目的制备人S100A9(hS100A9)重组腺病毒载体,为进一步研究人S100A9蛋白的分子生物学作用奠定基础。方法从pHAHA-hS100A9中PCR扩增hS100A9片段,亚克隆至穿梭质粒pAdTrack-TOX,获得重组穿梭质粒pAdTrack-TOX-hS100A9。酶切、聚合酶链反应(P...目的制备人S100A9(hS100A9)重组腺病毒载体,为进一步研究人S100A9蛋白的分子生物学作用奠定基础。方法从pHAHA-hS100A9中PCR扩增hS100A9片段,亚克隆至穿梭质粒pAdTrack-TOX,获得重组穿梭质粒pAdTrack-TOX-hS100A9。酶切、聚合酶链反应(PCR)及测序鉴定正确后经PmeⅠ酶切电转入AdEasier感受态细胞,获得重组腺病毒质粒pAdhS100A9,该质粒经PacⅠ酶切后由脂质体转染至HEK293细胞中包装扩增重组腺病毒并进行滴度测定,最后通过PCR及蛋白印迹法(Western blot)鉴定重组腺病毒。结果正确扩增出hS100A9基因;重组穿梭质粒pAdTrack-TOX-hS100A9均及重组腺病毒质粒pAdhS100A9构建成功,并在HEK293细胞中成功包装且扩增后滴度为1 010 I U/mL;重组腺病毒AdhS100A9在HEK293细胞中成功转录和表达。结论用pAdEasy系统成功构建hS100A9重组腺病毒载体,为探讨其生物学作用奠定了基础。展开更多
OBJECTIVE: To study the effects of a decoction of Fuzhengzengxiao formula on lung adenocarcinoma regarding the inflammatory protein S100A9 known to enhance cancer cell sensitivity.METHODS: A nude mouse model of human ...OBJECTIVE: To study the effects of a decoction of Fuzhengzengxiao formula on lung adenocarcinoma regarding the inflammatory protein S100A9 known to enhance cancer cell sensitivity.METHODS: A nude mouse model of human lung adenocarcinoma was established. The mice were randomly divided into four groups using the random number table method: Group Ⅰ, control;Group Ⅱ, treatment with a decoction of the Fuzhengzengxiao formula alone; Group Ⅲ, treatment with radiotherapy alone; and Group Ⅳ, treatment with radiotherapy plus a decoction of Fuzhengzengxiao formula. When the tumor body was 1 cm^3 in diameter, the tumor bearing mice in GroupsⅢ and Ⅳ were irradiated at a single dose of 10 Gy and the tumor inhibition rate was evaluated.The expression of S100A9 was determined using Western blotting and q-PCR(Real-time Quantitative PCR Detecting System). The sensitivity of cells containing RNAi S100A9 to radiotherapy was evaluated using the Click multiple target model,and the cell cycle was analyzed using flow cytometry.RESULTS: Relative to the control group,the expression of S100A9 in the tumors in each treatment group was decreased,especially in Group Ⅳ. The sensitizing enhancement ratio(SER) Dq was >1 after RNAi S100A9; it decreased the surviving fraction after a 2 Gy dose exposure,and also the D_0 and Dq of the tumor cells; in addition, the radiosensitivity of G_2/M cells was significantly increased.CONCLUSION: The decoction of the Fuzhengzengxiao formula downregulated the expression of S100A9 in lung adenocarcinoma cells.展开更多
文摘目的制备人S100A9(hS100A9)重组腺病毒载体,为进一步研究人S100A9蛋白的分子生物学作用奠定基础。方法从pHAHA-hS100A9中PCR扩增hS100A9片段,亚克隆至穿梭质粒pAdTrack-TOX,获得重组穿梭质粒pAdTrack-TOX-hS100A9。酶切、聚合酶链反应(PCR)及测序鉴定正确后经PmeⅠ酶切电转入AdEasier感受态细胞,获得重组腺病毒质粒pAdhS100A9,该质粒经PacⅠ酶切后由脂质体转染至HEK293细胞中包装扩增重组腺病毒并进行滴度测定,最后通过PCR及蛋白印迹法(Western blot)鉴定重组腺病毒。结果正确扩增出hS100A9基因;重组穿梭质粒pAdTrack-TOX-hS100A9均及重组腺病毒质粒pAdhS100A9构建成功,并在HEK293细胞中成功包装且扩增后滴度为1 010 I U/mL;重组腺病毒AdhS100A9在HEK293细胞中成功转录和表达。结论用pAdEasy系统成功构建hS100A9重组腺病毒载体,为探讨其生物学作用奠定了基础。
基金Supported by a Grant from the National Natural Science Foundation of China:the Effect of TCM Combined Radiotherapy and RNAi Suppression on the Protein Expression Changes of S100A9 & Cyclophilin A and Radiosensitivity in Lung Adenocarcinoma(No.81072925)
文摘OBJECTIVE: To study the effects of a decoction of Fuzhengzengxiao formula on lung adenocarcinoma regarding the inflammatory protein S100A9 known to enhance cancer cell sensitivity.METHODS: A nude mouse model of human lung adenocarcinoma was established. The mice were randomly divided into four groups using the random number table method: Group Ⅰ, control;Group Ⅱ, treatment with a decoction of the Fuzhengzengxiao formula alone; Group Ⅲ, treatment with radiotherapy alone; and Group Ⅳ, treatment with radiotherapy plus a decoction of Fuzhengzengxiao formula. When the tumor body was 1 cm^3 in diameter, the tumor bearing mice in GroupsⅢ and Ⅳ were irradiated at a single dose of 10 Gy and the tumor inhibition rate was evaluated.The expression of S100A9 was determined using Western blotting and q-PCR(Real-time Quantitative PCR Detecting System). The sensitivity of cells containing RNAi S100A9 to radiotherapy was evaluated using the Click multiple target model,and the cell cycle was analyzed using flow cytometry.RESULTS: Relative to the control group,the expression of S100A9 in the tumors in each treatment group was decreased,especially in Group Ⅳ. The sensitizing enhancement ratio(SER) Dq was >1 after RNAi S100A9; it decreased the surviving fraction after a 2 Gy dose exposure,and also the D_0 and Dq of the tumor cells; in addition, the radiosensitivity of G_2/M cells was significantly increased.CONCLUSION: The decoction of the Fuzhengzengxiao formula downregulated the expression of S100A9 in lung adenocarcinoma cells.
