| In recent years, the use of stem cells has shown broad application prospects in regenerative medicine, cell replacement therapy and drug screening because of their characteristics of both self-renewal and multilineage differentiation potential. With the further study of stem cells and tissue engineering, stem cells have showed good therapeutic effect in some diseases. However, there are still many diseases which cannot be completely cured with modern medical treatment, some of them associated with cell, tissue and organ necrosis such as diabetes, immune dysfunction, Alzheimer’s disease, Parkinson and so on. The appearance of stem cells and gradually further study brings new hope for the cure of these diseases. At present, the main sources and potential sources of stem cells include embryo, fetal tissue, umbilical cord blood and adult tissues. In clinical trials and application of a variety of stem cells, the basic theory and technology of hematopoietic stem cells are relatively mature. Hematopoietic stem cell transplantation is a radical treatment method with great potential for most malignant hematological system diseases and some immunodeficiency diseases and has been widely used in clinical. Hematopoietic stem cell transplantation mainly includes two kinds: autologous transplantation and allogeneic transplantation. In the aspect of source of hematopoietic stem cells, bone marrow and peripheral blood used to be ideal sources, but they are derived from adult and show low level of stemness. Researchers have been unremittingly looking for more ideal and easily available sources of stem cells. In the 1980’s, umbilical cord blood attracted researchers’ attention because of its rich content of hematopoietic stem cells. Cells derived from umbilical cord blood are developing rapidly in clinic application and have an ideal effect on hematopoietic reconstruction and immune system recovery, which expand of the source of hematopoietic stem cells. Umbilical cord blood stem cells have rich sources and low immunogenicity but the quantity of cells in umbilical cord blood is smaller and thus limits their clinical application. Therefore, to find a more extensive, more suitable sources of cells has become breakthrough in the clinical application of hematopoietic stem cell transplantation.The placenta is a disk-shaped organ which embedded in the wall of the uterus. It supports a variety of material exchange between fetus and mother during pregnancy and is with endocrine function. The placenta is composed of decidua basalis, chorion frondosum and amnion. Usually, the placenta belongs to the waste generated during delivery process, however, in recent years many studies have found a variety of stem cells and progenitor cells in the placenta and brings more and more attention and exploration. A large number of stem and progenitor cell groups’ existence in the placenta shows its enormous potential in regenerative medicine in the future. Many studies have shown that many different types of stem cells are contained in the placenta, however, in recent years, studies mainly focused on mesenchymal stem cells but less on hematopoietic stem cells. Based on previous experimental research and results, we can infer that further study on the placenta derived cells and understanding of their biological characteristics are playing a crucial role in selecting and promoting their earlier clinical application. Besides, the potential use of placenta can solve the cell source shortage or incompatible problems in various related clinical diseases. Compared with somatic cells, placenta derived cells are more primitive and may obtain better clinical efficacy.The main purpose of this study is to detect the total number of cells in the placenta, the number of CD34+ cells and their proportion and compared with the number of cells in cord blood; observe colony formation of placenta and umbilical cord blood derived cells; examine HLA type of placenta derived cells, maternal blood cells and umbilical cord blood cells; analyze the chimerism of placenta derived cells, maternal blood and umbilical cord blood cells by STR-PCR technique to provide relevant theoretical basis for future clinical application. Our research is mainly divided into the following two parts.1. Study on the total number of cells and the proportion of CD34+ cells in the human placenta and umbilical cord blood. The colony forming condition of placenta and umbilical cord blood derived cells cultured in vitro.Pregnant women and their family members were informed and signed the informed consent. Our research had obtained the approval of the medical ethics committee of the 307 th Hospital of PLA. During the delivery process, we extracted umbilical cord blood and placed it into disposable plastic blood bag temporarily, later we sent it for centrifugation and collect the cells. Placenta was washed by physiological saline repeatedly to remove the residual blood and impurity on the surface. We collected the blood in the placental vascular. After that, we infused placental vascular system with saline containing 10g/L AMD3100, occluded the umbilical artery and incubated for 30 minutes, collected the perfusion. Took the placental vascular blood and perfusion fluid for centrifugal and counted the cells. The remaining part of placenta tissue were cut and dealt with enzymatic digestion and grinding, filtrated by the 100 mesh steel mesh. Centrifugalized and washed the filtration liquid and counted the cell number. Plus the cell numbers contained in placental vascular blood, perfusion fluid and tissue.Flow cytometry was used to detect the number of CD34+ cells and the proportion in human umbilical cord blood cells and placenta cells. 5x104 cells derived from umbilical cord blood and placenta were solely added into 1.1m L culture medium and put into petri dish. The dishes were kept under circumstance with 5%CO2 and temperature of 37℃.The research findings showed that the number of total cells, CD34+ cells and their proportion obtained from the placenta are significantly higher than those from umbilical cord blood. We could observe the emergence of colony on the sixth day after inoculation in placenta and umbilical cord blood colony culture dishes and good colony forming on the fourteenth day. However, with the same inoculum density, placenta derived cells formed less colonies than umbilical cord blood cells. The number of cells contained in the placenta had obvious advantages, which suggested us that placenta might have the potential to be the source of stem cells transplantation for the treatment of various diseases in the future. Colony formation experiment revealed that placenta derived cells have the ability of formation different colony, while the colony forming ability was an important evaluation content of differentiation and proliferation potential of hematopoietic stem cells and progenitor cells.2. Detection of HLA type and chimerism of placenta derived cells, umbilical cord blood cells and maternal blood cells.We extracted 5m L peripheral blood since the basilic vein before the mothers getting into the delivery room, centrifugalized the blood and took the cells. Cells from the placenta, maternal blood cells and umbilical cord blood were detected on HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1. HLA testing results showed that two of the five placentas contained matrix composition due to HLA locus, the remaining three cases showed that placenta derived cells were completely consistent with cells from umbilical cord blood on locus. The results of STR-PCR detecting chimerism showed that all five placental samples were with maternal chimeric components, while the chimerism was especially high in the two cases whose HLA detection suggesting maternal component. Therefore, we assumed that the different detection results of HLA may be associated with the detection sensitivity and the deviation of placenta tissue structure with naked eyes observation. But the STR detection results reminded us that, with present separation method, the placenta derived cells were really mixed with maternal source components. However, whether the maternal components could influence further clinical effect of stem cell transplantation still need further research.The innovation of this research is that: umbilical cord blood, placenta and maternal blood derived cells were systematically detected by the same method and obtained some results with certain significance. At the same time, HLA detection and STR-PCR detection were both used in the chimerism detection of placental cells with maternal and fetal components. More accurate detection of chimeric ratio laid the foundation of method selection and feasibility of potential clinical application. |
PDF Full Text Request