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1.
Stra 8基因的激活与精原干细胞的特异性分化研究 总被引:2,自引:0,他引:2
视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现,在雄性生殖腺发育过程中有一组基因,它们可以被视黄酸特异性的诱导活化,称为Stra(Stimulated by Retinoic Acid)基因。从鼠源分离得到的Stra8基因编码一种细胞质蛋白,该基因只特异性的在成熟雄性生殖细胞中表达,其功能被认为与精子形成有关。为研究Stra8基因的表达特性,我们从小鼠的基因组中克隆了Stra8基因的启动子序列(1.4kb)。将Stra8基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前,构建成由Stra8基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中,如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等,通过荧光显微镜观察发现,绿色荧光蛋白只在小鼠精原干细胞中表达,表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞,经G418筛选2周后,用视黄酸诱导,12h培养后,有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8的转录,还有生殖细胞特异表达基因CyclinA8和Oct4的转录,这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。 相似文献
2.
视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现,在雄性生殖腺发育过程中有一组基因,它们可以被视黄酸特异性的诱导活化,称为Stra(Stimulated by Retinoic Acid)基因。从鼠源分离得到的Stra8 基因编码一种细胞质蛋白,该基因只特异性的在成熟雄性生殖细胞中表达,其功能被认为与精子形成有关。为研究Stra8 基因的表达特性,我们从小鼠的基因组中克隆了Stra8 基因的启动子序列(1.4kb)。将Stra8 基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前,构建成由Stra8 基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中,如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等,通过荧光显微镜观察发现,绿色荧光蛋白只在小鼠精原干细胞中表达,表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞,经G418筛选2周后,用视黄酸诱导,12h培养后,有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8 的转录,还有生殖细胞特异表达基因CyclinA8 和Oct4的转录,这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。 相似文献
3.
视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现,在雄性生殖腺发育过程中有一组基因,它们可以被视黄酸特异性的诱导活化,称为Stra(Stimulated by Retinoic Acid)基因。从鼠源分离得到的Stra8 基因编码一种细胞质蛋白,该基因只特异性的在成熟雄性生殖细胞中表达,其功能被认为与精子形成有关。为研究Stra8 基因的表达特性,我们从小鼠的基因组中克隆了Stra8 基因的启动子序列(1.4kb)。将Stra8 基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前,构建成由Stra8 基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中,如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等,通过荧光显微镜观察发现,绿色荧光蛋白只在小鼠精原干细胞中表达,表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞,经G418筛选2周后,用视黄酸诱导,12h培养后,有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8 的转录,还有生殖细胞特异表达基因CyclinA8 和Oct4的转录,这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。 相似文献
4.
Figla基因过表达促进小鼠胚胎干细胞向雌性生殖细胞分化 总被引:1,自引:0,他引:1
生殖系a因子(Figla)是最早表达的生殖细胞特异性转录因子之一,对卵泡的发育、Zp基因的表达和透明带的形成具有调节作用. Figla基因异常会引起卵巢早衰的发生. 本研究通过 PCR自小鼠基因组中扩增出Figla基因,将其克隆到真核报告载体pDsRed1 N1,构建了携带609 bp 的Figla重组载体pDsRed1 N1 Figla. 用该载体转染小鼠胚胎干细胞(mESCs)系J1、小鼠成纤维细胞系NIH 3T3、小鼠畸胎瘤细胞P19和小鼠精原细胞系GC1,在荧光显微镜下观察红色荧光蛋白(RFP)在细胞中的表达,同时检测转染细胞中Figla基因及其它生殖细胞特异性基因的表达. 结果显示,转染2 d,mESCs内Figla总表达量明显增加,且内源性表达量亦有所提高,即转入的外源性Figla基因可以促进内源性Figla的启动和表达. 免疫荧光染色显示,表达RFP 的细胞同时表达生殖特异性基因Vasa,减数分裂特异性基因Stra8、Scp3及卵母细胞标志基因Zp3. 通过QRT PCR检测发现,在转染3 d的细胞中,Vasa、Scp3和Zp1的表达较对照组均有明显上调,而Oct4和Stra8的表达量下降. 研究表明,Figla基因对生殖特异性基因的表达具有调控作用,可以激活雌性生殖基因表达,为更清楚地了解Figla基因在生殖细胞生长发育过程中的调控机制,以及发现该基因在生殖细胞中的新功能奠定了基础. 相似文献
5.
目的:研究microRNA-18a (miR-18a) 对缺氧引起的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,
hPASMCs)增殖的调控作用及其可能机制。方法:体外培养hPASMCs,分为未转染组、miR-18a 模拟物对照组、miR-18a 模拟物组、
miR-18a 抑制剂对照组、miR-18a 抑制剂组、siRNAcontrol组、siHIF-1-alpha组、miR-18a 抑制剂和siHIF-1-alpha共转染组。分别于常氧(21%
O2)和低氧(3%O2)作用24小时。采用CCK-8 法检测细胞的增殖情况,萤光素酶报告基因系统验证缺氧诱导因子-1-alpha(HIF-1alpha)是
否为miR-18a 的靶基因,并通过western-blot 以及实时荧光定量PCR 技术检测相关蛋白和基因的表达。结果:缺氧可促进
hPASMCs 增殖,使miR-18a 表达减少;miR-18a 模拟物可抑制hPASMCs 增殖,而miR-18a 抑制剂可促进hPASMCs 增殖;抑制
miR-18a 可使HIF-1-alpha的表达上调。同时抑制miR-18a 和HIF-1-alpha,可使miR-18a 对hPASMCs 增殖调控的能力消失。结论:缺氧通
过抑制miR-18a,上调HIF-1-alpha的表达,促进hPASMCs增殖。 相似文献
6.
《现代生物医学进展》2016,(16)
目的:研究microRNA-18a(miR-18a)对缺氧引起的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,hPASMCs)增殖的调控作用及其可能机制。方法:体外培养hPASMCs,分为未转染组、miR-18a模拟物对照组、miR-18a模拟物组、miR-18a抑制剂对照组、miR-18a抑制剂组、si RNA control组、si HIF-1α组、miR-18a抑制剂和si HIF-1α共转染组。分别于常氧(21%O2)和低氧(3%O2)作用24小时。采用CCK-8法检测细胞的增殖情况,萤光素酶报告基因系统验证缺氧诱导因子-1α(HIF-1α)是否为miR-18a的靶基因,并通过western-blot以及实时荧光定量PCR技术检测相关蛋白和基因的表达。结果:缺氧可促进hPASMCs增殖,使miR-18a表达减少;miR-18a模拟物可抑制hPASMCs增殖,而miR-18a抑制剂可促进hPASMCs增殖;抑制miR-18a可使HIF-1α的表达上调。同时抑制miR-18a和HIF-1α,可使miR-18a对hPASMCs增殖调控的能力消失。结论:缺氧通过抑制miR-18a,上调HIF-1α的表达,促进h PASMCs增殖。 相似文献
7.
为监测成体干细胞向生殖细胞分化的过程,分析生殖细胞特异性DEAD-box家族ATP依赖RNA解旋酶vasa在不同日龄山羊睾丸组织中的表达情况并构建山羊生殖细胞特异性报告载体p VASA-EGFP。通过免疫荧光及RT-PCR法监测vasa的表达情况,利用分子技术构建报告载体p VASA-EGFP,脂质体法转染山羊骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs),经过视黄酸(Retinoic acid,RA)诱导后,观察绿色荧光蛋白表达情况以鉴定该报告载体的有效性。免疫荧光结果显示,Vasa在性成熟不同阶段的山羊睾丸组织中均有表达,RT-PCR结果表明vasa基因在3月龄、10月龄山羊睾丸组织中显著高于10日龄组。测序及酶切鉴定结果表明,扩增的vasa基因启动子片段成功连至N1载体,转染BMSCs后经4 d的RA诱导,发现有绿色荧光蛋白表达,表明成功构建了山羊vasa基因启动子调控的报告载体p VASA-EGFP。以上结果表明,vasa基因在不同日龄山羊睾丸组织中均有表达,所构建的山羊生殖细胞特异性报告载体p VASA-EGFP具有示踪山羊成体干细胞向生殖细胞分化过程的能力,为下一步监测山羊BMSCs向生殖细胞分化的过程提供了鉴定和筛选方法。 相似文献
8.
《中国组织化学与细胞化学杂志》2019,(6)
目的探讨miR-143在人颈动脉粥样硬化患者斑块组织中的表达变化及可能的作用机制。方法收集我院血管外科因颈动脉狭窄行颈动脉内膜剥脱术(CEA)切除的颈动脉斑块组织及斑块旁内膜组织56例,根据术前颈动脉超声检查及术后斑块病理学检查将所取得的斑块组织又分为稳定斑块组26例和不稳定斑块组30例。另外选择同期年龄、性别相匹配的在我院因外伤、门脉高压或原发性血小板增多症而行脾脏切除术患者的正常的脾动脉内膜组织20例作为正常对照组,通过实时荧光定量PCR(qRT-PCR)检测组织中miR-143的表达水平。体外培养人主动脉血管平滑肌细胞(HAVSMCs),分别转染mi R-143模拟物、模拟物阴性对照、miR-143抑制物、抑制物阴性对照,采用qRT-PCR检测转染后HAVSMC细胞miR-143表达水平的变化,CCK-8法和Transwell迁移实验分别检测转染后细胞增殖活性和迁移能力的变化。通过生物信息学预测miR-143的可能靶基因,双荧光素酶报告基因实验观察miR-143与ADAMTS4的靶向调控关系,Western blot观察miR-143对ADAMTS4表达水平的影响。结果颈动脉粥样硬化斑块组织中miR-143表达水平明显低于斑块旁内膜组织和正常动脉血管内膜组织,并且不稳定斑块组患者斑块组织中miR-143的表达水平明显低于稳定斑块组斑块组织。转染miR-143模拟物后,HAVSMCs中miR-143表达水平明显升高,增殖活性和迁移能力明显降低;转染miR-143抑制物后,HAVSMCs中miR-143表达水平明显降低,增殖活性和迁移能力明显升高。双荧光素酶报告基因显示ADAMTS4是miR-143的直接靶基因,Western blot显示miR-143能够在转录后水平负向调控ADAMTS4表达。结论 miR-143在颈动脉粥样硬化斑块组织中表达降低,其可能通过靶向调控ADAMTS4影响血管平滑肌细胞增殖和迁移能力,与颈动脉斑块的形成和不稳定性有关。 相似文献
9.
目的:检测胶质瘤中miR-146a的表达水平,并研究miR-146a对胶质瘤细胞增殖的影响。方法:应用实时定量PCR的方法检测胶质瘤组织和癌旁组织中miR-146a的表达水平,采用脂质体细胞转染miRNA模拟物的方式过表达miR-146a,MTT法检测转染后细胞的增殖率,利用在线软件targetScan预测miRNA可能的靶基因。结果:miR-146a在胶质瘤组织中表达明显降低(P〈0.01),相对表达水平为癌旁组织的35%,细胞转染miR-146a模拟物后,miR-146a表达明显增加,癌细胞增殖率明显降低(P〈0.01),仅为原细胞的47%。Notch1基因是miR-146a影响胶质瘤细胞增殖活力的可能靶基因。结论:miR-146a可能通过抑制Notch1基因的表达调控胶质瘤细胞的增殖。 相似文献
10.
目的:检测胶质瘤中miR-146a的表达水平,并研究miR-146a对胶质瘤细胞增殖的影响。方法:应用实时定量PCR的方法检测胶质瘤组织和癌旁组织中miR-146a的表达水平,采用脂质体细胞转染miRNA模拟物的方式过表达miR-146a,MTT法检测转染后细胞的增殖率,利用在线软件targetScan预测miRNA可能的靶基因。结果:miR-146a在胶质瘤组织中表达明显降低(P<0.01),相对表达水平为癌旁组织的35%,细胞转染miR-146a模拟物后,miR-146a表达明显增加,癌细胞增殖率明显降低(P<0.01),仅为原细胞的47%。Notch1基因是miR-146a影响胶质瘤细胞增殖活力的可能靶基因。结论:miR-146a可能通过抑制Notch1基因的表达调控胶质瘤细胞的增殖。 相似文献
11.
Mingzhao Li Meng Yu Chao Liu Haijing Zhu Jinlian Hua 《Cell biochemistry and function》2013,31(4):281-288
Reproduction is required for the survival of all mammalian animals. Spermatogenesis is an essential and complex developmental process that ultimately results in production of haploid spermatozoa. Recent studies demonstrated that Boule and stimulated by retinoic acid 8 (Stra8) played important roles in initiation meiosis in male germ cells. miR‐34c is indispensable in the late steps of spermatogenesis; remarkably, the main function of miR‐34c is to reduce cell proliferation potentiality and promote cellular apoptosis. The objectives of this study were to investigate the expression patterns of Boule, Stra8, P53 and miR‐34c in dairy goat testis and their relationship in male germ line stem cells (mGSCs). The results first revealed the expression patterns of Boule, Stra8, P53 and miR‐34c in 30 dpp, 90 dpp and adult testes of dairy goats. The expression levels of Boule, Stra8, P53 and miR‐34c in adult dairy goat testes were significantly higher than that of 30 dpp. Overexpression of Boule and Stra8 promoted the expression of miR‐34c in dairy goat mGSCs. In our previous study, we showed that miR‐34c was P53 dependent in mGSCs. These results have shown that the up‐regulation of miR‐34c was not due to P53 protein activation but which might be caused by the up‐regulation of Boule and Stra8 promoting the advance of meiosis. In addition, we found retinoic acid would decrease the expression of P53 and miR‐34c, however, did not change the expression of c‐Myc greatly. It suggested that the function of driving differentiation of dairy goat mGSCs by retinoic acid might not be caused by P53. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献
12.
13.
MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology. 相似文献
14.
15.
Silva C Wood JR Salvador L Zhang Z Kostetskii I Williams CJ Strauss JF 《Molecular reproduction and development》2009,76(1):11-21
Cells that morphologically and functionally resemble male germ cells can be spontaneously derived from ES cells. However, this process is inefficient and unpredictable suggesting that the expression pattern of male germ cell associated genes during spontaneous ES cell differentiation does not mimic the in vivo profiles of the genes. Thus, in the present study, the temporal profile of genes expressed at different stages of male germ cell development was examined in differentiating ES cells. The effect of all-trans retinoic acid (RA) which is a known inducer of primordial germ cell (PGC) proliferation/survival in vitro and testosterone which is required for spermatogenesis in vivo on the expression of these genes was also determined. Each of the 12 genes analyzed exhibited one of four temporal expression patterns in untreated differentiating ES cells: progressively decreased (Dppa3, Sycp3, Msy2), initially low and then increased (Stra8, Sycp1, Dazl, Act, Prm1), initially decreased and then increased (Piwil2, Tex14), or relatively unchanged (Akap3, Odf2). RA-treated cells exhibited increased expression of Stra8, Dazl, Act, and Prm1 and suppressed expression of Dppa3 compared to untreated controls. Furthermore, testosterone increased expression of Stra8 while the combination of RA and testosterone synergistically increased expression of Act. Our findings establish a comprehensive profile of male germ cell gene expression during spontaneous differentiation of murine ES cells and describe the capacity of RA and testosterone to modulate the expression of these genes. Furthermore, these data represent an important first step in designing a plausible directed differentiation protocol for male germ cells. 相似文献
16.
Objectives
Recent lines of evidence have indicated that miR‐34c can play important roles in regulation of the cell cycle, cell senescence and apoptosis of mouse and human tumour cells, spermatogenesis, and male germ‐cell apoptosis. However, there is little information on the effects of miR‐34c on proliferation and apoptosis of livestock male germ cells. The dairy goat is a convenient domestic species for biological investigation and application. The purpose of this study was to investigate the effects of miR‐34c on apoptosis and proliferation of dairy goat male germline stem cells (mGSCs), as well as to determine the relationship between p53 and miR‐34c in this species.Materials and methods
Morphological observation, miRNA in situ hybridisation (ISH), bromodeoxyuridine staining, flow cytometry, quantitative‐RT‐PCR (Q‐RT‐PCR) and western blotting were utilized to ascertain apoptosis and proliferation of mGSCs, through transfection of miR‐34c mimics (miR‐34c), miR‐34c inhibitor (anti‐miR‐34c), miR‐34c mimics and inhibitors co‐transfected (mixture) compared to control groups.Results
Results manifested that miR‐34c over‐expression promoted mGSCs apoptosis and suppressed their proliferation. Simultaneously, a variety of apoptosis‐related gene expression was increased while some proliferation‐related genes were downregulated. Accordingly, miR‐34c promoted apoptosis in mGSCs and reduced their proliferation; moreover, expression of miR‐34c was p53‐dependent.Conclusions
This study is the first to provide a model for study of miRNAs and mechanisms of proliferation and apoptosis in male dairy goat germ cells.17.
Embryonic stem cells (ESCs) have the capacity to differentiate into nearly all sorts of cell types, including germ cells, which were regarded as one type of highly specialized cells in mammals, taking the responsibility of transferring genetic materials to the next generation. Studies on induction differentiation of murine embryonic stem cells (mESCs) into male germ cells, but with a low efficiency, basic reason is that the regulation mechanism of germ cell development in mammals is still unclear. miRNA might play an important role in spermatogenesis in mammals. In this study, several miRNAs, which might be related to spermatogenesis, were initially selected and detected in the mouse tissues by semi‐polymerase chain reaction (PCR) and quantitative real time (qRT)‐PCR to find a testis‐specific miRNA. To study its effect on mESCs differentiation into male germ cells, miR‐34c mimics were synthesized and pri‐miR‐34c‐GFP plasmid was constructed, transfected into mESCs and combined with retinoic acid induction. The effects of miR‐34c were analysed by morphology, alkaline phosphatase staining, qRT‐PCR_and immunofluorescent staining. The results showed that miR‐34c promoted mESCs differentiation into male germ‐like cells, to some extent. Then miR‐34c targeted genes were predicted by bioinformatics; Retinoic acid receptor gamma (RARg) was selected, and two dual‐luciferase reporter vectors contained the normal and mutated 3′untranslated region of RARg were constructed, respectively. By miRNA mimics and vector co‐transfection experiment, the predicted target gene‐RARg was confirmed. In conclusion, we found a mammalian male germ cell specific miRNA—miR‐34c, and it might be pivotal in mESCs differentiation into male germ cells through its target—RARg. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
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19.
《Cell cycle (Georgetown, Tex.)》2013,12(5):656-664
During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway. 相似文献
20.
Hailong Mu Jiang Wu Haijing Zhu Na Li Furong Tang Xi Yao Churong Yang Sha Peng Guangpeng Li Jinlian Hua 《Cell biochemistry and function》2013,31(8):629-635
During sequential stages of meiosis, numerous cytoplasmic and nuclear events take place in which many germline and non‐germline genes involved. It is demonstrated that the germline gene Stra8 and synaptonemal complex protein 3 (Scp3) play an important role in the meiosis. Recently, studies showed Msx1, a DNA‐binding protein taking part in the skeletal development, also having a functional attractive factor to Stra8 and Scp3 in the meiosis. In this study, we cloned the gene Msx1 then transfected the Msx1 constructed recombination plasmid, pMsx1‐Ires2‐AcGFP, into the dairy goat germline stem cells (male germline stem cells) and analysed the effects of Msx1 on the expression of Stra8 and Scp3. The results showed that Msx1 could enhance the expression of Stra8 and Scp3 and promote the meiosis in goat testicular cells. Bmp4 activated the expression of Msx1 and Stra8. This study suggests that Msx1 plays an important role in spermatogenesis and meiosis. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献