Tripterygium wilfordii polysaccharide induces differentiation of pancreatic stem cells into islet-like cell clusters

doi:10.3969/j.issn.2095-4344.0443

Abstract

Abstract:

BACKGROUND: Inadequate sources of islet cells mean that islet cell transplantation for diabetes cannot meet the clinical demand. Therefore, in vitro induction of pancreatic stem cells to differentiate into islets has become a focus of research.

OBJECTIVE: To study the effect of Tripterygium wilfordii polysaccharides on the differentiation of pancreatic stem cells from islets in mice, so as to explore the effect of traditional Chinese medicine on the differentiation of pancreatic stem cells into pancreatic beta cells.

METHODS: Tripterygium wilfordii polysaccharide was used to induce the differentiation of purified mouse pancreatic stem cells into islets in vitro. The islet-like cell clusters then underwent morphologic observation, dithizone (DTZ) staining, and western blot analysis.

RESULTS AND CONCLUSION: Cell morphology, cell growth characteristics and immunocytochemical staining showed that mouse pancreatic stem cells were obtained. They were induced by Tripterygium wilfordii polysaccharide into spherical islet-like structures, which had a spindly pedicle connected with the bottom of the culture flask, and were DTZ-stained to iron red. Western blot assay detected β-cytokine proteins in the islet-like cell clusters. These findings confirm that mouse pancreatic stem cells can be induced to differentiate into islet-like cell clusters containing β cells in vitro by Tripterygium wilfordii polysaccharide.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, Islets of Langerhans

CLC Number:

R394.2

Cen Yan-hui, Li Zhong-hua, Jia Wei, Yang Rui, Bao Juan, He Guo-zhen, Wu Xiao-jun, Zhong Jing, Deng Hui-feng, Shi Lei. Tripterygium wilfordii polysaccharide induces differentiation of pancreatic stem cells into islet-like cell clusters[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 729-735.

Figures/Tables 1

Stem cells derived from the endocrine pancreas were obtained by discontinuous density gradient centrifugation After the discontinuous Percoll density gradient centrifugation, the pancreatic tissue cells were distributed into three different density interfaces. The DTZ staining showed that 80%–90% of the cells from the first and second density interfaces were red iron (Figure 1), indicating that those cells were derived from the endocrine pancreas. Therefore, cells from both density interfaces were collected for primary culture. Stem cells derived from the endocrine pancreas had significant morphologic features At 48 hours of primary culture, cells derived from the endocrine pancreas showed some big, round, mononuclear cells surrounding the islet-like cell clusters. These cells had strong refraction of cytoplasm and a high nuclear-cytoplasmic ratio, showing adherence growth (Figure 2). In addition, these cells reflected the morphologic properties of pancreatic stem cells, so we named them pancreatic stem-like cells. Continuous cell culture was conducted after 7 days in primary culture. Islet-differentiated pancreatic stem cells were identified via a series of methods Growth characteristics of pancreatic stem cells Pancreatic stem-like cells began to obviously divide and grow during subculture, and soon formed a large colony (Figure 3). After passage at 96 hours, when these cells covered more than 80% of the orifice aperture area, and passage continued. In continuous passage, the cells remained large and round, with a single core, a high nuclear-cytoplasm ratio, a strong proliferative capacity, and other characteristics, at each passage (Figure 4). Detection of pancreatic stem cell-specific molecular markers of PDX-1 Immunocytochemical staining detecting the expression of PDX-1 showed positive results; the claybank-stained cytoplasm confirmed that the products were endocrine pancreas-derived stem cells (Figure 5). Pancreatic stem cells were induced into islet-like cell clusters by Tripterygium wilfordii polysaccharide Cell morphology and DTZ staining after induction After continuous induction and differentiation via Tripterygium wilfordii polysaccharide for 7 days, some of the cells began to gather, but there were no significant changes in cell morphology. These cells were divided and produced more budding cells to grow upward, forming a spherical islet-like cell structure after approximately 1 month, had spindly pedicles connected with the bottom of the culture flask, and floated in the culture medium as shown in Figure 6. The DTZ staining of the islet-like cell clusters was iron red, while the DTZ staining of the surrounding cells was negative (Figure 7). Islet-like cell clusters formed by induction expressed β-cytokine protein Western blot results revealed that the induced islet-like cell clusters expressed β-cytokine protein with a relative molecular mass of approximately 32 000, as shown in Figure 8. GAPDH was used as an internal control, and the emergence of a target band confirmed the high quality of the extracted total cellular protein. "

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