E prostanoid receptor 4 를 발현하는 큰포식세포의 Cxcl1 생산을 통한 염증성 대장염의 점막회복 유도 기전 연구

학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2020. 2. 석승혁.Restoration of epithelial barrier function prevents chronic inflammation upon facing intestinal microflora when tissue damages have occurred. Dysfunctional tissue regeneration is commonly considered to be a cause of inflammatory bowel diseases (IBDs). Macrophage is a fundamental component of wound healing, however, the key elements for generating a tissue regenerating phenotype of intestinal macrophage remain elusive. Here I found that E prostanoid receptor 4 (EP4) expression in macrophages is critically required for tissue regeneration in a damaged intestinal milieu. Csf1r-Cre/Esr1EP4fl/fl mice did not go through appropriate tissue regeneration with shortened colon length and prolonged disease activity index after recovery phase of dextran sodium sulfate induced colitis model. EP4+ macrophages are marked by high CD206 expression and high intracellular cAMP level at the resolution phase of colitis, implicating a functional significance of EP4 in the formation of wound healing macrophages. RNA sequencing from sorted macrophages identified a Cxcl1 secreted from EP4+ intestinal macrophages as a driving factor for the epithelial proliferation from regenerating crypts via the PGE2/EP4/MAPK signaling axis. These studies thus define an important role of EP4 expression on intestinal macrophages to revive damaged epithelium.장 조직이 손상되었을 때 장 상피세포 장벽의 회복은 장관내 미생물 균총을 격리하여 만성적인 염증이 일어나는 것을 막아준다. 따라서 장 조직의 재생이 제대로 일어나지 않으면 장관내 미생물에 대한 면역반응이 지속적으로 일어나 염증성 대장염을 야기하게 되며 질환으로부터의 완전한 회복을 위해서는 손상된 장 조직이 적절한 재생을 거쳐 해부학적 장벽을 형성해야 한다. 큰포식세포는 상처 치유의 기본적인 요소 중에 하나로 잘 알려져 있으나 장에 있는 큰포식세포의 조직 재생 능력에 기여하는 주요한 요소가 무엇인지에 대해서는 잘 알려져 있지 않다. 본 연구에서는 큰포식세포에서 지질매개인자인 PGE2의 수용체 중 하나인 EP4 수용체의 발현이 손상 받은 장 조직 재생에 핵심적으로 관여하는 것을 증명하였다. 먼저 큰포식세포 특이적으로 EP4를 발현하지 않는 마우스 (Csf1r-Cre/Esr1EP4fl/fl)를 제작하여 대장염을 일으킨 후 장염의 회복기에 여전히 짧은 장 길이 및 높은 질병 활성도 등을 확인하여 장염의 회복이 잘 일어나지 않음을 확인하였다. 또한 EP4 를 발현하는 큰포식세포는 CD206를 높게 발현하고 있었으며 세포 내 cAMP 를 많이 발현하고 있었다. 이는 EP4가 상처치유 큰포식세포 형성에 중요한 기능을 하고 있음을 암시하고 있다. 더 나아가 회복기 시기의 큰포식세포에 대한 대단위 유전체 분석을 통하여 EP4 신호전달 체계에 의한 큰포식세포의 조직재생능력 향상과 연관 있는 분자생물학적 기전을 확립하고자 하였다. 대단위 유전체 분석을 통하여 Cxcl1 의 발현이 Csflr-EP4-/- 마우스 유래 장 큰포식세포에서 크게 감소하는 것을 확인하였으며, Cxcl1 이 PGE2/EP4/MAPK 신호전달 체계를 통해서 crypt 의 분열을 도와 장 상피세포 분열을 촉진시킬 수 있음을 확인하였다. 본 연구결과로 염증성 대장염 모델의 조직 수복 시기에 조직의 적절한 재생이 이루어지기 위하여 EP4에 의한 큰포식세포의 PGE2/EP4/MAPK 신호전달체계 활성이 필요함을 확인하고 장 손상에 대한 재생 유도 반응의 전체적인 세포간 네트워크에서 큰포식세포가 Cxcl1생산을 통하여 관여하는 분자생물학적 기전을 새롭게 밝혔다.Introduction 1 Materials and Methods 4 Results 12 Discussion 58 References 63 Abstract in Korean 67Docto

Effects of medial septum deep brain stimulation for memory recovery in a memory impaired rat model established by 192 IgG-saporin injection

의과대학/박사The possibility of using deep brain stimulation (DBS) for memory enhancement was recently reported; however, the mechanisms underlying its effects are not precisely understood. This study was performed to find the therapeutic effects and mechanism of DBS using a memory impaired rat model. First, a spatial memory impaired rat model mimicking the cholinergic deficit in Alzheimer’s disease was established. Second, the effects of medial septum (MS)-DBS on this memory impaired rat model were investigated. Furthermore, to detect the stage of the memory process that was affected, DBS was delivered at different times. In the first experiment, 192 IgG-saporin was infused into the cerebral ventricle to cause selective cholinergic damage. Two weeks later, cholinergic denervation caused spatial memory impairment in the Morris water maze test and glucose hypometabolism of the cingulate cortex in the [F-18] fluorodeoxyglucose PET study. In the second experiment, a 60-Hz stimulation was delivered to the MS to confirm the therapeutic effects of MS-DBS. During the 2-week stimulation, MS-DBS enhanced spatial memory and increased the number of doublecortin immunopositive cells in the hippocampus. Next, to detect the stage of the memory process affected by MS-DBS, stimulation was delivered at different times: pre-stimulation, 5days of before the water maze test; training-stimulation, 5days after the start of the training phase for the water maze; probe-stimulation, 2 h of stimulation shortly before the probe test of the water maze. The most effective memory restoration occurred in the pre-stimulation group in which the latency of the training phase was similar to that of the normal group. Moreover, the pre-stimulation group had a better recalled of the platform position than the other stimulation groups. An increase in the level of brain derived neurotrophic factor (BDNF) was observed in the pre-stimulation group that was maintained in both the frontal cortex and hippocampus. The level of glutamic acid decarboxylase 65/67, an enzyme that catalyzes the decarboxylation of glutamate to GABA, was reduced in the hippocampus. However, acetylcholinesterase activity in the pre-stimulation group was not different from the lesion group. These results suggest that the spatial memory improvement associated with MS-DBS is mainly correlated with increased BDNF expression in the frontal cortex and hippocampus, rather than with direct electrical stimulation of cholinergic or GABAergic neurons in the hippocampus. Taken together, the memory impairment caused by cholinergic denervation could be improved by MS-DBS (60Hz). Additionally, it has a significant correlation with the up-regulation of BDNF expression and neurogenesis. Based on the results of this study, the use of MS-DBS during the early stage of disease is thought to restore spatial memory impairments.ope

Which one will you choose; open, laparoscopic, or robotic transduodenal ampullectomy?: a case report

Transduodenal ampullectomy (TDA) is the treatment of choice for large premalignant lesions of the ampulla of Vater (AoV). With the development of surgical techniques, various methods, including the open, laparoscopic, and robotic approaches, for performing TDA have emerged. Herein, we report four consecutive cases treated with open, laparoscopic, and robotic TDA, with technical pitfalls and future perspectives of TDA in treating premalignant lesions of the AoV. The surgical techniques and principles for TDA were the same regardless of the surgical approaches. After surgery, none of the patients showed any abnormal findings or complications, except for digestive problems. All these surgical approaches are appropriate for patients requiring TDA; however, minimally invasive TDA, particularly the robotic approach is ideal. Considering the surgical complexity of TDA, the robotic approach is considered to be effective.ope

Improvements in Memory after Medial Septum Stimulation Are Associated with Changes in Hippocampal Cholinergic Activity and Neurogenesis

Deep brain stimulation (DBS) has been found to have therapeutic effects in patients with dementia, but DBS mechanisms remain elusive. To provide evidence for the effectiveness of DBS as a treatment for dementia, we performed DBS in a rat model of dementia with intracerebroventricular administration of 192 IgG-saporins. We utilized four groups of rats, group 1, unlesioned control; group 2, cholinergic lesion; group 3, cholinergic lesion plus medial septum (MS) electrode implantation (sham stimulation); group 4, cholinergic lesions plus MS electrode implantation and stimulation. During the probe test in the water maze, performance of the lesion group decreased for measures of time spent and the number of swim crossings over the previous platform location. Interestingly, the stimulation group showed an equivalent performance to the normal group on all measures. And these are partially reversed by the electrode implantation. Acetylcholinesterase activity in the hippocampus was decreased in lesion and implantation groups, whereas activity in the stimulation group was not different from the normal group. Hippocampal neurogenesis was increased in the stimulation group. Our results revealed that DBS of MS restores spatial memory after damage to cholinergic neurons. This effect is associated with an increase in hippocampal cholinergic activity and neurogenesis.ope

Iroquois Homeobox 1 Acts as a True Tumor Suppressor in Multiple Organs by Regulating Cell Cycle Progression

Iroquois homeobox 1 (IRX1) belongs to the Iroquois homeobox family known to play an important role during embryonic development. Interestingly, however, recent studies have suggested that IRX1 also acts as a tumor suppressor. Here, we use homozygous knockout mutants of zebrafish to demonstrate that the IRX1 gene is a true tumor suppressor gene and mechanism of the tumor suppression is mediated by repressing cell cycle progression. In this study, we found that knockout of zebrafish Irx1 gene induced hyperplasia and tumorigenesis in the multiple organs where the gene was expressed. On the other hands, overexpression of the IRX1 gene in human tumor cell lines showed delayed cell proliferation of the tumor cells. These results suggest that the IRX1 gene is truly involved in tumor suppression. In an attempt to identify the genes regulated by the transcription factor IRX1, we performed microarray assay using the cRNA obtained from the knockout mutants. Our result indicated that the highest fold change of the differential genes fell into the gene category of cell cycle regulation, suggesting that the significant canonical pathway of IRX1 in antitumorigenesis is done by regulating cell cycle. Experiment with cell cycle blockers treated to IRX1 overexpressing tumor cells showed that the IRX1 overexpression actually delayed the cell cycle. Furthermore, Western blot analysis with cyclin antibodies showed that IRX1 overexpression induced decrease of cyclin production in the cancer cells. In conclusion, our in vivo and in vitro studies revealed that IRX1 gene functionally acts as a true tumor suppressor, inhibiting tumor cell growth by regulating cell cycle.ope

Short and long-term outcomes of minimally invasive central pancreatectomy: Comparison with minimally invasive spleen-preserving subtotal distal pancreatectomy

Background: Central pancreatectomy(CP) is more complex surgery and higher complication rate than distal pancreatectomy(DP). However, with the development of minimally invasive surgery, CP has become a safer surgery technique. In this study, we compare minimally invasive CP(MI-CP) and Minimally invasive spleen-preserving subtotal DP(MI-SpSTDP) to figure out the short-term and long-term outcomes of MI-CP. Methods: From March 2007 to June 2020, 36 cases of MI-SpSTDP and 23 cases of MI-CP were performed for benign and borderline malignant pancreatic tumors in Severance hospital. The occurrence of postoperative pancreatic fistula(POPF) and Clavian-Dindo classification grade 3 or more in the two group was investigated, and the Controlling nutritional status scores(CONUT score) before and 1-year after surgery were compared to determine the long-term outcomes of exocrine function. Results: There was no difference in postoperative complications including POPF between the two groups(17.4% vs 5.1%, p = 0.294). And there were no statistical differences in either the MI-CP group (0.74 ± 0.75 vs. 0.78 ± 0.99, p = 0.803) or the MI-SpSTDP group (0.86 ± 0.83 to 0.61 ± 0.59, p = 0.071). Conclusions: MI-CP had longer operation time and hospital stay and is safe and effective in preserving endocrine and exocrine functions in treatment of benign or borderline tumors located at the neck or proximal body of the pancreas.ope

Aberrant Hedgehog ligands induce progressive pancreatic fibrosis by paracrine activation of myofibroblasts and ductular cells in transgenic zebrafish

Hedgehog (Hh) signaling is frequently up-regulated in fibrogenic pancreatic diseases including chronic pancreatitis and pancreatic cancer. Although recent series suggest exclusive paracrine activation of stromal cells by Hh ligands from epithelial components, debates still exist on how Hh signaling works in pathologic conditions. To explore how Hh signaling affects the pancreas, we investigated transgenic phenotypes in zebrafish that over-express either Indian Hh or Sonic Hh along with green fluorescence protein (GFP) to enable real-time observation, or GFP alone as control, at the ptf1a domain. Transgenic embryos and zebrafish were serially followed for transgenic phenotypes, and investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry. Over-expression of Ihh or Shh reveals virtually identical phenotypes. Hh induces morphologic changes in a developing pancreas without derangement in acinar differentiation. In older zebrafish, Hh induces progressive pancreatic fibrosis intermingled with proliferating ductular structures, which is accompanied by the destruction of the acinar structures. Both myofibroblasts and ductular are activated and proliferated by paracrine Hh signaling, showing restricted expression of Hh downstream components including Patched1 (Ptc1), Smoothened (Smo), and Gli1/2 in those Hh-responsive cells. Hh ligands induce matrix metalloproteinases (MMPs), especially MMP9 in all Hh-responsive cells, and transform growth factor-ß1 (TGFß1) only in ductular cells. Aberrant Hh over-expression, however, does not induce pancreatic tumors. On treatment with inhibitors, embryonic phenotypes are reversed by either cyclopamine or Hedgehog Primary Inhibitor-4 (HPI-4). Pancreatic fibrosis is only prevented by HPI-4. Our study provides strong evidence of Hh signaling which induces pancreatic fibrosis through paracrine activation of Hh-responsive cells in vivo. Induction of MMPs and TGFß1 by Hh signaling expands on the current understanding of how Hh signaling affects fibrosis and tumorigenesis. These transgenic models will be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases which are induced by Hh signaling activation.ope

GLRX3, a novel cancer stem cell-related secretory biomarker of pancreatic ductal adenocarcinoma

Background: Cancer stem cells (CSCs) are implicated in carcinogenesis, cancer progression, and recurrence. Several biomarkers have been described for pancreatic ductal adenocarcinoma (PDAC) CSCs; however, their function and mechanism remain unclear. Method: In this study, secretome analysis was performed in pancreatic CSC-enriched spheres and control adherent cells for biomarker discovery. Glutaredoxin3 (GLRX3), a novel candidate upregulated in spheres, was evaluated for its function and clinical implication. Results: PDAC CSC populations, cell lines, patient tissues, and blood samples demonstrated GLRX3 overexpression. In contrast, GLRX3 silencing decreased the in vitro proliferation, migration, clonogenicity, and sphere formation of cells. GLRX3 knockdown also reduced tumor formation and growth in vivo. GLRX3 was found to regulate Met/PI3K/AKT signaling and stemness-related molecules. ELISA results indicated GLRX3 overexpression in the serum of patients with PDAC compared to that in healthy controls. The sensitivity and specificity of GLRX3 for PDAC diagnosis were 80.0 and 100%, respectively. When GLRX3 and CA19-9 were combined, sensitivity was significantly increased to 98.3% compared to that with GLRX3 or CA19-9 alone. High GLRX3 expression was also associated with poor disease-free survival in patients receiving curative surgery. Conclusion: Overall, these results indicate GLRX3 as a novel diagnostic marker and therapeutic target for PDAC targeting CSCs.ope

Integrative analysis of multiple genomic data from intrahepatic cholangiocarcinoma organoids enables tumor subtyping

As genomic analysis technology has advanced, it has become possible to sub-classify intrahepatic cholangiocarcinoma (ICC) at the histological or molecular level. Here, we verify the recently suggested two subgroups of ICC in the organoids model, compare the characteristics between types. ICC patients are subclassified into small-duct (SD) and large-duct (LD) subtype according to histological characteristics. ICC organoids are established, and unsupervised principal component analysis clustering separates each type of ICC. Differential gene expression reveals enrichment on KRAS, TGFβ and ERBB2 signaling pathways in LD-type compared with SD-type (P < 0.05). Gene set enrichment analysis demonstrates that the cholangiocarcinoma class 2 signature, defined by Andersen et al., is enriched in the LD-type (enrichment Score = 2.19, P < 0.001). A protein-protein interaction network analysis identifies ZNF217 as a significant hub protein (odds ratio = 4.96, P = 0.0105). We perform prospective modeling of histological subtype using patient-derived organoids. Moreover, gene expression profiling of ICC organoids enables identification of type-specific targetable pathways.ope

온곳 결합 돌개와 상온 이온성 액체의 동역학

학위논문(박사) --서울대학교 대학원 :물리학부,2007.Docto