| 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. |
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