| Since1893, autologous fat transplantation has been used for the correction of tissue defects by Neuber, which has been proved to be a new way to repair atrophy or loss of tissues. What’s more, autologous fat transplantation following liposuction has become the most popular dermal filler to correct the soft tissue defect theoretically due to high adipose availability, low donor site morbidity, low complication rate and almost none immunological rejection after80s of the twentieth Century when liposuction had been invented. Later, a lot of experimental and clinical studies confirmed the safety and reliability of this technology. However it has been shown that the problem of low survival rate of grafted fat and less adipose tissue remained after transplantation cannot be solved effectively because that a certain proportion of grafted autologous fat may be liquefaction necrosis by a large number of clinical and experimental researches. The etiology of the phenomenon has been explained that the grafted tissues were unable to establish effective blood circulation in at least3days after transplantation, so that the survival of them had to rely on the permeation of nutrients in tissue fluid under ischemia and hypoxia micro environment. Lots of solutions have been put forward by researches to reduce the absorption rate in order to improve the clinical results of the transportation (eg, mix the transplant with cell factors, improve lipid extraction and injection techniques), but none of them seems satisfactory.Mesenchymal stem cells (mesenchymal stem cells, MSCs) refer to a class of pluripotent stem cells derived from the early growth stage of the mesoderm and ectoderm. Its differentiation ability is restricted compared with embryonic stem cells, which guarantees that mesenchymal stem cells have higher security than embryonic stem cells. Otherwise, widely available, relatively high ability of differentiation, low tumorigenicity trend and none immune rejection, all these biological characteristics are the reasons why mesenchymal stem cells are widely used in treatment of a variety of cell and gene level in recent years.Since MSCs derived from adipose tissue acquired through liposuction by Zuk in2001, adipose derived mesenchymal stem cells (Adipose-derived stem cells, ASCs) as it called, has been used more and more in the tissue engineering and cell therapy due to its easily acquisition, in vitro amplification, grafting convenient, and biological safety of high potential of seed cells. At the same time, ASCs have also been widely applied as adipogenic, osteogenic and other sources of seed cells regeneration in tissue engineering because of the ability of differentiation and proliferation of adipose derived mesenchymal stem cells.Recently, a new method has been proposed by some domestic and foreign scholars that extraction of ASCs with a little bit autologous fat before graft, and amplification of it in vitro. Using a combination of it in fat transplantation, can significantly improve the survival rate of grafted fat and reduce the number of supplementary injection after operation. It is generally believed that grafted autologous fat with ASCs works in two ways:1. secret cytokines (VEGF-A,FGF-2,HGF,IL-1,TGF-P,ect.)2.invoke hypoactive.But currently, the adipose derived mesenchymal stem cells extracted are culured in vitro with ordinary oxygen concentration (21%O2) which is so much different with the physiological conditions of oxygen concentration in vivo. According to this kind of situation, the studys about ASCs can not represent the ability of differentiation and proliferation of them under ischemia and hypoxia micro environment.Hypoxia inducible factor-1(hypoxia inducible factor-1, HIF-1) was found by Semenza and Wang in1992, which commonly exists in human and other mammalian cells. It can generally be expressed by cells under ordinary oxygen concentration (21%O2), but degradated by the oxygen dependent ubiquitin proteasome degradation pathway rapidly. Therefore, HIF-1can only be stably expressed under hypoxic conditions. Besides, many studies have shown that, HIF-1is upregulated in tumor cells and plays a key role in tumor progression and metastasis under hypoxia environment surrounding the tumor. This means that HIF-1can be a factor at the regulation of cellular proliferation, migration, differentiation in hypoxia condition. Some scholars even suggest that HIF-1can also have an effect on stem cell in migration and differentiation aspect. As a result, moderate preconditioning hypoxia before the bone marrow derived mesenchymal stem cells (BMSCs) used for transplantation can get better clinical results.The cobalt chloride (CopCl2),a mature hypoxia mimetic agent, can inhibit the PHD activity, inhibit the hydroxylation of HIF-1α and reduce the degradation of HIF-1a under normoxic condition. Compared to the three gas incubator, hypoxia controllability manufactured by cobalt chloride is cheaper and better, and more suitable for mass cell hypoxia model. In this experiment, we create a gradient of hypoxic cell culture environment in vitro by adjusting the concentration of cobalt chloride and CoCl2. We will detect the HIF-1α expression of ASCs in such environment, and the changes in the capacity of proliferation, differentiation and migration of ASCs through a series of experiments.To sum up, we built a cultivation hypoxia for ASCs by cobalt chloride and verified the reliability of this hypoxia model and observed the changes of proliferation, differentiation and chemotaxisability in this condition. On one hand, we reveals the changes of cells in a hypoxic microenvironment after transplantation, on the other hand, we observed the physiological changes of ASCs in vitro hypoxia environment, which can provide some theoretical basis to improve the utilization of ASCs.Method:1. Cell identification:ASCs were cultured by adherence method in vitro (3-4generatbns), and observed by inverted microscope. The cell growth curve was analyzed. The characteristics of differentiation of BMSCs were identified by inducing adipogenie and osteogenic, and surface markers (CD29、CD3、CD44、CD45) were examined with flow cytometry (FCM).2Hypoxia and normoxia preconditioning protocol:For HP, cells were incubated in cell culture mediums with different concentrations of CoCl2(final concentration of CoCl2were0,50,100,200,400umol/L) with10%fetal bovine serum.3MTT assay to detect the growth inhibition rates of ASCs:To establish the best model of CoCl2induced hypoxic preconditioning, we used MTT assay for the ASCs culture mediums with different concentrations of CoCl2 (final concentration of CoCl2were0,50,100,,200,400umol/L) in different culture time (Oh,6h,24h,48h,72h) proliferation of cultured conditions of ASCs, assay the inhibition rate of each concentration.4Western blot expression of HIF-1α protein in different groups in different time:The protein expressions of HIF-1α of ASCs were detected by western blotting.5Oil red staining to detect the differentiation trend of cell extraction: After hypoxia treatment, adipose derived stem cells were observed every day under the microscope, and stained by oil red O in7,14days, concussion decolonization of10min, the intracellular staining triglyceride fully dissolved, enzyme mark instrument540nm the light absorption value.6Crutch wound healing assay and Transwell assay test cells on stromal cell derived factor-1(SDF-1) migration:After blocking CXCR4by its antagonist AMD3100, ASCs were divided into four groups, including normal culture group, hypoxia culture group, and hypoxia AMD3100group. The migration ability of BMSCs with hypoxic preconditioning was analyzed by scratch wound healing assay and transwell assay.7Statistical analysis:The student’s t-test was used to compare data between the two experimental groups. A one-way analysis of variance test was used to compare data among the three experimental groups. Data were expressed as the mean±SD. A p-value of less than0.05was considered statistically significant. The SPSS software package version13.0(SPSS, USA) was used for the statistical tests.ResultsIsolation of ASCs:Most of the primary cells were in a shuttle and irregular shape; these cells grew in parallel or vortex with a similar fibroblastic spindle-shaped morphology after several passages.Identification of ASCs:Cultured ASCs had the capacity for adipogenic and osteogenic differentiation and highly expressed CD29and CD44, and were negative for CD35and CD45.Effects of CoCl2on the proliferation of ASCs:MTT showed that compared with control group, ASCs proliferation had been inhibited when they cultured in400umol/L and200umol/L concentration of CoCl2(p<0.05); while the100umol/L and50umol/L groups of adipose derived stem cell proliferation did not produce significant results(p>0.05).Western blot expression of HIF-la protein in different groups in different time:Western blot showed that HIF-1α express in50umol/L and100umol/L group in the cells, and were higher than control group, the100umol/L group was significantly higher than the50umol/L.Oil red staining to detect the differentiation trend of cell extraction:Compared to the control group, CoCl2group showed more fatty drops under inverted microscope. Compared to control group, ASCs in CoCl2group exhibit more obvious adipogenic trend by oil red O staining quantitative measurement (p<0.05).The migration ability of ASCs:Compared with control group, the migration ability of ASCs in hypoxic preconditioning group was higher(P<0.05), after silencing HIF-la or knocking CXCR4out by AMD3100, the migration ability of ASCs in hypoxic preconditioning group was decreased and had no difference with control group(P>0.05).Conclusion1. ASCs can be isolated and expanded in vitro by adherent cultivation. Cells with high expression of CD29and CD44, low expression of CD34and CD45obtained after induction condition criteria can be induced to differentiate into osteoblasts and adipocytes in vitro, which meet the requirements.2. Using chemical hypoxia agent CoCl2to establish a hypoxic preconditioning model of BMSCs, we found that cell proliferation and the high expression of HIF-la won’t be influence with CoC12concentration100umol/L by measuring the cell inhibition rate.3. CoCl2induced chemical hypoxia preconditioning can promote ASCs to differentiate into adipocytes. And the formation rate and size of lipid droplet was positively related to the concentration of CoCl2.4. CoCl2induced chemical hypoxia preconditioning can promote ASCs migration in vitro. The mechanism may be related to the up regulation of HIF-1α expression and the subsequently increased expression of CXCR4after hypoxic preconditioning. |
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