Effects Of Vitamin D Receptor Gene On Biological Function Of Endothelial Progenitor Cells

Backgrounds:Kidney has the richest blood supply in human body, and it carries out its important functions via its vessels, such as the progress of glomerular filtration, tubular reabsorption etc.It is reported that vascular lesions have close relationship with prognosis of renal diseases, therefore, keeping normal structure and functions of vessels has significance in the integal function of kidney. On the early stage,vascular lesions presents that injuried of vascular endothelial cells(VEC),migration of vascular smooth muscle cells,accumulation of matrix and so on,among which VEC injuried is the initial step.On the late stage,anatomical structure of vessels changed,for example,malformation,stenosis,ruptured or blocked,which affect blood supply seriously. Treatments of vascular injury include etilogical treatment,drugs and surgery.Drugs,such as antioxidants,antihypertensive drugs,angiotensin converting enzeme inhibitors,angiotensin receptor blocker etc,with its poor effects,can not radically solve the already exist vascular lesions.Surgery,such as stent implantation,angioplasty etc,was applied to cure vascular lesions with its defects that the trauma is severe,risk is high and intervention dispalys on the late stage.Researchers found that,once VEC injuried,Endothelial Progenitor Cells(EPCs) located in bone marrow were immediately mobilized,migrated,homing,differentiate into VEC,and replace injured VEC to form new vessels. EPCs transplant is the promising method for vascular diseases, for it has been demonstrated to be effective in ischemic diseases through in vitro and in vivo animal and clinical studies, and it has advantages that the trauma is smaller, and it displays on much earlier stage.However,this technique still has lots of problems,for example,for quantities of EPCs are needed in vascular repair,the number was few in bone marrow and amplification in vitro can not meet the demands,and the angiogenic ability is not stable.Therefore,researches about proliferation and angiogenic ability of EPCs will certainly be focused.Recent researches showed that there is close relationship between Vitamin D defect and vascular diseases,and beneficial results came out after treated with Vitamin D,but the specific mechanism is still unclear.It was recently reported that Vitamin D Receptor(VDR) was also expressed in EPCs,and Vitamin D improved proliferation and angiogenic activity of EPCs,which were suggested VDR-dependent,indicating that there were connections between VDR and the proliferation and angiogenic activity of EPCs.But which one plays the leading role, Vitamin D or VDR?Can VDR displays regulatory role in EPCs without Vitamin D? If transfected VDR gene into EPCs, are there advantages for EPCs transplant? There are no related studies.Objective:Study the effects of different level VDR gene on cellular proliferation, apoptosis, ageing, cell cycyle, and migration of EPCs,in order to provide new perspective and theoretical basis for EPCs transplant.Methods:(1) Isolated rat bone marrow-derived mononuclear cells, cultured, and identified with immunofluorescent staining.(2) Constructed Ad-VDR and Ad-VDR shRNA.Transfected Ad-VDR or Ad-VDR shRNA in bone marrow-derived EPCs, in which EPCs without dispose were used as control,and detected the relative expression of VDR mRNA by Real-time PCR and VDR protein level by Western Blot.(3) Set6groups as Normal level Group which stood for EPCs without any dispose,High level Group in which transfected with Ad-VDR,Low level Group in which transfected with Ad-VDR shRNA,Normal level+Rapamycin Group,High level+Rapamycin Group and Low level+Rapamycin Group. Cellular proliferation, apoptosis, ageing, cell cycle, and migration of EPCs were detected respectively.Results:(1)The cultured cells were CD34+/CD133+/VEGFR2+cells,which were widely accepted as EPCs.(2) After transfected with Ad-VDR,the relative expression of VDR mRNA and protein level were much higher than control,while that were much lower in group which transfected with Ad-VDR shRNA.(3)Compaired to control groups,both value of OD and BrdU LI increased in High level groups, while which both decreased in low level groups.The expression of Caspase3decreased in high level groups,while ratio of apoptosis detected by FACS and expression of Caspase3both increased in low level groups. In the detection of ageing, the number of ageing cells increased in low level groups, while there were no significant differences revealed between high level groups and normal level groups. The cell ratio in G1phase decreased, and that in S phase increased in high level groups, while that in S phase decreased, even there was no cell in S phase in low level+Rapamycin group, and that in G2phase increased in low level groups. In detection of migration, the number of migrated cells increased in high level groups, while no significant differences revealed between low level groups and normal level groups.Conclusions:The perfect level of VDR gene is necessary to maintain normal biological function of EPCs. When VDR gene was under expression in EPCs, proliferation was suppressed, apoptosis and ageing were increased, and transition of G2/M was blocked.High level VDR gene improved cellular proliferation, inhibited apoptosis,improved G1/S transition,and improved migration of EPCs,but showed no significance effects on ageing of EPCs.