胰腺多肽纳米抗体的制备及结合活性分析

胰腺多肽(pancreatic polypeptide,PP)是一种含36个氨基酸的胰腺激素,在评估胰腺功能和辅助诊断相关疾病中发挥重要作用。以PP为靶点,筛选特异性的PP纳米抗体,评估其与PP抗原是否具有结合活性。利用原核表达系统制备了高纯度的PP抗原,经免疫羊驼后构建了高库容和高丰度的胰腺多肽免疫的纳米抗体噬菌体文库,并通过噬菌体展示技术进行文库筛选,获得8株胰腺多肽纳米抗体。选取1株纳米抗体(PP-VHH)构建原核表达体系,经IPTG诱导表达、纯化与鉴定,通过间接ELISA和双抗夹心ELISA分别检测和评价抗原抗体的结合活性和血清中PP含量,结果显示,PP-VHH与PP具有结合活性,且PP-VHH可用于检测鸡和人血清中的PP。建立的双抗夹心ELISA法拟合曲线R^(2)为0.9868,可检测不同个体鸡血清中PP浓度为48~55 pg/mL。而人血清中PP浓度范围波动较大。综上所述,本研究筛选到的1株纳米抗体PP-VHH,可用于检测鸡和人血清中的PP含量,为评估胰腺功能异常或诊断相关疾病提供基础。

重组人组织激肽释放酶与猪胰腺组织激肽释放酶的理化特性比较

为了研究表达在蛋清中的重组人组织激肽释放酶（rhK1）是否与天然组织激肽释放酶具有相似的理化特性，对鸡输卵管暂态生物反应器表达的rhK1与猪胰腺组织激肽释放酶（pK）的理化特性进行比较鉴定．Western blotting检测结果显示，表达在蛋清中的rhK1能被人组织激肽释放酶（hK1）特异抗体识别，与pK无交叉反应，rhK1酶原和成熟酶的分子量约为43kDa和37kDa．理化鉴定结果显示，rhK1在pH7～10的范围内活性稳定，而pK在pH8～9的范围内活性较稳定；rhK1和pK均能被Cu^2＋、Fe^3＋和Al^3＋完全抑制；rhK1和pK的热稳定性相似，但rhK1在37℃孵育20min后被显著激活、以上试验结果表明，表达在蛋清中的rhK1分子量正确，理化特性与猪的天然酶相似，具有开发利用价值．

5例胰腺血管活性肠肽瘤患者的护理

总结5例胰腺血管活性肠肽瘤患者的护理,术前做好腹泻和低钾血症的护理,术后严密监测血糖和电解质变化,做好生物治疗、靶向治疗后不良反应的护理,促进患者康复。

胰激肽释放酶治疗76例周围血管疾病的临床分析

胰激肽释放酶治疗７６例周围血管疾病的临床分析中国医学科学院协和医科大学血液学研究所血液病医院（天津３０００２０）王书桂，李尚珠，华国勋，黄平平，高继平，李洪钧周围血管疾病是一种常见病、慢性病，较顽固性疾病，对其治疗目前均采用综合性治疗，有手术、药物、...

急性重症胰腺炎38例临床分析

目的：探索急性重症胰腺炎治疗方法。方法：选择2008年1月到2011年1月期间本院收治的38名急性重症胰腺炎患者，常规治疗后采取奥曲肽静脉注射。结果：38名患者在奥曲肽治疗后，18例患者具有显著疗效，占47．4％，总有效人数34人，占89．5％。结论：奥曲肽临床治疗有效，值得推广。

Parenteral versus early intrajejunal nutrition:Effect on pancreatitic natural course,entero-hormones release and its efficacy on dogs with acute pancreatitis

AIM: To evaluate the effect of early intrajejunal nutrition (EIN) on the natural course, entero-hormone secretion and its efficacy on dogs with acute pancreatitis.METHODS: An acute pancreatitis model was induced by injecting 1 ml/kg of combined solution (2.5% sodium taurocholate and 8 000-10 000 BAEE units trypsin/mi) into the pancreas via pancreatic duct. Fifteen dogs were divided into parenteral nutrition (PN) group and EIN group. Two groups were isonitrogenous and isocaloric. EIN was used at postoperative 24 h. Serum glucose, calcium, amylase and lysosomal enzymes were determined before and 1, 4, 7 d after acute pancreatitis was induced. All the dogs were injected 50 uCi 125I-BSA 4 h before sacrificed on the 7th day.The 125I -BSA index of the pancreas/muscle, pancreas/blood,and pancreas pathology score (PPS) were determined. The peripheral plasma cholecystokinin (CCK), secretin (SEC) and gastrin were measured by ELISA and RIA, and was quantitative analysis of pancreatic juice and amylase,pancreatolipase and HCO3-, Cl-, Na+ and K+ performed by an autochemical analyzer at 30, 60, 120 and 180 min after beginning PN or EIN on the first day.RESULTS: There was no difference between two groups in the contents of serum calcium, amylase and lysosomal enzymes, 125I-BSA index of pancreas/muscle and pancreas/blood and PPS. The contents of CCK and gastrin in EIN were higher than those in PN group at 60 and 120 min (P＜0.05).The content of SEC post-infusion of nutrition solution was higher than that of pre-infusion of nutrition solution in both groups, and only at 60 min SEC in EIN group was higher than that in PN group. The content of gastrin in EIN was higher than that in PN group at 120 and 180 min (P＜0.05).The changes of pancreatic juice, amylase, pancreatolipase and HCO3-, Cl-, Na+ and K+ between two groups did not reach significantly statistical difference (P＞0.05).CONCLUSION: EIN does not stimulate entero-hormone and pancreatic juice secretion, and enzyme-protein synthesis and release. EIN has no effect on the natural course of acute pancreatitis.

胰腺小分子活性肽对H_2O_2诱导体外大鼠胰岛细胞凋亡的影响

目的研究胰腺小分子活性肽对H2O2诱导体外大鼠胰岛细胞凋亡的影响。方法采用胶原酶消化和组织培养法分离纯化大鼠胰岛细胞,检测其纯度与活性。对照组加入普通培养基,实验组培养基中加入不同质量浓度(100、200、300、400μg/ml)胰腺小分子活性肽,阳性对照组培养基中加入100nmol/L艾塞那肽。每组细胞做5个复孔(n=5),培养5d后每组均接受H2O2100μmmol/L处理1h。采用Hoechst33258核染色形态学观察;AnnexinV-FITC/PI双染色流式细胞仪检测各组细胞凋亡率;荧光定量PCR法检测bax、bcl-2及caspase3mRNA的表达;Western-bolt检测bax、bcl-2蛋白的表达。结果 300、400μg/ml胰腺小分子活性肽和阳性对照组胰岛细胞凋亡率小于对照组(P<0.05);300、400μg/ml胰腺小分子活性肽组胰岛细胞bcl-2蛋白表达高于对照组(P<0.05),bax与caspase3蛋白表达低于对照组(P<0.05);300、400μg/ml胰腺小分子活性肽组胰岛细胞bcl-2mRNA的表达高于对照组(P<0.05),且bax mRNA的表达低于对照组(P<0.05)。结论胰腺小分子活性肽在浓度为300、400μg/ml时对大鼠胰岛细胞凋亡有明显保护作用。

胰腺血管活性肠肽瘤临床特点和诊治

胰腺血管活性肠肽瘤(vasoactive intestinal peptide tumors,VlPoma)为罕见的胰腺内分泌肿瘤,有典型的临床表现,如水样腹泻、低钾血症、胃酸缺乏。胰体尾部是病变的主要部位。临床筛查,早期诊断,恰当的处理方式,可影响病人的存活率并提高生存质量。典型症状和血浆血管活性肠肽(vasoactive intestinal polypeptide,VIP)值是诊断的关键。手术切除是最佳的治疗方式。药物治疗首选生长抑素类似物,可以使疾病症状(即使是未行手术者)长期缓解。

A nuclear import inhibitory peptide ameliorates the severity of cholecystokinin-induced acute pancreatitis

AIM: To assess the effect of our novel cell-permeable nuclear factor-kappaB (NF-κB) inhibitor peptide PN50 in an experimental model of acute pancreatitis. PN50 was produced by conjugating the cell-penetrating penetratin peptide with the nuclear localization signal of the NF-κB p50 subunit.METHODS: Pancreatitis was induced in male Wistar rats by administering 2×100 μg/kg body weight of cholecystokininoctapeptide (CCK) intraperitoneally (IP) at an interval of 1 h. PN50-treated animals received 1 mg/kg of PN50 IP 30 min before or after the CCK injections. The animals were sacrificed 4 h after the first injection of CCK.RESULTS: All the examined laboratory (the pancreatic weight/body weight ratio, serum amylase activity,pancreatic levels of TNF-α and IL-6, degree of lipid peroxidation, reduced glutathione levels, NF-κB binding activity, pancreatic and lung myeloperoxidase activity) and morphological parameters of the disease were improved before and after treatment with the PN50 peptide.According to the histological findings, PN50 protected the animals against acute pancreatitis by favoring the induction of apoptotic, as opposed to necrotic acinar cell death associated with severe acute pancreatitis.CONCLUSION: Our study implies that reversible inhibitors of stress-responsive transcription factors like NF-κB might be clinically useful for the suppression of the severity of acute pancreatitis.

Clinical value of rapid urine trypsinogen-2 test strip, urinary trypsinogen activation peptide, and serum and urinary activation peptide of carboxypeptidase B in acute pancreatitis

AIM: To assess the usefulness of urinary trypsinogen-2 test strip, urinary trypsinogen activation peptide (TAP),and serum and urine concentrations of the activation peptide of carboxypeptidase B (CAPAP) in the diagnosisof acute pancreatitis.METHODS: Patients with acute abdominal pain and hospitalized within 24 h after the onset of symptoms were prospectively studied. Urinary trypsinogen-2 was considered positive when a clear blue line was observed (detection limit 50 μg/L). Urinary TAP was measured using a quantitative solid-phase ELISA, and serum and urinary CAPAP by a radioimmunoassay method.RESULTS: Acute abdominal pain was due to acute pancreatitis in 50 patients and turned out to be extrapancreatic in origin in 22 patients. Patients with acute pancreatitis showed significantly higher median levels of serum and urinary CAPAP levels, as well as amylase and lipase than extrapancreatic controls. Median TAP levels were similar in both groups. The urinary trypsinogen-2 test strip was positive in 68% of patients with acute pancreatitis and 13.6% in extrapancreatic controls (P<0.01). Urinary CAPAP was the most reliable test for the diagnosis of acute pancreatitis (sensitivity 66.7%, specificity 95.5%, positive and negative predictive values 96.6% and 56.7%, respectively), with a 14.6 positive likelihood ratio for a cut-off value of 2.32 nmol/L.CONCLUSION: In patients with acute abdominal pain,hospitalized within 24 h of symptom onset, CAPAP in serum and urine was a reliable diagnostic marker of acute pancreatitis. Urinary trypsinogen-2 test strip showed a clinical value similar to amylase and lipase.Urinary TAP was not a useful screening test for the diagnosis of acute pancreatitis.

Neutrophil depletion-but not prevention of Kupffer cell activation-decreases the severity of cerulein-induced acute pancreatitis

AIM： To determine whether neutrophil depletion and Kupffer cell inhibition might combine their protective effects to decrease the severity of acute pancreatitis. METHODS： Nice had cerulein administration to induce acute pancreatitis and were pretreated with either anti-mouse neutrophil serum or gadolinium chloride （GdCh） to prevent Kupffer cell activation, or both treatments. Injury was assessed in pancreas and lungs. Myeloperoxidases （MPO） assessed neutrophil infiltration. Interleukin-6 （IL-6） and IL-10 were measured in serum, pancreas, lungs and liver. RESULTS： In mice with acute pancreatitis, neutrophil depletion reduced the severity of pancreatitis and pancreatitis-associated lung injury. Kupffer cell inactivation by GdCh had less protective effect, although IL-6 and IL-10 concentrations were significantly decreased. The protective treatment brought by neutrophil depletion was not enhanced by Kupffer cell inactivation and both treatments did not combine their protective effects. CONCLUSION： Our results confirm the role of activated neutrophils in aggravating organ injury in acute pancreatitis while the role of Kupffer cell activation is less obvious.

Hyperlipidemia intensifies cerulein-induced acute pancreatitis associated with activation of protein kinase C in rats

AIM： To investigate the effects of hyperlipidemia on acute pancreatitis （AP） and the possible mechanisms. METHODS： Rat models of hyperlipidemia and AP were established by Triton WR1339 and cerulein respectively. Human albumin was used to treat AP complicated by hyperlipidemia. In each group, we compared the histological score, volume of ascites, ratio of pancreatic wet/dry weight, serum amylase （AMY） and pancreatic acinar cell apoptosis. The level of protein kinase C （PKC） membrane translocation in pancreatic tissue was detected by Western blot.RESULTS： In the hyperlipidemia model established by Triton WR1339, triglyceride （TG） increased remarkably and reached its peak 6 h after injection, and most rats developed mild acute pancreatitis. Histological score, volume of ascites, ratio of wet/dry weight and serum AMY in AP animals with hyperlipidemia were obviously higher than those in AP animals （P 〈 0.05） and decreased after albumin therapy but not significantly （P 〉 0.05）. Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） increased in AP animals with hyperlipidemia and did not change distinctly after albumin therapy. PKC membrane translocation level increased in AP animals with hyperlipidemia and decreased remarkably after albumin therapy （P 〈 0.05）.CONCLUSION： Hyperlipidemia may induce AP or intensify pancreatic injury. Albumin therapy can not alleviate pancreatic lesion effectively. PKC activation may be one mechanism by which AP is intensified by hyperlipidemia.

Presence of CCK-A, B receptors and effect of gastrin and cholecystokinin on growth of pancreatobiliary cancer cell lines

AIM: To investigate the effects of gastrin and cholecystokinin (CCK) and their specific antagonists on the growth of pancreatic and biliary tract cancer cell lines. METHODS: Five pancreatic and 6 biliary cancer cell lines with 2 conrtol cells were used in this study. Cell proliferation study was done using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test and direct cell count method. Reverse transcription-polymerase chain reaction (RT-PCR) and slot blot hybridization were performed to examine and quantify the expression of hormonal receptors in these cell lines. RESULTS: SNU-308 showed a growth stimulating effect by gastrin-17, as did SNU-478 by both gastrin-17 and CCK-8. The trophic effect of these two hormones was completely blocked by specific antagonists (L-365, 260 for gastrin and L-364, 718 for CCK). Other cell lines did not respond to gastrin or CCK. In RT-PCR, the presence of CCK-A receptor and CCK-B/gastrin receptor mRNA was detected in all biliary and pancreatic cancer cell lines. In slot blot hybridization, compared to the cell lines which did not respond to hormones, those that responded to hormones showed high expression of receptor mRNA. CONCLUSION: Gastrin and CCK exert a trophic action on some of the biliary tract cancers.

Caerulin-induced pancreatitis in rats: Histological and genetic expression changes from acute phase to recuperation

AIM： To study the histological and pancreatitis-associated protein mRNA accumulation changes of pancreas from acute phase of caerulin-induced pancreatitis to recuperation in rats. METHODS： Acute pancreatitis was induced by caerulein in male Wistar rats and followed up for 90 d by histological and mRNA analyses of pancreas. Pancreases were dissected at 0, 9, 24 h and 3, 5, 15, 30, 60, 90 d post-induction. Edema （E）, polymorphonuclear neutrophU （pIVlN） infiltration, cytoplasmic vacuolization （V）, zymogen granule depletion （ZD） and acinar disorganization （AD） were microscopically evaluated. Accumulation of pancreatitis- associated protein （PAP） and L13A mRNAs were quantified by real-time PCR. RESULTS： The main histological changes appeared at 9 h post-induction for PMN infiltration and cytoplasmic V, while at 24 h and 3 d for E and ZD, respectively. All the parameters were recovered after 5 d, except for ZD which delayed more than 30 d. The main AD was observed after 15 d and values returned to normal after 30 d. Similarly to histological changes, accumulation of the PAP mRNA was increased at 9 h with the highest accumulation at 24 h and differences disappeared after 5 d. CONCLUSION： From the acute phase to recuperation of pancreatitis, regeneration and re-differentiation of pan- creas occur and PAP expression is exclusively an acute response of pancreatitis.

Neoadjuvant peptide receptor radionuclide therapy for an inoperable neuroendocrine pancreatic tumor

Pancreatic endocrine tumors are rare but are among the most common neuroendocrine neoplasms of the abdomen.At diagnosis many of them are already advanced and diff icult to treat.We report on an initially inoperable malignant pancreatic endocrine tumor in a 33-year-old woman,who received neoadjuvant peptide receptor radionuclide therapy(PRRT)as firstline treatment.This resulted in a signif icant downstaging of the tumor and allowed its subsequent complete surgical removal.Follow-up for eighteen months revealed a complete remission.This is the first report on neoadjuvant PRRT in a neuroendocrine neoplasm with subsequent successful complete resection.

Effects of octreotide on acute necrotizing pancreatitis in rabbits

AIM:To assess the role of oxygen-derived free radicals and cytokines in the pathogenesis of taurocholic acid-induced acute pancreatitis,and to evaluate the preventive effects of octreotide towards the development of acute pancreatitis. METHODS:Acute pancreatitis was induced in male New Zealand white rabbits by retrograde injection of 0.8 mL/kg·b.m,of 50 g/L sodium taurocholate (NaTC) in the pancreatic duct.Sham- operated animals served as control.Octreotide i mg/kg·b.m. was administered subcutaneously before the induction of pancreatitis.Blood was taken from the jugular vein before and at 1,3,6,12 and 24 h after pancreatitis induction. Serum activities of amylase,IL-6 and TNF-α and levels of malonyl dialdehyde (MDA),glutathione (GSH),glutathione peroxidase (GPx),catalase and superoxide dismutase (Mn-, Cu-,and Zn-SOD) in pancreatic tissue were measured. RESULTS:Serum TNF-α and IL-6 levels increased significantly 3 h after the onset of pancreatitis,and then returned to control level.The tissue concentration of MDA was significantly elevated at 24 h,while the GSH level and GP-x,catalase,Mn-SOD,Cu-,Zn-SOD activities were all significantly decreased in animals with pancreatitis as compared to the control.Octreotide pretreatmnent significantly reversed the changes in cytokines and reactive oxygen metabolites.Octreotide treatment did not alter the serum amylase activity and did not have any beneficial effects on the development of histopathological changes. CONCLUSION:Oxygen-derived free radicals and proinflammatory cytokines are generated at an early stage of NaTc-induced acute pancreatitis in rabbits.Prophylactic octreotide treatment can prevent release of cytokines and generation of reactive oxygen metabolites,but does not have any beneficial effects on the development of necrotizing pancreatitis.

Octreotide reverses shock due to vasoactive intestinal peptide-secreting adrenal pheochromocytoma: A case report and review of literature

Vasoactive intestinal peptide-producing tumors （VIP-oma） usually originate in the pancreas and are chara-cterized by diarrhea, hypokalemia, and achlorhydria （WDHA syndrome）. In adults, nonpancreatic VIPoma is very rare. Herein, we report an unusual case of VIP-producing pheochromocytoma marked by persistent shock, fushing, and watery diarrhea and high sensitivity to octreotide. A 53-year-old woman was hospitalized for sudden-onset hypertension with convulsions, which then rapidly evolved to persistent shock, fushing, and watery diarrhea. Abdominal computed tomography indicated a left adrenal mass, accompanied by bleeding;and marked elevations of both plasma catecholamine and VIP concentrations were documented via laboratory testing. Surprisingly, all clinical symptoms responded swiftly to octreotide treatment. Once surgically treated, hormonal levels normalized in this patient, and the clinical symptoms dissipated. Postoperative pathological and immunohistopathological studies confrmed a VIP-secreting pheochromocytoma with strong, diffuse positivity for somatostatin receptor type 2. During a 6-mo follow-up period, she seemed in good health andwas symptom-free.

INTRAPANCREATIC CHOLECYSTOKININ MEDIATES VAGALLY STIMULATED EXOCRINE SECRETION FROM THE RAT PANCREAS

Although cholecystokinin is localized within neuronal fibres of the pancreas, a physiological role for intrapancreatic cholecystokinin has not been identified. The strategy of this study was to elicit pure vagal stimulation electrically, and to use specific receptor antagonists to identify the mediators of exocrine pancreatic secretion. We conclude that vagal stimulation of the rat pancreas involves ganglionic neurotransmission and release of acetylcholine and cholecystokinin from intrapancreatic, postganglionic fibres. To our knowledge, this is the first study to demonstrate a physiological role for intrapancreatic cholecystokinin.

Selective cyclooxygenase-2 inhibitor ameliorates cholecystokinin-octapeptide-induced acute pancreatitis in rats

AIM： To investigate the effect of selective Cyclooxygenase-2 （COX-2） inhibitor 4-[5-（4-Chloro-phenyl）-3- （trifluoromethyl）- 1H-pyrazol- 1-yl] benzenesulfonamide （SC-236）, on the cholecystokinin （CCK）-octapeptideinduced acute pancreatitis （AP） in rats. METHODS： Wistar rat weighing 240 g to 260 g were divided into three groups. （1） Normal DMSO treated group, （2） SC-236 at 4 mg/kg treated group; SC-236 systemically administered via the intravenous （i.v.） catheter, followed by 75 μg/kg CCK octapeptide subcutaneously three times, after 1, 3 and 5 h. This whole procedure was repeated for 5 d. （3） Dimethyl sulfoxide （DMSO） treated group： an identical protocol was used in this group as in the SC-236 cohort （see 2. above）. Repeated CCK octapeptide treatment resulted in a typical experimentally induced pancreatitis in the Wistar rats. RESULTS： SC-236 improved the severity of CCK- octapeptide-induced AP as measured by laboratory criteria [the pancreatic weight/body weight （p.w/ b.w） ratio, the level of serum amylase and lipase]. The SC-236 treated group showed minimal histologic evidence of pancreatitis and a significant reduction in myeloperoxidase activity. SC-236 also increased heat shock protein （HSP）-60 and HSP72 compared with the DMSO-treated group in the CCK-octapeptide-induced AP and also reduced the pancreatic levels of COX-2. Furthermore, SC-236 reduced proinflammatory cytokine synthesis and inhibited NF-KB activation compared with the DMSO-treated group in the CCK-octapeptide-induced AP. CONCLUSION： Our results suggested that COX-2 plays pivotal role in the development of AP and COX-2 inhibitors may play a beneficial role in preventing AP.

Hypercalcemia Appeared in a Patient with Glucagonoma Treated with Octreotide Acetate Long-acting Release

PABCREATIC neuroendocrine tumours are uncommon neoplasms of the pancreas.They may cause a clinical syndrome due to hormone overproduction.Glucagonoma is a rare kind of pancreatic tumors. Here we report a case of glucagonoma. Hypercalcemia occurred when the patient underwent octreotide acetate long-acting release.