| The first part The establishment of the culture of humanumbilical cord mesenchymal stem cells, identification,cryopreservation, and spinal cord injury modelPurpose1.Explore human umbilical cord mesenchymal stem cells in primary culture andidentification, and to observe the biological characteristics.2. Explore stem cell the climbing film culture, the expression of GDNF and BDNFfluorescence.3. Explore the injured spinal cord model created correctly.Method1.Primary cell culture and flow identification1.1Fresh, sterile full-term, healthy Caesarean maternal newborn umbilical cord,extracted from human umbilical cord Waldorf-through plastic or called Wharton the glue(Whartoncs jelly, WJ), line organization adherent method, application the DMEM/F12vitroprimaryculture and subculture and its separation, purification and identification.1.2Will the umbilical cord Waldorf pass glue cut approximately1mm×1mm×1mmsize, line organization the adherent law placed in6-well plates, followed by1.5hours on a37°C incubator adherent culture plates. Out of the tissue adherent prefabricated stainlesssteel mesh (1mm×1mm size box diameter) placed above the tissue blocks, addingDMEM/F12culture observed a stainless steel mesh on the number of cells in the cultureprocess. 1.3Flow cytometry was used to identify CD24, CD34, CD44, CD90expression in thestem cell surface.1.4And cryopreservation of cells (4°C refrigerator30min'-20℃30min'-80℃low temperature refrigerator freezer overnight'-194℃stored in liquid nitrogen.) Followthe principle of slow the freeze-speed financial. Cell culture passage to two generations instrict accordance with the steps principle cells frozen and stored in liquid nitrogen;transplant remove recovery train passage to the4th generation cell markers transplant.2.Human umbilical cord mesenchymal stem cells seeded culture, the expression ofGDNF and BDNF fluorescence.3.Odel of spinal cord injury3.1Wistar rat model of spinal cord injury. With reference to production methods asWamil, injured by a falling body principle made an impact device, the impact body headend3mm, length3.5cm, the colliding body weight8g dropping height2cm impact the bodyinjury impact8g×3.5cm×2cm. Rats weighing about250g, a dose of10%chloral hydrate0.7ml/100g line intraperitoneal injection of anesthesia, the rat limbs fixed to the fixing plateof homemade. Iodophor disinfection surgical area, followed by incision exposed spinal cord,install homemade blow positioning T10spinal cord above the combat injuries of the spinalcord, the line according to plan.Rat spinal cord hit model operation and results of the modeling are consistent fromgroup B, group C, group D, E group immediately after the BBB score are0.00±0.00, whileobserving rats sanity consciousness, mental status, heart rate, breathingand limb movementflexibility, while observing whether the abnormal adverse reactions after injection.3.2Collect stem cellsObserved stem cell fusion is up to80%-90%0.25%trypsin-EDTA digestion andcentrifuged1000rpm for5minutes, re-added to the medium, the cells were counted underthe microscope, the control in a concentration of1×106cells/ml, collected standbytransplantation.Result1.Umbilical cord Waldorf gum tissue (Whartoncs jelly, WJ) adherent methodDMEM/F12vitro cultured7days visible cells to swim out, radial growth, diamond-shapedor cord-like, prolate full cell colony proliferation were cultureintegration of more than90%to about20days the most exuberant cell; proliferation of cell passage more than90%faster,2-3days cell fusion, continue to line the cell passage.2.Use stainless steel mesh adherent culture, cells significantly accelerated swim out, significantly increased the number of stem cells, cell proliferation passage time; no effect onits morphological changes.3.Flow cytometry stem cell high expression of CD44, CD90; expression or lowexpression of CD24, CD34,4.Strictly in accordance with the principle of financial follow the slow freezing speed(4°C refrigerator30min'-20℃30min'-80℃low temperature refrigerator freezerovernight'-194℃liquid nitrogen storage.) Cells and cryopreservation; recovery afterstem cell morphologyconsistent structure, growth, proliferation and cryopreservation.5.Expression of BDNF and GDNF immunofluorescence staining of human umbilicalcord mesenchymal stem cells to whole cells have obvious expression, uniform distribution,the most obvious expression in the cytoplasm; appear green, the nucleus is not obvious;rendering tan expression,nucleus visible.6.With reference to production methods as Wamil, falling body injury principle madean impact device; according to the rat spinal cord against the modeling success criteria:spinal cord to combat the damage seen after retraction of the rat hind legs and torso archedtwitch, flick, followed by flaccid paralysishindlimb muscle relaxation, group B, group C,group D, E group immediately after successful modeling are0.00±0.00, BBB rated.7.Line DMEM/F12and4th generations of human umbilical cord mesenchymal stemcells were injected immediately after successful modeling, were group B, group C, group D,E group transplanted rat the Theosophical consciousness, mental status, heart rate, breathingandlimb movement flexibility indicators revealed no abnormalities, and no adverse reactionsafter injection.Conclusion1.Using tissue adherent method can be obtained a sufficient hucm stem cells. Plusstainless steel mesh tissue adherent culture method may further increase the number of cells,separation, purification and flow identification can increase cell purity.2.The good morphology of stem cells can also be obtained after the recovery of thecells stored at-80℃.3.Human umbilical cord mesenchymal stem cells may be obvious expression of BDNFand GDNF.4.Homemade blow against rat spinal cord can be successfully produced a rat model ofspinal cord injury, further experiments. THE SECOND PART EFFICACY OF HUMAN UMBILICALCORD MESENCHYMAL STEM CELLS SUBARACHNOIDTRANSPLANTATION IN THE TREATMENT OF SPINALCORD INJURYPurposeExplore subarachnoid transplantation efficacy of spinal cord injury.Method1.Randomization:Group A for the sham group: rats with conventional surgical spinalcord exposed, not to combat damage, do not do any transplant. Group B transplantDMEM/F12purely to the local spinal: After successful modeling of the hit model of acutespinal injury in rats, hemostasis make vision clearly,inject stem cells at a distance of six T10injury zone head point, at2mm end and both sides of the damage regional centers; Withprepared10μL Microinjector inject1.5mm DMEM/F122.5μL into the pin deviate fromthe spinal cord midline0.75mm, inject5min slowly using a Hamilton syringe.Localbleeding,needle retention after the3min injection, followed by suture paraspinal muscles,fascia, skin and subcutaneous tissue.Group C is DMEM/F12subarachnoid transplantation group: After successful modelingof the hit model of acute spinal injury in rats, hemostasis make vision clearly.With prepared50μL microinjector injected DMEM/F12liquid15μL slowly, injection time is5min.3min later of injection, bleeding, suture paraspinal muscles, followed by fascia, skin andsubcutaneous tissue. Group D is the local transplant group of stem cells in the spinalcord:transplant10μL hUC-MSCs suspension with the same way of group B, to the localspinal cord. Group E is subarachnoid transplantation group:transplant25μL hUC-MSCssuspension using the same method as the Group C to the local spinal cord.2. Observation of rats’ security general and death situation after transplantation.3.After injury1d、1w、2w、3w、4w、5w、6w、7w、8w carry out the BBB actsof school sports function score contrast, observed effect of transplantation of two ways.4. After injury1M,2M of immunohistochemical staining (GFAP, NSE),observedhuman umbilical cord mesenchymal stem cell migration and differentiation in rat spinalcord.5. After injury1M,2M with immunohistochemical staining (GFAP, NSE), observedthe expression of the human umbilical cord mesenchymal stem cells rat spinal cord. Result1. General observation and DeathA group of rats postoperative mental state, gait, limb movement, pain stimuli reflectioncheck no obvious abnormalities. Group A no deaths, the death of the B group, D group andgroup C, E group is basically the same. The successful modeling rats complications: Undernormal circumstances, the postoperative rats in2-3hours can be sober,4-6hours that maybe a small amount of food and water.Major complications after surgery: hematuria, urinaryretention, urinary incontinence, lungs and urinary tract infection, incision and spinalinfection, limb edema, self-mutilation. In the course of the experiment, A group of death0Bgroup died three group C4died,2D group of death,3E group died, the survival rate was80%.2.BBB scoring results of the postoperative periodWe regularly observe rats after rats1d,1w,2w,3w,4w,5w,6w,7w,8wBBB ratedobserve time to8W A group was21.00±0.00, preoperative and postoperativeno change; Bgroup was8.08±0.56, group C9.84±1.53, B group, C group results is basically the same,no significant difference; D group was16.58±1.88and E group was18.33±0.79D groupand E group recoveryThe faster, the effect is obvious; group B, group C significantdifference statistically significant (p <0.05).3. GFAP expression in the spinal cordImmunohistochemical staining1M,2M: The results showed no positive expressiongroup A, group B and group C very small part of GFAP weak expression exists, the resultsare not typical; A group, B group, C group of the three groups showed no significantdifference; group D, group E positive expression obviously, of GFAP in the organization ofexpression mainly to the vertical distribution, concentrated in the gray matter within thewhite matter expression results significantly reduce; with group A, group B, C group threegroup comparison there are obvious differences, havestatistically significant.A group, B group, C group three groups showed no significant difference in group D, Egroup fluorescence staining found: of GFAP positive expression area concentrated in thespinal cord gray matter, white matter expressed less positive expression area was thedevelopment of longitudinal; application goat anti-rabbit-FITC-antibody, dry cells appearsobvious yellow green fluorescence results have significantly with the expression; dropwiseaddition, goat anti-mouse-Cy3-antibody stem cells appear obvious bright red fluorescence,results have significantly with the expression; with group A, B of groupgroup C three groups have more significant difference; stem cells in the spinal cord injury site long-termsurvival was significantly fluorescent.4. NSE expression in the spinal cord1M,2M Immunohistochemical staining showed that umbilical cord stem celltransplantation NSE positive cells around the spinal cord injury District of Group D andGroup E than in group A, group B, C group increased significantly positive expression.Distribution and GFAP consistent, statistically significant.Group D and Group E fluorescent staining microscopic examination found: NSEpositive expression area concentrated in the spinal cord gray matter and white matterexpression significantly reduce the positive expression area mainly was the development ofthe vertical line; application goat anti-rabbit-FITC-antibodies, stem cells appear obviousyellow greenthe fluorescence expression results significant; dropwise addition of goatanti-mouse-Cy3antibody stem cells appeared bright red fluorescence, the expression resultssignificantly.5.hUC-MSCs migration and differentiation in the host spinal cordD、E two sets of umbilical cord stem cells after transplantation1M,2M microscopy,visible to a large number of stem cells in the host spinal cord survived and was verticalmigration the migration distance beyond the area of the injury, can reach farther distancedamage zone shows a large number of dyeingpositive cells in the gray matter within theperformance up to the most obvious, is mainly concentrated in this area within the whitematter less positive expression. By microscopic examination found within the group Eexpression and distribution of positive cells was more obvious than in group D, and GroupD partial transplant microenvironment changes may have a great relationship; while nosignificant differentiation.Conclusion1.Human umbilical cord mesenchymal stem cells subarachnoid transplantation foracute spinal cord injury better therapeutic effect, with intramedullary local transplant was nosignificant difference, significantly better than the control group, the difference wasstatistically significant.2.Immunohistochemical staining color a lot of survival of human umbilical cordmesenchymal stem cells in the host spinal cord and longitudinally migration, to play the roleof nerve repair.3.Human umbilical cord mesenchymal stem cell transplantation for high security,found no obvious rejection. |
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