摘要
目的验证同源基因A10(hoxA10)对10号染色体缺失的磷酸酶、张力蛋白同源基因(pten)、磷脂酰肌醇-3-激酶(pi3k)和蛋白激酶B(Akt)信号通路的影响。方法设计SD大鼠骨髓间充质干细胞hoxA10基因的短发夹RNA(shRNA)靶序列,并构建pLVSHG01-Rat HoxA10重组慢病毒。用敲低型慢病毒感染SD大鼠的骨髓间充质干细胞(BMSCs),将BMSCs分为感染组(感染hoxA10敲低型慢病毒)和空白对照组(未感染)。用嘌呤霉素筛选出感染成功的细胞株后,以蛋白质印迹法检测感染后BMSCs中HoxA10蛋白表达,用实时荧光定量反转录-PCR法检测各组骨髓间充质干细胞中hoxA10、pten、pi3k和akt基因的表达水平。用双染流式细胞凋亡试剂盒检测各组细胞的凋亡率和周期。结果成功构建了hoxA10低表达的SD大鼠骨髓间充质干细胞,空白对照组和感染组的hoxA10基因表达量分别为6.22±0.20和3.09±0.06;这2组的pten基因表达量分别为1.97±0.18和5.09±0.07;这2组的pi3k基因表达量分别为6.06±0.05和2.72±0.10;这2组的akt基因表达量分别为5.98±0.09和2.48±0.35。上述指标:空白对照组与感染组比较,差异均有统计学意义(均P<0.001)。空白对照组的细胞凋亡率(18.88±1.71)%明显低于感染组(36.01±0.45)%,组间差异有统计学意义(P<0.05)。结论HoxA10基因能够负向调控pten/pi3k/akt信号通路。
Objective To verify the effect of homeotic genes A10(hoxA10)on the pathway of chromosome 10 by phosphatase and tensin homolog deleted on chromosome ten(pten),phosphatidylin-ositol-3-kinase(pi3k),protein kinase B(Akt).Methods The short hair pin RNA(shRNA)target sequence of hoxA10 gene of SD rat bone marrow mesenchymal stem cells was designed,and the recombinant lentivirus PLVSHG01-rathoxA10 was constructed.Bone marrow mesenchymal stem cells(BMSCs)were then infected,and the cells were divided into infected group(infected with hoxA10 knockdown lentivirus)and blank control group(uninfected).We use knockdown lentivirus to infected BMSCs.After successfully infected cell lines were screened by purinomycin,the expression of HoxA10 protein in infected BMSCs was detected by Western-blot,and the expression levels of hoxA10,pten,pi3k and akt genes in BMSCs were detected by real-time fluorescence qua nt itative reverse transcription polymerase chain reaction.The apoptosis rate and cycle of each group were detected by Annexin V-FITC/PI double-dye flow cytometry apoptosis detection kit.Results Bone marrow mesenchymal stem cells with low hoxa10 expression in SD rats were successfully constructed.HoxA10 gene expression in blank control group and infected group were 6.22±0.20 and 3.09±0.06,respectively.The expression of pten gene in the two groups were 1.97±0.18 and 5.09±0.07,respectively.Pi3k gene expression levels in the two groups were 6.06±0.05 and 2.72±0.10,respectively.The expression of akt gene in the two groups was 5.98±0.09 and 2.48±0.35,respectively.The above indicators:the difference between the infected group and the blank control group was statistically significant(all P<0.001).The apoptosis rate of blank control group(18.88±1.71)%was significantly lower than that of infection group(36.01±0.45)%,and the difference between groups was statistically significant(P<0.05).Conclusion HoxA10 gene can negatively regulate pten/pi3k/akt signal pathway.
作者
刘燕
邵芷若
李玥
吕妍
宋阳
关永格
LIU Yan;SHAO Zhi-ruo;LI Yue;LÜYan;SONG Yang;GUAN Yong-ge(Third Clinical Medical College,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;School of Nursing,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China;The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510360,Guangdong Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2021年第22期3068-3071,共4页
The Chinese Journal of Clinical Pharmacology
基金
国家自然科学基金资助项目(81303000)
广东省科技计划基金资助项目(2017A020215119)
广东省普通高校特色创新基金资助项目(2017KTSCX041)。
