【作者】 马淑芳；

【导师】 林少达；

【作者基本信息】 汕头大学 ， 内科学， 2009， 硕士

【摘要】 背景与目的无论1型糖尿病还是2型糖尿病,诊断时β细胞已丧失50-80%。β细胞数量减少与细胞凋亡有关,糖尿病时胰岛β细胞凋亡加速;另外,糖尿病患者长期处于高血糖状态,使“高糖毒性”作用诱导的β细胞凋亡进一步加重,形成恶性循环。β细胞凋亡主要包括外源性(死亡受体介导的信号途径)、内源性凋亡途径(线粒体途径)以及穿孔素/颗粒酶B途径,其中bcl-2家族不仅通过改变线粒体外膜通透性而调控线粒体活化,在线粒体诱导的内源性凋亡途径中起重要作用,还起着连接β细胞三条凋亡途径的作用。动物实验研究显示生长激素释放肽(ghrelin)可阻止β细胞破坏所致的糖尿病;其他研究也表明ghrelin可抑制细胞饥饿、阿霉素及细胞因子等诱导的胰岛β细胞及其细胞株的凋亡。目前ghrelin对高糖环境下诱导的β细胞凋亡的作用尚不清楚。本研究拟ghrelin预处理高糖培养的小鼠胰岛β细胞株NIT-1细胞,检测β细胞凋亡率及其bcl-2、bax mRNA表达水平,探讨ghrelin对高糖诱导下胰岛β细胞凋亡的作用。材料与方法(1)葡萄糖浓度为5.6mmol/L的DMEM常规培养NIT-1细胞,倒置显微镜下观察细胞培养过程中的形态学特点,生长方式。(2)Annexin V-FITC/PI双染和流式细胞仪联合测定不同处理组:对照组(葡萄糖浓度5.6mmol/L,C)、ghrelin预处理组(10-7mmol/L ghrelin,GP)、高浓度葡萄糖诱导凋亡组(葡萄糖浓度33.3mmol/L,HG)、ghrelin预处理+高浓度葡萄糖组(GP+HG)的NIT-1细胞早期凋亡率。(3)Hoechst 33258标记细胞核,在荧光显微镜下检测不同处理组浓集核和碎裂核的数目,计算各组的细胞总凋亡率。(4)RT-PCR分别测定不同处理组bcl-2、bax基因mRNA表达量的变化,电泳后进行灰度值分析,得出不同组bcl-2、bax的相对表达量。结果(1) NIT-1细胞为多边形上皮样细胞,单层簇状生长,排列紧密。(2)流式细胞仪Annexin V-FITC/PI双染及Hoechst 33258测定细胞凋亡率显示,与对照组NIT-1细胞早期凋亡率及总凋亡率比较,Ghrelin预处理组无显著差异(P均>0.05)、高糖组增加(P均<0.01);与单纯高糖组比较,Ghrelin预处理+高糖组细胞早期凋亡率及总凋亡率降低(P<0.01,P<0.05)。(3) RT-PCR结果显示:与对照组比较,Ghrelin预处理组bax、bcl-2、bcl-2/bax水平无变化,而高糖组bcl-2、bcl-2/bax水平下降(P<0.05);与单纯高糖组比较,Ghrelin预处理+高糖组bcl-2、bcl-2/bax水平有升高趋势,但无统计学差异;各组bax表达水平无差别。结论(1) Ghrelin对正常葡萄糖浓度培养的胰岛β细胞凋亡无影响,但可抑制高浓度葡萄糖诱导的NIT-1胰岛β细胞凋亡增加。提示高浓度的葡萄糖对β细胞有毒性作用,Ghrelin可能对高浓度葡萄糖诱导的β细胞凋亡有保护作用。(2) Ghrelin抑制β细胞凋亡的同时,Bcl-2 mRNA及bcl-2/bax表达水平有升高趋势,但无统计学差异,这可能与β细胞类型、刺激因子的不同等有关,也不排除与其他细胞凋亡途径相关因子有关。更多还原

【Abstract】 BACKGROUND AND OBJECTIVE Regardless of type 1 or type 2 diabetes being newly diagnosed,βcell mass has lost about 50-80%.Decreasedβcell mass is associated with its apoptosis,andβcell apoptosis is accelerated in diabetes.Moreover,chronic hyperglycemia makes glucotoxicity-inducedβcell apoptosis more severe in diabetes, forming a vicious circle.βcell apoptosis effects mainly through the following three pathophysiological pathways:extrinsic apoptotic pathway (death receptors mediated signal pathway),intrinsic apoptotic pathway (mitochondrial pathway) and perforin/granzyme B pathway.Bcl-2 family not only regulates mitochondrial activation through changing mitochondrial outer membrane permeability,and plays an important role in mitochondrion induced intrinsic pathway,it also connects the three apoptotic pathways. It is found that ghrelin,also called as growth hormone release peptide,may prevent development of diabetes resulted fromβcell damage.And ghrelin can also inhibitβcell apoptosis induced by serum starvation,doxorubicin,and some cytokines.However,it is unclear about the effects of ghrelin on high glucose-inducedβcell apoptosis.This study in vitro is to pretreat high glucose cultured mouse pancreatic isletβ-cell line NIT-1 cell with ghrelin,and determine cell apoptotic rates and expression of bcl-2 and bax.Then,to investigate the effects of ghrelin on high glucose-inducedβcell apoptosis.METHODS(1) NIT-1cells were cultured in DMEM culture medium,which containing 5.6mmol/L glucose.To observe the morphology characteristics and its growth style under inverted microscope.(2) NIT-1 cells were divided into four groups,control group(5.6mmol/L glucose,C),ghrelin pretreatment group(10-7mmol/Lghrelin,GP),high glucose-induced apoptosis group (33.3mmol/L glucose,HG), ghrelin pretreatment +high glucose group(GP+HG).NIT-1 cell early apoptotic rates were tested by Annexin V-FITC/PI double staining and flowcytometry technique.(3) NIT-1 cell total apoptotic rates were evaluated by Hoechst 33258. NIT-1 cells were stained with Hoechst 33258 to detect apoptotic nuclei showing features of condensation and fragmentation under fluorescence microscope. (4) Expression of Bcl-2 and Bax in four groups were assayed by reverse transcription ploymerase chain reaction (RT-PCR) method. After DNA gel eletrophoresis,then to analyze the gray value and reach the relative expression of bcl-2 and bax.RESULTS(1) NIT-1 cells is polygon epithelial-like cells,growing monolayer-clustered and arranged densely.(2) Both Annexin V/PI FACS and Hoechst33258 results indicated that ghrelin inhibits high glucose-induced NIT-1 cell apoptosis.The early apoptotic rates and total apoptotic rates were no significant difference in GP group(P>0.05),but increased significantly in HG group (P<0.01),as compared with C group.However,the early and total apoptotic rates were significantly lower in GP+HG group than in HG group(P<0.01,P<0.05). (3) RT-PCR results show that ghrelin had no effects on bcl-2 and bax expression in high glucose-induced NIT-1 cell. The level of bax mRNA was no significant difference in all groups. The level of bcl-2 mRNA, bcl-2/bax were decreased significantly in HG group(P<0.05), but no significant difference in GP group (P>0.05), as compared with C group. Unexpectedly, The level of bcl-2 mRNA, bcl-2/bax were no significant difference between GP+HG group and HG group,just an increasing trend was seen in the former.CONCLUSIONS(1) Ghrelin has no effects on pancreatic isletβcell apoptosis cultured in normal glucose,however,it can inhibit high glucose-induced NIT-1 cell apoptosis.It is indicated high glucose has toxic effects onβcells,and ghrelin may have proctective effects on high glucose- inducedβcell apoptosis.(2) As ghrelin inhibitingβcell apoptosis,there is still an increasing trend of bcl-2 and bcl-2/bax expression inβcell,although it is no statistic significance.We speculates that ghrelin’s effects onβcell apoptosis maybe relates to theβcell types and stimulating factors.However, it could not be precluded that ghrelin inhibitingβcell apoptosis is through other apoptotic pathway associated factors.更多还原