灵芝孢子油对肺腺癌癌性胸水中原代肿瘤细胞的抗肿瘤作用

目的：研究灵芝孢子油对肺腺癌的癌性胸水中原代肿瘤细胞的抗癌作用。方法：体外培养肺腺癌细胞系A549,用CCK-8法检测灵芝孢子油对A549细胞的体外抗肿瘤作用。收集12例经病理确诊的肺腺癌患者的癌性胸水分离原代肿瘤细胞,用CCK-8法检测灵芝孢子油及临床常用化疗药物对原代肿瘤细胞的药物敏感性;流式细胞仪检测灵芝孢子油对原代肿瘤细胞的凋亡诱导作用,RT-PCR检测灵芝孢子油对凋亡相关蛋白基因bax及bc l-2 mRNA表达的影响。结果：灵芝孢子油对5例癌性胸水中原代肿瘤细胞的IC50平均值为0.28±0.04μl/m l,与对A549细胞的IC500.46±0.08μl/m l无显著差异（P〉0.05）。在各测试药物浓度（TDC）下,灵芝孢子油、多西紫杉醇、顺铂和健择对原代肿瘤细胞的抑制率分别为48.7%±3.3%,31.6%±14.1%,30.2%±11.6%,51.4%±5.6%,灵芝孢子油与这些化疗药物的抗肿瘤作用无显著差异（P〉0.05）。灵芝孢子油显著促进原代肿瘤细胞凋亡,对原代肿瘤细胞中凋亡相关蛋白bax mRNA有明显上调作用,对bc l-2 mRNA有明显下调作用。结论：灵芝孢子油对肺腺癌的癌性胸水原代肿瘤细胞有较好的抗癌作用。     

汉防己甲素抑制恶性腹腔积液中原代肿瘤细胞作用的观察

目的:研究中药单体汉防己甲素对恶性腹腔积液中原代肿瘤细胞的抗肿瘤作用。方法:体外培养肿瘤细胞系BGC-823、LOVO和SMMC-7721,WST-8法检测汉防己甲素在细胞系中的抗肿瘤作用。收集21例经病理确诊的恶性腹腔积液并分离原代肿瘤细胞,用WST-8法检测原代肿瘤细胞中汉防己甲素及化疗药的药物敏感性,荧光定量RT-PCR法检测汉防己甲素对凋亡相关蛋白基因Survivin及Bcl-2mRNA表达的作用。结果:汉防己甲素对21例恶性腹腔积液中原代肿瘤细胞IC50平均值为32.96μm,与三株细胞系中的IC50差异无统计学意义,P>0.05。汉防己甲素对胃癌、大肠癌及其他消化道肿瘤来源的腹水中原代肿瘤细胞的抗肿瘤作用差异无统计学意义,P>0.05。2例对汉防己甲素耐药的原代肿瘤细胞对多西紫杉醇、氟尿嘧啶和顺铂存在交叉耐药,而对化疗药物耐药的原代肿瘤细胞对汉防己甲素无交叉耐药。汉防己甲素对部分细胞系及原代细胞中凋亡相关蛋白Survivin及Bcl-2mRNA有明显下调作用,P<0.05。结论:汉防己甲素对恶性腹腔积液原代肿瘤细胞,尤其对化疗药物耐药的肿瘤细胞有一定的抗肿瘤作用。     

灵芝孢子油对肺腺癌癌性胸水中原代肿瘤细胞的抗肿瘤作用

目的:研究灵芝孢子油对肺腺癌的癌性胸水中原代肿瘤细胞的抗癌作用。方法:体外培养肺腺癌细胞系A549,用CCK-8法检测灵芝孢子油对A549细胞的体外抗肿瘤作用。收集12例经病理确诊的肺腺癌患者的癌性胸水分离原代肿瘤细胞,用CCK-8法检测灵芝孢子油及临床常用化疗药物对原代肿瘤细胞的药物敏感性;流式细胞仪检测灵芝孢子油对原代肿瘤细胞的凋亡诱导作用,RT-PCR检测灵芝孢子油对凋亡相关蛋白基因bax及bc l-2 mRNA表达的影响。结果:灵芝孢子油对5例癌性胸水中原代肿瘤细胞的IC50平均值为0.28±0.04μl/m l,与对A549细胞的IC500.46±0.08μl/m l无显著差异(P>0.05)。在各测试药物浓度(TDC)下,灵芝孢子油、多西紫杉醇、顺铂和健择对原代肿瘤细胞的抑制率分别为48.7%±3.3%,31.6%±14.1%,30.2%±11.6%,51.4%±5.6%,灵芝孢子油与这些化疗药物的抗肿瘤作用无显著差异(P>0.05)。灵芝孢子油显著促进原代肿瘤细胞凋亡,对原代肿瘤细胞中凋亡相关蛋白bax mRNA有明显上调作用,对bc l-2 mRNA有明显下调作用。结论:灵芝孢子油对肺腺癌的癌性胸水原代肿瘤细胞有较好的抗癌作用。     

Flavopiridol sensitivity of cancer cells isolated from ascites and pleural fluids.

PURPOSE: We examined the efficacy of flavopiridol, a cyclin-dependent kinase inhibitor that is undergoing clinical trials, on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. EXPERIMENTAL DESIGN: Metastasized cancer cells were isolated from the pleural fluids (n = 20) or ascites (n = 15) of patients, most of whom were refractory to chemotherapy. These primary cancer cells were used within 2 weeks of isolation without selecting for proliferative capacities. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to characterize the response of these cancer cells to commonly used chemotherapeutic agents, and their response to flavopiridol was compared with rapidly dividing cultured cell lines. RESULTS: The primary cancer cells displayed phenotypes that were different from established cell lines; they had very low replication rates, dividing every 1 to 2 weeks, and underwent replicative senescence within five passages. These primary tumor cells retained their resistance to chemotherapeutic drugs exhibited by the respective patients but did not show cross-resistance to other agents. However, these cancer cells showed sensitivity to flavopiridol with an average LD50 of 50 nmol/L (range, 21.5-69 nmol/L), similar to the LD50 in established cell lines. Because senescent cells also showed similar sensitivity to flavopiridol, it suggests that the mechanism of action is not dependent on the activity of cyclin-dependent kinases that regulate the progression of the cell cycle. CONCLUSION: Using cancer cells isolated from the ascites or pleural fluids, this study shows the potential of flavopiridol against cancer cells that have developed resistance to conventional chemotherapeutic agents.     

正交试验法优化胸腹腔积液细胞学检查条件

目的优选胸腹腔积液细胞学检查的条件。方法在单因素试验结果的分析基础上,考察送检标本量、送检次数、原发部位是否确诊对胸腹腔积液细胞学检查阳性检出率的影响。结果最佳检查条件为在有临床诊断依据的情况下,送检40ml,送检4次。结论胸腹腔积液细胞学检查的优化结果满意、确实可行。     

防己诺林碱对肺癌H1299和A549细胞的作用及其分子机制研究

目的 探究防己诺林碱在肺癌中的作用及其分子机制。方法 MTS法检测经10、15、20、30、40和60 μM/L防己诺林碱处理的肺癌H1299和A549细胞的增殖情况,通过Caspase3、Caspase8和Caspase9活性检测试剂盒和TUNEL试剂盒检测细胞凋亡情况。Western blot检测MAPK信号通路(p-Akt、PI3K、mTOR和Akt蛋白)、增殖和转移(MMP-2、MMP-9、PCNA、Cyclin D1和P21蛋白)、周期和凋亡(Cyclin D2、Bcl2、MCL-1、Bax和p53蛋白)相关蛋白的表达情况,通过Real-time PCR检测PI3k、mTOR、MMP-2、MMP-9、PCNA、Cyclin D1、Cyclin D2、MCL-1、Bax、Bcl2和TP53基因表达情况。结果 防己诺林碱会抑制肺癌H1299和A549细胞的增殖,诱导细胞凋亡,增加Caspase3、Caspase8和Caspase9酶活性。经防己诺林碱处理后,PI3K、p-Akt、mTOR、MMP-2、MMP-9、PCNA、Cyclin D1、Cyclin D2和Bcl2蛋白表达降低,P21、MCL-1、Bax和p53蛋白表达升高,并且表现为剂量依赖性,但对Akt蛋白表达无影响;PI3K、mTOR、MMP-2、MMP-9、PCNA、Cyclin D1、Cyclin D2和Bcl2基因表达降低,TP53、MCL-1和Bax基因表达增加。结论 防己诺林碱通过调控MAPK信号通路、增殖和转移、周期和凋亡相关蛋白和基因的表达,从而抑制细胞增殖,诱导细胞凋亡。     

白头翁皂苷B4、粉防己碱对奥沙利铂耐药的结肠癌细胞的耐药逆转作用及其机制

背景与目的：奥沙利铂(oraliplatin,L-OHP)是结直肠癌最常用的化疗药物之一,但临床上常见结直肠癌L-OHP化疗耐药。本研究旨在探讨中药单体白头翁皂苷B4(anemoside B4,AB4)以及粉防己碱(Tetrandrine,Tet)对人结肠癌L-OHP耐药细胞LoVo/L-OHP耐药的逆转作用,并初步研究其机制。方法：采用浓度梯度实验筛选无毒剂量的AB4、Tet,无毒剂量的AB4、Tet作用于LoVo/L-OHP细胞48 h后,检测LoVo/L-OHP细胞对L-OHP耐药的变化,检测处理后细胞凋亡、细胞周期变化情况,通过RT-PCR及Western blot检测相关基因P-gp、zDHHC9和SMAD4的mRNA及蛋白的表达水平。结果：AB4及Tet作用48 h后对LoVo/L-OHP细胞的5%抑制率浓度分别为0.71及0.45 μg/mL,以此作为无毒剂量。L-OHP对LoVo/L-OHP细胞IC50为(112.5±23.6)μg/mL,分别加入0.71 μg/mL AB4及0.45 μg/mL Tet后,L-OHP对LoVo/L-OHP细胞的IC50值分别为 (62.8±21.4)及(58.9±26.3) μg/mL,IC50值均显著下降(P值均<0.05)。细胞周期检测发现,AB4及Tet处理后G1期细胞略有减少,S期细胞略有增加,但差异无统计学意义(P>0.05)。细胞凋亡检测发现AB4及Tet处理后凋亡率略有上升,但差异均无统计学意义(P>0.05)。RT-PCR检测显示耐药细胞相对于亲本细胞,P-gp及zDHHC9基因表达升高,SMAD4表达降低(P值均<0.05),经过AB4处理后,耐药细胞P-gp基因表达下降。Western blot检测相关蛋白水平后发现,耐药细胞经过AB4处理后,P-gp蛋白表达下降,经过Tet处理后zDHHC9蛋白表达下调。结论：AB4及Tet能逆转LoVo/L-OHP耐药细胞对L-OHP的耐药,AB4逆转机制可能与降低P-gp表达有关,Tet逆转机制与降低zDHHC9表达水平有关。     

血清IL-2水平与恶性胸/腹水IL-2治疗疗效的关系

目的：对恶性胸/腹水患者血清白细胞介素2（interleukin-2,IL-2)水平进行测定，了解血清IL-2水平与胸/腹腔灌注IL-2治疗恶性胸/腹水的疗效之间的关系。以期获得判定恶性胸/腹水治疗疗效的指标。方法：88例恶性非肝癌性胸/腹水患者，均接受IL-2胸/腹腔灌注治疗，治疗前应用ELISA法检测血清IL-2水平，将IL-2测定值较高的44例患者分为一组，IL-2测定值较低的44例患者分为另一组，比较两组患者的治疗效果。结果：血清IL-2测定值较低组IL-2胸/腹腔灌注治疗的有效率为72.7%(32/44)。明显高于IL-2测定较高组的52.3%(23/44)。结论：恶性胸/腹水患者IL-2胸/腹腔灌注治疗前血清IL-2水平高低与IL-2治疗的疗效明显相关。原有IL-2水平较低的患者IL-2胸/腹腔灌注治疗的疗效较好。血清IL-2水平可作为判定IL-2胸/腹腔灌注治疗疗效的指标。     

乳腺原发癌和转移淋巴结肿瘤浸润淋巴细胞体外抗乳腺癌研究

目的探讨同个体乳腺原发癌和转移淋巴结肿瘤浸润淋巴细胞(TIL)抗瘤活性。方法从35例乳腺癌伴有腋窝淋巴结转移患者的乳腺原发癌组织及转移淋巴结组织中分离出乳腺原发癌TIL和转移淋巴结TIL,观察不同部位获得的TIL体外对不同部位乳腺癌细胞的杀伤活性。结果乳腺原发癌TIL对乳腺原发癌癌细胞与转移淋巴结TIL对转移淋巴结癌细胞均具有很高的杀伤活性[杀伤率分别为(71.31±3.11)%和(69.38±2.51)%],明显高于乳腺原发癌TIL对转移淋巴结癌细胞和转移淋巴结TIL对乳腺原发癌癌细胞的杀伤活性[杀伤率分别为(50.31±2.89)%和(49.80±3.21)%]。结论同一个体乳腺癌其原发癌和转移淋巴结TIL抗瘤活性有差别。     

汉防己甲素抑制血管生成的作用

背景与目的:血管生成在肿瘤从良性向恶性转变、癌细胞进入血液循环、转移灶发展和破裂中都起着重要作用.本研究探讨汉防己甲素对体外、体内血管生成的抑制作用及其可能的机制.方法:采用M1_r法观察汉防己甲素对人脐静脉血管内皮细胞(human umbilical vein endothelial cell HUVEC)和人肠癌LoVo细胞增殖的影响.通过Transwell小室趋化实验、体外小管形成实验观察汉防己甲素对HUVEC迁移、成血管能力的影响.建立裸鼠kIVo细胞皮下移植瘤模型.给予汉防己甲素灌胃,观察用药对肿瘤微血管密度的影响.结果:2-8 μg/mL的汉防己甲素作用48 h时对HUVEC的细胞增殖抑制率为24.6%-76.9%,对LoVo细胞增殖抑制率为11.6%～14.0%;体外小管形成实验发现.2～8 μg/mL的汉防己甲素作用24 h时HUVEC小管形成数目减少,且管腔不完整,与对照组比较差异有统计学意义(P值均＜0.001).经2-8 μg/mL的汉防己甲素处理12 h后HUVEC迁移数明显少于对照组(P值均＜O.001).在裸鼠皮下移植瘤体内实验中,80 mg/kg汉防己甲素作用于裸鼠LoVo细胞皮下移植瘤后其微血管密度与生理盐水对照组比较.差异具有统计学意义(p0.035).结论:汉防己甲素在体外能有效抑制血管生成,其机制可能与抑制HUVEC增生、迁移和小管形成,诱导HUVEC凋亡,抑制HUVEC DNA的合成有关.汉防己甲素在体内对裸鼠LoVo移植瘤具有抗血管生成作用.     

Synergistic interaction between tetrandrine and chemotherapeutic agents and influence of tetrandrine on chemotherapeutic agent-associated genes in human gastric cancer cell lines

PURPOSE: Tetrandrine (Tet), a bis-benzylisoquinoline alkaloid that was isolated from the dried root of Hang-Fang-Chi (Stephania tetrandra S. Moore), is well known as processing a marked antitumor effect in vitro and in vivo. The aim of this study was to assess the interaction between tetrandrine and chemotherapeutic agents widely used in gastric cancer treatment, and to investigate the influence of tetrandrine on chemotherapeutic agent-associated gene expression and apoptosis. METHODS: Synergistic interaction on human gastric cancer BGC-823 and MKN-28 cells was evaluated using the combination index (CI) method. The double staining with both Annexin-V-FITC and PI was employed to distinguish the apoptotic cells from living cells. Expression of chemotherapeutic agent-associated genes, i.e., excision repair cross-complementing 1 (ERCC1), thymidylate synthase (TS), class III beta-tubulin (beta-tubulin III) and tau, of BGC-823 cells with or without tetrandrine treatment were measured by real-time quantitative PCR. RESULTS: Tetrandrine had a synergistic effect on the cytotoxicity of chemotherapeutic agents in both two gastric cancer cell lines. The combination of tetrandrine and chemotherapeutic agents could also induce apoptosis in a synergistic manner. Tetrandrine could suppress the mRNA expression of ERCC1, TS, beta-tubulin III and tau. Most prominently, ERCC1, TS and beta-tubulin III mRNA levels were markedly suppressed at 0.29-, 0.12- and 0.60-fold, respectively, by the presentation of tetrandrine. CONCLUSION: Tetrandrine appears a promising candidate for combining with three chemotherapeutic agents. The possible mechanisms might be the synergistic apoptotic effect and the downregulation of chemotherapeutic agent-associated genes.     

Juvenile granulosa cell tumor of the ovary presenting with pleural effusion and ascites

Juvenile granulosa cell tumor (GCT) is a rare tumor, and the majority (90%) are reported in the prepubertal or under-30-year age group, in contrast to the adult type, which is more common in the fifth decade. On histopathological examination, juvenile GCTs are distinct from the adult type of GCT, and have a lower risk for late recurrences than the latter. Being solid tumors, they may be associated with ascites and pleural effusion (Meigs’ syndrome), which resolve after surgical removal of the tumor. Tumor markers for GCT are still investigational (inhibin) and of not much use in making a preoperative diagnosis, unlike in the case of germ cell tumors. In most of the reports about the initial surgical management of GCT, retroperitoneal lymph node sampling was not performed, and it was not done in the patient we report here. However, lymph node sampling is advocated for complete staging of these tumors, as a significant number of recurrences are reported in the retroperitoneum, as well as in incompletely staged patients. In the present patient, because of the association of Meigs’ syndrome, a preoperative diagnosis of benign tumors such as fibroma/thecoma was also considered. We report this rare tumor with an aim of reviewing the diagnosis and management from the reported literature.     

Chemotherapy sensitivity testing on ovarian cancer cells isolated from malignant ascites

Background: In epithelial ovarian cancer (EOC), 15–20% of the tumors do not respond to first-line chemotherapy (paclitaxel with platinum-based therapy), and in recurrences this number increases. Our aim is to determine the feasibility of cell proliferation assays of tumor cells isolated from malignant ascites to predict in vitro chemotherapy sensitivity, and to correlate these results with clinical outcome.Materials and Methods: Ascites was collected from twenty women with advanced EOC. Cell samples were enriched for tumor cells and EOC origin was confirmed by intracellular staining of CK7, surface staining of CA125 and EpCAM, and HE4 gene expression. In vitro sensitivity to chemotherapy was determined in cell proliferation assays using intracellular ATP content as an indirect measure of cell number. In vitro drug response was quantified by calculation of the drug concentration at which cell growth was inhibited with 50%. Clinical outcome was determined using post-treatment CA125 level.Results: Cell samples of twenty patients were collected, of which three samples that failed to proliferate were excluded in the analysis (15%). Three other samples were excluded, because clinical outcome could not be determined correctly. In twelve of the fourteen remaining cases (86%) in vitro drug sensitivity and clinical outcome corresponded, while in two samples (14%) there was no correspondence.Conclusions: Our study demonstrates the feasibility of drug sensitivity tests using tumor cells isolated from ascites of advanced EOC patients. Larger observational studies are required to confirm the correlation between the in vitro sensitivity and clinical outcome.     

粉防己碱对人肝癌细胞株7402的放射增敏作用

目的:观察粉防己碱(Tet)对人肝癌细胞株7402的放射增敏作用。方法:以人肝癌细胞株7402为研究对象,应用MTT比色法、克隆形成实验检测Tet的细胞抑制效应;应用克隆形成实验观察Tet对7402细胞株的放射敏感性的影响;流式细胞术测定照射后7402细胞株细胞周期的再分布并观察Tet能否去除放射引起的细胞周期阻滞;Western blot检测CyclinB1、Cdc2和Cdc25C磷酸化形式的表达水平;细胞分裂指数实验观察Tet对照射后细胞分裂指数的影响。结果:不同浓度的Tet作用于人肝癌7402细胞株24h后,其细胞毒性呈剂量依赖性。Tet(0.5μg/ml)能明显降低放射后7402细胞的克隆形成率,其放射增敏比(SERDq)为1.76。流式细胞术结果显示,照射明显导致7402细胞株G2期阻滞,Tet能够去除放射引起的7402细胞G2期阻滞。Western blot显示细胞在受到X射线照射后,CyclinB1表达水平明显降低,Cdc2和Cdc25磷酸化形式的表达水平明显增加,分裂指数也明显降低;经Tet处理后,CyclinB1表达水平明显增高,Cdc2和Cdc25磷酸化形式的表达水平明显下降,分裂指数则增高。结论:Tet对7402细胞株有放射增敏作用,其作用机制可能与Tet去除放射引起的G2/M期阻滞有关。     

汉防己甲素联合GP方案治疗晚期非小细胞肺癌的随机对照临床研究

目的：评价汉防己甲素注射液联合吉西他滨（gemcitabine,G）、顺铂（cisplatin,P）方案治疗晚期非小细胞肺癌（a—NSCLC）的临床疗效、不良作用。方法：240例a—NSCLC患者随机分为汉防己甲素联合GP方案组和单纯GP方案组。吉西他滨1000mg／m。于第1、8天静滴,顺铂75mg／m2于第1天静滴,治疗组同时加用汉防己甲素注射液150mg／d静滴,第1—10天连续给药,对照组单纯化疗,每21天为1个周期。按照RE—CIST标准评价近期疗效,参照Kamofsky评分及体重变化评价生活质量,按照NCICTC3．0版标准评价毒性反应。结果：219例患者可评价疗效。治疗组近期客观有效率36．1％,对照组为24．3％,P=0．057;治疗组近期疾病控制率63．9％,对照组为52．3％,P=0．081;1年生存率两组分别为45．7％、31．3％,P=0．059;治疗后两组KPS评分提高率分别为49．1％、32．4％,P=0．012;体重增加率分别为28．7％、16．2％,P=0．027。治疗组的2—4度白细胞、血小板减少及恶心、呕吐等发生率分别为38．0％、19．4％、46．3％、16．7％,均低于对照组53．2％、34．2％、63．0％、27．9％,均P〈0．05,而局部刺激为2．8％,略高于对照组0．O％,P〉0．05;上述不良反应均可耐受及预防。结论：汉防己甲素可能具有提高a—NSCLC患者化疗的近期疗效、延长生存期的作用,无明显不良作用,同时可减轻化疗不良反应、改善患者的生活质量,在一定程度上提高机体对化疗的耐受性,是a—NSCLC综合治疗的较好选择。     

新生霉素对人肝癌细胞的抑制作用

 目的　研究新生霉素 (NOVO)对人肝癌细胞的抗癌及其对临床常用抗癌药的调节作用。方法　采用抗癌药敏感试验 (MTT法 ) ,测定NOVO和 5种临床常用抗癌药单独应用对手术切除的原发性肝癌病人的肝癌细胞的抑制作用 ,同时测定NOVO和此 5种抗癌药联合应用的抑制作用。结果　NOVO对人肝癌细胞有抗癌作用 ,与 5种抗癌药联合应用对肝癌细胞的抑制率在低、中、高浓度均高于 5种抗癌药单独应用 ,差异均有显著性 ,P <0 .0 5。结论　结果表明NOVO对人肝癌细胞有较强的抗癌作用 ,并对诸如MMC、5 FU、DDP、CTX、ADM等有加强作用 ;NOVO可能有较大的临床应用价值     

44例结直肠癌合并恶性胸腹腔积液患者的预后分析

目的探讨结直肠癌合并恶性胸腹腔积液的预后因素。方法回顾性分析2001年1月至2010年12月中国医学科学院肿瘤医院内科结直肠癌合并恶性胸腹腔积液患者的临床资料。结果 44例患者中,治疗后积液完全消失2例,减少16例,稳定6例,增多20例。中位总生存时间为8个月。单因素分析结果显示,KPS评分、肝转移、治疗模式、积液控制率、白蛋白以及胆红素水平与总生存时间有关。结论 KPS评分≥80分、无肝转移、全身化疗、积液控制好、白蛋白和胆红素水平正常的患者预后更好。     

乳腺癌术后胸膜转移性胸水TIL的细胞毒活性研究

目的：探讨乳腺癌术后胸膜转移引起的胸水中肿瘤浸润淋巴细胞（TIL）毒活性。方法：采用生物技术，提纯乳腺癌胸膜转移所致胸水中的TIL和相应的肿瘤细胞，经过一定时间诱导培养后，在7天，14天，21天和28天分别检测TIL对自身肿瘤细胞和K562细胞的杀伤活性。结果：该TIL对自身肿瘤细胞和K562细胞的杀伤活性无显性差异，21天时细胞毒活性最强。结论：TIL诱导培养21天时具有较强的生物活性，此时进行临床应用可能取得最佳疗效。