SRBC膜提取物对猪PBMNC第二信使的影响

Study of the intracellular cAMP and Ca2+ of porince peripheral blood mononucleocytes

胰酶水解绵羊红细胞（ＳＲＢＣ）释放膜表面活性蛋白组分Ⅲ（ＴＲＦ－Ⅲ），单独作用可使猪外周血单个核细胞（ＰＢＭＮＣ）胞内ｃＡＭＰ水平升高，以１００μｇ／ｍｌ浓度刺激达最高峰，由对照组０．９４±０．１４ｐｍｏｌ／Ｌ升高到２．７５±０．２５ｐｍｏｌ／Ｌ（Ｐ＜０．０１）。如果ＰＢＭＮＣ事先与腺苷酸环化酶（ＡＣａｓｅ）抑制剂ＬｉC１孵育后再以ＴＲＦ－Ⅲ或ＰＡＨ刺激。ｃＡＭＰ增高受到抑制（Ｐ＜０．０１）；而ＥＤＴＡ－２Ｎａ（一种磷酸二酯酶ＰＤＥ抑制剂）对此ｃＡＭＰ升高无影响。结果提示，此ｃＡＭＰ升高主要是通过活化ＡＣａｓｅ水解ＡＴＰ生成ｃＡＭＰ，而不像是抑制ＰＤＥ减少ｃＡＭＰ降解引起的。ＴＲＦ－Ⅲ诱导猪ＰＢＭＮＣ胞内Ｃａ^(２＋)浓度升高，以１００μｇ／ｍｌ刺激２分钟升高最多，由对照组的２４２±７ｎｍｏｌ／Ｌ升高到３２３±１５ｎｍｏｌ／Ｌ（Ｐ＜０．０１）。以ＥＧ－ＴＡ除去胞外Ｃａ^(２＋)再以ＴＲＦ－Ⅲ或ＰＡＨ刺激，仅观察到小范围［Ｃａ^(２＋)］ｉ升高。看来这一过程包括了刺激胞内Ｃａ^(２＋)释放和胞外Ｃａ^(２＋)内流两种方式。以上结果证明，ＴＲＦ－Ⅲ对淋巴细胞功能影响与细胞内第二信使有关。

In PBMNC , the [cAMP] i levels induced by TRF- Ⅲand PHA was compared in this study. The results showed that both of them increased the [cAMP] i levels and the former reached its peak (2.75±0.25pmol/L ) from baseline level (0.94±0.14pmol/L ) when 100μg/ml TRP-Ⅲ was added. For further study , PBMNC incubated with LiCl , an A Case inhibitor , a significant inhibition of increment of [cAMP]i (P<0.01 ) was observed. Contrary to these results ,EDTA-2Na ,a PDE inhibitor ,incubated with PBMNC was assayed in combination with TRF-Ⅲ or PHA , there was an unsignificant enhancement of TRF-Ⅲ /pHA-induced increment of [cAMP] i detected (P>0.05) . Mainly by activating ACasc ,TRF-Ⅲ. as well as PHA induced an increase of [cAMP] i level. In Ca2+ mobilization experiments ,TRP-Ⅲ alone induced elevation of [Ca2+]i dose-dependently and elevated to its peak level ( 323±15nmol/L ) from baseline level (242±7nmol/L ) when 100μg/ml of TRF-Ⅲ was added. Removal of extracellular Ca2+ with EDTA ,we only observed a small rise in [Ca2+]i after addition of TRF-Ⅲ or pHA . These results indicated that TRF-Ⅲ as well as pHA induced the rise of Ca2+ not only by release of stored Ca2+ but also by influx from extracellular Ca2+ . These and previous results suggest that TRF-Ⅲ contains the activity of E receptor natural ligand .