牛胰腺中活性蛋白质的液相色谱分离纯化方法进展

牛胰腺中含有多种活性蛋白,其中许多蛋白已被开发为有利于人类健康的药物。从牛胰腺中分离纯化得到的蛋白药物是一种有高附加值的高技术产品。现代生物技术中所用的大多数有价值的活性蛋白产品的制备仍然依赖于不同的液相色谱法。本文综述了牛胰腺中活性蛋白质的提取方法以及以色谱分离为主的分离与纯化技术,为开展从天然产品中提取并应用蛋白质提供一定的参考。

在线单柱二维液相色谱法快速纯化牛胰腺中细胞色素C

用二维(弱阳,疏水)色谱柱首次完成了在线单柱二维液相色谱法快速纯化牛胰腺中的细胞色素C.在将牛胰腺粗提液进样到该二维色谱柱后,在弱阳离子交换模式下,以梯度洗脱方式进行一维色谱分离,并将分离得到的细胞色素C样品液收集到色谱仪的附加样品储液管内.然后将储液管中样品液全部排出,并二次进样到同一根二维色谱柱中,与此同时也完成了对该样品液的缓冲溶液交换,按疏水色谱(HIC)分离模式进行分离.最终对细胞色素C完成了第二维的HIC纯化.上述全部操作均为在线,在一具有正压的封闭体系中进行并可在52分钟内完成.细胞色素C的最终产品纯度高达94.7%(RSD=1.91%),质量回收率为80.5%(RSD=2.20%).预计此在线单柱二维液相色谱法也可能用于牛胰腺中其他功能蛋白的快速纯化,并可能将其放大到制备和生产规模.

Pioglitazone attenuates the severity of sodium taurocholate-induced severe acute pancreatitis

AIM： To determine the effect of pioglitazone, a specific peroxisome proliferator-activated receptor-γ, （PPARγ） ligand, on development of severe acute pancreatitis （SAP） and expression of nuclear factor-kappa B （NF-κB） and intercellular adhesion molecule-1 （ICAM-1） in the pancreas. METHODS： Male Sprague-Dawley （SD） rats （160-200 g） were randomly allocated into three groups （n = 18 in each group）： severe acute pancreatitis group, pioglitazone group, sham group. SAP was induced by retrograde infusion of 1 mL/kg body weight 5% sodium taurocholate （STC） into the biliopancreatic duct of male SD rats. Pioglitazone was injected intraperitoneally two hours piror to STC infusion. Blood and ascites were obtained for detecting amylase and ascitic capacity. Pancreatic wet/dry weight ratio, expression of NF-κB and ICAM-1 in pancreatic tissues were detected by immunohistochemical staining. Pancreatic tissue samples were stained with hematoxylin and eosin （HE） for routine optic microscopy. RESULTS： Sham group displayed normal pancreatic structure. SAP group showed diffuse hemorrhage, necrosis and severe edema in focal areas of pancreas. There was obvious adipo-saponification in abdominal cavity. Characteristics such as pancreatic hemorrhage, necrosis, severe edema and adipo-saponification were found in pioglitazone group, but the levels of those injuries were lower in pioglitazone group than those in SAP group. The wet/dry pancreatic weight ratio, ascetic capacity, serum and ascitic activities of anylase in the SAP group were significantly higher than those in the sham group and pioglitazone group respectively （6969.50 ± 1368.99 vs 2104.67 ± 377.16, 3.99 ± 1.22 vs 2.48 ± 0.74, P 〈 0.01 or P 〈 0.05）. According to Kusske criteria, the pancreatic histologic score showed that interstitial edema, inflammatory infiltration, parenchyma necrosis and parenchyma hommorrhage in SAP group significantly differed from those in the sham group and pioglitazone group （7.17 ± 1.83 vs 0.50 ± 0.55, 7.67 ± 0.82 vs 6.83 ± 0.75, P 〈 0.01, P 〈 0.05. The expression of NF-κB and ICAM-1 in sham group was lower than that in SAP group and pioglitazone group （0.50 ± 0.55 vs 33 ± 1.21, P 〈 0.01）. There was a significant difference in the expression of NF-κB and ICAM-1 between SAP group and pioglitazone group （7.50 ±1.05 vs 11.33 ± 1.75, 0.80 ± 0.53 vs 1.36 ± 0.54, P 〈 0.01 or P 〈 0.05） at 12 h after the induction of pancreatitis. CONCLUSION： Pioglitazone attenuates the severity of SAP. The beneficial effect of pioglitazone is multifactorial due to its anti-inflammatory activities, most likely through the inhibition of ICAM-1 expression and NF-κB activation. Specific ligands of PPARy may represent the novel and effective means of clinical therapy for SAP.

A simple taurocholate-induced model of severe acute pancreatitis in rats

AIM: To investigate gut barrier damage and intestinal bacteria translocation in severe acute pancreatitis (SAP), a simple rat model of SAP was induced and studied.METHODS: Pancreatitis was induced by uniformly distributed injection of 3.8% Na taurocholate (1 mL/kg) beneath the pancreatic capsule. Rats in the control group were injected with normal saline in the identical location. RESULTS: Serum amylase, plasma endotoxin, intestinal permeability, and pancreatitis pathology scores were all markedly higher in the pancreatitis group than in the control group (P < 0.01). The bacterial infection rate was signif icantly higher in the SAP group than in the control group (P < 0.01), observed in parallel by both bacterial culture and real-time polymerase chain reaction. Acute damage of the pancreas was observed histologically in SAP rats, showing interstitial edema, leukocyte infiltration, acinar cell necrosis and hemorrhage. The microstructure of the intestinal mucosa of SAP ratsappeared to be destroyed with loose, shortened microvilli and rupture of the intercellular junction, as shown by electron microscopy. CONCLUSION: Significant gut barrier damage and intestinal bacterial translocation were def initely observed with few potential study confounders in this SAP rat model, suggesting that it may be an appropriate animal model for study of gut barrier damage and bacterial translocation in SAP.

Induction of pancreatic duct cells of neonatal rats into insulin-producing cells with fetal bovine serum: A natural protocol and its use for patch clamp experiments

AIM： To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS： The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine ceils with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels （KATP）, voltage-dependent potassium channels （Kv）, and voltage-dependent calcium channels （KcA） in β-cells were identified by patch clamp technique. RESULTS： After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly （MTT value from 0.024 ±0.003 to 3.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L）. Then MTT value and insulin secretion increased quickly from d 5 to d 10 （MTT value from 0.028 ±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L）, then reached high plateau （MTT value 〉0.052±0.008, insulin secretion 〉18.3±2.6 mIU/L）. In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d （MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L）, but afterwards they reduced gradually （MTT value 〈0.031 ±0.011, insulin secretion 〈8.2±1.5 mIU/L）, and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, Kv, and KCA. CONCLUSION： Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.

Effect of Chaiqinchengqi decoction on inositol requiring enzyme 1α in alveolar macrophages of dogs with acute necrotising pancreatitis induced by sodium taurocholate

OBJECTIVE: To investigate the effect of Chaiqinchengqi decoction(CQCQD) on inositol requiring enzyme 1α(IRE1α) in alveolar macrophages(AMs)of the dog model of acute necrotising pancreatitis(ANP) induced by sodium taurocholate.METHODS: Fifteen beagle dogs were randomised into a control group,ANP group and CQCQD group(n = 5 per group). ANP was induced by a retrograde duct injection of 50 mg/kg of 5% sodium taurocholate. The dogs in the control group received injections of the same volume of saline as the sodium taurocholate. After the models were induced,the dogs in the CQCQD group were administered 10 m L/kg CQCQD every 2 h for 6 h. Two hours after the last administration of either CQCQD or saline,they were sacrificed by anaesthesia. AMs were collected to determine the IRE1α and Interleukin-1β(IL-1β)m RNA and protein expression,and pancreatic tissues were collected for histopathology analysis.RESULTS: Compared with the ANP group,the m RNA and protein expression of IRE1α and the protein expression of IL-1β of AMs in the CQCQD group were significantly down-regulated,and the pancreatic histopathology score of the CQCQD group also was lower. There was no significant difference in the m RNA expression of IL-1β of AMs between the two groups.CONCLUSION: The CQCQD-induced down-regulation of the IL-1β protein expression may involve the down-regulation of the m RNA and protein expression of IRE1α in AMs.