应用siRNA沉默共培养生精细胞uPAR基因表达的研究
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摘要
精子发生是个复杂、多步骤的过程,可分为三个过程:精原细胞的增殖更新、精母细胞的成熟分裂和精子细胞变态为精子三个阶段。目前发现在曲细精管中uPAR主要表达在精子细胞,可能与精子的排放有关。Tong Zhang等应用原位杂交技术检测发现猕猴睾丸支持细胞在精子发生的特定阶段(VII～VIII期)表达uPA mRNA;uPAR mRNA由睾丸生精细胞产生,但是不同发育阶段及不同生殖细胞表达的差异表明uPA/uPAR系统可能在精子发生过程中的特定阶段起作用。Vassalli认为支持细胞这种时期特异性表达的uPA与生殖细胞uPAR结合后可能是通过激活uPAR介导的信号传导通路作为生长因子而起作用。但是uPAR是通过激活何种信号传导通路来调节精子的发生过程目前尚不清楚。uPAR是否还通过信号传导来刺激生精细胞的发生及分化功能呢?因此,uPAR在精子发生中的作用机制尚待进一步研究。
然而由于睾丸组织结构和功能的复杂性,一般情况下很难在整体情况下对生殖细胞的生理生化功能和与其他细胞的相互作用进行深入研究。生精细胞培养技术的建立为深入地了解生精细胞发育过程,研究生精细胞自身及与其它生精细胞的关系,提供了一种新的、强有力的工具。RNA干扰(RNAi)是一种由双链小干扰RNA(siRNA)引发的转录后基因沉默(PTGS)新型机制,可导致靶基因mRNA的降解及特异性基因沉默信号的扩增。目前siRNA已经成为选择性沉默体外培养的哺乳细胞基因表达的一个有力工具。
本实验首先应用实时RT-PCR和Western Blot检测出生后不同发育阶段大鼠睾丸中uPAR基因及蛋白表达的变化,然后建立的生精细胞体外培养体系,最后应用siRNA沉默技术阻断体外培养生精细胞uPAR的基因表达,为研究uPAR在精子发生中作用机制及信号传导通路的研究提供新的重要研究手段和技术平台。
一、uPAR在大鼠睾丸第一精子发生波中的表达变化
目的:为探讨uPAR在大鼠精子发生中的作用,研究在第一个精子发生波中大鼠睾丸组织uPAR的mRNA表达及蛋白表达变化。方法:将SD大鼠按生后年龄分组,以出生当天为d0,取出生后d0、d5、d10、d15、d21、d28、d35、d42、d49、d56大鼠睾丸组织,用实时荧光定量分析方法检测各年龄组大鼠uPAR mRNA表达,并用Western印迹法检测各年龄组大鼠uPAR蛋白表达。结果:大鼠睾丸组织uPAR mRNA表达与蛋白表达表现出相似的趋势:刚出生时表达水平较高,后逐渐下降,约在d15天达最低,到d28时开始增加,到d35时到达高峰,随后d42下降,后保持一定的水平。双变量回归相关分析显示uPAR mRNA表达与蛋白表达为中度正相关。结论:大鼠睾丸uPAR表达在第一精子发生波中出现两个高峰,第一高峰在刚出生时,这个时期生殖母细胞向基底部迁移的时期,这在精子以后的发生中起着至关重要的作用,说明uPAR与精子发生的启动有着密切联系。第二高峰是在第5周升高明显,第5周为精子变形并开始向管腔排放的时期,这说明uPAR可能参与精子排放过程中的组织重塑及精子变态过程。
二、大鼠睾丸生殖细胞培养体系的建立
大鼠睾丸生殖细胞培养体系的建立是体外研究睾丸精子发生过程中各种调控因素的的重要工具。本实验通过建立支持细胞及生精细胞两种培养体系,为进一步研究uPAR在精子发生中的作用奠定基础。
(一)大鼠睾丸支持细胞的分离纯化和鉴定
目的培养高纯度的大鼠睾丸支持细胞,并应用检测ABP mRNA原位杂交方法鉴定分离培养的支持细胞。方法选用18～22天龄SD雄性大鼠睾丸,采用0.25%胰蛋白酶、0.1%透明质酸酶、0.1%胶原酶三酶依次消化法分离支持细胞,放于32℃5%CO2的培养箱培养,48小时后用20mmol Tris—HCl低渗处理培养细胞。培养一周后应用伊红染色、吖啶橙荧光染色、Feulgen染色对所培养支持细胞进行鉴定,同时应用原位杂交检测ABP mRNA方法鉴定分离培养的支持细胞。结果培养一周后所获培养的支持细胞纯度达95%以上。分离培养的支持细胞ABP mRNA表达阳性,其形态结构特征与用其它方法鉴定为支持细胞的形态结构特征一致。结论采用三酶连续消化及低渗处理法分离培养的支持细胞纯度高,而且应用原位杂交方法检测ABP mRNA是一种新的、特异、有效的鉴定支持细胞及其功能的方法。
(二)大鼠睾丸支持细胞/生精细胞共培养系统的建立
目的建立生精细胞体外分化培养体系,为uPAR在精子发生中作用的体外研究提供一个很好的研究模型。方法取20～22天龄SD雄性大鼠睾丸,去被膜及血管,放在预冷的Hank’s液中洗两次,然后用加1 mg/ml胶原酶的F12/DMEM液32℃消化15～20min。用眼科剪将睾丸剪碎成非常小的片断,再重复用1 mg/ml胶原酶消化5～10min。细胞用内含10%胎牛血清及各种营养因子的HamF12/ DMEM培养液悬浮,约按1*106CELL/cm2的密度接种于双室培养槽或放有盖玻片的六孔板中,在32℃、5%CO2条件下培养。相差显微镜下动态观察细胞生长、形态等变化,及定期应用伊红染色、Brdu染色法对所培养生精细胞/支持细胞进行染色鉴定。结果在生精细胞/支持细胞共培养体系中,双室培养2周后,部分生精细胞胞体一端有鞭毛出现,培养4周后,培养体系中仍有一定数量的生精细胞附着在支持细胞上,部分生精细胞上可见鞭毛。单室培养的生精细胞在培养第4-5天开始出现明显脱落,培养1周后大多数生精细胞发生脱落死亡,生精细胞上未见鞭毛出现,但在培养体系中可见次级精母细胞及精子细胞。结论:应用双室培养,生精细胞可存活达一月之久,而且部分生精细胞尾部出现鞭毛,从形态上发生了减数分裂及精子变态过程。
三、应用siRNA沉默共培养生精细胞uPAR的基因表达
目的利用小分子干扰RNA(siRNA)技术沉默共培养生精细胞uPAR的基因表达,为uPAR在精子发生中的作用研究提供一个很好的研究工具。方法siRNA混合鸡尾酒是根据ShortCut RNAi Kit手册来制备。简单的说,首先通过RT-PCR应用加有T7启动子的特异性引物来合成DNA模板,然后DNA模板通过体外转录过程产生双链RNA,最后通过ShortCut RNaseIII酶消化来制备siRNAs混合物。在培养48小时用Transfect转染试剂分别将15nM、30nM siRNA混合物转染到共培养的生精细胞,48小时应用实时荧光定量RT-PCR方法检测共培养生精细胞uPAR的基因表达。该试验设立两个对照组:空白对照(未加siRNA及转染试剂)及阴性对照组(只加转染试剂,无siRNA)。结果15nM及30nM siRNA转染组uPAR mRNA表达量均明显低于阴性对照组及空白对照组(p<0.05),其中以30nMsiRNA转染组更为明显;相对于阴性对照组,15nM及30nM siRNA转染组uPAR的基因表达抑制率分别为63.5%及76.7%。结论应用ShortCut RNase III法制备的siRNAs鸡尾酒能有效地抑制共培养生精细胞uPAR基因的表达。
五、结论
在本研究中我们发现uPAR在第一精子发生波中表达最高峰为出生后35天,此时为圆形精子转变为伸长精子,并开始向管腔排放的时期。在我们的共培养体系中,来自20～22天大小的大鼠睾丸组织的生精细胞在培养二周后能分化成精子细胞及变形成为伸长型精子。而这些变化相应于体内uPAR的表达出现高峰期所发生的变化。因此在本培养体系中,应用siRNA沉默uPAR的表达,可以为研究uPAR在精子发生特定时期中的作用及机制提供一个很好的研究工具,如其信号传导通路等。此研究模型的建立也有助于减数分裂及精子变态过程的其它调控因素的研究。
Spermatogenesis is a complex differentiation process that consists of three major phases: a proliferation phase of spermatogonial cells by mitosis, a meiotic phase of spermatocytes in which recombination of genetic materials and reductive division occur and a transformation phase which produces mature spermatozoa from haploid spermatids. Tong Zhang et al investigated the expression and localization of mRNAs for urokinase PA(uPA), uPA receptor(uPAR) in monkey testes by using in-situ hybridization, the results demonstrated that uPA mRNA was expressed stage-specifically in Sertoli cells of adult testis, and uPAR mRNA was localized in germ cells of mature testis. The expression levels differed among different germ cells and at different developmental stages indicated uPA/uPAR system may play an important role in the specifical stage of spermatogenesis. The stage-specifically expressed uPA in Sertoli cells may bind to its receptor on germ cells and act as serine protease, or as growth factor through activation of signal transduction pathways by uPA receptor. It is still unclear which signal transduction pathways can be activated by uPAR in spermatogenesis. Can uPAR modulate growth and differentiation of spermatogenic cell by its signal transduction pathway? So the role of uPAR and its mechanism in spermatogenesis is worthy of further study.
Because of complex of structure and function of testitular, the physiology and biochemistry function of spermatogenic cell is difficult to further research. Establish of spermatogenic cell culture system can provide a powerful tool to study the relationship between spermatogenic cell and other cell and investigate gene regulation in spermatogenesis. RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. siRNA pairs with its cognate mRNA, leading to degradation of target mRNA and amplification of gene-specific silencing signals. siRNA has become a powerful tool for selectively silencing gene expression in cultured mammalian cells.
In present study, firstly gene and protein expression of uPAR in rat testis at first wave of spermatogenesis was investigated by real time RT-PCR and Western Blot respectively, secondly germinal cell culture system was set up, finally siRNA cocktails were used to silience the gene expression of uPAR in the co-cultured germinal cells. This research provide a new study method and technology platform to investigate the role and signal pathways of uPAR in spermatogenesis.
1.Expression of uPAR in rat testis at first wave of spermatogenesis
Objective: To elucidate the role of urokinase-plasminogen activator receptor (uPAR) system in rat spermatogenesis,gene and protein expression of uPAR in rat testis at first wave of spermatogenesis was investigated. Method: The day of birth was designated as postnatal d0 ,the male rat were divided into 10 groups according to different postnatal stages, that is, d0, d5, d10, d15, d21, d28, d35, d42, d49, d56. The gene and protein expression of uPAR in rat testis was quantitated by Real-time polymerase chain reaction (PCR) and western blot methods separately. Result: Gene and protein expression of uPAR had similar tendency in testis of rats at first wave of spermatogenesis: Express level of uPAR situate at high relatively at postnatal d0, declined to a lowest level at postnatal d15, then increased obviously at postnatal d28 and reached peak at postnatal d35, then declines at postnatal d42 and retained low afterwards. Regression and correlation analysis showed that there was a moderately positive correlation between the gene and protein expression levels of uPAR. Discussion:Expression of uPAR exhibited two peak in testis of rats at first wave of spermatogenesis: first peak was at short after birth, when gonacyte migrated to the basilar part of seminiferous tubules and was crucial to initiate spermatogenesis, which indicated uPAR had intimate relationship whit onset of spermatogenesis. Second peak was at postnatal d35 when round spermatids transformed to elongate spermatids and began to spermiation, which indicated uPAR may be involved in spermiation and/or spermiogenesis.
2.Establish of germinal cell culture system of rat testis
Establishment of germinal cell culture system of rat testis is an important instrument to investigate the regulator involved in spermatogenesis. This experiment is to set up both sertoli / spermatogenic cells coculture system for further research of role of uPAR in spermatogenesis.
2.1 Isolation、purification and identification of sertoli cells from rat testis
Objective To obtain highly pure cultured sertoli cells from rat testis and identificate cultured cell by in situ hybridization for ABP mRNA. Methods Testis from 18～22-day-old SD rats were removed and decapsulated, then chopped and sequentially digested with three enzymes at 37℃: first with 0.25% trypsin for 15 minutes, then with 0.1% hyaluronidase for 30 minutes, last with 0.1% collagenase V for 2～3 hours. The isolated cells were incubated at 32℃in a humidified atmosphere of 5% CO2. To increase the purity of the Sertoli cells, cultured cells were subjected to hypotonic shock(treatment) with 20 mmol Tris—HCl after 48 hours incubation. After a week incubation, cultured cells were identificated by HE staining、AO fluorescence staining、Feulgen staining, at the same time by in situ hybridization with digoxin-labeled rat ABP cDNA probe. Conclusion Over 95% cultured cells were sertoli cells. Most of cultured cells expressed ABP mRNA and their structural features were the same as sertoli cells identificated by other methods. Results Highly pure sertoli cell can obstained by sequentially digested with three enzymes and hypotonic shock. As a specifical secreted protein by sertoli cells in testes, ABP mRNA detected by in situ hybridization is a new、specific、effective identification method of sertoli cells from testis. Meanwhile as an index of changes in sertoli cell function, this method can be used to study cultured sertoli cell function.
2.2 Coculture of Rat Sertoli/Spermatogenic Cells
Object To further study the role of uPAR in spermatogenesis in vitro, rat Sertoli/Spermatogenic cells coculture system was established. Method: The testis were removed from 20～22 day old SD rats and decapsulated. The tissues were washed twice with ice-cold Hank’s solution, were digested at 32℃in F12/DMEM containing 1 mg/ml collagenase at 32℃for 15～20 min, then were cut into small fragments and by 1 mg/ml collagenase again for 5～10 min. The cells were resuspended in culture medium supplemented various nutrition factors and 10% fetal calf serum, and seeded at about 106 cells/cm2 in bicameral chambers or culture plates covered with glass. Coculture was carried out at 32℃in a water-saturated atmosphere of 95% air and 5% CO2. The growth and morphology of coculture cells were monitored daily under contrast phase microscope, and were identificated by HE staining、Brdu staining at specific day. Results In bicameral culture system, flagella were seen emerging from one end of some spermatogenic cells after two-week coculture and a certain number of spermatogenic cells still attached on the surface of sertoli cell after four-week cultue while some of them has a short flagellum at one end. In unicameral culture system, spermatogenic cell began obviously to fall off the surface of sertoli cell after 4～5 day culture, there are only a few spermatogenic cells attached the surface of sertoli cell after one week coculture. But the second spermatocyte and spermatid were observed except for flagellum in this unicameral culture system. Conclusion: In bicameral culture system, the spermatogenic cells survived as long as four week, and some of them generated flagella at one end, which indicated that the process of meiosis and spermiogenesis could taken place in this culture system.
3. Inhibit uPAR gene expression by siRNA cocktails in co-cultred spermatogenic cells
Objective To futher study the role of uPAR in spermatogenesis, uPAR gene expression were inhibit by siRNA cocktails in co-cultred spermatogenic cell. Methods siRNA cocktails were generated according to shortcut RNAi Kit handbook. Biefly,suitable DNA templates were generated by RT-PCR using specific primers with appended T7 promoters, then the DNA template produces double-stranded RNA by in vitro transcription, finally digested by ShortCut RNase III digestion to produces a mixture of siRNAs. 15nM or 30nM siRNA cocktails were transfected in co-cultured spermatogenic cell using RNAiFect Transfection Reagent at 48 hours of culture. After 48 hours transfected, expression of uPAR mRNA were ananysized by real time RT-PCR.. The study consisted of two control groups; blank control group treated with neither siRNA nor transfection reagent and negative control group treated only with transfection reagent. Results The expression level of uPAR mRNA with 15nM and 30 nM of siRNA cocktails were significantly lower than those of negative control and blank control group(p<0.05); The inhibition rate silencing with 15nM and 30 nM of the siRNA cocktails were 63.5% and 76.7% separately compared with negative control group. Conclution siRNA cocktail producted by shortcut Rnase III can efficially inhibited the gene expression of uPAR in co-cultured spermatogenic cell.
Conclusion
In present study, expression of uPAR reached peak at postnatal d35 in testis of rats at first wave of spermatogenesis, when round spermatids transformed to elongate spermatids and began to spermiation. In our co-culture system, spermatogenic cell from 20-22 day old rat testis differentiated into spermatids and transformed to elongate spermatid during two-week co-culture. These changes were corresponding to those in vivo when expression of uPAR reached the peak. Hence, silencing uPAR gene expression with siRNA cocktails on this culture system can provide a powerful tool to further study the role and mechanism of uPAR involved in specific stages of spermatogenic cell development, such as its signal transduction in spermatogenesis. In the meantime, this model can also be helpful to research for other regulator invovled in meiosis and spermiogenesis.
然而由于睾丸组织结构和功能的复杂性,一般情况下很难在整体情况下对生殖细胞的生理生化功能和与其他细胞的相互作用进行深入研究。生精细胞培养技术的建立为深入地了解生精细胞发育过程,研究生精细胞自身及与其它生精细胞的关系,提供了一种新的、强有力的工具。RNA干扰(RNAi)是一种由双链小干扰RNA(siRNA)引发的转录后基因沉默(PTGS)新型机制,可导致靶基因mRNA的降解及特异性基因沉默信号的扩增。目前siRNA已经成为选择性沉默体外培养的哺乳细胞基因表达的一个有力工具。
本实验首先应用实时RT-PCR和Western Blot检测出生后不同发育阶段大鼠睾丸中uPAR基因及蛋白表达的变化,然后建立的生精细胞体外培养体系,最后应用siRNA沉默技术阻断体外培养生精细胞uPAR的基因表达,为研究uPAR在精子发生中作用机制及信号传导通路的研究提供新的重要研究手段和技术平台。
一、uPAR在大鼠睾丸第一精子发生波中的表达变化
目的:为探讨uPAR在大鼠精子发生中的作用,研究在第一个精子发生波中大鼠睾丸组织uPAR的mRNA表达及蛋白表达变化。方法:将SD大鼠按生后年龄分组,以出生当天为d0,取出生后d0、d5、d10、d15、d21、d28、d35、d42、d49、d56大鼠睾丸组织,用实时荧光定量分析方法检测各年龄组大鼠uPAR mRNA表达,并用Western印迹法检测各年龄组大鼠uPAR蛋白表达。结果:大鼠睾丸组织uPAR mRNA表达与蛋白表达表现出相似的趋势:刚出生时表达水平较高,后逐渐下降,约在d15天达最低,到d28时开始增加,到d35时到达高峰,随后d42下降,后保持一定的水平。双变量回归相关分析显示uPAR mRNA表达与蛋白表达为中度正相关。结论:大鼠睾丸uPAR表达在第一精子发生波中出现两个高峰,第一高峰在刚出生时,这个时期生殖母细胞向基底部迁移的时期,这在精子以后的发生中起着至关重要的作用,说明uPAR与精子发生的启动有着密切联系。第二高峰是在第5周升高明显,第5周为精子变形并开始向管腔排放的时期,这说明uPAR可能参与精子排放过程中的组织重塑及精子变态过程。
二、大鼠睾丸生殖细胞培养体系的建立
大鼠睾丸生殖细胞培养体系的建立是体外研究睾丸精子发生过程中各种调控因素的的重要工具。本实验通过建立支持细胞及生精细胞两种培养体系,为进一步研究uPAR在精子发生中的作用奠定基础。
(一)大鼠睾丸支持细胞的分离纯化和鉴定
目的培养高纯度的大鼠睾丸支持细胞,并应用检测ABP mRNA原位杂交方法鉴定分离培养的支持细胞。方法选用18～22天龄SD雄性大鼠睾丸,采用0.25%胰蛋白酶、0.1%透明质酸酶、0.1%胶原酶三酶依次消化法分离支持细胞,放于32℃5%CO2的培养箱培养,48小时后用20mmol Tris—HCl低渗处理培养细胞。培养一周后应用伊红染色、吖啶橙荧光染色、Feulgen染色对所培养支持细胞进行鉴定,同时应用原位杂交检测ABP mRNA方法鉴定分离培养的支持细胞。结果培养一周后所获培养的支持细胞纯度达95%以上。分离培养的支持细胞ABP mRNA表达阳性,其形态结构特征与用其它方法鉴定为支持细胞的形态结构特征一致。结论采用三酶连续消化及低渗处理法分离培养的支持细胞纯度高,而且应用原位杂交方法检测ABP mRNA是一种新的、特异、有效的鉴定支持细胞及其功能的方法。
(二)大鼠睾丸支持细胞/生精细胞共培养系统的建立
目的建立生精细胞体外分化培养体系,为uPAR在精子发生中作用的体外研究提供一个很好的研究模型。方法取20～22天龄SD雄性大鼠睾丸,去被膜及血管,放在预冷的Hank’s液中洗两次,然后用加1 mg/ml胶原酶的F12/DMEM液32℃消化15～20min。用眼科剪将睾丸剪碎成非常小的片断,再重复用1 mg/ml胶原酶消化5～10min。细胞用内含10%胎牛血清及各种营养因子的HamF12/ DMEM培养液悬浮,约按1*106CELL/cm2的密度接种于双室培养槽或放有盖玻片的六孔板中,在32℃、5%CO2条件下培养。相差显微镜下动态观察细胞生长、形态等变化,及定期应用伊红染色、Brdu染色法对所培养生精细胞/支持细胞进行染色鉴定。结果在生精细胞/支持细胞共培养体系中,双室培养2周后,部分生精细胞胞体一端有鞭毛出现,培养4周后,培养体系中仍有一定数量的生精细胞附着在支持细胞上,部分生精细胞上可见鞭毛。单室培养的生精细胞在培养第4-5天开始出现明显脱落,培养1周后大多数生精细胞发生脱落死亡,生精细胞上未见鞭毛出现,但在培养体系中可见次级精母细胞及精子细胞。结论:应用双室培养,生精细胞可存活达一月之久,而且部分生精细胞尾部出现鞭毛,从形态上发生了减数分裂及精子变态过程。
三、应用siRNA沉默共培养生精细胞uPAR的基因表达
目的利用小分子干扰RNA(siRNA)技术沉默共培养生精细胞uPAR的基因表达,为uPAR在精子发生中的作用研究提供一个很好的研究工具。方法siRNA混合鸡尾酒是根据ShortCut RNAi Kit手册来制备。简单的说,首先通过RT-PCR应用加有T7启动子的特异性引物来合成DNA模板,然后DNA模板通过体外转录过程产生双链RNA,最后通过ShortCut RNaseIII酶消化来制备siRNAs混合物。在培养48小时用Transfect转染试剂分别将15nM、30nM siRNA混合物转染到共培养的生精细胞,48小时应用实时荧光定量RT-PCR方法检测共培养生精细胞uPAR的基因表达。该试验设立两个对照组:空白对照(未加siRNA及转染试剂)及阴性对照组(只加转染试剂,无siRNA)。结果15nM及30nM siRNA转染组uPAR mRNA表达量均明显低于阴性对照组及空白对照组(p<0.05),其中以30nMsiRNA转染组更为明显;相对于阴性对照组,15nM及30nM siRNA转染组uPAR的基因表达抑制率分别为63.5%及76.7%。结论应用ShortCut RNase III法制备的siRNAs鸡尾酒能有效地抑制共培养生精细胞uPAR基因的表达。
五、结论
在本研究中我们发现uPAR在第一精子发生波中表达最高峰为出生后35天,此时为圆形精子转变为伸长精子,并开始向管腔排放的时期。在我们的共培养体系中,来自20～22天大小的大鼠睾丸组织的生精细胞在培养二周后能分化成精子细胞及变形成为伸长型精子。而这些变化相应于体内uPAR的表达出现高峰期所发生的变化。因此在本培养体系中,应用siRNA沉默uPAR的表达,可以为研究uPAR在精子发生特定时期中的作用及机制提供一个很好的研究工具,如其信号传导通路等。此研究模型的建立也有助于减数分裂及精子变态过程的其它调控因素的研究。
Spermatogenesis is a complex differentiation process that consists of three major phases: a proliferation phase of spermatogonial cells by mitosis, a meiotic phase of spermatocytes in which recombination of genetic materials and reductive division occur and a transformation phase which produces mature spermatozoa from haploid spermatids. Tong Zhang et al investigated the expression and localization of mRNAs for urokinase PA(uPA), uPA receptor(uPAR) in monkey testes by using in-situ hybridization, the results demonstrated that uPA mRNA was expressed stage-specifically in Sertoli cells of adult testis, and uPAR mRNA was localized in germ cells of mature testis. The expression levels differed among different germ cells and at different developmental stages indicated uPA/uPAR system may play an important role in the specifical stage of spermatogenesis. The stage-specifically expressed uPA in Sertoli cells may bind to its receptor on germ cells and act as serine protease, or as growth factor through activation of signal transduction pathways by uPA receptor. It is still unclear which signal transduction pathways can be activated by uPAR in spermatogenesis. Can uPAR modulate growth and differentiation of spermatogenic cell by its signal transduction pathway? So the role of uPAR and its mechanism in spermatogenesis is worthy of further study.
Because of complex of structure and function of testitular, the physiology and biochemistry function of spermatogenic cell is difficult to further research. Establish of spermatogenic cell culture system can provide a powerful tool to study the relationship between spermatogenic cell and other cell and investigate gene regulation in spermatogenesis. RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. siRNA pairs with its cognate mRNA, leading to degradation of target mRNA and amplification of gene-specific silencing signals. siRNA has become a powerful tool for selectively silencing gene expression in cultured mammalian cells.
In present study, firstly gene and protein expression of uPAR in rat testis at first wave of spermatogenesis was investigated by real time RT-PCR and Western Blot respectively, secondly germinal cell culture system was set up, finally siRNA cocktails were used to silience the gene expression of uPAR in the co-cultured germinal cells. This research provide a new study method and technology platform to investigate the role and signal pathways of uPAR in spermatogenesis.
1.Expression of uPAR in rat testis at first wave of spermatogenesis
Objective: To elucidate the role of urokinase-plasminogen activator receptor (uPAR) system in rat spermatogenesis,gene and protein expression of uPAR in rat testis at first wave of spermatogenesis was investigated. Method: The day of birth was designated as postnatal d0 ,the male rat were divided into 10 groups according to different postnatal stages, that is, d0, d5, d10, d15, d21, d28, d35, d42, d49, d56. The gene and protein expression of uPAR in rat testis was quantitated by Real-time polymerase chain reaction (PCR) and western blot methods separately. Result: Gene and protein expression of uPAR had similar tendency in testis of rats at first wave of spermatogenesis: Express level of uPAR situate at high relatively at postnatal d0, declined to a lowest level at postnatal d15, then increased obviously at postnatal d28 and reached peak at postnatal d35, then declines at postnatal d42 and retained low afterwards. Regression and correlation analysis showed that there was a moderately positive correlation between the gene and protein expression levels of uPAR. Discussion:Expression of uPAR exhibited two peak in testis of rats at first wave of spermatogenesis: first peak was at short after birth, when gonacyte migrated to the basilar part of seminiferous tubules and was crucial to initiate spermatogenesis, which indicated uPAR had intimate relationship whit onset of spermatogenesis. Second peak was at postnatal d35 when round spermatids transformed to elongate spermatids and began to spermiation, which indicated uPAR may be involved in spermiation and/or spermiogenesis.
2.Establish of germinal cell culture system of rat testis
Establishment of germinal cell culture system of rat testis is an important instrument to investigate the regulator involved in spermatogenesis. This experiment is to set up both sertoli / spermatogenic cells coculture system for further research of role of uPAR in spermatogenesis.
2.1 Isolation、purification and identification of sertoli cells from rat testis
Objective To obtain highly pure cultured sertoli cells from rat testis and identificate cultured cell by in situ hybridization for ABP mRNA. Methods Testis from 18～22-day-old SD rats were removed and decapsulated, then chopped and sequentially digested with three enzymes at 37℃: first with 0.25% trypsin for 15 minutes, then with 0.1% hyaluronidase for 30 minutes, last with 0.1% collagenase V for 2～3 hours. The isolated cells were incubated at 32℃in a humidified atmosphere of 5% CO2. To increase the purity of the Sertoli cells, cultured cells were subjected to hypotonic shock(treatment) with 20 mmol Tris—HCl after 48 hours incubation. After a week incubation, cultured cells were identificated by HE staining、AO fluorescence staining、Feulgen staining, at the same time by in situ hybridization with digoxin-labeled rat ABP cDNA probe. Conclusion Over 95% cultured cells were sertoli cells. Most of cultured cells expressed ABP mRNA and their structural features were the same as sertoli cells identificated by other methods. Results Highly pure sertoli cell can obstained by sequentially digested with three enzymes and hypotonic shock. As a specifical secreted protein by sertoli cells in testes, ABP mRNA detected by in situ hybridization is a new、specific、effective identification method of sertoli cells from testis. Meanwhile as an index of changes in sertoli cell function, this method can be used to study cultured sertoli cell function.
2.2 Coculture of Rat Sertoli/Spermatogenic Cells
Object To further study the role of uPAR in spermatogenesis in vitro, rat Sertoli/Spermatogenic cells coculture system was established. Method: The testis were removed from 20～22 day old SD rats and decapsulated. The tissues were washed twice with ice-cold Hank’s solution, were digested at 32℃in F12/DMEM containing 1 mg/ml collagenase at 32℃for 15～20 min, then were cut into small fragments and by 1 mg/ml collagenase again for 5～10 min. The cells were resuspended in culture medium supplemented various nutrition factors and 10% fetal calf serum, and seeded at about 106 cells/cm2 in bicameral chambers or culture plates covered with glass. Coculture was carried out at 32℃in a water-saturated atmosphere of 95% air and 5% CO2. The growth and morphology of coculture cells were monitored daily under contrast phase microscope, and were identificated by HE staining、Brdu staining at specific day. Results In bicameral culture system, flagella were seen emerging from one end of some spermatogenic cells after two-week coculture and a certain number of spermatogenic cells still attached on the surface of sertoli cell after four-week cultue while some of them has a short flagellum at one end. In unicameral culture system, spermatogenic cell began obviously to fall off the surface of sertoli cell after 4～5 day culture, there are only a few spermatogenic cells attached the surface of sertoli cell after one week coculture. But the second spermatocyte and spermatid were observed except for flagellum in this unicameral culture system. Conclusion: In bicameral culture system, the spermatogenic cells survived as long as four week, and some of them generated flagella at one end, which indicated that the process of meiosis and spermiogenesis could taken place in this culture system.
3. Inhibit uPAR gene expression by siRNA cocktails in co-cultred spermatogenic cells
Objective To futher study the role of uPAR in spermatogenesis, uPAR gene expression were inhibit by siRNA cocktails in co-cultred spermatogenic cell. Methods siRNA cocktails were generated according to shortcut RNAi Kit handbook. Biefly,suitable DNA templates were generated by RT-PCR using specific primers with appended T7 promoters, then the DNA template produces double-stranded RNA by in vitro transcription, finally digested by ShortCut RNase III digestion to produces a mixture of siRNAs. 15nM or 30nM siRNA cocktails were transfected in co-cultured spermatogenic cell using RNAiFect Transfection Reagent at 48 hours of culture. After 48 hours transfected, expression of uPAR mRNA were ananysized by real time RT-PCR.. The study consisted of two control groups; blank control group treated with neither siRNA nor transfection reagent and negative control group treated only with transfection reagent. Results The expression level of uPAR mRNA with 15nM and 30 nM of siRNA cocktails were significantly lower than those of negative control and blank control group(p<0.05); The inhibition rate silencing with 15nM and 30 nM of the siRNA cocktails were 63.5% and 76.7% separately compared with negative control group. Conclution siRNA cocktail producted by shortcut Rnase III can efficially inhibited the gene expression of uPAR in co-cultured spermatogenic cell.
Conclusion
In present study, expression of uPAR reached peak at postnatal d35 in testis of rats at first wave of spermatogenesis, when round spermatids transformed to elongate spermatids and began to spermiation. In our co-culture system, spermatogenic cell from 20-22 day old rat testis differentiated into spermatids and transformed to elongate spermatid during two-week co-culture. These changes were corresponding to those in vivo when expression of uPAR reached the peak. Hence, silencing uPAR gene expression with siRNA cocktails on this culture system can provide a powerful tool to further study the role and mechanism of uPAR involved in specific stages of spermatogenic cell development, such as its signal transduction in spermatogenesis. In the meantime, this model can also be helpful to research for other regulator invovled in meiosis and spermiogenesis.
引文
[1] Mondino A, Blasi F. uPA and uPAR in fibrinolysis, immunity and pathology. Trends Immunol. 2004;25(8):450～5.
[2] Das A, Boyd N, Jones TR, et al. Inhibition of choroidal neovascularization by a peptide inhibitor of the urokinase plasminogen activator and receptor system in a mouse model. Arch Ophthalmol. 2004;122(12):1844～9.
[3] Stoppelli MP, Corti A, Soffientini A, et al. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci USA 1985; 82: 4939–4943.
[4] Vassalli JD. The urokinase receptor. Fibrinolysi 1994,8(Supply);172
[5] Ragno P. The urokinase receptor: a ligand or a receptor? Story of a sociable molecule. Cell Mol Life Sci. 2006; 63(9): 1028-37.
[6] Chaurasia P, Aguirre-Ghiso JA, Liang OD, et al. A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth. J Biol Chem. 2006; 281(21): 14852-63.
[7] Alfano D, Franco P, Vocca I, el. The urokinase plasminogen activator and its receptor: Role in cell growth and apoptosis. Thromb Haemost. 2005;93(2):205～11.
[8]丁晓芳,熊承良.尿激酶型纤溶酶原激活因子在精子趋化运动中的作用.中华男科学. 2005; 11(6): 409-12, 418.
[9]郑萍,邹如金,刘以训.猕猴精浆纤溶酶原激活因子的来源及在精子获能中的作用.生理学报. 2001; 53(1): 45-50.
[10]王玲,官黄涛,田永红,等.少弱精子症和正常生育男性精液中尿激酶及其受体含量的研究.中华男科学杂志. 2006; 12(9):791-793
[11] Zhang Z, Renfree MB, Short RV. Successful Intra- and Interspecific Male Germ Cell Transplantation in the Rat. Biol Reprod, 2003, 68:961-967.
[12] Ellis PJ, Furlong RA, Wilson A, et al. Modulation of the mouse testis transcriptome during postnatal development and in selected models of male infertility. Mol Hum Reprod. 2004; 10(4):271-81.
[13] Vigueras-Villasenor RM, Moreno-Mendoza NA,et al. The effect of estrogen on testicular gonocyte maturation. Reprod Toxicol. 2006; 22(3):513-20.
[14] Killian J, Pratis K, Clifton RJ, et al. 5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development. Biol Reprod. 2003; 68(5): 1711-8.
[15] Jahnukainen K, Chrysis D, Hou M, et al. Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biol Reprod. 2004; 70(2): 290-6.
[16]石建莉,徐晨.支持细胞与其周围细胞之间的相互作用.生殖与避孕,2003,22(1):42-5.
[17]Zhou H, Vassalli JD. The receptor for urokinase-type plasminogen activator is expressed during mouse spermatogenesis. FEBS Lett. 1997; 413(1): 11-5.
[18]Zhang T, Zhou HM, Liu YX. Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Mol Hum Reprod.1997; 3(3): 223–231.
[19]Teng X, Yang J, Xie Y, et al. A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis. Biochem Biophys Res Commun. 2006; 342(4): 1223-7
[20] Aguirre-Ghiso JA, Estrada Y, et al. ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK). Cancer Res. 2003;63(7):1684～95.
[21] Cao DJ, Guo YL, Colman RW. Urokinase-type plasminogen activator receptor is involved in mediating the apoptotic effect of cleaved high molecular weight kininogen in human endothelial cells. Circ Res. 2004;94(9):1227～34.
[22] Kusch A, Tkachuk S, Haller H, et al. Urokinase stimulates human vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk2 interaction. J Biol Chem. 2000;275(50):39466～73.
[23] Guerrero J, Santibanez JF, Gonzalez A, et al. EGF receptor transactivation by urokinase receptor stimulus through a mechanism involving Src and matrix metalloproteinases. Exp Cell Res. 2004;292(1):201～8.
[24] Canipari R, Galdieri M. Retinoid modulation of plasminogen activator production in rat Sertoli cells. Biol Reprod. 2000;63(2):544-50.
[1]熊承良,吴明章,刘继红,等.人类精子学〔M〕.武汉:湖北科学技术出版社,2002,33:414.
[2] Kokk K, Verajankorva E, Wu XK, et al. Expression of insulin signaling transmitters and glucose transporters at the protein level in the rat testis. Ann N Y Acad Sci. 2007;1095:262-73.
[3] Kim SK, Yoon YD, Park YS, et al. Involvement of the Fas-Fas ligand system and active caspase-3 in abnormal apoptosis in human testes with maturation arrest and Sertoli cell-only syndrome. Fertil Steril. 2007;87(3):547-53.
[4]丁训诚,蒋学之,顾祖维,等.男性生殖毒理学〔M〕.北京:中国人口出版社,1997.37-65.
[5] Wang RS, Yeh S, Chen LM, et al. Androgen receptor in sertoli cell is essential for germ cell nursery and junctional complex formation in mouse testes. Endocrinology. 2006;147(12):5624-33.
[6]Kodani M, Kodani K. The in vitro cultivation of mammalian Sertoli cells. Proc Natl Acad Sci U S A. 1966 Oct;56(4):1200-6.
[7] Guo J, Tao SX, Chen M, et al. Heat treatment induces liver receptor homolog-1 expression in monkey and rat sertoli cells. Endocrinology. 2007;148(3):1255-65.
[8] Noguchi J, Ohnuma K, Ozawa M,et al. Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord. J Reprod Dev. 2006;52(3):383-9.
[9] SHANGGUAN Fang-fang, SHI Xiao-lin. The promotive effect of sertoli cell on neurons in vitro. Acta Anatomica Sinica,2003,34(5):510-513.
[10] Boulogne B, Habert R, Levacher C. Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids, triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells. Mol Reprod Dev,2003,65(2):194-203.
[11] Shamekh R, Newcomb J, Mallery J, et al. Survival of rat or mouse ventral mesencephalon neurons after cotransplantaion with rat sertoli cells in the mouse striatum.Cell Transplant,2005,14(8):551-64.
[12] Valdes-Gonzalez R, Silva-Torres L, Ramirez-Gonzalez B,et al. Method for evaluating quality of cultured neonatal pig Sertoli cells. Xenotransplantation,2005,12(4):316-23.
[13] Teng Y, Xue WJ, Ding XM,et al. Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro. Chin Med J (Engl),2005,118(22):1857-62.
[14] Anway MD, Folmer J, Wright WW, Zirkin BR. Isolation of Sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function. Biol Reprod,2003, 68(3):996-1002.
[15] DING Xun-cheng,JIANG Xue Zhi, GU Zhu Wei, et al. Male Reproduction Toxicology[M].Beijing: Chinese Population Press. 1997,1997,230.
[16] Dufour JM, Rajotte RV, Seeberger K, et al. Long-term survival of neonatal porcine Sertoli cells in non-immunosuppressed rats. Xenotransplantation,2003, 10(6):577-86.
[17] DUAN Xiu-qing, SONG Chun, XU Ping, et al. Seperation and identification of rat FasL+ sertoli cells. Basic&Clinical Medicine,2004,24(1):88-91.
[18] Cameron DF, Hushen JJ, Dejarlais T, et al. A unique cytoplasmic marker for extratesticular Sertoli cells. Cell Transplant, 2002, 11(6):507-12.
[19] Hemendinger RA, Gores P, Blacksten L, et al. Identification of a specific Sertoli cell marker, Sox9, for use in transplantation. Cell Transplant, 2002, 11(6):499-505.
[20] Saxlund MA, Sadler-Riggleman I, Skinner MK. Role of Basic Helix-Loop-Helix (bHLH) and CREB Transcription Factors in the Regulation of Sertoli Cell Androgen-Binding Protein Expression. Mol Reprod Dev, 2004, 68(3):269-78.
[21] DING Yun-ming, YE Shi-juan,CHEN Yong-mei,et al. Postnatal change of androgen binding protein mRNA in rat sertoli cell.Chinese Journal of Andrology,1998,12(1):2-6.
[22] Cristina Esteban, Anne Gerard, Sara Larrib, et al. Sertoli cell-spercific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages. Mol Cell Endocrinol ,1997,132(1-2):127-36.
[1]熊承良,吴明章,刘继红,等.人类精子学〔M〕.武汉:湖北科学技术出版社,2002,33:414.
[2] Sacher F, Moller C, Bone W,et al. The expression of the testis-specific Dyrk4 kinase is highly restricted to step 8 spermatids but is not required for male fertility in mice. Mol Cell Endocrinol. 2007;267(1-2):80-8.
[3] Li MW, Xia W, Mruk DD, et al.Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes. J Endocrinol. 2006;190(2):313-29.
[4] Pareek TK, Joshi AR, Sanyal A, et al. Insights into male germ cell apoptosis due to depletion of gonadotropins caused by GnRH antagonists. Apoptosis. 2007 Feb 1; [Epub ahead of print]
[5] Li W, Zhang J, Liu X, et al. Correlation of appearance of metastasis-associated protein1 (Mta1) with spermatogenesis in developing mouse testis. Cell Tissue Res. 2007 Apr 2; [Epub ahead of print]
[6] Qiu GF, Ramachandra RK, Rexroad CE , et al. Molecular characterization and expression profiles of cyclin B1, B2 and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout (Oncorhynchus mykiss). Anim Reprod Sci. 2007 Mar 7; [Epub ahead of print]
[7] STEINBERGER A, STEINBERGER E. Differentiation of rats seminiferouse epithelium in organ culture。Differentiation of rat seminiferous epithelium in organ culture. J Reprod Fertil. 1965;9:243-8
[8] Hue D. Staub C. Perrard-Sapori MH, et al .Meiotic differentiation of germinal cells in three- week cultures of whole cell population from rat seminiferous tubules. Biol Reprod.1998;59(2):379-387.
[9]Sousa M, Cremades N, Alves C,et al. Developmental potential of human spermatogenic cells co-cultured with sertoli cells. Hum Reprod. 2002;17(1):161-72.
[10] Magueresse-Battistoni BL. Gerard N. Jegou B. Pachytene spermatocytes can achieve meiotic process in vitro. Biochem Biophys Res Commun.1991;179(2):1115-1121.
[11] Kierszenbaum AL. Mammalian spermatogenesis in vivo and in vitro: a partnership of spermatogenic and somatic cell lineages. Endocr Rev, 1994;15(1):116-134.
[12] Orth JM, McGuinness MP, Qiu J, et al. Use of in vitro systems to study male germ cell development in neonatal rats. Theriogenology. 1998;49(2):431-9.
[13]刘锋,邹彦,邓志华,等.人类生精细胞体外培养的初步研究.中国男科学杂志,2006;20( 3):32-37
[14]Tesarik J, Balaban B. In vitro spermatogenesis resumption in men with maturation arrest: relationship with in vivo blocking stage and serum FSH. Hum Reprod 2000;15(6):1350-1354.
[15]黄宇烽,许瑞吉主编.男科诊断学[M].上海:第二军医大学出版社,1999.14-39.
[16] Bellve AR, Cavicchia JC, Millette CF,et al. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol. 1977;74(1):68-85.
[17] Staub C. Hue D. Nicolle JC, et al .The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells. Exp Cell Res. 2000;260(1) : 85-95.
[18]丁训诚,蒋学之,顾祖维,等.男性生殖毒理学〔M〕.北京:中国人口出版社,1997.221-236.
[1] Kiyan J, Kiyan R, Haller H, et al. Urokinaseinduced signalling in human vascular smooth muscle cells is mediated by PDGFR-b. EMBO J. 2005;24: 1787–1797
[2] Shetty S. Regulation of urokinase receptor mRNA stability by hnRNP C in lung epithelial cells. Mol. Cell Biochem. 2005;272: 107–118
[3] Li Y, Lawrence DA, Zhang L. Sequences within domain II of the urokinase receptorcritical for differential ligand recognition.J Biol Chem. 2003; 278(32): 29925-32.
[4] Colman RW, Pixley RA, Najamunnisa S, et al. Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor. J Clin Invest. 1997;100: 1481–1487
[5] Chaurasia P, Aguirre-Ghiso JA, Liang OD, et al. A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth. J Biol Chem. 2006; 281(21): 14852-63.
[6]熊承良,黄勋彬,夏文家,等.人精子中尿激酶型纤溶酶原激活因子及其受体的分布.生殖与避孕,1996,16(6):422 424.
[7]王玲,官黄涛,田永红,熊承良.少弱精子症和正常生育男性精液中尿激酶及其受体含量的研究.中华男科学杂志,2006,12(9):791-793
[8]丁晓芳,熊承良.尿激酶受体在尿激酶诱导小鼠精子趋化运动中的作用.生殖医学杂志,2005,4(5):297-299.
[9] Zhou H, Vassalli JD. The receptor for urokinase-type plasminogen activator is expressed during mouse spermatogenesis. FEBS Lett. 1997; 413(1): 11-5.
[10] Tong Zhang,Hong-Ming Zhou,Yi-Xun Liu et al. Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Molecular Human Reproduction.1997; 3(3): 223–231.
[11]Fire A,Xu S,Montgomery MK, et a1.Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature,1998,391(6669):806-811.
[12] Chakraborty C. Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing. Curr Drug Targets. 2007; 8(3): 469-82.
[13] Zhang SL, Lu YM, Meng LR, et al. Inhibition of invasive and chemotactic abilities of SKOV3 cells by human epithelial growth receptor-2 small interfering RNA.Zhonghua FuChan Ke Za Zhi. 2007; 42(1):48-53.
[14] Kalota A, Opalinska JB. Design of antisense oligonucleotides and short interfering RNA duplexes (siRNA) targeted to BCL6 mRNA: Towards rational drug development for specific lymphoma subsets.Blood Cells Mol Dis. 2007;38(3):199-203.
[15] Jain KK. Commercial potential of RNAi.Mol Biosyst. 2006;2(11):523-6.
[16] Kabilova TO, Vladimirova AV, Repkova MN, et al. Silencing of c-myc gene expression by enzymatically and chemically synthesized siRNAs. Mol Biol (Mosk). 2006;40(6):1037-46.
[17] Fan Y, Xin XY, Chen BL, et al. Knockdown of RAB25 expression by RNAi inhibits growth of human epithelial ovarian cancer cells in vitro and in vivo. Pathology. 2006;38(6):561-7.
[18] Mohapatra JK, Sanyal A, Hemadri D, et al. Evaluation of in vitro inhibitory potential of small interfering RNAs directed against various regions of foot-and-mouth disease virus genome. Biochem Biophys Res Commun. 2005;329(3):1133-8.
[19] Li L, Yang L, Scudiero DA, et al. Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells. Gene Ther. 2007 Mar 1; [Epub ahead of print]
[20] Tang G, Reinhart BJ, Bartel DP, et al. A biochemical framework for RNA silencing in plants.Genes Dev. 2003;17(1):49-63.
[21] Yang D, Buchholz F, Huang Z, et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci U S A. 2002; 99(15): 9942-7.
[22]Dun Yang, Frank Buchholz, Zhongdong Huang,et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells。Proc Natl Acad Sci U S A. 2002 ; 99(15): 9942–9947.
[1] Vassalli JD,Baccino D,Belin D. A cellular binding site for the Mr55,000 form of the human plasminogen activator,urokinase.J Cell Biol,1985, (1):86-92
[2] Sitrin RG, Pan PM, Blackwood RA, et al. Cutting edge: evidence for a signaling partnership between urokinase receptors (CD87) and L-selectin (CD62L) in human polymorphonuclear neutrophils. J Immunol. 2001;166(8):4822-5.
[3] Ploug M, Ronne E, Behrendt N, et al. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem, 1991,266:1926-1933.
[4] Ploug M, Kjalke M, Ronne E, et al. Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. J Biol Chem, 1993,268: 17539–17546.
[5] Behrendt N, Ploug M, Patthy L, et al. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem,1991,266:7842–7847.
[6] Llinas P, Le Du MH, Gardsvoll H, et al. Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.EMBO J. 2005, 24: 1655–1663.
[7] Stoppelli MP, Corti A, Soffientini A, et al. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci USA.1985, 82: 4939–4943.
[8] Behrendt N, Ronne E, Dano K. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains. J Biol Chem.1996;271(37):22885-94.
[9] Ploug M. Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site. Biochemistry. 1998, 37: 16494–16505
[10] Behrendt N, Ploug M, Patthy L, et al. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem. 1991,266 (12): 7842–7847.
[11] Hoyer-Hansen G, Ronne E, Solberg H, et al. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem. 1992,267: 18224–18229.
[12] Resnati M, Guttinger M, Valcamonica S, et al. Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. EMBO J. 1996, 15 (7): 1572–1582.
[13] 24 Andolfo A, English WR., Resnati M, et al. Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in the intact soluble receptor. Thromb. Haemost. 2002, 88: 298–306
[14] Montuori N, Visconte V, Rossi G, et al. Soluble and cleaved forms of the urokinase-receptor: degradation products or active molecules? Thromb. Haemost. 2005, 93: 192–198.
[15] Montuori N, Carriero MV., Salzano S, et al.Rossi G. and Ragno P. The cleavage of the urokinase receptor regulates its multiple functions. J. Biol. Chem. 2002, 277: 46932–46939.
[16] Zhou H. M., Nichols A., Meda P. and Vassalli J. D. (2000) Urokinase-type plasminogen activator and its receptor synergize to promote pathogenic proteolysis. EMBO J. 19: 4817–4826
[17] Wilhelm O. G., Wilhelm S., Escott G. M., Lutz V., Magdolen V., Schmitt M. et al. (1999) Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression. J. Cell Physiol. 180:225–235
[18] Pannell R, Gurewich V. Activation of plasminogen by single-chain urokinase or by two-chain urokinase-A. Blood, 1987,69:22
[19] Gardsvoll H., Dano K. and Ploug M. (1999) Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis. J. Biol. Chem. 274: 37995–38003
[20] Heron Yu, Schultz RM. Relationship between secreted urokinase plasminogen activator activity and metastatic potential in murine B16 cells transfected with human urokinase senseand antisense genes. CancerRes, 1990,50:7623
[21] Li Y, Lawrence DA, Zhang L. Sequences within domain II of the urokinase receptor critical for differential ligand recognition.J Biol Chem. 2003;278(32):29925-32.
[22] Colman RW, Pixley RA., Najamunnisa S, at al. Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor. J Clin Invest. 1997; 100: 1481–1487
[23] Sidenius N, Blasi F. Domain 1 of the urokinase receptor (uPAR) is required for uPAR-mediated cell binding to vitronectin. FEBS Lett. 2000; 470: 40–46
[24] Sidenius N, Andolfo A, Fesce R, et al. Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization. J. Biol. Chem. 2002; 277: 27982–27990
[25]Cunningham O, Andolfo A, Santovito ML, et al. Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions. EMBO J. 2003; 22: 5994–6003
[26] Chavakis T, Kanse SM, Lupu F, et al. Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor-dependent interactions. Blood. 2000; 96: 514–522
[27] Dellas C, Loskutoff DJ. Historical analysis of PAI-1 from its discovery to its potential role in cell motility and disease. Thromb. Haemost. 2005;93: 631–640
[28] Docheva D, Popov C, Mutschler W, et al. Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system.J Cell Mol Med. 2007;11(1):21-38.
[29] Wei Y, Lukashev M, Simon DI, et al. Regulation of integrin function by the urokinase receptor. Science. 1996; 273: 1551–1555
[30] Xue W, Mizukami I, Todd RF, et al. Urokinase-type plasminogen activator receptors associate withβ1 andβ3 integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res. 1997; 57: 1682–1689
[31] Xue W, Kindzelskii AL, Todd RF, et al. Physical association of complement receptor type 3 and urokinase-type plasminogen activator receptor in neutrophil membranes. J Immunol. 1994; 152: 4630–4640
[32] Bohuslav J, Horejsi V, Hansmann C, et al. Urokinase plasminogen activator receptor, b2-integrins and Src-kinases within a single receptor complex of human monocytes. J Exp Med. 1995; 181: 1381–1390
[33] Simon DI., Wei Y, Zhang L, et al. Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function. J Biol Chem. 2000; 275: 10228–10234
[34] May AE, Kanse SM., Lund LR., et al. Urokinase receptor (CD87) regulates leukocyte recruitment via b2 integrins in vivo. J Exp Med. 1998; 188: 1029–1037
[35] Pluskota E, Soloviev DA, Plow EF. Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin aMb2 as well as by the urokinase receptor regulates cell adhesion and migration. Blood. 2003;101:1582–1590
[36] Pluskota E, Soloviev DA, et al. Integrin aMb2 orchestrates and accelerates plasminogen activation and fibrinolysis by neutrophils. J. Biol. Chem. 2004; 279: 18063–18072
[37] Chapman HA, Wei Y, Simon DI, et al. Role of urokinase receptor and caveolin in regulation of integrin signalling. Thromb. Haemost. 1999,82: 291–7
[38] Wei Y, Eble JA, Wang Z, et al. Urokinase receptors promote b1 integrin function through interactions with integrin a3 b1. Mol. Biol. Cell. 2001; 12: 2975–2986
[39] Aguirre Ghiso JA, Kovalski K, Ossowski L. Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signalling. J. Cell Biol. 1999; 147: 89–104
[40]Aguirre-Ghiso JA, Liu D, Mignatti A, et al. Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo. Mol. Biol. Cell. 2001;12: 863–879
[41] Monaghan E, Gueorguiev V, Wilkins-Port C, et al. The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts. J. Biol. Chem. 2004; 279: 1400–1407
[42]Tarui T, Andronicos N, Czekay RP, et al. Critical role of integrinα5β1 in urokinase (uPA)/urokinase receptor (uPAR, CD87) signalling. J. Biol. Chem. 2003; 278: 29863–29872
[43]Degryse B, Resnati M, Czekay RP, et al. Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signalling activity:generation of a new integrin inhibitor. J. Biol. Chem. 2005;280:24792–24803
[44]Montuori N, Rossi G, Ragno P. Cleavage of urokinase receptor regulates its interaction with integrins in thyroid cells. FEBS Lett. 1999; 460: 32–36
[45] Le Y, Li B, Gong W, et al. Novel pathophysiological role of classical chemotactic peptide receptors and their communications with chemokine receptors. Immunol. Rev. 2000; 177: 185–94
[46] Le Y., Murphy P. M. and Wang J. M. Formyl-peptide receptors revisited. Trends Immunol. 2002, 23: 541–8
[47] Fazioli F, Resnati M, Sidenius N, et al. A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity. EMBO J. 1997; 16: 7279–7286
[48] Resnati M, Pallavicini I, Wang JM, et al. The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor。FPRL1/LXA4R. Proc. Natl. Acad. Sci. USA. 2002; 99: 1359–1364
[49] Furlan F, Orlando S, Laudanna C, et al. The soluble D2D3(88–274) fragment of the urokinase receptor inhibits monocyte chemotaxis and integrin-dependent cell adhesion. J. Cell Sci. 2004; 117: 2909–2916
[50]de Paulis A, Montuori N, Prevete N, et al. Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2. J. Immunol. 2004; 173: 5739–48
[51] Selleri C, Montuori N, Ricci P, et al. Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization. Blood. 2005; 105: 2198–2205
[52] Gargiulo L, Longanesi-Cattani I., Bifulco K, et al. Cross-talk between fMLP and vitronectin receptors triggered by urokinase receptor-derived SRSRY peptide. J. Biol. Chem. 2005; 280: 25225–25232
[53] Behrendt N, Ploug M, Patthy L, et al. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J. Biol.Chem. 1991; 266: 7842–7847
[54]Hoyer-Hansen G, Pessara U, et al. Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor. Biochem J. 2001; 358: 673–679
[55] de Paulis A, Montuori N, Prevete N, et al. Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2. J. Immunol. 2004; 173: 5739–48
[56] Ali H, Richardson R M, Haribabu B, et al. Chemoattractant receptorcross-desensitization. J Biol Chem. 1999; 274: 6027–6030
[57] Le Y, Shen W, Li B, et al. A new insight into the role of‘old’chemotactic peptide receptors FPR and FPRL1: down-regulation of chemokine receptors CCR5 and CXCR4. Forum. 1999;9: 299–314
[58] Li B. Q, Wetzel MA, Mikovits JA, et al. The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1. Blood. 2001;97: 2941–2947
[59] Selleri C, Montuori N, Ricci P, et al. Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization. Blood. 2005, 105: 2198–2205
[60] Comoglio PM, Boccaccio C, Trusolino L. Interactions between growth factor receptors and adhesion molecules: breaking the rules. Curr Opin Cell Biol. 2003;15: 565–571
[61] Aguirre Ghiso JA, Kovalski K, Ossowski L. Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signalling. J Cell Biol. 1999; 147: 89–104
[62] Liu D, Aguirre Ghiso J, Estrada Y, et al. EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell. 2002;1: 445–457
[63] Jo M, Thomas KS, O’Donnell DM, et al. Epidermal growth factor receptor-dependent and–independent cell-signalling pathways originating from the urokinase receptor. J Biol Chem. 2003; 278: 1642–1646
[64] Jo M, Thomas KS., Marozkina N, et al. Dynamic assembly of the urokinasetype plasminogen activator signalling receptor complex determines the mitogenic activity of urokinase-type plasminogen activator. J Biol Chem. 2005; 280: 17449–17457
[65] Kiyan J, Kiyan R, Haller H, et al. Urokinaseinduced signalling in human vascular smooth muscle cells is mediated by PDGFR-b. EMBO J. 2005; 24: 1787–1797
[66] Soravia E, Grebe A, De Luca P, et al. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene.Blood. 1995; 86: 624–635
[67] Hapke S, Gawaz M, Dehne K, et al. b(3)A-integrin downregulates the urokinase-type plasminogen activator receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the u-PAR promoter. Mol Cell Biol. 2001; 21: 2118–2132
[68] Wang Y, Dang J, Wang H, et al. Identification of a novel nuclear factor-kappaB sequence involved in expression of urokinase-type plasminogen activator receptor. Eur J Biochem. 2000; 267: 3248–3254
[69] Wang H, Yang L, Jamaluddin MS, et al. The Kruppel-like KLF4 transcription factor, a novel regulator of urokinase receptor expression, drives synthesis of this binding site in colonic crypt luminal surface epithelial cells. J Biol Chem. 2004; 279: 22674–22683
[70]Montuori N, Rossi G,Ragno P. Post-transcriptional regulation of gene expression in the plasminogen activation system. Biol Chem. 2002; 383: 47–53
[71] Wang GJ, Collinge M, Blasi F, et al. Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement. Proc. Natl Acad Sci USA. 1998; 95: 6296–6301
[72]Tran H, Maurer F, Nagamine Y. Stabilization of urokinase and urokinase receptor mRNAs by HuR is linked to its cytoplasmic accumulation induced by activated mitogenactivated protein kinase-activated protein kinase 2. Mol Cell Biol. 2003; 23: 7177–7188
[73] Shetty S. Regulation of urokinase receptor mRNA stability by hnRNP C in lung epithelial cells. Mol Cell Biochem. 2005; 272: 107–118
[74] Shetty S, Kumar A, Idell S. Posttranscriptional regulation of urokinase receptor mRNA: identification of a novel urokinase receptor mRNA binding protein in human mesothelioma cells. Mol Cell Biol. 1997; 17: 1075–1083
[75] Shetty S, Muniyappa H, Halady P K. et al. Regulation of urokinase receptor expression by phosphoglycerate kinase. Am J Respir Cell Mol Biol. 2004; 31: 100–106
[76] Montuori N, Mattiello A, Mancini A, et al. Urokinase-type plasminogen activatorupregulates the expression of its cellular receptor through a post-transcriptional mechanism. FEBS Lett. 2001; 508: 379–384
[77] Montuori N, Mattiello A, Mancini A, et al. Urokinase-mediated posttranscriptional regulation of urokinase-receptor expression in non small cell lung carcinoma. Int J Cancer . 2003:105: 353–360
[78]Zannetti A, Del Vecchio S, Carriero MV, et al. Coordinate up-regulation of Sp1 DNA-binding activity and urokinase receptor expression in breast carcinoma. Cancer Res. 2000,60: 1546–1551
[1] Blasi F,Carmeliet P. uPAR: a versatile signaling orchestrator. Nat. Rev. Mol. Cell Biol. 2002; 3: 932–943
[2] Myohanen H, Vaheri A. Regulation and interactions in the activation of cell-associated plasminogen. Cell Mol Life Sci. 2004 Nov;61(22):2840-58.
[3]Edwards S, Lalor PF, Tuncer C, et al. Vitronectin in human hepatic tumours contributes to the recruitment of lymphocytes in an alpha v beta3-independent manner. Br J Cancer. 2006; 95(11):1545-54.
[4] Chaurasia P, Aguirre-Ghiso JA, Liang OD, et al. A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth. J Biol Chem. 2006;281(21):14852-63.
[5] Zhou H, Vassalli JD. The receptor for urokinase-type plasminogen activator is expressed during mouse spermatogenesis. FEBS Lett. 1997; 413(1): 11-5.
[6] Tong Zhang, Hong-Ming Zhou、Yi-Xun Liu, et al. Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Molecular Human Reproduction .1997; 3(3): 223–231.
[7] Teng X, Yang J, Xie Y, et al. A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis. Biochem Biophys Res Commun. 2006; 342(4): 1223-7.
[8] Huang Donghui, Zhao hu,Li Honggang, et al. Gene expression of urokinase plasminogen activator and urokinase plasminogen activator receptor in rat testis at postnatal stages. Asian Journal of Andrology,2007
[9]熊承良,黄勋彬,夏文家,等.人精子中尿激酶型纤溶酶原激活因子及其受体的分布[J].生殖与避孕,1996,16(6):422 424.
[10]王玲,官黄涛,田永红,等.少弱精子症和正常生育男性精液中尿激酶及其受体含量的研究。中华男科学杂志,2006,12(9):791-793
[11]熊承良,沈继云.尿激酶对弱精症患者精子活力影响的研究.生殖医学杂志,1995,4(3):164-166.
[12] oliveira RG,Tomasi L,Rovasio RA,et a1.Increased velocity and induction of chem otactic response in mouse spermatozoa by follicular and oviductal fluids[J]. J Reprod Fe~il,1999,115(1):23-27.
[13] Georgina F,Roberto AR,Silvia C,et a1.Chemotaxis of capacitated rabbit sperm atozoa to follicular fluid revealed by a novel directionality-based assay[J].Biol Reprod,2002,67(5):1565—1571.
[14]丁晓芳,熊承良.尿激酶受体在尿激酶诱导小鼠精子趋化运动中的作用.生殖医学杂志,2005, 14( 5):297-299.
[15] Gabler C, Killian GJ, Einspanier R. Differential expression of extracellular matrix components in the bovine oviduct during the oestrous cycle. Reproduction. 2001;122(1):121-30.
[16] Ming Li, Eli G, Karakji, Rosie Xing, et al. Expression of Urokinase-Type Plasminogen Activator and Its Receptor during Ovarian Follicular Development. Endocrinology. 1997;138(7):2790-9.
[17] Dow MP, Bakke LJ, Cassar CA, et al. Gonadotropin Surge-Induced Up-Regulation of the Plasminogen Activators (Tissue Plasminogen Activator and Urokinase PlasminogenActivator) and the Urokinase Plasminogen Activator Receptor Within Bovine Periovulatory Follicular and Luteal Tissue. Biol Reprod. 2002;66(5):1413-21.
[18] Teesalu T, Blasi F, Talarico D. Embryo implantation in mouse: fetomaternal coordination in the pattern of expression of uPA, uPAR, PAI-1 and alpha 2MR/LRP genes. Mech Dev. 1996 ;56(1-2):103-16.
[19] Solberg H, Rinkenberger J, Dano K, et al. A functional overlap of plasminogen and MMPs regulates vascularization during placental development. Development. 2003; 130(18): 4439-50
[20] Pierleoni C, Samuelsen GB, Graem N, et al. Immunohistochemical identification of the receptor for urokinase plasminogen activator associated with fibrin deposition in normal and ectopic human placenta. Placenta. 1998;19(7):501-8.
[2] Das A, Boyd N, Jones TR, et al. Inhibition of choroidal neovascularization by a peptide inhibitor of the urokinase plasminogen activator and receptor system in a mouse model. Arch Ophthalmol. 2004;122(12):1844～9.
[3] Stoppelli MP, Corti A, Soffientini A, et al. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci USA 1985; 82: 4939–4943.
[4] Vassalli JD. The urokinase receptor. Fibrinolysi 1994,8(Supply);172
[5] Ragno P. The urokinase receptor: a ligand or a receptor? Story of a sociable molecule. Cell Mol Life Sci. 2006; 63(9): 1028-37.
[6] Chaurasia P, Aguirre-Ghiso JA, Liang OD, et al. A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth. J Biol Chem. 2006; 281(21): 14852-63.
[7] Alfano D, Franco P, Vocca I, el. The urokinase plasminogen activator and its receptor: Role in cell growth and apoptosis. Thromb Haemost. 2005;93(2):205～11.
[8]丁晓芳,熊承良.尿激酶型纤溶酶原激活因子在精子趋化运动中的作用.中华男科学. 2005; 11(6): 409-12, 418.
[9]郑萍,邹如金,刘以训.猕猴精浆纤溶酶原激活因子的来源及在精子获能中的作用.生理学报. 2001; 53(1): 45-50.
[10]王玲,官黄涛,田永红,等.少弱精子症和正常生育男性精液中尿激酶及其受体含量的研究.中华男科学杂志. 2006; 12(9):791-793
[11] Zhang Z, Renfree MB, Short RV. Successful Intra- and Interspecific Male Germ Cell Transplantation in the Rat. Biol Reprod, 2003, 68:961-967.
[12] Ellis PJ, Furlong RA, Wilson A, et al. Modulation of the mouse testis transcriptome during postnatal development and in selected models of male infertility. Mol Hum Reprod. 2004; 10(4):271-81.
[13] Vigueras-Villasenor RM, Moreno-Mendoza NA,et al. The effect of estrogen on testicular gonocyte maturation. Reprod Toxicol. 2006; 22(3):513-20.
[14] Killian J, Pratis K, Clifton RJ, et al. 5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development. Biol Reprod. 2003; 68(5): 1711-8.
[15] Jahnukainen K, Chrysis D, Hou M, et al. Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis. Biol Reprod. 2004; 70(2): 290-6.
[16]石建莉,徐晨.支持细胞与其周围细胞之间的相互作用.生殖与避孕,2003,22(1):42-5.
[17]Zhou H, Vassalli JD. The receptor for urokinase-type plasminogen activator is expressed during mouse spermatogenesis. FEBS Lett. 1997; 413(1): 11-5.
[18]Zhang T, Zhou HM, Liu YX. Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Mol Hum Reprod.1997; 3(3): 223–231.
[19]Teng X, Yang J, Xie Y, et al. A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis. Biochem Biophys Res Commun. 2006; 342(4): 1223-7
[20] Aguirre-Ghiso JA, Estrada Y, et al. ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK). Cancer Res. 2003;63(7):1684～95.
[21] Cao DJ, Guo YL, Colman RW. Urokinase-type plasminogen activator receptor is involved in mediating the apoptotic effect of cleaved high molecular weight kininogen in human endothelial cells. Circ Res. 2004;94(9):1227～34.
[22] Kusch A, Tkachuk S, Haller H, et al. Urokinase stimulates human vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk2 interaction. J Biol Chem. 2000;275(50):39466～73.
[23] Guerrero J, Santibanez JF, Gonzalez A, et al. EGF receptor transactivation by urokinase receptor stimulus through a mechanism involving Src and matrix metalloproteinases. Exp Cell Res. 2004;292(1):201～8.
[24] Canipari R, Galdieri M. Retinoid modulation of plasminogen activator production in rat Sertoli cells. Biol Reprod. 2000;63(2):544-50.
[1]熊承良,吴明章,刘继红,等.人类精子学〔M〕.武汉:湖北科学技术出版社,2002,33:414.
[2] Kokk K, Verajankorva E, Wu XK, et al. Expression of insulin signaling transmitters and glucose transporters at the protein level in the rat testis. Ann N Y Acad Sci. 2007;1095:262-73.
[3] Kim SK, Yoon YD, Park YS, et al. Involvement of the Fas-Fas ligand system and active caspase-3 in abnormal apoptosis in human testes with maturation arrest and Sertoli cell-only syndrome. Fertil Steril. 2007;87(3):547-53.
[4]丁训诚,蒋学之,顾祖维,等.男性生殖毒理学〔M〕.北京:中国人口出版社,1997.37-65.
[5] Wang RS, Yeh S, Chen LM, et al. Androgen receptor in sertoli cell is essential for germ cell nursery and junctional complex formation in mouse testes. Endocrinology. 2006;147(12):5624-33.
[6]Kodani M, Kodani K. The in vitro cultivation of mammalian Sertoli cells. Proc Natl Acad Sci U S A. 1966 Oct;56(4):1200-6.
[7] Guo J, Tao SX, Chen M, et al. Heat treatment induces liver receptor homolog-1 expression in monkey and rat sertoli cells. Endocrinology. 2007;148(3):1255-65.
[8] Noguchi J, Ohnuma K, Ozawa M,et al. Successful long term culture of immature porcine sertoli cells in the reconstructed testicular cell cord. J Reprod Dev. 2006;52(3):383-9.
[9] SHANGGUAN Fang-fang, SHI Xiao-lin. The promotive effect of sertoli cell on neurons in vitro. Acta Anatomica Sinica,2003,34(5):510-513.
[10] Boulogne B, Habert R, Levacher C. Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids, triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells. Mol Reprod Dev,2003,65(2):194-203.
[11] Shamekh R, Newcomb J, Mallery J, et al. Survival of rat or mouse ventral mesencephalon neurons after cotransplantaion with rat sertoli cells in the mouse striatum.Cell Transplant,2005,14(8):551-64.
[12] Valdes-Gonzalez R, Silva-Torres L, Ramirez-Gonzalez B,et al. Method for evaluating quality of cultured neonatal pig Sertoli cells. Xenotransplantation,2005,12(4):316-23.
[13] Teng Y, Xue WJ, Ding XM,et al. Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro. Chin Med J (Engl),2005,118(22):1857-62.
[14] Anway MD, Folmer J, Wright WW, Zirkin BR. Isolation of Sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function. Biol Reprod,2003, 68(3):996-1002.
[15] DING Xun-cheng,JIANG Xue Zhi, GU Zhu Wei, et al. Male Reproduction Toxicology[M].Beijing: Chinese Population Press. 1997,1997,230.
[16] Dufour JM, Rajotte RV, Seeberger K, et al. Long-term survival of neonatal porcine Sertoli cells in non-immunosuppressed rats. Xenotransplantation,2003, 10(6):577-86.
[17] DUAN Xiu-qing, SONG Chun, XU Ping, et al. Seperation and identification of rat FasL+ sertoli cells. Basic&Clinical Medicine,2004,24(1):88-91.
[18] Cameron DF, Hushen JJ, Dejarlais T, et al. A unique cytoplasmic marker for extratesticular Sertoli cells. Cell Transplant, 2002, 11(6):507-12.
[19] Hemendinger RA, Gores P, Blacksten L, et al. Identification of a specific Sertoli cell marker, Sox9, for use in transplantation. Cell Transplant, 2002, 11(6):499-505.
[20] Saxlund MA, Sadler-Riggleman I, Skinner MK. Role of Basic Helix-Loop-Helix (bHLH) and CREB Transcription Factors in the Regulation of Sertoli Cell Androgen-Binding Protein Expression. Mol Reprod Dev, 2004, 68(3):269-78.
[21] DING Yun-ming, YE Shi-juan,CHEN Yong-mei,et al. Postnatal change of androgen binding protein mRNA in rat sertoli cell.Chinese Journal of Andrology,1998,12(1):2-6.
[22] Cristina Esteban, Anne Gerard, Sara Larrib, et al. Sertoli cell-spercific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages. Mol Cell Endocrinol ,1997,132(1-2):127-36.
[1]熊承良,吴明章,刘继红,等.人类精子学〔M〕.武汉:湖北科学技术出版社,2002,33:414.
[2] Sacher F, Moller C, Bone W,et al. The expression of the testis-specific Dyrk4 kinase is highly restricted to step 8 spermatids but is not required for male fertility in mice. Mol Cell Endocrinol. 2007;267(1-2):80-8.
[3] Li MW, Xia W, Mruk DD, et al.Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes. J Endocrinol. 2006;190(2):313-29.
[4] Pareek TK, Joshi AR, Sanyal A, et al. Insights into male germ cell apoptosis due to depletion of gonadotropins caused by GnRH antagonists. Apoptosis. 2007 Feb 1; [Epub ahead of print]
[5] Li W, Zhang J, Liu X, et al. Correlation of appearance of metastasis-associated protein1 (Mta1) with spermatogenesis in developing mouse testis. Cell Tissue Res. 2007 Apr 2; [Epub ahead of print]
[6] Qiu GF, Ramachandra RK, Rexroad CE , et al. Molecular characterization and expression profiles of cyclin B1, B2 and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout (Oncorhynchus mykiss). Anim Reprod Sci. 2007 Mar 7; [Epub ahead of print]
[7] STEINBERGER A, STEINBERGER E. Differentiation of rats seminiferouse epithelium in organ culture。Differentiation of rat seminiferous epithelium in organ culture. J Reprod Fertil. 1965;9:243-8
[8] Hue D. Staub C. Perrard-Sapori MH, et al .Meiotic differentiation of germinal cells in three- week cultures of whole cell population from rat seminiferous tubules. Biol Reprod.1998;59(2):379-387.
[9]Sousa M, Cremades N, Alves C,et al. Developmental potential of human spermatogenic cells co-cultured with sertoli cells. Hum Reprod. 2002;17(1):161-72.
[10] Magueresse-Battistoni BL. Gerard N. Jegou B. Pachytene spermatocytes can achieve meiotic process in vitro. Biochem Biophys Res Commun.1991;179(2):1115-1121.
[11] Kierszenbaum AL. Mammalian spermatogenesis in vivo and in vitro: a partnership of spermatogenic and somatic cell lineages. Endocr Rev, 1994;15(1):116-134.
[12] Orth JM, McGuinness MP, Qiu J, et al. Use of in vitro systems to study male germ cell development in neonatal rats. Theriogenology. 1998;49(2):431-9.
[13]刘锋,邹彦,邓志华,等.人类生精细胞体外培养的初步研究.中国男科学杂志,2006;20( 3):32-37
[14]Tesarik J, Balaban B. In vitro spermatogenesis resumption in men with maturation arrest: relationship with in vivo blocking stage and serum FSH. Hum Reprod 2000;15(6):1350-1354.
[15]黄宇烽,许瑞吉主编.男科诊断学[M].上海:第二军医大学出版社,1999.14-39.
[16] Bellve AR, Cavicchia JC, Millette CF,et al. Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J Cell Biol. 1977;74(1):68-85.
[17] Staub C. Hue D. Nicolle JC, et al .The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells. Exp Cell Res. 2000;260(1) : 85-95.
[18]丁训诚,蒋学之,顾祖维,等.男性生殖毒理学〔M〕.北京:中国人口出版社,1997.221-236.
[1] Kiyan J, Kiyan R, Haller H, et al. Urokinaseinduced signalling in human vascular smooth muscle cells is mediated by PDGFR-b. EMBO J. 2005;24: 1787–1797
[2] Shetty S. Regulation of urokinase receptor mRNA stability by hnRNP C in lung epithelial cells. Mol. Cell Biochem. 2005;272: 107–118
[3] Li Y, Lawrence DA, Zhang L. Sequences within domain II of the urokinase receptorcritical for differential ligand recognition.J Biol Chem. 2003; 278(32): 29925-32.
[4] Colman RW, Pixley RA, Najamunnisa S, et al. Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor. J Clin Invest. 1997;100: 1481–1487
[5] Chaurasia P, Aguirre-Ghiso JA, Liang OD, et al. A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth. J Biol Chem. 2006; 281(21): 14852-63.
[6]熊承良,黄勋彬,夏文家,等.人精子中尿激酶型纤溶酶原激活因子及其受体的分布.生殖与避孕,1996,16(6):422 424.
[7]王玲,官黄涛,田永红,熊承良.少弱精子症和正常生育男性精液中尿激酶及其受体含量的研究.中华男科学杂志,2006,12(9):791-793
[8]丁晓芳,熊承良.尿激酶受体在尿激酶诱导小鼠精子趋化运动中的作用.生殖医学杂志,2005,4(5):297-299.
[9] Zhou H, Vassalli JD. The receptor for urokinase-type plasminogen activator is expressed during mouse spermatogenesis. FEBS Lett. 1997; 413(1): 11-5.
[10] Tong Zhang,Hong-Ming Zhou,Yi-Xun Liu et al. Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Molecular Human Reproduction.1997; 3(3): 223–231.
[11]Fire A,Xu S,Montgomery MK, et a1.Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans.Nature,1998,391(6669):806-811.
[12] Chakraborty C. Potentiality of small interfering RNAs (siRNA) as recent therapeutic targets for gene-silencing. Curr Drug Targets. 2007; 8(3): 469-82.
[13] Zhang SL, Lu YM, Meng LR, et al. Inhibition of invasive and chemotactic abilities of SKOV3 cells by human epithelial growth receptor-2 small interfering RNA.Zhonghua FuChan Ke Za Zhi. 2007; 42(1):48-53.
[14] Kalota A, Opalinska JB. Design of antisense oligonucleotides and short interfering RNA duplexes (siRNA) targeted to BCL6 mRNA: Towards rational drug development for specific lymphoma subsets.Blood Cells Mol Dis. 2007;38(3):199-203.
[15] Jain KK. Commercial potential of RNAi.Mol Biosyst. 2006;2(11):523-6.
[16] Kabilova TO, Vladimirova AV, Repkova MN, et al. Silencing of c-myc gene expression by enzymatically and chemically synthesized siRNAs. Mol Biol (Mosk). 2006;40(6):1037-46.
[17] Fan Y, Xin XY, Chen BL, et al. Knockdown of RAB25 expression by RNAi inhibits growth of human epithelial ovarian cancer cells in vitro and in vivo. Pathology. 2006;38(6):561-7.
[18] Mohapatra JK, Sanyal A, Hemadri D, et al. Evaluation of in vitro inhibitory potential of small interfering RNAs directed against various regions of foot-and-mouth disease virus genome. Biochem Biophys Res Commun. 2005;329(3):1133-8.
[19] Li L, Yang L, Scudiero DA, et al. Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells. Gene Ther. 2007 Mar 1; [Epub ahead of print]
[20] Tang G, Reinhart BJ, Bartel DP, et al. A biochemical framework for RNA silencing in plants.Genes Dev. 2003;17(1):49-63.
[21] Yang D, Buchholz F, Huang Z, et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci U S A. 2002; 99(15): 9942-7.
[22]Dun Yang, Frank Buchholz, Zhongdong Huang,et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells。Proc Natl Acad Sci U S A. 2002 ; 99(15): 9942–9947.
[1] Vassalli JD,Baccino D,Belin D. A cellular binding site for the Mr55,000 form of the human plasminogen activator,urokinase.J Cell Biol,1985, (1):86-92
[2] Sitrin RG, Pan PM, Blackwood RA, et al. Cutting edge: evidence for a signaling partnership between urokinase receptors (CD87) and L-selectin (CD62L) in human polymorphonuclear neutrophils. J Immunol. 2001;166(8):4822-5.
[3] Ploug M, Ronne E, Behrendt N, et al. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol. J Biol Chem, 1991,266:1926-1933.
[4] Ploug M, Kjalke M, Ronne E, et al. Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. J Biol Chem, 1993,268: 17539–17546.
[5] Behrendt N, Ploug M, Patthy L, et al. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem,1991,266:7842–7847.
[6] Llinas P, Le Du MH, Gardsvoll H, et al. Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.EMBO J. 2005, 24: 1655–1663.
[7] Stoppelli MP, Corti A, Soffientini A, et al. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci USA.1985, 82: 4939–4943.
[8] Behrendt N, Ronne E, Dano K. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains. J Biol Chem.1996;271(37):22885-94.
[9] Ploug M. Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site. Biochemistry. 1998, 37: 16494–16505
[10] Behrendt N, Ploug M, Patthy L, et al. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J Biol Chem. 1991,266 (12): 7842–7847.
[11] Hoyer-Hansen G, Ronne E, Solberg H, et al. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain. J Biol Chem. 1992,267: 18224–18229.
[12] Resnati M, Guttinger M, Valcamonica S, et al. Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. EMBO J. 1996, 15 (7): 1572–1582.
[13] 24 Andolfo A, English WR., Resnati M, et al. Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in the intact soluble receptor. Thromb. Haemost. 2002, 88: 298–306
[14] Montuori N, Visconte V, Rossi G, et al. Soluble and cleaved forms of the urokinase-receptor: degradation products or active molecules? Thromb. Haemost. 2005, 93: 192–198.
[15] Montuori N, Carriero MV., Salzano S, et al.Rossi G. and Ragno P. The cleavage of the urokinase receptor regulates its multiple functions. J. Biol. Chem. 2002, 277: 46932–46939.
[16] Zhou H. M., Nichols A., Meda P. and Vassalli J. D. (2000) Urokinase-type plasminogen activator and its receptor synergize to promote pathogenic proteolysis. EMBO J. 19: 4817–4826
[17] Wilhelm O. G., Wilhelm S., Escott G. M., Lutz V., Magdolen V., Schmitt M. et al. (1999) Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression. J. Cell Physiol. 180:225–235
[18] Pannell R, Gurewich V. Activation of plasminogen by single-chain urokinase or by two-chain urokinase-A. Blood, 1987,69:22
[19] Gardsvoll H., Dano K. and Ploug M. (1999) Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis. J. Biol. Chem. 274: 37995–38003
[20] Heron Yu, Schultz RM. Relationship between secreted urokinase plasminogen activator activity and metastatic potential in murine B16 cells transfected with human urokinase senseand antisense genes. CancerRes, 1990,50:7623
[21] Li Y, Lawrence DA, Zhang L. Sequences within domain II of the urokinase receptor critical for differential ligand recognition.J Biol Chem. 2003;278(32):29925-32.
[22] Colman RW, Pixley RA., Najamunnisa S, at al. Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor. J Clin Invest. 1997; 100: 1481–1487
[23] Sidenius N, Blasi F. Domain 1 of the urokinase receptor (uPAR) is required for uPAR-mediated cell binding to vitronectin. FEBS Lett. 2000; 470: 40–46
[24] Sidenius N, Andolfo A, Fesce R, et al. Urokinase regulates vitronectin binding by controlling urokinase receptor oligomerization. J. Biol. Chem. 2002; 277: 27982–27990
[25]Cunningham O, Andolfo A, Santovito ML, et al. Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions. EMBO J. 2003; 22: 5994–6003
[26] Chavakis T, Kanse SM, Lupu F, et al. Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor-dependent interactions. Blood. 2000; 96: 514–522
[27] Dellas C, Loskutoff DJ. Historical analysis of PAI-1 from its discovery to its potential role in cell motility and disease. Thromb. Haemost. 2005;93: 631–640
[28] Docheva D, Popov C, Mutschler W, et al. Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system.J Cell Mol Med. 2007;11(1):21-38.
[29] Wei Y, Lukashev M, Simon DI, et al. Regulation of integrin function by the urokinase receptor. Science. 1996; 273: 1551–1555
[30] Xue W, Mizukami I, Todd RF, et al. Urokinase-type plasminogen activator receptors associate withβ1 andβ3 integrins of fibrosarcoma cells: dependence on extracellular matrix components. Cancer Res. 1997; 57: 1682–1689
[31] Xue W, Kindzelskii AL, Todd RF, et al. Physical association of complement receptor type 3 and urokinase-type plasminogen activator receptor in neutrophil membranes. J Immunol. 1994; 152: 4630–4640
[32] Bohuslav J, Horejsi V, Hansmann C, et al. Urokinase plasminogen activator receptor, b2-integrins and Src-kinases within a single receptor complex of human monocytes. J Exp Med. 1995; 181: 1381–1390
[33] Simon DI., Wei Y, Zhang L, et al. Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function. J Biol Chem. 2000; 275: 10228–10234
[34] May AE, Kanse SM., Lund LR., et al. Urokinase receptor (CD87) regulates leukocyte recruitment via b2 integrins in vivo. J Exp Med. 1998; 188: 1029–1037
[35] Pluskota E, Soloviev DA, Plow EF. Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin aMb2 as well as by the urokinase receptor regulates cell adhesion and migration. Blood. 2003;101:1582–1590
[36] Pluskota E, Soloviev DA, et al. Integrin aMb2 orchestrates and accelerates plasminogen activation and fibrinolysis by neutrophils. J. Biol. Chem. 2004; 279: 18063–18072
[37] Chapman HA, Wei Y, Simon DI, et al. Role of urokinase receptor and caveolin in regulation of integrin signalling. Thromb. Haemost. 1999,82: 291–7
[38] Wei Y, Eble JA, Wang Z, et al. Urokinase receptors promote b1 integrin function through interactions with integrin a3 b1. Mol. Biol. Cell. 2001; 12: 2975–2986
[39] Aguirre Ghiso JA, Kovalski K, Ossowski L. Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signalling. J. Cell Biol. 1999; 147: 89–104
[40]Aguirre-Ghiso JA, Liu D, Mignatti A, et al. Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo. Mol. Biol. Cell. 2001;12: 863–879
[41] Monaghan E, Gueorguiev V, Wilkins-Port C, et al. The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts. J. Biol. Chem. 2004; 279: 1400–1407
[42]Tarui T, Andronicos N, Czekay RP, et al. Critical role of integrinα5β1 in urokinase (uPA)/urokinase receptor (uPAR, CD87) signalling. J. Biol. Chem. 2003; 278: 29863–29872
[43]Degryse B, Resnati M, Czekay RP, et al. Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signalling activity:generation of a new integrin inhibitor. J. Biol. Chem. 2005;280:24792–24803
[44]Montuori N, Rossi G, Ragno P. Cleavage of urokinase receptor regulates its interaction with integrins in thyroid cells. FEBS Lett. 1999; 460: 32–36
[45] Le Y, Li B, Gong W, et al. Novel pathophysiological role of classical chemotactic peptide receptors and their communications with chemokine receptors. Immunol. Rev. 2000; 177: 185–94
[46] Le Y., Murphy P. M. and Wang J. M. Formyl-peptide receptors revisited. Trends Immunol. 2002, 23: 541–8
[47] Fazioli F, Resnati M, Sidenius N, et al. A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity. EMBO J. 1997; 16: 7279–7286
[48] Resnati M, Pallavicini I, Wang JM, et al. The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor。FPRL1/LXA4R. Proc. Natl. Acad. Sci. USA. 2002; 99: 1359–1364
[49] Furlan F, Orlando S, Laudanna C, et al. The soluble D2D3(88–274) fragment of the urokinase receptor inhibits monocyte chemotaxis and integrin-dependent cell adhesion. J. Cell Sci. 2004; 117: 2909–2916
[50]de Paulis A, Montuori N, Prevete N, et al. Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2. J. Immunol. 2004; 173: 5739–48
[51] Selleri C, Montuori N, Ricci P, et al. Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization. Blood. 2005; 105: 2198–2205
[52] Gargiulo L, Longanesi-Cattani I., Bifulco K, et al. Cross-talk between fMLP and vitronectin receptors triggered by urokinase receptor-derived SRSRY peptide. J. Biol. Chem. 2005; 280: 25225–25232
[53] Behrendt N, Ploug M, Patthy L, et al. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator. J. Biol.Chem. 1991; 266: 7842–7847
[54]Hoyer-Hansen G, Pessara U, et al. Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor. Biochem J. 2001; 358: 673–679
[55] de Paulis A, Montuori N, Prevete N, et al. Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2. J. Immunol. 2004; 173: 5739–48
[56] Ali H, Richardson R M, Haribabu B, et al. Chemoattractant receptorcross-desensitization. J Biol Chem. 1999; 274: 6027–6030
[57] Le Y, Shen W, Li B, et al. A new insight into the role of‘old’chemotactic peptide receptors FPR and FPRL1: down-regulation of chemokine receptors CCR5 and CXCR4. Forum. 1999;9: 299–314
[58] Li B. Q, Wetzel MA, Mikovits JA, et al. The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1. Blood. 2001;97: 2941–2947
[59] Selleri C, Montuori N, Ricci P, et al. Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization. Blood. 2005, 105: 2198–2205
[60] Comoglio PM, Boccaccio C, Trusolino L. Interactions between growth factor receptors and adhesion molecules: breaking the rules. Curr Opin Cell Biol. 2003;15: 565–571
[61] Aguirre Ghiso JA, Kovalski K, Ossowski L. Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signalling. J Cell Biol. 1999; 147: 89–104
[62] Liu D, Aguirre Ghiso J, Estrada Y, et al. EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell. 2002;1: 445–457
[63] Jo M, Thomas KS, O’Donnell DM, et al. Epidermal growth factor receptor-dependent and–independent cell-signalling pathways originating from the urokinase receptor. J Biol Chem. 2003; 278: 1642–1646
[64] Jo M, Thomas KS., Marozkina N, et al. Dynamic assembly of the urokinasetype plasminogen activator signalling receptor complex determines the mitogenic activity of urokinase-type plasminogen activator. J Biol Chem. 2005; 280: 17449–17457
[65] Kiyan J, Kiyan R, Haller H, et al. Urokinaseinduced signalling in human vascular smooth muscle cells is mediated by PDGFR-b. EMBO J. 2005; 24: 1787–1797
[66] Soravia E, Grebe A, De Luca P, et al. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene.Blood. 1995; 86: 624–635
[67] Hapke S, Gawaz M, Dehne K, et al. b(3)A-integrin downregulates the urokinase-type plasminogen activator receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the u-PAR promoter. Mol Cell Biol. 2001; 21: 2118–2132
[68] Wang Y, Dang J, Wang H, et al. Identification of a novel nuclear factor-kappaB sequence involved in expression of urokinase-type plasminogen activator receptor. Eur J Biochem. 2000; 267: 3248–3254
[69] Wang H, Yang L, Jamaluddin MS, et al. The Kruppel-like KLF4 transcription factor, a novel regulator of urokinase receptor expression, drives synthesis of this binding site in colonic crypt luminal surface epithelial cells. J Biol Chem. 2004; 279: 22674–22683
[70]Montuori N, Rossi G,Ragno P. Post-transcriptional regulation of gene expression in the plasminogen activation system. Biol Chem. 2002; 383: 47–53
[71] Wang GJ, Collinge M, Blasi F, et al. Posttranscriptional regulation of urokinase plasminogen activator receptor messenger RNA levels by leukocyte integrin engagement. Proc. Natl Acad Sci USA. 1998; 95: 6296–6301
[72]Tran H, Maurer F, Nagamine Y. Stabilization of urokinase and urokinase receptor mRNAs by HuR is linked to its cytoplasmic accumulation induced by activated mitogenactivated protein kinase-activated protein kinase 2. Mol Cell Biol. 2003; 23: 7177–7188
[73] Shetty S. Regulation of urokinase receptor mRNA stability by hnRNP C in lung epithelial cells. Mol Cell Biochem. 2005; 272: 107–118
[74] Shetty S, Kumar A, Idell S. Posttranscriptional regulation of urokinase receptor mRNA: identification of a novel urokinase receptor mRNA binding protein in human mesothelioma cells. Mol Cell Biol. 1997; 17: 1075–1083
[75] Shetty S, Muniyappa H, Halady P K. et al. Regulation of urokinase receptor expression by phosphoglycerate kinase. Am J Respir Cell Mol Biol. 2004; 31: 100–106
[76] Montuori N, Mattiello A, Mancini A, et al. Urokinase-type plasminogen activatorupregulates the expression of its cellular receptor through a post-transcriptional mechanism. FEBS Lett. 2001; 508: 379–384
[77] Montuori N, Mattiello A, Mancini A, et al. Urokinase-mediated posttranscriptional regulation of urokinase-receptor expression in non small cell lung carcinoma. Int J Cancer . 2003:105: 353–360
[78]Zannetti A, Del Vecchio S, Carriero MV, et al. Coordinate up-regulation of Sp1 DNA-binding activity and urokinase receptor expression in breast carcinoma. Cancer Res. 2000,60: 1546–1551
[1] Blasi F,Carmeliet P. uPAR: a versatile signaling orchestrator. Nat. Rev. Mol. Cell Biol. 2002; 3: 932–943
[2] Myohanen H, Vaheri A. Regulation and interactions in the activation of cell-associated plasminogen. Cell Mol Life Sci. 2004 Nov;61(22):2840-58.
[3]Edwards S, Lalor PF, Tuncer C, et al. Vitronectin in human hepatic tumours contributes to the recruitment of lymphocytes in an alpha v beta3-independent manner. Br J Cancer. 2006; 95(11):1545-54.
[4] Chaurasia P, Aguirre-Ghiso JA, Liang OD, et al. A region in urokinase plasminogen receptor domain III controlling a functional association with alpha5beta1 integrin and tumor growth. J Biol Chem. 2006;281(21):14852-63.
[5] Zhou H, Vassalli JD. The receptor for urokinase-type plasminogen activator is expressed during mouse spermatogenesis. FEBS Lett. 1997; 413(1): 11-5.
[6] Tong Zhang, Hong-Ming Zhou、Yi-Xun Liu, et al. Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes. Molecular Human Reproduction .1997; 3(3): 223–231.
[7] Teng X, Yang J, Xie Y, et al. A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis. Biochem Biophys Res Commun. 2006; 342(4): 1223-7.
[8] Huang Donghui, Zhao hu,Li Honggang, et al. Gene expression of urokinase plasminogen activator and urokinase plasminogen activator receptor in rat testis at postnatal stages. Asian Journal of Andrology,2007
[9]熊承良,黄勋彬,夏文家,等.人精子中尿激酶型纤溶酶原激活因子及其受体的分布[J].生殖与避孕,1996,16(6):422 424.
[10]王玲,官黄涛,田永红,等.少弱精子症和正常生育男性精液中尿激酶及其受体含量的研究。中华男科学杂志,2006,12(9):791-793
[11]熊承良,沈继云.尿激酶对弱精症患者精子活力影响的研究.生殖医学杂志,1995,4(3):164-166.
[12] oliveira RG,Tomasi L,Rovasio RA,et a1.Increased velocity and induction of chem otactic response in mouse spermatozoa by follicular and oviductal fluids[J]. J Reprod Fe~il,1999,115(1):23-27.
[13] Georgina F,Roberto AR,Silvia C,et a1.Chemotaxis of capacitated rabbit sperm atozoa to follicular fluid revealed by a novel directionality-based assay[J].Biol Reprod,2002,67(5):1565—1571.
[14]丁晓芳,熊承良.尿激酶受体在尿激酶诱导小鼠精子趋化运动中的作用.生殖医学杂志,2005, 14( 5):297-299.
[15] Gabler C, Killian GJ, Einspanier R. Differential expression of extracellular matrix components in the bovine oviduct during the oestrous cycle. Reproduction. 2001;122(1):121-30.
[16] Ming Li, Eli G, Karakji, Rosie Xing, et al. Expression of Urokinase-Type Plasminogen Activator and Its Receptor during Ovarian Follicular Development. Endocrinology. 1997;138(7):2790-9.
[17] Dow MP, Bakke LJ, Cassar CA, et al. Gonadotropin Surge-Induced Up-Regulation of the Plasminogen Activators (Tissue Plasminogen Activator and Urokinase PlasminogenActivator) and the Urokinase Plasminogen Activator Receptor Within Bovine Periovulatory Follicular and Luteal Tissue. Biol Reprod. 2002;66(5):1413-21.
[18] Teesalu T, Blasi F, Talarico D. Embryo implantation in mouse: fetomaternal coordination in the pattern of expression of uPA, uPAR, PAI-1 and alpha 2MR/LRP genes. Mech Dev. 1996 ;56(1-2):103-16.
[19] Solberg H, Rinkenberger J, Dano K, et al. A functional overlap of plasminogen and MMPs regulates vascularization during placental development. Development. 2003; 130(18): 4439-50
[20] Pierleoni C, Samuelsen GB, Graem N, et al. Immunohistochemical identification of the receptor for urokinase plasminogen activator associated with fibrin deposition in normal and ectopic human placenta. Placenta. 1998;19(7):501-8.