| Objective:1.Establish the method for the isolation and cultivation of rat bone MSCs.2. Explore the method of constructing high-copy plasmid total expressing BDNF and the method of BDNF gene transfected to bone MSCs.3.Investigate the changes of rat behavior and tissue pathology of CA1 region of hippocampus when grafting MSCs, empty plasmid modified bone MSCs, plasmid carrying BDNF gene modified bone MSCs respectively by the way of lateral cerebral ventricle of AD rat model.4. Investigate the levels of BDNF and TrkB in CA1 region of hippocampus when grafting MSCs,empty plasmid modified bone MSCs,plasmid carrying BDNF gene modified bone MSCs respectively by the way of lateral cerebral ventricle of AD rat model.Methods:1.Bone MSCs were isolated from thigh bone marrow of SD rat and purified through adherence ability. These cells were identified as MSCs by their surface-antibody CD29, CD44, CD34.2. pEGFP-BDNF plasmid was reconstructed into pIRESneo-EGFP-BDNF plasmid which was transfected to MSCs with electroproation. The transfected MSCs were bolted by G418 and were observed by blue light excited by green light under fluorescence inverted microscope. BDNF proteins were measured by Western blot in the transfected bone MSCs.3. Forty healthy male SD rats were selected and then divided into five groups randomly, which were Sham group, AD group, MSCs group, CON group, BDNF group. The AD model was set up by injection with beta-amyloid peptide 25～35(Aβ25～35) into the hippocampus of both sides stereotacticly. The AD models were treated by bone MSCs, empty plasmid modified bone MSCs, plasmid carrying BDNF gene modified bone MSCs respectively transplanted through cerebral lateral ventricle, and then behavioral changes were observed in place navigation test and in spatial probe test, and then tissue pathological changes were observed by HE stain and Nissl stain in light microscope, and ultrastructural changes of neurons and synapses in CA1 region of hippocampus were observed under electron microscope.4. The expressions of BDNF and TrkB in CA1 region of hippocampus were analyzed by the methods of RT-PCR and Western blot, while the AD models treated by bone MSCs, empty plasmid modified bone MSCs, plasmid carrying BDNF gene modified bone MSCs respectively.Result:1. The cells selected by their adherence ability had basic phenotypical properties of MSCs, which were with good activity and high purity by subculturing.2. pIRESneo-EGFP-BDNF plasmid was identified by double digestion for carrying BDNF gene and carrying EGFP gene as report gene. High efficacy of BDNF transfected to MSCs by electroporation was obtained after bolted by G418.3. Results of Morris Water Maze test:The AEL was significantly prolonged and the frequency of crossing over the platform position was significantly decreased in AD Group compared to Sham Group(P<0.05 or P<0.01). The AEL was significantly shortened and the frequency of crossings over the platform position was significantly increased in MSCs Group, CON Group, BDNF Group respectively compared to AD Group, and in BDNF Group compared to CON Group or MSCs Group (P<0.05或P<0.01), but these were no significant difference between MSCs Group and CON Group (P>0.05). The results of tissue pathology:The number of neurons in hippocampus was reduced obviously. The array of neurons in hippocampus was irregular obviously. The damage of cell organs was very serious and the number of synapses in hippocampus was significantly reduced. The gaps of most of synapses in CA1 were obscure notably. Those changes above were obviously lessened in MSCs Group, CON Group, BDNF Group, especially in BDNF Group.4. The results of RT-PCR:The expressions of BDNFmRNA and TrkBmRNA in hippocampus tissue were existed in all groups. The levels of BDNFmRNA and TrkBmRNA in hippocampus tissue were obviously lower in AD Group compared to Sham Group (P<0.01). The levels of BDNFmRNA and TrkBmRNA in hippocampus tissue were obviously higher in MSCs Group,CON Group,BDNF Group respectively compared to AD Group, and in BDNF Group compared to CON Group or MSCs Group (P<0.05), but there was no significant difference between MSCs Group and CON Group (P>0.01). The results of Western blot:The expressions of BDNF protein and TrkB protein in hippocampus tissue were existed in all groups. The levels of BDNF protein and TrkB protein in hippocampus tissue were obviously lower in AD Group compared to Sham Group (P<0.01). The levels of BDNF protein and TrkB protein in hippocampus tissue were obviously higher in MSCs Group, CON Group, BDNF Group respectively compared to AD Group, and in BDNF Group compared to CON Group or MSCs Group (P<0.05), but there was no significant difference between MSCs Group and CON Group (P>0.01).Conclusion:1. Bone MSCs with good activity and high purity were obtained through their adherence ability and subculture.2. Electroporation for transfecting stem cells was a good method which can improve transfection efficiency.2. The AD model which was set up by injection with beta-amyloid peptide 25～35 (Aβ25～35) into the hippocampus of both sides stereotacticly was successful and convenient. The learning and memory of AD rat models could be improved through bone MSCs modified by plasmid carrying BDNF gene modified bone MSCs or bone MSCs transplanted by the way of cerebral lateral ventricle, especially in the former group. The pathological changes of neurons and synapses in hippocampus were improved too. So BDNF played an important role in recovery of nervous system.4 The expressions of BDNF and TrkB in hippocampus of AD rat model were improved obviously by bone MSCs modified by plasmid carrying BDNF gene or bone MSCs transplanted through directly sereotactic injection into cerebral lateral ventricle. especially in the former group. Perhaps it was one of mechanisms which treated AD. Brilliant future for treating AD is not far. |
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