The Research Of Pre Induction Culture Of Human Peripheral Blood Mononuclear Cells And Its Survival And Function In Mice With Retinal Degeneration

Stem cells are a class of cells that have the ability to maintain self-renewal and potential differentiation(1).In adult peripheral blood mononuclear cells(hPBMCs),there is a small cluster of heterogenous stem cells.As having the potential of multi-directional differentiation,easy access,a wide range of sources,Stem cells are an ideal resource for stem cell therapy.Retinal degeneration(RD)is a group of genetic disease with common manifestation of loss of light sensitive photoreceptors and retinal pigment epithelial progressively.RD diseases are a major cause of untreatable blindness in the world.The disease has not yet effective threapy,and stem cell transplantation for the treatment of retinal degenerative diseases provides a new way.It is a prerequisite for stem cell therapy to have a curative effect that they can be transplanted into the body and prolonged survival.The primary purpose of this research was to observe the survival of the pre-induced adult hPBMCs transplanted into subretinal space of retinal degeneration mice(rd1 and rds mice)for 5-6 months.The experimental process is as follows: At the beginning,the peripheral blood was drawn from the healthy adult vein.The hPBMCs was extracted from the peripheral blood by lymphocyte isolation.The harvested hPBMCs were cocultured with the retina of the neonatal rats for 4 days.And the expression of the markers in the nerve and retina after pre-induced culture was detected by real-time quantitative PCR and flow cytometry.Furthermore,hPBMCs were injected into the subretinal space of retinal degeneration mice.Finally,the retinal electroretinogram was measured by retinal electrophysiological test at 5 months after cell transplantation.Sixth months after cell transplantation,the survival of the transplanted hPBMCs was analyzed by immunofluorescence,flow cytometry,and RT-PCRThe results of immunofluorescence in RDS mice eye biopsy show that the transplanted cells exists in the inner nuclear layer and ganglion cell layer,and some transplanted cells express neural stem cell marker Nestin,neural progenitor cell marker Vimentin,Tubulin beta III,neural cell markers MAP2 and photoreceptor cell marker Rhodopsin.Flow cytometry showed that 19.35±10.33% of the cells expressed anti-human mitochondria-specific antibody signal in the eyes of the experimental group compared with the blank control group,and 11.55±1.52% of the cells in the non-operated eyes of the experimental group.PCR results showed that the human cytochrome b gene still expressed in the mouse eyes after six months of transplantation.Full-field electroretinogram(ERG)results showed that after 5 months of transplantation,experimental eyes in some treated mice recovered to respond to light.These results suggest that adult peripheral blood mononuclear cells(hPBMCs)transplanted into the subretinal space can survive and regenerate in nerve cells and light-sensitive cells within 6 months after pre-induction culture.