| BackgroundProfessor Lin Yuan has put forward a new hypothesis-fasiaology. In fasciaology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of undifferentiated cells of unspecialized connective tissues. The other is the function system, which is consisted of differentiated cells and is enclosed by the supporting-storing system. That is "two system theory". The undiffferented stem cells in the supporting-storing syetem incessantly differentiate into functional cells. The supporting-storing system throughout the body regulates the functional and living status of differentiated cells and provides a stable internal environment for the survival of these cells. Loose connective tissue is an important part of the supporting-storing system, and adipose-derived stem cells(ADSCs) are main stem cells reserved in this syetem.At present, the study about adiposed stem cells has obtained a signifigant progress. adipose-derived stem cells are a kind of adult stem cells, which derive from adipose tissue and possess multiplex differentiation potentials. Zuk first isolated the processed lipoaspirate cells which can proliferate stably in vitro and has the ability to differentiate into a veriety of kinds of cells from human adipose tissue. Compared with other kinds of adult stem cells, PLA cells have the advantages of extensive sourses, convenient draw of materials, little trauma and no existance of immune rejection. The experiment demonstrated that ADSCs can differentiate into mesoderm-derived stem cells such as adipocytes, osteoblasts, chondrocytes and cardiocytes, ectoderm-derived stem cells such as nerve cells, entoderm-derived stem cells such as endothelial cells and hepatocytes. However, could adipose-derived stem cells differentiate into germ cells?Genn cells are cells that can reproduce offsprings in multicellular organisms including primordial germ cells and final differentiated germ cells. At the third weekend of human embryonic development, the embryo generates three original germ layers(entoderm, mesoderm, ectoderm). At the fourth weekend of embryonic development, ectoderm induces the adjacent epiblast to generate PGCs. PGCs begin its sexual differentiation.Embryonic germ cells have pluripotency, and their cell markers are consistent with ES cells. Because alkaline phosphatase and Oct4are expressed in embryonic germ cells, they are usually used to detect the specialized PGCs in the mouse tissue.At6.25d of embryo, Vasa is RNA helicase that is dependent on ATP and specific in germ cells. RNA binding proteins coded by Vasa are located in cytoplasm and expressed in PGCs of sex-ridge. Vasa is the specific gene marker of germ cells that have entered gonad during the later migration stage. Vasa is expressed in the tissue of ovary and testis and can not be detected in adult tissues, therefore, it is the specific marker of the highly differentiated germ cells. So far, Vasa is the only entirely specific gene for germ cells. In most species, the expression of Vasa in cells means germ cell specialization. As the marker of PGC, tissue non-specificalkaline phosphatase can be used to observe the migration of PGC from allantois to sex-ridge. In the formation of sperms and oocytes, Stra8regulates the start of meiosis, and meiosis is initiated by Stra8induced by RA.There are three methods that can induce stem cells to differentiate in vitro:①to add inducing factors into culture medium;②to cultivate the stem cells with the target cells;③to thansfect the target genes into the stem cells to make them express the genes. In our experiment, we add inducer RA and testosterone to induce adipose-derived stem cells. RA is a basic regulator in the gametogenesis. It has been decided that RA is derived from vitamin A and essential for germ cells to enter meiosis. The lack of vitamin A will lead this process to cease. After the sexuality of gonad in female mouse is determined, the meiosis will be initiated quickly. Because meiosis is realized by Stra8, this initiation will be blocked by RA antagonist or by knocking off Stra8. Stra8is mainly expressed in germ cells before meiosis, and once entering meiosis, the level of Stra8in germ cells will greatly reduce or disappear. Testosterone is a kind of steroid hormone and the primary androgen which is related to the development of secondary sex characteristic and important in the process of spermatogenesis.This experiment is based on fasciological theory and determine whether adipose-derived stem cells have the potentials to differentiate into germ cells in vitro. We will examine whether germ cell markers are expressed in adipose-derived stem cells under the condition of induction in vitro.ObjectiveTo explore whether ADSCs express germ cell related genes stra8and vasa to determine whether ADSCs have the potentials to differentiate into germ cells. To explore the change of ADSCs’ cell cycle when ADSCs are induced by testosterone and RA respectively or altogether.1. To determine whether there are undifferentiated mesenchymal stem cells in nonspecific connective tissue by isolating subcutaneous adipose tissue and culturing cells;2. To verify whether ADSCs express the germ cell related genes Stra8and Vasa by RT-PCR to further determine whether ADSCs have the potentials to differentiate into germ cells;3. To verify whether ADSCs express the germ cell related genes Oct4, Dazl, Nobox and Piwil by real-time PCR to further determine whether ADSCs have the potentials to differentiate into germ cells;4. To explore the change of ADSCs’ cell cycle when ADSCs are induced by testosterone and RA respectively or altogether. Methods1. To detach the ADSCs from the2month old female Wistar rats’ inguinal adipose tissue; to cultivate the cells in vitro by the enzyme digestion; to identify whether ADSCs in non-specific fascia connective tissue consistent with the characteristics of MSCs by observing their formation, means of growth, morphological, functional study, flow cytometry and biological markers on the cells’ membrane; To judge whether connective tissue contains ADSCs.2. To induce passage4th ADSCs differentiation into adipogenic and osteogenesis, in order to conform their differentiation potential. After ADSCs were cultured under adipogenic and osteogenic condition, The morphological characterization of the inductive cells was observed via alizarin red and alkaline phosphatase staining for mineralization nodules and oil red O staining for lipid accumulation. Meanwhile, to detect part of the surface antigens of passage4th ADSCs by flow cytometry so that identify the stem cell characteristics of ADSCs.3.The total RNA are extracted from passage4th ADSCs for RT-PCR, to detect the expression of mRNA of Stra8and Vasa, with the positive contrast of the expression of Stra8and Vasa in testis cells. To detect the expression of alkaline phosphatase which is the marker of primordial germ cells.4. ADSCs are devided into six groups:①control group,②ADSCs indu ced with10-5mol/L RA for2weeks,③ADSCs induced with10-5mol/L RA for4weeks,④ADSCs induced with10-6mol/L RA for2weeks,⑤ADSCs induce d with10-6mol/L RA for3weeks,⑥ADSCs induced with10-6mol/L RA for4weeks, then the germ cell related genes Oct4, Dazl, Nobox, Piwil were examin ed from the six groups.5. ADSCs are devided into six groups:①control groupk,②10-5mol/L RA group,③10-6mol/L RA group,④10-5mol/L RA+testosterone group,⑤10-6mo1/L RA+testosterone group,⑥testosterone group, the cell cycle was detected n these groups of cells by flow cytometry after thirty-six hours of cultivation.Results 1. ADSCs can be extracted from the rats’fat tissue. In primary culture, the isolated ADSCs show a fibroblast-like morphology. The two passage cells were adherent and have a spindle-shaped morphology in the days3rd; the mostly adherent ADSCs presented spindle shape or polygon, cell size uniformity under optical microscope;2. Flow cytometric analysis of surface antigens demonstrated that ADSCs were uniformly expressed CD29, CD90and CD106, and negative for CD49d, CD11b and CD45;3. The undifferentiated cells were cultured for14days under adipogenic conditions to induced the formation of lipid-filled vesicles that were stained red by oil-red-O staining and were presented characteristics of adipocytes, while the red staining of lipid droplets were not observed in the negative control wells.. Induction of osteogenic differentiation of the cells for21days resulted in the deposition of mineralized nodules that were stained red by Alizarin red staining and were presented the characteristics of osteoblasts. negative control wells were not dyed pink calcium nodules;4. The OD value we detected in ADSCs treated with10-5mol/L,10-6mol/L,10-7mol/L RA for7days are0.585±0.04,0.268±0.07,0.151±0.03(the control group is0.068±0.01), for14days are0.417±0.02,0.341±0.01,0.187±0.02,(the control group is0.067±0.01). This demonstrated that RA can dramatically improve the ALP expression in ADSCs (P<0.01), and the expression is dose-dependent. The expression of Vasa mRNA in ADSCs induced with10-5mol/L RA for7days is negative. However, the germ cell marker stra8and vasa are expressed in testis cells of positive control group.5. The real-time PCR result showed that ADSCs can express oct4, dazl, nobox and piwil. The three genes oct4, dazl and nobox have the same expression profile, and they are all expressed with the highest level when the cells are induced with10"6mol/L RA for3weeks. However, piwil is epressed with the highest level when cells are induced with10-6mol/L RA for4weeks.6. Flow cytometry showed that the cell proportion in phase G1of all groups are respectively80.3%,82.0%,85.2%,81.0%,65.1%,68.5%,and the cell proportion in phase S of all groups are respectively14.7%,12.7%,9.2%,13.5%,28.6%,22.7%.Conclusion1. Through this experiment, the author consider that there are multipotent stem cell in adult Wister rat’s adipose tissue of the connective tissue. Furthermore, ADSCs can be successfully obtained by the digested-adherent-passage programs. The experimental support are provided to the view that fascia connective tissue of the body play a supporting-storing role for the functional system(stem cell reserve). In other word, ADSCs existed in fascia connective tissue suplly stem cells source to repair body damage.2. ADSCs with the characteristics of mesenchymal stem cells and multipotent differentiation potential, can be induced into the osteogenic and adipogenic differentiation.3.ADSCs can express alkaline phosphatase which is the marker of primordial germ cells, but ADSCs treated with RA for7days can not express stra8and vasa which are the markers of germ cells. Testis cells can express stra8and vasa which are the markers of germ cells.4. ADSCs induced with RA can express germ cell related genes oct4, dazl, nobox, piwil, this demenstrated that ADSCs have the potentials to differentiate into germ cells when induced with RA.5. Retinoic acid can inhibit the proliferation of adipose-derived stem cells, while testosterone can promote the proliferation of adipose-derived stem cells.In summary, this study investigate ADSCs from morphological features, adipogenic and osteogenic differentiation, surface markers etc., by using cells isolation and culture, flow cytometry, adipogenic, osteogenic induction culture and detection techniques. The experiment conform there are MSCs in adult rat’s connective tissue and show fascia connective tissue distributed all over the body is probably the inherent pool of stem cell. The stem cells are easy isolation and culture, and have a good proliferation and differentiation capabilities. We consider ADSCs are main reserve stype of MSCs. more importantly, the fascia connective tissue plays the role of support and reserve, and supply stem cells to repair injury.Through trace enzyme standard instrument, we detected that the level of alkaline phosphatase in ADSCs treated with RA increased greately. By RT-PCR, we demonstrated that ADSCs treated with RA for7days could not express stra8and vasa which are germ cell markers. ADSCs can express germ cell related genes oct4, dazl, nobox and piwil when induced with RA, this demensrtated that ADSCs have the potentials to differentiate into germ cells. And RA can inhibit the proliferation of ADSCs. |
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