Bone Marrow-derived Mesenchymal Stem Cells Expressing The Shh Transgene Promotes Functional Recovery After Spinal Cord Injury In Rats

Spinal cord injury (SCI) is one of the problems of the medical challenge, whichdue to injury, traffic accident, landslides, natural disasters and etc. SCI, which canlead to loss of cells and damage of neural tracts, therefore result in many tissular andorganic dysfunction, is one of the most common disability and lethal disease. Patientsafter SCI will not only bring serious physical and mental damage, but also cause ahuge economic burden to the whole society, therefore seriously affect the quality ofhuman life.At present the main treatment of spinal cord injury include surgery removingoppression, steroids, protein kinase, metalloproteinases, suppression and regenerationtechnology and etc, however the effect of treatment is limited. In recent years, how torepair spinal cord injury has achieved great progress. Stem cell gene transplantation isrecognized as a promising treatment, and is the focus for the treatment of spinal cordinjury.Previous studies have shown that bone marrow mesenchymal stem cells(BMSCs), which have widely source, a good ability of proliferation anddifferentiation, easy to obtain and cultivation, low immune rejection, and don’tinvolve the ethical issues, are the ideal carrier of the gene therapy. In their treatmentfor spinal cord injury, BMSCs can secrete neurotrophic factors, improve themicroenvironment of the spinal cord injury, differentiate into neurons which replacenecrosis or death of nerve cells, have the bridge role which can provide the axonregeneration with a good connection channel, promote the formation of new blood vessels and target spinal cord lesions. However, most BMSCs die aftertransplantation as a result of ischemia and hypoxia in the region of SCI.Growth factor is essential for repairing spinal cord injury. Sonic hedgehog (Shh),which is produced by the notochord and floor plate, is a member of the family ofhedgehog proteins and is essential for the development and differentiation of thenervous system. Shh works on several aspects of spinal cord regeneration: it inducesthe production of motor neurons and oligodendrocytes, enhances the delivery ofneurotrophic factor to improve the microenvironment in BMSCs, facilitates neuronalsurvival, promotes axonal growth, and prevents activation of the astrocyte lineageafter SCI. However, an injection of Shh protein after SCI does not have a good effecton functional behavioral recovery because of the fast clearance of Shh protein fromthe spinal cord.In our study, we speculated that transplantation of BMSCs transfected with Shh(Shh-BMSCs), which can provide long-term stable expression and secretion of highlevels of Shh, could conquer the difficulties of inherent in treatment with Shh alone,to facilitate repair and recovery after SCI.In this study we referred to cell and animal research. The research mainly coversthe following three parts:1. To isolate, culture and identification of rat BMSCs2. To construct Shh carrier, use the lentiviral vector technology to transfect BMSCsand identify the transduction effect.3To establish rat spinal cord injury model, treat SCI using Shh-BMSCs via thesubarachnoid injection and observe the therapeutic effects after SCI. Section one Isolation, culture and identification of rat bonemarrow mesenchymal stem cellsObjectiveTo establish a method of isolation, cultivation of rat bone marrow mesenchymalstem cells (BMSCs) in vitro, to identify cell morphology, cell proliferation, cellsurface markers, osteogenetic and adipogenic differentiation capacity.Research methods1.AnimalsAdult female Sprague Dawley (SD) rats,160-200g, were purchased fromAnimal Center of Chinese People’s Army Military Medical and Scientific Academy,Beijing, China.2. Isolation of BMSCs and cultivation of BMSCs in primarycultureAnimals were anesthetized with10%chloral hydrate. Take the rat femur andtibia rapidly and wash them using PBS. Use scissor to cut off the epiphyseal end,expose the long bone marrow cavity, and wash them using L-DMEM mediumcontaining100U/ml penicillin streptomycin until the marrow cavity was white. Thesingle cell suspension was centrifuged by1200r/min for5minutes and cultured bySD MSC complete medium at37°C under a5%CO2atmosphere. The inoculationconcentration is6-8×l06/ml.3. The cultivation, purification and passage of BMSCsIn the process of primitive culture, change a half quantity medium after48hoursand follow a full volume replacement every three days. When BMSCs were coveredabout80%, use trypsin-EDTA solution to digest them. The ratio of subculture was1:3.4. The cell morphology of BMSCs To observe the cell morphology and growth condition using inverted microscopeand photograph them.5. To detect cell proliferation using MTT methodsAt passage3, BMSCs were cultured in96-well culture plate under a5%CO2atmosphere. The inoculation density was5x103/hole. Take out one plate and detectit every day by MTT method. Detect the optical density of palte for seven days usingenzyme-linked immune detector absorption value, and the wavelength was490nm.The observation time was for the horizontal axis and the value of optical density wasthe vertical axis.6. To detect the cell surface markers of BMSCs using flowcytometric analysisAt passage3, BMSCs were trypsinized and centrifuged by1200r/min for5minutes at4°C. Count the cells and join the monoclonal antibody CD29, CD34,CD44, and CD45for30min at room temperature. After washing cells using PBS toremove the uncombined antibody, join the second antibody labeling FITC and PE in adark place for30min, and detect them using flow cytometric analysis.7. Osteogenetic and adipogenic differentiation capacity ofBMSCsAt passage3, BMSCs were cultured in6-well culture plate. When BMSCs werecovered about80%, join the osteogenic and adipogenic media and change a fullvolume replacement every three days, at the same time set up a blank control withoutjoining induction media. After cells was fixed by40g/L paraformaldehyde at roomtemperature for10minutes, we evaluated osteogenic differentiation by alizarin redstaining at day28and the adipogenic differentiation was assessed at day23bystaining with oil red-O solution. Observe and photograph them using inverted microscope.Results1. The morphological observation of BMSCsBMSCs were spindle and showed radial colony arrangement. Cells kept goodgrowth and could passage in continuous over10passages.2. The cell growth curve of BMSCsCell growth curve demonstrated that BMSCs were consistent with the growthcharacteristics and strong activity of normal cells.3. The expression of BMSCs cell surface markersAt passage3, BMSCs were found to express cell markers CD29and CD44, butCD34and CD45were not detected.4. The identification of osteogenic and adipogenicdifferentiationAfter osteogenic and adipogenic differentiation, alizarin red and oil red-Ostaining were positive.SummaryThe whole bone marrow adherence method is simple, and can isolate, purify andamplify BMSCs in vitro. The obtained cells have general biologicalcharacteristics ofbone mesenchymal stem cells, and also have potentiality of osteogenic andadipogenic differentiation. This experimental way has important practicalsignificance to provide suffient source of carrier cells for gene engineering. Section two Construction of bone marrow-derivedmesenchymal stem cells expressing the Shh transgene andidentificationObjectiveTo construct the Shh gene modified BMSCs using a lentiviral vector in vitro andthen observe the identification of transduction efficiency, provide a potential stablestem cell source for the treatment of SCI.Research methods1. Construction of Shh carrierFirstly amplify attB1-Shh-attB2using overlapping PCR; then constructpDown-Shh using Gateway Technology; finally structure pLV.EX3d.P/puro-EF1α>Shh>IRES/DsRed(red fluorescent protein)Express2using Gateway Technology.2. Constuction of lentiviral vector and BMSCs expressing theShh transgene using themTransfection of293T cells using Lipofectamine2000. Detect the titers oflentivirus using hole-by-hole dilution method. Infect BMSCs with shh lentiviralvector and detect the transfection efficiency through the fluorescence.3. Detect the shh mRNAexpression of Shh-BMSCs usingReal-time PCR analysisTotal RNA was extracted from the cells. Total RNA was reverse transcribed intocDNA under a PCR-reaction system and the condition of Reverse transcription.Record the value of Ct, amplification curve and dissolve curve. Calculate theexperimental results (2-ΔΔCT) using relative quantitative methods. 4. Detect the shh proteinn expression of Shh-BMSCs usingWestern blot analysisAfter the proteins were collected from cells, protein concentrations were testedby the BCA method. Configuration of12%SDS-polyacrylamide gel, on sample,electrophoresis, transfer the membrane, blocked, reaction of primary and secondaryantibody, electrochemiluminescence detection.Results1. Construction of BMSCs expressing the Shh transgeneThe shh lentiviral vector was constructed. It was entirely correct by enzymedigestion and sequencing identification, and it could transfect293T cells and achieveexpression. The lentiviral titer was2-4×108TU/ml. The fluorescence expressionincreased significantly at5d following transduction2. Shh-BMSCs increased the shh expressionShh-BMSCs could long-term stable mRNA and protein expression of high levelsof Shh using lentiviral vector comparing with the uninfected BMSCs.SummaryConstruction of Shh lentiviral vector successfully and Shh-BMSCs, which canlong-term stable expression of high levels of Shh. BMSCs were the basis for thesubsequent experiments. Section three Effect of bone marrow-derived mesenchymalstem cells expressing the Shh transgene on recovery after spinalcord injury in ratsObjectiveTo establish the spinal cord injury model and observe the BMSCs survival andfunctional recovery via transplantation of Shh-BMSCs at7days after SCI.Research methods1.AnimalsAdult female Sprague Dawley (SD) rats,200-220g, were purchased fromAnimal Center of Chinese People’s Army Military Medical and Scientific Academy,Beijing, China.2. Spinal cord injury modelAnimals were anesthetized with10%chloral hydrate (0.3ml/100g,intraperitoneal injection); then followed by Allen’s methods. In brief, the spinal cordwas exposed by performing T10laminectomy. SCI was induced by dropping a10-grod (diameter,2.5mm) from a height of25mm onto an impounder positioned on thespinal cord.3. Treatment of Shh-BMSCs after SCI via intrathecal injectionSeven days after SCI, a second laminectomy was performed at lumbar level4.We therefore microinjected Shh-BMSCs into the model rats intrathecally. In thecontrol group, BMSCs or PBS was injected.4. Detect the shh protein expression after treatment ofShh-BMSCs in rats with SCI To detect the shh protein expression using Western blot analysis at28d aftertreatment.5. Detect the neuroprotection after treatment of Shh-BMSCs inrats with SCI5.1The expression of neurotrophic factorsTo detect the basic fibroblast growth factor (bFGF) and vascular endothelialgrowth factor (VEGF) protein expression using Western blot analysis at28d aftertreatment.5.2Shh-BMSCs promoted the BMSCs survivalTo observe the lesion epicenter using a fluorescence microscope at28d aftertreatment and analyze the number of BMSCs using Image J software.5.3Detection of neurofilament200(NF200) and glial fibrillaryacidic protein (GFAP) immunofluorescence stainingThe sections around the lesion epicenter were washed, blocked, incubated byprimary antibodies (NF200and GFAP) and secondary antibody and stained by DAPIat28d after treatment. The expressions of GFAP and NF200were observed using afluorescence microscope and analyzed by Image J software.5.4Nissl’s staining for the motoneuronsThe sections around the lesion epicenter were stained in1%solvent blue at60°Cfor40min, washed three times using distilled water, differentiated in95%ethanol,dehydrated in100%ethanol,cleared in xylene and covered by using a resinousmedium. Photograph and count the motoneurons using a inverted microscope.Analyze them by Image J software.5.5Evaluate the lesion cavity using HE staining The sections around the lesion epicenter were stained in Harris hematoxylin for7min and0.5%eosin for2-4min. The volumes of lesion cavity were photographedusing a fluorescence microscope and analyzed by Image J software.6. Evaluate the behavioral recovery after treatment ofShh-BMSCs in rats with SCIThe Basso–Beattie–Bresnahan (BBB) locomotor rating score is widely used toevaluate hind-limb motor function. Rats were examined by three trained examinersfor6weeks after SCI in a double-blinded manner. The BBB results were averaged.7. Statistical analysisAll data were expressed as mean±SD. One-way analysis of variance (ANOVA)was used to compare mean values. A repeated measure ANOVA was used to analyzeweekly BBB scores in different groups. Statistical analyses were performed usingSPSS15.0software. Statistical evaluations were considered significant at P <0.05.Results1. The model of spinal cord injury was successfully establishedRat tail swing and hind limb extremity paralysis indicated that induction of SCIwas successful. Manual bladder expression was performed before recovery ofautonomous urination. All rats were injected penicillin intraperitoneally and survivedbetter.2. Shh-BMSCs promoted the expression of Shh in vivoShh expressions of spinal cord were detected by Western blot analysis at28dafter treatment. The protein expression of Shh was significantly greater afterShh-BMSCs treatment than the other two interruptions. Differences were consideredstatistically significant (P <0.05). 3. Shh-BMSCs increased the neuroprotection3.1Shh-BMSCs promoted the expression of bFGF and VEGFThe bFGF and VEGF protein levels were determined using western blot analysisat day28after treatment.The protein levels of bFGF and VEGF in the Shh-BMSCsgroup were markedly higher than those in the PBS and BMSCs groups, and the bFGFand VEGF protein expression in the BMSCs group increased significantly by day28in comparison with the levels in the PBS group. Differences were consideredstatistically significant (P <0.05).3.2Shh-BMSCs enhanced the BMSCs survivalBMSCs survival was detected using a fluorescence microscope at28d aftertreatment. The number of BMSCs present at this time point in the Shh-BMSCs groupwas significantly more than those in the BMSCs group on day28after transplantation.Differences were considered statistically significant (P <0.05).3.3Shh-BMSCs increased the expression of NF200and reducedthe expression of GFAPThe expressions of NF200and GFAP were detected using immunofluorescencestaining. On day28after transplantation, the expression of NF200in the Shh-BMSCsgroup was significantly higher than that in the other two groups, and the staining inthe PBS group was significantly lower than that in the BMSCs group. The expressionof GFAP on day28after transplantation was markedly weaker in the Shh-BMSCsgroup versus the other two groups, and the staining between the PBS and BMSCsgroups did not differ significantly. Differences were considered statisticallysignificant (P <0.05).3.4Shh-BMSCs increased the number of motoneurons after SCI Nissl’s staining at the ventral horns was used to evaluate the number ofmotoneurons at28d after treatment. Injection of Shh-BMSCs significantly increasedthe number of motoneurons in the ventral horns, compared with injection of BMSCsor PBS. The BMSCs group had more motoneurons than the PBS group at the ventralhorns. Differences were considered statistically significant (P <0.05).3.5Shh-BMSCs reduced the area of lesion cavity after SCIHE staining was used to evaluate the area of lesion cavityat28d after treatment.Injection of Shh-BMSCs significantly reduced the area of lesion cavity, comparedwith injection of BMSCs or PBS. The BMSCs group had less area of lesion cavitythan the PBS group. Differences were considered statistically significant (P <0.05).4. Shh-BMSCs promoted significant functional recoveryBBB tests showed highly significant motor improvement in rats that had beentreated with Shh-BMSCs compared with those treated with PBS or BMSCs, from2wto6w after SCI. The BBB score of the BMSCs group was significantly greater thanthose of the PBS group (P <0.05).Differences were considered statisticallysignificant.SummaryBMSCs that have been transfected with a Shh transgene, which can providelong-term stable expression and secretion of high levels of Shh and so enhance thesurvival of BMSCs, would promote the neuroprotection and facilitate repair andrecovery after SCI and could be a potential valuable therapeutic intervention for SCIclinically.