Study On Cell Protective Function Of Protein SUMO Modification Under The Conditions Of Low Temperature And High Temperature Stress

Objective:To study the variation characteristics and regular patterns of SUMOs in low temperature and heat stress,and then applied its neuroprotective effect to treat the central nervous system diseases.Methods:(1)(1)The mouse derived BMSCs were primary cultured and its marker proteins expression were detected by flow cytometry.(2)BMSCs were cultured at 37°C(normal body temperature),33 °C(mild hypothermia)and 18 °C(deep hypothermia),its cell proliferation activity were detected by MTT methods,BMSCs cell cycle were detected by flow cytometry,and the ability of BMSCs differentiation into neurons and glial cells were detected by immunofluorescence method.(3)The neuron OGD model was established,then cell apoptosis of BMSCs were detected by cell apoptosis in situ assay,and the content of LDH was tested by ELISA assay.The covalent binding state and free state of SUMO1 and SUMO2/3 protein expression changes by detectd by western blot assay,and theirs cellular localization was detected by immunofluorescence assay.(4)The siRNA technology was used to induce the silence of SUMO1/2/3 gene,and spectomycin B1 was used to inhibite the expression of Ubc9 gene in BMSCs.Then the changes of cell morphology,cell proliferation,cell differentiation and tolerance to OGD of BMSCs were observed.The expression level of PCNA,Oct4,P53 and HIF-1αprotein was detectd by western blot method.And the possible mechanism of BMSCs increasing their tolerance to adverse environmental in low temperature was analyzed.(2)(1)The genotype of SUMO1/2/3 gene knockout and SUMO1/2/3transgenic mice was identified by double immunofluorescent staining method and western blot method.(2)The mouse cerebral cortex neurons were isolated and cultured,and which are respectively positioned at 37 °C(normal body temperature),42 °C(high fever)and 47 °C(super high fever)culture environment,the expression level of SUMO1 and SUMO2/3 protein was detected by western blot method,and theirs cellular localization was detected by immunofluorescence assay.(3)Then cell apoptosis was detected by cell apoptosis in situ assay,and the content of LDH was examined by ELISA method.(4)Then mouse model of febrile seizures were established,seizure score was tested by five point scale method,and convulsion percentage,latency and duration of seizures was recorded.Results:(1)(1)The mesenchymal stem cell specific protein CD71 and CD105 were high expression,but less expression of hematopoietic stem cell specific protein CD34 and CD4 in BMSCs.(2)The BMSCs grew slowly at low temperature,and their ability of differentiation into neural cells gradually decreased as the temperature decreases.BMSCs under the condition of OGD shows a large number of apoptotic cells,accompanied by a high level of lactate dehydrogenase release,and low temperature can improve the situation.(3)A large number of proteins can be modified by SUMOs when BMSCs explored in low temperature conditions,with the characteristics of significantly higher level of covalent binding states of SUMO1 and SUMO2/3,and much lower level of the free SUMO1 and SUMO2/3,accompanied by the translocation into nucleus from cytoplasm.(4)The BMSCs started aging with the silencing of SUMO1/2/3 gene,their cell growth slowed down,cell cycle arrested,and undergoed terminal differentiation into nerve cells.The spectinomycin B1 can inhibit Ubc9 expression in a certain extent,thereby reducing the protein level of covalent binding state of SUMO1 and SUMO2/3,as well as they reduce the expression level of PCNA,Oct4,P53,HIF-1α protein.And also low temperature can increase the tolerance of BMSCs to adverse environmental.(2)(1)The results of the double immunofluorescent staining method and western blot method showed that there was stable genetype expression in both SUMO1/2/3 gene knockout and SUMO1/2/3 transgenic mice.(2)There was no obvious changes of SUMO1 protein expression both in covalent and free states,however,the expression level of covalent binding state of SUMO2/3 protein increased significantly,and the expression level of free state of SUMO2/3 protein decreased,accompanied by the translocation into nucleus from cytoplasm,when neurons explored at high fever and super high fever environment.The above phenomenon can be recovered when those neurons were reback to 37°C from 42°C,however can not be recovered when they were reback to 37°C from 47°C.(3)Compared with neurons derived from wild type mouse,there had a higher level of apoptosis and LDH in neurons from SUMO1/2/3 knockout mouse,while those neurons from SUMO1/2/3 transgenic mouse could significantly increase their tolerance to heat stress.(4)The results from heat convulsion model test showed that,compared with wild type mice,there was the highest convulsion seizure score,percentage of seizures,latency and duration of seizures in SUMO1/2/3 knockout mice group,while the lowest convulsion seizure score,percentage of seizures,latency and duration of seizures in SUMO1/2/3 transgenic mice.Conclusion:(1)Hypothermia can inhibits proliferation and differentiation potential to neural cells of BMSCs,and increases its tolerance to hypoxia microenvironment.(2)Hypothermia can increase the level of SUMO modification of a large number of proteins in BMSCs,and induce the translocation of SUMO1 and SUMO2/3 from cytoplasm to nucleus.(3)The activation of the SUMOs pathway is essential for BMSCs proliferation and stemness maintenance.(4)The spectinomycin B1 can inhibit the SUMOs pathway by inhibiting the expression of Ubc9 gene,and then reduce tolerance of BMSCs to the adverse hypoxic microenvironment.(5)PCNA,Oct4,P53 and HIF-1αSUMOylation were regulated by low temperature,and participate in BMSCs proliferation,stemness maintenance and resistance to apotosis.(6)SUMO-2/3,rather than SUMO1,is involved in the early response of neurons to heat stress.(7)The covalent modification of SUMO2/3 induced by a short period of high heat stress was reversible,whereas the covalent modification of SUMO2/3 induced by the super high temperature heat stress was irreversible.(8)Increase of SUMOs expression in neurons can tolerate higher levels of heat stress and reduce the risk of febrile seizures.