Study On Characteristics,Cryopreservation And Gene Transfection Of Goat Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) are a kind of adult stem cell that characterized by their abilities of high proliferation, self-renewal and multipotent differentiation into various lineages of tissue cells. At present, BMSCs from human, mouse, rat, rabbit, pig and sheep and many other species can be induced in vitro to differentiate into a variety of cells such as cardiomycyte, myotube, chondrocyte, osteoblast, dermal cell, adipocyte, tendon ligament, neuron proved under certain conditions. In addition, BMSCs also have the higher degree of synchronization and easy to be re-programmed. so BMSCs are one of promising seed cells for the cell/gene therapy and tissue engineering in future. However, there were so few reports about systemic study on goat BMSCs on the isolation, culture, cryopreservation, transgene, and so on. Therefore, this experiment targeted Huanghuai goat, more systematic and comprehensive study such as the isolation, culture, proliferation and growth characteristics, surface markers, in vitro differentiation, cryopreservation and transfection efficiency were been conducted. The concrete contents are as follows:1. The isolation, cell culturation, biological characteristics of goat BMSCs.The biological characteristics of goat BMSCs mainly included the proliferation, gene expression and differentiation potential. The primary cells were separated using whole bone marrow culture method. The cells had shuttle shape, a typical morphological fibroblast and were confluent in single layer after being plated for 9 days. The growth curves of P1 and P7 were drew by cell-counting method. The curves showed "S" pattern, according to the general law of cells growth. P5 cell cycles were analyzed by flow cytometry 2 days after contact inhibition. The cells in G0/G1 phase were up to 89.69%, which means high synchronization ability. Goat BMSCs expressed CD29, CD44, CD90, CD106 and CD166; but did not express CD45, the surface markers of hematopoietic stem cells. Finally, to testify differentiation ability, BMSCs were induced to differentiate into osteoblast and adipocyte.2. Study on cryopreservation of goat BMSCs.This part optimized the frozen liquid type, concentration and cryopreservation procedures. Firstly, analysis on the effect of cryopreservation on the biological characteristics of goat BMSCs and we found that cells growth after thawing are still the "S" pattern curve, while cell cycles were analyzed by flow cytometry 2 days after contacted inhibition, G0/G1 phase showed cells reached 89.45%, with no difference with that before freezing. Study of the DMSO concentrations, sucrose concentrations and freezing procedures of cryopreservation as follows:(1) with using of cell progressive freezing method, we detected the freezing effect on goat BMSCs of the freezing liquid with 2.5%,5% and 10% DMSO. as a result, when the DMSO content was 2.5%, live cells after thawing were (97.76±1.3)%, significantly higher than 5% and 10% groups, but the adherence rate of three groups after 24 h had no difference; (2) adopting progressive freezing method,0.05 M,0.15 M and 0.25 M sucrose was added to the cryopreservation solution containing 10% DMSO. As a result, we found that the difference of living cells rate after thawing and adherence rate after 24 h was not significant among the three experimental groups; (3) with the frozen solution containing 10% DMSO, three differed cryopreservation procedures of goat BMSCs were compared:progressive freezing, isopropanol freezing box and directly-80℃freezing, we found in directly -80℃group freezing living cells obtained the highest rate of recovery, but the 24 h adherence rate was not significant in the three experimental groups.3. Study on gene transfection of goat BMSCsStudy on transfection efficiency of goat BMSCs. The plasmid of pEGFP-C 1 was transfected into the goat ear fibroblast cells and goat BMSCs. The results showed that goat BMSCs are sensitive to liposome LTX, so their cell-specific transfection protocols were as followed:100μL DMEM-LG was mixed with 0.25μg plasmid,0.5 pL PLUSTM Reagents and 1.25μL LipofectamineTM LTX, the efficiency of transfection is up to (67.91±5.3)%, significantly higher than that of the goat ear fibroblast cells. The results suggest that goat BMSCs can be used as a kind of high efficient donor cells of transgenic nuclear transplantation.