Experimental Study On The Cell Factors-modified MSCs Combined With Rhodiola Rosea To Improve The Recovery Of Spinal Cord Injury

Objective：At hypoxic environment, female rats spinal cord injury model were treatedwith Rhodiola rosea extract combined with or without keratinocyte growth factor (KGF)and/or hypoxia inducible factor (HIF) gene-modified bone marrow mesenchymal stem cells(MSCs) with followed by dynamic assessment of spinal cord injury healing and functionalrecovery of motor neuron were assessed, which provides insight into the therapeuticprospective of spinal cord regeneration and functional reconstruction under hypoxicenvironment.Method：The bone marrow mesenchmal stem cells from male Winstar rats were isolatedby density gradient centrifugation, and transfected with either control adenovirus or KGF andHIF-carrying adenovirus. The female rat spinal cord injury model was established withmodified Allen method and70Winstar rats randomly divided into7groups: MSCstransplantation group, Ad-HH-MSCs transplantation group, Ad-HH-MSCs transplantationcombined Rhodiola group, saline control group, and the Rhodiola only group. The successfultransplantation of MSCs was determined with in situ hybridization of sex-determining geneSry gene. The spinal cord hemorrhage and edema were observed under light microscopy afterHE staining. The swelling, degeneration, and necrosis of nerve cells were analyzed by nerveNissl methylene blue staining. The distribution and participation of injury repair oftransplanted cells and the effect of Rhodiola were evaluated by detecting the expression ofKGF and HIF protein by immunohistochemistry and examining the levels of adenovirus andsry by fluorescence quantitative real-time PCR. Data were expressed as x±s. Thedifferences between groups were analyzed with student t-test using SPSS19.0statisticalsoftware. A p-value less than0.05was considered statistically significant.Result:1、HE staining observation: no significant difference was observed among groups oneweek after transplantation; three and six weeks after injury, saline control group had morecavities whereas neuronal regeneration and tissue repair were observed in the spinal cord ofrats received MSCS transplant;12weeks after injury, the spinal cord tissue of saline controlgroup rats were darker with more cavities many microcapsules in the white matter, necrosis of nerve fibers and cells, large vacuoles in the vicinity of transection while that from ratsreceived MSCS transplant and/or Rhodiola was lighter with no significant cavities, vacuolesless and small, and denser nerve fibers and more neurons. The combined treatment ofAd-HH-MSCs transplantation and Rhodiola had the best results.2、Nerve cell swelling, degeneration, necrosis and Nissl body changes were analyzedunder light microscopy with nerve Nissl methylene blue staining. There was no significantdifference among groups1and3weeks after injury. The number of neurons was slightly morein treatment groups compared to saline control. At week12post-injury, serious neuronaldamage, neuron reduction, and increased vacuoles were observed in saline control whereasgradual neuronal regeneration was seen in rats received MSCs and/or Rhodiola.3、Immunohistochemical staining was employed to detect the KGF and HIF proteinlevels in the spinal cord of rats at1,2, and3weeks after injury. The KGF and HIF proteinlevels were low in saline control and Rhodiola group while there were significantlyincreased in MSCs, Ad-HH-MSCs, and Ad-HH-MSCs plus Rhodiola groups, the levels ofthose proteins peaked in the first week. The difference among groups was statisticallysignificant.4、The expression levels of adenoviral gene and sry were analyzed by fluorescencequantitative PCR. There was no significant difference at week1and3after injury, at week6and12, the expression of adenoviral gene and sry was undetectable in saline control andRhodiola groups but strongly expressed in Ad-HH-MSCs and Ad-HH-MSCs combined withRhodiola groups. The difference among groups was statistically significant.5、Sry gene expression in injured rat spinal cord was visualized by in situ hybridization.Sry mRNA was detected in MSCs, Ad-HH-MSCs, and Ad-HH-MSCs plus Rhodiola groupsand the abundance in those groups was in ascending order. Sry mRNA was not detected insaline and Rhodiola groups.Conclusions：1、MSCs are successfully isolated through density gradient centrifugation. Once infectedby recombinant adenovirus, they can express HIF and KGF, two foreign genes that canexpress biological activities.2、pinal cord injury model are successfully established. 3、It is tested and verified preliminarily that in the spinal cord jury model of rats in lowoxygen environment, for rats with HIF and KGF gene modification, the control scheme ofMSCs with rhodiola rosea is effective to the reparation of the injury.4、It has been found that HIF and KGF have regional expression and transplanted cellsexist in injury repairing mechanism, both of which indicate that genes and cells havefunctioned in spinal cord injury repairing.