ABSTRACT
Objective @# To investigate the effect of SHP2 protein phase separation induced by activation mutation on cell proliferation and its mechanism through construct a mouse model of SHP2E76K mutation . @*Methods @#Hybrid PTPN11 E76K⁃NEO/ + C57BL/6J mouse were hybridizedwith Mx1 ⁃cre tool mice to obtain the required Mx1 ⁃cre;Ptpn11 + / + and Mx1 ⁃cre;Ptpn11 E76K/ + . The later genotype mice were injected with pI⁃pC to induce the expression of Cre enzyme and mutate Ptpn11E76K in bone marrow mesenchymal stem cells(MSC) . The Mx1 ⁃cre ; Ptpn11 + / + and Mx1 ⁃cre;Ptpn11 E76K/ + genotype mice ′s cells were isolated and cultured in vitro and identified as MSCs by immunofluorescence staining . With Ptpn11 + / + MSC as the control group and Ptpn11 E76K/ + MSC as the experimental group , the two kinds of cells were divided into 6 groups by adding drugs : Ptpn11 + / + group ; Ptpn11 + / + + SHP099 group ; Ptpn11 + / + + ET070 group ; Ptpn11 E76K/ + group ; Ptpn11 E76K/ + + SHP099 group ; Ptpn11 E76K/ + + ET070 group . The differences of SHP2 protein phase separation in the six groups were observed by immunofluorescencestaining, and the differences of SHP2 protein expression were detectedby Western blot . CCK⁃8 was used to observe the changes of cell proliferation after phase separation was affected . Western blot was used to detect the expression levels of ERK/ cell proliferation after phase separation was affected . Western blot was used to detect the expression levels of ERK/ p ⁃ERK , AMPK/p⁃AMPK , mTOR/p⁃mTOR and other molecules between the six groups . @*Results @# Genotypes Mx1 ⁃cre ; Ptpn11 E76K/ + and Mx1 ⁃cre ; Ptpn11 + / + mice were obtained by genotyping , and the primary MSCs were isolated . Compared with Ptpn11 + / + group , SHP2 proteins in the Ptpn11 E76K/ + group produced more phase separation condensates , and compared with Ptpn11 E76K/ + group , the SHP2 proteins in the Ptpn11 E76K/ + + SHP099 and Ptpn11 E76K/ ++ ET070 groups produced less phase separation condensates . No difference in SHP2 protein expression levels between groups was detected by Western blot . Compared with Ptpn11 + / + group , the proliferation ability of MSC in Ptpn11 + / + + SHP099 and Ptpn11 + / + + ET070 groups decreased , the expression of p ⁃ERK and p ⁃mTOR decreased, and the expression of p ⁃AMPK protein increased . Compared with Ptpn11 E76K/ + group , the proliferation ability of MSC in Ptpn11 E76K/ + + SHP099 and Ptpn11 E76K/ + + ET070 groups decreased , the expression of p ⁃ERK and p ⁃ mTOR decreased , and the expression of p⁃AMPK protein increased . @*Conclusion @# SHP2 phase isolation is involved in the alteration of proliferative capacity of SHP2E76K ⁃activated MSCs by stimulating the expression of AMPK⁃mTOR signaling pathway .