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A Preliminary Study of BMSCs Characteristics and Its Transplantation Mediated by Gelatin Sponge into Nervous System

Author: ZhangHaiZuo
Tutor: FengJi
School: Hebei Medical University
Course: Surgery
Keywords: Bone marrow mesenchymal stem cells Gelatin sponge Carrier Differentiation Transplant
CLC: R329
Type: Master's thesis
Year: 2009
Downloads: 40
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Abstract

Purpose of the preliminary study of the first part: bone marrow mesenchymal stem cell separation, culture, and identification of their characteristics: the experimental separation of mesenchymal stem cells in the bone marrow, culture, identification, and to study its own characteristics, as well as under step gelatin sponge carrier equipped with a cell equipped with transplantation experiments. Method: First, the use of differential adherent cultured bone marrow mesenchymal stem cells isolated from the bone marrow of rats being purified. After using the flow cytometry method of bone marrow mesenchymal stem cell surface markers to be identified. Identified as mesenchymal stem cells in the bone marrow to C6 cell line as a control to draw its growth curve in different algebraic period to study its tumorigenic. Studied by immunofluorescence studies bone marrow mesenchymal stem cells under certain conditions, characteristics of spontaneously transformed into nerve cells. Use of the the Transwell method to study the bone marrow mesenchymal stem cells can be directed migration to the damaged nerve tissue characteristics. Finally, the study mesenchymal stem cells in the bone marrow and easy method of cryopreservation and resuscitation to save. The results: out of the mixed cells from rat bone marrow cavity, inverted phase contrast microscope, appear as small round cells with a large number of individual or group is evenly distributed. 4 to 8 hours after cell sedimentation and fitted to the dish bottom. Observed 24 hours after the shop in a petri dish at the bottom of most of the cells become visible flat and gradually extended protrusions. 3 days after the non-adherent cells and other medullary cavity material medium was changed most of the removal. After adherent cells constantly amplified, pooled into a film shows a large number of cells to 10 to 14 days, different forms, into the group growth. These visible large flat shape, polygon, star, fusiform other cell morphology. Gradually moving towards a single after the passaged cell morphology and type, to the 5th generation cell the same size, shape fusiform, has basically been purified. Detection using flow cytometry methods, the cell-free system for the expression of the surface markers CD31, CD34, CD45 negative, and expression of the surface markers CD71 positive. Its different periods of the growth curve and C6 cell lines compared obvious depress bone marrow mesenchymal stem cell growth curve. By inverted phase contrast microscope observation and detection of immunofluorescence learn, under certain conditions, found that bone marrow-derived mesenchymal stem cells spontaneously differentiate into neural-like cells express neural markers GFAP, NSE. Transwell experiments, can be observed in mesenchymal stem cells through the rate was significantly higher bone marrow mesenchymal stem cells in the bone marrow injury group within the same time through the rate of the statistical P lt; 0.05 significant difference. Mesenchymal stem cells in the bone marrow frozen after the easy way at least -80 ℃ survive for 3 months and stored in liquid nitrogen can survive for at least six months. Mesenchymal stem cells in the bone marrow after the number of recovery after there will be a large loss, but survive as long as the recovery of bone marrow-derived mesenchymal stem cells reach a certain number and density of these cells are still rapidly amplify and get a lot in a short period of time , stable, and can take advantage of the bone marrow mesenchymal stem cells. Conclusion: The most common application of differential adherent culture method can be very stable culture of mesenchymal stem cells in the bone marrow, 5th generation cells have reached the purification requirements. Can be determined through the identification of cell surface markers for bone marrow mesenchymal stem cells. 10 generations within the bone marrow mesenchymal stem cells to the tumorigenicity is minimal or non-existent, can spontaneously differentiate into neurons and glial cells. In addition, it has a unique homing can migrate to the damaged nerve tissue. Bone marrow mesenchymal stem cells through the simple cryopreservation and recovery method to a long time stability of preservation, reserve cells for subsequent application. Part II: mesenchymal stem cells in the bone marrow after gelatin sponge carrier equipped with the purpose of the preliminary study of the feasibility of nerve tissue transplantation: whether the survival rate of bone marrow mesenchymal stem cells gelatin sponge carrier equipped by the impact and its characteristics have been changed and further study of bone marrow mesenchymal stem cells after gelatin sponge carrier equipped with the ability to coexist with the nerve cells. Method: First, observe whether bone marrow mesenchymal stem cell morphology by gelatin sponge equipped to change the statistics of the number of mesenchymal stem cells in the bone marrow after transplantation gelatin sponge equipped adherent survival and without carrying cells compared to whether there are differences. Then, without carrying 5th generation bone marrow mesenchymal stem cells as the control draw equipped with the growth curve of bone marrow mesenchymal stem cells, and to study its tumorigenicity. After induced differentiation toward the nerve cells were observed after equipped with equipped with bone marrow mesenchymal stem cells without cell morphology after induction different, and statistics whether there are differences in the proportion of both differentiated neurons. Finally, the establishment of in vitro nerve cell culture system, and observed by inverted phase contrast microscope study whether mesenchymal stem cells by the bone marrow equipped gelatin sponge co-cultured nerve cells. Results: inverted phase contrast microscope gelatin sponge group compared with the control group, no significant change in cell morphology, multi-fusiform, flat aging phenomenon is rare. In between the the gelatin sponge Central or the edge of the Ministry had bone marrow mesenchymal stem cells adherent and adherent cell number in the middle of the gelatin sponge some more. Gelatin sponge group and the control group was not adherent cell count compare P gt; 0.05, not statistically significant. Equipped with 5th generation cell equipped with 5th generation bone marrow mesenchymal stem cell growth curve and without growth curve compared to no observed significant changes. Bone marrow mesenchymal stem cells can be observed in the induction of 6 to 8 hours after the cell body began to shrink and projecting projections, some projections are interconnected to form a network-like structure. The cells can be detected by immunofluorescence learn to express glial cell marker GFAP and neuronal cell markers NSE. Gelatin sponge group and the control group expressed NSE-positive rate by flow cytometry method of comparing the statistical P gt; 0.05, no significant difference. Most of the cells after 12 to 24 hours after the planting of the neonatal rat nerve cells adherent and extended projections. With prolonged incubation time the neuron synapses trunk and branches significantly prolong the formation of a dense network, the growing number of glial cells. Primary neural cells can be maintained in vitro and cultured for 14 to 30 days or more. After equipped with bone marrow mesenchymal stem cells transplanted into the neural cell culture system, inverted phase contrast microscope observed the two can work together growth, reproduction. Conclusion: mesenchymal stem cells in the bone marrow after gelatin sponge equipped adherent cells survived not been significantly affected. Its low induced neoplastic proliferative properties and characteristics that can differentiate into nerve cells did not change significantly. Gelatin sponge equipped transplantation of bone marrow mesenchymal stem cells can coexist with nerve cells.

A Preliminary Study of BMSCs Characteristics and Its Transplantation Mediated by Gelatin Sponge into Nervous System

Abstract

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