人类肿瘤细胞分化中细胞周期素的表达规律及作用机制

目的：探讨诱导人类肿瘤细胞分化过程中，细胞周期素（Cyclin）的变化在细胞周期调控机制中的作用。方法：应用全反式维甲酸（ATRA）处理HL－60细胞，流式细胞仪行细胞周期分析，应用十二烷基磺酰钠－聚丙烯胺凝胶电泳（SDS－PAGE）及Western Blot检测细胞周期素D1、E的蛋白表达量改变，使用流式细胞仪检测细胞周期素D1、E、A的表达量变化及细胞周期素B的时相性改变。结果：HL－60细胞ATRA诱导后，80％左右的细胞被祖滞在G0／G1期，S期细胞减少到2．81％，WesternBlot显示，诱导后，CyclinD1表达量明显降低，CyclinE表量高。流式细胞仪检测CyclinD1、CyclinE蛋白表达变化与WesternBlot结果相同，CyclinA表达量升高，CyclinB表达特征由经放导前的非时相性表达转为诱导分化后的时相性表达。结论：细胞周期素D1、E、A、B与肿瘤细胞分化的细胞周期调控有着密切的关系。     

全反式维甲酸对人结肠癌细胞亚株SW480/M5增殖和凋亡的影响

目的 观察全反式维甲酸(ATRA)对人结肠癌细胞亚株SW480/M5增殖和凋亡的影响.方法 噻唑蓝(MTT)比色法检测1、2、4、8μmol/L ATRA作用24、48、72 h对SW480/M5细胞的抑制率.流式细胞仪检测8 μmol/L ATRA作用24、48、72 h及2、4μmol/L ATRA作用48 h的肿瘤细胞周期和凋亡率.结果 ATRA抑制SW480/M5细胞增殖作用呈一定时效和量效依赖性;8μmol/LATRA作用72 h明显抑制肿瘤细胞增殖,抑制率接近30％.2、4μmol/L ATRA作用SW480/M5细胞48 h或8μmol/L作用24 h,肿瘤细胞G1期比例开始降低,S期比例增加(P＜0.05).8umol/L ATRA作用48 h后,S期比例进一步增高,G2/M期细胞比例不增加;作用72 h后,G1期细胞比例进一步下降,伴G2/M期细胞比例增加(P＜0.05).8μmol/L ATRA作用24 h无明显诱导SW480/M5细胞凋亡作用,作用48 h或72 h可诱导肿瘤细胞凋亡,但凋亡率差异无统计学意义(P＞0.05).结论 8μmoL/LATRA作用48 h或72 h通过阻滞SW480/M5细胞在S期或(和G2/M)期,并诱导肿瘤细胞凋亡,可明显抑制肿瘤细胞增殖.     

反义寡聚核苷酸抑制人膀胱癌T24细胞系端粒酶活性的研究

目的研究反义寡聚核苷酸(asONs)能否抑制人膀胱肿瘤T24细胞系端粒酶活性和增殖活性。方法设计并合成2条针对端粒酶RNA模板区的asONs片段,在其作用于T24细胞第5天时,用7复孔法用聚合酶链反应-酶标法(PCR-ELISA)检测其对端粒酶活性的影响;分别在实验的第1、3、5、7天,用7复孔四唑盐比色试验(MTT)法检测其对细胞增殖活性的影响。结果经不同浓度(0、5、10μmol/L)处理5d后T24细胞端粒酶活性受到抑制,吸光度(A)值分别为0.79±0.10、0.24±0.14;经10μmol/L处理1、3、5、7d,T24细胞的增殖活性逐渐下降,A值分别为0.59±0.11、0.39±0.09、0.21±0.02、0.19±0.07,各组分别比较差异有非常显著性(P＜0.001)。结论asONs能够同时抑制人膀胱肿瘤T24细胞系端粒酶及细胞增殖活性,提示asONs可能通过抑制端粒酶活性达到抑制人膀胱肿瘤T24细胞系的增殖。     

端粒酶反义寡核苷酸对乳腺癌端粒酶活性及细胞生长的影响

目的　探讨端粒酶反义寡核苷酸对人乳腺癌细胞端粒酶活性的影响及抑制端粒酶活性以控制乳腺癌细胞生长的可能性。方法　采用 10 μmol/L与端粒酶催化亚基 (hTRT )mRNA互补的反义寡核苷酸处理乳腺癌细胞 ;于处理后 2 4、72h用端粒重复序列扩增及酶联免疫吸附方法检测细胞端粒酶活性 ;于处理后 72h以流式细胞术测定细胞周期 ;于处理后 12 0h采用原位末端标记法检测细胞凋亡。结果　经hTRT反义寡核苷酸作用后 ,细胞端粒酶活性明显受到抑制 ,至作用后 72h ,端粒酶活性较对照组降低 65 .6% (P <0 .0 5 ) ;细胞生长明显受到抑制 ;细胞周期发生显著变化 :G0 /G1期细胞数增多 ,S期及G2 /M期细胞数则减少 ;细胞增殖指数由 0 .46降低至 0 .2 5 ;并可观察到凋亡细胞的出现 ,凋亡发生率为 12 .5 % ;而无义组及空白对照组以上各指标均无明显变化 (P >0 .0 5 )。结论　端粒酶反义寡核苷酸可明显抑制乳腺癌细胞端粒酶活性 ,抑制细胞生长和增殖 ,诱导细胞凋亡 ;表明应用反义技术抑制端粒酶活性治疗乳腺癌具有广阔的前景。     

全反式维甲酸对人胰腺癌细胞PC-3诱导分化作用及其对Survivin表达的影响

维甲酸类药物是一类细胞诱导分化剂，能够抑制多种肿瘤细胞的增殖，调节着细胞的生长和分化，全反式维甲酸（A—TRA）作为维甲酸类药物的代表，具有显著的抗肿瘤作用。我们用ATRA诱导人胰腺癌细胞PC-3，观察细胞周期的改变和Survivin基因表达的变化，以及ATRA对人胰腺癌细胞增殖的抑制作用，为维甲酸类药物临床应用于胰腺癌的治疗提供实验依据。     

维甲酸对人骨肉瘤细胞抑制增殖和诱导分化的影响

目的探讨维甲酸对人骨肉瘤（HOS）细胞抑制增殖和诱导分化的影响。方法不同浓度的维甲酸作用HOS细胞,采用噻唑蓝法、倒置相差显微镜观察药物对细胞生长的影响并绘制细胞生长曲线;软琼脂集落形成实验观察细胞生长和侵袭能力的变化;流式细胞仪测定细胞周期变化。结果维甲酸可明显抑制HOS细胞增殖,并呈现剂量和时间的依赖性;细胞形态呈明显的良性分化,瘤细胞的软琼脂集落形成率明显降低;维甲酸能够延缓细胞周期G1～S进程,阻滞细胞于G1期。细胞周期测定结果显示：未经诱导物处理的对照组细胞处于G0/G1期的细胞比例为31.19%;经维甲酸处理之后,实验组细胞G0/G1期的细胞比例上升为57.94%。结论维甲酸对HOS细胞有明显的抑制增殖和诱导分化作用。     

反义人端粒酶RNA亚基对胆囊癌端粒酶活性及细胞生长的抑制作用

目的探讨反义RNA技术对胆囊癌细胞端粒酶活性的影响及对胆囊癌细胞生长增殖的作用。方法根据测定的胆囊癌人端粒酶RNA亚基(hTR)基因的序列结果，并根据真核表达载体多克隆酶切位点的物理图谱，体外合成反义RNA及正义RNA的基因序列，并构建入pTriEx-4真核表达载体，酶切鉴定正确后，采用脂质转染法导入胆囊癌细胞。TRAP法检测端粒酶活性。流式细胞仪检测细胞周期及凋亡情况，电镜观察细胞微观形态变化。结果实验组胆囊癌细胞的端粒酶活性较对照组明显减低，正义组、转染空载体及单纯脂质体转染的细胞端粒酶活性与未转染细胞比较则变化不明显。反义RNA基因作用后，G0／G1期细胞比例明显增高，S期细胞比例明显降低。细胞的分裂、增殖受到明显抑制。反义RNA基因转化后，10d凋亡率为11．10％。13d后凋亡率为29．02％。电镜下可见明显凋亡细胞。结论反义RNA技术对胆囊癌细胞端粒酶活性有明显的抑制作用；并抑制胆囊癌细胞的生长，促进细胞的凋亡；且克服了反义寡核苷酸作用时间短的缺点。     

端粒酶活性在人膀胱肿瘤组织中表达的临床意义

目的：探讨不同临床病理类型的膀胱肿瘤及癌旁组织端粒酶活性表达及临床意义，方法：以改良TRAP法测定91例膀胱肿瘤组织标本端粒酶活性表达。结果：83例膀胱移行细胞癌组织中78例检测到端粒酶活性，阳性率为94％，其对应的癌旁组织也有14％的检出率，8例膀胱乳头状瘤组织中4例检测到端粒酶活性，阳性率为50％，其对应的癌旁组织检出率为12％，端粒酶活性在不同临床病理类型的膀胱肿瘤及癌旁组织中表达差异无显著性意义（P＞0．05），结论：膀胱肿瘤及癌旁组织端粒酶活性的检测对肿瘤的诊断有重要意义。     

苯乙酸对人结直肠癌细胞HCT-8的G1细胞周期阻滞及增殖抑制作用

摘要：目的 探讨诱导分化剂苯乙酸（PA）对人结直肠癌细胞系HCT 8细胞周期及增殖的影响。 方法 应用MTT比色法，以1.0，2.0，3.0，4.0，5.0 mmol/L的PA作用于体外培养的HCT 8细胞，分别于24，48，72h后对细胞增殖进行检测；流式细胞术分析细胞周期。结果 随着PA浓度的增加（1～5 mmol/L）或药物作用时间的延长（24～72h），肿瘤细胞生长抑制率明显增加。PA1~5mmol/L作用24h细胞生长抑制率为5.1%-24.3%，48h为16.7%-72.3%，72h为30.2%-93.4%。PA作用细胞72h后，G0/G1期比例显著下降，S期比例相对升高，组间差异显著（P＜0.05）。结论 PA在诱导分化结直肠癌HCT 8细胞过程中，可诱导G1细胞周期阻滞，抑制细胞增殖。     

三种分化诱导剂对人胆管癌细胞株QBC939的作用

目的观察二甲基亚砜(DMSO)、全反式维甲酸(ATRA)和9顺式维甲酸(9cisRA)对人胆管癌细胞株QBC939的作用,筛选对它有效的分化诱导剂。方法以DMSO、ATRA和9cisRA作用于培养的胆管癌细胞株,描绘生长曲线,观察细胞形态,双层软琼脂培养法观察细胞恶性度,流式细胞术(FCM)检测细胞周期动力学变化。结果10-6mol/L9cisRA作用2d后开始抑制QBC939细胞生长,抑制率为39.4%～80.7%(P<0.05)。细胞软琼脂集落形成能力较对照组下降80.9%(P<0.01)。细胞形态向正常细胞转化。9cisRA作用1、3、5、7dG0/G1期细胞比例分别为55.56%、63.92%、73.38%、76.92%。ATRA对QBC939细胞无明显作用,DMSO主要引起细胞死亡。结论9cisRA可诱导QBC939细胞向良性表型分化,对该细胞株具有有诱导分化作用。     

端粒酶反义RNA转染促进膀胱癌T24细胞凋亡的研究

目的　探讨端粒酶反义RNA对膀胱癌T2 4细胞恶性表型的抑制及促进其凋亡的作用。　方法　采用脂质体转染法将转录出端粒酶反义RNA质粒导入膀胱癌T2 4细胞。应用PCR ELISA法测定转染后T2 4细胞的端粒酶活性 ;光镜、电镜、MTT及流式细胞术 (FCM )等方法观察端粒酶反义RNA对T2 4细胞生长及凋亡的影响。　结果　端粒酶反义RNA能显著抑制T2 4细胞的端粒酶活性 ,转染T2 4细胞后使其生长受到抑制。形态学观察 ,转染后T2 4细胞出现典型的凋亡现象。FCM检测发现G1期前出现凋亡峰。　结论　转染端粒酶反义RNA能抑制膀胱癌T2 4细胞的恶性表型 ,促进其凋亡     

Inhibition of growth and telomerase activity by novel cationic ceramide analogs with high solubility in human head and neck squamous cell carcinoma cells.

OBJECTIVES: Head and neck squamous cell carcinoma (HNSCC) is notoriously resistant to chemotherapy. The sphingolipid ceramide and its analogs have been demonstrated to exert antitumor activity in many cell types; however, the effectiveness of these analogs has been limited by potency and solubility. This study focuses on the effects of novel highly soluble cationic pyridinium-ceramides, alone and in combination with various chemotherapeutic agents, on cell survival, telomerase activity, and cell cycle arrest in HNSCC cell lines in vitro. METHODS: The concentration of pyridinium-ceramides and chemotherapeutic agents that inhibited cell growth by 50% (IC50) was determined by MTT cell survival assays. The cell cycle profiles were determined by flow cytometry. Telomerase activity was determined by telomerase repeat amplification protocol (TRAP) assay. RESULTS: Treatment of the human UM-SCC-22A (SCC of the hypopharynx) cells, as well as various other HNSCC cell lines, with C6-Pyr-Cer resulted in the inhibition of cell survival with an IC50 concentration of approximately 250 to 300 nM at 96 hours, whereas its IC50 was greater than 1000 nM in noncancerous Wi-38 human lung fibroblasts, and adult human epidermal keratinocytes. Moreover, treatment with C6-Pyr-Cer also resulted in cell cycle arrest in G0/G1, which correlated with a significant inhibition of telomerase activity in UM-SCC-22A cells. Additional results demonstrated that the combination of C6-Pyr-Cer with gemcitabine (GMZ) or doxorubicin (DOX), which have the lowest IC50 concentrations among various chemotherapeutic drugs in these cells, enhances the effects of these drugs in the inhibition of telomerase and cell growth. CONCLUSIONS: These data suggest that the novel C6-Pyr-Cer with high solubility and bioavailability may lead to the development of new therapeutic strategies that target telomerase for the treatment of HNSCC.     

侵袭性骨肿瘤端粒酶活性检测及其意义

目的：检测端粒酶在侵袭性骨肿瘤中的活性及评估其作为骨肿瘤恶性诊断的意义。方法：采用非放射性同位素ＴＲＡＰ－银染方法从侵袭性骨肿瘤组织及骨源细胞系／株两方面进行端粒酶活性检测，并将此方法与ＴＲＡＰ－ＥＢ染色进行了比较。结果：５３例侵袭性骨肿瘤组织端粒酶活性检出率８３．０％（４４／５３）；同时检测的１９例同一病人的肿瘤旁对照组织和１例骨的瘤样病变—动脉瘤样骨囊肿均未检测到端粒酶活性；３例正常的骨膜培养细胞，１例已经３次化学致癌剂４－ＮＱＯ处理，传至１０２代的动脉瘤样骨囊肿细胞及１例人骨肉瘤细胞株（ＯＳ７３２）未能检出端粒酶活性，但另１例人骨肉瘤细胞株（ＨＯＳ）却表达有端粒酶活性。结论：端粒酶活性可能是骨肿瘤恶性的一种潜在生化标志物，并在骨肿瘤的发生发展过程中可能起到重要的作用。结果还表明非放射性同位素ＴＲＡＰ－银染方法是一种简便、安全、快速而有效的检测端粒酶活性的方法     

Telomere Length and Telomerase Activity in Bladder and Prostate Cancer Cell Lines

Background: Specific repeats of oligonucleotides at the ends of chromosomes (telomeres) are shortened by cell division in somatic cells and are thought to be related to aging. Immortal cells such as germ-line cells or cancer cells have demonstrated increased activity of the telomere-elongating enzyme (telomerase). The length of the telomeres of these cells is stable regardless of cell division. We examined the telomere length and telomerase activity in 3 bladder (JTC30, JTC32, and T24) and 2 prostate cancer (LNCaP and DU145) cell lines.
Methods: Telomere lengths were evaluated by Southern blot analysis with a oligonucleotide probe, (TTAGGG)_S and telomerase activities were detected with a polymerase chain reaction-based assay method.
Results: Telomerase activity was detected in all of the cell lines. Each cell line had a specific telomere length. In 2 bladder cancer cell lines (JTC30 and JTC32), the telomere length decreased with increased passage of the cells.
Conclusion: The presence of telomerase may be a biological character of bladder and prostate cancers as well as other malignancies, although it does not always compensate telomere shortening.     

Postmeiotic modifications of spermatogenic cells are accompanied by inhibition of telomerase activity

We investigated whether testicular telomerase activity is due to telomerase expression in all cells or expression in a limited number of cells. Telomerase activity was assayed in highly purified fractions of spermatogonia cells plus primary spermatocytes, secondary spermatocytes plus round spermatids, secondary spermatocytes plus spermatids plus spermatozoa, round spermatids, or spermatozoa prepared from healthy or cryptorchid animals. Telomerase activity was additionally assayed in testicular tissue of prepubertal animals and animals with Sertoli cell only pathophysiology. Telomerase activity was detected in fractions containing primary spermatocytes and/or secondary spermatocytes and/or spermatids. Fractions enriched in round spermatids were positive for telomerase activity. In contrast, spermatozoa or Sertoli cell fractions were negative for telomerase activity. Using the relative telomerase activity assay and the sensitive quantitative telomerase assay to quantify telomerase activity, we showed that induction of cryptorchidism does not result in quantitative alterations in testicular tissue telomerase activity. In addition, elimination of round spermatids does not lead to significant alterations in testicular tissue telomerase activity. The present results suggest that the male gamete telomerase activity is inhibited during spermiogenesis. Furthermore, it appears that spermatogonia/primary spermatocytes are the main sources of telomerase activity in the testis. Received: 29 October 1997 / Accepted: 20 October 1998     

大肠癌肝转移肿瘤中端粒酶活性和p16基因纯合缺失

目的 研究人大肠癌肝转移肿瘤中端粒酶活性和p16基因纯合缺失及其临床意义。方法 采用TRAP银染方法和半定量多重PCR检测24例大肠癌转移肿瘤组织中端粒酶活性及p16基因的纯合缺失情况,并结合临床病理参数,进行统计学分析。结果 本组24例大肠癌肝转移肿瘤组织中检测到19例端粒酶阳性,阳性率为79．2％。端粒酶活性与转移瘤大小、肝内转移灶数目、分化程度、HBsAg以及是否伴有纤维化无关。在24例转移瘤组织中有9例标本中检测到p16基因的缺失,阳性率为37．5％,p16基因的失与端粒酶活性相关。结论 大肠癌肝转移肿瘤中端粒酶活性和p16基因异常对阐明大肠癌转移的生物学行为有重要的意义。     

反义人类端粒酶 RNA逆转录病毒载体构建及其对结直肠癌细胞的抑制作用

目的 研究反义人类端粒酶RNA逆转录病毒载体对结直肠癌HT29细胞端粒酶活性及细胞生长的抑制作用。探讨以端粒酶为酸点的结直肠癌基因治疗的可能性。方法 将端粒酶hTR的cDNA反向插入逆转录病毒载体pLXRN中，转染包装细胞PT67后获得反义重组病毒，感染结直肠癌HT29细胞，采用RT-PCR检测hTR表达。端粒酶重复扩增法（telomerase repeat amplification protocol,TRAP)检测端粒酶活性，绘制生长曲线了解细胞生长状态，倒置显微镜观察和DNA片段电泳检测细胞凋亡。结果 反义hTR作用后的结直肠癌细胞hTR表达下降，端粒酶活性和细胞生长受到明显抑制，细胞出现凋亡。结论 反义hTR对结直肠癌HT29细胞的生长和端粒酶活性具有明显的抑制人舰艇可能以hTR为靶点对结直肠癌进行基因治疗。     

Telomerase activity in human pleural mesothelioma

BACKGROUND: Gradual telomere erosion eventually limits the replicative life span of somatic cells and is regarded as an ultimate tumour suppressor mechanism, eliminating cells that have accumulated genetic alterations. Telomerase, which has been found in over 85% of human cancers, elongates telomeres and may be required for tumorigenesis by the process of immortalisation. Malignant mesothelioma is an incurable malignancy with a poor prognosis. The disease becomes symptomatic decades after exposure to carcinogenic asbestos fibres, suggesting the long term survival of pre-malignant cell clones. This study investigated the presence of telomerase in pleural malignant mesothelioma, which may be the target for future anti-telomerase drugs. METHODS: Telomerase activity was semiquantitatively measured in extracts from 22 primary pleural mesotheliomas, two benign solitary fibrous tumours of the pleura, four mesothelioma cell lines, and six short term mesothelial cell cultures from normal pleura using a non-isotopic dilution assay of the telomeric repeat amplification protocol. RESULTS: Twenty of the 22 primary mesotheliomas (91%) and all tumour derived mesothelioma cell lines were telomerase positive. Different levels of enzyme activity were observed in the tumours of different histological subtypes. Telomerase activity could not be detected in the six normal mesothelial cell cultures or in the two mesotheliomas. Both benign solitary fibrous tumours showed strong telomerase activity. CONCLUSIONS: Telomerase activity is found in a high proportion of mesotheliomas and anti-telomerase drugs might therefore be useful clinically. The results are consistent with the hypothesis that telomerase activity may be a feature of carcinogenesis in mesotheliomas and possibly in many other cancers.