The role of mitochondria,cytochrome c and caspase-9 in embryonic lens fibre cell denucleation

During the differentiation of secondary lens fibre cells from the lens epithelium, the fibre cells lose all of their cytoplasmic organelles as well as their nuclei. The fibre cells, containing crystallins, which confer optical clarity, then persist in the adult lens. The process of denucleation of these cells has been likened to an apoptotic event which is not followed by the plasma membrane changes that are characteristic of apoptosis. We have examined the expression and subcellular translocation of molecules of the apoptotic cascade in differentiating lens epithelial cells in culture. In this culture system, the epithelial cells differentiate into lentoids composed of lens fibre cells. We find that caspase-9, which is expressed and activated before embryonic day 12 in intact lenses, is localized in the cytosol outside mitochondria in non-differentiating cultured cells. In lentoid cells, caspase-9 migrates into mitochondria after the latter undergo a membrane permeability transition that is characteristic of apoptotic cells. At the same time, caspase-9 co-localizes with cytochrome c in the cytosol. The cytochrome c is apparently released from the mitochondria in lentoid cells after the mitochondrial membrane permeability transition and during the period of nuclear shrinkage. Also during this time, the mitochondria aggregate around the degenerating nuclei. Cytochrome c disappears rapidly, while mitochondrial breakdown occurs approximately coincident with the disappearance of the nuclei, but mitochondrial remnants persist together with cytochrome c oxidase, which is a mitochondrial marker protein. Apaf-1, another cytosolic protein of the apoptotic cascade, also migrates to the permeabilized mitochondria and also co-localizes with caspase-9 and cytochrome c in the cytosol or mitochondria of denucleating cells, thus providing evidence for the formation of an 'apoptosome' in these cells, as in apoptotic cells. At no time did we observe the translocation of molecules between cytoplasmic compartments and the nucleus in differentiating lentoid cells. We suggest that the uncoupling of nuclear and membrane apoptotic events in these cells may be due to the early permeability changes in the mitochondria, resulting in the loss of mitochondrial signalling molecules, or to the failure of molecules to migrate to the nucleus in these cells, thus failing to activate nuclear-plasma membrane signalling pathways.     

miR-486-5p在氧化应激引起人骨髓间充质干细胞凋亡中的作用

目的:观察microRNA-486-5p(miR-486-5p)在氧化应激引起人骨髓间充质干细胞(h MSCs)凋亡中的作用并探讨其作用机制。方法:h MSCs经培养鉴定后分为5组:空白对照组、H2O2组、miR-486-5p模拟物+H2O2组、抑制物(αnti-miR)+H2O2组及相应的阴性对照(scrambled control)+H2O2组。荧光定量PCR(real-time PCR)检测氧化应激诱导h MSCs凋亡过程中miR-486-5p的表达变化。用脂质体分别转染miR-486-5p的模拟物、抑制物及阴性对照到h MSCs。应用MTT、Hoechst标记和流式细胞术的方法检测miR-486-5p对氧化应激介导细胞活性下降及凋亡效应的影响,Western blotting检测凋亡相关蛋白、Akt与其磷酸化水平,采用试剂盒测定caspase-3活性。结果:H2O2诱导h MSCs凋亡过程中miR-486-5p的表达较对照组显著下降(P0.05)。与阴性对照组相比,在h MSCs中过表达miR-486-5p,能使细胞在氧化应激情况下活性显著下降,凋亡发生率增高,蛋白Bcl-2/Bax比值、caspase-3酶原含量及Akt磷酸化水平降低,caspase-3活性增强;而使用抑制物阻遏miR-486-5p的作用后,细胞在氧化应激条件下活性增加,凋亡发生率降低,蛋白Bcl-2/Bax比值及Akt磷酸化水平升高,caspase-3活性下降。结论:过表达miR-486-5p促进氧化应激引起的h MSCs凋亡,阻遏miR-486-5p的作用抑制氧化应激条件下的h MSCs凋亡,其中作用机制可能与调控Akt通路有关。     

低剂量米非司酮对人卵巢黄素化颗粒细胞凋亡的影响

目的：观察不同低剂量米非司酮对人卵巢黄素化颗粒细胞凋亡的影响，确定米非司酮作为口服避孕药的理论基础。方法:体外培养颗粒细胞。利用免疫荧光、脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记 (TUNEL)及流式细胞技术检测不同低剂量米非司酮处理下颗粒细胞的凋亡形态、凋亡率及caspase-3荧光强度。结果:免疫荧光检测显示处理组中颗粒细胞呈现染色质浓缩，边缘化的凋亡形态。TUNEL表明，米非司酮处理各组均与对照组差异显著，P<0.01；米非司酮处理各组之间两两比较，除1.25 μmol/L与2.50 μmol/L外，均有显著差异，P<0.01。流式细胞检测发现，米非司酮各处理组中caspase-3的荧光强度与对照组均有显著差异（P<0.01），各处理组之间比较与TUNEL结果相一致。结论:低剂量米非司酮能诱导颗粒细胞凋亡，1.25 μmol/L浓度的米非司酮也能诱导颗粒细胞凋亡。米非司酮可能通过影响caspase-3的活性变化来调节颗粒细胞的凋亡。     

Age-dependent changes in the expression of superoxide dismutases and catalase are associated with ultrastructural modifications in human granulosa cells

Limited knowledge exists about changes in follicle quality associated with age. The aim of this work was to investigate whether ageing may cause oxidative stress-mediated alterations in human granulosa cells (GCs) from periovulatory follicles. GCs employed in this study were obtained from follicular aspirates of 20 younger women (range 27-32 years) and 20 older women (range 38-41 years) undergoing an IVF treatment. Results obtained from comparative RT-PCR analysis revealed that the mean relative levels of mRNAs coding for superoxide dismutases, Cu, ZnSOD (SOD1), MnSOD (SOD2) and catalase were significantly decreased in women > or =38 years (P < 0.05, Student's t-test). These changes were associated with a reduced expression of SOD1, SOD2 and catalase at the protein level. When examined at an ultrastructural level, most of the GCs from this group showed defective mitochondria and fewer lipid droplets than those observed in the younger group. These results indicate that GCs from older patients suffer from age-dependent oxidative stress injury and are taken as an evidence for reduced defence against reactive oxygen species (ROS) in GCs during reproductive ageing.     

角质细胞生长因子及其受体在人卵泡膜细胞和颗粒细胞的分布

目的　探讨角质细胞生长因子 (KGF)及其受体 (KGFR)在人卵泡膜细胞和颗粒细胞的表达。方法　采用原位杂交和免疫组化方法分别观察KGF、KGFR在人卵泡膜细胞和颗粒细胞的分布。结果　KGFmRNA存在于正常人卵泡膜细胞和颗粒细胞中 ,KGFmRNA的表达为卵泡膜细胞高于颗粒细胞 (P <0 .0 5 ) ,KGFR的表达为颗粒细胞高于卵泡膜细胞 (P<0 .0 5 ) 。结论　人卵泡膜细胞分布有KGF ,颗粒细胞分布有KGFR ,两者可能存在旁分泌的调节作用。     

Pro-caspase-3 is constitutively expressed in luteinized granulosa cells from women undergoing controlled ovarian stimulation for in vitro fertilization

Apoptosis regulation in luteinized granulosa cells (LGC) during assisted reproduction procedures is still controversial. Caspase-3 is a major apoptosis mediator encoded by CASP3 and formed through cleavage of its precursor pro-caspase-3. The aim of this study was to investigate the expression patterns of pro-caspase-3 (mRNA and protein) and cleaved caspase-3 in human LGC. Thirty-five women undergoing controlled ovarian stimulation for in vitro fertilization were prospectively enrolled in the study. LGC were isolated from follicular fluid during oocyte pickup and evaluated by immunocytochemistry for pro-caspase-3 and cleaved caspase-3, and by real-time PCR for CASP3 mRNA expression. We found a positive staining of pro-caspase-3 in 77 % of the LGC (95 % confidence interval [CI] 60%–84%), whereas cleaved caspase-3 was found in only 4% of the cells (95 % CI 3%–6%). The abundance of cells expressing pro-caspase-3 was independent from CASP3 mRNA levels (r = 0.24, p = 0.255) and did not correlate with the amount of cleaved caspase-3 (r = -0.24, p = 0.186). Multivariable logistic regression showed that pro-caspase-3 positivity was not influenced by clinical characteristics such as age, cause or length of infertility, antral follicle count or hormonal drugs used to induce ovulation. These findings suggest that pro-caspase-3 is constitutively expressed in LGC, allowing quick cleavage into active caspase-3 and apoptosis triggering whenever needed in the course of gonadotropin-induced follicular development.     

The regulatory role of FSH in steroid formation by luteinized human granulosa cells in culture

Granulosa cells were aspirated from human pre-ovulatory folliclesfollowing a combined clomiphene-gonadotrophin stimulation inan in-vitro fertilization (IVF) programme. The cells were culturedfor 8 days in medium M199 containing 10% bovine fetal calf serumunder 5% CO₂ in air. Pure human FSH and human LH were addedalone or in combination to the culture in various concentrationsand the progesterone (P) and oestradiol-17 (E₂) levels in themedium were measured every second day by a conventional RIAtechnique. In the presence of FSH or LH the formation of P increased2- to 3-fold with the pronounced effect after 4 to 6 days inculture. Addition of testosterone (T) (3 ? 10^–7 M) tothe culture medium affected neither basal nor gonadotrophinstimulated P formation. In this system, only minute amountsof E₂ were formed and neither FSH nor LH stimulated its formation.When the medium was fortified with T, basal E₂ formation increased50- to 100-fold. FSH further stimulated this conversion significantlyafter 6 and 8 days of culture, while LH had no significant influence.     

Characteristics of populations of granulosa cells from individual follicles in women undergoing 'coasting' during controlled ovarian stimulation (COS) for IVF

BACKGROUND: The aim of this study was to evaluate the functional characteristics of granulosa cell populations of individual follicles of women undergoing controlled ovarian stimulation (COS) for IVF/ICSI in whom gonadotrophin had been withheld ('coasted') for the prevention of OHSS. METHODS: Follicular fluid and granulosa cells were isolated from 224 individual follicles in 41 women who had been coasted and from 257 individual follicles in 50 women who had a 'normal' response to COS. Cells were cultured at 10,000 cells per well, to evaluate progesterone secretion. Follicular fluid was assayed for progesterone and estradiol (E2). RESULTS: No significant differences were observed between the two groups with respect to granulosa cell number or follicular fluid progesterone and E2 and follicle size, the retrieval of an oocyte and the subsequent fertilization of the oocyte. However, the granulosa cells derived from the coasted group showed a higher rate of progesterone secretion per cell at 72 h which was sustained for longer. Differences were also seen at 72 and 120 h of culture with a loss of correlation between progesterone secretion and follicle diameter in the coasted group. CONCLUSIONS: Our findings suggest that coasting has an effect on the functional capacity of the granulosa cells and the duration of their function. It is likely that in women at risk of OHSS who are not coasted, the granulosa cells have the capacity to produce significantly more chemical mediators per cell and for a more prolonged period of time.     

喜树碱诱导的HL-60细胞凋亡过程中线粒体的变化

目的:研究喜树碱(CPT)诱导的人早幼粒细胞性白血病细胞HL-60凋亡过程中线粒体的质量和膜电势的变化。方法:以CPT诱导HL-60细胞凋亡为模型,利用膜联蛋白V(annexinV)-FITC/PI双染流式细胞术,研究HL-60细胞的凋亡和坏死。用DiOC6(3)染色流式细胞术,检测线粒体的膜电势(△ψm)。用NAO染色流式细胞术,检测线粒体的质量。结果:在4×10-6mol/LCPT的诱导下,HL-60细胞(12h)早期的凋亡率为(12.75±4.61)%,对照组为(2.88±2.49)%,二者相比较差异显著(P<0.01);CPT组坏死比率为(3.48±1.67)%,对照组为(0.71±1.10)%(P<0.01)。PI染色的结果显示,HL-60细胞(12h)晚期凋亡细胞的百分率,CPT组为(3.52±1.07)%,对照组为(0.46±1.06)%(P<0.01)。同时观察到,G2/M期细胞出现阻滞,G2/M期细胞的百分率,对照组为(22.46±2.19)%,CPT组为(13.45±1.91)%(P<0.01)。在12h时间点,CPT组线粒体的质量显著低于对照组(P<0.01),低线粒体质量的细胞所占百分率,对照组为(4.53±1.26)%,CPT组为(25.74±2.09)%。同时,CPT组线粒体的膜电势显著下降(P<0.01),CPT组线粒体膜电势降低的比率为(17.71±5.23)%,对照组为(1.64±2.00)%。结论:CPT诱导HL-60细胞凋亡过程中,线粒体的质量和膜电势均有所下降,但其去极化作用增强。     

Sterigmatocystin induced apoptosis in human pulmonary cells in vitro

Sterigmatocystin (ST) is generally recognized as a potential carcinogen, mutagen and teratogen. Studies showed that ST could induce adenocarcinoma of lung in mice in vivo and DNA damage, cell cycle arrest in a human immortalized bronchial epithelial cell line (BEAS–2 B cells) and a human lung cancer cell line (A549 cells) in vitro. Besides, ST could induce G₂ arrest (cell cycle arrest in G₂ phase) in several other cells. Cell cycle arrest may be one of the common toxic effects of ST. As cells may undergo apoptosis or death due to cell cycle arrest, we wondered whether apoptosis is another common effect of ST in different cells in vitro. In the present study, we studied the effects of ST on proliferation and apoptosis in A549 cells and BEAS–2 B cells with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis (FCM). The MTT results showed that proliferation inhibition following ST treatment for 24 h was observed in both A549 and BEAS–2 B cells in vitro. And increased apoptosis by FCM was also found after ST treatment. Down-regulation of Bcl-2, up-regulation of Bax and the activation of caspase-3 after ST treatment were detected by western blotting analyses. The results in the present study are consistent with our previous results, which indicated that inducing apoptosis may be a common effect of ST in different cells in vitro.     

神经酰胺介导粒细胞凋亡机理初探

目的 观察神经酰胺对ＨＬ－６０及中性粒细胞（ＰＭＮ）凋亡产生的影响,探讨其在粒细胞发生过程中可能的调节作用。方法：采用ＤＮＡ凝胶电泳,流式细胞术,光镜及ＲＴ－ＰＣＲ等技术对细胞进行凋亡鉴定及相关基因ｂｃｌ－２,ｃ－ｍｙｃ,ｍＲＮＡ表达分析。结果 神经酰胺（ｃ２－ｃｅｒａｍｉｄｅ）肿瘤坏死因子（ＴＮＦα）及长春新碱（Ｖｉｎｃｒｉｓｔｉｎｅ）均可不同程度地诱导ＨＬ－６０及成熟ＰＭＮ产生凋亡,ＣＰＫ激活     

Cultured human granulosa cells as a model for corpus luteum function: relative roles of gonadotrophin and low density lipoprotein studied under defined culture conditions.

In primates, corpus luteum development involves both gonadotrophin stimulation and exposure to low density lipoprotein (LDL) delivered through vascularization of the granulosa cell-derived layer. These regulatory influences were modelled in vitro using granulosa cells obtained during in-vitro fertilization (IVF) cycles controlled with gonadotrophin releasing hormone (GnRH) analogue, human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG). Granulosa cells were cultured in defined medium on extracellular matrix. Without gonadotrophin or LDL in the medium, progesterone production declined progressively. With LDL alone, there was a short-lived elevation of progesterone output which subsequently declined. Culture with HCG alone resulted in a relatively unchanged rate of steroid production over 5 days despite morphological development. This contrasted with a marked and sustained increase in progesterone output over the same time when granulosa cells were cultured with combined HCG/LDL. Cultures were challenged with combined HCG/LDL on day 5. Where initial incubation included HCG, the challenge resulted in a recovery of progesterone output to values comparable to those of granulosa cells exposed to continuous HCG/LDL. Initial incubation without gonadotrophin led to a reduced response. Results suggest that LDL delivery to granulosa cells of the early corpus luteum causes a short-lived period of progesterone production. Sustained luteinization of granulosa cells and maintenance of gonadotrophin responsiveness requires continued exposure to gonadotrophin in the luteal phase.     

脂肪分化相关蛋白真核表达载体的构建及其对H9c2心肌细胞增殖和凋亡的影响

目的：构建编码脂肪分化相关蛋白（adipose differentiation-related protein, ADRP）基因全长序列的真核表达载体,探讨ADRP过表达对软脂酸诱导的H9c2心肌细胞凋亡有何影响。方法：(1) RT-PCR扩增编码ADRP全长的DNA 序列,重组入pEGFP-C1质粒表达载体中,酶切、测序鉴定后转化大肠埃希菌DH5α,挑取阳性克隆,扩增后提取质粒并进行鉴定;采用脂质体转染法将鉴定后的重组质粒稳定转染H9c2心肌细胞;荧光显微镜下观察细胞绿色荧光蛋白表达情况;RT-qPCR和Western blotting检测ADRP表达情况;(2) 采用MTT比色法和流式细胞术检测不同浓度软脂酸对细胞增殖和凋亡的影响。结果：(1) 成功构建了真核质粒表达载体pEGFP-C1-ADRP,转染H9c2细胞后,荧光显微镜下观察发现细胞内有绿色荧光蛋白表达;RT-qPCR和Western blotting显示重组质粒转染H9c2细胞ADRP mRNA和蛋白表达水平明显高于空质粒转染组和正常对照组（P<0.01）;(2) 经不同软脂酸刺激后,重组质粒转染H9c2细胞增殖抑制率和凋亡率均明显低于空质粒转染组和正常对照组（P<0.05）。结论：(1) 成功获得了稳定表达ADRP的H9c2心肌细胞株;(2) 软脂酸可抑制H9c2心肌细胞增殖并可诱导其凋亡,而ADRP高表达可对抗软脂酸的这一作用,表明ADRP对高脂环境中心肌细胞可能具有一定的保护作用。     

Effects of in-vitro exposure to HCG on subsequent HCG-responsiveness of human granulosa cells obtained following treatment with GnRH analogue and gonadotrophins: an in-vitro model for luteal phase support

The pregnancy rate in patients undergoing assisted conception treatment following pituitary desensitization with GnRH analogue and ovarian stimulation with gonadotrophins has been reported to be higher when ovarian function is supported in the luteal phase by exogenous human chorionic gonadotrophin (HCG). In the present study, we have examined the effects of culturing monolayers of granulosa cells, collected from such patients at oocyte retrieval, for various time intervals in the presence or absence of HCG on their subsequent ability to secrete progesterone (P4) either spontaneously or in response to further challenge with HCG. When cultured in the absence of HCG, granulosa cells demonstrated a rapid decline in both the spontaneous P4 secretion rate and the ability to secrete P4 in response to HCG. Maintenance in the presence of HCG inhibited the rapid decline in ability to secrete P4 spontaneously and also significantly enhanced the ability to respond to subsequent HCG stimulation. These results suggest that HCG support in the luteal phase in GnRH analogue-treated patients may have a cellular basis for its action both in maintenance of P4 secretion and also in rendering the corpus luteum more sensitive to rescue by conceptus-derived HCG.     

钙/钙调蛋白依赖性蛋白激酶Ⅱ通过介导凋亡加重H9c2心肌细胞缺氧/复氧损伤

目的:探讨钙/钙调蛋白依赖性蛋白激酶II(Ca~(2+)/calmodulin-dependent protein kinase II,Ca MKII)在H9c2心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤中的作用。方法:H9c2心肌细胞随机分为4组:正常对照(control)组、Ca MKII特异性抑制剂KN-93(1μmol/L)对照(KN-93)组、H/R组和KN-93+H/R组,其中KN-93组及KN-93+H/R组先用1μmol/L KN-93预处理2 h后,再进行其它处理。倒置显微镜观察各组H9c2心肌细胞的形态变化,CCK-8法检测各组H9c2心肌细胞的活力,乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒检测各组培养基中LDH的活性,Western blot法检测Ca MKII及其底物受磷蛋白(phospholamban,PLN)的磷酸化水平以及凋亡相关蛋白cleaved caspase-3的表达,TUNEL染色和流式细胞术观察细胞凋亡。结果:与control组相比,KN-93组各项指标均无显著性差异;H/R组细胞皱缩,大量死亡,细胞活力明显降低,培养基中LDH活性明显升高(P0.01),Ca MKII及PLN的磷酸化水平和cleaved caspase-3的蛋白水平显著增加(P0.01),TUNEL染色阳性细胞数和流式细胞凋亡率显著增加(P0.01);1μmol/L KN-93干预H/R可明显改善细胞形态,增加细胞活力,降低培养基中LDH活性(P0.01),减少Ca MKII和PLN的磷酸化水平以及cleaved caspase-3的蛋白水平(P0.05),降低TUNEL染色阳性细胞数和流式细胞凋亡率(P0.01)。结论:Ca MKII通过介导细胞凋亡参与H9c2心肌细胞缺氧/复氧损伤。     

Anti-proliferative effect of levamisole on human myeloma cell lines in vitro

Levamisole has been employed as an immunomodulatory agent in conjunction with 5-fluorouracil in the treatment of colon cancer relapse. At high doses, levamisole has been shown to have both anti-cancer and immunosuppressive activities. In vitro, levamisole has been shown to potentiate the anti-proliferative effect of 5-fluorouracil in several types of tumor cell lines; however, its mechanism of cytotoxic action and its molecular targets in cells remains to be elucidated. Here, the effect of levamisole on the proliferative response of the human multiple myeloma cell lines RPMI 8226 and U266B1 was studied in vitro. Treatment of both lines with varying concentrations of levamisole for 48 and 72?h in culture resulted in a significant inhibition of proliferation (unstimulated) in a dose-dependent manner, as assessed by an 3-[(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide dye assay. Furthermore, measurements of cell viability (using a trypan blue dye exclusion assay) clearly showed that the levamisole was cytotoxic. The preliminary evaluation of the mechanism of this cytotoxic effect revealed that this drug induced apoptosis in the myeloma cells, as evidenced by increases in the levels of DNA fragmentation, release of cytochrome c into the cytoplasm, and the activation of caspase-3 activity in the cells. The results of these studies strongly suggest that levamisole could be a potent anti-myeloma agent and might be considered in the treatment of multiple myeloma in the future.     

Sequential antigen-specific growth of T cells in the T zones and follicles in response to pigeon cytochrome c

The sites of accumulation and growth of antigen-specific T cells was assessed during the Vα11/Vβ3 T cell receptor-restricted response of IE^k+ mice to pigeon cytochrome c. In the T zone, the response was early but transient; Vα11/Vβ3⁺ T cell numbers fell to background levels as germinal centers developed. The follicles were a major site of specific T cell growth, but Vα11/Vβ3⁺ T cells were not obvious in foci of extrafollicular B cell growth in the red pulp. As germinal centers waned, Vα11/Vβ3⁺ T cells in the T zones again rose above background levels and some persisted in the follicles. After the initial stage of T cell priming, specific T cell growth seems to occur where specific interaction can occur with B cells that are presenting processed antigen.     

Progesterone production by human granulosa cells cultured in serum free medium: effects of gonadotrophins and insulin-like growth factor I (IGF-I).

There is evidence that insulin-like growth factor I (IGF-I) is a potent regulator of oestradiol synthesis by human granulosa and luteal cells; however, the question of whether IGF-I regulates progesterone synthesis by these cell types has yet to be answered. As a first step towards this goal, we have compared the effects of IGF-I, follicle stimulating hormone (FSH), and human chorionic gonadotrophin (HCG) on progesterone production by human granulosa cells obtained from individual dominant and cohort follicles, and granulosa luteal cells from preovulatory follicles of patients undergoing in-vitro fertilization (IVF). Granulosa cells from normal, unstimulated follicles cultured in serum-free medium as controls (no additions) produced some progesterone spontaneously. In all cases, FSH stimulated basal progesterone levels (10-fold average increase) and the effect was dose-dependent (ED50 of FSH = 9.1 +/- 3.9 ng/ml). Similar effects were observed when granulosa cells from large follicles were incubated with HCG (ED50 of HCG = 6.9 +/- 2.8 ng/ml). By comparison, the effects of IGF-I on progesterone production were not marked, being absent in 80% of the follicles tested. However, granulosa cells from healthy follicles co-incubated with IGF-I and FSH or HCG produced more progesterone compared with cells treated with the gonadotrophins alone; this effect of IGF-I was dose dependent (ED50 of IGF-I = 10 ng/ml). When the effect of each agonist was tested on IVF granulosa luteal cells, HCG but not FSH or IGF-I stimulated basal progesterone levels but the HCG effect required a two-day lag phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

线粒体损伤在创伤弧菌诱导树突状细胞凋亡中的作用

 目的：探讨线粒体损伤在创伤弧菌（Vibrio vulnificus,Vv）诱导树突状细胞（dendritic cell,DC）凋亡过程中的作用及其可能机制。方法：建立Vv 1.1758与DC2.4细胞混合培养模型。采用扫描电镜和透射电镜观察创伤弧菌侵入细胞方式和细胞线粒体病变情况。荧光探针DCFH-DA和Fluo-8/AM分别检测侵入细胞内活性氧（ROS）和Ca2+离子水平。流式细胞术检测细胞线粒体膜电位和细胞凋亡情况。采用Western blotting检测NF-κB p65和TNF-α蛋白的表达。结果：Vv 1.1758可诱导DC2.4细胞凋亡。Vv 1.1758以菌体一端与细胞表面结合的方式侵入细胞,侵入细胞的线粒体有明显病变,细胞内ROS和Ca2+水平升高,线粒体膜电位降低。共培育1 h,NF-κB p65蛋白即开始升高,5 h达高峰,6 h稍有下降;TNF-α蛋白则在共培育2 h开始增高,6 h达高峰。结论：线粒体损伤在Vv诱导DC凋亡中发挥作用,其作用机制可能与细胞内ROS和Ca2+水平升高、线粒体膜电位降低有关,NF-κB p65和TNF-α可能是细胞凋亡过程中的重要信号分子。     

Role of stromal cells and their products in protecting young and aged B-lineage precursors from dexamethasone-induced apoptosis

Bone marrow stromal cells are potent providers of stimuli that induce proliferation of B-cell precursors. We proposed that stromal cells play a role in protecting B-lineage cells from corticosteroid-induced apoptosis. We found that stromal cells protected B-cell precursors from dexamethasone-induced apoptosis, but this did not strictly correlate with interleukin-7 (IL-7) production. To determine if stromal-derived factors were involved in protection of B-cell precursors from apoptosis, we examined the activity of three lymphopoietic growth factors: IL-7, stem cell factor (SCF), and insulin-like growth factor-1 (IGF-1). Either IL-7 or IGF-1 alone protected B-cell precursors from dexamethasone-induced apoptosis. The combined activities of IGF-1 and IL-7 were additive rather than synergistic. SCF did not protect B-cell precursors from apoptosis. Aging altered the ability of B-cell precursors to respond to protective stimuli induced by IL-7 and IGF-1. Precursors from aged animals were deficient in ability to modulate expression of apoptosis regulatory genes Bax, Bcl-2, and Bcl-x in comparison to B-cell precursors from young animals. Taken together, these results suggest that stromal cells can protect B-lineage precursors from a corticosteroid-induced apoptotic signal, protection is mediated by stromal-derived cytokines, and aging decreases the ability of B-cell precursors to respond efficiently to protective stimuli.