摘要
目的:观察骨髓间充质干细胞在体外不诱导条件下,移植于链脲佐菌素所致糖尿病大鼠后在胰腺的定位及胰岛素分泌情况。方法:实验于2004-08/2006-03在2级实验室唐山工人医院中心实验室和具有国家认证的屏障动物环境实验室华北煤炭医学院动物中心完成。①实验材料:青年清洁级SD大鼠购自华北煤炭医学院动物中心(SCXK2002-0003),4周龄,体质量180～250g,动物级别:SPF/VAF。②实验方法:体外培养雄性SD大鼠骨髓间充质干细胞,并采用流式细胞仪鉴定。选取雌性SD大鼠,按65mg/kg剂量腹腔注射链脲佐菌素建立糖尿病大鼠模型。给药后二三天,大鼠血糖升高,当血糖≥16.7mmol/L且稳定2d,认为糖尿病模型建立成功。将建模成功的40只大鼠完全随机分为对照组、实验组,每组20只。对照组大鼠右侧肾包囊注射磷酸缓冲液0.2mL,实验组大鼠右侧肾包囊下注射骨髓间充质干细胞0.2mL,细胞数为4×105L-1。于移植前及移植后第3,5,7天采大鼠尾静脉血检测血糖。③实验评估:于移植前及注射后7d采大鼠心脏血检测C-肽值,移植后7d取大鼠胰腺组织作石蜡切片,应用原位杂交技术检测Y染色体细胞。结果:40只大鼠全进入结果分析。①骨髓间充质干细胞鉴定:骨髓间充质干细胞呈梭形、放射状,形成数个细胞克隆时排列整齐,原代培养7～10d即可长满,按1∶3传代,生长四五天达融合。流式细胞术表面标志鉴定显示,培养的原代骨髓间充质干细胞表面标志CD90阳性,基质细胞表面标志CD44阳性,造血细胞表面标志CD45及内皮细胞表面标志CD31阴性。②大鼠尾静脉血血糖值、心脏血C-肽值分析:血糖结果显示,对照组治疗后第3,5,7天血糖值及C-肽值与治疗前相比,无明显变化(P>0.05);实验组治疗后第3,5,7天血糖值较治疗前明显下降,治疗后第7天C-肽值明显上升,与对照组对应时间比较,差异均显著[实验组治疗前:(21.75±2.44)mmol/L,(0.21±0.01)μg/L;实验组治疗后:(12.23±1.95)mmol/L,(14.00±2.85)mmol/L,(12.75±2.86)mmol/L,(0.26±0.01)μg/L;对照组治疗后:(21.63±1.34)mmol/L,(23.03±1.11)mmol/L,(22.48±1.92)mmol/L,(0.21±0.01)μg/L,P均<0.01]。③原位杂交技术检测Y染色体细胞:对照组大鼠胰腺组织切片中未见黄绿荧光Y染色体,实验组大鼠胰腺组织切片中可见黄绿荧光Y染色体,存在于胰腺腺泡间隙,或胰腺组织中。结论:雄性大鼠骨髓间充质干细胞在肾包囊注射下可到达雌性糖尿病大鼠胰腺组织,使糖尿病大鼠血糖下降,C-肽值升高,具有分化为胰岛素分泌细胞的可能性。
AIM： To trace the location and insulin secretion of bone marrow mesenchymal stem cells （MSCs） without in vitro induction after transplanted into streptozotocin-induced diabetic rats by nephric cyst injection. METHODS： Between August 2004 and March 2006, the experiment was conducted in the Second Grade Central Laboratory of Tangshan Worker Hospital and animal center of North China Coal Medical College. （1）Young male SPFNAF SD rats of 4 weeks old and 180-250 g were purchased from the animal center of North China Coal Medical College （number： SCXK2002-2003）. 92）Male＇ rat MSCs were cultured and identified by flow cytometry. Meanwhile, the diabetes models were made by intraperitoneally injecting 65 mg/kg streptozotocin in the female rats. The models were judeged to be successfully established if the blood glucose levels rose over 16.7 mmol/L after administration for 2 to 3 days and stabilized for 2 days. Forty modeled rats were randomly divided into control group, and experiment group with 20 animals in each group. In the control group, 0.2 mL phosphate buffer was injected in the right nephric cyst, while in the experiment group, 0.2 mL MSCs （cell count 4×10^5 L 1） were injected. The blood glucose level was tested before and 3, 5, and 7 days after transplantation. （3）C-peptide level was measured before transplantation and on the seventh day after injection. Seven days after transplantation, the pancreatic tissue of rats from each group was harvested to prepare paraffin sections, and Y-chromosome tagged pancreatic tissue cells were identified by in situ hybridization. RESULTS： All 40 rats were involved in the result analysis. （1）MSCs grew like fusiform or radial, and aligned when several cell clones formed. Primary culture overgrew 7 to 10 days later. Passage at the ratio of 1： 3, and cellular fusion occurred after 4-5 days. Identification of surface markers by flow cytometry suggested that CD90 was positive in MSCs of primary culture, CD44 positive in matrix cells, CD45 negative in hematopoietic cell, and CD31 negative in endothelial cell. Blood glucose test showed that on the third, fifth, and seventh day, the blood glucose and C-peptide levels in the control group had no obvious change compared with those pre-injection （P 〉 0.05）. However, on the third, fifth, and seventh days, the blood glucose level in the experiment group had decreased remarkably and the C-peptide level on the seventh day was evidently enhanced compared with pre-injection. There were significant differences in the blood glucose and C-peptide levels between the experiment group and control group at the same time point [the experiment group： pre-injection： （21.75±2.44） mmol/L, （0.21±0.01） p,g/L; post-injection： （12.23±1.95） mmol/L, （14.00±2.85） mmol/L, （12.75± 2.86） mmol/L, （0.26±0.01） p,g/L; the control group： post-injection： （21.63±1.34） mmol/L, （23.03±1.11） mmol/L, （22.48＋ 1.92） mmol/L, （0.21±0.01） p,g/L, P〈 0.01]. （3）In the experiment group, Y-chromosome tag （SPY positive cell） with yellow green fluorescent light was found in the pancreatic tissue or intercellular space of pancreas acinus in the paraffin section of rat pancreatic tissue, but not found in the control group. CONCLUSION： Male rat-derived MSCs could migrate to the pancreas of female diabetic rats after nephric cyst injection, leading to a decrease of blood glucose level and increase of C-peptide, and display the possibility to differentiate into insulin-secreting cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第20期3924-3927,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
