Study On The Differentiation Of MSCs And Effect Of MSCs With RAAV-GDNF On The Function Recovery Of Spinal Cord After SCI In Rats

Part Ⅰ: The isolation, culture of the MSCs and study on theirbiological features in vitroObjective: To isolate MSCs from rat bone marrow, then culture and identify the obtained cells by detecting it's biological features and ultrastructure, found a base for using of MSCs in tissue engineering. Methods: MSCs were isolated from bone marrow suspension by discontinuous gradient centrifugation on Percoll(mass concentration: 1073g/L),then cultured on theL-DMEM medium containing 10% fetal boving serum(FBS). Invert phase-contrast microscopy was used to observe morphology change of MSCs each day. The ultrastructures of the cultured cells were observed by transmission electron microscopy(TEM). Results: The morphologies of each passage of MSCs are stable ,most of which appear fibroblast-like. Under TEM ,MSCS express the features of early ,young cells with little organelles and little cytoplasm. Conclusion: In our present study , MSCs are stable after culturing 20 passages ,most of which appear fibroblast-like and they are still in the early stage.Part Ⅱ: The experimental study on rat MSCs induced to differentiate into neural cells in vitroObjective: MSCs are multipotential and have the ability to self-renew. In addition, MSCs are easy to be obtained and good for self-transplantation. Therefore MSCs are considered as one kind of promising candidates for tissue-engineering and cell therapy . To achieve better efficiency of cell therapy, the problems such as, to get enough MSCs ,and to induce them into specified neurons, should be solved before transplantation. In present study, cell culture, immunohistochemistry and TEM were used to detect the capacity of MSCs to be differentiated to neuron-like cells and found a base for further investigation and using of MSCs in the field of neuron transplant. Methods: MSCs were induced with BHA/DMSO. The treatment protocol: MSCs were pretreated with preinduction media consisting of DMEM/20% FBS/10ng/ml bFGF for 24h, then were induced with induction media consisting of DMEN/2%DMSO/200 μmol/L BHA. The differentiating cells were observed by phase-constrast microscopy and TEM and detected by immunocytochemistry. Results: 1.After induced, more than 80% of MSCs exhibited neuron-like characteristics morphologically and were immunopositive for NESTIN, NSE, NF, GFAP, MAP, synaptophysin and these cells have large quantity of Nissil's body in the cytoplasm. Some induced cells expressed CHAT, TH, GAD. 2. Under TEM, neuron-like cells nucleuses are big, global with obvious nucleolus. They were rich in organelles. The cytoplasm showed obvious Golgimplex , rough endoplasmic reticula and numerous mitochondria. Conclusion: MSCs can be induced to differentiate into neuron-like cells in vitro, the neuron-like cells had the capacity of neurotransmitter synthesis, and neuron-like cells express the ultrastructure features of early neuron.PartHI: A study on the expression of pAAV-GDNF inthe MSCs in vitroIn order to identify the AAV vector plasmid (pAAV-LacZ, pAAV-GDNF) was constructed successfully, the restriction fragments were analysed with agarose gel elctrophoresis. The results showed that the fragments of both LacZ and GDNF had the same size with the expected. After plasmid purification, 293 cells, packaging cell line, were cotransfected by the calcium phosphate coprecipitation method with the vector plasmid (pAAV-LacZ, pAAV-GDNF, pHelper and RC). AAV vectors were then harvested and purified bytwo sequential continuous CsCl gradient ultracentrifugations. The vector titer was judged by the infectious efficiency in the HT1080 cell line with a serial dilution of the pAAV-LacZ, and was routinely 1012 particles per milliliter.In order to detect the expression of pAAV-GDNF in the cultured marrow stromal cells (MSCs). The MSCs were transduced with an appropriate amount pAAV-GDNF using calcium phosphate coprecipitation method. The expression of the pAAV-GDNF in the cultured MSCs was detected by anti-GDNF immunocytochemistry and Western analysis. The results of anti-GDNF fluorescent immunocytochemistry showed that a widespread, diffuse distribution of the GDNF throughout the cell body and the processes was seen with fluorescent microscopy. A 16kD molecular weight species, the expected size of GDNF could be observed by Western blot probed with antibody against GDNF. The results of this present study indicated that the recombinant vector, pAAV-GDNF, could express GDNF in the cultured MSCs.