正常人及肝细胞癌患者肝脏及血浆中丙酮酸激酶同工酶免疫测定

 分别从人肝及鼠肝中高度纯化L型和M_1型丙酮酸激酶(Pyk),制备相应的免抗血清。从抗血清中纯化IgG并水解成F(ab')_2,进而还原成Fab',后者与辣根过氧物酶(HRP)交联成Fab'-HRP,再利用这两种复合物建立了各自的高灵敏夹心ELISA来免疫定量L及M_2型(与鼠M_1-Pyk有交叉免疫)Pyk,结果发现:正常人肝中95％的Pyk为L型,M_2-Pyk仅占5％,而肝细胞癌中L-Pyk降至正常肝的3.5％,M_2-Pyk却增至正常的14.6倍,以致M_2-Pyk占总量的95.5％。免疫组化研究进一步证实定量的结果,正常人肝L-Pyk染色呈强阳性。M_2-Pyk染色较弱,而肝细胞癌恰好相反,L-Pyk染色明显减退,而M_2-Pyk不仅癌组织染色很深,癌周组织也明显深于正常,而且愈近癌巢,染色愈深。测定血浆Pyk,发现正常人L-Pyk占89.9％,M-Pyk(以M_2计)仅占10.1％。肝细胞癌患者血浆L-Pyk略见降低,为正常值的79.6％,p值在0.02～0.05之间,但血浆M型Pyk明显升高至正常的4.59倍,占Pyk总量的39.6％,并证明主要来源于肝癌组织中的M_2,故M_2-Pyk有可能成为肝细胞癌诊断的新指标。

Immunoiogicai Determination of Pyruvate Kinase Isozymes in Liver and Plasma of Normal Human and Hepatocarcinoma Patients

L and M1-type pyruvte kinase(Pyk)were highly purified from human liver and rat muscle respectivly and their antiserums were raised in rabbits. The purified IgCs from both anti-serum were hydrolyzed to F(ab')2s and further reduced to Fab's that were cross-linked with HRP, producing Fab'-HRP conjugates.Using both conjugates, highly sensitive sandwich ELISA were developed to quantitatively determine the L and M2 Pyk(the latter was immuno-cross reactive with M1-Pyk of rat).The results showed that in normal human liver, 95% of total Pyk was in L form,and only 5% was M2-Pyk, whereas in hepatocellular carcinoma (HCC), the liver L-Pyk was decreased to 3.5% and M2-Pyk was increased to 14.6 times of normal value and beca-me 95.5% of total Pyk. Immunohistochemical studies further confirmed the immun-oquantitative results, revealing that in normal liver L-Pyk was strongly, but M2-Pyk was very weakly stained. Conversely, in the liver of HCC patients, the staining of L-PyK was significantly decreased, whereas M2-PyK was strongly stained not only in cancer tissues, but also in the region adjacent to the cancer area.The more the region was closed to cancer area, the higher M2-PyK was found. When the PyK was determined in the plasma, it was found that in normal adults 89.9% PyK in plasma was L-form, only 10.1% was M2-form. In the plasma of HCC patients, L-PyK was slightly decrease to 79.6% of normal value, P value was between 0.02-0.05, but M2-PyK was markly elavated to 4.89 times of normal level and 39.6% of total PyK in plasma.lt was proved that M2-PyK was originated from the cancer tissue.Therefore, M2-PyK may become a new dignostic index of HCC.