Fura－2荷载血小板胞内游离钙浓度的连续测定

KINETIC MESUREMENT OF PLATELET [Ca2+] iCONCENTRATION:ESTABLISHMENT OF METHODOLOGY

以Ｃａ２＋荧光指示剂Ｆｕｒａ－２荷载血小板，连续测定血小板胞内游离Ｃａ２＋（［Ｃａ２＋］ｉ）浓度动态变化。在生理胞外Ｃａ２＋浓度时。正常成人血小板［Ｃａ２＋］ｉ静息水平为１０８．３±３．６ｎｍｏｌ／Ｌ（－ｘ±ｓ－ｘｎ＝１５，下同），以凝血酶０．２Ｕ／ｍｌ作诱导，［Ｃａ２＋］ｉ迅速升至峰值７７０．３±２７．３ｎｍｏｌ／Ｌ，其下降幅度在４ｍｉｎ时为４０．９％±１．８％；以ＴＭＢ８４００μｍｏｌ／Ｌ阻滞胞内Ｃａ２＋释放，［Ｃａ２＋］ｉ的峰值（４４３．７±２７．７ｎｍｏｌ／Ｌ）明显下降（Ｐ＜０．００１），而４ｍｉｎ的下降幅度（４７．７％±３．８％）则无明显差异（Ｐ＞０．０５）。在胞外Ｃａ２＋＜１０－８ｍｏｌ／Ｌ时，［Ｃａ２＋］ｉ静息水平７０．２±７．５ｎｍｏｌ／Ｌ，作同样的诱导，［Ｃａ２＋］ｉ的峰值（３２１．２±１７．８ｎｍｏｌ／Ｌ）及下降幅度（９９．２％±３．６％）均有显著变化（ｐ＜０．００１）。结果表明，本文所建立的方法能连续测定血小板［Ｃａ２＋］ｉ的动态变化。

he human platelet were loaded with fura-2 by incubating platelet rich plasma (PRP) at 37℃ for 45 min with 2 μmol/L fura-2 and resuspended to 2× l08/ml in Hepes buffer. Fuores- ence reading were recorded at 37℃ using a Hitachi spectrophotofluorometer (model 850) . In order to record the alteration of fluorescence intensity continuously , the instrument was adapted. Thhe results show the mean value for resting [Ca2+]i from fura-2-loaded mormal human platelets in Hepes buffer was 1 08. 3± 3. 6nmol/L (x±s-x ,n= 1 5 ) .In the presence of 1 mmol/L extracellular Ca￣2+,thrombin (0.2U/ml)induced an influx of extracellular calcium and calcium release from intra-cellular stores,[Ca2+]I increased to 770.3+27.nmol/L(x+sx,n=15),and then declined to a plateou still well above the basal level within 4 min. Calcium releasse wa studied in the presence of 2mmol/L,and then returned rapidly to the basal level within 4 min.After preincubation with 400 mol/L of TMB8,an intracellular calcium blocker,[Ca2+]ireached a peak of 443.7+27.7nmol/L,but did not decline back to the basal level.