α-1抗胰蛋白酶减轻小鼠胰腺外分泌细胞对移植胰岛的损伤

A1AT alleviates pancreas exocrine cells damage to transplanted islets in mice

目的探讨α-1抗胰蛋白酶（A1AT）减轻胰腺外分泌细胞对移植胰岛的损伤及促进胰岛口细胞增殖。方法（1）体外实验：选10只BALB／c小鼠，挑取所有的胰岛并收集胰腺外分泌细胞。纯化胰岛组（n=6），每孔100个胰岛，常规培养；实验组（n=6），每孔100个胰岛和等体积外分泌细胞同时培养，并加入A1AT（0．5mg／m1）；对照组（n=6），每孔100个胰岛和等体积外分泌细胞同时培养。48h后，收集各孔上清液，分别检测胰岛素含量及胰蛋白酶浓度；然后分别换低糖、高糖RPMI-1640培养液，行胰岛素释放实验。（2）体内实验：链脲佐菌素腹腔注射诱导BALB／c小鼠成为糖尿病小鼠。实验组（n=10）和对照组（n=10）分别于小鼠左肾被膜上下级同时移植胰岛细胞250个和等体积的胰腺外分泌细胞，术后实验组按腹腔注射A1AT83mg／kg（28d），对照组注射等量生理盐水（28d），同时两组小鼠分别按0．5btg／g腹腔注射EDU（28d）。1个月后切除左肾，免疫组织化学染色法检测左肾包膜下抗淀粉酶抗体的表达，免疫荧光染色法检测移植胰岛B细胞增殖情况，并继续检测血糖。结果（1）纯化胰岛组胰岛素含量为（1．53±0．33）μg／孔，实验组为（1．26±0．13）μg／孔，对照组为（0．82±0．24）μg／孔，各组间的差异均有统计学意义（P〈0．01）。纯化胰岛组胰蛋白酶浓度为（1．05±0．22）ng／ml，实验组为（2．42±0．44）ng／ml，对照组为（5．73±1．12）ng／ml，各组间的差异均有统计学意义（P〈0．01）。纯化胰岛组胰岛素刺激指数为4．11±1．45，实验组为2．53±1．52，对照组为1．32±1．12，各组间的差异均有统计学意义（P〈0．01）。（2）胰岛移植后，实验组和对照组血糖均降至正常，对照组较实验组血糖恢复正常时间延迟，组间差异有统计学意义（P〈0．05）。切除两组小鼠左肾3dN，两组小鼠随机血糖均〉21mmol／L。对照组移植物周围可见大量抗淀粉酶抗体阳性颗粒，实验组较对照组明显减少。免疫荧光检测显示实验组移植胰岛Insulin’／EDU’8细胞数较对照组明显增多。结论（1）胰岛细胞与胰腺外分泌细胞在体外共同培养时加入A1AT可以缓解胰腺腺泡细胞分泌的蛋白酶对胰岛的破坏。（2）胰岛细胞与胰腺外分泌细胞共同移植在糖尿病小鼠体内，腹腔注射AIAT可以明显减少腺泡细胞胰蛋白酶的分泌，减轻胰腺外分泌细胞对移植胰岛的损伤，促进胰岛B细胞的增殖。

Objective To investigate the effect of alpha 1-antitrypsin （A1AT） concerning in reducing the injury of transplanted islets by pancreas exocrine cells and promoting proliferation of the pancreas B cells. Method The pancreases of mice were digested with eollagenase, islets were isolated artificially, and pancreatic exocrine cells were collected. In purified islet group （n = 6）, 100 islets were seeded into a 6-well culture plate. In experimental group（n = 6）, 100 islets were co-cultured with equal volume of pancreas exocrine cells, and 0. 5 mg/mL A1AT was added into a 6-well culture plate. In control group（n = 6）, 100 islets were co-cultured with equal volume of pancreas exocrine cells. After 48 h, insulin content of islets in each well and trypsin concentration in the supernatant of each well were measured. The islets were cultured in low sugar and high sugar 1640 medium, then glucose stimulated insulin secretion （GSIS） test was carried out. In vivo, 8-9-week old male BALB/C mice were induced with STZ （190 mg/kg body weight, i. p） to establish the diabetic model and randomly divided into two groups. In experimental（n = 10） and control（n = 10） groups, 250 islets and the equal volume of pancreatic exocrine cells were transplanted into different regions of left kidney subcapsule, resepctively. The experimental group was injected with A1AT （83 mg/kg, qd, i. p） for 28 days after operation, and the control group was injected with the same amount of normal saline （qd, i. p） for 28 days. Both two groups were given EDU （5μg/g, qd, i. p） for 28 days. The blood glucose level was monitored continually. Nephrectomies were performed after 28 days. The expression of anti-amylase antibodies in the renal subcapsule was detected by immunohistochemical staining, and the proliferation of islet beta cells was examined using immunofluorescence staining. Result Insulin levels and insulin stimulation index in the control group were decreased as compared with those in the purified islet group; those in the experimental group were higher than in the control group, but lower than in the purified islet group. Trypsin concentration in the control group was increased as compared with the purified islet group, that in the experimental group was lower than the control group, but higher than in the purified islet group （all P〈 0. 01 ）. After islets transplantation, the blood glucose levels in control and experimental groups were normal, but those in the control group recovered later than in the experimental group （P〈0. 01）. At 3rd day after nephrectomy, the blood glucose levels were 〉21 mmol/L in both two groups. A large number of anti-amylase antibody-positive cells were found in the renal subcapsule in the control group while little seen in the experimental group after 28 days. The irnmunofluorescence showed that the insulin ＋/EDU ＋ B cells in the experimental group were more than those in the control group. Conclusion Conclusion Co-culture of islets and pancreatic exocrine cells with A1AT can prevent islet cells from damage caused by trypsin. A1AT could inhibit the secretion of pancreatic amylase from pancreatic acinar cells and promote proliferation of islet beta cells.