CAJ | 학술논문

目的：研究原矛头蝮蛇毒(PMV)及其组分作用于血液循环系统的部分生物学活性,进一步探讨其毒性机制。方法采用 Sephadex G-75凝胶层析方法分离不同分子质量范围的蛋白组分 FⅠ, FⅡ和FⅢ。贫血小板血浆调节富血小板血浆使血小板为3×1011 L-1,血小板悬液分别与 PMV, FⅠ, FⅡ和 FⅢ0.03 g??L-1预孵育5 min,血小板聚集仪测定 PMV 及其组分诱导血小板聚集活性；PMV, FⅠ, FⅡ和 FⅢ0.05 g??L-1分别与纤溶酶原0.1 U??L-1预孵育10 min,采用单一时间点测定和酶动力学测定 PMV 及其组分酶切发色底物S-2251的作用；将大鼠血浆与 PMV, FⅠ, FⅡ和 FⅢ1.0 g??L-1分别孵育5和30 min,测定凝血酶时间(TT)、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和纤维蛋白原(FlB)水平；PMV, FⅠ, FⅡ和 FⅢ10,50和250 mg??L-1处理微血管内皮细胞24 h,倒置相差显微镜观察细胞形态,MTT 法检测细胞存活；PMV, FⅠ, FⅡ和 FⅢ0.1 g??L-1与含卵磷脂和不含卵磷脂的豚鼠红细胞悬液分别孵育不同时间,计算溶血率。结果与对照组相比,PMV 和高分子质量蛋白组分 FⅠ(>71 ku)诱导血小板聚集率显著升高〔(61.0±5.8)%和(56.9±5.9)% vs(12.4±4.1)%〕(P<0.01)。酶切发色底物 S-2251结果显示,PMV 与中分子质量组分 FⅡ(18～37 ku)具有酶切发色底物的作用(P<0.01)。 PMV 和 FⅠ可引起血浆凝固。与对照组相比,FⅡ组分和低分子质量蛋白组分 FⅢ(<10 ku)明显使 TT, APTT 和 PT 的时间延长(P<0.01)。PMV, FⅠ及 FⅡ导致内皮细胞解离悬浮,与对照组相比,细胞存活率下降,分别为(56.8±3.6)%,(71.6±3.8)%和(58.2±5.5)%。在无卵磷脂条件下,PMV 和 FⅡ可缓慢地引起红细胞轻度溶血；在卵磷脂参与下, PMV 和FⅡ引起豚鼠红细胞急剧溶血,与正常对照组相比,在0.5 min 内溶血率从(17.7±1.0)%分别升高至(81.0±4.0)%和(81.0±1.0)%(P<0.01)。结论 PMV 在体外表现出多方面的血循毒性质的活性,不同分子质量蛋白组分活性机制及作用不同。目的：研究原矛頭蝮蛇毒(PMV)及其組分作用于血液循環繫統的部分生物學活性,進一步探討其毒性機製。方法採用 Sephadex G-75凝膠層析方法分離不同分子質量範圍的蛋白組分 FⅠ, FⅡ和FⅢ。貧血小闆血漿調節富血小闆血漿使血小闆為3×1011 L-1,血小闆懸液分彆與 PMV, FⅠ, FⅡ和 FⅢ0.03 g??L-1預孵育5 min,血小闆聚集儀測定 PMV 及其組分誘導血小闆聚集活性；PMV, FⅠ, FⅡ和 FⅢ0.05 g??L-1分彆與纖溶酶原0.1 U??L-1預孵育10 min,採用單一時間點測定和酶動力學測定 PMV 及其組分酶切髮色底物S-2251的作用；將大鼠血漿與 PMV, FⅠ, FⅡ和 FⅢ1.0 g??L-1分彆孵育5和30 min,測定凝血酶時間(TT)、活化部分凝血活酶時間(APTT)、凝血酶原時間(PT)和纖維蛋白原(FlB)水平；PMV, FⅠ, FⅡ和 FⅢ10,50和250 mg??L-1處理微血管內皮細胞24 h,倒置相差顯微鏡觀察細胞形態,MTT 法檢測細胞存活；PMV, FⅠ, FⅡ和 FⅢ0.1 g??L-1與含卵燐脂和不含卵燐脂的豚鼠紅細胞懸液分彆孵育不同時間,計算溶血率。結果與對照組相比,PMV 和高分子質量蛋白組分 FⅠ(>71 ku)誘導血小闆聚集率顯著升高〔(61.0±5.8)%和(56.9±5.9)% vs(12.4±4.1)%〕(P<0.01)。酶切髮色底物 S-2251結果顯示,PMV 與中分子質量組分 FⅡ(18～37 ku)具有酶切髮色底物的作用(P<0.01)。 PMV 和 FⅠ可引起血漿凝固。與對照組相比,FⅡ組分和低分子質量蛋白組分 FⅢ(<10 ku)明顯使 TT, APTT 和 PT 的時間延長(P<0.01)。PMV, FⅠ及 FⅡ導緻內皮細胞解離懸浮,與對照組相比,細胞存活率下降,分彆為(56.8±3.6)%,(71.6±3.8)%和(58.2±5.5)%。在無卵燐脂條件下,PMV 和 FⅡ可緩慢地引起紅細胞輕度溶血；在卵燐脂參與下, PMV 和FⅡ引起豚鼠紅細胞急劇溶血,與正常對照組相比,在0.5 min 內溶血率從(17.7±1.0)%分彆升高至(81.0±4.0)%和(81.0±1.0)%(P<0.01)。結論 PMV 在體外錶現齣多方麵的血循毒性質的活性,不同分子質量蛋白組分活性機製及作用不同。
목적：연구원모두복사독(PMV)급기조분작용우혈액순배계통적부분생물학활성,진일보탐토기독성궤제。방법채용 Sephadex G-75응효층석방법분리불동분자질량범위적단백조분 FⅠ, FⅡ화FⅢ。빈혈소판혈장조절부혈소판혈장사혈소판위3×1011 L-1,혈소판현액분별여 PMV, FⅠ, FⅡ화 FⅢ0.03 g??L-1예부육5 min,혈소판취집의측정 PMV 급기조분유도혈소판취집활성；PMV, FⅠ, FⅡ화 FⅢ0.05 g??L-1분별여섬용매원0.1 U??L-1예부육10 min,채용단일시간점측정화매동역학측정 PMV 급기조분매절발색저물S-2251적작용；장대서혈장여 PMV, FⅠ, FⅡ화 FⅢ1.0 g??L-1분별부육5화30 min,측정응혈매시간(TT)、활화부분응혈활매시간(APTT)、응혈매원시간(PT)화섬유단백원(FlB)수평；PMV, FⅠ, FⅡ화 FⅢ10,50화250 mg??L-1처리미혈관내피세포24 h,도치상차현미경관찰세포형태,MTT 법검측세포존활；PMV, FⅠ, FⅡ화 FⅢ0.1 g??L-1여함란린지화불함란린지적돈서홍세포현액분별부육불동시간,계산용혈솔。결과여대조조상비,PMV 화고분자질량단백조분 FⅠ(>71 ku)유도혈소판취집솔현저승고〔(61.0±5.8)%화(56.9±5.9)% vs(12.4±4.1)%〕(P<0.01)。매절발색저물 S-2251결과현시,PMV 여중분자질량조분 FⅡ(18～37 ku)구유매절발색저물적작용(P<0.01)。 PMV 화 FⅠ가인기혈장응고。여대조조상비,FⅡ조분화저분자질량단백조분 FⅢ(<10 ku)명현사 TT, APTT 화 PT 적시간연장(P<0.01)。PMV, FⅠ급 FⅡ도치내피세포해리현부,여대조조상비,세포존활솔하강,분별위(56.8±3.6)%,(71.6±3.8)%화(58.2±5.5)%。재무란린지조건하,PMV 화 FⅡ가완만지인기홍세포경도용혈；재란린지삼여하, PMV 화FⅡ인기돈서홍세포급극용혈,여정상대조조상비,재0.5 min 내용혈솔종(17.7±1.0)%분별승고지(81.0±4.0)%화(81.0±1.0)%(P<0.01)。결론 PMV 재체외표현출다방면적혈순독성질적활성,불동분자질량단백조분활성궤제급작용불동。
OBJECTIVE To investigate the effect of Protobothrops mucrosquamatus venom (PMV) and its fractions on functions of the circulatory system in vitro in order to better understand its toxicity mechanism. METHODS PMV was isolated to three fractions FⅠ, FⅡ and FⅢ with a different molecular mass range by Sephadex G-75 gel filtration chromatography. Platelet rich plasma was adjusted to 3×1011 L-1 by platelet poor plasma. Platelet suspension was incubated with PMV and its fractions 0.03 g.L-1 for 5 min, respectively, and platelet aggregation was determined on an LBY-NJ4 aggregometer. PMV and its fractions 0.05 g.L-1 were preincubated with plasminogen 0.1 U.L-1 for 10 min before chromogenic substrate cleavage activity was measured by endpoint and enzyme kinetics determination. PMV and its fractions 1.0 g.L-1 were incubated with rat plasma for 5 or 30 min, and thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FlB) content were assayed. The microvascular endothelial cells were exposed to PMV and its fractions 10, 50 and 250 mg.L-1 , respectively, for 24 h, while the morphological change was observed using an inverted phase contrast microscope, and the cell viability was determined by MTT method. PMV and its fractions were incubated with guinea pig red blood cell suspension in the presence or absence of lecithin for different time, and hemolysis was measured. RESULTS Compared with normal control, platelet aggregation rate was significantly increased by PMV and FⅠ (>71 ku)〔(12.4±4.1)%,(61.0±5.8)% and (56.9±5.9)%〕(P<0.01). PMV and FⅡ (18-37 ku) significantly hydrolyzed chromogenic substrate S-2251(P<0.01). PMV and FⅠ caused plasma coagulation. Compared with normal control, FⅡ and FⅢ (<10 ku) remarkably prolonged TT, APTT and PT( P<0.01). Morphological observation revealed that PMV, FⅠ and FⅡdetached the adherent cells. Compared with normal control group, PMV, F Ⅰ and F Ⅱ inhibited cell viability, and the survival rate of the cells decreased to (56.8±3.6)%,(71.6±3.8)% and(58.2±5.5)%, respectively. PMV and FⅡ slowly caused slight hemolysis in absence of lecithin. PMV and FⅡ caused significant hemolysis in the presence of lecithin, and the hemolytic rate increased to (81.0±4.0)% and (81.0±1.0)%( P <0.01) in 0.5 min, respectively, compared with (17.7±1.0)% of the control group. CONCLUSION PMV possesses different activities that affect the functions of the circulatory system in vitro, and the fractions play different roles in toxicity mechanisms.