【摘要】目的探讨p16基因、蛋白失活在小儿急性淋巴细胞白血病（ALL）中的临床意义。方法应用免疫细胞化学方法检测52例初治小儿ALL P16蛋白的表达水平。差异PCR技术检测42例初治小儿ALL p16基因的纯合缺失。结果p16基因总的纯合缺失率38%（16/42），p16基因缺失与T系表型、高白细胞总数（＞25×10⁹/L）、高肿瘤负荷、髓外浸润等影响预后的高危临床特征显著相关。高危型ALL p16基因缺失率（67%）远高于标危型ALL（10%），P＜0.01。在预后好者中，p16基因纯合缺失率为10%（2/19）；在预后差者中，p16基因纯合缺失率为54%（6/11），二组差异有显著性，P＜0.05。P16蛋白表达缺失率为58%(30/52)，P16蛋白缺失者除多见髓外浸润外，与其它临床高危因素及预后无关。结论p16基因、蛋白失活在小儿ALL的发病过程中起重要作用。 p16基因缺失者预后不良，与ALL复发可能有关，可作为评估预后，指导治疗的指标之一。

The expression of P16 protein in childhood acute lymphoblastic leukemia

ZHU YipingLIAO QingkuiLI Qinbo

（Second Affiliated Hospital, West China University of Medical Sciences, Chengdu 610041, China）

【Abstract】ObjectiveThe tumor suppressor gene p16 mapped at 9p21. as a negative regulatory protein of cell cycle plays a role in inhibition of cell division and proliferation. Recently, it has been reported that the homozygous deletion of p16 gene was much higher in childhood acute lymphoblastic leukemia(ALL), especially in T-ALL, than other types of leukemia. But it is difficult to find the abnormalities of the gene regulation sequence with the ordinary molecular biological techniques. Study on protein activity could finally reflect gene function in tumor cells. Thereafter, we detected the expression of P16 protein, and explored the relationship with immunophenotypes in childhood ALL and the significance in the pathogenesis of ALL.MethodsFifty-two recently diagnosed childhood ALL patients aged from 1 year 2 months to 14 years including 31 boys and 21 girls were selected as experiment group. Twenty healthy children aged from 2 months to 9 years, 13 boys and 7 girls, were in the control group. The patients included 10 T-ALL, 9 early pre-B ALL, 14 C-ALL, 4 pre-B ALL, and 4 B-cell ALL. The biotin-streptavidin method was used to detect the P16 protein. Cell samples were pre-incubated with hydrogen peroxide (3%) for 20 min at room temperature, and with non-immune goat serum (10%) for 20 min at 37℃. Primary antibodies (polyclonal antibody, Santa Crue, dilution 1:50) were applied and incubated for 12hr at 4℃. The secondary antibodies were biotinylated goat anti-rabbit IgG (1∶50 dilution)and incubated for 30 min at 37℃, after that the streptavidin-biotinylated-peroxidase (SP) complex (dilution 1:200, 30 min, 37℃) was added. Finally, peroxidase activity was visualized by adding DAB, which formed a yellowish-brown product, counterstain was performed with hematoxylin. The staining was graded as negative (-), weakly positive (+), positive(++) and strongly positive (+++).ResultsThe positive expression rate of P16 protein in childhood ALL patients was 42%, obviously lower than that in normal group (95%, P＜0.001). The lower level expression (+) of P16 protein in normal group was 80%, while in ALL, the ratio of strongly positive (+++) over positive expressions was 73% (16/22), the ratio of weak positive (+) over positive expression was 5% (1/22). The deletion of P16 protein expression was found in 8/10 of T-lineage ALL (80%) and in 18/31 of B-lineage ALL (58%) patients, respectively (P＞0.05). In the subtype of B-lineage ALL, the deletion of P16 protein expression occurred in 3/9 of early pre-B ALL (33%), 7/14 of C-ALL (50%), 4/4 of pre-B ALL (100%) and 4/4 of B-cell ALL (100%) . The degree of P16 protein expression in pre-B ALL and B-cell was lower than that in pre-B ALL and C-ALL by Ridit analysis. The strong positive expression of P16 protein was revealed in 77% of B-lineage ALL.ConclusionThe P16 inactivation could suppress the function of G1/S checkpoint in cell cycle, which plays a very important role in the leukemogenesis and progression of leukemia. The enhanced expression of P16 protein may also be related to the leukemogenesis in some groups of B-lineage ALL by via inhibition of apotosis; in contrast, P16 inactivation which results in the higher proliferative activity may be associated closely with the development of cancer in T-lineage ALL.

【Key words】Leukemia, lympholcytic, acute;Genes p16; protein P16;Gene deletion

p16基因是1994年克隆和定位于染色体9p21的抑癌基因，野生型P16蛋白在细胞周期调控途径中起抑制作用^［1］。早在p16基因发现以前，人们已注意到急性淋巴细胞白血病（ALL）染色体9p21的畸变与某些临床高危因素有关。由于p16基因是9p21异常的靶基因之一，其改变与临床表现及预后的关系已引起广泛关注。但因样本量少，随访期短，各家研究结论不一，国内未见报道。我们在检测了42例小儿ALL p16基因的纯合缺失与52例P16蛋白表达状况的基础上，分析了p16基因、蛋白失活与临床高危特征及预后的关系。

对象和方法

一、 对象

1997年5月～1999年1月在本院儿科门诊和病房收治的初治ALL患儿。P16蛋白检测者52例，男31例，女21例；年龄1.2～14岁，中位年龄6.5岁。复发病例3例，均为男性，年龄7～9岁。p16基因检测者42例，男27例，女15例；年龄2～14岁，中位年龄6岁。经FAB形态学确诊。对44例作了免疫学分型，其中T-ALL 10例，早期前B型ALL (early pre B-ALL) 9例 ，普通型ALL(C-ALL) 14 例，前B型ALL (PreB-ALL) 4例，B-ALL 4例，杂合性白血病3例(T、B双表型1例，淋粒双表型2例)。对照组取非白血病且骨髓象正常者骨髓20份。

二、方法

1. P16蛋白测定：采用常规SP免疫组化染色法。标本置3%H₂O₂室温处理20 min，依次加10%羊血清，I抗（兔抗人P16多克隆抗体，1∶50稀释，Santa Cruz产品），生物素标记II抗（1∶200稀释）及SP复合物（1∶200稀释），滴加DAB显色液，苏木素复染。以淋巴瘤阳性片为阳性对照，以PBS液代替I抗为阴性对照。阳性染色呈棕黄色细颗粒状，分布于细胞核或细胞浆上。油镜下计数至少500个白血病细胞， 阳性细胞数＜5%为（-）；5%～25%为（+）；＞25%～50%为（++）；＞50%为强阳性（+++）。

2. p16基因分析: (1) 提取DNA：酚-氯仿法。用TE液溶解，调整浓度为0.1 μg/μl。(2) 差异PCR检测p16基因缺失：P16目的基因与β-珠蛋白内参照基因在同一试管内扩增，通过图像分析，得出二者的比率，判断结果。

引物(美国生命技术公司生产)^［2］：外显子1 上游引物：5′-GGGAGCAGCATGGAGCCG-3′，下游引物： 5′-AGTCGCCCGCCATCCCCT-3′，扩增片段长度为204 bp；外显子2 上游引物： 5′-AGGTCATGATGATGG-GCAG-3′，下游引物 5′-TCTGAGCTTTGGAAGCTCT-3′，扩增片段长度为320 bp； β-珠蛋白基因上游引物：5′-GCACTGACCTCCCACATTC-3′， 下游引物：5′-TATC-ATGCCTCTTTGCACCATTC-3′ 扩增片段长度为527 bp。

操作： 在25 μl反应体系中加入P16外显子1或外显子2，引物各0.5 μmol/L，β-珠蛋白引物各0.3 μmol/L，10×dNTP 250 μmol/L，KCl 50 mmol/L，MgCl₂ 1.6 mmol/L，Taq酶1.5U，模板DNA 100 ng。反应条件：95℃预变性5 min，95℃、1 min；60℃ 45 s （外显子1），58℃ 45 s （外显子2）；72℃ 45 s，循环30次；72℃延伸10 min。电泳：取PCR扩增产物10 μl，在1.5%琼脂糖凝胶上电泳，80 V、30 min，缓冲液0.5×TBE。EB染色，凝胶图像分析仪照像并分析。阳性对照：正常小儿外周血单个核细胞。阴性对照：含p16基因纯合缺失的K562细胞株。结果判定：计算P16/β-珠蛋白基因扩增量测定值（峰面积）比值。无p16基因扩增带或P16/β-珠蛋白比值＜20%（低于该患儿标本正常细胞百分数），确定为p16基因纯合缺失^［2］。

三、临床资料

1. 临床分型：根据发病年龄、临床特点及实验室检查结果分为高危和标危两型。总积分＜3分者为标危型，≥3分者为高危型。

2. 髓外浸润：有如下之一者为阳性：(1)肝和(或)脾肿大＞5 cm；(2)中枢神经系统白血病；(3)纵膈肿块；(4)睾丸白血病。

3. 白血病细胞负荷危险因素评分（risk factor RF）^［3］：RF=0.2×log（外周血白血病细<