3周龄断奶仔猪胰酶发育规律的研究

以 3周龄断奶仔猪为试验材料 ,研究了断奶对仔猪胰酶活性的影响。试验表明 ,3周龄断奶可以显著降低仔猪胰脏中胰淀粉酶、胰蛋白酶、胰糜蛋白酶和胰脂肪酶的活性 ,且在断奶后 2周继续维持断奶时水平。断奶降低了小肠内容物中胰淀粉酶和胰脂肪酶的活性 ,但空肠和回肠胰蛋白酶、胰糜蛋白酶的活性在断奶后并未下降。     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Inhibition study of red rice polyphenols on pancreatic α-amylase activity by kinetic analysis and molecular docking

This study sought to investigate the possible inhibition mechanism of red rice polyphenols (RRP) on pancreatic α-amylase (PA) activity. RRP showed strong inhibition against PA activity and the half-inhibitory concentration (IC₅₀) value was 3.61 μg/mL. The fluorescence quenching of PA by RRP was a combination of static quenching and dynamic quenching. RRP could aggregate with PA and the physiochemical properties of the aggregates were closely related to the concentration of RRP. Kinetic analysis suggested that the inhibition mode of RRP on PA was reversible inhibition, which was a mixing of competitive inhibition and noncompetitive inhibition. Molecular docking speculated that RRP could form hydrogen bonds with PA by binding to the catalytic active sites (ASP197, GLU233 and ASP300) and the microenvironments of TRP58 and TRP59 were altered, thus inhibiting PA activity.     

牛肌肉卫星细胞向胰岛素分泌细胞的诱导转分化研究

肌肉卫星细胞是源自中胚层的肌源干细胞,具有增殖分化融合成肌管并形成肌细胞的能力.在体内条件下,肌肉卫星细胞不可能跨胚层分化为内胚层来源的胰腺细胞.本研究从牛(Bos taurus)胎儿肌肉中分离培养得到肌肉卫星细胞,并在体外条件下将其诱导成胰岛素分泌细胞.在诱导过程中,分析了与胰腺发育相关的胰十二指肠同源基因盒1基因(pancreatic and duodenal homeobox 1,PDX1)、神经原素3基因(neurogenin 3,NGN3)、淀粉酶基因(Amylase)和胰岛素基因(Insulin,INS)的动态表达情况.结果表明,PDX1作为决定胰腺分化发育和胰岛功能的主要调节基因,在诱导分化的第2天能够检测到其mRNA的表达,在第3天表达量达到了最大,第4天以后维持在较低水平.NGN3是内分泌细胞形成非常重要的转录因子,是能够将胰岛素分泌到胞外的关键基因,其mRNA在分化诱导的第3天开始表达,第4天达到最高水平,之后逐渐下降.Amylase是胰腺发育中细胞外分泌相关的基因,Amylase mRNA在第6天才开始表达,8d后达到最高.INS mRNA表达量在11～12 d最高,酶联免疫吸附分析(enzyme-linked immunosorbent assay,ELISA)发现,诱导形成的胰腺细胞能够正常分泌胰岛素,培养液中胰岛素的浓度在诱导的11～12 d达到最高值.向培养液中添加葡萄糖后,可使细胞的胰岛素分泌量显著提高,说明诱导得到的细胞类似于成体胰岛的功能,其胰岛素分泌受外界葡萄糖浓度的调节.上述结果表明,肌肉卫星细胞可以被诱导转分化为胰岛素分泌细胞,并对培养环境中的葡萄糖刺激做出应答反应.本研究结果为进一步的研究和临床应用提供基础资料.     

Pancreatic pseudocyst causing extrahepatic biliary obstruction in a dog

A 3-year-old Rhodesian Ridgeback was examined because of recurrent pancreatitis of 2 months duration. The dog had signs of abdominal pain and jaundice. Blood biochemical findings were consistent with extrahepatic bile duct obstruction, but on abdominal ultrasonography no cause of obstruction was identified. At surgery a pancreatic pseudocyst was found in the body of the pancreas. Cystoduodenostomy, cystic omentalisation and biliary diversion resulted in excellent long-term recovery.     

Nanoparticle for siRNA delivery and its pancreatic cancer targeting ability

AIM: To synthesize a safe, efficient and targeted nanoparticulate carrier for siRNA delivery to pancreatic cancer cells. METHODS: Iron oxide nanocrystal with carboxylic acid group-polyethyleneimine (IONP-PEI) was synthesized and investigated as a nonviral carrier of siRNA to the pancreatic cells. The size, surface and charge using zeta potential were characterized. The perfect charge ratio between amino groups of IONP-PEI and phosphate groups of siRNA (N/P) was determined by the transfection efficiency detection, gel retardation assay and MTS assay. An antibody-directed nonviral vector, scFvCD44v6-IONP-PEI nanoparticle attaching to the cancer-associated CD44v6 single-chain variable fragment, was constructed as a cancer-targeting nanocarrier for siRNA delivery. Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFvCD44v6-IONP-PEI/siRNA complexes in the cells. The transfection efficiency, fluorescence intensity and the expression of KRAS at mRNA and protein levels in the cells transfected by IONP-PEI/siRNA and scFvCD44v6-IONP-PEI/siRNA were detected by flow cytometry, fluorescence microscopy, real-time PCR and Western blotting, respectively. RESULTS：The mass ratio of IONP to PEI was 0.75. The suitable ratio of N/P was 20. The averaged size and surface zeta potential of IONP-PEI/siRNA in deionized water were (51.3±2.2)nm (diameter) and (21.73±8.07)mV,respectively. Red fluorescence was seen in both targeting and nontargeting groups, which clearly revealed the intracellular distribution of siRNA and delivery agents. Transfection efficiencies in targeting and nontargeting groups were (89.75±1.81)% and (59.87±4.52)%, respectively. Down-regulation of the KRAS mRNA in Panc-1 cells transfected with siKRAS by scFvCD44v6-IONP-PEI and IONP-PEI was up to (34.02± 6.15)% and (51.09±6.70)%, respectively. The protein level of KRAS was lower in targeting group than that in nontargeting group. CONCLUSION：scFvCD44v6-IONP-PEI is a safe and efficient nanoparticulate carrier for gene delivery. It is more effective to transfer siRNA into the cells and mediate gene silencing effect in vitro than the nontargeting group.     

Inhibitory effect of 5-fluorouracil on ultra-microstructure and insulin secretion of pancreatic β cells

AIM: To investigate the molecular mechanisms of β cell dysfunction induced by 5-fluorouracil (5-FU) in islet β cell line (NIT-1 cells). METHODS: The NIT-1 cells were treated with different concentrations of 5-FU. The content of insulin in the culture medium was determined by radioimmunoassay. Cell apoptosis was observed by flow cytometry with annexin V/PI staining. The ultra-microstructural changes of NIT-1 cells were observed under transmission electron microscope. The expression of pancreatic and duodenal homeobox protein 1(PDX-1) at mRNA and protein levels in NIT-1 cells was examined by RT-PCR and Western blotting, respectively. RESULTS: Exposed to the low glucose concentration (5.6 mmol/L), insulin secretion in NIT-1 cells was not significantly decreased following a 24 h treatment with 5.0 to 40.0 mg/L 5-FU (P>0.05). On the contrary, the high glucose (16.7 mmol/L)-stimulated insulin secretion in NIT-1 cells was inhibited by 5.0 to 40.0 mg/L of 5-FU in a dose-dependent manner after 24 h of incubation (P<0.01). The apoptosis rate of NIT-1 cells was significantly increased as compared to those in the control levels(P<0.05). The structural changes of mitochondria were the main apoptotic changes under transmission electron microscope. Significant down-regulation of PDX-1 expression at mRNA and protein levels was observed in NIT-1 cells treated with 5-FU at the concentration of 10.0 mg/L to 40.0 mg/L(P<0.05).CONCLUSION: 5-FU inhibits the insulin secretion in islet β cell induced by high glucose. A relative deficiency in insulin secretion following 5-FU treatment is related to the changes of β cell ultra-microstructure and the reduction of β cell numbers, by which an increase in apoptosis of pancreatic β cells is induced. Down-regulation of PDX-1 expression may play a pivotal role in increasing the apoptosis of pancreatic β cells induced by 5-FU in high-glucose condition.     

Somatostatin receptor imaging in vivo by planar scintigraphy facilitates the diagnosis of canine insulinomas

Somatostatin receptors expressed by insulinomas in 5 dogs were imaged in vivo by means of indium in 111 pentetreotide (OctreoScan) scintigraphy. The diagnosis in each dog was supported by the presence of hypoglycemia (<60 mg/dL), hyperinsulinemia (>20 microU/mL), and histopathologic review of neoplastic tissue. All insulinomas expressed high-affinity somatostatin receptors of subtype sst2, as shown by receptor autoradiography in vitro using 125I-[tyrosine3]-octreotide and 125I-[leucine8, Dtryptophan22, tyrosine25]-somatostatin-28 with an sst2 subtype-selective analogue. Scintigrams were obtained at 1, 4, 12, and 24 hours after the i.v. administration of 74-222 MBq of OctreoScan to each patient. Abnormal foci of activity were 1st observed from 1 hour after administration of the radioligand in dog 3, to 24 hours after its administration in dog 4; in dogs 1 and 2, abnormal foci of activity were visible from 12 hours. Dog 5 showed a questionable abnormal focus of activity at 12 hours, but not at 24 hours. Scintigraphy enabled accurate prediction of the anatomical location of the primary tumor in 1 of 4 dogs, but was unable to differentiate a right- from a left-pancreatic lobe tumor, or vice versa, in 3 dogs; the 5th dog had equivocal results. 111In-pentetreotide scintigraphy is a useful diagnostic adjunct to the clinical evaluation of the insulinoma patient, but is unable to localize the tumor in some cases.     

Successful treatment of a pancreatic pseudocyst by omentalisation in a dog

CASE HISTORY A 3-year-old male Labrador retriever was presented with a history of dietary indiscretion followed by vomiting and abdominal pain.

CLINICAL FINDINGS AND TREATMENT: Abdominal ultrasonography revealed the presence of a fluid-filled cystic structure in the region of the pancreas. Flocculent, dark fluid was drained from the cystic structure during exploratory celiotomy prior to resection of the lateral cystic wall. Omentum was sutured into the cystic cavity to provide drainage and enhance immune response. A jejunostomy feeding tube was placed. Post-surgical care consisted of antibiotic therapy. The dog was clinically normal at 1 and 7 months post-operatively and the pancreas appeared normal on final ultrasonographic examination.

CLINICAL RELEVANCE: This case describes the use of omentum to provide physiological drainage of a pancreatic pseudocyst. This surgical technique may help clinicians manage patients with this condition in the future.