多聚(ADP-核糖)聚合酶与细胞损伤

多聚(ADP-核糖)聚合酶[poly(ADP-ribose)polymerase, PARP],又称多聚(ADP-核糖)合成酶[poly(ADP-ribose)synthetase, PARS],或多聚(ADP-核糖)转移酶[poly(ADP-ribose)transferase pADPRT],广泛存在于真核细胞核内,具有蛋白修饰和核苷酸聚合作用.环境刺激、自由基或氧化剂(如过氧化氢、羟自由基和ONOO-)和基因毒性介质所引起的DNA链的缺口或断裂均可引起PARP活化[1].PARP在机体中的作用有赖于DNA的损伤程度,少量的DNA损伤引起的PARP活化时,通过裂解底物NAD+生成ADP-核糖,引起包括PARP自身在内的多种蛋白酶发生多聚(ADP-核糖)基化,参与DNA链的修复;但大量DNA损伤时PARP过度活化,利用NAD+形成大量的(ADP-核糖)聚合物,利用尼克酰胺重新生成NAD+时将消耗ATP,能量耗竭将使细胞严重损伤终导致细胞死亡[2].大量DNA损伤、断裂引起PARP过度活化导致细胞死亡的概念已被近年众多研究所证实,新近报道PARP在细胞凋亡过程中也起重要作用.本文拟就PARP在细胞损伤中的作用做一综述. 1 PARP的结构及功能特性 PARP在许多生理过程中如染色质解聚、DNA复

JAK/STAT信号转导途径活化和Mcl-1表达上调介导IL-6在人骨髓瘤细胞系XG-7上的凋亡抑制效应

背景和目的:IL-6能够抑制多种因素诱发的骨髓瘤细胞凋亡反应,在此过程中涉及到胞浆内一系列信号蛋白分子的参与.本研究旨在确定能够介导IL-6在人骨髓瘤细胞系XG-7上凋亡抑制效应的Bcl-2家族抗凋亡蛋白(Bcl-2,Bcl-xL,Mcl-1)和IL-6信号转导途径(JAK/STAT、Ras/MAPK、PI-3K/Akt途径).方法:采用碘化丙腚(PI)染色和流式细胞术分析XG-7细胞的凋亡情况;采用免疫印迹方法检测Bcl-2家族分子在XG-7细胞中的表达情况;分别采用针对JAK/STAT、Ras/MAPK和PI-3K/Akt途径的特异性抑制剂AG490、PD98059和LY294002处理XG-7细胞,从而确定哪条信号转导途径能够介导IL-6在XG-7细胞上的凋亡抑制效应.结果:IL-6能够抑制XG-7细胞凋亡,同时上调Bcl-2家族抗凋亡蛋白Mcl-1表达.AG490处理的XG-7细胞中Mcl-1表达上调被明显抑制;但PD98059和LY294002处理后对Mcl-1表达没有显著影响.结论:IL-6在XG-7细胞上的凋亡抑制效应是通过上调Mcl-1表达和激活JAK/STAT途径而介导的,与Ras/MAPK和PI-3K/Akt途径的活化状态无关.

Low-density lipoprotein receptor-related protein 1 (LRP1, also known as CD91), a multifunctional endocytic and cell signaling receptor, is widely expressed on the surface of multiple cell types such as hepatocytes, ifbroblasts, neu-rons, astrocytes, macrophages, smooth muscle cells, and malignant cells. Emerging invitro and invivo evidence demonstrates that LRP1 is critically involved in many processes that drive tumorigenesis and tumor progression. For example, LRP1 not only promotes tumor cell migration and invasion by regulating matrix metalloproteinase (MMP)-2 and MMP-9 expression and functions but also inhibits cell apoptosis by regulating the insulin receptor, the serine/threonine protein kinase signaling pathway, and the expression of Caspase-3. LRP1-mediated phosphorylation of the extracellular signal-regulated kinase pathway and c-jun N-terminal kinase are also involved in tumor cell proliferation and invasion. In addition, LRP1 has been shown to be down-regulated by microRNA-205 and methylation ofLRP1 CpG islands. Furthermore, a novel fusion gene,LRP1-SNRNP25, promotes osteosarcoma cell invasion and migration. Only by understanding the mechanisms of these effects can we develop novel diagnostic and therapeutic strategies for cancers mediated by LRP1.

Ubiquitination is crucial for cellular processes, such as protein degradation, apoptosis, autophagy, and cell cycle progression. Dysregulation of the ubiquitination network accounts for the development of numerous diseases, including cancer. Thus, targeting ubiquitination is a promising strategy in cancer therapy. Both apoptosis and autophagy are involved in tumorigenesis and response to cancer therapy. Although both are categorized as types of celldeath, autophagy is general y considered to have protective functions, including protecting cells from apoptosis under certain cellular stress conditions. This review highlights recent advances in understanding the regulation of apoptosis and autophagy by ubiquitination.

In the research community, resistance to apoptosis is often considered a hallmark of cancer. However, pathologists who diagnose cancer via microscope often see the opposite. Indeed, increased apoptosis and mitosis are usualy observed simultaneously in cancerous lesions. Studies have shown that increased apoptosis is associated with cancer aggressiveness and poor clinical outcome. Furthermore, overexpression of Bcl-2, an antiapoptotic protein, is linked with better survival of cancer patients. Conversely, Bax, CD95, Caspase-3, and other apoptosis-inducing proteins have been found to promote carcinogenesis. This notion of the role of apoptosis in cancer is not new; cancer cells were found to be short-lived 88 years ago. Given these observations, resistance to apoptosis should not be considered a halmark of cancer.

In Vitro Study on Lethal Effect of Human Choroidal Melanoma OCM-1 Cell Line by Repeating-70℃Freeze Thawing

Objective: To investigate the effects of repeating -70℃ freeze thawing on human choroidal melanoma cell line OCM-1.Methods: OCM-1 cells were frozen by repeating -70℃ freeze thawing with various durations and frequencies. Then the inhibit rate of cells was examined by MTT essay.The cell viability was measured by monoclonal formation assay. We also used the HE staining, immunohistochemistry staining and the laser-scanning confocal microscopy (LSCM) to investigate the morphological changes of the cells.Results: The growth of OCM-1 cells was inhibited by repeating -70℃ freeze thawing in time-dependent and frequency-dependent manners (P ＜ 0.01). Different morphous including necrosis and apoptosis of the cells could be observed after -70℃ freeze thawing by the LSCM.Conclusion: Repeating -70℃ freeze thawing can not only kill cells directly and induce considerable cells to apoptosis, but also inhibit the growth of the survivals. The kill and wound ratio of the cells disposal with different times and frequencies present variance. And the distinction when treated with different frequencies during the same time is much more significant than different times with the same frequency, which guide clinical workers to choose repeating cryotherapy with short term method instead of single cryotherapy with long term in choroidal melanoma treatment.

Effect of Pyruvate on Polyol Pathway and Lens Epithelial Cells Apoptosis in Diabetic Rats

Purpose: To investigate the effect of polyol pathway on lens epithelial cells apoptosis and the activity of caspase-3 and its reversal by pyruvate in diabetic rats.Methods: 220 Wister rats were divided into 3 groups: control group, model group and treatment group. After streptozotocin (STZ) induced cataract, the treatment group received 2% pyruvate in the diet and drinking. The opacification of lens was detected by microscope every 2 weeks. On 4W, 8W and 12W of the experiment, glucose and sorbitol in the lens were quantified by high-performance liquid chromatography. The percentage of lens epithelial cells undergoing apoptosis was measured by Annexin V/PI staining. The activity of caspase-3 was analyzed by Western-blot.Results: Studies show that there was significant increase of glucose, sorbitol in lens of model group, the apoptosis rate and caspase-3 activity of lens epithelial cells were also gradually increase. Pyruvate treatment decreased the levels of sotbitol, glucose, lens epithelial cells apoptosis and caspase-3 activity. The progress of cataract was also significantly delayed.Conclusions: Polyol pathway, possibly through regulation of the activity of caspase-3,can induce apoptosis of lens epithelial cell. Pyruvate ingested orally can effective inhibit diabetic cataractogenesis in rats through inhibit polyol pathway.

Effect of Chaihu Shugan decoction on gastric smooth muscle cell apoptosis in rats with functional dyspepsia

Objective:To investigate the effect of Chaihu Shugan decoction (CSD) on gastric smooth muscle cells (GSMCs) apoptosis in rats with functional dyspepsia (FD).Methods:48 Sprague Dawley (SD) rats were randomly as signed into six groups:a normal control group,a model group,a positive control (domperidone) group and low-,middle-and high-dose CSD groups.A rat model of FD was established by constantly squeezing their tails.The rats were administered CSD (0.16 g/mL,0.32 g/mL,0.64 g/mL) or domperidone (0.3 g/L) via intragastric gavage for four weeks.The gastric emptying rate was detected at 4 weeks post-administration.Apoptosis of GSMCs was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and the mitochondrial morphology was observed by transmission electron microscopy.The expres sion of Bcl 2 and Bax was measured by immunohistochemistry.Results:FD resulted in marked reduction of gastric emptying rate,severe gastric tissue damage and mitochondria in jury,but were reversed by CSD treatment (P ＜0.05).The apoptosis-induced protein Bax was markedly down-regulated by CSD,whereas the expression of the anti-apoptotic Bcl 2 protein was notably increased (P ＜ 0.05).Furthermore,CSD could protect the FD rats against GSMCs apoptosis manifested by a decreased in TUNEL positive cells (P ＜0.05).Conclusion:CSD could alleviate GSMCs apoptosis in FD rats,possibly by the modulation of Bcl-2 and Bax expression,and the suppression of mitochondria injury.

Expression and function of transcription factor RUNX2in synovial tissues of rheumatoid arthritis

Objective:This study aimed to explore the expression profile of transcription factor Runt-related transcription factor 2 (RUNX2)in the synovial tissues of rheumatoid arthritis (RA) and its effect on the apoptosis of RA fibroblast-like synovial cells (RA-FLS),and to screen new targets for the diagnosis,treatment and prognosis of RA.Methods:The expression and localization of mRNA and protein of RUNX2 in the synovial tissues were detected by real-time quantitative PCR (qPCR)and immunohistochemical staining,respectively.The effect of the expression of exogenous RUNX2 on the apoptosis process of RA-FLS cell line MH7A was investigated by flow cytometry,and the activation of NF-κB signaling pathway was detected by western blotting.Results:The expression of RUNX2 mRNA was significantly higher in the synovial tissues from RA patients,compared to that in the OA patients (P＜0.05).RUNX2 protein was mainly localized in the nuclear of the RA synovial cells.Overexpression of exogenous RUNX2 could notably decrease the apoptosis of RA-FLS,which was substantially reversed by pretreatment with SN50,a specific inhibitor of NF-κB pathway.Compared with blank group and negative control group (pCMV6-AC-GFP-vector),the apoptotic rate of exogenous RUNX2 overexpressing (pCMV6-AC-GFP-RUNX2) MH7A cells was significantly decreased (P＜0.05).NF-κB signaling pathway activity was significantly increased (P＜0.05),and this effect could be effectively reversed by NF-κB signal pathway-specific inhibitor SN5.Conclusion:The high expression of UNX2 in RA synoviocytes could significantly inhibit the spontaneous apoptosis of cells,and it was related to the abnormal activation of NF-κB signaling pathway.In-depth studies of RUNX2/NF-κB signaling pathways will help to identify novel therapeutic targets for RA.

Apoptosis of Macrophages Induced by Human Papollimavirus Type 18 E2 Protein

Objective To investigate the effect of over-expression of E2 protein GFP-E2,GFP-TAD(N-extremity domain of HPV18 E2)and GFP-DBD(C-extremity domain of HPV18 E2)fusion proteins on the apoptosis of macmphages in uterine cervix cancer was studied.Methods TAD or DBD gene was amplified by PCR from pEGFP-C1/HPV18 E2,and cloned into pEGFP-C1 vector.Then the subclone of pEGFP-C1/TAD or pEGFP-C1/DBD was screened respectively.Forty-elght hours after transfection of pEGFP-C1/HPV18 E2,pEGFP-C1/TAD,pEGFP-C1/DBD or pEGFP-C1 into macrophages,the localization or expression of them was analyzed by fluorescent micmscope or western blot,respectively.The apoptotic rate of macrophages was detected by flow cytometry.Results The plasmid of pEGFP-C1/TAD or pEGFP-C1/DBD was constructed respectively.Macmphages transfected with different plasmid,over-expression of GFP-E2 or GFP-TAD fusion protein up-regulated apoptotic rate of macrophages.Furthermore,the effect of GFP-TAD was more powerful than GFP-E2.But the GFP-DBD over-expressed had not the same effects.Conclusion Over-expression of GFP-E2 or GFPTAD fusion protein could induce apoptosis of macrophages.

FOXO1真核表达载体构建及其对TNF-α介导的A549细胞凋亡的影响

目的 构建FOXO1真核表达质粒,明确FOXO1对TNF-α介导的Ⅱ型肺泡上皮细胞凋亡的影响及其可能存在的调控机制.方法 根据GenBank中人FOXO1CDS序列设计并合成引物,提取A549细胞总RNA,通过RT-PCR获得FOXO1目的基因片段;通过酶切、连接,构建GV230-FOXO1真核表达质粒并进行测序分析鉴定;经鉴定后的表达载体瞬时转染入A549细胞,荧光显微镜及Western blot法检验FOXO1蛋白表达.体外培养A549细胞,利用脂质体LipofectamineTM 2000将本次合成的GV230-FOX01质粒转染入A549细胞,给予10 ng/ml TNF-α刺激24 h,流式细胞术检测细胞凋亡率,Western blot检测凋亡相关蛋白Bim的表达.结果 成功构建GV230-FOXO1荧光真核表达质粒;FOXO1组细胞凋亡率明显高于TNF-α组和阴性对照组(P＜0.05),FOX01组蛋白Bim的表达明显高于TNF-α组和阴性对照组(P＜0.05).结论 FOXO1在TNF-α介导的A549细胞损伤中起促进细胞凋亡的作用,其作用机制可能为通过上调促凋亡蛋白Bim的表达,从而促进细胞凋亡.

Botulinum toxin A (BTXA) has been used in several clinical trials to treat excessive glandular secretion;however, the precise mechanism of its action on the secretory function of salivary gland has not been fully elucidated. In this study, we aimed to investigate the effect of BTXA on secretion of submandibular gland in rabbits and to identify its mechanism of action on the secretory function of salivary gland. At 12 weeks after injection with 5 units of BTXA, we found a significant decrease in the saliva flow from submandibular glands, while the salivary amylase concentration increased. Morphological analysis revealed reduction in the size of acinar cells with intracellular accumulation of secretory granules that coalesced to form a large ovoid structure. Expression of M3-muscarinic acetylcholine receptor (M3 receptor) and aquaporin-5 (AQP5) mRNA decreased after BTXA treatment, and distribution of AQP5 in the apical membrane was reduced at 1, 2 and 4 weeks after BTXA injection. Furthermore, BTXA injection was found to induce apoptosis of acini. These results indicate that BTXA decreases the fluid secretion of submandibular glands and increases the concentration of amylase in saliva. Decreased expression of M3 receptor and AQP5, inhibition of AQP5 translocation, and cell apoptosis might involve in BTXA-reduced fluid secretion of submandibular glands.

15-deoxy-△12,14-prostaglandin J2对ECV304细胞增殖与凋亡的影响

AIM: To investigate the effects of 15-deoxy-△12,14-prostaglandin J2 (15d-PGJ2) on cell proliferation and apoptosis in ECV304 endothelial cells and related molecular mechanism. METHODS: MTT, Hoechst33258, TUNEL, Flow cytometry, DNA ladder, RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA) analysis were employed. RESULTS: The 15d-PGJ2 induced apoptosis in ECV304 endothelial cells in a dose-dependent manner (the percentage of apoptosis was enhanced from 10.0 %+1.3 % to 32.8 %+1.6%), which was accompanied by inhibition of NF-κB and AP-1 DNA binding activity, down-regulation of c-myc, upregulation of Gadd45 and p53,and activation of p38 kinase. However, the expression of p21 was found no significant change. CONCLUSION:peroxisome proliferator-activated receptor gamma ligand, 15d-PGJ2, can inhibit proliferation and induce apoptosis in ECV304 endothelial cells through different mechanisms.

野生型p53增强药物耐药的人肝癌细胞Bel7402/5-FU的化疗敏感性

AIM: To study the effect of wild type (wt) p53 gene transfection on drug resistant human hepatocellular carcinoma (HCC) cells induced by 5-Fluorouracil (5-FU). METHODS: The cytotoxicity of anticancer drugs on Be17402 and Be17402/5-FU cells was assessed using SRB assay. p53 expression was detected at its mRNA level by RT-PCR assay and at its protein level Western blot or immunocytochemistry assay in Be17402/5-FU cells transfected with either control vector or wt p53. AnnexinV-FITC/PI double labeled assay was performed to detect apoptosis. The chemosensitivity of Be17402/5-FU cells transfected with wt p53 was assessed using SRB assay. RESULTS: Be17402/5-FU cells exhibited cross-resistance to vincristine, doxorubicin, paclitaxel, and so on. wt p53 gene transfection upregulated the expression of p53 in Be17402/5-FU cells. wt p53 was able to greatly inhibit cell proliferation and significantly induce apoptosis in Bel7402/5-FU cells. Moreover, wt p53 gene transfection increased the chemosensitivity of Be17402/5-FU cells to some anticancer drugs. CONCLUSION: These results indicated that the wt p53 gene transfection not only induced suppression of cell growth, but also increased the sensitivity of Be17402/5-FU cells to 5-FU, vincristine, and doxorubicin.

GM-CSF,IL-3和GM-CSF/IL-3融合蛋白对辐射致髓样白血病细胞株Tf-1凋亡的影响

AIM: To observe the effects of three cytokines on the apoptosis of Tf- 1 cells induced by γ irradiation and investigate the relationship between apoptosis and caspase-3 activity. METHODS: Different cytokines GM-CSF, IL-3 and GM-CS/IL-3 fusion protein were added into the irradiated Tf-1 cells. MTT assay, morphology, flow cytometry,and DNA fragmentation assay were used to observe the effects of cytokines on apoptosis. The caspase-3 activity was determined with a fluorocytometer. RESULTS: Irradiated Tf-1 cells showed typical morphological characteristic of apoptosis demonstrated by transmission electron microscopy and were accumulated in G0/G1 phase. In the groups treated with growth factors after irradiation, three cytokines significantly increased the viability rate, distinctly decreased the apoptosis rate and the proportion of DNA fragmentation. When Tf- 1 cells were irradiated by γ irradiation, caspase-3 activity was increased at different time points. In comparison with the control group in which no growth factor was added after the cells were irradiated, the caspase-3 activity of irradiated Tf-1 cells was significantly inhibited by addition of the above cytokines. Thirty-six hours after irradiation, in the control group,GM-CSF, IL-3, GM-CSF and IL-3 in combination, and two GM-CSF/IL-3 fusion protein groups, the apoptosis rate was 73 %, 11%, 15 %, 13 %, 12 %, and 13 %. The percent of fragmented DNA was 36 %, 19 %, 18 %, 14 %,13 %, and 14 %. The fluorescence intensity was 16923, 5529, 6581, 5322, 5426, and 5485. CONCLUSION:GM-CSF, IL-3, and GM-CSF/IL-3 fusion protein could protect Tf-1 cells from apoptosis induced by γ irradiation.After Tf-1 cells were irradiated, the caspase-3 activity was significantly increased but was dramatically decreasedby the above cytokines. The remarkable inhibition of caspase-3 activity may be one of the mechanisms of thesehematopoietic growth factors exerting their anti-apoptotic effects.

吴茱萸碱通过不同途径诱导肿瘤细胞死亡:凋亡和坏死

AIM: To study the different death pathways in human cervical cancer HeLa and melanoma A375-S2 cells initiated by evodiamine. METHODS: Viability of evodiamine-induced HeLa and A375-S2 cells was measured by MTT assay. Apoptotic cells with condensed or fragmented nuclei were visualized by Hoechst 33258 staining. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Proportion of cell death through apoptotic and necrotic pathways was determined by LDH activity-based cytotoxicity assays. Cell cycle distribution was observed by flow cytometry. RESULTS: Evodiamine induced HeLa and A375-S2 cell death dose- and time-dependently.Caspase-3 and-8 were activated in apoptosis induced by evodiamine 15 μmol/L. However, over 24- h incubation of A375-S2 cells, evodiamine 15 μmol/L initiated necrosis related to p38 and ERK (extracellular signal-regulated kinases)activities. Evodiamine-induced HeLa cell death was preceded by an accumulation of cells at the G2/M phase of the cell cycle, but there was no significant effect of evodiamine on A375-S2 cell cycle. CONCLUSION: Evodiamine induces caspase-3,8-dependent apoptosis in HeLa cells which is related to G2/M arrest of the cell cycle. On the other hand, in A375-S2 cells, evodiamine initiates caspase-3,8-mediated apoptosis at early stages and the induction of MAPK-mediated necrosis at later stages of cell culture.

CMV-hFasL转基因小鼠容易被小剂量的链尿菌素诱导至糖尿病

AIM: To investigate the role of Fas-FasL pathway in the pathogenesis of streptozotocin (STZ)-induced type I diabetes mellitus. METHODS: Low dose injections of STZ were used to induce type I diabetes mellitus in the CMV-hFasL transgenic mice. Blood glucose concentration was measured with Glucotrand Plus blood glucose test strips. Expression of hFasL was detected by RT-PCR and Western blotting. The severity of insulitis was determined by histologicalexamination. Expressions of IL- 1 β and TNF-α mRNA in the pancreas were detected by semi-quantitative RT-PCR analysis. Fas expression in apoptotic RIN-5F cells was also confirmed by RT-PCR in vitro. RESULTS: hFasL was expressed in the islets of CMV-hFasL transgenic mice. The transgenic mice were sensitive to diabetic induction than the control WT mice. IL-1 β and TNF-α expressions in the pancreas of CMV-hFasL transgenic mice were far more than that in WT mice. We also found STZ and IL-1β could both induce higher expression of Fas in RIN-5F. The combining of Fas-FasL could lead to the apoptosis of β cells in the CMV-hFasL transgenic mice. CONCLUSION: Fas-FasL interaction plays a significant role in the pathogenic mechanism of type I diabetes mellitus.

CDK4,pRB和E2F1参与人参皂苷Rg1对淀粉样β蛋白诱导的大鼠皮层神经元凋亡的抑制作用

AIM: To explore the possible mechanism of β-amyloid (Aβ)-induced apoptosis in rat cortical neurons and the protective effect of ginsenoside Rg1. METHODS: AO-EB staining was used to quantify the apoptotic cells. DNA fragmentation was observed by gel electrophoresis. The levels of cyclin-dependent kinases-4 (CDK4) and phosphorylated pRB were detected by Western blot. RT-PCR was used to examine the expression of E2F1 mRNA. RESULTS: Treatment with A1-40 at the concentration of 20, 40, 80 mg/L for 48 h induced rat cortical neuron apoptosis from 12.5 %+1.5 % (control) to 22.3 %+1.4 %, 38.8 %+1.3 %, 36.7 %+1.4 %, respectively. Pretreat ment with Rg1 at the dose of 0.5, 1, 2, 4, 8, 16 μmol/L for 24 h, then treatment with A~-40 40 mg/L for 24 h, the percentage of apoptotic neurons decreased from 38.8 %+1.3 % to 14.5 %+1.3 %, 13.3 %+1.0 %, 11.6 %+0.29 %, 11.8 %+ 1.0 %, 6.2 %+0.8 %, 5.8 %+0.8 %, respectively. After treatment with Aβ1-40 40 mg/L for 24 h, there were transient increases in CDK4 and phosphorylated pRB protein level, as well as the expression of E2F1 mRNA. However, the above levels decreased markedly after pretreatment with Rg1 8 μmol/L for 24 h. CONCLUSION: Ginsenoside Rg1 attenuated Aβ1-40-induced apoptosis in rat cortical neurons via inhibiting the activity of CDK4, decreasing the phosphorylation of pRB and downregulating the expression of E2F1 mRNA.

吗多明和N-硝基-L-精氨酸甲基酯抑制小鼠胚胎着床以及子宫内膜的细胞调亡

AIM: To investigate the possible effect of nitric oxide on receptivity and apoptosis of mouse endometrium and the possible pathway. METHODS: Female pregnant mice were treated with either molsidomine, a generator of nitric oxide (NO), or N-omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. The pregnancy rates of each group were calculated; 3'-end-labeling was used to detect DNA fragmention of apoptotic cells; immunohistochemistry, in situ hybridization, and Western blot were applied respectively to estimate expression levels of Fas/FasL proteins and mRNA. RESULTS: The pregnancy rate in the drug treated group was reduced in a dose-dependent manner; apoptosis, Fas protein and mRNA levels in the endometrium of drug treated mice were correlatively decreased during the peri-implantation period. CONCLUSION: The decreased pregnant rate in mice by abnormal levels of nitric oxide may be brought about by inhibiting the normally occurrence of apoptosis in the receptive endometrium.

下调survivin表达对HL-60耐多柔比星细胞株耐药性的逆转作用

AIM: To investigate the ability of an antisense RNA eukaryotic expression plasmid pcDNA3.1/survivin in downregulating the expression level of survivin mRNA and survivin protein and reversed multidrug resistance (MDR) in adriamycin-resistant HL-60/ADR cell line. METHODS: The expression of survivin mRNA was measured by RTPCR and the expression of survivin protein was measured by Western blot. Caspase-3 activity was determined by Phar Mingen colorimetric assay kit. Apoptosis was assessed by flow cytometry. The chemosensitivity of HL-60/ ADR ceils to adriamycin (ADR) was measured by MTT assay. RESULTS: pcDNA3.1/survivin down-regulated the expression level of survivin mRNA and survivin protein obviously, and induced apoptosis of HL-60/ADR cells in a time-dependent manner during 12-48 h. After transient transfection with pcDNA3.1/survivin for 48 h, survivin mRNA decreased by 67 %, survivin protein decreased by 57 %, caspase-3 activity increased 4.37 times, and the apoptosis rate increased by 4.41% compared with control. Compared with ADR alone, pcDNA3.1/survivin significantly reversed MDR in HL-60/ADR cells, the chemosensitivity of HL-60/ADR cells to ADR was increased to 5.36folds. CONCLUSION: pcDNA3.1/survivin down-regulated the expression level of survivin mRNA and survivin protein obviously, the threshold of apoptosis was decreased and MDR was reversed.