CN1098462C - 测定人免疫状态抑制基因成分的方法及其试剂盒 - Google Patents

测定人免疫状态抑制基因成分的方法及其试剂盒 Download PDF

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CN1098462C
CN1098462C CN95192634A CN95192634A CN1098462C CN 1098462 C CN1098462 C CN 1098462C CN 95192634 A CN95192634 A CN 95192634A CN 95192634 A CN95192634 A CN 95192634A CN 1098462 C CN1098462 C CN 1098462C
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I·N·高拉维斯考夫
L·Y·戈沙拉娃
K·A·阿里戈汉塔夫
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Abstract

测定人免疫状态抑制基团成分的方法，包括收集外周血，获得单核细胞(MNCs)悬浮液，将所述悬浮液分为两等份，在不含抑制基团激活剂下培养第一份MNCs，在所述抑制基团激活剂(TBG)存在下培养第二份MNCs，从培养基中洗出MNCs，阻断增殖，将由正常供体分离的MNCs(已用植物血凝素刺激)以等量加到各个MNC部分中以获得试验培养物，培养所述试验培养物，进一步评价所述试验培养物中的增殖情况并基于试验培养物中增殖水平之比来测定抑制值。

Description

测定人免疫状态抑制基因成分的方法及其试剂
本发明涉及医学，特别是涉及诊断评价T-抑制基因活性的方法，即涉及测定人免疫状态抑制基因成分的方法，也涉及其实现手段。
来源于胎盘的β-I-糖蛋白是已知的，它是用作造血细胞的生长和增殖刺激因子的滋养性β-I-糖蛋白(TBG)的类似物(US专利5169835，Cl.A61K 35/50，1989)。
然而，该已知的化合物未被用于测定人免疫状态抑制基因成分。
将TBG用于诊断和预示怀孕过程是已知的(见L.G.Sotnikova等，The Role of β-I-glycoprotein Trophoblast in Diagnosing andPrognpsticating Pregnancy，Metodicheskije Recomendatsii，Moscow，1984)。然而所述研究未公开将TBG用于诊断人免疫状态抑制基因成分的可能性。
测定人免疫状态抑制基因成分的方法是已知的，它包括收集外周血，获得用来培养有抑制基因激活剂和无此激活剂的试验培养物的纯淋巴细胞悬浮液，并进一步评价增殖水平(见Dutton R.W.Inhibitory andStimulatory Effects of Concanavalin A on the Response of Mouse Spleencells Suspensions to Antigen.J.Exp.Med.，V.138，P.14961505，1973)。
然而，已知方法需要一种很难得到且昂贵的外来制剂，那就是作为抑制基因激活剂的伴刀豆球蛋白A。
测定人免疫状态抑制基因成分的方法是已知的，它包括收集外周血，获得单核细胞(MNCs)悬浮液，将所述悬浮液分为两等份，在无抑制基因激活剂存在下培养第一份MNCs并在抑制基因激活剂存在下培养第二份MNCs，从培养基中洗出MNCs并阻断增殖，将从正常供体中新鲜分离的MNCs加到上文所述的各份MNCs中，用等份的植物血凝素来刺激而获得试验培养物，培养它们并进一步评价在所述试验培养物中的增殖情况，基于在所述各种试验培养物中增殖水平之比来确定抑制值(见Lien Shov，Stanley A.Schwarts and Robert A Good Suppressor cellActivity after Concanavalin A treatment of Lymphocytes from NormalDonors J.Exp.Med.，1976，v.143，n.5，p.1100-1110)。
然而，所述已知方法也需要很难得到的且昂贵的外来制剂，即伴刀豆球蛋白A。
本发明的主要目的是提供一种使用易于获得的制剂来测定人免疫状态抑制基因活性的低费用方法，所述制剂具有免疫校正活性且不引起过敏反应。
通过将滋养性β-I-糖蛋白(TBG)用作测定人免疫状态抑制基因成分的试剂来完成所述目的，而且本发明的测定人免疫状态抑制基因成分的方法包括以每毫升MNC悬浮剂3-120μg的剂量使用滋养性β-I-糖蛋白，该方法包括收集外周血样，获得单核细胞(MNCs)悬浮液，将所述悬浮液分为两份，在不存在抑制基因激活剂下培养第一份，在含抑制基因激活剂下培养第二份，从培养基中洗出MNCs，阻断增殖，将从正常供体获得的新鲜分离的MNCs加到各个所述MNC部分中，用等份的植物血凝素刺激来产生试验培养物，培养所述培养物，进一步评价所述培养物的增殖情况并基于试验培养物中增殖水平之比来确定抑制值。
由细胞可制备MNC悬浮液，该细胞是从phycoll-urotrust Single-stepgradient(番考尔-乌拉斯特一步梯度)中分离而获得的，MNCs培养进行48小时，用丝裂霉素C处理MNCs来阻断增殖，并可将各个试验培养物培养72小时。
实验和临床试验已经显示TBG作为用来测定人免疫状态抑制基因成分的抑制基因激活剂的新特性。
第一步是由细胞制备MNC悬浮液，该细胞是由phycoll-urotrust Single-stage density gradient(番考尔-乌拉斯特一步密度梯度)(Boyum方法)中分离而获得的。
用静脉穿刺术从病人体中取出外周血，同时放入含肝素溶液的管中(1ml血液＝20-30肝素单位)。然后，将该血样用不含Ca++和Mg++的Hanks溶液以1∶2稀释并用番考尔-乌拉斯特梯度(密度为1.078)分层。
然后在400g下离心30分钟。将界面的MNC分散液放于离心管中，加入不含Ca++和Mg++的Hanks溶液，并进行3次连续的离心(每次10分钟)把细胞从番考尔-乌拉斯特溶液洗出。第三次离心后，将MNC残余物重新悬浮在1ml的199介质中，使用Goryajev照相机记录单核细胞的数量。
第二步是将MNCs分为两等份，第一份在不含抑制基因激活剂下培养，而第二份在抑制基因激活剂存在下培养，并用滋养性β-I-糖蛋白(TBG)作为所述激活剂。
在37℃下，在有#14.5橡皮塞的青霉素容器中培养MNCs。培养基为具有20％IV(AB)型血清和300mg谷氨酰胺的PPMI-1640。
每个容器中含有5×106个细胞，存在于2.0ml完全培养基中。
以3-120μg的剂量将TBG加到培养物中以诱导抑制基因基因。
将细胞培养48小时。然后将MNCs从培养基中洗出，用丝裂霉素C处理(40μg/ml，37℃下，30分钟)来阻断增殖。用含5％IV(AB)血清(冷冻的)的199介质洗涤三次。重新悬浮细胞残余物，记录含核细胞的数目，用0.1％锥虫蓝溶液测定细胞生存力的百分数，并将所得悬浮液稀释到所需浓度。所有操作都要分别针对对照细胞和TBG刺激的细胞进行，用聚硅氧烷盘来洗涤细胞。
下一步是将从正常供体新鲜分离的淋巴细胞(该淋巴细胞是预先用植物血凝素(PHA)刺激的且用作应答的试验细胞)以等量加到对照部分和TBG刺激的淋巴细胞中(0.5×106∶0.5×106细胞/ml)以便获得试验培养物。培养进行72小时。再用N3-胸苷评价试验培养物的增殖情况并通过增殖减小程度来评价抑制情况。用下式计算抑制指数：
Figure C9519263400051
为了评价正常供体的抑制基因成分，使用一组正常供体(100人)来进行已知方法的诊断研究(见Lien shov，stanley A.Schwarts andRobert A.Good.Suppressor Cell Activity after Concanavalin ATreatment of Lymphocytes from Normal Donors.J.Exp.Med.，1976，V.143，n.5，p.1100-1110)，以及本发明方法的诊断研究。基于由此所获得的结果，在伴刀豆球蛋白A诱导下T-抑翻基因活性的正常值为56.8％±4％，而按本发明方法测定的值为63.4％±4.7％。
在下列实施例中提供本发明进一步公开的内容。
实施例1
病人V.，30，来到医院诊断为急性期的“多发性硬化，脑脊髓型”。在伴刀豆球蛋白A诱导下的外周血的T-抑制基因活性为15％，该值是按已知方法来测定的，该方法是用来评价人免疫状态抑制基因成分的。同时采用本发明的方法(TBG诱导下)测定同一病人外周血的T-抑制基因活性，由此获得的值为17％。
实施例2
病人S(女)，40，来医院诊断为急性期的“多发性硬化，脑脊髓型”。伴刀豆球蛋白A诱导下外周血的T-抑制基因活性为16％(按已知方法测定)。同时，使用本发明方法(TBG诱导下)测定同一病人外周血的T-抑制基因活性，由此获得的值为19％。
研究了150名患有多发性硬化和类风湿性关节炎的病人。已经显示由本发明方法测定的T-抑制基因活性与由已知方法获得的值相一致。
所以，已证实TBG作为测定人免疫状态抑制基因成分的试剂的高效性。
本发明测定人免疫状态抑制基因成分的方法的上述优点以及所使用手段的那些优点都能将所述方法广泛用于临床上的科学目的。
也应当注意，使用伴刀豆球蛋白A治疗上述疾病因其毒性而产生相反的作用，而TBG(由人滋养细胞产生)不显示毒性且不引起过敏反应。这一事实能使所述制剂用作药物。

Claims (7)

1.一种测定人免疫状态抑制基因成分的试剂，它包含滋养性β-I-糖蛋白。
2.测定人免疫状态抑制基因成分的方法，它包括：
-收集外周血；
-获得单核细胞悬浮液并将其分为两等份；
-在不含抑制基因激活剂下培养第一份单核细胞，在用作抑制基因激活剂的滋养性β-I-糖蛋白的存在下培养第二份单核细胞；
-从培养基中洗出单核细胞并阻断增殖；
-将从正常供体中新鲜分离的单核细胞以等量加到所述各个单核细胞部分中以获得试验培养物，所述新鲜分离的单核细胞已用植物血凝素刺激，并培养所述试验培养物；
-评价所述试验培养物中的增殖情况并基于各试验培养物中增殖水平之比来测定抑制值。
3.权利要求2的方法，其特征在于以每1ml单核细胞悬浮液3～120μg的剂量使用滋养性β-I-糖蛋白。
4.权利要求2的方法，其特征在于由细胞产生单核细胞悬浮液，该细胞是通过番考尔-乌拉斯特一步梯度分离而获得的。
5.权利要求2的方法，其特征在于单核细胞培养进行48小时。
6.权利要求2的方法，其特征在于用丝裂霉素C处理单核细胞来阻断增殖。
7.权利要求2的方法，其特征在于各试验培养物被培养72小时。
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