The effect of DPP-4 inhibitor on angiogenic regeneration by bone marrow mesenchymal stem cell in hind limb ischemia injury model

의과대학/석사Background: Mesenchymal stem cells (MSCs) are known to have a therapeutic potential for severe limb ischemia. However, poor survival of implanted MSCs in the target tissue remains as an important factor that attenuates the angiogenic potential of the cell therapy. Thus, we investigated whether sitagliptin, a DPP-4 inhibitor, may enhance the angiogenic efficacy of MSC in a hind limb ischemia murine model by increasing production of SDF-1. Methods and Results: Mice with induced hind limb ischemia were devided into 4 groups; group 1 treated with oral saline and local injection of saline, group 2 treated with oral sitagliptin and local injection of saline, group 3 treated with oral saline and local injection of MSCs, and group 4 treated with oral sitagliptin and local injection of MSCs. Angiogenic responses were measured by laser-Doppler perfusion imaging, muscle capillary density, and protein and mRNA expression of SDF-1, CXCR4, and vascular endothelial growth factor VEGF growth factors and compared among the treatment groups. The combined treatment of oral sitagliptin and local injection of MSCs achieved more effective angiogenic response than oral sitagliptin administration or local MSC transplantation alone in a mouse hind limb ischemia model. The combination therapy also demonstrated increased expression of VEGF, SDF-1 and CXCR4 in hind limb ischemia models. Conclusion: The combination therapy of oral sitagliptin and local transplantation of MSCs was more effective in enhancing angiogenic responses to ischemia than oral sitagliptin or local transplantation of MSCs alone possibly due to up-regulation of SDF-1.ope

파이토케미칼의 유용 효능과 응용 방법에 대한 연구

학위논문 (석사)-- 서울대학교 국제농업기술대학원 국제농업기술학과, 2017. 8. 김도만.To characterize inhibitory activities of phenolic compounds against NS2B-NS3 protease of ZIKA virus (ZIKV NS2B-NS3pro) and their structure activity relationship, in this study, ZIKV NS2B-NS3pro was expressed in E. coli BL21(DE3) as 35 kDa protein. The enzyme was displayed a Km of 26.3 µM with fluorogenic peptide Dabcyl-KTSAVLQSGFRKME-Edan, and purified ZIKV NS2B-NS3pro was used for inhibition and kinetic assays with twenty-two phenolic compounds. These phenolic compounds inhibited the activity of ZIKV NS2B-NS3pro by 6.2% to 87.9%. The IC50 of seven phenolic compounds ranged from 22.2 ± 0.2 to 112.0 ± 5.5 µM. Myricetin showed mixed type inhibitory pattern against ZIKV NS2B-NS3pro protease with IC50 values of 22.2 ± 0.2 µM and Ki value of 8.9 ± 1.9 µM. Relationships between chemical structures and inhibitory activities against ZIKV NS2B-NS3pro can be further explored to develop highly selective inhibitors against ZIKV NS2B-NS3pro. Solubility plays a key role of bioavailability in human body. Rubusoside is a steviol glycoside sweetener in herbal tea and widely known for enhancing solubility in water. Stevioside, also a steviol glycoside sweetener, was commonly cultivated in many countries compared with rubusoside. β-galactosidase from Thermus thermophilus presented hydrolytic activity on β-1,2 linkage of stevioside, and can convert from stevioside to rubusoside. In this study, to industrialize production of rubusoside, firstly, β-galactosidase from T. thermophilus was expressed in E. coli BL21(DE3)plysS using lactose induction. Enzyme immobilization has several advantages for enzyme reaction. Immobilized enzyme could be repeatedly used without another method of separation and the decrease in its activity was not largely observed. Sodium alginate was used for enzyme immobilization. Stevioside conversion for free enzyme and immobilized enzyme were shown over 77%. Enzyme reaction was carried out in the vertical double-jacket glass column reactors thermostatted at 70°C for continuous reaction. Immobilized enzyme was stable for 31 days in double-jacket glass column reactors and activity of immobilized enzyme was remained 51.4% after 31 days.Chapter 1. Inhibitory effect of phenolic compounds against ZIKA virus NS2B-NS3 protease and their structure activity relationship 1 1.1 Introduction 1 1.1.1. Zika virus 1 1.1.2. Zika virus NS2B-NS3 protease 2 1.1.3. Phenolic compounds 2 1.2. Materials and methods 4 1.2.1. Preparation of ZIKV NS2B-NS3 pro 4 1.2.2. Characterization of ZIKV NS2B-NS3 pro 7 1.2.3. Inhibition assay 8 1.3. Results and discussion 10 1.3.1. Expression of ZIKV NS2B-NS3 pro 10 1.3.2. Phenolic compounds inhibition against the activity of ZIKV NS2B-NS3 pro 14 1.3.3. Relationship between chemical structure of inhibitor and ZIKV NS2B-NS3 pro activity 21 1.4. Conclusion 23 Chapter 2. Production of rubusoside from stevioside using β-galactosidase from Thermus thermophilus 24 2.1 Introduction 24 2.1.1. Steviol glycosides 24 2.1.2. β-galactosidase from Thermus thermophilus 25 2.1.3. Immobilization of enzymes 25 2.2. Materials and methods 27 2.2.1. Chemicals 27 2.2.2. Selection of expression strains 27 2.2.3. Optimization of lactose concentration for induction 27 2.2.4. β-galactosidase hydrolytic activity assay 28 2.2.5. Fermentation and purification of β-galactosidase 28 2.2.6. Immobilization of β-galactosidase 29 2.2.7. Production of Rubusoside mixture using double jacket columns 29 2.2.8. Purification of rubusoside 30 2.3. Results and discussion 31 2.3.1. Optimization for the expression of β-galactosidase 31 2.3.2. Fermentation of recombinant β-galactosidase 34 2.3.3. Production of rubusoside using immobilized β-galactosidase on double jacket columns 36 2.4. Conclusion 39 References 40 Abstract in Korean 46Maste

(The) correlation between health condition and preparation for the old-aged in the middle-aged.

간호학과/석사[한글] 오늘날 지속적인 생활수준의 향상과 보건의료기술의 발달로 평균수명이 연장되어 노인 인구의 증가가 두드러지게 나타나고 있다. 그러나 사회복지제도가 제대로 확립되지 못한 우리나라에서 노년기를 맞이할 지금의 중년들은 노후에 대한 사회적 의식의 변화와 함께 개인 스스로 노후에 대한 준비가 절실한 상황이다.중년층은 신체적으로 노화현상이 일어나는 갱년기이며 여러 가지 만성질환과 악성질환이 발병될 수 있고 자녀들을 독립시키고 새로운 인생을 시작해야 하는 역할상의 변화를 경험하는 시기이기 때문에 건강관리 측면에서는 위기라고 볼 수 있다. 중년층의 건강상태에 따라 생활수준과 수명이 달라질 것이고, 이는 노후준비에 유의한 영향을 미칠 것이라고 예상된다. 따라서 본 연구에서는 중년층을 대상으로 하여 건강상태와 노후준비 정도를 알아보고, 건강상태가 노후준비에 미치는 영향에 대해서 연구해 보고자 한다. 본 연구는 서술적 상관관계 연구로서 서울특별시에 거주하는 만 40세에서 59세 사이의 중년층을 대상으로 2002년 10월 14일부터 11월 19일까지 구조화된 설문지를 이용하여 자료를 수집하였다. 건강상태를 측정하기 위한 연구도구는 Derogatis(1977)가 개발한 증상체크리스트-90을 기초로 하여 오가실(1993)이 개발한 건강증상조사서-48를 수정, 보완하여 사용하였다. 노후준비를 측정하기 위한 연구도구는 배계희(1988)와 황승일(2000)이 개발한 도구를 수정, 보완하여 사용하였다. 수집된 자료는 SPSS 11.0 program을 이용하여 t-test, ANOVA, Pearson Correlation coefficients, Multiple regression 방법으로 분석하였다.연구 결과는 다음과 같다. 1. 중년층의 건강상태 정도는 171.8점(평균평점 3.6점)으로 비교적 좋은 것으로 나타났으며, 영역별로는 신체적 건강상태, 정서적 건강상태, 사회적 건강상태가 모두 평균평점 3.6점으로 동일하였다. 2. 중년층의 노후준비 정도는 99.0점(평균평점 2.6점)으로 보통 수준으로 나타났으며, 영역별로는 신체적 노후준비(2.5점), 정서적 노후준비(2.7점), 경제적 노후준비(2.6점), 여가 노후준비(2.7점)가 비슷하였다. 3. 노후준비는 건강상태와 순상관관계를 보여(r=.27, p=.000), 중년층의 건강상태가 좋을수록 노후준비를 더 잘하는 것으로 나타났다. 이를 세부항목별로 살펴보면 노후준비는 신체적 건강상태(r=.21, p=.000), 정서적 건강상태(r=.28, p=.000), 사회적 건강상태(r=.20, p=.001)와 모두 순상관관계를 보였고, 그 중 정서적 건강상태와 가장 상관관계가 높음을 알 수 있었다.4. 중년층의 노후준비에 대한 관련 요인은 건강상태가 노후준비의 7%를 설명하였고, 직업이 6%, 월수입이 3%를 더 설명하여 이 세 요인이 노후준비를 총 16% 설명하였다. 이를 건강상태의 세부 항목인 신체적 건강상태, 정서적 건강상태, 사회적 건강상태로 분류하여 살펴본 결과 정서적 건강상태가 좋을수록, 전업주부의 경우, 월수입은 201만원 이상인 경우, 학력은 전문대 졸업 이상인 경우 노후준비를 더 잘하는 것으로 나타났다. 정서적 건강상태가 노후준비의 8%를 설명하였고, 직업이 6%, 월수입이 3%, 학력이 1%를 더 설명하여 이 네 요인이 노후준비를 총 18% 설명하였다. 이상의 연구 결과를 종합하여 볼 때 건강상태는 노후준비와 밀접한 관련이 있다는 것을 알 수 있었다. 특히 건강상태 중에서도 정서적 건강상태가 노후준비와 높은 상관관계가 있으므로 정서적 건강상태의 향상을 통해 중년층의 노후준비를 증진시킬 수 있다고 하겠다. [영문] The old population is remarkably increasing due to the continuous improvement of living standard and health care technology, which make the average life span prolonged. However, the current mid-life, who will enter into their old age in this country where social welfare system is not established well, keenly have to provide against the declining years by themselves, along with social awareness on the later life.The middle-aged is a crucial moment from the viewpoint of health care, because it is climacteric showing symptoms of aging effect, has the potential to outbreak of chronic & malignant diseases, and also experiences changes in their role of parents to start their own life after their children''s marriage. It is expected that living standard and life span will be changed according to the health condition of the middle-aged, which will effect on the preparation for the later life. Therefore, this study is performed to investigate health condition and the standard of preparation against the old age and research how health condition affects the preparation for the old age, aimed at the middle age.As a descriptive correlation, this study collected information through structural questionnaire form Oct 14 to Nov 19, 2002, targeting the middle age from 40 to 59. Based on the Symptom Checklist-90 Rivision(SCL-90-R) developed by Derogatis(1977), the Symptom Checklist-48 designed by Oh, Ga-Sil(1933) was used as a study tool to measure health condition after some correction and supplement. To measure the preparation against the old age, a tool developed by Bae, Kye-Hee(1988) & Hawang, Seung-Il(2000) was applied with correction and supplement.The data collected was analyzed by the SPSS 10.0 program using t-test, ANOVA, Pearson Correlation coefficients and Multiple regression. The results are drawn as follows;1. The health condition of the middle-aged comparatively showed good. In parts, physical, emotional, social healths were equal as 3.6 point.2. The preparation standard of the elderly indicated medial (ordinary) as 99.0 point (2.6 in average). In parts, physical (2.5), emotional (2.7), economic (2.6), and leisure (2.7) preparation for the elderly were presented much the same. 3. Providing for the old age showed correlation with health condition(r=.27, p=.000), the healthier middle age prepares for their old better. In detail, preparation for the old age indicated correlation with all the physical(r=.21, p=.000), emotional(r=.28, p=.000) and social health condition(r=.20, p=.001), especially, emotional showing the highest relation. 4. Health condition explained 7% of preparation for the old age, occupation 6% and monthly income 6%, which showed 16% in total. By looking it in details as physical, emotional, and social health condition, the healthier showed better preparation against the elderly for those who are housewives or have monthly income over 2,010K(KRW) or are more than college graduate. The emotional health condition explained 8% of preparation for the old age, occupation 6%, monthly income 3%, and education 1%, which indicated 18% in total.Overall, it is proposed that health condition is closely related with preparation for the old age. Amongst all factors in health condition, emotional health showed the closest relation with the preparation for the old age. Therefore, it is concluded that the preparation for the old age of the middle-aged can be promoted through improving emotional health condition.ope

독소루비신 유도 심근 독성 모델에서 YAP/TAZ 신호 전달 체계 조절을 통한 아토르바스타틴의 심근 보호 효과

Yes-Associated Protein (YAP) and Transcriptional Co-activator with PDZ-binding Motif (TAZ) are involved in proliferation and cell survival and are transcriptional co-factors regulated by LATS/MST in the Hippo pathway. In addition, in anticancer drug toxicity studies, YAP and TAZ play an important role in regulating cell apoptosis. Doxorubicin is an anthracycline anticancer drug, and when it is used in the long term, it causes myocardial toxicity, which leads to heart failure. Atorvastatin lowers the occurrence of cardiovascular disease by reducing blood cholesterol and the cardiotoxicity of anticancer drugs in cancer patients. So, we investigated whether YAP and TAZ mediate the protective effect of statin against doxorubicin-induced cardiotoxicity. YAP and its downstream target Park2 decreased when H9c2 cardiomyocytes were treated with doxorubicin. Also, TAZ and its downstream target survivin were decreased. In contrast, atorvastatin treatment restored reduced YAP, TAZ, Park2, and survivin mRNA and protein levels in doxorubicin-induced cardiotoxicity. Furthermore, atorvastatin was decreased expression of cleaved-caspase3 (apoptotic marker) in doxorubicin-induced apoptosis. However, when YAP and TAZ were knocked down by siRNA, atorvastatin could not reduce doxorubicin-induced apoptosis. Also, Park2 and survivin were not restored by knockdown of YAP and TAZ despite the pre-treatment of atorvastatin in doxorubicin-induced cardiotoxicity. In addition, knockdown of Park2 or survivin attenuated the effect of atorvastatin in inhibiting doxorubicin-induced apoptosis. In chronic or acute doxorubicin-induced cardiomyopathy mice model, oral administration of statin decreased apoptosis of cardiomyocytes. In conclusion, atorvastatin can prevent doxorubicin-induced cardiotoxicity, and these results showed that atorvastatin exerts antiapoptotic effects by increasing the expression of YAP and TAZ and activating their downstream target, Park2 and survivin. YAP 과 TAZ는 세포 증식 및 세포 생존에 관여하며 Hippo 신호 전달 경로에서 LATS/MST 에 의해 조절되는 전사인자이다. 또한 항암제 독성 연구에서 세포 사멸을 조절하는 중요한 역할을 한다. 독소루비신은 안트라사이클린계 항암제로서 장기간 사용시 심근독성을 일으켜 심부전을 유발한다. 스타틴은 혈중 콜레스테롤을 낮춰서 심혈관 질환의 발생을 줄일 뿐 아니라 일부 암 환자들에서 항암제 독성을 줄이는 효과가 밝혀져 있다. YAP 과 TAZ는 전사 인자로서 세포사멸을 억제하는 능력을 가지고 있으며, 독소루비신 유도 심근 독성 유발 기전에 관여한다. 따라서 본 연구에서는 YAP 과 TAZ가 독소루비신에 의한 심장 독성에 대한 아토르바스타틴의 보호 효과에 관여하는지에 대해 조사했다. H9c2 심근세포에 독소루비신을 처리하면 YAP 이 감소하고 그 하위 시그널인 Park2가 감소하였다. 또한 독소루비신 처리 후 TAZ가 감소하고 그 하위 시그널인 survivin이 감소하였다 반면에, 독소루비신을 처리한 H9c2 심근세포에서 감소되어 있던 YAP, TAZ, Park2 그리고 survivin은 아토르바스타틴에 의해서 mRNA와 단백질 발현이 회복되었다. 그리고 독소루비신에 의해 증가하였던 세포사멸과 이에 관련된 인자들이 아토르바스타틴에 의해서 감소하였다. 그러나 siRNA를 이용하여 YAP/TAZ의 발현을 저하시키면,아토르바스타틴은 독소루비신으로 유도된 세포사멸을 줄이지 못하였다. 또한 독소루비신을 처리한 H9c2 심근 세포에 아토르바스타틴 을 전 처리하여도, siRNA를 이용하여 YAP/TAZ의 발현을 저해 시킨 경우 Park2/survivin의 발현이 회복되지 못하였다. 게다가 siRNA를 이용하여 Park2나 survivin의 발현을 저해 시키면 아토르바스타틴은 독소루비신에 의한 세포사멸을 저해하지 못하였다. 만성 또는 급성 독소루비신 유발 심근병증 동물 모델에서 아토르바스타틴의 경구 투여는 심근 세포 사멸을 감소 시켰으며, 독소루비신으로 인해 저하된 심장기능이 회복되는 것을 cardiac MRI를 통해 확인하였다. 결론적으로 아토르바스타틴은 독소루비신으로 유도된 심장독성을 예방할 수 있었다. 이러한 아토르바스타틴의 심장 보호 작용의 기전으로는 아토르바스타틴이 YAP 및 TAZ의 발현을 증가시키고 그 하 위 신호인 Park2 및 survivin을 활성화시킴으로써 항-세포사멸 효과를 나타냄을 제시한다.open박