소의 뇌 미세혈관 내피세포를 이용한 생체외 혈액뇌관문 모델의 확립

손동섭(Dong-Suep Sohn),김경용(Kyung-Yong Kim),김대진(Dae-Jin Kim),김기정(Ki-Jeong kim),임인자(In-Ja Lim),김성수(Sung-Su Kim),이원복(Won-Bok Lee),김대중(Dae-Joong Kim),방숭규(Seung-Kyu Bang),이희상(Hee-Sang Lee) 대한체질인류학회 2003 대한체질인류학회지 Vol.16 No.4

소의 뇌 겉질에서 미세혈관 내피세포를 분리, 배양하여 영태학적, 기능적 관찰을 통해 생체외 혈액뇌관문 모델을 확립하였다. 배양을 시작한 후 뇌 미세혈관 내피세포는 방추모양으로 자랐으며 배양 후 6-7일 경에 세포들이 소용돌이치는 형태의 특정적인 단층을 형성하였다. 내피세포사이에는 생체내에서와 같이 치밀이음부가 행성되어 있었으며 내피세포 단층의 전기저항은 배양 후 11일 경까지 증가하다가 그 후에 감소하는 양상을 나타냈다. γ GTP 활성도 (1791.5±397.8nmol/min/mg protein)는 높은 연이었다. 형광면역염색에서 anti-vWF, anti-Zo-lanti-vimentin anti-fibronectin 항체에 대한 반응이 있었으나 별아교세포 희소돌기아교세포, 신경세포에 각각 특정적 A로 반응하는 anti-GFAP, anti-GalC, anti-neurofilament l60/200kD 항체에 대한 면역반응은 없었다. 세포간이 동모델약물의 이동도는 mannitol> sucrose> PEG-4000 순이었으며 세포막 이동모델약물인 progesterone 과 propranolol 의 이동도는 10-5cm/sec 대의 높은 이동도를 나타냈다. 이 연구의 결과로서 다공막에 배양된 소의 뇌 미세혈관 내피세포의 단층이 생체외 혈액뇌관문 모델로 충분히 이용될 수 있으며 향후 약물의 개발이나 혈액뇌관문을 통한 약물의 이동 기전을 연구하는데 유용하게 쓰일 수 있 다고 판단된다. The In Vitro blood-brain barrier (BBB) model was established with bovine brain iniciovessel endothelial cells (BBMECs) The charactenstics of BBMECs were identified by morphological and functional studies BBMECs began to grow as spindle shaped cells and distinctly formed the monolayer of whirling appearance by 6 to 7 days after plating Transendothelial electrical resistance (TEER) of the monolayer increased through 11 days and then started to decrease The Y-GTP activity (1791.5±397.8 nmol/min/mg of protein) and alkaline phosphatase activity (15.0±7.8 μmol/mm/mg of piotein) were high BBMECs in culture were characterized by the binding of anti-vWF anti-Z0-1, anti-vimentin, and anti-fibronectin antibodies They failed to react with anti-GFAP, anti-Gaic, and anti-neuiofilament l60/200kD antibodies markers for astrocyte oligodendrocyte and neuron respectively Decreasing older of the permeability through the BBMEC monolayers of paracellular transprt model drugs was mannitol, suciose, and PEG-4000 The permeability of transcellular transport model drugs, progesterone and propranolol was much higher than that of paracellular transpoit model drugs The BBMECs cultured on porous membrane have been qualified as an In Vitro BBB model and also can be used for the BBB transport study m the drug development and for the BBB transport mechanism of drugs

전립선암세포주에서 Granulocyte Macrophage Colony-Stimulating Factor에 의한 MMP-7, MMP-13의 증가

김태형(Tae Hyoung Kim),명순철(Soon Chul Myung),김영선(Young Sun Kim),강군현(Goon Hyun Kang),임인자(In Ja Lim),방효원(Hyo Weon Bang) 대한비뇨기종양학회 2004 대한비뇨기종양학회지 Vol.2 No.2

이가 양이온이 토끼 위 유문동 근세포의 외향성 포타슘 전류에 미치는 영향

임인자,조성일,방효원,엄대용,이상돈 중앙대학교 의과대학 의과학연구소 1994 中央醫大誌 Vol.19 No.3

Using the whole-cell mode patch-clamp techniques at 20~23℃, membrane currents were recorded from single smooth muscle cells enzymatically isolated from the rabbit gastric antrum. Outward currents were activated with a threshold around-30 mV and carried by K^+, which showed apparent outward rectification. The outward current was potentiated by high (4 mM) calcium, but reduced by EGTA and cadmium in bath solution. The outward current was potentiated by caffeine above -10 mV by test pulses, however, not changed below -10 mV. External TEA(2~4 mM) inhibited the outward current in a concentration-dependent manner. The outward current was slightly increased by 4-AP(5 mM) above -10 mV by test pulse but decreased below -10 mV in normal Tyrode solution. In the presence of 10 mM TEA to block outward K^+ current, the residual outward current was further blocked by 4-AP on the whole voltage range of test pulses. Outward K^+ current could be divided into two components : (1)a Ca^2+- and voltage-dependent oscillatory current. It was blocked by TEA of low concentration and slightly increased by 4-AP. Delayed rectifier K^+ current was Ca^2+-independent, but voltage-dependent and it was insensitive to TEA(2~4 mM) and could be blocked by 4-AP in the presence of TEA(10 mM) to block Ca^2+ -activated K^+ current. When Mg^2+ was applied extracellularly or into intrapipette solution, outward K^+ currents were decreased in a dose-dependent manner and the degree of block was increased with depolarization. Ca^2+ -activated K^+ current was potentiated by elevation of extracellular calcium but this potentiation had bell-shaped concentration dependency. External barium, which was known to block the K^+ currents, potentiated Ca^2+ -activated K^+ current in normal Tyrode solution. But is blocked the residual outward current in the presence of 10 mM TEA to block Ca^2+ -activated K^+ current. The above results suggested that magnesium might have blocked outward K^+ current by inhibiting the Ca^2+ -activated K^+ current and the calcium-induced calcium release. And calcium increased the Ca2+ -activated K^+ current up to 2~4 mM and then decreased it probably by the calcium channel inactivation in higher concentration. Barium might have been a charge carrier for Ca^2+ -activated K^+ current and blocked only the delayed rectifier K^+ current.

토끼의 위 유문동 근세포의 지연성 외향 포타슘 전류

임인자 건국대학교 1996 學術誌 Vol.40 No.2

To isolate and characterize the delayed rectifier K+ current of the single rabbit gastric antral myocytes, we used the whole cell mode of patch clamp techniques and manipulated the composition of bath and pipette solutions so that the other interfering currents would be minimized. CdCl2(1 mM) in the Ca2+-free Tyrode extracellular solution served to block calcium current and transient outward potassium current and EGTA(10 mM) in the pipette chelated intracellular calcium to inhibit the calcium-dependent activation of Ca2+-activated potassium currents. Test potentials ranged from -30mV to +40 mV were used because the Ca2+-activated potassium current is carried through a voltage-dependent change that can open at high potentials( > 60 mV) even in the absence of intracellular calcium. The Ca2+-activated potassium current is blocked by extracellular TEA in low doses(Beech and Bolton, 1989). So we added the TEA to the bath solution in various concentrations for blocking of Ca2+-activated potassium current. From the current-concentration relationship curve, 2∼ 4 mM TEA to the bath solution was sufficient to block Ca2'-activated potassium current. As we known, the delayed rectifier potassium current is not reduced by TEA of low dose(Beech and Bolton,1989). Thus we used 2∼4 mM TEA to the bath solution in all experiments for isolation of delayed rectifier potassium current only. These isolated currents share many characteristics with delayed rectifier potassium currents of the other tissues. The representative superimposed currents display less fluctation than Ca2+-activated potassium current and voltage-dependent activation in current-voltage curve The predominant charge carrier was the potassium ion because the reverasl potential (-75.1±2.15 mV, n=5) was similar to calculated Ek+(-78 mV). The outward potassium current were not inactivated by long duration pulses(5 sec) and were not changed by calcium addition(4 mM) to the bath solution. Thus these outward potassium currents did not show time-dependent inactivation and calcium-dependent properties. The delayed rectifier potassium current was blocked by low dose 4- AP in many other tissues. And in these experiments the outward potassium currents were reduced by 1 mM 4-AP. The well known specific Ca2+-activated potassium channel blocker, charybdotoxin was examined to block the residual Ca2+-activated potassium current in these outward potassium current. Therefore we concluded that the outward currents recorded from rabbit gastric antral myocytes in these experiments were delayed rectifier K+ currents and they had common characteristics with that of the other tissues. And 2∼4 mM TEA and 1 mM CdCl2 in bath solution and 10 mM EGTA in the pipette is sufficient to isolate the delayed rectifier K+ current with no use of charybdotoxin.

Papaverine이 저분극된 토끼 유두근의 수축력과 활동전압에 미치는 영향

임인자,조성일,엄대용,이상돈 중앙대학교 의과대학 의과학연구소 1991 中央醫大誌 Vol.16 No.3

In Partially deppolarized, isolated papillary muscle from rabbit heart, the effect of papaverine on contractile force and calcium mediated slow action potential was investigated. Results were as follows; 1. Papaverine induced the negative inotropic effect dose-dependently in the majority of preparations, although some preparations showed the opposite namely positive inotropic response. The significant correlation was found between the degree of depression in tension and increase that of in drug concentration. 2. The contractile force depressed by papaverine was restored by calcium(4 mM). 3. In partially depolarized papillary muscle, papaverine reduced slightly both action potential amplitude and upstroke velocity (dV/dt_max). The duration of action potential (duration of 90% repolarization) was also increased by papaverine in a dose-dependent manner. 4. The resting membrane potential was not altered in low concentrations (10^-7∼5X 10^-6M) of papaverine, however, it was significantly decreased in high concentration (10^-5∼5X 10^-5M) of the drug. The slightly increased overshoot potential was also observed in the above at high concentration range. From these results, it is suggested that in papillary muscle of rabbit heart, inhibitory effect of papaverine on the contractility may be attributable to its antagonistic action on the calcium current rather than its inhibitory effect on the phosphodiesterase activity. The local anesthetic property of the drug may also contribute to the papaverine induced decreases in both contractile force and upstroke velocity. The prolongation of action potential duration brought by papaverine appears to be induced not by increase in calcium currents but by a decrease in K^+ conductance.

세포외 pH 변화가 토끼 문정맥의 장력과 Ca2+-activated K+ 전류에 미치는 영향

임인자,조성일 건국대학교 의과학연구소 1996 건국의과학학술지 Vol.5 No.-

We investigated the effects of changes in external pH on tension of smooth muscle and outward currents of single myocytes from the portal vein of the rabbit at room temperature. When external pH altered tension in high-K(??) depolarized small strips of portal vein, increasing external pH(pH 7.4→7.9) increased the tension of the tissue(139.8±18.6%, mean±S.D.) and decreasing external pH(pH 7.4→6.4) decreased it(72.5±8.4%, mean±S.D.). Using whole-cell mode of patch clamp techniques, outward currents elicited by depolarizing voltage steps beyond -20mV recorded. This outward current exhibited properties of outward rectification and oscillation. And this current had no time-dependent decay. The outward currents sensitive to external Ca²(??) and Ca²(??) channel blocker Cd²(??)(2mM) and were blocked by external tetraethylammonium(TEA). But 4-aminopyridine(4-AP) did not reduce the outward current. From these results suggest Ca²(??) activated K(??) current contributed to the outward current. Changes in external pH(pH 6.4∼7.9) strongly affected the amplitude of the Ca²(??) activated K(??) current. An increases of external pH from 7.4 to 7.9 produced an increases the amplitude of the outward current. Acidification to 6.4 reduced the outward current in a ph-dependent manner. The above results suggested that the effects of a change of a external pH on vascular smooth muscle tone are mediated via a change of Ca²(??) activated K(??) currents in the rabbit portal vein.

백서 척수 후근 신경절에 존재하는 Mechanosensitive Two-pore Domain 포다슘 통로의 전기생리학적 특성

임인자,강경태,방효원 중앙대학교 의과대학 의과학연구소 2001 中央醫大誌 Vol.26 No.2

Mechanosensitive two-pore potassium channel families have been newly identified only within the last few years. They are found in different types of cells and species. Three potassium channels named TRAAK, TREK-1 and TREK-2 are activated by applying negative pressure or unsaturated free fatty acids to the cell membrane. We investigated the exstence and electrophysiological characteristics of mechanosenstive two-pore potassium channels in rat dorsale root ganglion neurones(DRGs). TRAAK,TREK-1 AND TREK-2 mRNA ARE EXPRESSED IN drg neurones DRG neurones as judged by reverse transcriptase-polymerase chain reaction. The expression level of TREK-2 mRNA is 30% compare to that of TRAAK and TREK-1. On the electrophysiological recording, however, the most common type of the currets in DRG neurones were activated by membrane stretch. The mechanosensitive pot assium channels of DRG neurones were also activated by arachidonic acid and alkalic pH. These results show that mechanosensitive potassium channels-TRAAK, TREK-1 and TREK-2 are expressed in DRG neurnes and they may exist as more complicated forms than the cloned potassium channels.

토끼의 위 유문동 평활근 포타슘이온통로의 활성조절에 미치는 에스트로젠의 효과

임인자,양수진 중앙대학교 의과대학 의과학연구소 2001 中央醫大誌 Vol.26 No.2

Large conductance Ca^2+-activated K^+(maxi-K) channel was studied using insideout patches of smooth muscle cell from gastric antrum of rabbit. Single smooth muscle cell were obtained through proteolytic enzyme digestion(collagenase)and patch clamp technique was used. The maxi-K channel showed a single channel conductance of about 180 pS in physiological concentration of K^+ on both sides of patch. The channel activity was dependent on intracellular Ca^2+ concentration change. Estrogen increased the channel activity and the effects were voltage-and intracellular calcium-dependent. From these results, it is strongly suggested that maxi-K channels of rabbit gastric antral myocyte were activated by estrogen through membrance-delimited pathway.

엘리트 운동선수의 성별에 따른 안정시 심전도 특성

임인자,김태호,김찬 대한스포츠의학회 2000 대한스포츠의학회지 Vol.18 No.1

We have investigated resting 12 lead electrocardiogram of 1181 elite athletes with 173 age and sex matched sedentary controls and splitted according to sex to analyze the influence of gender and exercise. We found that males had a significant lower heart rate(p=0.046), prolonged conduction times(PR and QRS intervals), shortened repolarization times(QT intervals), increased maximal Q and T wave amplitudes, indices of right and left hypertrophy, higher maximal ST segment elevation and more ST segment depression than females(p<0.0001). Males had significant higher frequency in junctional rhythm, sinus bradycardia, first degree AV block, early repolarization and tall T wave(p<0.0001). But sinus rhythm, coronary sinus rhythm, sinus arrythmia were more frequent in females(p<0.0001). The differences in electrocardiogram parameters between males and females were also persisted when the athletes and control groups were investigated separately. We think that the gender is highly important factor for most parameters in the resting EKG, so we need the knowledge of the different upper normal limits of parameters according to gender for differential diagnosis between well trained athlete's heart and organic heart diseases.

인간 정관 평활근의 포타슘 이온통로 활성조절에 미치는 프로제스톤의 효과

임인자,함형석 중앙대학교 의과대학 의과학연구소 2001 中央醫大誌 Vol.26 No.2

Large conductance Ca^2+-activated K^+(maxi-K) channels were studied using inside-out patches of smooth muscle cells from human Vas deferens. Single smooth muscle cells were obtained through preteolytic enzyme digestion and the patch clamp technique was used. The maxi-K channle showed a single channel conductance of about 150 pS in physiological concentration of K^+ on both sides of patch. The channel activity was dependent on intracellular Ca^2+ concentration change. Progesterone hardly affected the channel activity. As the application time incresed the channel activity was small increased a little but the effects were not statistically significant. Macroscopic recording results were almost the same as single channel recording results. From these results, it is suggested that maxi-K channel activities of human Vas deferens cells were independent of progesterone action.