| Background Tumor, trauma, infection, congenital disease, osteomyelitis operation debridement in bone and cartilage defects is the clinical common disease, and provides a new idea of bone tissue engineering for clinical treatment of bone and cartilage defect, bone marrow-derived stem cells with strong self-renewal ability and very good plasticity, can differentiate into osteoblast, chondroblast, nerve cells, fat cells, oligodendrocytes dendritic cells, muscle cells, cell differentiation, but the bone marrow-derived stem cells in the body have a limited number of patients, were made into great pain, the cost is higher, a time to become the bottleneck bone tissue engineering to repair bone defect and cartilage development. In2001Zuk [1] suspension isolate stem cells in the body fat, the adipose-derived stem cells [3] and bone marrow-derived stem cells have similar biological characteristics, can also be osteoblasts, cartilage cells, nerve cells, fat cells, oligodendrocytes dendritic cells, muscle cells, cell differentiation adipose-derived stem cells, and extensive source, easily, in patients suffering caused by small, can be used as seed cells to repair bone defects of articular cartilage defect, ideal, become a research hotspot in recent years.Objective1isolation of rabbit adipose-derived stem cells. Cell growth and the best condition and biological characteristics of the2.study of adipose-derived stem.3. the positive rate of detection of adipose-derived stem cells CD29, CD34, CD44, CD45by flow cytometry.4. drawing adipose-derived stem cells and cell growth curve.5.study of rabbit adipose-derived stem cells in the bone induction under the conditions of the osteogenic potential, stained with naphthol Gomori phosphate staining and Von Kossa and test results, bone induction.6.study of rabbit adipose-derived stem cells in cartilage induction under the conditions of the chondrogenic differentiation potential of [4], and alcian blue staining and saffron-light green staining, check chondrogenic induction results.7. screening of miRNA cartilage differentiation related, analysis of chondrogenic differentiation process first off the miRNA in adipose derived stem cells into chondrocytes expression pattern in the process of.Methods l.Stem cells isolated from fat source Anesthesia in rabbits with intraperitoneal injection of chloral hydrate, skin preparation, sterilization, drape, aseptic operation along the abdomen paramedian2cm cut the size of about5cm incision, exposed the subcutaneous white adipose tissue, cut off the superficial fascia and small blood vessels, with PBS buffer washing, into the ampoule bottle, in the clean workbench for Department of Ophthalmology shear crushed into a paste, adding2times volume of type0.1%I collagenase, in37℃constant temperature incubator digestion of60min, digestion after adding DMEM/F12containing10%fetal bovine serum to stop digestion,200mesh filter,1000r/min centrifugal10min, go to the supernatant, adding10%FBS containing DMEM/F12medium, the gentle wind and percussion uniform, culture flasks inoculated in25C square meters, with37℃,5%CO2constant temperature incubator, every3D change of liquid1times daily, in the inverted microscope was used to observe the morphology and growth of cells, the cell fusion about80%removing the culture liquid, adding0.25%trypsin digestion, according to1:2passage culture.2adipose-derived stem cells were detected by flow cytometry.Collect the fifth generation of adipose mesenchymal stem cells by trypsin digestion method, adjusting the density of109/L, CD29-PE, CD34-PE, CD44-PE, CD45-PE monoclonal antibody5ul, cell suspension was100μL, mixing dark incubation of15min, plus PBS500μl suspended, flow cytometry.3drawing cell growth curve.Third,5,10,15ADSCs, density adjustment is1×107/L, cultured in96well plates, each hole is200μL, each generation of4holes. Daily intake of1plates, each hole to join5g/L MTT20μL,37℃incubating for5h, plus DMSO150μL, gently shake10min, microplate absorbance at the wavelength of490nm (A) value, even in cells9D, the absorbance value (A) as the ordinate, time (d) as abscissa, growth curve.4. adipose-derived stem cells into osteoblasts differentiation When primary cells at the third passage and cover the bottom of the bottle about80%, the bottom of the bottle cells were collected and digested, seeded in6well plates,3holes for the experimental group, the remaining3holes for the control group,24h after the replacement for DMEM/F12,10%FBS,0.1μmol/L dexamethasone,μg/ml ascorbic acid,10mmol/L β-Sodium glycerophosphate osteogenesis induced liquid, the control group with ordinary medium, induced by2weeks after cell climbing piece of naphthol Gomori phosphate assay and Von Kossa staining, regular morphological changes were observed by inverted microscope and photographed.5. adipose-derived stem cells into chondrocytes induced differentiation When the adipose-derived stem cells in primary cells to the third generation and cover the bottom of the bottle about80%, the bottom of the bottle cells were collected and digested, seeded in6well plates,3holes for the experimental group, the remaining3holes for the control group,24h after the replacement for DMEM/F12,0.1μmol/L dexamethasone,50μg/ml vitamin C10ng/ml, TGF-β1,6.μg/ml to6.25μg/ml insulin, transferrin chondrogenic induction liquid, the control group with ordinary medium, induced by2weeks after cell climbing piece of Alcian blue staining and saffron-light green staining, regular morphological changes were observed by inverted microscope and photographed records.6gene chip screening chondrogenic related miRNA isolation, culture of adipose-derived stem cells from rabbit abdominal subcutaneous adipose tissue.The application of gene chip technique in screening of adipose-derived stem cartilage induction and expression of miRNA showed significant differences between cells, using the technology of RT-PCR miRNA to detect differences in cartilage induction, seventh days, fourteenth days, twenty-first heaven and downregulation of relative expression intensity, with the corresponding time point ELISA kit to detect the expression of cartilage associated protein.Results1.Cell morphological observation of stem fat source The6h primary cells most adherent, rounded, short fusiform or polygonal, uniform distribution, most of the36h cells to grow fusiform, cell volume increased significantly, the cytoplasm was granular,72h after the change of liquid medium, to seventh Tianda80%fusion, culture trypsin for subculture,24h were fibroblast-like morphology, swirl, radial or parallel arrangement, distribution,3-4d80%fusion. With the increase of the number of generations, no details change cell morphology, cell than primary cells increased significantly.2.adipose-derived stem cells by flow cytometry detection results Flow cytometry showed different algebraic cell phenotype detection results of CD29, CD44expression is higher, the positive rate was above95%, and with the increase of algebra without obvious decline trend, CD34, CD45expression was low, positive rate is less than5%.3adipose-derived stem cell growth curve The results showed that third,5,10,15generation of adipose mesenchymal stem cell growth curve had no significant difference, is a "S" shape, about4days in logarithmic growth phase, about6Tianda peak, growth curve did not change significantly with the increase of algebra.4adipose-derived stem cells into osteogenic differentiation and identification Second,4,6,8generation of cells with liquid differentiation inducing bone was measured by Gomori naphthol phosphate staining (ALP staining, Von Kossa staining). With the prolongation of induction time, ALP staining for alkaline phosphatase positive cells gradually increased, manifested as brown particles, Von Kossa mineralized node staining calcium nodules were black, the performance of the Von Kossa positive control group, and join the common medium were negative expression.5. adipose-derived stem cartilage differentiation and identification of second,4,6,8generation of cells with liquid differentiation of cartilage was measured by alcian blue staining and saffron-light green staining cells With the induction time extension, alcian blue staining after induction cells into blue, alcian blue staining was positive; saffron-bright green staining after induction of cell into red, not green, a saffron-light green staining.6chondrogenic induction before and after miRNA gene chip screening results Adipose-derived adipose-derived stem cells can obtain high uniformity in the fourth generation after stem cell; gene chip screening technology chondrogenic differentiation and differential expression of10miRNA significantly, of which6were up-regulated,4fall; quality concentration of cartilage related protein collagen type Ⅱ collagen, x, Aggrecan was significantly increased in chondrogenic differentiation of seventh days, fourteenth Tianda peak, twenty-first days decreased slightly.Conclusions Adipose-derived stem cells was detected by flow cytometry showed different algebraic cell phenotype detection results of CD29, CD44expression is higher, the positive rate was above90%, and with the increase of algebra without obvious decline trend, CD34, CD45expression was low, positive rate is less than10%, of which CD29plays a key role in blood vessel development in the process, CD44played an important role in extracellular matrix of new generation process, the test results and the bone marrow-derived stem cells, the detection results are consistent, in the expression of cell surface molecules on adipose-derived stem cells and bone marrow-derived stem cells have very similar. Draw the growth curve, the results showed that the third,5,10,15generation of adipose-derived stem cell growth curve had no significant difference, is a "S" shape, about4days in logarithmic growth phase, about6Tianda peak, growth curve did not change significantly with the increase of that algebra, adipose-derived stem cells increased value the characteristics of stable, repeated utilization.6h primary cells most adherent, rounded, short fusiform or polygonal, uniform distribution, most of the36h cells to grow fusiform, cell volume increased significantly, the cytoplasm was granular,72h after the change of liquid medium, to seventh Tianda80%fusion, cultured with trypsin passaged in fiber,24h like the pattern, whirlpool, radial or parallel arrangement, distribution,3-4d80% fusion. With the increase of the number of generations, no details change cell morphology, cell than primary cells increased significantly, many cultured adipose-derived stem cell biological characteristics are stable, easy to be isolated and cultured by cryopreservation in liquid nitrogen, and after recovery, no changes of biological property. The experiment proves that adipose-derived stem cells with osteogenic differentiation potential in inducing conditions, after passage of the biological characteristics without significant decline, biological stability, cutting easy, small trauma, can be used as seed cells to repair bone defects of ideal. Adipose-derived stem cells in cartilage induction conditions, respectively by alcian blue staining and saffron-light green staining, positive, the secretion of acid mucopolysaccharides, and main functions of chondrocytes is the secretion of acid mucopolysaccharides and collagen, the experiment with the prolongation of time, the staining results have aggravated trend, increasing content of secretory acid mucopolysaccharide and collagen, proved that adipose-derived stem cells have the potential of differentiation into chondrocytes in cartilage induction conditions, can be used as ideal seed cells for cartilage defect repair. |
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