摘要
目的探讨大鼠骨髓间充质干细胞(BMSCs)与胰岛细胞共培养体系下分化为胰岛样细胞团的可能性。方法无菌条件下从Wistar大鼠的股骨及胫骨分离纯化BMSCs,流式细胞仪检测BMSCs表面标志物CD_(44)和CD_(90)表达;行成骨方向及成脂肪方向诱导分化,采用茜素红染色和油红O染色对诱导后的细胞进行鉴定。分离纯化大鼠胰腺的胰岛细胞,采用双硫腙染色鉴定。使用ELISA法检测胰岛细胞培养液的基础胰岛素水平;分别加入含葡萄糖5.6 mmol/L(低糖)和25.0 mmol/L(高糖)的培养液,ELISA法检测胰岛素释放量。取第5代BMSCs和胰岛细胞,随机分为干细胞单独培养组(干细胞组)、干细胞-胰岛细胞共培养组(共培养组)及胰岛细胞单独培养组(胰岛组),使用倒置相差显微镜观察各组BMSCs的形态学变化;使用ELISA法检测各组基础胰岛素分泌量及高、低葡萄糖刺激后胰岛素释放量;免疫细胞化学染色法检测共培养组诱导形成的胰岛样细胞团的胰岛素蛋白表达;透射电镜观察胰岛样细胞超微结构。结果BMSCs表面标志物CD_(44)和CD_(90)的阳性率分别为99.48%、99.50%,经成骨方向诱导分化形成多个钙结节,经成脂肪方向诱导分化胞质内形成较多脂滴。双硫腙染色显示胰岛中β细胞呈棕红色,每个胰腺可获得胰岛约450个,平均纯度达80%。低糖、高糖组刺激后胰岛的胰岛素释放量分别为(7.105±1.551)mIU/ml和(20.231±1.592)mIU/ml,差异有统计学意义(P<0.05)。共培养组培养7 d后,见局部干细胞开始聚拢并以出芽的方式向上生长成小团块,直至最后形成球形的胰岛样细胞团结构。干细胞组基础胰岛素分泌量<0.5 mIU/L;胰岛组培养5 d胰岛素分泌量达峰值,随后逐渐下降,13 d时降至最高值的20%;共培养组胰岛素水平在5 d时达到峰值,13 d时仍维持在峰值水平的40%左右,胰岛组与共培养组8、10、13 d基础胰岛素分泌量差异有统计学意义(P值均<0.05),但
Objective To examine the possibility of the differentiation into islet-like cell clusters from the co-culture system of bone marrow mesenchymal stem cells(BMSCs)and islet cells.Methods Rat BMSCs from the femur and tibia of Wistar rats were isolated and purified taken under aseptic conditions;the surface markers CD_(44) and CD_(90) expressions of BMSCs were detected by flow cytometry;and alizarin red staining and oil red O staining were used to identify the cells induced in the osteogenic direction and adipogenic direction,respectively.Rat islet cells from the pancreas of Wistar rats were isolated and purified;and dithiazone staining was performed for validation.The basal insulin level of the culture was detected by ELISA method.5.6mmol/L(low glucose)and 25.0 mmol/L(high glucosa)glucose were added to the culture,respectively,and insulin release was detected by ELISA.5-generation BMSCs and islet cells were collected and divided randomly into stem cell culture alone group(stem cell group),stem cell-islet co-culture group(co-culture group),and islet culture alone group(islet group).The morphological changes of BMSCs during co-culture were observed using an inverted phase contrast microscope;basal insulin secretion and insulin secretion stimulated by low and high glucose were tested by ELISA.Insulin protein expression in induced islet-like cell masses in co-culture group were detected by immunocytochemical staining.The ultrastructure of islet-like cells was observed by using transmission electron microscopy.Results The positive rates of CD_(44) and CD_(90) were 99.48%and 99.50%,respectively;BMSCs were induced the formation of multiple calcium nodules outside the differentiation cells in the osteogenic direction,and many lipid droplets in the cytoplasm of differentiated cells in the adipogenic direction.Dithiazone staining showed thatβcells in pancreatic islet were brown red and about 450 islets could be obtained per pancreas with a mean purity up to 80%.The insulin release in the low sugar group and the high sugar gr
作者
刘鑫
岑妍慧
贾微
杨瑞
李森
姜娜
黄泽萍
宁志莹
黄威
Liu Xin;Cen Yanhui;Jia Wei;Yang Rui;Li Sen;Jiang Na;Huang Zeping;Ning Zhiying;Huang Wei(Department of Histology and Embryology,Guangxi University of Chinese Medicine,Nanning 530200,China)
出处
《中华胰腺病杂志》
CAS
2022年第6期439-445,共7页
Chinese Journal of Pancreatology
基金
广西自然科学基金面上项目(2020GXNSFAA259011、2019JJA140246)
广西高校中青年教师科研基础能力提升项目(2021KY0291)
广西一流学科建设开放课题(2019XK137)
广西研究生教育创新计划项目(YCSZ2022003)
广西壮瑶药重点实验室子项目(GXZYKF2019-5、GXZYKF-201708)
2020年大学生创新创业训练计划项目(202010600018)
广西中医药大学校级科研项目(2019MS001)
广西一流学科建设项目重点课题(2018XK023,2018XK024)。
关键词
骨髓间充质干细胞
胰岛
共同培养技术
细胞分化
胰岛样细胞团
Bone marrow mesenchymal stem cells
Islets
Coculture techniques
Cell differentiation
Islet-like cell cluster
