| Chapter 1 Biology research mice adipose stem cellBackground:Stem cell research has been becoming more and more popular in the world. Characteristics of prolifemiceion and differentiation of stem cells have attracted more attention from the researchers. Among many adult stem cells, adipose-derived stem cells gradually highlight in the field of tissue engineering because of their unique advantages.Objective:Through cultivation of mice adipose stem cell lines in vitro, to understand its biological chamiceeristics, and provide experimental materials for follow-up study.Methods:Adipose tissue from abdominal subcutaneous tissue and the groin in 15 adult Kunming mice as experimental material, digested with type I collagenase under the condition of 37 ℃ for 1 h to get adipose-derived stem cells. Screening culture system of adipose-derived stem cells in vitro, the application of immunohistochemistry was used to analyze mice adipose stem cell surface markers, to analyze the mice adipose-derived stem cells in vitro amplification, as well as chromosome karyotype during in vitro culture.Results:Twelve mice adipose cell lines from 15 adult kunming mice were obtained, Primary culture cells were short, round or ovoid, high luminose when started to culture,24 hours later, a small amount of cells were observed to attached to plate wall, and 48 hours later more cells were observed adherent, until cells were full of culture plate and tended to cell fusion. In this stage, cells become long and thin spindle, form for long fusiform. During 120 days of culture, it was found that mice adipose stem cell growth curve is typical of the "S" type, PDT was 42.14 hr, 46.32hr and 43.34 hr. Cell prolifemiceion process constitues of incubation period, logarithmic phase and platforms phase, which implies mouse adipose-derived stem cells has strong ability of self-renewal, easy to cultivate in vitro augmentation. Immunohistochemical identification of adipose-derived stem cells:CD13, CD29, CD44, CD71, CD73, CD90, CD105 and CD166 is positive expression, the MAP-2, GFAP and Nestin is negative expression. The MAP-2, GFAP and Nestin are markers for nerve cells, shows that fat stem cells do not express nerve-cells-like related proteins. Mice adipose-derived stem cells in the diploid chromosome number is 2 n=40, including 19 pairs of autosomal and 1 pair of sex chromosomes, the sex chromosomes are X, Y.Conclusion:Mice adipose-derived stem cells isolated in these experiments are stable under the culture condition in vitro, show strong expansion ability, fast prolifemiceion after passage, good mophology, lowl level of mice adipose stem cell aging, and in the surface markers analysis without express specific markers of nervous system, is good experimental materials for cells inductio of nerve cells and repair of spinal cord injury.Chapter 2 The research of adipose-derived stem cells differentiation into nerve cellsBackground:Previous studies suggests that the nerve cells of the nervous systems doesn’t genemicee new cells after initial genemicein of nerve cells. This means that adult nerve cells in the brain and spinal cord can only be gradually reduced and cannot be regnemiceed. Acuqired central nervous system damage, such as brain injury and spinal cord injury, neurological system is not able to selfrepair. Nerve cells from stem cells as a new nerve cell in the donor site, bring a new dawn for nerve cell damage repair.Objective:Provide seed cells for cell therapy of spinal cord injury through in vitro inducing mice adipose-derived stem cells to differentiate into nerve cells. Methods:Taking the third genemiceion of cultured mice adipose-derived stem cells, the 5x 105/mL cell concentmiceion incubated in 25 cm2 culture bottle. Using 20 ng/ml bFGF and 20 ng/ml, EGF and 2% of B27 medium at 37℃ and 5% CO2 incubator to culture. Every three days to change half amount of culture medium, in 5-7 days there were neural cells appeared. Nestin was identified and analyed with immunofluorescence staining and PCR.Results:The mice adipose-derived stem cells in the 3-4 days of induction culture, started to gather in the center, to 5-7 days cells started to have many small synaptic, and clearly distinguish between the cell body and protuberant, in immunofluorescence staining and RT-PCR analysis nestin is positive. Culture in culture medium containing 2% B27 for 10 days, some of the cells show typical change of nerons, cells are round, with neucleus, slender and long or net-like cell processes. Immunofluorescence staining and RT-PCR analysis show cells can express the MAP-2 and GFAP. The diameter of the cells go up to 100 at the 14th day of culture.Conclusion:In specific and controled environment, mouse adipose-derived stem cells can differentiate to neural cells and differentiated into neurons and astrocytes later after induction.Chapter 3 Evaluation of treatment on spinal cord injury with adipose-derived stem cellsBackground:In recent years, stem cells have been used in the research of treatment of brain, spinal cord, peripheral nerve injury and other disease. Currently the main problem to use stem cells is that the cell source, ethical, moral, legal issues, the occurrence of tumors, neuropathic pain and immune rejection, etc.. In this study, we use use autologous fat tissue sepamiceion to obtain adipose-derived stem cells and differentiate into nerve cells by the directional differentiation, and injected the induction of neural cells into the spinal injury mouse model, in order to use the cell transplantation treatment in spinal injury, and to provide new theoretical proofs for stem cell therapy.Objective:To assess the ability of nerve cells from induction of adipose-derived stem cells to repair spinal cord injury by injection of differentiatied stem cells into mouse model of spinal cord injury.Methods:The acute spinal cord injury model in mice. Mice was anesthesized with 10% chloral hydmicee (3 ml/kg) intraperitoneal injection. In the position of T10 segments, put the plastic gasket ((3 mm x 3 mm) under T10 segment of spinal cord, use modified Allen’s device, to give a 5g*5cm vertical hit, mice tail swing spasmodicly, double lower limbs and body retraction flutter, impplied model was successfully set up. ADSCs was marked. Using ADSCs in good condition, after nerve cells change was observed, the induced cells were transfected with the liposome with GFP report gene. ADSCs transplantation was carried out 9 d after spinal cord injury. After anesthesia, along the original cut through each layer of tissues,, reveal the corresponding damage of spinal cord segment, in the area, with micro syringe transplant 10 ul cell suspension (1.0 x 105 cells/ul) or same amount of PBS in the control group, close the incision afer transplantation. Routine nursing after opemiceion. Cyclosporine A (10 mg/kg/d) injection from the 1 d prior to the opeation to the end of the entire opemiceion. At 4 weeks after transplantation, with 4% paraformaldehyde in heart perfusion, spinal cord segment was isolated and frozen section for testing was prepared.Results:Immunohistochemistry test was performed after 4 weeks. In the transplantation group, the cells gathered in the damage ara of spinal cord, regenemiceion of nerve cells were observed, and connected the two ends of broken spinal cord. No GFP positive cells were observed in PBS control group, there was still cavity in the damage area. To evaluate the movement in mice after transplantation:The movement capability and recover is better in the experimental group than the other two control groups. In BBB score system, the experimental group is also better significantly than the other two groups. SPSS data analysis showed that mice in different periods after cell transplantation compared with PBS group, the value was significantly different (P< 0.05).Conclusion In this study, with transplantation of nerve cells from redirected induction and differentiation of adipose-derived stem cells, the cells can survive and grow in the damage area of spinal cord, and rebuld neural circuits in the surrounding tissue, finally results in the reconstruction and improvement of body function. |
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