丁酸钠增强U937细胞凋亡敏感性的分子机制研究

目的 探讨丁酸钠 (NaBu)对细胞周期检测点效应及对U937细胞凋亡的敏感性。方法以U937 ASPI3K(ATM阴性 )、U937 pZeosv2 (+) (野生型ATM基因 )两种U937的变异细胞系作为细胞模型。用免疫沉淀及激酶活性测定p38MAPK、ERK1的激酶活性。用免疫印迹分析Bad磷酸化灭活。结果 经NaBu预处理的U937 pZeosv2 (+)细胞经13 7Cs照射后 ,细胞凋亡敏感性呈NaBu剂量依赖性增强。这种增强效应可被p38MAPK阻滞剂OLM阻断 ,但不能被p34cdc2激酶的特异性抑制剂ALP及CDK2阻滞剂CDK2 Ⅰ阻断。经NaBu预处理的U937 ASPI3K细胞经13 7Cs照射后 ,细胞凋亡敏感性进一步增强 ,这种增强效应可被OLM阻断。放射线可显著增强p38MAPK激酶活性 ,抑制ERK1激酶活性 ;NaBu预处理与放射线联用后 ,对p38MAPK激酶活性的增强有极其显著的协同效应。放射线诱导U937 ASPI3K细胞Bad蛋白磷酸化灭活 ,在NaBu协同作用下 ,Bad蛋白磷酸化灭活效应进一步增强。结论 NaBu通过p38MAPK激酶活性 ,增强细胞凋亡敏感性 ,该效应与ATM基因是否缺失无关 。

Fas死亡受体及FLIP对大肠癌细胞凋亡的影响

目的:比较不同大肠癌细胞Fas受体及其下游通路抑制因子FLIP的表达差异,探讨Fas、FLIP对大肠癌细胞凋亡的影响. 方法:体外培养大肠癌细胞,应用直接免疫荧光流式细胞术检测细胞表面Fas表达率,半定量RT-PCR法测定FLIP mRNA的水平,并采用Annexin V法评价细胞对Fas介导的凋亡的敏感性. 结果:大肠癌细胞表面Fas表达率不同,其中HT-29细胞的表达率为42.46±4.32%,明显高于其他3株细胞.SW620 和HT-29细胞FLIP mRNA含量较高,Colo205居中,Lovo 则呈阴性;且相对于任何一株FLIP表达阳性的细胞,FLIPL的表达水平均高于FLIPs.给予凋亡诱导型抗Fas抗体(CH-11)刺激后,4株大肠癌细胞凋亡敏感性均较低. 结论:不同的大肠癌细胞株可能通过不同途径逃避Fas介导的凋亡,其中包括下调Fas的表达和上调FLIP的含量.

TRAIL介导的细胞凋亡与肝纤维化

1996年，Pitti等从人胎盘cDNA文库中发现了一种与人细胞凋亡因子受体[FasL（Apo-1L）3很相似的肿瘤坏死因子（TNF）超家族成员，命名为Apo-2L，也称为TNF相关的凋亡诱导配体（TNF-related apoptosis-inducing ligand，TRAIL）。近年研究发现，在活化的肝星状细胞（hepatic stellate cell，HSC），表面TRAIL受体蛋白表达增加，且HSC对TRAIL介导的细胞凋亡敏感性也随之加强。正常HSC为静止型，其主要生理功能是摄取和储存维生索A；肝损伤后HSC激活为活化型，出现基因表达及细胞表型的改变，大量合成肝细胞外基质（extracelluar matrix，ECM）及分泌基质金属蛋白酶抑制剂（tissue inhibitor of metalloproteinase，TIMP），进而导致肝纤维化的发生发展。肝间质中的HSC是肝细胞外基质（extracellular matrix，ECM）的主要来源细胞，如何诱导和促进活化型HSC凋亡是目前治疗肝纤维化的热点。现就TRAIL、TRAIL受体与肝纤维化关系的研究进展概述如下。

肿瘤坏死因子相关的凋亡诱导配体及其受体与肝纤维化治疗

肿瘤坏死因子相关的凋亡诱导配体（TNF-related apoptosis-inducing ligand，TRAIL），也称Apo-2L，是1995年发现的一种肿瘤坏死因子超家族成员。近年研究表明，在活化的肝星形细胞（hepatic stellate cell，HSC）表面TRAIL受体表达增加，且HSC对TRAIL介导的细胞凋亡敏感性也随之增强。激活状态HSC的清除对肝纤维化的逆转起着关键性作用，

Down-regulation of survivin expression by small interfering RNA induces pancreatic cancer cell apoptosis and enhances its radiosensitivity

AIM： To investigate the inhibitory effect of small interfering RNA （siRNA） on the expression of survivin in pancreatic cancer cell line PC-2 and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity. METHODS： A siRNA plasmid expression vector against survivin was constructed and transfected into PC-2 cells with LipofectamineTM 2000. The down regulation of survivin expression was detected by semi-quantitive RT-PCR and immunohistochemical SP method and the role of siRNA in inducing PC-2 cell apoptosis and enhancing its radiosensitivity was detected by flow cytometry. RESULTS： The sequence-specific siRNA efficiently and specifically down-regulated the expression of survivin at both mRNA and protein levels. The expression inhibition ratio was 81.25% at mRNA level detected by semlquantitive RT-PCR and 74.24% at protein level detected by immunohistochemical method. Forty-eight hours after transfection,apoptosis was induced in 7.03% cells by siRNA and in 14.58% cells by siRNA combined with radiation. CONCLUSION： The siRNA plasmid expression vector against survivin can inhibit the expression of survivin in PC-2 cells efficiently and specifically. Inhibiting the expression of survivin can induce apoptosis of PC-2 cells and enhance its radiosensitivity significantly. RNAi against survivin is of potential value in gene taerapy of pancreatic cancer.

Experimental study of gemcitabine combination with radiation in vitro on a highly metastatic human ovarian cancer cell line HO-8910PM

Objective： The aim of the study was to investigate the mechanism of gemcitabine （GEM） combination with radiation on the high metastasis human ovarian cancer cell line （HO-8910PM）. Methods： Human ovarian cancer cell line HO- 8910PM was treated with different concentrations of gemcitabine for 24 h, then the cells were counted. In the study of GEM combination with radiation, an efficiency of colony formation was observed; the cell cycle and apoptosis were analyzed by flow cytometry; the experiment of depend on the time and its radio sensitivity were observed by using mitotic index with the cells for each 24, 48, 72 and 96 h after experiment. Results： It suggested that the GEM had an inhibition effect on the human ovarian cancer cell line. The alive cell numbers were decreased by following a height of GEM concentration. When GEM in combina- tion with a radiation, the suppression was significantly increased than that of single GEM therapy. The efficiency of colony formation was significantly lower, under this condition the cell could be arrested at G0-G1 phase and could be decreased to enter into the S phase; the apoptosis percentage could be significantly increased; especially, under the 4 Gy and 6 Gy doses the cell apoptosis was more obvious. GEM combination with radiation had depended on the time to the cells; mitotic index of the calls in combination group was observed significantly lower than that of single GEM therapy or single radiation, and this showed that it had an effect of radiosensitivity. Conclusion： The GEM has a significant growth inhibition on the human ovarian cancer cells, GEM combination with radiation could induce HO-8910PM cell occurred arrested and apoptosis. It has depended on the time and has a radiosensitivity effect. The result shows that it is a better method to treat the human ovarian cancer by using radiotherapy combined with gemcitabine.

Adenovirus-mediated PTEN gene transfection suppresses growth and promotes chemosensitivity of endometrial carcinoma

Objective： To determine the potential of sustained transgene expression by intratumoral injection of Ad-PTEN in the nude mouse model of endometrial carcinoma. Methods and Results： We constructed recombinant adenovirus carrying the wild-type PTEN gene （Ad-PTEN）. RL95-2 cells, an endometrial carcinoma cell line lacking PTEN function, was infected with Ad-PTEN and showed increased expression of PTEN and chemosensitivity to doxorubicin, decreased proliferation rate, and elevated apoptosis and Go/G1 arrest. Furthermore, the tumorigenicity of these cells was also completely suppressed. These results indicated that gene therapy with Ad-PTEN could significantly inhibit the endometrial carcinoma xenografts growth in nude mice by intratumoral injection, induce apoptosis of tumor cells, and reduce expression of proliferating cell nuclear antigen （PCNA）. Immunohistochemistry analysis also showed that the expression of progesterone receptors （PR） in Ad-PTEN treated tumor cells were induced, while P-glycoproteins （P-gp） and estrogen receptors （ER） decreased significantly. Conclusion： PTEN may play an important role in the development of endometrial carcinoma. Our findings cast new lights for treatment ofendometrial carcinoma.