【作者】 林树梅；

【导师】 胡建民；

【作者基本信息】 沈阳农业大学 ， 食品科学与工程， 2009， 博士

【摘要】 目的:研究牛磺酸合成限速酶CSD基因在大鼠胰岛细胞中的表达及糖尿病情况下表达量变化,并在此基础上观测治疗性添加牛磺酸(Tau)对于链脲佐菌素(STZ)所致糖尿病大鼠糖脂代谢、抗氧化能力、胰岛细胞凋亡以及胰岛内分泌细胞等方面的影响,探讨牛磺酸治疗糖尿病的作用机制,为应用牛磺酸治疗糖尿病提供理论依据。方法:1.将250-300g雄性Wistar大鼠麻醉后,胆总管原位灌注胶原酶V,38℃水浴消化胰腺组织,Histopaque 1077密度梯度离心纯化胰岛细胞,DTZ检测纯度,葡萄糖刺激检测胰岛细胞活性。2.提取分离纯化后的大鼠胰岛细胞总RNA,根据NCBI上的大鼠脑部CSD基因序列,设计一对特异性的引物,进行RT-PCR扩增。将扩增产物回收后,与PMD18-T克隆载体连接,并转化到Ecoli DH5α感受态细胞,通过对菌液PCR鉴定后测序,并将测序结果与大鼠脑部CSD核苷酸序列进行比对。3.向胰岛细胞的培养基中加入一定量STZ,并用半定量PCR的方法测定STZ组和对照组胰岛细胞CSD基因的相对表达量。4.从120只Wistar雄性大鼠中随机取15只作为正常对照组,其余105只大鼠按50mg/kg体重一次性腹腔注射链脲佐菌素(STZ)。72h后用血糖仪测鼠尾静脉空腹血糖。血糖值≥16.7mmol/L,且出现明显的”三多”症状为标准,确认为糖尿病模型。5.造模成功后随机分成7组,正常对照组(Control组)、糖尿病模型组(DM模型组)、高剂量牛磺酸治疗组(4.3 g/kg bw Tau治疗组)、中剂量牛磺酸治疗组(3 g/kg bwTau治疗组)、低剂量牛磺酸治疗组(1.7 g/kg bw Tau治疗组)、罗格列酮治疗组(4mg/kgbw RSG治疗组)、胰岛素治疗组(20 iu Ins治疗组)。连续治疗70d,期间动态观测空腹血糖。治疗第8周时杀鼠,测定血清中C-肽(C-P)、胰高血糖素(Glu)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、总抗氧化能力(T-AOC)、总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDL)的含量。6.取大鼠胰腺组织做石蜡切片,分别用HE染色观察胰岛组织形态,TUNEL原位末端标记法检测胰岛细胞的凋亡百分率,用免疫组化法染色观察胰岛细胞中Fas、Bax、Bcl-2的蛋白表达量。结果:1.每只大鼠可获得约800个胰岛细胞,其纯度在95%以上,所得胰岛细胞具有良好的活性。2.成功扩增出长为2044bp的基因片段,其编码区在18-1433bp,共编码470个氨基酸,相对分子质量为53KDa。测序结果显示该基因片段与参考的大鼠脑部CSD基因序列同源性达到99%。3.STZ处理后胰岛细胞中CSD基因表达量明显下降,其水平显著低于对照组。4.105只正常大鼠经STZ腹腔注射72h后,90只造模成功。在随后2个月实验过程中,DM模型组大鼠体重持续下降,Tau组体重明显高于DM模型组,RSG组和Ins组与DM模型组相比差异不显著。5.Tau组和Ins组血糖明显降低,而DM模型组血糖一直维持在高水平,RSG组血糖有一定程度降低,但作用不明显。经统计学检验,Tau组与DM模型组比较,血糖均有显著降低(P＜0.05)。6.Tau组C-肽水平显著升高,胰高血糖素显著降低。同时Tau能不同程度的降低血清TG,TC,MDA含量;升高SOD,GSH-Px,T-AOC,HDL值。7.(1)HE光镜下观察DM模型组胰岛萎缩,胰岛数量减少,大部分胰岛细胞完全融合,出现玻璃样变性,核溶解。Tau组与其他治疗组相比胰岛结构尚完整,细胞变性程度较轻。(2)胰岛素免疫组化观察,糖尿病模型组中β细胞表达胰岛素显著减少,牛磺酸治疗组β细胞表达胰岛素量明显高于模型组,并表现一定的剂量依赖性,罗格列酮组和胰岛素组与低剂量牛磺酸组接近。(3)TUNEL标记及计数,DM模型组残存的胰岛内有大量胰岛细胞凋亡,Tau高剂量治疗组仅发现少量凋亡细胞,凋亡率明显低于其他治疗组,低剂量组胰岛内亦可见大量凋亡胰岛细胞,中剂量治疗组较少,罗格列酮组和胰岛素组与DM模型组相比差异不显著。(4)凋亡相关因子的免疫组化结果表明:Control组Bcl-2高表达,Bax、Fas低表达;DM模型和Ins组较正常对照组Bcl-2表达降低,而Bax、Fas表达明显增高(P＜0.05)。Tau组明显抑制STZ所致的Bcl-2表达降低和Bax、Fas表达增高(P＜0.05)。RSG组也抑制了Bcl-2表达降低和Bax、Fas表达增高,但是不如Tau组明显。结论:1.大鼠胰岛细胞中存在CSD基因的表达。2.STZ处理的大鼠胰岛细胞CSD基因的相对表达量下降。3.给糖尿病模型大鼠治疗性灌胃牛磺酸能够调控胰岛内分泌,改善与糖尿病相关的血液生化指标,并通过调节凋亡相关基因Fas和Bcl家族成员间的平衡,发挥抗凋亡作用。更多还原

【Abstract】 Aim:This research aims to investigate the effects of taurine on lipid metabolism, oxidation resistance ability and the function of islet endocrine cell in streptozotocin-induced diabetes mellitus rats, approach the mechanism of taurine on diabetes, and provide a theoretical basis for using taurine as a treatment of diabetes.Method:1. 250-300 rats were in situ perfusion with collagenase V in bile common duct after anesthesia. Pancreatic tissues were digested using aqueous bath under 38 centigrade, islet cells were purified by Histopaque 1077 density gradient centrifugation, then detected the purity by DTZ, detected the cytoactive by glucose stimulation.2. Designed the specific primer of purified islet cell total RNA according to CSD gene sequence in the brain of rats listed in NCBI and then amplified it by RT-PCR. Conjugated the product with PMD18-T cloning vehicle after extraction, and transformed it to competent cell. Then aligned the results of sequencing with CSD nucleotide in rats brain after identification of germ solution PCR.3. Added a certain amount of STZ into medium of islet cells, and determined the relative expression of islet cell CSD gene in STZ and control groups by way of Semi-Quantitative PCR.4. Randomly selected 15 rats from 120 male Wistar rats as normal control group, the others were intraperitoneal injected with 50mg/kg streptozotocin (STZ). Detected the fasting blood glucose of rats vena caudalis by glucometer after 72 hours. The diabetic models were affirmed according to obvious "poly three" symptom as well as the blood glucose was more than 16.7mmol/L.5. The rats were randomly divided into seven groups, normal control group (control group), diabetic model group (DM model group), high dose taurine treatment group (4.3 g/kg bw Tau treatment group), median dose taurine treatment group (3 g/kg bw Tau treatment group), low dose taurine treatment group (1.7 g/kg bw Tau treatment group), rosiglitazone treatment group (4mg/kg bw RSG treatment group), insulin treatment group (20 iu INS treatment group). Observe the fasting blood glucose for days. The rats were killed at the 8 th week of treatment, and detected the serum C-peptide, glucagon, superoxide dismutase (SOD), glutathion peroxidase (GSH-Px), malondialdehyde (MDA), total antioxidation capability (T-AOC), total cholesterol (TC), triglyeride (TG) and high density lipoprotein (HDL).6 . Took the pancreatic tissue and made into paraffin section, observed the Histomorphological Changes after HE staining, detected the apoptosis percentage of islet cells by TUNEL in situ end labeling, observed the protein expression of Fas, Bax and Bcl-2 by way of immunohistochemistry.Results:1. Each rat could get about 800 islet cell, the purity of which could above 95%, and the cells have good activity.2. 2044bp genetic fragment were successfully amplified, the coding region of which was between 18-1433bp and could encode 470 amino acid, the relative molecular mass of which was 53 KDa. The result of sequencing showed that the sequence homology of this genetic fragment and the CSD gene in rats brain could be 99%.3. The gene expression of CSD gene in islet cells after treated with STZ decreased significantly compared with the control group.4. 72 hours after intraperitoneal injected with STZ, 90 rats were successfully modeled diabetes. During the 2 months of experiment, the body weight of rats in DM model group decreased, the body weight of Tau groups were significantly higher than that of the DM group, that of the RSG and INS groups had no significant differences when compared with the DM model group.5. Blood glucose of Tau and INS groups significantly decreased, while that of the DM model group was still kept on a high level, the RSG group decreased to a certain degree, but not so obvious.6. The level of c peptide in taurine group increased significantly, the level of glucagon decreased significantly. At the same time, taurine could decrease serum TG, TC and MDA, and increase serum SOD, GSH-Px, T-AOC and HDL.7. (1) It was observed under light microscope after HE stain that islet of DM model group were atrophied, the number of islet decreased, most of the islet cells were fully fused, glassy degeneration and caryolysis could also be observed. In TAU group, the structure of islet was integrity, the degree of cell degeneration was lighter, the morphology of islet was almost normal when compared with other groups.(2) It was observed by immunohistochemistry that insulin expressed byβcells in diabetic model group decreased significantly, that of the taurine treatment groups were significantly higher than that of the model group, and there was some dose dependent. The expression of rosiglitazone and insulin groups were almost similar to that of the taurine groups.(3) It was found by TUNEL labeling and counting that there were a large amount of apoptosis islet cells in DM model group, while there were only a few in TAU high-dose group, the apoptosis rat in which was significantly lower compared with other groups. The number of apoptosis in low-dose groups was also large, while in medium dose group was small. There were no significant differences between that of the rosiglitazone group, the insulin groups and DM model groups.(4) It was observed by immunohistochemistry that the expression of Bcl-2 was high, while the expression of Bax and Fas were low in control group. The expressions of Bcl-2 in DM model group and INS group were lower compared with the normal group, while the expressions of Bax-2 and Fas were increased. The expression of Bcl-2 was also inhibited in ROS group, while the expressions of Bax and Fas increased, but not as obvious as that in TAU group.Conclusion:1. There were CSD gene expression in rats islet cells.2. The relative CSD gene expression in islet cells treated by STZ decreased.3. Taurine, given curatively to rats with diabetes, could accommodate the endocrine of islet positively, improve the relative biochemical indexes, and take the effect of antiapoptosis by way of regulating the balance of apoptosis related gene Fas with Bcl family member.更多还原