RGD肽保护大鼠胰岛活性和功能的实验研究

目的探讨RGD肽对分离纯化后的胰岛活性和功能的影响。方法将实验大鼠分成两组：1640组和RGD组。采用Ficoll不连续密度梯度离心纯化胰岛，浓度分别为：27％、25％、23％、20．5％和11％。1周后，用丫啶橙／溴乙啶（AO／EB）荧光染色观察RGD肽对胰岛活性的影响。胰岛素分泌功能检测：采用放免法检测培养液中胰岛素含量，计算胰岛素释放指数（SI）。流式细胞仪检测：caspase-9和磷酸化Akt阳性细胞比例，观察RGD肽对caspase-9及Akt的影响。结果采用改良梯度离心法，平均每只大鼠可获约600IEQ～700IEQ胰岛，YO／EB荧光染色，结果显示刚分离后胰岛活率〉95％。1640组培养1周后胰岛分泌指数为1．64±0．28，RGD组胰岛分泌指数为2．28±0．16，较1640组明显增加，差异具有统计学意义（P〈0．05）。流式细胞仪检测结果表明胰岛1640组培养1周后，激活的caspase-9为（22．66±3．56）％，RGD组激活的caspase-9为（10．54±1．96）％，较1640组明显降低13％，且其差异具有统计学意义（P〈0．05）；检测RGD对Akt磷酸化的影响，胰岛1640组培养1周后，Akt磷酸化占（31．47±4．08）％，RGD组磷酸化Akt占（61．054±6．03）％，较1640组明显升高，其差异具有统计学意义（P〈0．05）。结论RGD可通过Akt／PKB的磷酸化抑制细胞凋亡。

Protective effects of RGD peptides on islet viability and function

Objective To investigate the effect of argenin-glycin-aspartic acid (RGD) peptides on islet viability and function. Methods Rats were randomly divided into 2 groups: 1640 group and RGD group. Ficoll was used to purify islet in a discontinuous-density-gradient way. Islet concentration is 27% ,25% ,23% ,20.5% and 11% respectively. Acridine orange/ethidium bromide(AO/EB) fluorescent staining method was adopted to observe the effect of RGD peptides on islet viability. Radioimmunoassay was adopted to detect insulin level and measure insulin secretion index (SI). Caspase-9 cells and phospho-Akt 473-positive cells were determined by flow cytometry to investigate the influence of RGD peptides on caspase-9 activity and Akt. Results 600-700 IEQ islet was extracted from each rat by Ficoll purification through modified gradient centrifugation. AO/EB stain showed islet survival rate was more than 95% immediately after separation. Islets cultured in the medium of RPMI-1640 showed SI of 1.64 ±0.28 after 1 week, while islets cultured in the medium of RPMI-1640 containing RGD peptides showed SI of 2.28 ± 0.16 (P ＜ 0.05 ). Flow cytometry showed the level of activated caspase-9 was ( 22.66 ± 3.56 ) % if islet was cultured in RPMI-1640 alone for 1 week while the level was( 10.54 ± 1.96) % if islet was cultured in RPMI-1640 containing RGD peptides. There was statistical difference ( P ＜ 0. 05 ). The effect of RGD on Akt phosphorylation was also detected. Akt phosphorylation proportion was (31.47 ±4.08)% 1 week after cultured in RPMI-1640 while the value was(61. 054 ±6.03)% if cultured in RPMI-1640 containing RGD peptides. The difference had statistical significance (P ＜ 0. 05). Conclusion RGD peptides could inhibit apoptosis through the phosphorylation of Akt/PKB.