Effect Of HTRA1 Gene Mutation On Cellular Function And Redox Reaction In Vascular Smooth Muscle Cell

CARASIL is a single gene disease which directly effect on cerebral small vessel.It is caused by mutations of HTRA1 gene encoding serine peptidase/protease 1(HTRA1).CARASIL occurs more often in men with early onset.The exact incidence of CARASIL is not clear.The main clinical manifestations of CARASIL are ischemic stroke or progressive deterioration of brain function,dementia,premature baldness,severe back pain,spondylosis deformans and prolapse of intervertebral.CT and MRI of the brain showed the imaging characteristics with diffuse white matter changes and multiple lacunar infarctions in the basal ganglia and thalamus.On histopathology of CARASIL,small perforating artery arteriosclerosis was observed with intimal thickening,loss of vascular smooth muscle cells and hyaline degeneration of media.HTRA1 gene is located in the region of 10q26,containing 9 exons.CARASIL related allelic variants appeared in exon 1,3,4 and 6.It has a total of 8 mutations,including 5 missense mutations,1 frameshift mutation and 2 nonsense mutations.HTRA1 is a secreted serine protease,which can promote degeneration of extracellular matrix protein.HTRA1 protease has important physiological effects,associated with arthritis,cancer,familial ischemic cerebral small vessel disease,age-related macular degeneration and Alzheimer's disease.The loss of HTRA1 protease function result in CARASIL,but the exact mechanism is still not clear.In 2007,we reported the first CARASIL family in china,and then we discover a new HTRA1 gene mutation locus which is missense mutations in exon 6: 1091T>C(point mutation).Previous researches has found that the HTRA1 gene mutation can lead to a reduction in the expression of HTRA1 m RNA and protein,and upregulate expression of TGF-?1/Smads/CTGF signaling pathway.In this experiment,using the HTRA1 mutant and wild gene lentivirus vector infecting into human cerebral vascular smooth muscle cells,we observed the cell function and redox reaction of HBVSMCs,in order to explore the pathogenesis of CARASIL.Part ? Construction and packaging of lentivirus vector of HTRA1 geneObjective: To construction the lentivirus vector of HTRA1 gene and 1091T>C mutant gene(HTRA1-Mut).Methods: We designed and constructed the primer of HTRA1 gene by utilizing NCBI Genbank database.Obtaining the target gene by PCR from the plasmids containing wild type and mutant HTRA1 gene,enzyme cutting the target vector GV287,exchanging the recombinant plasmid after electrophoresis recovering the Enzyme cutting product,transformed the product to the bacteria competent cell.The positive clone which is identified by PCR needs analysis of sequencing and comparison.Correct comparison of positive clone means the construction of lentivirus vector is successful.Recombinant virus plasmids of HTRA1 wild gene and HTRA1 mutant gene,as well as two auxiliary packaging original vector plasmids need respectively highly purified extraction without endotoxin,then co-infected into 293 T cells.The cell supernatant was collected with rich lentivirus particles,and then concentrated to high titer lentivirus concentrated liquid,calibrated the virus titer in 293 T cells.Results: We obtained the objective fragments of HTRA1 wild gene and HTRA1 mutant gene by enzyme digesting and PCR amplification,which are transformed to bacteria competent cells after enzyme digesting with GV287.Analysis of sequencing and comparison of positive PCR identified clone showed consistent with HTRA1 wild gene sequence and HTRA1 mutant gene sequence.We can observe fluorescence in cells after HTRA1/HTRA1-Mut expression vector infecting 293 T cells.The viral titer was 2E+8 TU/ml.Conclusions: HTRA1 gene and HTRA1-Mut gene eukaryotic expression vectors were constructed successfully;HTRA1 and HTRA1-Mut lentivirus were packaging and titer detected successfully.Part ? Model of vascular smooth muscle cell infected by lentivirus vector of HTRA1 geneObjective: The lentivirus vectors of HTRA1 wild gene and HTRA1 mutant gene were infected into human brain vascular smooth muscle cells(HBVSMC).Methods: The HBVSMCs were cultured,phenotypic identified by ?-SMA antibody fluorescence staining,and infected by HTRA1 and HTRA1-Mut lentivirus vector.Results: The HBVSMCs are in good growth state.We can observe normal cell morphology under the light microscope,and we also observed the cell fluorescence color is good after ?-SMA antibody staining.HBVSMCs had expression of fluorescence after infected by HTRA1 wild gene and HTRA1 mutant gene with lentivirus vector.Fluorescence rate reaches above 80%.Conclusions: It is successful that HBVSMCs are cultured and identified.The models of vascular smooth muscle cells infected by HTRA1 and HTRA1-Mut gene lentivirus vectors are established successfully.Part ? Effect of HTRA1 gene mutation on proliferation,migration and apoptosis of human brain vascular smooth muscle cellsObjective: To detect changes of proliferation,migration and apoptosis of HBVSMCs after infected by HTRA1 and HTRA1-Mut gene lentivirus vectors.Methods: HBVSMCs were divided into three groups,normal human brain vascular smooth muscle cells(NC),mutant gene lentivirus infected cells(OE-MU),and wild gene lentivirus infected cells(OE-WT).All of the three groups of cells were performed to detect cell proliferation by CCK-8,cell migration by Transwell,and cell apoptosis by flow cytometry.Results: Compared with the HTRA1 wild gene lentivirus infected cells(OE-WT),the proliferation multiple of HTRA1 mutant gene lentivirus infected cells slowed down,the transfer rate were decreased,the apoptosis were affected.Conclusions: Proliferative capacity and migration activity of HBVSMCs reduced after infection caused by HTRA1 mutant gene lentivirus vectors,and the apoptosis of the cells was affected.Part ? Effect of HTRA1 gene mutation on oxidation reaction of human brain vascular smooth muscle cellObjective: Detection of changes on oxidative stress levels of HBVSMCs after infected by HTRA1 wild and HTRA1 mutant gene lentivirus vectors.Methods: HBVSMCs which were infected by HTRA1 wild gene and HTRA1 mutant gene lentivirus vectors were divided into three groups,NC group,OE-WT group and OE-MU group.DCFH-DA method was used to detect the levels of intracellular reactive oxygen species in three groups.The total RNA and total protein of three groups of cells were collected at a given time,and detected expression level of NOX4 m RNA and NOX4 protein by Real-time PCR and Western Blot.Results: The level of ROS in normal HBVSMCs was low,while increased after infection of HTRA1 wild gene and HTRA1 mutant gene lentivirus vectors.It was more obvious in the mutant lentivirus infected cells.In the part of Rt-PCR,NOX4 gene expression abundance in OE-MU group was 2.015 times higher than NC group.About Western blot,the level of NOX4 protein expression in normal human vascular smooth muscle cells was low,while was higher in HTRA1 wild gene and HTRA1 mutant gene infected HBVSMCs.Conclusions: Intracellular ROS production of HBVSMCs was increased after infected by HTRA1 mutant gene lentivirus vector.The expression level of NOX4 m RNA in HTRA1 mutant gene cells had no significant difference compared with the wile type of HTRA1 gene.The expression level of NOX4 protein was higher than the other two groups.