| IntroductionBone defects are a common problem needed to solve in clinical orthopedics. At present, Autograft, allograft and biological bone substitute are not the ideal treatment in bone defects. However, the bone tissue engineering provides us an ideal way in treatment, which cultures the seed cells having osteogenic potential in biomaterial in vitro and then graft the newly growth bone into the defects sites.In the past, most bone tissue engineering researches on seed cells were focused on the BMSC cells, however these kinds of stem cells are normally short in the total amount and basically hard to obtain. Moreover these cells cultured in vitro tend to age and the patients are not easy to accept. Adipose-derived stem cells bone taking form the patients'own, on the other hand, are easy to obtain, large in amount, multiple in differentiation, no graft against host reaction and stable in expression. From the above reasons, Adipose-derived stem cells as a vector could be superior in the gene therapy.In present study, we successfully extracted adipose stem cells from fat tissue. The target gene was BMP2 and the Adipose-derived stem cells were the target cell. Transfection was carried out by using trans-gene technique. The expression of the Adipose-derived stem cells was stable. The osteogenic characteristic of Adipose-derived stem cells was studied in vitro. This study proved that the target genes could successfully be transferred into adipose-derived stem cells, which has a good capability in bone formation.There are all together three parts in this study.1. The isolated culture, identification and osteogenic differentiation of adipose-derived stem cells.Object: To culture adipose stem cells, to identify them and confirm their osteogenic potential.Methods: CollagenaseⅠdigested the fat tissue and centrifuged for sediment cells. Cultured these cells in 10% FCS-DMEM within incubator with 37℃5% CO2. The shape of the cell growth and the amount of the cells were recorded, and then the growth proliferation curve were drawn. Calculated the population doubling time of the cell colony and the immunohistologic stain was applied to identify the expression of the protein. CD29 CD31 CD34 CD49d CD105and CD106 on the ADSCs surfaces were detected through flow cytometry. The third generation of these cultured cells was transferred to the osteogenic culture medium. The osteogenic capabilities were detected by ALP immunohistologic stain, Von Kossa stain and CollagenaseⅠstain.Results: ADSCs could be successfully extracted from human fat tissue and the amount of cell proliferation was considerable, which was 5×106 cells from 10ml fat tissue. 4-6h after inoculation, the original ADSCs started to deposit and adheres. 48h later, most cells adhered.5d later, these cells grew in a colony and in a fabric cell shape. After 15th generation of the original cells, there were no cell aging signs. P3 cells proliferated in a'S'shape, and the population doubling time was 43h. After osteogenic cultivation, ADSCs proliferated slowly and the growth cycle became longer. The population doubling time of the cell colony was 60h. The immunocytochemistry stain showed the expression of the ADSCs was positive, which indicates that ADCS originates from mesoderm and belong to the mesenchymal cell. Flow cytometry detection showed that the expression of CD29 CD49d CD105 were positive. The expression of these factors in the third generation cells was above 85%. After osteogenic induction, ADSCs shapes changed remarkably, the cell body enlarged and cytoplasm became abundant. The brown particles in the cytoplasm could be seen in the ALP stain. The brown black calcified node could be seen in the central part of the cell colony in Von Kossa stain. The cellular nucleus were blue and brownish particles appeared in the cytoplasm in CollagenaseⅠstain. The above results proved that ADSCs has the osteogenic capability.2. Ad-BMP2 transfected ADSCs and the detection of ADSCs osteogenic capabilityObject: To construct the BMP-2 adenovirus vector and infect ADSCs; to detect the osteogenic capability of the infected ADSCs.Methods: PCA13-hBMP2 and pBHGE3 plasmid coinfected 293 cells through Lipofectamine2000 to construct Ad5-hBMP2 adenovirus vector. After vector accreditation, the proliferation started and the virus titer was calculated. Ad-EGFP infected ADSCs to detect the adenovirus vector transfection efficiency and confirm the best MOI. Ad-BMP2 infected ADSCs in vitro. The cell growth shape, the cell amount and cell proliferation curve were recorded and the population doubling time was calculated. BMP-2 expression was detected through Western blot, the expression of BMP-2 on the mRNA lever were detected by RT-PCR. These cells were divided into Ad-hBMP2 infected group, mineral liquid induction group and non-transfection group. The ALP quantitation was detected on 4d, 1W, 2W, 3W, 4W, 5W, 6W respectively. The expression of OCN and CollagenⅠwere detected by RT-PCR on 3d, 1W, 2W, 3W, 4W, 5W, 6W respectively.Results: The Ad5-hBMP2 adenovirus vector was successfully constructed. The virus titer was 1.669×1010pfu.mL-1. Ad-EGFP infected ADSCs, when MOI was 100, more than 90%cells were fluorescent. MOI=100 was best. 4w after virus infection, the cells still could be fluorescent under scope observation, which indicated the reconstructed Adenovirus had a high biologic activity and could effectively transduce target gene for a period. Shortly after the transfection, some cells shrinks, some even died. After 24h these cells stated to recover to the original shapes. The curve of cells proliferation was in a'S'shape, the delay time lasted to 4d.The population doubling time was longer than the P3 cells, which was about 46h. BMP2 immunohistologic stain showed that there were brown particles in the cytoplasm of the tansinfected cells. RT-PCR detection showed that the expression of BMP-2 gene started in 48h right after transinfection. The expression enhanced in 1w, 2w, 3w and the peak time was on 2w. The expression weakened gradually in 3w. Western blot showed the expression of BMP-2 in ADSCs could be detected in 1w and the expression intensed in the second and third week. The expression weakened in the fourth week and the fifth week it decreased remarkably. The above results indicated the reconstructed adenovirus could transfect ADSCs cells effectively and the target gene could express in a high level for a certain period time. The ALP quantity detection was carried out on 4d,1W,2W,3W,4W,5W,6W in Ad-hBMP2 infected group, mineral liquid induce and non-transinfection group. The results showed that ALP active expression could be detected in the Ad-hBMP2 infected cells on the fourth day, after which the ALP expression kept increasing. The peak value appeared on the third week and the value maintained in a high level for the following three weeks. The ALP activity started to get low on the sixth week. In the mineral liquid induce group, the ALP activity was not detected on the fourth day, which was detected on the first week. From then on, the expression level kept increasing. And the peak value also appeared on the third week. In the following time, the expression level maintained highly except for the sixth week. The ALP expression of mineral liquid induce group were lower than that of Ad-hBMP2 infected group on every time points. There were significant in statistics between these two groups. The OCN and collagenⅠwere detected respectively on 3d, 1W, 2W, 3W, 4W, 5W, 6W. The results showed the OCN could not be detected on the third day and the first week in the Ad-hBMP2 infected group. The OCN started to express on the second week. However the expression was in a relatively low level. From the second week, the OCN expression kept in a high level. From the fifth week, the OCN expression began to decrease. The OCN active expression could be detected on the fourth week in the mineral liquid induce group and the detection was negative on the other time points. The OCN expression of the Ad-hBMP2 infected group was higher than that of the mineral liquid induce group on the fourth week. There was statistic difference between these two groups. There was no OCN expression in the non-transinfection group. CNI expression could be detected on the third day in the Ad-hBMP2 infected group. In the following time, the expression kept in a high level, peak appeared on the third week and decreased from the sixth week. In the mineral group, the CNI expression could be detected on the third day. Expression peak appeared in the second week and kept a high level to the sixth week. In the non-transfection group, the CNI expression could be detected on every time point, however the expression level was relatively low and there was no peak time appeared. The CNI expression between Ad-hBMP2 and mineral group was no significant difference. The CNI expression of these two groups were both higher than that of non transfection group and there significant difference existed. The above results indicated that the BMP2 infected ADSCs and the mineral ADSCs both have the osteogenic potential and the BMP2 infected ADSCs osteogenic potential was higher that of solo mineral ADSCs. 3. Ad-BMP2 infected ADSCs ectopia ossification in situ in the naked mouse.Object: To observe the ectopia ossification capability of BMP2 infected ADSCs.Methods: Four groups were divided as: A group BMP2 infected ADSCs+ chitosan scaffold; B group mineral liquid induce ADSCs+ chitosan scaffold; C non-transfection ADSCs+ chitosan scaffold; D chitosan scaffold. the combination of ADSCs and chitosan scaffold was put in the muscle fissure of femoral lateral M.and femoral posterior M. the mouse were killed on the fourth and eighth week and the experiment material was obtained. The histolgic observation of ectopia ossification was taken through HE stain, BMP2 immunohistologic was used to observe BMP2 expression in vitro.Results: the HE stain was positive in A group on the fourth week and negative in the other groups. On the 8W all specimen were negative, which showed the expression of ADSCs could lasted for more than four weeks and there were no expression on the 8W. The ectopia ossification in A B groups exist on the 4W and 8w. The osteogenic capability of A group is stronger than that of B group.Conclusion:ADSCs could be successfully extracted from human fat tissue and the amount of cell proliferation was considerable, which was 5×106 cells from 10ml fat tissue.ADSCs has stable growth in vitro, and the proliferation capability is stable, even to the 15th generation, there is no remarkable aging sign.The third generation of ADSCs can express high level surface agent which is related to stem cells. The unified quality of these cells is high.ADSCs has osteogenic potential in the induce culture medium, and the population doubling time delays, however the proliferation is stable.The Ad-BMP2 adenovirus vector was successfully constructed and the target gene BMP2 can express effectively.The Ad-BMP2 infected ADSCs has a efficient gene infection rate and the expression can be lasting for a certain time with a high level.The Ad-BMP2 infected ADSCs appears strong osteogenic potential, which is stronger than that of ADSCs in the induce culture medium. ADCS under normal culture seems no osteogeni capability.Ectopia ossification happens in the Ad-BMP2 infected ADSCs+ chitosan scaffold group and mineral liquid induce ADSCs+ chitosan scaffold group in the Naked mouse, the Ad-BMP2 infected ADSCs osteogenic potential is higher than that of ADSCs in the induce culture medium. There is no ectopia ossification in ADSCs under normal cultivation.Present study proves that the target genes can be transferred to the fat stem cells though the combination of tissue engineering and gene therapy technology. The infected fat tissue cells have a good osteogenic capability in vivo. The ADSCs as the seed cells have unique superiority. ADSCs are promising cells to be used as a new stem cells bank. In the future research, we will focus on the bone defects repair through bone tissue engineering in order to lay a foundation for the clinic application.
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