The Experimental Study On Treatment Of Spinal Cord Injury By Overexpression Of EGF And CNTF Induced Reactive Astrocytes De-differentiation

Neural stem cells (NSCs) are regards as a perspective candidate for neural treatment inclinical therapies for central nerve system injury and neurological discorders.Unfortunately, NSCs sources supply, tumor formation and persistence of pronouncedimmue response make this technique face great challenge for its therapeutic applications.Astrocytes within injured central nerve system region resulting from mechanical,chemical and pathological unjury may undergo a process of de-differentiation, thesede-differentiated astrocytes express nestin, the marker of neural progenitor cells, orsometimes possess neural precursor/stem cells characteristics. How the astrocytes makede-differentiation following injury is largely unclear. It presumption that astrocytesde-differentiation may correlate to intimate microenviroment around them. The data invitro shows astrocytes from injured spinal cord may acquire NSCs in culture conditions,the approved viewpoint is of astrocytes de-differentiation profiles. The property ofastrocytes de-differentiation offers a potential useful method for treatment of centralnerve system injury. The purpose of this experiment is to bulid the eukaryoticexpression vector of epidermal growth factor (EGF) and ciliary neurotrophic factor(CNTF). The recombinant vectors were co-transfected into reactive astrocytes (RAS) toobserve its effect on RAS de-differentiation in vitro. Then the recombinant vectors wereco-transfected with a spinal cord injury in rats, to observed its effect on RASde-differentiation in vivo and promote the repair of spinal cord injury.The study is divided in three parts. In the first experiment, a SD rat aged3weeks was selected and killed by dislocation of neck, the submandibular gland and sciatic nervewere quickly taken out under cold condition, then dissected the submandibular glandand sciatic nerve, after that, the total RNA of the submandibular gland and sciatic nervewas extracted under the condition without RNA enzyme contamination with Trizolreagents. Using revers transcription PCR, the cDNA fragments of EGF and CNTF geneswere amplified from total RNAs respectively. After the end of electrophoretic analysis,the purified PCR products were collected. Then the amplified fragments wererespectively inserted into eukaryotic expression vector pSecTag2/Hygro B to constructthe recombined plasmid that encoded EGF and CNTF cDNA. The plasmids carryingEGF and CNTF genes were transfected alone respectively into competence JM109E.coli. After preliminary screening by PCR, the two positive clone were digested bydouble restriction enzyms BamH Ⅰ and Hind Ⅰ. The vectors which build correctly werenamed pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF. The purified plasmidscarrying EGF and CNTF genes were transfected alone respectively or cotransfected intocos-7cells by liposome method. After transfecting48hours, collecting cell culturemedium and then the expression proteins were detected by Western blot. The resultsshowed that, a specific positive band was detected in6kD in signal transfectionpSecTag2/Hygro B-EGF group, a specific positive band was detected in22kD in signaltransfection pSecTag2/Hygro B-CNTF group, co-transfected group in both6kD and22kD, the control group showed a negative result. These results confirmed that therecombinanted EGF and CNTF protein were correctly expression in cos-7cells.In the second experiment, we first isolated and cultured rat spinal cord astrocytes invitro, then established the RAS cell model by scratch method. The immunofluorescencestaining showed that the RAS GFAP and nestin expression in double positive, thisconfirmed that astrocytes ungo de-differentiation by stimulation, and expression ofneural stem cell specific protein, which in consistent with the preliminary findings of other reserachs. Then, we on-transfected the successful constracted eukaryoticexpression vector pSecTag2/Hygro B-EGF and pSecTag2/Hygro B-CNTF into RAS tooverexpression recombinant EGF and CNTF proteins. As time goes on, RAS expressnestin constantly enhance the strength, at6d, the field of view are basically GFAP andnestin double positive cells, the expression of nestin also significantly enhanced. But thenormal astrocytes do not appear nestin positive cells with co-transfection eukaryoticexpression vectors. The RAS which transfected with blank plasmid only a small numberof GFAP-positive cells express nestin, and the expression intensity is relatively weak.And the semiquantitative analysis found that no significant expression of nestin innoramal astrocytes. The RAS transfected with blank plasmind expression nestin isrelatively weak. From2to6day, the nestin expression intensity gradually rising inco-transfection group, at the6th day, the nestin expression reach the highest. Set therelative optical density value of1in blank plasmid transfection group, at2day therelative optical density value is1.03±0.13, the4d is1.14±0.16, the6d is1.23±0.27.These results suggest that the recombinant EGF and CNTF eukaryotic expression vectorwere co-transfected the RAS cell model in vitro, that can up-regulated nestin expressionin RAS, and promote RAS de-differentiation, but the specific molecular mechanismneeds further research.In the third experiment, Ninety-six rats were randomly divided into four groups, andeach group contained24rats. The rats in EGF uni-transfection group (group A), CNTFuni-transfection group (group B), EGF and CNTF co-transfection group (group C) andcontrol group (group D). The rats received a spinal cord contusive injury at T10levelusing modified Allen’s method. EGF and CNTF recombinant plasmid and blankplasmid were transfected into the damaged areas of exprimental group and controlgroup respectively by Alzet pumps. At1,2,4,6weeks, Basso-Beattle-Bresnahan (BBB)Rating Scale was used to observe the recovery of motor function. The expressions of EGF and CNTF DNA and protein in injured spinal cord were detected by RT-PCR andWestern blot techniques. And double immunofluorescence and histopathologicexaminations were performed to study the proliferation and differentiation of thereactive astrocyte and pathological change after SCI. The results showed that, At2,4,6weeks after SCI, the BBB scores in the group C was significantly higher than that inother three groups (P<0.05). RT-PCR and Western blot showed that the DNA andprotein expressions of EGF and CNTF were observed in group A, B, C and noexpression was seen in group D. Histologic observation showed that the morphology ofspinal cord and neurons in group C was better than that in the other three groups andwas close to the normal tissue. The number of Nestin+astrocytes in group C wassignificantly more than that in other three groups (P<0.05). These results suggested thatthe recombinant EGF and CNTF in vivo environment by promoting the RASde-differentiation neurological function recovery in rats with spinal cord injury.In summary, overexpression of EGF and CNTF protein in vitro model of RAS cells cancontribute to the process of de-differentiation of the RAS. Overexpression of EGF andCNTF protein can promote RAS de-differentiation and promotion of spinal cord injuryin rats recovery of neurological function in spinal cord injury in rats.