총설 : 유전자 치료의 현황과 전망

변종회 한국생화학분자생물학회 (구 한국생화학회) 2005 생화학분자생물학회 소식 Vol.25 No.4

Poster Presentation : Synergistic Effects of Naked DNA Vectors Encoding VEGF and bFGF on Angiogenesis and Blood Flow Recovery in Ischemic Hindlimb

이정선,변종회,김정민,김경리,이재영,장형석,신인순,서원희,김채영,김병문,정지형,장양수,김덕경 한국지질동맥경화학회 ( 구 한국지질학회 ) 2005 한국지질동맥경화학회 추계학술발표논문집 Vol.1 No.-

Monocrotaline-induced pulmonary hypertension correlates with upregulation of connective tissue growth factor expression in the lung

이영삼,변종회,김정아,이정순,김경리,서연림,김정민,장형숙,이재영,신인숙,서원희,전은석,강대희 생화학분자생물학회 2005 Experimental and molecular medicine Vol.37 No.1

Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular sm ooth m uscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 µg/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH. Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular sm ooth m uscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 µg/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.

Cardiac expression profiles of the naked DNA vectors encoding vascular endothelial growth factor and basic fibroblast growth factor

이정선,변종회,김정민,김채영,김병문,정지형,장양수,김덕경 생화학분자생물학회 2005 Experimental and molecular medicine Vol.37 No.5

We investigated expression profiles and biological effects of the naked DNA vectors in the heart. To this end, naked DNA vector was injected into the apex of the beating rat heart after thorocotomy. When the expression of LacZ reporter was examined by reverse transcription-PCR and histochemical staining for β -galactosidase, LacZ expression was detected only in the heart, suggesting limited dissemination of the injected vector in vivo. Even within the heart, LacZ expression was limited to the injection area (apex). Similar observations were made with other transgenes such as VEGF and basic fibroblast growth factor (bFGF), where 77% and 69% of the total transgene exprssion were detected in the heart segments containing the apex. Although VEGF and bFGF expressions were de-tected until 2 weeks after DNA injection, the highest levels of VEGF and bFGF were observed on day 5 and day 1, respectively. The optimal doses of the vectors were 10 µg and 25 µg for the VEGF and bFGF vectors, respectively. Interestingly, injection of bFGF vector led to 50% increase in the level of endogenous murine VEGF expression. Consistent with this finding, the number of vessels that stained positive for α -smooth muscle actin was increased in the bFGF vector-injected heart. These results suggest that simple injection of naked DNA vector may be sufficient to induce significant angio-genesis in the myocardium and that naked DNA gene therapy may be a feasible approach for the treatment of ischemic heart disease.

Improved expression by cytomegalovirus promoter/enhancer and behavior of vascular endothelial growth factor gene after myocardial injection of naked DNA

JinOkJeong,변종회,Eun-SeokJeon,권현철,YoungShinLim,JangwonPark,Seon-JuYeo,YoungJooLee,김선영,김덕경 생화학분자생물학회 2002 Experimental and molecular medicine Vol.34 No.4

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart.

Cardiac Ankyrin Repeat Protein의 과량발현이 혈관내피세포에서 갖는 혈관신생 촉진 효과

공훈영,변종회,Kong, Hoon-Young,Byun, Jong-Hoe 한국미생물학회 2008 미생물학회지 Vol.44 No.4

혈관이 좁아지거나 막혀서 생기는 허혈은 심장, 뇌, 다리와 같은 인체의 여러 장기에 영향을 미친다. 최근 혈관신생 분야에서 많은 진전이 있어 기존 치료법으로 치료가 되지 않는 허혈성 환자들의 치료 가능성에 대한 기대가 많아졌다. 혈관형성은 여러 개의 인자들과 세포들이 관여하는 복잡한 과정이기 때문에, 한 개의 인자보다는 여러 인자들을 병합하는 요법이 점점 많이 시도되고 있는데, 이런 병합요법의 한 예로 전사인자를 전달하는 전략을 생각할 수 있다. 이에 본 연구에서는 cardiac ankyrin repeat protein (CARP)의 유전자를 대상으로 그 혈관신생 능력을 혈관내피세포에서 조사하였다. 아데노바이러스 벡터 내에 human CARP의 cDNA를 클로닝하여 재조합 아데노바이러스를 제조하였으며, 이를 이용한 유전자 전달실험 결과, CARP 유전자 전달 군에서 유의하게 혈관내피세포의 중식과 모세관 구조 형성, 그리고 vascular endothelial growth factor의 발현 등을 증가시킴을 확인하였다. 본 연구 결과는 CARP가 혈관신생 연구의 새로운 목표 유전자로서 그 기능에 대한 많은 연구가 필요함을 뒷받침해준다. Tissue ischemia resulting from the constriction or obstruction of blood vessels leads to an illness that may affect many organs including the heart, brain, and legs. In recent years, considerable progress has been made in the field of therapeutic angiogenesis and the new approaches are expected to cure those "no-option patients" who are unsuited to conventional therapies. Although single angiogenic growth factor may be successful in inducing angiogenesis, combination of multiple growth factors is increasingly sought these days to augment the therapeutic responses. This trend is proper in light of the fact that blood vessel formation is a complex and multi-step process that requires the actions of many different factors. To meet the growing need for functionally significant blood flow recovery in the ischemic tissues, a novel strategy that can provide concerted actions of multiple factors is required. One way to achieve such a goal is to use a transcription factor that can orchestrate the expression of multiple target genes in the ischemic region and thus induce significant level of angiogenesis. Here, a putative transcription factor, cardiac ankyrin repeat protein (CARP), was evaluated in adenoviral vector context for angiogenic activity in human umbilical vein endothelial cells. The results indicated significant increase in proliferation, capillary-like structure formation, and induction of vascular endothelial growth factor, a typical angiogenic gene. Taken together, these results suggest that CARP represents itself as a novel target for therapeutic angiogenesis and warrants further investigation.

Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

강훈영,변종회 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.2

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2’-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2’-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.

Syntheses and Characterizations of Serine and Threonine Capped Water-Dispersible ZnS:Mn Nanocrystals and Comparison Study of Toxicity Effects on the growth of E. coli by the Methionine, Serine, Threonine, and Valine Capped ZnS:Mn Nanocrystals

임은주,박상현,변종회,황청수 대한화학회 2012 Bulletin of the Korean Chemical Society Vol.33 No.5

Water-dispersible ZnS:Mn nanocrystals were synthesized by capping the surface of the nanocrystals with conventional aminoacids ligands: serine and threonine. The aminoacids capped ZnS:Mn nanocrystal powders were characterized by XRD, HR-TEM, EDXS, ICP-AES and FT-IR spectroscopy. The optical properties were also measured by UV/Vis and solution photoluminescence (PL) spectroscopies in aqueous solvents. The solution PL spectra showed broad emission peaks around 600 nm with PL efficiencies of 9.7% (ZnS:Mn-Ser) and 15.4% (ZnS:Mn-Thr) respectively. The measured particle sizes for the aminoacid capped ZnS:Mn nanocrystals by HR-TEM images were about 3.0-4.0 nm, which were also supported by Debye-Scherrer calculations. In addition, cytotoxic effects of four aminoacids capped ZnS:Mn nanocrsystals over the growth of wild type E. coli were investigated. Although toxicity in the form of growth inhibition was observed with all the aminoacids capped ZnS:Mn nanocrystals at higher dose (1 mg/mL), ZnS:Mn-Met and ZnS:Mn-Thr appeared non-toxic at doses less than 100 μg/mL. Low biological toxicities were seen at doses less than 10 μg/ mL for all nanocrystals.

항암제 치료에 있어서 영상 생체표지자로서의 초음파 조영제 영상검사: 파크리탁셀과 이종이식 종양 모델을 이용한 예비연구

이학종,황성일,변종회,공훈영,정현숙,강미라 대한초음파의학회 2011 ULTRASONOGRAPHY Vol.30 No.2

Purpose: We wanted to assess tumor angiogenesis of human prostate cancer cells (PC3) implanted in mice before and after paclitaxel injection via contrast-enhanced ultrasonography (CEUS). Materials and Methods: Twelve mice were injected with human prostate cancer cells (PC3) on the back or hind limbs. The mice were grouped into two groups; one was the paclitaxel treated group (n = 6) and the other was the control group, which was treated with normal saline (n = 6). Before injection of paclitaxel into the peritoneal cavity, baseline CEUS was performed by the administration of 500 μl (1×108microbubbles) of contrast agent. The area under the curve (AUC) up to 50 seconds after contrast injection was derived from the time-intensity curves. After injection of paclitaxel or saline, one week follow up CEUS studies were performed. The changes of the tumor volume and the AUC in both two groups were evaluated. After CEUS, the mice were sacrificed and the microvessel density (MVD) was compared. Results: In the paclitaxel treated group, the AUC from CEUS showed a significant decrease one week after paclitaxel administration (p = 0.03), even though the tumor volume showed no significant changes (p = 0.116). In the control group, there was no significant decrease of the AUC (p = 0.173). Pathologically, there was a significant difference of microvessel density in both groups (p = 0.002). Conclusion: The AUC from the time intensity curve derived from CEUS showed early change in response to the anti-cancer drug treatment in advance of a tumor size response. The findings of CEUS could be an imaging biomarker for assessing the tumor response to anti-cancer drug treatment.

Biological Toxicity Changes of Mercaptoacetic Acid and Mercaptopropionic Acid Upon Coordination onto ZnS:Mn Nanocrystal

공훈영,황청수,변종회 대한화학회 2012 Bulletin of the Korean Chemical Society Vol.33 No.2

Mercaptoacetic acid (MAA) and mercaptopropionic acid (MPA) capped ZnS:Mn nanocrystals were synthesized and their physical characteristics were examined by XRD, HR-TEM, EDXS, and FT-IR spectroscopy. The optical properties of the MPA capped ZnS:Mn nanocrystals dispersed in aqueous solution were also measured by UV/Vis and solution photoluminescence (PL) spectra, which showed a broad emission peak around 598 nm (orange light emissions) with calculated relative PL efficiency of 5.2%. Comparative toxicity evaluation of the uncoordinated ligands, MAA and MPA, with the corresponding ZnS:Mn nanocrystals revealed that the original ligands significantly suppressed the growth of wild type E. coli whereas the ligandcapped nanocrystals did not show significant toxic effects. The reduced cytotoxicity of the conjugated ZnS:Mn nanocrystals was also observed in NIH/3T3 mouse embryonic fibroblasts. These results imply that potential toxicities of the capping ligands can be neutralized on ZnS:Mn surface.