泡的体积密度及表面积密度均明显小于对照组，且随着吸收剂量的增大逐渐减小（Ｐ＜００１）；间质的体积密度则随着剂量的增大而明显增大（Ｐ＜００１）；胶体的半径、体积和表面积随剂量的增大而明显变小；１３１Ⅰ、１３２Ⅰ各剂量组胶体半径的频率或概率分布的峰值均随照射剂量的增大而明显左移，即分布范围变窄，胶体的最大半径Ｘｍａｘ和期望值Ｅ（ｘ）显著变小。上述结果说明，胶体或滤泡的体积变小萎缩。用体积密度、表面积密度、胶体平均半径、体积和表面积等参数与吸收剂量的回归方程式计算出的半减或半增剂量Ｄ５０造成此相同效应１３１Ⅰ所需剂量为１３２Ⅰ的７～２６倍；因而，以１３１Ⅰ作为参考辐射造成此生物终点的１３２Ⅰ的相对生物效应（ＲＢＥ）为７～２６。     

肺癌组织中黑色素瘤抗原-A3基因mRNA的表达及其序列点突变分析(英文)

目的检测肺癌组织中黑色素瘤抗原-3基因(MAGE-A3)mRNA 的表达。方法用逆转录-套式聚合酶链反应(RT-PCR)对31例肺癌患者癌组织和相应癌旁组织 MAGE-A3 mRNA 表达情况进行测定;PE-377DNA 测序仪对5例10个 RT-PCR 扩增产物中的目的基因片段进行 DNA 序列测定。结果 31例肺癌患者癌组织中26例表达 MAGE-A3 mRNA,阳性率为83.9%;相应的癌旁组织均未表达。DNA 序列测定证明 PCR扩增产物中目的基因片段均为 MAGE-A3 cDNA 序列,所测5例样本中4例样本有两个相同位置的碱基发生了点突变(C2773→32773;G2807→A2807),导致一个氨基酸残基改变(E143→K)。结论 MAGE-A3mRNA 在肺癌中呈高比例表达,提示此抗原有可能作为肺癌患者免疫治疗的靶抗原。我国肺癌患者中存在 MAGE-A3基因个别位点的变异。     

邻苯二甲酸二(2-乙基己)酯对大鼠卵巢组织凋亡基因和癌基因表达水平的影响

目的： 探讨邻苯二甲酸二(2-乙基己)酯(DEHP)对大鼠卵巢组织凋亡基因和癌基因表达水平的影响,为评价DEHP的雌性生殖毒性提供科学依据。方法：用不同剂量DEHP灌胃染毒雌性SD大鼠6周,设对照组(玉米油)、低剂量组(100 mg/kg)、中剂量组(500 mg/kg)、高剂量组(1 500 mg/kg),利用荧光定量PCR技术检测卵巢组织凋亡基因(Bcl-2、Caspase-3、Caspase-8、Caspase-9)和癌基因(c-fos、k-ras)mRNA表达的变化。结果：Bcl-2 mRNA表达水平在DEHP高剂量组显著降低,与对照组比较差异具有统计学意义(P<0.01);Caspase-3、Caspase-8和Caspase-9 mRNA表达水平随DEHP染毒剂量升高呈上升趋势,与对照组比较显著升高,差异均有统计学意义(P<0.05或P<0.01)。c-fos mRNA表达量与对照组间的差异无统计学意义(P>0.05),k-ras mRNA表达量在DEHP中剂量组和高剂量组显著高于对照组(P<0.01)。结论：DEHP可能通过线粒体途径激活Caspase通路诱导卵巢细胞凋亡,进而损害大鼠卵巢功能和生殖内分泌功能。且DEHP可激活原癌基因异常表达,具有一定的潜在致癌风险。     

Prognostic value of the KRAS G12V mutation in 841 surgically resected Caucasian lung adenocarcinoma cases

Background:

Identifying patients who will experience lung cancer recurrence after surgery remains a challenge. We aimed to evaluate whether mutant forms of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) (mEGFR and mKRAS) are useful biomarkers in resected non-small cell lung cancer (NSCLC).

Methods:

We retrospectively reviewed data from 841 patients who underwent surgery and molecular testing for NSCLC between 2007 and 2012.

Results:

mEGFR was observed in 103 patients (12.2%), and mKRAS in 265 (31.5%). The median overall survival (OS) and time to recurrence (TTR) were significantly lower for mKRAS (OS: 43 months; TTR: 19 months) compared with mEGFR (OS: 67 months; TTR: 24 months) and wild-type patients (OS: 55 months; disease-free survival (DFS): 24 months). Patients with KRAS G12V exhibited worse OS and TTR compared with the entire cohort (OS: KRAS G12V: 26 months vs Cohort: 60 months; DFS: KRAS G12V: 15 months vs Cohort: 24 months). These results were confirmed using multivariate analyses (non-G12V status, hazard ratio (HR): 0.43 (confidence interval: 0.28–0.65), P<0.0001 for OS; HR: 0.67 (0.48–0.92), P=0.01 for TTR). Risk of recurrence was significantly lower for non-KRAS G12V (HR: 0.01, (0.001–0.08), P<0.0001).

Conclusions:

mKRAS and mEGFR may predict survival and recurrence in early stages of NSCLC. Patients with KRAS G12V exhibited worse OS and higher recurrence incidences.     

消化道肿瘤病人外周血TSmRNA和DPDmRNA的表达

[目的]探讨胸苷酸合成酶（thymidylate synthase，TS）mRNA和二氢嘧啶脱氢酶（dihydropyrimidine dehydrogenase，DPD）mRNA在癌组织和外周血中的表达及其相互关系。[方法]采用RT—PCR检测36例胃肠道癌及外周血中TSmRNA和DPDmRNA的表达水平，同时检测32例对照组外周血中表达水平。[结果]在癌组织中TSmRNA检出52．8％（19／36），DPDmRNA检出44．4％（12／36），高于外周血的检出率30．5％（11／36）和25．0％（9／36），但差异无统计学意义fD0．05）。肿瘤组织TSmRNA、DPDmRNA和外周血的表达结果相关良好，r值分别为0．627和0．645，在外周血中肿瘤组的表达高于对照组（P〈0．01）。[结论]外周血和肿瘤组织中的TSmRNA和DPDmRNA高度相关，用RT—PCR方法检测外周血TSmRNA和DPDmR—NA，操作简单、可反复检测，适于临床应用。     

KRAS G12C Metastatic Colorectal Cancer: Specific Features of a New Emerging Target Population

BackgroundKirsten rat sarcoma viral oncogene (KRAS) G12C mutation occurs in about 4% of colorectal cancers (CRCs). Recently, KRAS G12C was identified to be a potential drug target and predictor of response to the novel on AMG510 target treatment. We described the clinicopathologic features and prognosis of KRAS G12C-mutated metastatic CRCs compared to other KRAS mutation.Patients and MethodsClinicopathologic features and outcome data of KRAS-mutated metastatic CRC (mCRC) patients referred to 3 Italian oncology units from January 2010 to December 2018 were collected. A cohort of KRAS-mutant mCRC patients referred to the Department of Medical Oncology at Fondazione IRCCS Istituto Nazionale dei Tumori, Milan (Italy) within the same time frame was included as external validation.ResultsA total of 839 KRAS-mutated mCRC cases were included in the main patient population. A total of 145 patients (17%) had KRAS G12C mutation. Our analyses showed that patients harboring KRAS G12C mutation were more likely to be men and to present lung and liver metastases, and were less likely to have peritoneal spread. KRAS G12C mutation was associated with shorter overall survival compared to other KRAS mutations (hazard ratio, 1.32; 95% confidence interval, 1.07-1.63; P = .009). Such results were confirmed in the external validation cohort.ConclusionThe knowledge of the distinctive traits of KRAS G12C-mutated CRC patients is crucial to future translational research studies, clinical trial design, and proper interpretation of results.     

宫颈癌患者淋巴结CK19 mRNA的检测及其临床意义

背景与目的：采用RT—PCR技术检测CK19（cytokeratin 19）mRNA的转录，可在部分恶性肿瘤患者中检测到常规病理学方法不能检测到的隐性微转移。本研究旨在检测宫颈癌患者盆腔淋巴结组织特异性标志物（CK19mRNA）的表达，并探讨其临床意义。方法：采用RT—PCR技术检测Ⅰ～Ⅱ期宫颈癌患者盆腔淋巴结CK19 mRNA的转录。生存曲线用Kaplan—Meier转录。生存率比较采用Log—rank检验。结果：32例宫颈癌患者共检测淋巴结标本206枚，经常规病理组织学方法检测到24枚淋巴结有癌转移，阳性率为12％（24／206）；经RT—PCR法检测到44枚淋巴结表达CK19 mRNA，检出率为21％（44／206）。RT—PCR法与常规病理组织学方法比较，差异有显著性（P〈0．01）。常规HE染色阴性的182枚淋巴结中，29枚检测到CK19mRNA的特异性条带，微转移的检出率为15．9％（29／182）。低分化患者的淋巴结CK19检出率为87．5％（7／8）显著高于中～高分化患者的检出率33．3％（8／24）（P〈0．05）。而淋巴结CK19的检出率与年龄、临床分期、组织学类型、宫颈肿瘤大小、肌层浸润深度、宫旁浸润、脉管癌栓均无明显关系（P〉0．05）。淋巴结CK19 mRNA阳性患者的4年无瘤生存率为58％；而淋巴结CK19 mRNA阴性患者的4年无瘤生存率为76．5％，差异无显著性（P〉0．05）。结论：与传统的病理组织学比较，采用RT—PCR技术检测CK19 mRNA转录能显著提高淋巴结微转移的检出率，预后判断有待增大病例数进一步研究。     

Effect of 12-O-tetradecanoylphorbol-13-acetate on inhibition of expression of keratin 1 mRNA in mouse keratinocytes mimicked by 12(S)-hydroxyeicosatetraenoic acid

Differentiation of cultured keratinocytes is controlled by the calcium concentration of the medium and is marked by the expression of differentiation-specific keratins. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the normal differentiation program and suppresses keratin (K) 1 expression. Based on reported similarities in the effects of TPA and the arachidonic acid metabolite 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), we hypothesized that 12(S)-HETE might suppress K1 expression in mouse keratinocytes. We also investigated the effect of pretreatment with 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE) because others have reported that 13(S)-HODE prevents 12(S)-HETE–induced events. In our study, 100 nM 12(S)-HETE mimicked the effect of 500 nM TPA in suppressing K1 mRNA expression within 24 h of calcium-induced differentiation. Pretreatment with 100 nM 13(S)-HODE blocked the 12(S)-HETE effect but not the TPA effect. A role for protein kinase C (PKC) was suggested for both TPA and 12(S)-HETE based on the loss of response with the PKC inhibitors bryostatin-1 or RO-31-8220. Both TPA and 12(S)-HETE stimulated keratinocyte PKC activity. Pretreatment with 13(S)-HODE blocked the 12(S)-HETE–induced increase in PKC activity. Immunoblotting showed that whereas TPA caused a rapid, partial translocation of the PKCα isozyme, it had no effect on the distribution of PKCδ. Conversely, 12(S)-HETE had no effect on the distribution of PKCα but caused a complete translocation of PKCδ. Pretreatment with 13(S)-HODE prevented 12(S)-HETE–elicited translocation of PKCδ. We conclude that 12(S)-HETE mimics the effect of TPA on K1 mRNA and that the effect is mediated through different isoforms of PKC. Mol. Carcinog. 19:157–164, 1997. © 1997 Wiley-Liss, Inc.     

Real-World Study of Characteristics and Treatment Outcomes Among Patients with KRAS p.G12C-Mutated or Other KRAS Mutated Metastatic Colorectal Cancer

BackgroundThe KRAS p.G12C mutation has recently become an actionable drug target. To further understand KRAS p.G12C disease, we describe clinicopathologic characteristics, treatment patterns, overall survival (OS), and real-world progression-free survival (rwPFS) in patients with metastatic colorectal cancer (mCRC), KRAS p.G12C mutations (KRAS G12C), and other KRAS mutations (KRAS non-G12C) using a de-identified database.Patients and MethodsClinical and tumor characteristics, including treatments received, genomic profile, and clinical outcomes were assessed for patients from a US clinical genomic database with mCRC diagnosed between January 1, 2011, and March 31, 2020, with genomic sequencing data available.ResultsOf 6477 patients with mCRC (mCRC cohort), 238 (3.7%) had KRAS G12C and 2947 (45.5%) had KRAS non-G12C mutations. Treatment patterns were generally comparable across lines of therapy (LOT) in KRAS G12C versus KRAS non-G12C cohorts. Median (95% CI) OS after the first LOT was 16.1 (13.0-19.0) months for the KRAS G12C cohort versus 18.3 (17.2-19.3) months for the KRAS non-G12C cohort, and 19.2 (18.5-19.8) months for the mCRC overall cohort; median (95% CI) rwPFS was 7.4 (6.3-9.5), 9.0 (8.2-9.7), and 9.2 (8.6-9.7) months, respectively. The different KRAS non-G12C mutations examined did not affect clinical outcomes. Median OS and rwPFS for all cohorts declined with each subsequent LOT.ConclusionsPatients with KRAS p.G12C-mutant mCRC have poor treatment outcomes, and outcomes appear numerically worse than for those without this mutation, indicating potential prognostic implications for KRAS p.G12C mutations and an unmet medical need in this population.