MircoRNA-214对吉非替尼敏感及耐药细胞增殖和凋亡的影响

摘 要：［目的］ 研究mircoRNA-214（miR-214）对PC9肺腺癌及吉非替尼耐药细胞（PC9/GR）增殖和凋亡的影响。［方法］ 在PC9细胞中转染miR-214模拟物及PC9/GR中转染miR-214抑制剂,使用定量逆转录PCR（qRT-PCR）检测其表达。MTT检测细胞转染miR-214模拟物或其抑制剂后的存活及增殖。在PC9细胞中顺时转染miR-214模拟物以检测上调miR-214对PC9细胞耐药性的影响,在PC9/GR细胞中顺时转染miR-214抑制物以检测下调miR-214对PC9/GR细胞耐药性的影响,并用流式细胞仪检测细胞的凋亡。Western blotting检测PTEN在PC9和PC9/GR细胞中的表达。构建PTEN 3’-UTR荧光素酶报告质粒验证miR-214的靶基因;建立异种移植模型检测miR-214抑制物对肺癌移植瘤的影响。［结果］ PC9细胞中miR-214低表达,上调miR-214的表达后PC9细胞对吉非替尼的敏感性降低,并且抵抗吉非替尼诱导的凋亡;而在PC9/GR细胞中低表达miR-214后,下调miR-214增加PC9/GR细胞对吉非替尼的敏感性,并且可以增强吉非替尼诱导的凋亡作用。荧光素酶报告载体实验证实PTEN是miR-214在细胞内的靶基因。动物异种移植模型表明miR-214抑制物可以增强PC9/GR对吉非替尼的敏感性。［结论］ MiR-214可能通过靶基因PTEN调控吉非替尼的获得性耐药。     

TIP30逆转人非小细胞肺癌吉非替尼耐药的实验研究

  目的  本实验旨在研究TIP30能否逆转非小细胞肺癌（non-small cell lung cancer,NSCLC）的吉非替尼耐药,并探讨其可能的机制。  方法  慢病毒LV-TIP30转染NSCLC吉非替尼耐药株PC9/GR上调TIP30,以PC9/GR、PC9/GR-LVTIP30和PC9/GR-LVNC为研究对象,分别加或不加5 μmol/L吉非替尼处理共6组细胞。CCK8检测细胞增殖抑制率,划痕修复实验、Transwell实验检测细胞迁移侵袭能力,免疫印迹实验检测p-AKT、p-ERK、p-MEK以及核内EGFR蛋白表达水平。  结果  非吉非替尼处理组中,PC9/GR-LVTIP30细胞的增殖、迁移和侵袭能力与PC9/GR细胞相比均受到明显的抑制（P < 0.05）,吉非替尼处理组中PC9/GR-LVTIP30细胞较PC9/GR细胞抑制作用更明显（P < 0.05）;过表达TIP30后,蛋白p-MEK、p-ERK、p-AKT表达水平降低,核内EGFR表达水平较PC9/GR降低,差异均具有统计学意义（P < 0.05）。  结论  上调TIP30能够逆转人非小细胞肺癌PC9/GR细胞对吉非替尼的耐药性,其发挥作用的机制可能是通过抑制EGFR核内化,进而抑制下游信号通路相关蛋白p-AKT、p-ERK、p-MEK激活发挥作用。      

EGFR T790M突变丰度对奥希替尼治疗晚期非小细胞肺癌疗效的影响

摘 要：［目的］ 分析奥希替尼治疗晚期非小细胞肺癌疗效，并探索ddPCR方法外周血检测表皮生长因子（epidermal growth factor，EGFR）T790M突变的丰度与奥希替尼疗效之间的关系。［方法］ 回顾性分析104例接受奥希替尼治疗的Ⅲb~Ⅳ期非小细胞癌肺癌患者，采用ddPCR法测定外周血T790M突变丰度，采用Kaplan-Meier法和Cox模型进行生存预后分析，并探索疗效相关T790M突变丰度的界值。［结果］ 外周血EGFR T790M突变丰度中位值为0.89%（0.02%~35.10%）。将突变丰度分为＜5.00%和≥5.00%两组，客观缓解率分别为46.5%和88.9%（P=0.001），中位无进展生存期分别为8.80个月和21.83个月（P=0.039）。Cox回归分析显示，初治时基因类型（EGFR 19外显子缺失和21外显子L858R突变）、PS评分、外周血EGFR T790M突变丰度分组（＜5.00%和≥5.00%）是奥希替尼治疗患者PFS的独立影响因素。［结论］ EGFR T790M突变丰度可能可预测奥希替尼治疗的晚期EGFR T790M突变NSCLC患者的有效率和无进展生存期。     

PD-L1单抗对EGFR敏感性突变肺癌细胞PD-L1表达及对增殖的影响

摘 要：［目的］ 探讨PD-L1单抗对EGFR敏感性突变肺癌细胞PD-L1的表达及对增殖的影响。［方法］ 选取2017年5月至2018年8月在我院呼吸内科治疗的非小细胞肺癌患者60例为研究对象,同时选取同期来我院检查的健康人群30例为对照。采集外周静脉血,测定血清标本中游离PD-L1（sPD-L1）蛋白含量。建立肺癌细胞与T细胞共培养体系。［结果］ 肺鳞癌组与肺腺癌组sPD-L1的表达水平明显高于对照组（P<0.001）,而肺鳞癌组与肺腺癌组sPD-L1表达水平无统计学差异（t=2.584,P=0.086）;野生组与突变组sPD-L1表达水平亦无统计学差异（t=2.215,P=0.097）。sPD-L1表达量与患者性别、年龄、分期无相关性（P＞0.05）,而与患者的吸烟史、肿瘤转移相关（P＜0.05）。 EGFR敏感型sPD-L1表达率明显高于EGFR野生型（t=12.365,P<0.001）。加入PD-L1单抗后,EGFR的sPD-L1的表达水平有明显下降趋势,而EGFR野生型与治疗前无统计学差异（t=2.154,P=0.958）。PD-L1单抗使用后,PC9、HCC827等EGFR敏感型细胞株共同体系中T细胞明显增加。［结论］ sPD-1表达与EGFR敏感性无明显相关性,而与患者是否吸烟、肿瘤转移相关。与此同时,EGFR敏感突变肺癌细胞行PD-L1单抗干预后,sPD-L1的表达量明显降低,T细胞增殖明显。     

安罗替尼联合吉非替尼对吉非替尼耐药非小细胞肺癌PC9/GR细胞增殖的影响及其可能的作用机制

目的 探讨安罗替尼联合吉非替尼对吉非替尼耐药的人非小细胞肺癌PC9/GR（gefitinib resistance）细胞增殖的影响及其可能的作用机制。 方法 依据不同给药情况将PC9/GR细胞分为安罗替尼单药组、吉非替尼单药组、安罗替尼和吉非替尼联合用药组及阴性对照组,用MTT法检测各组细胞的增殖情况,流式细胞仪检测细胞的周期分布,Western blot检测p-ERK1/2和p-AKT蛋白的表达水平。 结果 安罗替尼和吉非替尼作用于PC9/GR细胞72 h的半数抑制浓度（half inhibitory concentration,IC₅₀）分别为（1.91±0.18） μmol/L和（4.83±0.15） μmol/L,两药均呈剂量依赖性的抗增殖作用,且两药联合时表现出明显的协同效应,联合指数（combination index,CI）小于1。安罗替尼和吉非替尼单药均可将PC9/GR细胞阻滞于G0/G1期（均P<0.05）。与各单药组比较,联合用药组表现出更明显的G0/G1期阻滞（均P<0.05）,且下调p-ERK1/2和p-AKT蛋白的表达水平（均P<0.05）。 结论 安罗替尼联合吉非替尼对非小细胞肺癌 PC9/GR细胞具有协同抗增殖作用,且可增强吉非替尼敏感性,其协同抗肿瘤机制可能与诱导细胞周期阻滞和下调p-ERK1/2和p-AKT蛋白的表达相关。     

MiRNA-21抑制物对人肺腺癌PC9/GR细胞吉非替尼耐药性的影响

目的:观察miR-21抑制物对人肺腺癌PC9/GR细胞株增殖、凋亡及迁移能力的影响,探讨miR-21抑制物在PC9/GR细胞吉非替尼耐药的生物学行为中的作用.方法:实验分为miR-21 inhibitor组、阴性对照组和正常细胞对照组3组.MiR-21 inhibitor组转染miR-21抑制物抑制其细胞内miR-21表达,阴性对照组转染microRNA inhibitor NC.加入吉非替尼处理后,采用MTT比色实验测定3组细胞第1、2、3、4、5天的吸光度(OD值),绘制生长曲线.采用流式细胞仪检测细胞凋亡,Transwell法检测细胞迁移.结果:两对照组间细胞的增殖速度无明显差异(P＞0.05),与对照组相比,实验组细胞增殖速度明显减慢(P＜0.05).实验组细胞凋亡率为(40.06 ±2.32)％,明显增高(P＜0.05);阴性对照组[(7.39±0.79)％]和空白对照组[(6.96±0.68)％]之间细胞凋亡无明显差异(P＞0.05).实验组细胞迁移力受到明显抑制(P＜0.05).空白对照组与阴性对照组细胞迁移力相比无明显差异(P＞0.05).结论:在PC9/GR细胞中,下调miRNA-21的表达后,PC9/GR细胞对GR的敏感性提高,凋亡程度增强,部分逆转了肺癌PC9/GR细胞对GR的耐药性.为miRNA-21与化疗联合治疗肺癌提供了实验依据.     

吉非替尼联合培美曲塞对EGFR-TKI获得性耐药的非小细胞肺癌细胞的协同效应

目的:探讨培美曲塞联合吉非替尼对体外诱导的表皮生长因子受体-酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)获得性耐药的人非小细胞肺癌PC9/吉非替尼耐药(gei tinib resistance,GR)细胞株的效应及其可能的机制。方法:吉非替尼和培美曲塞单药或联合作用于PC9/GR细胞后,MTT法检测各药物处理组细胞的增殖抑制率及药物的联合指数(combination index,CI),FCM法检测各组细胞的凋亡率,蛋白质印迹法检测各组细胞磷酸化AKT和Bcl-2蛋白的表达水平。结果:吉非替尼和培美曲塞联合应用对PC9/GR细胞的增殖抑制作用和促凋亡作用明显强于各单药组(P0.05);吉非替尼和培美曲塞的CI值1,表现出明显的协同效应。与未进行药物处理的对照组比较,培美曲塞联合吉非替尼可明显下调PC9/GR细胞中磷酸化AKT和Bcl-2蛋白的表达水平(P0.01)。结论:培美曲塞联合吉非替尼对PC9/GR细胞具有较好的协同作用,这协同作用可能与诱导细胞凋亡和下调磷酸化AKT蛋白表达有关。     

非小细胞肺癌患者外周血表皮生长因子受体T790M检测与奥希替尼疗效的相关性研究

摘 要：［目的］ 评估非小细胞肺癌患者第一代表皮生长因子受体-酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI）获得性耐药后外周血EGFR T790M突变的阳性率和用外周血T790M检测结果预测奥希替尼疗效的可靠性。［方法］ 回顾性分析2017 年3月至2018年6月经第一代EGFR-TKI治疗后获得性耐药且使用超级扩增阻滞突变系统(ultra-amplification refractory mutation system,Ultra-ARMS )进行外周血EGFR T790M检测的原发性非小细胞肺癌患者。评估血T790M阳性患者使用奥希替尼的疗效。［结果］共有103例符合标准的第一代EGFR-TKI获得性耐药患者,其中28例（27.2%,28/103）血T790M阳性,75例（72.8%,75/103）血T790M阴性。血T790M阳性患者中,接受奥希替尼治疗有23例：部分缓解（partial response,PR） 15例,疾病稳定（stable disease,SD）6例,疾病进展（progression of disease,PD） 2例。疾病控制率(disease control rate,DCR) 91.3%,客观有效率（objective response rate,ORR)65.2%。奥希替尼治疗的中位无进展生存时间（progression free survival,PFS） 12.5个月（95%CI：11.2~13.8）。有9例血T790M阴性患者后续进行了组织检测,3例在组织中检测到T790M突变。有6例血T790M阴性的患者虽未再行组织检测,但要求试用奥希替尼靶向治疗,1例患者获得了PR,1例SD（PFS超过5个月）。［结论］对第一代EGFR-TKI获得性耐药后未能再次行组织活检的非小细胞肺癌患者,Ultra-ARMS方法检测血T790M阳性可预测奥希替尼疗效。血T790M检测阴性的患者建议再次取组织进行T790M检测以排除假阴性。组织检测是T790M检测的金标准,血T790M检测可作为补充。     

非小细胞肺癌吉非替尼耐药相关miRNAs的筛选鉴定

  目的  miRNA是一类通过结合mRNA调节基因表达的非编码单链小分子RNA,本研究目的是探讨非小细胞肺癌（NSCLC）中miRNA与吉非替尼耐药的关系。  方法  CCK8法检测NSCLC吉非替尼耐药细胞PC9/GR相对于亲本细胞PC9的耐药倍数;miRNA芯片检测PC9/GR与PC9中miRNA的表达差异;RT-PCR验证miRNA芯片结果。将差异表达的miRNA模拟物/抑制剂转染至PC9/GR中,观察其对吉非替尼敏感性的影响。  结果  吉非替尼对PC9和PC9/GR的IC₅₀值分别为42.89 nmoL/L和3.87 μ moL/L,耐药倍数为90.23倍。miRNA芯片结果显示,PC9/GR与PC9比较55条有差异表达miRNAs（P < 0.01）,其中在PC9/GR上调的miRNAs有21条,包括miRNA-1 246、miRNA-125b等;下调的miRNAs有34条,包括miRNA-224、miRNA-125a~5p等。RT-PCR进一步验证其中9条miRNAs,有8条与芯片结果趋势一致。将上述8条miRNAs的模拟物/抑制剂转染至PC9/GR中,发现miRNA-125a~5p模拟物可降低吉非替尼敏感性。  结论  PC9/GR与PC9的miRNA表达存在差异,miRNA可能与NSCLC吉非替尼耐药相关,miRNA-125a~5p可促进PC9/GR对吉非替尼产生耐药。      

HGF、c-Met、p-Met在NSCLC中的表达及与EGFR表达的相关性

摘 要：［目的］ 通过检测非小细胞肺癌患者中HGF、c-Met和p-Met的表达情况,分析其与临床病理特征关系,并与EGFR表达的相关性。［方法］ 收集经手术切除非小细胞肺癌患者的标本100例,采用免疫组化方法检测HGF、c-Met、p-Met蛋白的表达水平,并收集这些患者既往检测EGFR的表达情况。［结果］ HGF阳性表达率为64%,c-Met阳性表达率为47%,p-Met阳性表达率为3%。HGF、c-Met的表达与吸烟状态、年龄、性别、临床分期、分化程度、病理类型均无统计学差异;p-Met阳性表达患者均为EGFR突变的HGF强表达的腺癌患者。HGF、c-Met的表达水平与EGFR表达水平无相关性。［结论］ HGF、c-Met的阳性表达与吸烟状态、年龄、性别、临床分期、分化程度、病理类型以及EGFR的表达水平均无关。p-Met的阳性表达患者均为HGF强表达的腺癌EGFR突变型。     

Prognostic Significance of Inflammation-associated Blood Cell Markers in Nonmetastatic Clear Cell Renal Cell Carcinoma

ObjectivesOur objective was to evaluate the effect of the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), and red blood cell distribution width (RDW) on the survival outcomes of nonmetastatic clear cell renal cell carcinoma (ccRCC).Materials and MethodsWe accessed our single-center, urologic-oncologic registry to extract the data for patients who had undergone nephrectomy for nonmetastatic ccRCC. The optimal cutoff for these markers was determined using X-tile software, and survival analyses using Cox regression were performed.ResultsA total of 687 patients had undergone nephrectomy. The optimal cutoffs for NLR, PLR, LMR, and RDW were 3.3, 210, 2.4, and 14.3%, respectively. The NLR, PLR, LMR, and RDW were significantly associated with a larger pathologic tumor size, and stage, more aggressive Fuhrman grade, and the presence of tumor necrosis. After adjusting for age, baseline Eastern Cooperative Oncology Group, pathologic tumor and nodal stage, and Fuhrman grade, only PLR remained an independent prognostic marker for both cancer-specific survival (hazard ratio, 2.69; 95% confidence interval, 1.36-5.33; P = .004) and overall survival (hazard ratio, 2.19; 95% confidence interval, 1.36-3.50; P = .001). When the PLR was included with the Leibovich score and University of California, Los Angeles, integrated staging system, the Harrell’s c-index increased from 0.854 to 0.876 and 0.751 to 0.810, respectively, for cancer-specific survival at 5 years after nephrectomy. When risk stratified by the Leibovich risk group and UCLA integrated staging system, PLR was a significant prognostic factor only within the intermediate- to high-risk groups.ConclusionsPLR is a robust prognostic marker in nonmetastatic ccRCC that clearly outperforms other inflammatory indexes in those who had undergone nephrectomy. However, its prognostic effect was limited in the low-risk category of ccRCC.     

Inhibition of Glioma Cell Proliferation by Neural Stem Cell Factor

Summary Neural stem cells (NSC) have unique differentiation-, proliferation-, and motility properties. To investigate whether they secrete factors that interfere with the proliferation of glioma cells, we grew glioma cells in conditioned medium (CM) obtained from cultures of neurospheres including neural stem / progenitor cells (NSPC) isolated from embryonic (E14)- or adult mouse brain or fetal human brain. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and BrdU-labeling assays showed that CM from NSPC (NSPC/CM) contained factor(s) that inhibited the proliferation of glioma cells by 28–87%. Filter-fractionation of NSPC/CM revealed that the 50,000–100,000 nominal molecular weight limit (NMWL) fraction contained the inhibitory activity. On the basis of these observations we transplanted 203G glioma cells and/or NSPC into the intrathecal space of the cisterna magna of mice to investigate whether NSPC interfere with the proliferation of glioma cells in vivo. Mice transplanted with both 203G and NSPC survived significantly longer than did mice transplanted only with 203G. We concluded that NSPC secrete factor(s) that may control glioma cell proliferation.     

High Occurrence of Non-Clear Cell Renal Cell Carcinoma in Oman

It is conventionally accepted that renal cell carcinoma (RCC) occurs in older patients and the clear cell type is the most common histology. However, ethnic variations exist and this study was carried out to determine the epidemiological pattern of RCC in Oman. Ninety RCC patients who presented to a tertiary care center in the Sultanate of Oman from 2010 to 2014 were studied. The main findings were that the median age of presentation was low, more patients presented with localized stage, and there was a higher incidence of non-clear (especially papillary) histology. Data from other Gulf countries and possible reasons for the different profile are discussed.     

Inhibition of Natural Killer Cell Cytotoxicity by Cell Growth-related Molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H- ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H- ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-γ clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-I antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')₂ fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.     

人干扰素对HL60,K562细胞生长及细胞周期的影响

用人干扰素(α和γ)与HL60、K562细胞共同培养后,对细胞生长有不同程度抑制作用。IFN_r对细胞生长抑制作用强于IFN_r,IFN_r和IFN_r联合应用有协同作用,在K562细胞,细胞在细胞周期中的分布也发生改变,G_0/G_1期细胞比例减少,S期细胞比例增高,在HL60细胞则无明显的细胞周期再分布情况。提示细胞在S期的堆积是细胞生长受抑的原因之一。     

Cell kinetics

Cell kinetic concepts have pervaded radiation therapy since the early part of the 20th century and have been instrumental in the development of modern radiotherapy. In this review, the fundamental radiobiological concepts that have been developed based on cell kinetic knowledge will be revisited and discussed in the context of contemporary radiation therapy. This will include how the proliferation characteristics, variation in sensitivity during the cell cycle and the extent of radiation-induced cell cycle delay translate into a variable time for the expression of damage, how cell kinetics interacts with hypoxia and how the response to fractionated radiation schedules is influenced by cell kinetics in terms of repair, redistribution, reoxygenation and repopulation. The promise of combining radiation with new biologically targeted agents and the potential of non-invasive positron emission tomography imaging of proliferation are areas where cell kinetics will continue to influence radiotherapy practice.     

Hematopoietic Stem Cell Transplantation in the Era of Engineered Cell Therapy

Purpose of Review

Cellular therapy using T cells modified to express chimeric antigen receptors (CAR-T cells) has had striking success in patients that have failed previous treatment for CD19⁺ B cell non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), or acute lymphoblastic leukemia (ALL). Curative therapy for this group of diseases has previously been limited to allogeneic hematopoietic cell transplantation HCT (alloHCT). The recent results of CAR-T cell therapy raise the question of how best to integrate CAR-T cell therapy and alloHCT in the care of these patients.

Recent Findings

Within the past 2 years, results from larger trials and increased follow-up of patients treated with CD19 CAR-T cell therapy suggest that some may achieve durable remission without transplant.

Summary

The balance of efficacy and toxicity for CAR-T cell therapy and alloHCT vary by disease type, disease status at the time of treatment, patient characteristics, and the specific therapy employed. There are early signals that subsequent transplantation of patients who have achieved remission with CAR-T may be a potentially viable (though expensive) strategy.