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Study on Rat Bone Marrow Stromal Cells Proliferation and Differentiation into Islet-Like Cell Cluster

Author: ZengMingHua
Tutor: ChenShuLin
School: Northwest University of Science and Technology
Course: Cell Biology
Keywords: Bone marrow stromal cells Islet-like cell Nestin Diabetes
CLC: Q813
Type: Master's thesis
Year: 2007
Downloads: 2
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Abstract

Islet transplantation is one of the effective therapies for the patients of type I diabetes, while the lack of donor cells and transplantation rejection hindered the clinical application of this therapy. Bone marrow stromal cells are one of the most important seed cells for islet induction because of its easy acquisition, proliferation ability and multi-potential. This study referred to and refined the methods of the former researchers and developed a new and effective method for inducing MSC into cell clusters with characteristics of islets. In this study, we first isolated MSC cells with uniform morphology and good proliferation capability from rats of different ages via commonly used method of Percoll density gradient centrifugation in conjunction with the attachment method and the low osmotic buffer treatment. The proliferation ability of the isolated cells was studied by microscopic observation and establishment of the proliferation curve. In the induction experiment, we exploited a two-step inducing method. In first step, we induced MSC into nestin positive cells by treatment with serum-free low glucose DMEM containing 1% DMSO for 24 h. In the second step, we changed the culture media to high glucose DMEM culture media containing 20% FBS and 20 mmol/L Nicotinamide and induced the nestin-positive cells into islet-like cell clusters. The nestin expression of the cells was detected by immunocytochemistry staining after pre-induction. After islets-like cell clusters formed, the cells were detected at different levels via DTZ staining, immunocytochemistry staining, RT-PCR. The glucose challenge test was conducted to detect the sensitivity of the cells to glucose and insulin secretion was measured by insulin RIA kit. Finally, the induced islet-like cells were transplanted under the capsule of kidneys into diabetic rats. The blood glucose levels of recipients were monitored after transplantation. On the 7th day post transplantation, the kidneys of the receipts were removed, fixed and sectioned for HE staining and insulin immunohistochemistry staining. The main results of the study are as follows:1.Isolated cells by Percoll density gradient centrifugation were mingled with many red cells. The amount of the mingled red cells reduced overtly after treatment with low osmotic buffer and the isolated cells still kept good viability. After cultured for 48 h, the suspending cells were removed and many shuttle like cells were observed attached on the culture flask. The proliferation curve of MSC revealed the MSC isolated from 4-week rats proliferated most rapidly with a reduplication time of 3 d and a proliferation cycle of 10 d. The reduplication times for 8-week rat and 12-week rat were 4 d and 5 d respectively; the cell cycle for 8-week and 12-week rat were 12 d and 15 d respectively.2.After pre-treatment with serum-free low glucose DMEM cotaining 1% DMSO for 24 h, the morphology of induced cells changed. Some cells shrinked; the brim of some cells became irregular; slim projects were observed on some cells. Nestin immunocytochemistry staining showed most cells were positively stained. After treatment with high glucose DMEM culture media containing 20% FBS and 20 mmol/L Nicotinamide for 24 h, cell clusters began to formed and more cells shrinked into taper-like cells with long projects. The ends of some projects formed filament-like pseudopod. Cultured for another two days, the cell clusters grew big and lined like spheral grapes with round cells inside containing abundant secretory granules. The cell clusters resembled the islets-like cell aggregation formed by induction of ESC. Some MSC cells were induced into nerve-like cells.3.The induced cell clusters were stained red and brown by DTZ solution which proved there were isletβcells in the clusters. Immunocytochemical staining showed the induced cells were insulin and glucagon positive. RT-PCR result showed there were transcription of islet specific genes including Insulin-1, Insulin-2, Glucagon and Somatostain in the induced cells. Glucose challenge test showed the induced cells were sensitive to the glucose concentration changes.4.The induced cells could remarkably decreased the glucose levels of diabetic rats that received the transplantation which proved the function and viability of the cells in receipts. Immunohistochemical staining indicated there were viable induced insulin positive cells at the transplantation sites.In conclusion, the method of isolation, proliferation and induction of MSC described in this experiment could provide large amount of functional islet-like cell clusters. Therefore, this study provides a new strategy for inducing MSC into islet cells.

Study on Rat Bone Marrow Stromal Cells Proliferation and Differentiation into Islet-Like Cell Cluster

Abstract

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