Summary： The deubiquitinating enzyme ubiquitin specific peptidase 15 （USP15） is regarded as a regulator of TGFβ signaling pathway. This process depends on Smad7, the inhibitory factor of the TGFβ signal, and type...Summary： The deubiquitinating enzyme ubiquitin specific peptidase 15 （USP15） is regarded as a regulator of TGFβ signaling pathway. This process depends on Smad7, the inhibitory factor of the TGFβ signal, and type Ⅰ TGFβ receptor （TβR- Ⅰ ）, one of the receptors of TGFβ. The expression level of USP 15 seems to play vital roles in the pathogenesis of many neoplasms, but so far there has been no report about USP15 in psoriasis. In this study, immunohistochemical staining of USP15, TβR- Ⅰ and Smad7 was performed in 30 paraffin-embedded psoriasis specimens and 10 normal specimens to investigate the expression of USP15, TβR- Ⅰ and Smad7 in psoriasis and to explore the relevance among them. And USP 15 small interfering RNA （USP 15 siRNA） was used to transfect Hacat cells to detect the mRNA expression of TβR- Ⅰ and Smad7. Of 30 cases of psoriasis in active stage, 28, 24 and 26 cases were positive for USP15, TβR- Ⅰ and Smad7 staining, respectively. The positive rates of USP15 and Smad7 were significantly higher in psoriasis specimens than in normal skin specimens （44.1%±26.0% vs. 6.1%±6.6%, 47.2%±27.1% vs. 6.6%±7.1%）, and positive rate of TβR- I （20.3%±22.2%） in psoriasis was lower than that in normal skin specimens （46.7%±18.2%）. There was a significant positive correlation between USP15 and Smad7 expression, and significant negative correlations between USP15 and TβR- Ⅰ expression, and between TβR- Ⅰ and Smad7 expression in psoriasis. After transfection of USP15 siRNA in Hacat ceils, the expression ofTβR- Ⅰ mRNA was up-regulated and that of Smad7 was down-regulated. It is concluded that USP15 may play a role in the pathogenesis of psoriasis through regulating the TβR- Ⅰ/Smad7 pathway and there may be other cell signaling pathways interacting with USP 15 to take part in the development of psoriasis.展开更多
背景:Sema7A是一种细胞表面蛋白,它可以促进破骨细胞的融合同时促进成骨细胞的迁移,从而影响骨的动态平衡。推测,Sema7A siR NA可能减弱钛微粒成骨细胞分化。目的:分析信号素7A(Sema7A)对钛颗粒抑制鼠MC3T3-E1前成骨细胞活性过程的影响...背景:Sema7A是一种细胞表面蛋白,它可以促进破骨细胞的融合同时促进成骨细胞的迁移,从而影响骨的动态平衡。推测,Sema7A siR NA可能减弱钛微粒成骨细胞分化。目的:分析信号素7A(Sema7A)对钛颗粒抑制鼠MC3T3-E1前成骨细胞活性过程的影响。方法:取第六七代生长状态良好的小鼠颅骨前成骨细胞(MC3T3-E1),分为4组培养。其中空白对照组为单独培养的细胞;标准对照组加入了钛微粒共培养;实验1组加入Sema7A过表达质粒,实验2组加入Sema7AsiR NA进行干扰。采用流式细胞仪检测细胞凋亡情况;Q-PCR检测骨涎蛋白、骨钙素、Ⅰ型胶原蛋白的mR NA表达情况;Western-blot检测骨涎蛋白、骨钙素、Ⅰ型胶原蛋白的蛋白表达情况;茜素红钙结节染色检测成骨细胞的矿化程度。结果与结论:结果提示标准对照组、实验1组检测到骨涎蛋白、骨钙素、Ⅰ型胶原蛋白表达明显减少而实验2组骨涎蛋白、骨钙素、Ⅰ型胶原蛋白则比标准对照组表达增加(P<0.05)。流式细胞仪结果提示实验1组成骨细胞凋亡率明显高于其他组(P<0.05),而实验2组则比标准对照组凋亡率降低(P<0.05)。茜素红染色检测结果显示实验1组未见明显的矿化结节,实验2组有矿化结节形成。结果表明通过基因干扰技术抑制Sema7A活性可以对钛颗粒抑制鼠MC3T3-E1成骨细胞分化的过程进行干扰,进而为临床上用生物技术治疗磨损微粒诱导的骨溶解问题提供了一条可行的思路。展开更多
Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm ...Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm Ⅰ recognition sites were introduced to both ends of the PCR product.This fragment was cloned into the Sma Ⅰ site of pUC18 or the linearized pUC18 with its polylinker deleted.The recombinant plamids were cleaved with Xcm Ⅰ.the larger fragments generated which had 3’T over hangs at both ends were used as T vctors.Genes of rotavirus VP7 and the plasminogen k5 were successfully cloned into these two T vectors with recombination efficiency (recombinants/transformants×100%) of 100%,no blue/white clolny screening assay was needed.展开更多
基金supported by grants from National NaturalScience Foundation of China(No.81101191 and No.30972654)
文摘Summary： The deubiquitinating enzyme ubiquitin specific peptidase 15 （USP15） is regarded as a regulator of TGFβ signaling pathway. This process depends on Smad7, the inhibitory factor of the TGFβ signal, and type Ⅰ TGFβ receptor （TβR- Ⅰ ）, one of the receptors of TGFβ. The expression level of USP 15 seems to play vital roles in the pathogenesis of many neoplasms, but so far there has been no report about USP15 in psoriasis. In this study, immunohistochemical staining of USP15, TβR- Ⅰ and Smad7 was performed in 30 paraffin-embedded psoriasis specimens and 10 normal specimens to investigate the expression of USP15, TβR- Ⅰ and Smad7 in psoriasis and to explore the relevance among them. And USP 15 small interfering RNA （USP 15 siRNA） was used to transfect Hacat cells to detect the mRNA expression of TβR- Ⅰ and Smad7. Of 30 cases of psoriasis in active stage, 28, 24 and 26 cases were positive for USP15, TβR- Ⅰ and Smad7 staining, respectively. The positive rates of USP15 and Smad7 were significantly higher in psoriasis specimens than in normal skin specimens （44.1%±26.0% vs. 6.1%±6.6%, 47.2%±27.1% vs. 6.6%±7.1%）, and positive rate of TβR- I （20.3%±22.2%） in psoriasis was lower than that in normal skin specimens （46.7%±18.2%）. There was a significant positive correlation between USP15 and Smad7 expression, and significant negative correlations between USP15 and TβR- Ⅰ expression, and between TβR- Ⅰ and Smad7 expression in psoriasis. After transfection of USP15 siRNA in Hacat ceils, the expression ofTβR- Ⅰ mRNA was up-regulated and that of Smad7 was down-regulated. It is concluded that USP15 may play a role in the pathogenesis of psoriasis through regulating the TβR- Ⅰ/Smad7 pathway and there may be other cell signaling pathways interacting with USP 15 to take part in the development of psoriasis.
文摘背景:Sema7A是一种细胞表面蛋白,它可以促进破骨细胞的融合同时促进成骨细胞的迁移,从而影响骨的动态平衡。推测,Sema7A siR NA可能减弱钛微粒成骨细胞分化。目的:分析信号素7A(Sema7A)对钛颗粒抑制鼠MC3T3-E1前成骨细胞活性过程的影响。方法:取第六七代生长状态良好的小鼠颅骨前成骨细胞(MC3T3-E1),分为4组培养。其中空白对照组为单独培养的细胞;标准对照组加入了钛微粒共培养;实验1组加入Sema7A过表达质粒,实验2组加入Sema7AsiR NA进行干扰。采用流式细胞仪检测细胞凋亡情况;Q-PCR检测骨涎蛋白、骨钙素、Ⅰ型胶原蛋白的mR NA表达情况;Western-blot检测骨涎蛋白、骨钙素、Ⅰ型胶原蛋白的蛋白表达情况;茜素红钙结节染色检测成骨细胞的矿化程度。结果与结论:结果提示标准对照组、实验1组检测到骨涎蛋白、骨钙素、Ⅰ型胶原蛋白表达明显减少而实验2组骨涎蛋白、骨钙素、Ⅰ型胶原蛋白则比标准对照组表达增加(P<0.05)。流式细胞仪结果提示实验1组成骨细胞凋亡率明显高于其他组(P<0.05),而实验2组则比标准对照组凋亡率降低(P<0.05)。茜素红染色检测结果显示实验1组未见明显的矿化结节,实验2组有矿化结节形成。结果表明通过基因干扰技术抑制Sema7A活性可以对钛颗粒抑制鼠MC3T3-E1成骨细胞分化的过程进行干扰,进而为临床上用生物技术治疗磨损微粒诱导的骨溶解问题提供了一条可行的思路。
文摘Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm Ⅰ recognition sites were introduced to both ends of the PCR product.This fragment was cloned into the Sma Ⅰ site of pUC18 or the linearized pUC18 with its polylinker deleted.The recombinant plamids were cleaved with Xcm Ⅰ.the larger fragments generated which had 3’T over hangs at both ends were used as T vctors.Genes of rotavirus VP7 and the plasminogen k5 were successfully cloned into these two T vectors with recombination efficiency (recombinants/transformants×100%) of 100%,no blue/white clolny screening assay was needed.
