Objective:To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells co-cultured with Sertoli cells.Methods:Tissues of neonate NMRI male mice testes were used for harv...Objective:To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells co-cultured with Sertoli cells.Methods:Tissues of neonate NMRI male mice testes were used for harvesting spermatogonial stem cells and Sertoli cells.After cell harvest,flow cytometry using promyelocytic leukemia zinc-finger(PLZF)protein antibody was used to assess the purity of the cells.The isolated testicular cells were cultured in the absence(the control group)or presence of soft agar-coated dishes(the experimental group)supplemented with leukemia inhibitory factor and glia cell line–derived neurotrophic factor for two weeks.Alkaline phosphatase activity was assessed in the colonies formed after two weeks of culture by alkaline phosphatase staining.On day 14 of culture,the expression levels of DNA-binding protein inhibitor(ID-4)and PLZF genes in the undifferentiated cells were evaluated by the detection of PLZF protein antibody using real-time PCR and immunocytochemistry techniques.The number and diameter of the colonies of spermatogonial stem cells were assessed by ImageJ software.Results:In the experimental group,the number and the diameter of colonies significantly increased as compared with those in the control group(P<0.05,P<0.01,respectively).In addition,the level of expression of ID-4 and PLZF genes in the undifferentiated cells significantly increased in the experimental group as compared with the control group(P<0.01).However,the expression of tyrosine-protein kinase kit(c-kit)gene in differentiated cells decreased in the experimental group as compared with the control group,but there was no significant difference between the two groups.Conclusions:Spermatogonial stem cells can be efficiently proliferated by culturing the stem cells in soft agar-coated dishes.The new protocol used in this study can be a valuable method for future studies.展开更多
背景与目的:FRZB(frizzled motif associated with bone development)是sFRP(secreted frizzled related protein)家族的成员,在胚胎发育过程中起重要作用。有文献报道FRZB的表达能抑制基质金属蛋白酶-2(matrix metalloproteinase 2,MMP...背景与目的:FRZB(frizzled motif associated with bone development)是sFRP(secreted frizzled related protein)家族的成员,在胚胎发育过程中起重要作用。有文献报道FRZB的表达能抑制基质金属蛋白酶-2(matrix metalloproteinase 2,MMP-2)和MMP-9表达而与前列腺癌的侵袭能力相关。本研究拟探讨FRZB基因对人胃癌SGC-7901细胞成瘤性和侵袭能力的影响,及其可能的机制。方法:构建FRZB真核表达载体并转染SGC-7901细胞,经G418筛选获得稳定表达克隆。用MTT法检测并绘制细胞增殖曲线,软琼脂培养和裸鼠皮下注射的体外和体内实验检测细胞成瘤性变化。用细胞免疫化学检测转染FRZB后细胞中MMP-2、MMP-7、MMP-9的表达变化,体外侵袭和粘附实验检测细胞侵袭能力变化。结果:筛选稳定表达细胞株经定量PCR检测,FRZB表达显著提高。体内外实验证实,转染FRZB的细胞(SGC-7901/FRZB)生长抑制率约为60%,克隆形成能力和成瘤性减弱。免疫细胞化学显示MMP-2、MMP-7、MMP-9在细胞浆内的阳性表达降低或接近阴性,对细胞外基质成分(CollagenI、IV、Vitronectin、Fibronectin、Laminin)粘附能力均增高,比空载体转染对照组(SGC-7901/vector)增加12.8%、19.8%、59.8%、26.7%、15.2%,差异具有显著性(P<0.05),而空载体转染对照组与亲本细胞间差异无统计学意义(P>0.05)。SGC-7901、SGC-7901/vector和SGC-7901/FRZB细胞侵袭能力分别是每高倍视野55.90±5.68、54.80±6.97、6.60±2.63,差异具有统计学意义(P<0.05)。结论:FRZB高表达在体内体外均可抑制SGC-7901细胞的增殖、成瘤性和侵袭能力,具有肿瘤抑制基因的功能。FRZB抑制肿瘤细胞的生长和抑制MMP-2、MMP-7、MMP-9的表达为抑癌功能相关机制之一。展开更多
Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferatio...Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.展开更多
基金This study was supported by Shahroud University of Medical Sciences(Grant No.98131).
文摘Objective:To investigate the effects of soft agar on in-vitro proliferation of neonate mouse spermatogonial stem cells co-cultured with Sertoli cells.Methods:Tissues of neonate NMRI male mice testes were used for harvesting spermatogonial stem cells and Sertoli cells.After cell harvest,flow cytometry using promyelocytic leukemia zinc-finger(PLZF)protein antibody was used to assess the purity of the cells.The isolated testicular cells were cultured in the absence(the control group)or presence of soft agar-coated dishes(the experimental group)supplemented with leukemia inhibitory factor and glia cell line–derived neurotrophic factor for two weeks.Alkaline phosphatase activity was assessed in the colonies formed after two weeks of culture by alkaline phosphatase staining.On day 14 of culture,the expression levels of DNA-binding protein inhibitor(ID-4)and PLZF genes in the undifferentiated cells were evaluated by the detection of PLZF protein antibody using real-time PCR and immunocytochemistry techniques.The number and diameter of the colonies of spermatogonial stem cells were assessed by ImageJ software.Results:In the experimental group,the number and the diameter of colonies significantly increased as compared with those in the control group(P<0.05,P<0.01,respectively).In addition,the level of expression of ID-4 and PLZF genes in the undifferentiated cells significantly increased in the experimental group as compared with the control group(P<0.01).However,the expression of tyrosine-protein kinase kit(c-kit)gene in differentiated cells decreased in the experimental group as compared with the control group,but there was no significant difference between the two groups.Conclusions:Spermatogonial stem cells can be efficiently proliferated by culturing the stem cells in soft agar-coated dishes.The new protocol used in this study can be a valuable method for future studies.
文摘背景与目的:FRZB(frizzled motif associated with bone development)是sFRP(secreted frizzled related protein)家族的成员,在胚胎发育过程中起重要作用。有文献报道FRZB的表达能抑制基质金属蛋白酶-2(matrix metalloproteinase 2,MMP-2)和MMP-9表达而与前列腺癌的侵袭能力相关。本研究拟探讨FRZB基因对人胃癌SGC-7901细胞成瘤性和侵袭能力的影响,及其可能的机制。方法:构建FRZB真核表达载体并转染SGC-7901细胞,经G418筛选获得稳定表达克隆。用MTT法检测并绘制细胞增殖曲线,软琼脂培养和裸鼠皮下注射的体外和体内实验检测细胞成瘤性变化。用细胞免疫化学检测转染FRZB后细胞中MMP-2、MMP-7、MMP-9的表达变化,体外侵袭和粘附实验检测细胞侵袭能力变化。结果:筛选稳定表达细胞株经定量PCR检测,FRZB表达显著提高。体内外实验证实,转染FRZB的细胞(SGC-7901/FRZB)生长抑制率约为60%,克隆形成能力和成瘤性减弱。免疫细胞化学显示MMP-2、MMP-7、MMP-9在细胞浆内的阳性表达降低或接近阴性,对细胞外基质成分(CollagenI、IV、Vitronectin、Fibronectin、Laminin)粘附能力均增高,比空载体转染对照组(SGC-7901/vector)增加12.8%、19.8%、59.8%、26.7%、15.2%,差异具有显著性(P<0.05),而空载体转染对照组与亲本细胞间差异无统计学意义(P>0.05)。SGC-7901、SGC-7901/vector和SGC-7901/FRZB细胞侵袭能力分别是每高倍视野55.90±5.68、54.80±6.97、6.60±2.63,差异具有统计学意义(P<0.05)。结论:FRZB高表达在体内体外均可抑制SGC-7901细胞的增殖、成瘤性和侵袭能力,具有肿瘤抑制基因的功能。FRZB抑制肿瘤细胞的生长和抑制MMP-2、MMP-7、MMP-9的表达为抑癌功能相关机制之一。
基金This work was supported by NationalNatural Science Foundation of China (No. 30200095).
文摘Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.
基金This work was supported by Returning Scholars Fund of Heilongjiang Province (No. LC04C02) the Department of Education Overseas Researcher Fund of Heilongjiang Province (No. 1054HZ013).
