[보고서]단백질공학 (protein engineering)을 이용한 발기부전의 근본적인 치료법 개발

류지간

보고서 정보

주관연구기관

인하대학교
InHa University

연구책임자

류지간

참여연구자

김호민

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2018-10

과제시작연도

2017

주관부처

보건복지부
[Ministry of Health & Welfare(MW)(MW)

등록번호

TRKO202100008683

과제고유번호

1465024021

사업명

질환극복기술개발

DB 구축일자

2021-08-21

키워드

발기부전.단백질의약품.구조기반 단백질설계.혈관생성.신경재생.Erectile Dysfunction.Protein Therapeutics.Structure Based Protein Engineering.Therapeutic Angiogenesis.Therapeutic Neural Regeneration.

표 연구 개념도: 단백질공학 (protein engineering)을 이용한 발기부전의 근본적인 치료법 개발
표 국내 발기부전 시장규모
표 HGF protein transfer restores erectile function by increasing endothelial and smooth muscle content in diabetic mice
표 HGF protein transfer decreases cavernous superoxide anion and nitrotyrosine production in diabetic mice
표 HGF protein transfer decreases apoptosis and cavernous oxidized LDL extravasation in cavernous endothelial cells of diabetic mice
표 HGF protein transfer enhances tube formation in primary cultured mouse cavernous endothelial cells (MCECs) exposed to high-glucose condition
표 HGF protein transfer induces proliferation and decreases apoptosis in mouse cavernous endothelial cells (MCECs) exposed to high-glucose condition
표 HGF protein transfer induces neurite outgrowth in cultured mouse pelvic ganglion (MPG) tissue exposed to high-glucose condition
표 Decrease in DKK2 expression under diabetic conditions
표 DKK2 overexpression is resistant to diabetes-induced angiopathy
표 Pericyte-derived DKK2 migrates into endothelial cells and regulates angiogenesis
표 DKK2 overexpression is resistant to diabetes-induced neuropathy
표 DKK2-mediated recovery of erectile function is dependent on the Ang1-Tie2 signaling pathway
표 Schematic diagram of a proposed mechanism in which DKK2 preserves erectile function in diabetic mice
표 DKK2 is mainly expressed in pericytes
표 DKK2 overexpression enhances proliferation of endothelial cells and pericytes
표 Preserved eNOS phosphorylation in DKK2-Tg mice under diabetic conditions
표 Preserved endothelial cell-cell junction proteins in DKK2-Tg mice under diabetic conditions
표 DKK2-Tg mice decrease the extravasation of oxidized-LDL under diabetic conditions
표 Establishment of a direct mixed MCEC-MCP co-culture system
표 DKK2 overexpression promotes migration of endothelial cells and pericytes
표 DKK2 overexpression is resistant to diabetes-induced erectile dysfunction
표 Angiopoietin-1 (Ang1) derived from mouse cavernous pericytes (MCPs) rather than mouse cavernous endothelial cells (MCECs) plays a crucial role in DKK2-mediated angiogenesis and neural regeneration
표 DKK2 restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve. (a, b) Representative ICP responses for the sham operation group or CNI mice stimulated at 1 or 2 weeks after intracavernous injections of PBS (20 μl) or DKK2 protein (days -3 and 0; 6 μg/20 μl). The stimulus interval is indicated by a solid bar. (c-f) Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group. Each bar depicts the mean (± SE) values from n = 6 animals per group. *P < 0.001 vs. sham operation group, #P < 0.05 vs. PBS-treated CNI group. CNI = cavernous nerve injury; DKK2 = dickkopf2
표 DKK2 induces neural regeneration. (a) nNOS (red) and neurofilament (NF, green) staining of penis tissue from sham operation group or CNI mice 1 week after receiving intracavernous injections of PBS (20 μl) or DKK2 protein (days -3 and 0; 6 μg/20 μl). Nuclei were labeled with DAPI (blue). Scale bar = 100 μm. (b-d) Quantitative analysis of cavernous nNOS- and neurofilament-immunopositive area in cavernous tissue or dorsal nerve bundle (DNB) was performed by an image analyzer. Each bar depicts the mean (± SE) values from n = 6 animals per group. *P < 0.05 vs. sham operation group, #P < 0.05 vs. PBS-treated CNI group. (e) Neurofilament (green) and nNOS (red) staining in mouse major pelvic ganglion (MPG) tissue, which were treated with PBS or DKK2 protein (300 ng/ml). Scale bar = 100 μm. (f, g) Quantitative analysis of neurofilament- or nNOS-immunopositive neurite length was performed by an image analyzer. Each bar depicts the mean (± SE) values from n = 4 independent experiments. *P < 0.001 vs. control group. CNI = cavernous nerve injury; DKK2 = dickkopf2; nNOS, neuronal nitric oxide synthase
표 DKK2 increases the expression of neurotrophic factors. (a, b) Representative Western blot for neurotrophic factors (NGF, BDNF, and NT-3) in penis tissue from sham operation group or CNI mice 1 week after receiving intracavernous injections of PBS (20 μl) or DKK2 protein (days -3 and 0; 6 μg/20 μl); and in PC12 cells exposed to lipopolysaccharide (LPS), which were treated with DKK2 protein (300 ng/ml). (c-h) Data are presented as the relative density of each protein compared with that of β-actin or GAPDH. Each bar depicts the mean (± SE) values from n = 4 independent samples. *P < 0.05 vs. sham operation group, #P < 0.05 vs. PBS-treated group. BDNF = brain-derived neurotrophic factor; CNI = cavernous nerve injury; DKK2 = dickkopf2; NGF = nerve growth factor; NT-3 = neurotrophin-3. All the blots/gels were presented by using cropped images (Full-length blots/gels images are presented in supplementary Figure S1)
표 DKK2 restores cavernous endothelial and pericyte content. (a) PECAM-1 (red) and NG2 (green) staining of cavernous tissue from sham operation group or CNI mice 1 week after receiving intracavernous injections of PBS (20 μl) or DKK2 protein (days -3 and 0; 6 μg/20 μl). Nuclei were labeled with DAPI (blue). Scale bar = 100 μm. (b, c) Quantitative analysis of cavernous endothelial cell and pericyte content was performed by an image analyzer. Each bar depicts the mean (± SE) values from n = 6 animals per group. *P < 0.05 vs. sham operation group, #P < 0.05 vs. PBS-treated CNI group. CNI = cavernous nerve injury; DKK2 = dickkopf2
표 DKK2 decreases cavernous endothelial cell apoptosis and enhances endothelial cell proliferation. (a, b) PECAM-1 (red) and cleaved caspase-3 (green) or PECAM-1 (green) and BrdU (red) staining of cavernous tissue from sham operation group or CNI mice 1 week after receiving intracavernous injections of PBS (20 μl) or DKK2 protein (days -3 and 0; 6 μg/20 μl). Nuclei were labeled with DAPI (blue). Scale bar = 100 μm. (c) Number of apoptotic cells in endothelial cells per high-power field (screen magnification ´200). Each bar depicts the mean (± SE) values from n = 6 animals per group. *P < 0.001 vs. sham operation group, #P < 0.001 vs. PBS group. (d) Number of BrdU-immunopositive endothelial cells per high-power field (screen magnification ´400). Each bar depicts the mean (± SE) values from n = 4 animals per group. *P < 0.01 vs. PBS-treated CNI group. CNI = cavernous nerve injury; DKK2 = dickkopf2
표 DKK2 enhances tube formation. (a) Tube formation assay in primary cultured mouse cavernous endothelial cells (MCEC) and pericytes (MCP) mono-culture system or in MCEC-MCP co-culture system, which were treated with PBS or DKK2 protein (300 ng/ml). (b-d) Number of tubes per high-power field. Each bar depicts the mean (± SE) values from n = 4 independent experiments. *P < 0.001 vs. control group. DKK2 = dickkopf2
표 DKK2-Tg mice are resistant to CNI-induced erectile dysfunction. (a) Representative ICP responses for the wild-type (WT) mice receiving sham operation, WT mice receiving CNI, or DKK2-Tg mice receiving CNI. The stimulus interval is indicated by a solid bar. The ICP was measured 2 weeks after CNI. (b, c) Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group. Each bar depicts the mean (± SE) values from n = 6 animals per group. *P < 0.001 vs. WT + sham operation group, #P < 0.05 vs. WT + CNI group. CNI = cavernous nerve injury; DKK2-Tg = dickkopf2-transgenic mice
표 DKK2-Tg mice are resistant to CNI-induced neuropathy and angiopathy. (a) nNOS (red) and neurofilament (NF, green) staining of penis tissue from wild-type (WT) mice receiving sham operation, WT mice receiving CNI, or DKK2-Tg mice receiving CNI. Nuclei were labeled with DAPI (blue). Scale bar = 100 μm. (b) PECAM-1 (red) and NG2 (green) staining of cavernous tissue from each group. (c-g) Quantitative analysis of nNOS- and nerofilament-immunopositive area in dorsal nerve bundle, and cavernous endothelial and pericyte content was performed by an image analyzer. Each bar depicts the mean (± SE) values from n = 4 animals per group. *P < 0.001 vs. WT + sham operation group, #P < 0.001 vs. WT + CNI group. CNI = cavernous nerve injury; DKK2-Tg = dickkopf2-transgenic mice
표 LRG1 expression in diabetic condition. (정상 및 당뇨조건의 음경조직. 음경 혈관내피세포, 음경 혈관 주위세포, 및 인간 혈관내피세포에서 LRG1의 발현차이는 없음. 인간 혈관주위세포에서 당뇨 조건하에서 LRG1의 발현이 감소함)
표 TGFb1 expression in diabetic condition. (정상 및 당뇨조건의 음경조직. 음경 혈관내피세포, 음경 혈관 주위세포, 및 인간 혈관내피세포에서 당뇨 조건하에서 TGFb1의 발현 증가함. 인간 혈관 주위세포에서 TGFb1의 발현차이는 없음)
표 LRG1 protein transfer restores erectile function in diabetic mice. (LRG1 단백질의 음경내 국소 투여가 당뇨성 발기부전모델에서 발기력을 회복시킴)
표 LRG1 protein regenerates endothelial cells and pericytes in diabetic mice. (3차원 영상 분석에서 LRG1 단백질이 당뇨 조건하에서 혈관주위세포 [NG2], 혈관내피세포 [CD31], 평활근세포 [α-actin]의 양을 회복함)
표 LRG1 protein regenerates nerve contents in diabetic mice. (3차원 영상 분석에서 LRG1 단백질이 당뇨 조건하에서 신경세포 [bIII tubulin]의 양을 회복함)
표 LRG1 increases tube formation in endothelial cells. MCECs, mouse cavernous endothelial cells; HUVEC, human umbilical vein endothelial cells. (음경 혈관내피세포 및 인간제대정맥 내피세포에서 LRG1 단백질이 고 글루코스 조건에 의해 저해된 튜브형성을 정상 글루코스 조건 수준으로 회복시킴)
표 LRG1 promotes microvessel sprouts from mouse aortic ring. (대동맥절편을 이용한 실험에서 LRG1 단백질이 고 글루코스 조건에 의해 저해된 내피세포 sprouting을 정상 글루코스 조건 수준으로 회복시킴)
표 LRG1 enhances endothelial cell migration. (음경 혈관내피세포 및 인간제대정맥 내피세포에서 LRG1 단백질이 고 글루코스 조건에 의해 저해된 세포이동을 정상 글루코스 조건 수준으로 회복시킴)
표 LRG1 enhances cavernous endothelial cell proliferation in diabetic mice in vivo and primary cultured mouse cavernous endothelial cells (MCECs) in vitro. (당뇨조건의 음경조직 및 음경 혈관내피세포에서 LRG1 단백질이 혈관내피세포의 증식을 촉진함)
표 LRG1 enhances cavernous pericyte proliferation. (당뇨조건의 음경조직 및 음경 혈관주위세포에서 LRG1 단백질이 혈관주위세포의 증식을 촉진함)
표 LRG1 decreases cavernous endothelial apoptosis. (당뇨조건의 음경조직 및 음경 혈관내피세포에서 LRG1 단백질이 혈관내피세포의 apoptosis을 억제함)
표 LRG1 transfer induces eNOS phosphorylation in diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경에서 eNOS 인산화를 촉진함)
표 LRG1 transfer decreases cavernous endothelial permeability in diabetic mice. (당뇨조건의 음경조직 및 음경 혈관내피세포에서 LRG1 단백질이 당뇨 또는 고 글로코스 조건에 의해 증가된 혈관내피세포 permeability를 정상 수준으로 회복함)
표 LRG1 transfer increases cavernous endothelial tight junction protein in diabetic mice. (당뇨조건의 음경조직에서 LRG1 단백질이 당뇨 의해 감소된 혈관내피세포 tight junction protein의 발현을 증가시킴)
표 LRG1 transfer decreases cavernous endothelial claudin-5 in STZ induced wild type or LRG1 Tg mice retina. (wild type 와 lrg1 tg 마우스 당뇨조건의 망막조직에서 LRG1 단백질이 당뇨 의해 감소된 혈관내피세포 tight junction protein의 발현을 증가시킴)
표 LRG1 transfer up-regulates endoglin expression in cavernous endothelial cells of diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경 혈관내피세포에서 LRG1의 작용에 중요한 역할을 하는 endoglin의 발현을 증가시킴)
표 LRG1 transfer up-regulates endoglin expression in mouse cavernous endothelial cells (MCECs) exposed to high-glucose condition. (음경 혈관내피세포에서 LRG1 단백질이 고 글로코스 조건에 의해 감소된 endoglin 발현을 정상 수준으로 회복함)
표 Inhibition of endoglin with siRNA blocks LRG1-medicated tube formation in mouse cavernous endothelial cells. (음경 혈관내피세포에서 endoglin siRNA를 전처리하였을 때 LRG1 단백질에 의한 튜브형성 촉진이 나타나지 않음. 이는 LRG1의 혈관신생 작용에 있어서 endoglin이 중요한 역할을 함을 의미함)
표 LRG1 transfer induces phosphorylation of Smad1/5 in diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경에서 혈관신생과 관련된 Smad1/5 인산화를 촉진함)
표 LRG1 transfer induces phosphorylation of Smad1/5 in huvec and mcecs. (LRG1 단백질이 음경혈관내피세포 및 인간제대정맥에서 혈관신생과 관련된 Smad1/5 인산화를 촉진함)
표 LRG1 transfer decreases phosphorylation of Smad2 in diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경에서 조직섬유화와 관련된 Smad2 인산화를 억제함)
표 LRG1 transfer restores neurofilament expression in diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경 배부신경에서 neurofilament 발현을 촉진함)
표 LRG1 transfer restores nNOS expression in the dorsal nerve bundle of diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경 배부신경에서 nNOS 발현을 촉진함)
표 LRG1 transfer restores nNOS expression in the corpus cavernosum of diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경 발기조직에서 nNOS 발현을 촉진함)
표 LRG1 transfer induces neurite sprouting in cultured mouse major pelvic ganglion (MPG). (주골반신경절을 이용한 실험에서 LRG1 단백질이 고 글루코오즈 조건에 의해 저해된 neurite sprouting을 정상 글루코오즈 조건 수준으로 회복시킴)
표 LRG1 transfer induces neurite sprouting in cultured mouse Dorsal Root Ganglion (DRG). (후근신경절을 이용한 실험에서 LRG1 단백질이 고 글루코오즈 조건에 의해 저해된 neurite sprouting을 정상 글루코오즈 조건 수준으로 회복시킴)
표 LRG1 transfer restores neurotrophic factors and their receptors in diabetic mice. (LRG1 단백질의 음경 내 국소 투여가 당뇨성 발기부전모델의 음경 발기조직에서 NGF, NT-3, BDNF 등의 신경재생인자와 NGF 수용체인 TrkA의 발현을 촉진함)
표 Inhibition of TGFb signaling blocks LRG1-mediated neurite sprouting in cultured mouse major pelvic ganglion (MPG). (주골반신경절을 이용한 실험에서 고 글루코오즈 조건에서 LRG1 단백질에 의해 촉진된 neurite sprouting이 TGFb 신호전달 억제제 [CB, LDN]에 의해 다시 억제됨)
표 Inhibition of TGFb signaling blocks LRG1-mediated erectile function recovery in diabetic mice. (당뇨성 발기부전모델에서 LRG1 단백질에 의해 촉진된 발기력이 TGFb blocking antibody [aTGFb]에 의해 다시 억제됨)
표 Inhibition of TGFb signaling blocks LRG1-mediated restoration of cavernous endothelial and pericyte contents in diabetic mice. (당뇨성 발기부전모델에서 LRG1 단백질에 의해 회복된 음경 내 혈관내피세포 및 혈관주위세포의 양이 TGFb blocking antibody [aTGFb]에 의해 다시 억제됨)
표 Inhibition of TGFb signaling blocks LRG1-mediated neural regeneration in diabetic mice. (당뇨성 발기부전모델에서 LRG1 단백질에 의해 회복된 음경 내 신경세포의 양이 TGFb blocking antibody [aTGFb]에 의해 다시 억제됨)
표 Inhibition of TGFb signaling blocks LRG1-mediated tube formation in mouse cavernous endothelial cells (MCECs). (마우스 음경혈관내피세포를 이용한 실험에서 고 글루코오즈 조건에서 LRG1 단백질에 의해 촉진된 튜브형성이 TGFb 신호전달 억제제 [aTGFb]에 의해 다시 억제됨)
표 Inhibition of TGFb signaling blocks LRG1-mediated neurite sprouting in cultured mouse major pelvic ganglion (MPG) (주골반신경절을 이용한 실험에서 TGFb 처리한 조건에서 LRG1 단백질에 의해 촉진된 neurite sprouting이 TGFb 신호전달 억제제 [aTGFb]에 의해 다시 억제됨)
표 LRG1 does not affect on the TGF-beta1-induced expression of extracellular matricx protein in human penile fibroblasts. (인간의 음경에서 일차 분리배양된 섬유모세포에서 LRG1 단백질이 TGFb1에 의해 유도된 extracellular matrix protein 발현 증가를 감소시키지 못함)
표 LRG1 restores cavernous endothelial content via ADGRL2 pathway in wild type mice and LRG1 Tg mice. (당뇨마우스에서 LRG1에 의한 발기력 개선효과가 ADGRL2를 억제하였을 때는 나타나지 않음)
표 LRG1-mediated restoration of cavernous endothelial and pericyte contents via ADGRL2 pathway in wild type and LRG1 Tg diabetic mice. (당뇨마우스에서 LRG1에 의한 혈관내피세포, 혈관주위세포의 재생이 ADGRL2를 억제하였을 때는 나타나지 않음)
표 LRG1-mediated neural regeneration via ADGRL2 pathway in wild type and LRG1 Tg diabetic mice. (당뇨마우스에서 LRG1에 의한 신경세포의 ADGRL2를 억제하였을 때는 나타나지 않음)
표 LRG1 induced angiogenesis via ADGRL2 pathway in MCECs. (음경 혈관내피세포에서 LRG1에 의한 튜브형성 촉진이 ADGRL2를 억제하였을 때는 나타나지 않음)
표 LRG1 restores erectile function in mouse model of bilateral cavernous nerve crushing
표 LRG1 increases cavernous endothelial cell and pericyte content in mouse model of bilateral cavernous nerve crushing
표 LRG1 increases neurofilament expression in dorsal nerve bundle of bilateral cavernous nerve crushing mice
표 LRG1 increases nNOS exprssion in dorsal nerve bundle of bilateral cavernous nerve crushing mice
표 LRG1 increases Beta Ⅲ Tubulin in dorsal nerve bundle of bilateral cavernous nerve crushing mice
표 LRG1 enhances nNOS expression in cavernous tissue of bilateral cavernous nerve crushing mice
표 LRG1 restores neurotrofic factors (NT-3, NGF) in PC12 cells exposed to LPS in in vitro
표 Increased proNGF and p75NTR expression under diabetic conditions. (A and B) PECAM-1 (red), βIII-tubulin (red), proNGF (green), or p75NTR (green) immunostaining in mouse cavernous tissue. (C) Representative Western blots for proNGF and p75NTR in mouse cavernous tissue. (D) vWF (red), proNGF (green), or p75NTR (green) immunostaining in human cavernous tissue. Scale bar = 100 μm (corpus cavernosum) or 25 μm (dorsal nerve bundle). (E-H) The proNGF or p75NTR immunopositive area in the corpus cavernosum and dorsal nerve bundle of control or diabetic mice was quantified by ImageJ (N = 6). *P < 0.05 vs. control group. (I and J) Normalized band intensity values for proNGF and p75NTR (N = 5). *P < 0.01 vs. control group. Data in graphs are presented as mean ± SE. DM, diabetes mellitus; VWF, von Willebrand factor
표 ProNGF-Ab restores erectile dysfunction under diabetic conditions. (A and B) Representative intracavernous pressure (ICP) responses for the age-matched control, diabetic mice stimulated at 2 weeks after repeated intracavernous injections of saline (S; days -3 and 0; 20 μL) or proNGF-Ab (pNAb; days -3 and 0; 20 μg/20 μL). The cavernous nerve was stimulated at 1V and 5V. The stimulus interval is indicated by a solid bar. (C and D) Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) stimulated at 1V (N = 6). *P < 0.05 vs. control group; #P < 0.05 vs. saline-treated diabetic group. (E and F) Ratios of mean maximal ICP and total ICP to MSBP stimulated at 5V (N = 6). *P < 0.05 vs. control group; #P < 0.05 vs. saline-treated diabetic group. Data in graphs are presented as mean ± SE. DM, diabetes mellitus; STZ, streptozotocin
표 ProNGF-Ab restores cavernous endothelial cell and pericyte content, and enhances tube formation under diabetic conditions. (A) PECAM-1 (red) or NG2 (green) immunostaining in cavernous tissue from age-matched control and diabetic mice 2 weeks after receiving repeated intracavernous injections of saline (S; days -3 and 0; 20 μL) or proNGF-Ab (pNAb; days -3 and 0; 20 μg/20 μL). Nuclei were labeled with DAPI (blue). Scale bar = 100μm. (B and C) Quantification of cavernous endothelial cell and pericyte contents by ImageJ (N = 6). *P < 0.05 vs. control group; #P < 0.05 vs. saline-treated diabetic group. (D) Tube formation assay in primary cultured mouse cavernous endothelial cells (MCEC) and mouse cavernous pericytes (MCP) exposed to normal-glucose (NG) or high-glucose (HG) condition, which were treated with proNGF-Ab or siRNA for p75NTR (sip75). (E and F) Number of tubes per field (screen magnification ´40) (N = 4). *P < 0.01 vs. NG group; #P < 0.01 vs. HG + PBS group; ##P < 0.05 vs. HG + PBS group. Data in graphs are presented as mean ± SE. DM, diabetes mellitus; STZ, streptozotocin
표 siRNA-mediated knockdown of p75NTR decreases endothelial cell and pericyte apoptosis under diabetic conditions. (A) TUNEL assay in primary cultured mouse cavernous endothelial cells (MCEC) exposed to normal-glucose (NG) or high-glucose (HG) condition, which were treated with siRNA for p75NTR (siP75). Nuclei were labeled with DAPI (blue). Scale bar = 100 μm. (B) Number of apoptotic cells per field (screen magnification ´40) (N = 7). *P < 0.05 vs. NG group; #P < 0.05 vs. HG + PBS group. (C) TUNEL assay in mouse cavernous pericytes (MCP). Scale bar = 100 μm. (D) Number of apoptotic cells per field (screen magnification ´40) (N = 4). *P < 0.05 vs. NG group; #P < 0.05 vs. HG + PBS group. Data in graphs are presented as mean ± SE
표 ProNGF-Ab restores cavernous endothelial cell-cell junction proteins under diabetic conditions. (A) Zonular ocludens-1 (ZO-1; green), occludin (green), or PECAM-1 (red) immunostaining in cavernous tissue from age-matched control and diabetic mice 2 weeks after receiving repeated intracavernous injections of saline (S; days -3 and 0; 20 μL) or proNGF-Ab (pNAb; days -3 and 0; 20 μg/20 μL). Scale bar = 100 μm. (B) Representative Western blots for occludin in mouse cavernous tissue. (C and D) Quantitative analysis of occludin and ZO-1 immunopositive area by ImageJ (N = 6). *P < 0.05 vs. control group; #P < 0.05 vs. saline-treated diabetic group. (E) Normalized band intensity values for occludin (N = 5). *P < 0.05 vs. control group; #P < 0.05 vs. saline-treated diabetic group. Data in graphs are presented as mean ± SE. DM, diabetes mellitus; STZ, streptozotocin
표 ProNGF-Ab induces cavernous eNOS phosphorylation under diabetic conditions. (A) Phospho-eNOS (p-eNOS, green) and PECAM-1 (red) immunostaining in cavernous tissue from age-matched control and diabetic mice 2 weeks after receiving repeated intracavernous injections of saline (S; days -3 and 0; 20 μL) or proNGF-Ab (pNAb; days -3 and 0; 20 μg/20 μL). Scale bar = 100 μm. (B) Representative Western blot for phospho-eNOS and eNOS in each group. Results were similar from three-independent experiments. (C) Quantitative analysis of phospho-eNOS immunopositive area by ImageJ (N = 6). *P < 0.05 vs. control group; #P < 0.05 vs. saline-treated diabetic group. Data in graphs are presented as mean ± SE. STZ, streptozotocin
표 ProNGF-Ab induces neural regeneration under diabetic conditions. (A) nNOS (red) and β III-tubulin (green) staining of penis tissue from age-matched control and diabetic mice 2 weeks after receiving repeated intracavernous injections of saline (S; days -3 and 0; 20 μL) or proNGF-Ab (pNAb; days -3 and 0; 20 μg/20 μL). Scale bar = 100 μm. (B) βIII-tubulin (red) staining of mouse major pelvic ganglion (MPG) tissue exposed to normal glucose (NG) or high glucose (HG) condition, which were treated with proNGF-Ab. Scale bar = 100 μm. (C) Phase image of cultured MPG. Scale bar = 200 μm. (D-G) Quantitative analysis of βIII-tubulin and nNOS immunopositive area in corpus cavernosum or dorsal nerve bundle by ImageJ (N = 6). *P < 0.05 vs. control group; #P < 0.05 vs. saline-treated diabetic group. (H) Quantitative analysis of βIII-tubulin immunopositive neurite length by ImageJ (N = 5). *P < 0.05 vs. NG group; #P < 0.05 vs. HG + PBS group. Data in graphs are presented as mean ± SE. DM, diabetes mellitus; nNOS, neuronal nitric oxide synthase
표 Experimental procedure for preparation of pericyte-derived exosome
표 Pericyte-derived exosome restores erectile function in diabetic mice
표 Pericyte-derived exosome increases cavernous endothelial cell and pericyte cell content in diabetic mice
표 Pericyte-derived exosome increases cavernous smooth muscle cell content in diabetic mice
표 Pericyte-derived exosome restores cavernous endothelial cell-cell junction proteins in diabetic mice
표 Pericyte-derived exosome induces cavernous eNOS phosphorylation in diabetic mice
표 Pericyte-derived exosome induces angiogenesis in primary cultured mouse cavernous endothelial cells and pericytes
표 Pericyte-derived exosome increases nerve cell content in diabetic mice
표 Pericyte-derived exosome induces neurite sprouting in cultured mouse major pelvic ganglion (MPG)
표 Design of multi-paratopic VEGF decoy receptors and their binding affinity to VEGFA, PlGF, and tumor markers
표 Cet-Grab and Tras-Grab inhibit EC migration and tube formation by suppressing VEGF signaling pathways
표 Cet-Grab and Tras-Grab induce cancer cell death by suppressing the EGFR family-mediated proliferative signaling
표 Specific-targeting of Cet-Grab and Tras-Grab toward tumor in the xenograft mouse model
표 Cet-Grab effectively suppress tumor growth in an A431 xenograft mouse model via tumor vessel blockage and EGFR signaling inhibition
표 Lipopolysaccharide transfer cascade to TLR4/MD2 complex via LBP and CD14
표 동물세포 CHO-K1을 활용한 LRG1 단백질 안정발현 시스템 구축 과정
표 LRG1 단백질 pH 정제 결과 (좌), LRG1 단백질 10X 농축 결과 (우)
표 곤충세포와 동물세포에서 생산한 LRG1-Fc 정제 결과와(위) 사이즈 배제 크로마토그래피 결과(아래)
표 LRG1 단백질의 초기 결정화 조건
표 최적화 과정을 통해 얻은 LRG1 단백질 결정(좌) 및 엑스선 회절 사진(우)
표 LRG1 단백질의 결정 데이터 Statistics (좌), LRG1 단백질 3차 분자구조 (우)
표 혈관성 발기부전 조직 RNA sequencing 후 LRG1 처리에 의해 감소한 유전자 heat-map
표 LRG1 처리에 의해 8배 이상 감소한 유전자 (Group1)
표 LRG1 처리에 의해 감소한 유전자 Pathway 분석 (Group1)
표 LRG1 처리에 의해 4배 이상 감소한 유전자 (Group 2)
표 Expi293 세포주 활용 LRG1-YFP 단백질 발현
표 LRG1과 결합하는 세포막 수용체가 많이 존재하는 세포주 탐색 위한 Binding assay
표 LRG1 결합 세포막 수용체 발굴을 위한 LRC-TriCEPS 기술 개요
표 LRG1-TriCEPS의 HEK293T 세포 표면 결합
표 LRG1-TriCEPS 기법을 활용하여 LRG1의 수용체 ADGRL 발굴
표 ADGRL 단백질 도메인 모식도와 도메인별 발현 테스트
표 LRG1과 ADGRL 단백질 결합 Minimal Binding 도메인 탐색
표 발기부전 치료제 특허 현황

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