Transfection of Mesenchymal Stem Cells with the FGF-2 Gene Improves Their Survival under Hypoxic Conditions

Bone marrow mesenchymal stem cells (MSCs) have shown potential for cardiac repair following myocardial injury, but this approach is limited by their poor viability after transplantation. To reduce cell loss after transplantation, we introduced the fibroblast growth factor-2 (FGF-2) gene ex vivo before transplantation. The isolated MSCs produced colonies with a fibroblast-like morphology in 2 weeks; over 95% expressed CD71, and 28% expressed the cardiomyocyte-specific transcription factor, Nkx2.5, as well as a-skeletal actin, Nkx2.5, and GATA4. In hypoxic culture, the FGF-2-transfected MSCs (FGF-2-MSCs) secreted increased levels of FGF-2 and displayed a threefold increase in viability, as well as increased expression of the anti-apoptotic gene, Bcl2, and reduced DNA laddering. They had functional adrenergic receptors, like cardiomyocytes, and exposure to norepinephrine led to phosphorylation of ERK1/2. Viable cells persisted 4 weeks after implantation of 5.0 ´ 105 FGF-2-MSCs into infarcted myocardia. Expression of cardiac troponin T (CTn T) and a voltage-gated Ca2+ channel (CaV2.1) increased, and new blood vessels formed. These data suggest that genetic modification of MSCs before transplantation could be useful for treating myocardial infarction and end-stage cardiac failure.ope

Corporate transformation through strategic balancing and mechanism building : case study of multinational firms

학위논문(박사)--서울대학교 대학원 :경영학과 경영학 전공,2003.Docto

Assessment of myocardial perfusion in humans using stress intravenous PES

의학과/석사[한글] 허혈성 심장질환은 다양한 원인에 의해 관상동맥의 내경이 감소됨으로써 기저 상태에서의 심근 관류량의 절대적인 감소와, 관류량의 증가가 요구될 때 이를 적절히 증가 시키지 못하여 발생한다. 따라서 허혈성 심장질환을 진단하는데 심근관류측정은 매우 중요한 평가방법이며 치료와 예후를 결정하는데 중요한 변수로 작용한다. 심근관류를 평가하는 방법으로는 고식적인 방법인 thallium-201 이나 Tc-99m sestamibi를 이용한 동위원소 관류스캔과 심근조영 심초음파검사(myocardial contrast echocardiography:MCE)가 있다. 심근관류를 평가한다는 점에서 두 검사방법은 비슷하지만 약간의 차이가 있다. 동위원소 관류스캔은 심근관류 뿐만 아니라 심근세포의 온전성을 평가하여 심근의 생존 여부를 판단할 수 있다. 심근조영 심초음파 검사는 4-2O㎛ 의 다양한 크기의 미세기포를 함유한 조영제를 투여한 후 미세혈관 내 관류 되는 미세기포에 반사되어 산란 되는 초음파의 성질을 이용하여 심근 미세혈관의 온전성을 통해 심근관류를 평가하는 방법이다. 심근조영 심초음파검사는 검사 비용이 적게 들며 방법이 비교적 간단하고 검사 후 즉시 육안적 평가가 가능한 것 외에도 심근의 이면성 영상을 동시에 관찰할 수 있기 때문에 동위원소 관류스캔 보다 공간 분포적 영상(spatial distribution image)에 있어 해상도가 뛰어나므로 관류되는 심근의 부위와 크기를 정확히 평가할 수 있는 장점이 있으나, 미세기포를 관상동맥 내로 직접 투여 하여야 하는 단점이 있었다. 그러나 최근 낮은 확산성과 낮은 용해도의 고분자기체를 이용하여 미세기포의 안정성을 향상 시키고, second harmonic imaging, intermittent triggering, pulse inversion harmonic imaging기법 등 새로운 기술의 개발로 미세기포를 정맥 내 주사 함으로서 단점을 극복 할 수 있게 되었다. 본 연구에서는 흉통을 주소로 내원한 환자 중에서 협심증으로 진단 받은 46명의 환자를 대상으로 하여 perfluorocarbon-exposed sonicated dextrose albumin(PESDA)의 정맥 내 지속적 점적 투여 영상을 이용한 심근조영 심초음파검사의 임상적 유용성을 Tc-99m-sestamibi single-photon emission computed tomography(Tc-99m sestamibi SPECT)와 비교 하였다. 관상동맥 조영술 결과 관상동맥 내경 협착이 70%이상인 경우를 의미 있는 관상동맥 협착증으로 정의하였을 때, 심근조영 심초음파검사의 경우 민감도 70.7%, 특이도 95.8%, 양성예측도 87.8%, 음성예측도 88.5%였으며, Tc-99m sestamibi SPECT의 경우는 민감도 75.6%, 특이도 98.9%, 양성예측도 96.8%, 음성예측도 90.6%였다. 관상동맥 조영술의 해부학적인 협착 정도가 아닌, 관상동맥 관류량을 반영하는 Tc-99m sestamibi SPECT 과 비교하였을 때, 전체 대상환자 46명중 두 검사법 모두에서 관류결손을 보인 경우가 16명, 두 검사법 모두에서 정상소견이 22명으로 진단 일치률은 82.6%(K=0.64) 였다. 전체 138 개 관상동맥 영역(vascular territory)에서는 진단일치률이 86.9%(K=0.63)였으며, 736개 심근 분절별(myocardial segment) 분석에서 는 진단일치률이 86.8%(K=0.55) 였다. 이상의 결과는 관상동맥 협착증에 의한 심근관류결손을 진단하는데 심근조영 초음파검사가 SPECT와 높은 진단일치율을 보이며 유사한 임상적 유용성을 갖는다는 것을 보여주었다. 그러나, 심첨부에서의 미세기포의 파괴, basal lateral wall의 약화현상(attenuation)등에 의한 위양성의 증가가 향후 보안 되어야 할 사항이며, 실제 임상에서 관상동맥 질환의 심근관류 평가에 보편적으로 이용되기 위해서는 미세기포의 투여 방법, 농도, 좀더 안정적인 미세기포의 개발 등 몇 가지 기술적인 문제점과 acoustic power, gain control, dynamic range등 적절한 영상을 얻기 위한 방법에 대한 더 많은 연구가 필요 하겠다. [영문] Objective; The object of this study was to assess the accuracy of dipyridamole stress intravenous( Ⅳ) PESDA myocardial contrast echocardiography(MCE) using Pulse Inversion Harmonic imaging in the detection of perfusion defect in patients with coronary artery disease in comparison with dipyridamole stress Tc-99m sestaMIBl SPECT. Methods: Total 46 patients (29 males, mean age 64 years old, 1 vessel disease: 12 patients, 2 vessel disease: 7 patients, 3 vessel disease: 5 patients, near normal coronary artery: 22 patients) were consecutively enrolled. Patients with prior myocardial infarction were excluded. MCE and Tc-99m sestamibi SPECT were performed at the same day during rest and after 0.56 or 0.84mg/㎏ DP infusion. Continuous Ⅳ infusion of PESDA(2-5mL/min) was administered while obtaining triggered (1:1, 1:3, 1:5) end-systolic apical 2 and 4 chamber and long axis views. Tc-99m sestamibi was injected 3 minutes after dipyridamole. Tc-99m sestamibi SPECT images were obtained one hour later. Coronary angiography was followed one or two days later in all patients. Tc-99m sestamibi SPECT images were matched to the sixteen segments of left ventricle according to ASE for segmental comparison. Both images were analyzed visually. Results : Using coronary angiography as the standard, MCE had an overall sensitivity of 70.7% and a specificity of 95.8%, positive predictve value (PPV) : 87.8%, negative predictive value (NPV):88.5%) for the detection of coronary atherosclerosis(>70%stenosis). Tc-99m sestamibi SPECT showed a sensitivity of 75.6% and a specificity of 98.9%(PPV: 96.8%, NPV: 90.6%). The overall concordance rate between MCE and Tc-99m sestamibi SPECT for the detection of perfusion defects was 86.9% ( Cohen's kappa value 0.63) according to coronary territory and 86.8% (Cohen's kappa value 0.55) according to segmental analysis. Conclusion: Dipyridamole stress Ⅳ PESDA MCE using Pulse Inversion Harmonic Imaging is comparable to Tc-99m sestamibi SPECT in identifying significant coronary stenosis and inducible myocardial ischemia in patients with coronary artery disease. MCE is a promising modality for assessing myocardial perfusion in the patient with suspected coronary artery disease.ope

戰略的 意志와 目標의 共有를 考慮한 學習組織 構築

학위논문(석사)--서울大學校 大學院 :經營學科 經營學專攻,1996.Maste

(The) effects of cell transplantation into the hearts of the rats with a cryoinjury : combined with gene therapy

의학과/박사[한글] 허혈성 심장 질환으로 인한 심근의 괴사는 관상동맥 재관류 요법이나 관상동맥 우회로술로는 손상 받은 조직의 손상 정도를 경감할 수 있으나, 심근세포를 재생할 수는 없다. 따라서 심근세포로 분화하는 줄기 세포(stem cell)를 이식하여 심근세포를 재생하려는 많은 시도가 이루어지고 있다. 본 연구에서는 골수 간엽 줄기세포(bone marrow-derived mesenchymal stem cells; MSCs)를 냉동 손상 받은 심근에 이식하여 이식된 세포의 생존여부를 관찰하고, 성장인자인 섬유아세포 성장인자(fibroblast growth factor-2: FGF-2)를 발현하는 유전자를 삽입한 경우에 이식된 세포의 생존능력과 분화정도에 차이가 있는지 관찰하고자 하였다. 백서의 골수세포를 획득하기 위하여, 백서의 대퇴골과 경골로부터 골수를 채취하고, Percoll gradient 방법으로 단핵구(mononuclear cell)를 분리 배양하면서 plate에 부착하지 않은 조혈모세포는 배제하고, 부착된 세포만 배양하여 골수 간엽 줄기 세포를 얻었으며, FGF-2 유전자를 삽입하고 이식 직전 DAPI처리를 통해 이식할 세포들을 표시하였다. 냉동손상은 액체 질소로 냉각된 cryo-probe을 노출시킨 심장의 좌심실 전벽에 충분한 시간동안 접촉시켜 괴사를 유도하였다. 냉동손상을 입은 부위의 주변부에 골수 간엽 줄기 세포를 이식하였으며, 이식한지 4주 뒤에 심장을 적출하여 이식한 세포의 생존 여부와 특성을 조사하였다. 연구 결과, DAPI-표지된 이식세포들이 이식부위에 생존하고 있음이 관찰되었고, 동일한 microscopic field에서 DAPI와 troponin T가 양성으로 염색되었으며, 심근세포에 특이적인 voltage-gated Ca2+ channel (Cav2.1)이 양성으로 염색되었다. 배양중인 골수 간엽 줄기 세포에서 atrial natriuretic peptide(ANP)와 brain natriuretic peptide(BNP) 유전자가 모두 발현되었고, DAPI-표지된 이식된 세포에서 myosin heavy chain과 α-skeletal actin 유전자의 발현 및 심근세포 특이적 전사 인자인 Nkx2.5와 GATA4 유전자의 발현이 관찰되어 이식된 세포가 분화된 심근 세포의 표현형을 보인다는 것을 알 수 있었다. 또한, FGF-2 유전자를 삽입한 경우 배양액으로 분비되는 FGF-2의 양이 대조군에 비해 증가함을 확인하였으며, troponin T와 voltage-gated Ca2+ channel의 발현이 대조군에 비해 증가되었고, 이식된 골수 간엽 줄기 세포의 생존율도 대조군에 비해 증가되었다. 결론적으로 본 연구에서 냉동 손상된 심근의 변연부에 골수 간엽 줄기세포를 이식할 경우 이식된 줄기 세포가 생존하며, 심근세포로 분화될 수 있는 가능성을 제시하였고, FGF-2 유전자 삽입은 이식된 세포의 생존율과 심근세포로의 분화를 향상시켰다. [영문]Loss of cardiomyocytes in the myocardial infarction leads to regional contractile dysfunction, and necrotized cardiomyocytes in infarcted ventricular tissues are progressively replaced by fibroblasts to form scar tissue. To restore dead myocardium, we have isolated bone marrow mesenchymal stem cells(MSCs) in vitro and the FGF-2 gene were transfected into the isolated cells to enhance cell survival and differentiation after implantation. In in vitro culture, the isolated stem cells formed the colonies with fibroblast-like morphology after 2-week cultivation. They expressed atrial natriuretic peptide, brain natriuretic peptide, isoforms of contractile protein genes, such as myosin heavy chain, and α-actin, and cardiomyocyte-specific transcription factors, Nkx 2.5/Csx and GATA4. For the promotion of survival of implanted MSCs, FGF-2 gene was transfected into the MSCs by LIPOFECTAMIN PLUSTM reagent. The level of secreted FGF-2 into the culture medium was increased in the MSCs containing FGF-2 gene compared with the control MSCs containing lacZ(150¡3/418pg/ml VS. 21¡3/45pg/ml). In in vivo studies, 4 weeks after implantation of the MSCs on the cryo-injured host myocardium, viable cells labeled with 4'',6-diamidino-2-phenylindole was identified in host myocardium. The survival rate of MSCs containing FGF-2 gene was increa- sed at the ratio of 60%, compared with the control MSCs containing lacZ and the expression levels of troponin T and voltage-gated Ca2+ channel were increased in the MSCs containing FGF-2 gene compared with the control. Our studies indicated that locally delivered MSCs containing FGF-2 gene can reconstitute dead myocardium effectively.ope

서투인1과 서투인3은 갑상선암들에서 유전독성물질에 대한 저항성에 기여한다

Dept. of Medicine/박사Introduction: Sirtuin is NAD+ dependent deacetylase, which plays great roles on gene silencing, DNA repair and apoptosis. The aim of study is to investigate whether the genotoxic stress such as etoposide treatment is able to induce SIRT1 and SIRT3 in thyroid cancer cell lines and to determine the effect of SIRT1 and SIRT3 induction on programmed cell death provoked by etoposide treatment and to determine the change of the apoptosis related genes ( p53, p21, Bcl-xL and Bax ).Materials and Methods: Protein expression, apoptosis rate, cell viability and mRNA expression of SIRT1, SIRT3 and apoptosis related genes ( Bax, Bcl-xL, p53, p21) were assessed in three thyroid cancer cell lines ( TPC-1, FTC-133 and FRO ) treated with 200 μM etoposide.Results: In western blot, protein induction of SIRT1 and SIRT3 was increased in TPC-1 and decreased in FTC-133 and FRO cell lines. FTC-133 and FRO cells showed significantly increased levels of apoptosis compared with TPC-1 cell via FACS analysis. In RT-PCR, only p21 expression was elevated in TPC-1 and Bax was highly elevated in FTC-133. In FRO, expression of all apoptosis related genes were decreased. Conclusion: The SIRT1 and SIRT3 inducibility by etoposide was also different in thyroid cancer cell lines. The ability to survive under the genotoxic stress was observed accordingly with SIRT1 and SIRT3 inducibility. In addition, SIRT1-Foxp3 signal might be involved in generation of resistance against genotoxic stress. Finally, the emerging role of p21 under genotoxic stress should be addressed in future studies to develop new drug target of thyroid cancer. SIRT1 and SIRT3 might confer the prerequisite resistance to genotoxic drug induced apoptosis.ope

Soluble tumor necrosis factor-related apoptosis-inducing ligand after percutaneous coronary intervention: A potential biomarker for vascular remodeling

[No abstract available

Decreased insulin sensitivity is associated with the extent of coronary artery disease in patients with angina

Background: Insulin resistance has been proposed as an important risk factor in the development of atherosclerosis. Aim: To evaluate the association of insulin resistance and coronary atherosclerosis, we investigated the correlation between insulin sensitivity and the degree of coronary stenosis in patients with angina pectoris. Methods: The study population consisted of 74 subjects with angina (54 men and 20 women) aged from 31 to 73 years. Coronary angiograms were evaluated by three semiquantitative scoring systems (vessel score, stenosis score and extent score) to estimate the extent of focal and diffuse coronary artery disease (CAD). Insulin sensitivity (KITT) was determined by the insulin tolerance test. Results: There were significant correlations existed between KITT and all three coronary scores. Multivariate analysis revealed significant and independent correlations of all three coronary scores with KITT (vessel score: β = -0.349, p = 0.004; stenosis score: β = -0.487, p < 0.001; extent score: β = -0.481, p < 0.001), even in patients without diabetes mellitus (vessel score: β = -0.387, p = 0.008; stenosis score: β = -0.469, p < 0.001; extent score: β = -0.559, p < 0.001). KITT was significantly lower in patient with diffuse CAD than without diffuse CAD (2.13 ± 0.66 vs. 2.57 ± 0.79%/min, p < 0.05). However, KITT was not different between patients with and without focal CAD. Conclusions: Insulin sensitivity has statistically significant and independent associations with the extent of coronary stenosis. These results suggest that insulin resistance may play a major role in the development of diffuse coronary artery stenosis. © 2004 Blackwell Publishing Ltd

Efficacy and safety of co-administered telmisartan/amlodipine and rosuvastatin in subjects with hypertension and dyslipidemia

Single risk factors, such as hypertension and dyslipidemia, can combine to exacerbate the development and severity of cardiovascular disease. Treatment goals may be more effectively achieved if multiple disease factors are targeted with combination treatment. We enrolled 202 patients who were randomly divided into the following three groups: telmisartan/amlodipine 80/5 mg + rosuvastatin 20 mg, telmisartan 80 mg + rosuvastatin 20 mg, and telmisartan/amlodipine 80/5 mg. The primary efficacy variables were changes from baseline in mean sitting systolic blood pressure (MSSBP) between telmisartan/amlodipine 80/5 mg + rosuvastatin 20 mg and telmisartan 80 mg + rosuvastatin 20 mg at 8 weeks, and the percent changes from baseline in low-density lipoprotein (LDL) cholesterol between telmisartan/amlodipine 80/5 mg + rosuvastatin 20 mg and telmisartan/amlodipine 80/5 mg at 8 weeks. The secondary efficacy variables were changes in MSSBP, mean sitting diastolic blood pressure (MSDBP), LDL cholesterol and other lipid levels at 4 weeks and 8 weeks, as well as observed adverse events during follow-up. There were no significant differences between the three groups in demographic characteristics and no significant difference among the three groups in terms of baseline characteristics for the validity evaluation variables. The mean overall treatment compliance in the three groups was, respectively, 98.42%, 96.68%, and 98.12%, indicating strong compliance for all patients. The Least-Square (LS) mean (SE) for changes in MSSBP in the two (telmisartan/amlodipine 80/5 mg + rosuvastatin 20 mg and telmisartan 80 mg + rosuvastatin 20 mg) groups were -19.3 (2.68) mm Hg and -6.69 (2.76) mm Hg. The difference between the two groups was significant (-12.60 (2.77) mm Hg, 95% CI -18.06 to -7.14,P < .0001). The LS Mean for the percent changes from baseline in LDL cholesterol in the two (telmisartan/amlodipine 80/5 mg + rosuvastatin 20 mg and telmisartan/amlodipine 80/5 mg) groups were -52.45 (3.23) % and 2.68 (3.15) %. The difference between the two groups was significant (-55.13 (3.20) %, 95% CI -61.45 to -48.81,P < .0001). There were no adverse events leading to discontinuation or death. Combined administration of telmisartan/amlodipine 80/5 mg and rosuvastatin 20 mg for the treatment of hypertensive patients with dyslipidemia significantly reduces blood pressure and improves lipid control. ClinicalTrials.gov identifier: NCT03067688