Chronická pankreatitída je závažný, multifaktoriálny, fibroinflamačný syndróm charakterizovaný opakovanými bolestivými exacerbáciami. Toto ochorenie má výrazný vplyv na kvalitu života pacientov, najmä z hľadiska bolesti, rozvoja diabetes mellitus a opakovaných záchvatov akútnej pankreatitídy. Autológna transplantácia Langerhansových ostrovčekov po totálnej pankreatektómii je potenciálnou možnosťou na zachovanie endogénnej sekrécie inzulínu u pacientov so závažnými, opakujúcimi sa bolestivými atakmi chronickej pankreatitídy. V našom prípade prezentujeme pacienta s chronickou pankreatitídou trpiaceho opakovanými bolestivými exacerbáciami. Pacient potreboval opakované hospitalizácie s opakovanými ERCP zákrokmi a stentážou pankreatického vývodu. Po genetickom potvrdení hereditárnej pankreatitídy a multidisciplinárnom zhodnotení bolo rozhodnuté o totálnej pankreatektómii s autotransplantáciou ostrovčekov. V tomto prípade prezentujeme prvú medzinárodnú autotransplantáciu Langerhansových ostrovčekov v Slovenskej republike.
Chronic pancreatitis with repeated painful relapses is a severe multifactorial, fibroinflammatory syndrome that significantly affects the quality of life of patients, mainly in terms of pain, development of diabetes mellitus, and repeated attacks of acute pancreatitis. Autologous islet transplantation after total pancreatectomy is a potential option in selected patients with severe, recurrent painful episodes of chronic pancreatitis. In our case, we present a patient with severe recurrent chronic pancreatitis. The patient required frequent hospitalizations with repeated stenting of the pancreatic duct. After the confirmation of a genetic mutation, the members of the multidisciplinary session decided to perform a total pancreatectomy with islet autotransplantation. In this case, we present the first international islet cell autotransplantation of Langerhans islets in Slovakia after total pancreatectomy in a patient with chronic hereditary pancreatitis.
Transplantace Langerhansových ostrůvků představuje zavedenou metodu léčby pacientů s diabetem 1. typu se syndromem porušeného vnímání hypoglykemie, u kterých selhává použití moderních technologických postupů. Nejčastěji se jedná o radiointervenční metodu, při které se suspenze pankreatických ostrůvků podává perkutánně zavedeným katétrem do větve portální žíly. Ačkoliv se na rozdíl od orgánové transplantace pankreatu jedná o výkon jen minimálně invazivní, řada technických problémů zůstává nevyřešena. Mezi možné komplikace patří zejména krvácení a trombóza portální žíly. Na rozdíl od svého přirozeného umístění disperzně ve tkáni exokrinního pankreatu jsou izolované transplantované ostrůvky v jaterním parenchymu vystaveny bezprostřední hypoxii, přímému působení toxinů a imunosupresivních léčiv. Přímý kontakt s krví příjemce vyvolává bezprostřední zánětlivou reakci, tzv. IBMIR (instant blood mediated inflammatory reaction), díky které i více než polovina transplantovaných buněk po aplikaci zaniká. Velikost ostrůvkového štěpu je proto často nedostatečná a řada pacientů potřebuje po výkonu určité množství exogenního inzulinu. To všechno jsou důvody, proč hledáme pro ostrůvkový štěp jiné místo transplantace s příznivějšími podmínkami pro dlouhodobé přežívání. V experimentálním i klinickém výzkumu byla zkoušena různá aplikační místa, jejichž výhody a nevýhody shrneme v tomto sdělení. Jako nejnadějnější se v současné době jeví transplantace do velkého omenta, která již byla na několika pracovištích klinicky použita.
Pancreatic islets transplantation is an established treatment method for type 1 diabetic patients with the hypoglycemia unawareness syndrome in whom a therapy with modern technologies fails. Islet transplantation is most commonly done using an interventional radiology method: a tissue suspension of pancreatic islets is applied into a branch of the portal vein through a percutaneously installed catheter. Although being minimally invasive unlike pancreas organ transplant, this method is associated with many technical difficulties. Possible complications of the procedure include hemorrhage and portal vein thrombosis. Unlike their natural dispersed localization in exocrine pancreas, isolated pancreatic islets are exposed to hypoxia, toxins and immunosuppressive drugs in the liver parenchyma. Direct contact with the recipient’s blood causes an instant blood mediated inflammatory reaction (IBMIR) resulting in the death of more than half of the pancreatic islets shortly after their application. Therefore the size of the islet graft is often insufficient and a number of transplanted patients require administration of exogenous insulin. All of these are reasons for seeking an alternative transplantation site with more hospitable conditions for long-term islet survival. Various transplantation sites have been tested in experimental and clinical research. The advantages and disadvantages of some of them are summarized in this paper. Currently, transplantation into the greater omentum seems most promising, which has already been used in clinical practice at several institutions.
- Klíčová slova
- syndrom porušeného vnímání hypoglykemie,
- MeSH
- diabetes mellitus 1. typu chirurgie MeSH
- lidé MeSH
- omentum MeSH
- transplantace Langerhansových ostrůvků * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system. The respective gene sequences were derived from C. histolyticum and modified by addition of a C-terminal polyhistidine tag. Harvested bacteria were lysed and the collagenase protein was affinity purified using a His-tag column. The purity, identity, integrity of the eluted collagenases G and H were determined by SDS electrophoresis and Western blot. The proteolytic activity of the collagenase G and H blend (rColGH) was determined by the standard FALGPA assay. The tissue dissociation activity was verified using a standardized method for isolation of rat pancreatic islets. Biocompatibility of the blend was validated by a standardized viability assay on the isolated islets. Two batches of rColGH were produced and compared to a commercially available collagenase. Based on our results, we conclude that rColGH is a functional and non-toxic novel recombinant collagenase worth further characterization and blend optimization in order to make it a competitive commercial product.
- MeSH
- Clostridium MeSH
- kolagenasy * MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Reprogramming of non-endocrine pancreatic cells into insulin-producing cells represents a promising therapeutic approach for the restoration of endogenous insulin production in diabetic patients. In this paper, we report that human organoid cells derived from the pancreatic tissue can be reprogrammed into the insulin-producing cells (IPCs) by the combination of in vitro transcribed modified mRNA encoding transcription factor neurogenin 3 and small molecules modulating the epigenetic state and signalling pathways. Upon the reprogramming, IPCs formed 4.6 ± 1.2 % of the total cells and expressed typical markers (insulin, glucokinase, ABCC8, KCNJ11, SLC2A2, SLC30A8) and transcription factors (PDX1, NEUROD1, MAFA, NKX2.2, NKX6.1, PAX4, PAX6) needed for the proper function of pancreatic β-cells. Additionally, we have revealed a positive effect of ALK5 inhibitor RepSox on the overall reprogramming efficiency. However, the reprogrammed IPCs possessed only a partial insulin-secretory capacity, as they were not able to respond to the changes in the extracellular glucose concentration by increasing insulin secretion. Based on the achieved results we conclude that due to the incomplete reprogramming, the IPCs have immature character and only partial properties of native human β-cells.
- MeSH
- antigen AC133 metabolismus MeSH
- beta-buňky cytologie účinky léků MeSH
- dospělí MeSH
- genetická transkripce účinky léků MeSH
- inzulin biosyntéza MeSH
- knihovny malých molekul farmakologie MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- organoidy cytologie MeSH
- přeprogramování buněk účinky léků genetika MeSH
- proliferace buněk MeSH
- proteiny nervové tkáně genetika metabolismus MeSH
- transkripční faktory bHLH genetika metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.
- Publikační typ
- časopisecké články MeSH
The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Differentiation of pancreatic β-cells is regulated by a wide range of signalling pathways. The aim of our current work was to evaluate the effect of the Jak/Stat signalling pathway on the differentiation of human non-endocrine pancreatic cells into insulin-producing cells. Activation of the Jak/Stat signalling pathway by leukaemia inhibitory factor (LIF) stimulated differentiation of C-peptide-negative human non-endocrine pancreatic cells into insulin-producing cells in 6.3 ± 2.0 % cells (N = 5) and induced expression of pro-endocrine transcription factor neurogenin 3, Notch signalling pathway suppressor HES6 and stimulator of β-cell neogenesis REG3A. The expression of the REG3A gene and increased rate of differentiation into insulin-producing cells (10.2 ± 2.1 %) were further stimulated by a combination of LIF with nicotinamide and dexamethasone. Glucose-stimulated (5 vs. 20 mM) C-peptide secretion confirmed proper insulin secretory function of trans-differentiated insulin-producing cells (0.51 vs. 2.03 pmol C-peptide/μg DNA, P < 0.05). Our results indicate that Jak/Stat signalling critically contributes to trans-differentiation of non-endocrine pancreatic cells into functional insulin-producing cells. The positive effect of the Jak/Stat signalling pathway on trans-differentiation is mediated by the key genes that activate differentiation of pancreatic β-cells.
- MeSH
- beta-buňky cytologie MeSH
- buněčná diferenciace účinky léků MeSH
- C-peptid MeSH
- imunohistochemie MeSH
- Janus kinasy genetika metabolismus MeSH
- kultivované buňky MeSH
- leukemický inhibiční faktor farmakologie MeSH
- lidé MeSH
- pankreas cytologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- signální transdukce účinky léků MeSH
- transkripční faktory STAT genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
