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BACKGROUND:Until now, little has been reported on establishment of pancreatic stem cell strains and lines,and the purification of pancreatic stem cells is difficult since the cell line establish rate is low.OBJECTIVE:To explore a more rational and effective technique of in vitro separation and continuous passage of pancreatic stem cells, with the hope to establish cell strains and even cell lines and to lay the foundation for the follow-up study of pancreatic stem cells in the treatment of diabetes.METHODS:Firstly, Percoll discontinuous density gradient centrifugation method was applied to separate the mouse pancreatic endocrine portion from the exocrine portion, then to obtain cell strains with highly proliferative ability and low differentiation from pancreatic endocrine portion-the islet. We used mouse embryonic fibroblasts treated with mitomycin C as a feeder layer, for in vitro continuous culture of islet-derived pancreatic stem cells under feeder layer conditions until they were transferred to the 30th passage to establish cell lines. Then pancreatic stem cell line derived from pancreatic islet was detected and identified by a series of tests including growth characteristic test, morphological observation, related molecular marker identification and differentiation characteristic identification.RESULTS AND CONCLUSION:In the continuous process of passage, pancreatic stem cells showed active proliferative ability, and maintained the typical morphological characteristics of stem cells and expression of pancreatic stem cell marker-Nestin. After induction, pancreatic stem cells showed insulin gene expression,reflecting their differentiation potential. Therefore, under the condition of feeder layer, the pancreatic stem cell line derived from Kunming mice was successfully established and the related identification was completed,which lays the foundation for the following research.
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BACKGROUND: There is no effective method to identify liver cancer stem cells until now, which has become one of the major challenges in this field. OBJECTIVE: To explore a more effective and suitable way for the identification of liver cancer stem cells in hepatocellular carcinoma cell lines. METHODS: Firstly, the single cell separation technique was used to obtain single cells, which were seeded into 96-wel plates one by one. Secondly, cell sublines derived from single cell colonies (tumorigenic colonies) were selected and obtained. At last, the cells from these clones were transplanted into the forelimb armpits of nude mice to observe the tumorigenic ability. RESULTS AND CONCLUSION: The number of holes of single cells obtained was 371 by the single cell separation technique, and the success rate was 96.4%. According to the growth of cell clone, tumorigenic colonies were selected to be transplanted to the forelimb armpits of nude mice, and the tumor formation rate of the colonies was 100%. The identification model of single-cell-transplant-tumorigenic-test for liver cancer stem cells is confirmed to be preliminarily established, which lays the technology and methodological basis for the follow-up research.
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BACKGROUND: In vivo experiments have confirmed that fibroblast growth factor can effectively protect gentamicin-induced renal tubular epithelial cell injury, but the effect on the in vitro cultured cells is still rare. OBJECTIVE: To explore the mechanisms of basic fibroblast growth factor (bFGF) at different concentrations on preventing nephrotoxity mediated by genamicin on the primarily cultivated renal tubular epithelial cell models. METHODS: By use of enzyme and mesh screening, renal tubular epithelial cells were isolated from Kunming mice and purified, adjusting the cell concentration of 1×10~8/L, then cell suspension was moved to a 96-well culture plate and divided into different groups for culture: blank control group: normal culture; gentamicin group: 10, 30, 50 μL/hola (ie, 400, 1 200, 2 000 U/holes)arerecorded as G1, G2, G3; bFGF group: 20, 50, 80 μL/hole (ie, 90,225, 360 ng/hole) are recorded as B1, B2, B3; gentamicin plus bFGF intervention group: after adding bFGF 12 hours, then added gentamicin 12 hours, assigned into 9 dose subgroups, namely, G1B1, G1B2, G1B3, G2B1, G2B2, G2B3, G3B1, G3B2, G3B3, each subgroup contained four-hole complex. Cell morphology and quantity was observed. RESULTS AND CONCLUSION: Gentamicin showed a dose-dependent effect on the renal tubular epithelial cell injury, epithelial cells in the medium and high concentration groups exhibited shrinkage, rounded, swelling, poor adhesion, severely damaged cytoplasm and structural disorder. In the low concentration group, the number change of cells was not obvious, and fibroblasts began to appear; In the bFGF groups, cells were full, exhibited strong refraction, the cell number increased significantly, these manifestations were significant in 50 μL/hole concentration, and there was no significant difference compared with 80 μL/hore concentration; in case of gentamicin plus bFGF intervention, cells with low concentrations of gentamicin had no obvious damage to cells, which increased, the damaged cells collapse was reduced in the group of low concentration of gentamicin, cell shrinkage and poor adhesion were slightly relieved, high concentrations of bFGF intervention could yield to good cell morphology, but high concentrations of gentamicin caused cell swelling and necrosis of injury, which could not be improved by any concentrations of bFGF intervention. 50 μL/hole bFGF has antagonistic effect on the nephrotoxicity mediated by medium and low concentrations of gentamicin, but has no protection on high concentration of gentamicin-induced nephrotoxicity.
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BACKGROUND: There is a great debate but little research addressing the cell suspension obtained from the digested mantle tissues can effective amplify and form pearl sac in vitro, thus producing pearl. OBJECTIVE: To establish an effective technique and method of in vitro separation and culture of mantle of the pearl oyster (Pinctada martensii), and to determine the optimal method of forming pearl sac with the intact structure and secretion function, thus producing pearl. DESIGN, TIME AND SETTING: Single sample observation was performed at the School of Basic Medicine, Guangxi Traditional Chinese Medical University, between August and December in 2008. MATERIALS: Pearl oyster (Pinctada martensii) aged 1.0 2.0 years, were offered by Yingpan Pearl Industrial Co., Ltd. Of Beihai City, China; the self-modified marine shellfish balanced salt solution; the self-prepared concha pteriae serum and concha pteriae body fluid; keratinocyte growth factor was purchased from Sigma, USA. METHODS: The mantle of pearl oyster (Pinctada martensii) was digested with 2.5 g/L trypsin, the harvested cells were cultured using M199 medium containing 10% fetal bovine serum and supplemented with 10 μg/L keratinocyte growth factor, 10% self-prepared concha pteriae serum and concha pteriae body fluid. The cultivation was performed for 30 days. MAIN OUTCOME MEASURES: Cell growth characteristics and growth state. RESULTS: The pearl mantle epithelial cells cultured in vitro were shown to proliferate rapidly, secrete productively, and the muscle cells showed a great proliferation, finally encapsulated the mantle epithelial cells to form pear sac with the intact structure and strong secretion function. CONCLUSION: Using the modified culture technology and culture system, the addition of keratinocyte growth factor can obtain the well growing and secreting pearl sac during in vitro culture of mantle cells.
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Objective An investigation of new cultivars of Amomum villosum Lour.(AVL) with high yield and good quality was carried out,thus to supply evidences for the identification of AVL cultivars in accordance with the morphological features of their flowers and fruits.Methods An investigation of AVL species from the genuine producing areas of Yangchun city of Guangdong province was performed.The morphological features of flowers and fruits of two cultivars(Changguo and Yuanguo) as well as one breeding type(Chunxuan type) were examined.Results Specific and significant features were screened out in different cultivars of AVL.Conclusion There exit specific features in flowers and fruits of different cultivars of AVL from Yangchun.