【作者】 路一芳；

【导师】 刘阁玲；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:对大鼠骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)进行分离培养、体外扩增,采用含不同葡萄糖浓度的培养基加尼克酰胺、Exendin-4进行诱导。采用免疫细胞化学染色、双硫腙染色、电镜、放射免疫分析等方法对BMSCs诱导分化的胰岛样细胞进行鉴定,并观测不同葡萄糖浓度及诱导剂诱导效果的差异。将诱导后的细胞通过尾静脉及肾包囊移植入糖尿病大鼠模型体内,通过对血糖的检测,观察胰岛样细胞对糖尿病大鼠模型的治疗效果。方法:1取3~4周龄的雄性SD大鼠,采用percoll密度梯度离心法结合贴壁培养法分离纯化BMSCs,传代扩增,倒置相差显微镜下进行形态学观察。取纯化度较高的第3代细胞进行诱导分化,按实验设计分为6组:①低糖对照组;②中糖对照组;③高糖对照组;④低糖诱导组;⑤中糖诱导组;⑥高糖诱导组。2诱导后进行细胞鉴定:倒置相差显微镜下观测细胞形态学变化;透射电镜观察细胞超微结构的改变;双硫腙染色法、免疫细胞化学染色法对胰岛样细胞进行鉴定。用放射免疫分析法观察诱导后细胞对葡萄糖刺激试验的反应。Real time-PCR检测诱导组及对照组细胞胰岛β细胞相关基因的表达。3将24只雌雄各半的SD大鼠通过腹腔注射链脲佐菌素破坏胰腺造成糖尿病大鼠模型,分为:①肾包囊对照组;②肾包囊组;③尾静脉对照组;④尾静脉组。将Brdu标记的细胞从大鼠肾包囊或尾静脉移植入糖尿病大鼠模型体内,观察临床疗效,免疫组织化学染色确定Brdu标记的细胞及胰岛素在心、肝、肺、脾、胃、肾以及胰腺的分布。结果:1 BMSCs接种初期可见大量悬浮细胞, 24小时后观察大部分细胞开始贴壁生长。贴壁生长的细胞呈单个分散存在或形成数个细胞克隆,逐渐伸角变形,呈圆形、多角形、长梭形等多种形态。传至3代,细胞纯化,形态较为单一,呈长梭形贴壁生长,以平行排列生长为主或呈漩涡状生长。2分低糖、中糖、高糖培养基培养后,细胞趋向形成细胞团簇。加入诱导剂尼克酰胺、Exendin-4后,成团趋势更加明显,细胞成团簇状分布。3双硫腙染色检测各组细胞胰岛素的合成。镜下观察,诱导组大部分细胞团被染成棕红色,而对照组则无染色。表明诱导的细胞胞浆中富含锌离子,证实胰岛素分泌细胞的存在。4免疫细胞化学染色法鉴定各组细胞胰岛素的表达。实验结果显示对照组、诱导组随着葡萄糖浓度的增加,积分光密度值不断增高,各对照组之间差异无统计学意义(p>0.05);低糖诱导组与中糖诱导组之间,低糖诱导组与高糖诱导组之间具有统计学意义(p<0.01);中糖诱导组和高糖诱导组两组之间差异无统计学意义(p>0.05)。诱导组比对照组积分光密度值明显增高,差异具有统计学意义(p<0.01)。5透射电镜观察诱导前后细胞超微结构变化。结果显示,与诱导前相比,诱导后细胞具备典型分泌细胞特点,胞浆内有可见大量圆形、椭圆形大小不等的空泡,粗大的内质网,线粒体,细胞核不太规则等改变。6各诱导组细胞经低糖和高糖刺激后用放射免疫分析法测均有胰岛素分泌,而相应对照组则无胰岛素分泌。低糖诱导组、中糖诱导组、高糖诱导组经低糖刺激后,胰岛素含量随着葡萄糖浓度的增加而增加,各组间差异具有统计学意义(p<0.01)。低糖诱导组、中糖诱导组、高糖诱导组经高糖刺激后,胰岛素含量随着葡萄糖浓度的增加而增加,组间差异具有统计学意义(p<0.01)。各诱导组细胞经高糖刺激后则胰岛素分泌量增加明显,高于低糖刺激(p<0.01)。7 Real time-PCR定量分析诱导期结束后检测各组细胞有不同程度的胰岛β细胞相关基因GK、PDX-1、ngn3、GLP-1 mRNA的表达,差异具有统计学意义(p<0.01)。GK、PDX-1 mRNA表达水平随培养基葡萄糖浓度相应增高(p<0.01),而ngn3表达情况相反(p<0.01);GLP-1 mRNA表达水平各组不同(p<0.01),与葡萄糖浓度无相关趋势。8腹腔注射链脲佐菌素(streptozotocin,STZ)3天后,检测大鼠血糖。造模组大鼠血糖≥16.7mmol/L且稳定两天,并可观察到大鼠有明显多饮、多尿等症状,可判断大鼠糖尿病模型造模成功。各组大鼠糖尿病模型均造模成功。移植细胞后,应用血糖仪检测尾静脉血糖,结果显示:肾包囊对照组、尾静脉对照组血糖较移植前无明显下降(P>0.05),肾包囊组、尾静脉组移植后第7天开始下降, 14天、21天、28天血糖水平下降较移植前明显(P <0.01)。肾包囊组与尾静脉组血糖下降程度相比,差异无统计学意义(P >0.05)9免疫细胞化学染色法检测胰岛样细胞Brdu标记率,Brdu标记的细胞胞核显色为棕红色,未被标记的细胞胞核复染为蓝色。镜下随机挑取五个高倍视野,计数100个细胞,求其阳性细胞率。Brdu标记率均在95%以上。10免疫组织化学方法检测移植后Brdu标记细胞及胰岛素抗体在各脏器的分布,Brdu标记的细胞在细胞核显色,为棕黄色或棕黑色,未被标记的细胞胞核复染为蓝色。胰岛素抗体主要显色在胞浆,为棕黄色。肾包囊组可在肾脏观察到Brdu标记的细胞,其胰岛素表达阳性;在其他脏器未发现标记细胞,胰岛素表达阴性,尾静脉组可在肺脏、肝脏、肾脏、胰腺观察到Brdu标记的细胞及相应的胰岛素表达阳性,在心脏、脾脏、胃则没有观察到。在对照组各个脏器都未发现Brdu标记的细胞及相应的胰岛素表达阳性。结论:1通过Percoll密度梯度离心法及贴壁筛选法联合应用,去除杂细胞效果好,在体外能获得纯化的BMSCs。2大鼠BMSCs在尼克酰胺和exendin-4作用下可以诱导分化为胰岛样细胞。加用诱导剂后,短期内观察,高糖培养基效果优于低糖组及中糖组。3诱导出的胰岛样细胞对葡萄糖刺激具有反应性,初步具有模拟体内β细胞的生理功能。4诱导细胞通过尾静脉和肾包囊途径移植入糖尿病大鼠体内,可降低大鼠血糖水平。5本试验研究为体外诱导BMSCs,体内移植治疗糖尿病提供了一定的实验基础和理论依据。更多还原

【Abstract】 Objective : Bone marrow mesenchymal stem cells (BMSCs) were proliferated and differentiated in vitro. Different dosage glucose culture medium and nicotinamide、Exendin-4 were used to induce the cells into islet-like cells. To verify if the bone marrow mesenchymal stem cells were transdifferentiated into islet-like cells ,we used the immunocytochemical stain、Dithizone (DTZ) stain、transmission electron microscope、radio-immunity analysis . We also wanted to know whether the dosage glucose culture medium and revulsiva have the same effect or not. We have transplanted these induced cells into streptozotocin-induced diabetic rats by caudal vein and renal encyst. The effect of islet-like cells to treat streptozotocin-induced diabetic rats was tested by blood glucose detection.Methods:1 BMSCs from the rats were separated and purified via gradient centrifugation with Percoll solution and adherence to the culture plastic, and then the cells were expanded by subculturing successively. Cell morphology and growth pattern were observed under invert microscope. The third-generation cells used for the experiment are divided into six groups: 1) low-glucose control group;2) mid-glucose control group;3) high-glucose control group;4) low-glucose induced group;5) mid-glucose induced group;6) high-glucose induced group.2 In order to identify the transdifferentiated cells, we detected them by several methods. Invert microscope was used to investigate the morphologic changes of the cells. Cellular ultrastructure was observed by electron microscope. The insulin-like cells were identified by dithizone (DTZ) and immunocytochemistry. After glucose stimulation, we estimated the induced cells by radio-immunity analysis (RIA).The expression levels of gene about the induced group cells and control group cells were measured by real-time quantitative PCR.3 24 SD rats were introperitonally injected with strep- tozotocin to set up animal model of diabetes and randomly divided into four groups. Each group was consisted 6 rats (3 males and 3 females): 1) renal encyst control group;2) renal encyst group;3) caudal vein control group;4) caudal vein group. The bromodeoxyuridine (Brdu) -labeled cells were transplanted into streptozotocin-induced diabetic rats by kidney capsule and caudal vein. And the clinical effects were observed. Meanwhile, We examined the Brdu-labeled cells distributions in the rats by immunohistochemistry . Result:1 After 24 hours the inoculation BMSCs could adhere to the flask, where single-dispersal cell and sheet-shaped cell clone were formed, and the cells displayed round、fusiform or multi-angle. The third generation cells were purified and most of them were spindle-shaped. They showed vortex shaped or parallel arrangement growth.2 After cultivated by low-glucose、mid-glucose、high- glucose, cell clusters were formed. Addition to nicotinamide、Exendin-4,this trend was obvious .3 Insulin in the induced cells was examined by DTZ stain. To observe the morphology using microscope, the induced group cells were stained and the control group not.4 Insulin secretion of induced cells in every group was detected by immunocytochemical method. With the glucose dosage increasing, the secretion and integral optical density were increased. The result had no statistics difference among different groups(P >0.05). There was significant difference between low- and mid-induced group or high-induced group(P <0.01). Compared mid- with high-induced group, there was no statistics difference(P >0.05).Integral optical density in induced and control groups had distinctness(P <0.01).5 Comparing the non-induced with the transdifferentiated cells ultramicrostructure by electron microscope, some changes were proposed. After being induced the cells showed some typical signs of secretory cell was rich in round or oval-shape cavitation , the mitochondrion in cytoplasm and the structure of rough endoplasmic reticulum was clear , cellular nucleus was unround.6 Insulin secreting was tested by radio-immunity analysis .Each induced group cells could secreted insulin by low- and high-glucose stimulated, and the control group could not. The low-、mid- and high-glucose group were stimulated by low glucose and high glucose, the insulin secreting was increasing(P <0.01).The high glucose was better stimulus than low glucose (P <0.01).7 Real time-PCR quantitative analysisAfter induced by revulsiva,theβ-cell related gene GK、PDX-1、ngn3、GLP-1mRNA expression of the cells were changed. With the additional dosage of glucose GK、PDX-1 mRNA expression were increasing(P <0.01),ngn3 mRNA expression were reducing(P <0.01);GLP-1 mRNA expression were not related to the glucose dosage(P <0.01)Theβ-cell related gene GK、PDX-1、ngn3、GLP-1mRNA expression of the insulin-like cells were changed. With the addition dosage of glucose GK、PDX-1 mRNA expression were increasing(P <0.01),ngn3 mRNA expression were reducing(P <0.01);GLP-1 mRNA expression were not related to the glucose dosage(P <0.01).8 On the third day after injection STZ, the rats whose blood glucose≥16.7 mmol/L were lasting 2 days. The model was successful when obviously polydipsia and hyperdiuresis characters. We applied the blood glucose instrument to detect the blood glucose levels by the caudal vein. The blood glucose were not changed in renal encyst and caudal vein control group(P >0.05),and the blood glucose were reduced at 7 day and obviously at 14、21、28 day in renal encyst and caudal vein group(P<0.01). And there were no significant difference between renal encyst and caudal vein group(P >0.05).9 The labeling efficiency of Brdu-labeled cells was tested by immunocytochemical stain. The Brdu-labeled cell nucleus were brownish red color,unlabeled were blue. Five high power microscopic (200) sights were selected randomly and observed with light microscope, then the percentage of positive cells (number of positive cells/total number of cells) in each high-power field was recorded. The labeling efficiency was more than 95%.10 We detected the distribution of Brdu-labeled cells in each organ by immunohistochemistry.The Brdu-labeled cells whose insulin-expression positive were tested in the renal encyst group, and the other organ were not found. In caudal vein group, the Brdu-labeled cells with insulin-expression positive were found at lung、liver、kidney、pancreas,but not the heart、spleen、gaster.Conclusions:1 It is better that BMSCs are separated and purified by means of gradient centrifugation with Percoll solution and adherence to the culture plastic. 2 The nicotinamide、Exendin-4 can transdifferentiated BMSCs into islet-like cells. BMSCs were induced in the high-glucose medium and revulsiva are best.3 The transdifferentiated islet-like cells is similar to theβ-cells capability can response to glucose stimulating.4 The transdifferentiated cells could reduced hyperglycemia in the streptozotocin- induced diabetic rats by renal encyst or caudal vein .5 Our research provides a possibility that BMSCs were transdifferentiated in vitro and transplanted those cells to therapy the diabetes in vivo.更多还原