Meloidogyne graminicola has emerged as one of the most destructive plant-parasitic nematodes affecting rice(Oryza sativa)production worldwide.Resistance to M.graminicola in rice could be the most effective option for ...Meloidogyne graminicola has emerged as one of the most destructive plant-parasitic nematodes affecting rice(Oryza sativa)production worldwide.Resistance to M.graminicola in rice could be the most effective option for its management.However,sources of germplasm with resistance to M.graminicola in rice remain limited.Here,we describe the root attraction,gall formation and genetic analysis of the resistance to M.graminicola in the rice variety Huidao 5.A nematode attraction assay showed that second-stage juveniles(J2s)of M.graminicola were attracted at the root tip of Huaidao 5 within 8 h without a significant reduction in attraction compared to the susceptible rice variety Nanjing 9108.Microscopic observation of the infection revealed that the J2s invaded root tissues 12 h after inoculation,but their subsequent movement to the root tip was hindered in Huaidao 5,resulting in decreased nematode number compared to Nanjing 9108.Additionally,we used the soil and hydroponic culture systems to simulate upland and flooding conditions in the paddy fields respectively,and found that gall number was significantly reduced,and nematode development was clearly suppressed in Huaidao 5.To investigate the genetic basis of this resistance,cross breeding was performed between the Huaidao 5 and Nanjing 9108 varieties.There was no reduction in the resistance of the F_(1) offspring to M.graminicola in the greenhouse or field trials,suggesting that a dominant gene could control resistance in Huaidao 5.In summary,this study provides a detailed characterization of a novel source of resistance to M.graminicola in rice,which is of great potential for use in crop breeding.展开更多
The effect ofAphelenchoides besseyi on 27 cultivars of rice （23japonica and 4 indica） was assessed in the field for two seasons during 2010 and 2011. The vigorous pathogenic nematodes culturing on Botrytis cinerea w...The effect ofAphelenchoides besseyi on 27 cultivars of rice （23japonica and 4 indica） was assessed in the field for two seasons during 2010 and 2011. The vigorous pathogenic nematodes culturing on Botrytis cinerea were used for this experiment. Inoculation was carried out at the tilling stage; the growth parameters and nematode population were recorded at the end of growth of rice plants. The results showed that the cultivars differed in their response to infection. Most of cultivars were lack of the characteristic symptom of white tip, which was seen less frequently than the other two symptoms, namely small grains and erect panicles; moreover, the expression of symptoms was probably hereditary. The infection lowered the values of all the measured biological parameters, namely length of the stem and of the panicle, the number of filled grains per panicle, and 100-grain weight, in all the cultivars. The final nematode population indicated that the threshold of economic damage had also been exceeded in 10 cultivars, and none of them was immune. Three japonica cultivars proved most vulnerable whereas Tetep, an indica type, showed a level of resistance potentially useful in controlling A. besseyi.展开更多
ObjectiveTo explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it’s down-stream mediators in colorectal cancer (CRC) cells.MethodsQuantitative polymerase chain reaction was ...ObjectiveTo explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it’s down-stream mediators in colorectal cancer (CRC) cells.MethodsQuantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues.ResultsPZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05).ConclusionThe mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21.展开更多
水稻干尖线虫是水稻上重要的寄生线虫,严重危害水稻安全生产。14-3-3蛋白调控生物体的细胞代谢、生长发育和逆境适应等一系列生物过程。本研究通过c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)分离获得了一个水稻干...水稻干尖线虫是水稻上重要的寄生线虫,严重危害水稻安全生产。14-3-3蛋白调控生物体的细胞代谢、生长发育和逆境适应等一系列生物过程。本研究通过c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)分离获得了一个水稻干尖线虫(Aphelenchoides besseyi)14-3-3基因,命名为Ab-14-3-3-a。Ab-14-3-3-a全长c DNA序列含有59 bp的5'非翻译区(UTR)、编码251个氨基酸756 bp的开放阅读框。Ab-14-3-3-a DNA序列包含4个外显子和3个内含子。Ab-14-3-3-a蛋白与其他植物寄生线虫14-3-3蛋白氨基酸序列高度相似,系统发育树显示与植物寄生线虫位于同一进化分支。原位杂交显示Ab-14-3-3-a特异定位于成虫的背食道腺细胞中,表明其在线虫取食和寄生过程中发挥潜在功能。qRT-PCR显示,Ab-14-3-3-a在水稻干尖线虫各个龄期均表达,其中在成虫表达水平最高,而在发育过程中幼虫表达水平最低。当线虫取食不同寄主时,Ab-14-3-3-a的表达水平具有差异性,线虫取食感病水稻早期,Ab-14-3-3-a表达水平上升1.5倍,而取食灰葡萄孢(Botrytis cinerea)时Ab-14-3-3-a表达可提高数百倍。利用体外RNA干扰技术将Ab-14-3-3-a的表达进行有效抑制,结果显著影响了线虫的产卵、成虫发育以及线虫种群数量。综上,Ab-14-3-3-a参与调控水稻干尖线虫的取食行为和发育进程。本研究相关结果有助于拓展植物寄生线虫14-3-3蛋白功能,为深入研究线虫与植物互作机制提供了理论依据。展开更多
It is well-known that risk for endometrial adenocarcinoma increases in patients with high level ofestrogen that is unopposed by progestin. And activation of extracellular signal-regulated kinase （ERK） and phosphatid...It is well-known that risk for endometrial adenocarcinoma increases in patients with high level ofestrogen that is unopposed by progestin. And activation of extracellular signal-regulated kinase （ERK） and phosphatidylinositol 3 kinase/protein kinase B （PI3K/PKB） pathway are responsible for hormone-dependent cell growth in endometrial carcinoma. PI3K produces phosphatidylinositol- 3-phosphates by phosphory-lating the D3 hydroxyl of phosphoinositides, leading to membrane translocation of PKB,展开更多
Background Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated...Background Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium （[Ca2±]c） was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-IA cells. Methods Cells were cultured with nifedipine （10 μmol/L） and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine （3-MA） （2.5 mmol/L） for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alphalD （Cavl.3） protein was detected. At last, cells were cultured and assigned to four groups with different treatment： untreated （control group）, 10 μmol/L nifedipine （N group）, 2.5 mmol/L 3-MA （3-MA group）, and 10 μmol/L nifedipine plus 2.5 mmol/L 3-MA （N±3MA group）. Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins （LC3, Beclinl and P70s6K） by Western blotting and monodansylcadaverine （MDC） labeled visualization. Results Proliferation of Hec-lA cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours （P=0.000 for each day）. The suppression of migration ability of the nifedipine-treated cells （94.0±8.2） was significantly different from that of the untreated cells （160.00±9.50, P=0.021 ）. The level of early period cell apoptosis induced by nifedipine was （2.21_±0.19）%, which was （2.90±0.13）% in control group （P=-0.052）, whereas the late period apoptosis level reached （10.38_±0.96）% and （4.40_±0.60）% （P=0.020）, respectively. The 3-MA group induced a slight increase in the Cavl.3 levels within 15 minutes, but significantly attenuated the Cavl.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group （20.63_±3.36） than the control group （6.29_±0.16, P=-0.015）. GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N±3MA group, 3-MA group were 2.80_±0.29, 2.30_±0.17, and 1.80±0.21, respectively. Cells in the N group showed significant augmentation of autophagy （P 〈0.05）. Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group （0.85±0.21） and N±3MA group （1.21±0.12） compared with nifedipine treatment （2.64±0.15, P 〈0.05）. The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N＋3-MA group （（11.22±0.91）%） differed significantly from that of the control group （（2.51±0.70）%） and N group （（3.47±0.39）%）. Similarly, the late period level of the N＋3-MA group （（55.19±2.51）%） differed significantly from that of the control group（（15.81±1.36）%） and the N group （（22.09±2.48）%, P 〈0.05）. The down-regulated expression of P70s6k and up-regulated expression of the Beclinl revealed significant differences between the N＋3-MA group and control group （P=0.025; Beclinl： P=-0.015）. Conclusions Proliferation and migration in vitro of endometrial carcinoma Hec-lA cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-lA cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cavl.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclinl and mTOR pathways.展开更多
Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, a...Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A （TSA） to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells. Methods Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase （DNMT）I, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA （SiRNAi） was used to knock down DNMT3B. Results HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 Iumol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, （90.0＋8.4）% of normal cell survive after treated with 1 IJmol/L of TSA. We compared 1 pmol/L group with untreated control with t-test. There was no significance between 1 pmol/L group and untreated control for normal cell （P 〉0.05）. HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B. Conclusions DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.展开更多
Total or near-total fertilization failure after intracytoplasmic sperm injection （ICSl） is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnan...Total or near-total fertilization failure after intracytoplasmic sperm injection （ICSl） is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnancy and live birth after calcium ionophore A23187 （A23187） activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia, and who had experienced repeated near-total fertilization failure after ICSI. In the second ICSI cycle, only one oocyte was fertilized while nine were unfertilized. Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer. A clinical pregnancy was achieved and a healthy baby was born. To our knowledge, this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte. This indicates that ＂rescue oocyte activation＂ on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.展开更多
Background Phenotypic and genotypic heterogeneity is a known feature of many cancers.Whether serum tumor marker kinds vary and change following chemotherapy is still unclear.The aim of this study was to investigate wh...Background Phenotypic and genotypic heterogeneity is a known feature of many cancers.Whether serum tumor marker kinds vary and change following chemotherapy is still unclear.The aim of this study was to investigate whether there is a change in the expression of serum tumor markers following chemotherapy,and the potential clinical significance in patients with epithelial ovarian carcinoma （EOC） or primary serous peritoneal carcinoma （PSPC）.Methods Samples were collected before surgery,during chemotherapy and during follow-up for enzyme-linked immunosorbent assay （ELISA）-based evaluation of serum CA-125,CA19-9 and CP2 levels in patients with EOC or PSPC who had received primary debulking surgery followed by adjuvant chemotherapy.In total,72 patients were examined,including 37 patients with recurrent lesions and 35 patients receiving first-line chemotherapy.Results In 35 de novo patients,20% （7/35） demonstrated a significant changed serum tumor marker kinds among whom the patients with mucinous carcinoma （57.1%,4/7） showed resistance to chemotherapy.In the 37 recurrent patients,51.4% （19/37） had changed serum tumor markers,of whom 57.9% （11/19） presented with serous carcinoma.There was no significant difference in median progression-free survival or overall survival in patients with drug-sensitive or drug-resistant recurrence in patients with changed tumor marker kinds relative to those with unchanged markers.However,for patients with changed serum tumor markers there was a trend towards prolonged survival compared with the unchanged serum tumor marker group.In the 17 patients with secondary recurrence,37.5% （6/17） had changed tumor marker levels.The ratios of CA-125/CP2 and CA-125/CA19-9 were significantly different after either chemotherapy or recurrence.Conclusions Serum tumor marker expression in patients with EOC or PSPC may change after chemotherapy or recurrence,indicating that in addition to the markers that are abnormal before surgery,those markers that are normalshould also be monitored during chemotherapy and follow-up.展开更多
基金the National Natural Science Foundation of China(31871943)the Jiangsu Agricultural Science and Technology Innovation Project,China(CX(21)1011)the General Program of Hebei Natural Science Foundation,China(C2019402344)。
文摘Meloidogyne graminicola has emerged as one of the most destructive plant-parasitic nematodes affecting rice(Oryza sativa)production worldwide.Resistance to M.graminicola in rice could be the most effective option for its management.However,sources of germplasm with resistance to M.graminicola in rice remain limited.Here,we describe the root attraction,gall formation and genetic analysis of the resistance to M.graminicola in the rice variety Huidao 5.A nematode attraction assay showed that second-stage juveniles(J2s)of M.graminicola were attracted at the root tip of Huaidao 5 within 8 h without a significant reduction in attraction compared to the susceptible rice variety Nanjing 9108.Microscopic observation of the infection revealed that the J2s invaded root tissues 12 h after inoculation,but their subsequent movement to the root tip was hindered in Huaidao 5,resulting in decreased nematode number compared to Nanjing 9108.Additionally,we used the soil and hydroponic culture systems to simulate upland and flooding conditions in the paddy fields respectively,and found that gall number was significantly reduced,and nematode development was clearly suppressed in Huaidao 5.To investigate the genetic basis of this resistance,cross breeding was performed between the Huaidao 5 and Nanjing 9108 varieties.There was no reduction in the resistance of the F_(1) offspring to M.graminicola in the greenhouse or field trials,suggesting that a dominant gene could control resistance in Huaidao 5.In summary,this study provides a detailed characterization of a novel source of resistance to M.graminicola in rice,which is of great potential for use in crop breeding.
基金supported by the Jiangsu Agriculture Science and Technology Innovation Project,China(CX(10)206)
文摘The effect ofAphelenchoides besseyi on 27 cultivars of rice （23japonica and 4 indica） was assessed in the field for two seasons during 2010 and 2011. The vigorous pathogenic nematodes culturing on Botrytis cinerea were used for this experiment. Inoculation was carried out at the tilling stage; the growth parameters and nematode population were recorded at the end of growth of rice plants. The results showed that the cultivars differed in their response to infection. Most of cultivars were lack of the characteristic symptom of white tip, which was seen less frequently than the other two symptoms, namely small grains and erect panicles; moreover, the expression of symptoms was probably hereditary. The infection lowered the values of all the measured biological parameters, namely length of the stem and of the panicle, the number of filled grains per panicle, and 100-grain weight, in all the cultivars. The final nematode population indicated that the threshold of economic damage had also been exceeded in 10 cultivars, and none of them was immune. Three japonica cultivars proved most vulnerable whereas Tetep, an indica type, showed a level of resistance potentially useful in controlling A. besseyi.
基金Supported by National Natural Science Foundation of China(Nos.81803882,82274188 and 82274148)Natural Science Foundation of Fujian Province(No.2020J06026)。
文摘ObjectiveTo explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it’s down-stream mediators in colorectal cancer (CRC) cells.MethodsQuantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues.ResultsPZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05).ConclusionThe mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21.
文摘水稻干尖线虫是水稻上重要的寄生线虫,严重危害水稻安全生产。14-3-3蛋白调控生物体的细胞代谢、生长发育和逆境适应等一系列生物过程。本研究通过c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)分离获得了一个水稻干尖线虫(Aphelenchoides besseyi)14-3-3基因,命名为Ab-14-3-3-a。Ab-14-3-3-a全长c DNA序列含有59 bp的5'非翻译区(UTR)、编码251个氨基酸756 bp的开放阅读框。Ab-14-3-3-a DNA序列包含4个外显子和3个内含子。Ab-14-3-3-a蛋白与其他植物寄生线虫14-3-3蛋白氨基酸序列高度相似,系统发育树显示与植物寄生线虫位于同一进化分支。原位杂交显示Ab-14-3-3-a特异定位于成虫的背食道腺细胞中,表明其在线虫取食和寄生过程中发挥潜在功能。qRT-PCR显示,Ab-14-3-3-a在水稻干尖线虫各个龄期均表达,其中在成虫表达水平最高,而在发育过程中幼虫表达水平最低。当线虫取食不同寄主时,Ab-14-3-3-a的表达水平具有差异性,线虫取食感病水稻早期,Ab-14-3-3-a表达水平上升1.5倍,而取食灰葡萄孢(Botrytis cinerea)时Ab-14-3-3-a表达可提高数百倍。利用体外RNA干扰技术将Ab-14-3-3-a的表达进行有效抑制,结果显著影响了线虫的产卵、成虫发育以及线虫种群数量。综上,Ab-14-3-3-a参与调控水稻干尖线虫的取食行为和发育进程。本研究相关结果有助于拓展植物寄生线虫14-3-3蛋白功能,为深入研究线虫与植物互作机制提供了理论依据。
基金This study was partially supported by a grant from the Scientific Research Fund for Capital Medicine Development (No.ZD 199911).
文摘It is well-known that risk for endometrial adenocarcinoma increases in patients with high level ofestrogen that is unopposed by progestin. And activation of extracellular signal-regulated kinase （ERK） and phosphatidylinositol 3 kinase/protein kinase B （PI3K/PKB） pathway are responsible for hormone-dependent cell growth in endometrial carcinoma. PI3K produces phosphatidylinositol- 3-phosphates by phosphory-lating the D3 hydroxyl of phosphoinositides, leading to membrane translocation of PKB,
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30973182）.
文摘Background Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium （[Ca2±]c） was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-IA cells. Methods Cells were cultured with nifedipine （10 μmol/L） and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine （3-MA） （2.5 mmol/L） for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alphalD （Cavl.3） protein was detected. At last, cells were cultured and assigned to four groups with different treatment： untreated （control group）, 10 μmol/L nifedipine （N group）, 2.5 mmol/L 3-MA （3-MA group）, and 10 μmol/L nifedipine plus 2.5 mmol/L 3-MA （N±3MA group）. Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins （LC3, Beclinl and P70s6K） by Western blotting and monodansylcadaverine （MDC） labeled visualization. Results Proliferation of Hec-lA cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours （P=0.000 for each day）. The suppression of migration ability of the nifedipine-treated cells （94.0±8.2） was significantly different from that of the untreated cells （160.00±9.50, P=0.021 ）. The level of early period cell apoptosis induced by nifedipine was （2.21_±0.19）%, which was （2.90±0.13）% in control group （P=-0.052）, whereas the late period apoptosis level reached （10.38_±0.96）% and （4.40_±0.60）% （P=0.020）, respectively. The 3-MA group induced a slight increase in the Cavl.3 levels within 15 minutes, but significantly attenuated the Cavl.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group （20.63_±3.36） than the control group （6.29_±0.16, P=-0.015）. GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N±3MA group, 3-MA group were 2.80_±0.29, 2.30_±0.17, and 1.80±0.21, respectively. Cells in the N group showed significant augmentation of autophagy （P 〈0.05）. Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group （0.85±0.21） and N±3MA group （1.21±0.12） compared with nifedipine treatment （2.64±0.15, P 〈0.05）. The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N＋3-MA group （（11.22±0.91）%） differed significantly from that of the control group （（2.51±0.70）%） and N group （（3.47±0.39）%）. Similarly, the late period level of the N＋3-MA group （（55.19±2.51）%） differed significantly from that of the control group（（15.81±1.36）%） and the N group （（22.09±2.48）%, P 〈0.05）. The down-regulated expression of P70s6k and up-regulated expression of the Beclinl revealed significant differences between the N＋3-MA group and control group （P=0.025; Beclinl： P=-0.015）. Conclusions Proliferation and migration in vitro of endometrial carcinoma Hec-lA cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-lA cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cavl.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclinl and mTOR pathways.
基金This article was supported by the grants from the National Natural Science Foundation of China，the “985” Project of the Peking University Health Science Center and the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry
文摘Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A （TSA） to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells. Methods Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase （DNMT）I, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA （SiRNAi） was used to knock down DNMT3B. Results HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 Iumol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, （90.0＋8.4）% of normal cell survive after treated with 1 IJmol/L of TSA. We compared 1 pmol/L group with untreated control with t-test. There was no significance between 1 pmol/L group and untreated control for normal cell （P 〉0.05）. HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B. Conclusions DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.
文摘Total or near-total fertilization failure after intracytoplasmic sperm injection （ICSl） is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnancy and live birth after calcium ionophore A23187 （A23187） activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia, and who had experienced repeated near-total fertilization failure after ICSI. In the second ICSI cycle, only one oocyte was fertilized while nine were unfertilized. Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer. A clinical pregnancy was achieved and a healthy baby was born. To our knowledge, this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte. This indicates that ＂rescue oocyte activation＂ on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.
文摘Background Phenotypic and genotypic heterogeneity is a known feature of many cancers.Whether serum tumor marker kinds vary and change following chemotherapy is still unclear.The aim of this study was to investigate whether there is a change in the expression of serum tumor markers following chemotherapy,and the potential clinical significance in patients with epithelial ovarian carcinoma （EOC） or primary serous peritoneal carcinoma （PSPC）.Methods Samples were collected before surgery,during chemotherapy and during follow-up for enzyme-linked immunosorbent assay （ELISA）-based evaluation of serum CA-125,CA19-9 and CP2 levels in patients with EOC or PSPC who had received primary debulking surgery followed by adjuvant chemotherapy.In total,72 patients were examined,including 37 patients with recurrent lesions and 35 patients receiving first-line chemotherapy.Results In 35 de novo patients,20% （7/35） demonstrated a significant changed serum tumor marker kinds among whom the patients with mucinous carcinoma （57.1%,4/7） showed resistance to chemotherapy.In the 37 recurrent patients,51.4% （19/37） had changed serum tumor markers,of whom 57.9% （11/19） presented with serous carcinoma.There was no significant difference in median progression-free survival or overall survival in patients with drug-sensitive or drug-resistant recurrence in patients with changed tumor marker kinds relative to those with unchanged markers.However,for patients with changed serum tumor markers there was a trend towards prolonged survival compared with the unchanged serum tumor marker group.In the 17 patients with secondary recurrence,37.5% （6/17） had changed tumor marker levels.The ratios of CA-125/CP2 and CA-125/CA19-9 were significantly different after either chemotherapy or recurrence.Conclusions Serum tumor marker expression in patients with EOC or PSPC may change after chemotherapy or recurrence,indicating that in addition to the markers that are abnormal before surgery,those markers that are normalshould also be monitored during chemotherapy and follow-up.
