胶质瘤中p53基因突变p16基因缺失细胞增殖和凋亡及与临床病理特征的相关性研究
효질류중p53기인돌변p16기인결실세포증식화조망급여림상병리특정적상관성연구
Correlations of p53 Mutation, p16 Deletion,Proliferation (PCNA) and Apoptosis with Clinicopathologic Features of Human Gliomas
  • 万方数据
    济宁医学院学报 제저의학원학보
    JOURNAL OF JINING MEDICAL COLLEGE
    2007年 1期 1-8 ,共8页

    崔文%孔霞%曹慧玲%王旭%高继发%王舟%任启伟%王学春%吴人亮

    최문%공하%조혜령%왕욱%고계발%왕주%임계위%왕학춘%오인량

    胶质瘤%基因突变%基因产物%增殖%凋亡 膠質瘤%基因突變%基因產物%增殖%凋亡
    효질류%기인돌변%기인산물%증식%조망
    Glioma%Gene Mutation%Gene Product%Proliferation%Apoptosis
    目的 探讨P53基因突变、p16基因缺失、肿瘤细胞增殖活性(PCMNA)和凋亡在胶质瘤发生发展中的作用、相互关系及其与临床病理特征的相关性.方法 应用LSAB免疫组化法检测96例不同类型胶质瘤中P53, P16蛋白表达及其肿瘤细胞增殖活性(PCNA);应用非同位素PCR-SSCP技术检测p53基因5-8外显子突变.应用PCR-DNA测序技术检测SSCP阳性标本中p53基因5-8外显子碱基突变谱和氨基酸顺序改变.应用TUNEL技术检测肿瘤细胞中凋亡的改变.结果 结果显示96例不同类型胶质瘤中P53蛋白阳性表达率为41.7%(40/96),其中Ⅱ级肿瘤阳性率为27.3%(13/53),Ⅲ、Ⅳ级肿瘤阳性率分别为62.5%(20/32)、63.6%(7/11),低分级与高分级胶质瘤中P53蛋白阳性表达率有显著性差异 (χ2 = 4.88, P＜0.05).SSCP检测结果显示34/96(35.4%)例胶质瘤出现p53基因异常移动的单链DNA电泳带,主要分布在5,7,8外显子.在40例P53蛋白阳性的病例中有32例呈现该基因的单链构象多态性改变,两种方法检测的符合率基本一致.DNA序列分析显示,17例SSCP有异常电泳带的标本中均存在p53基因突变,而且主要发生在5-8外显子,突变类型多数为点突变或碱基缺失,突变位点多分布在第5外显子130-175号密码子之间,第8外显子270-291密码子之间,突变类型主要为错义突变,且多为单碱基的改变,碱基突变以G→A或A→G最多.p16蛋白的表达缺失率为60.4%(58/96),其中Ⅱ级肿瘤为39.6%(21/53),Ⅲ、Ⅳ级肿瘤为81.2%(26/32)、100%(11/11),在高级别胶质瘤中P16表达缺失率均显著高于低级别的肿瘤.复合表达研究显示,p53蛋白的表达与p16缺失之间存在负相关性(P＜0.05).96例胶质瘤细胞增殖活性及凋亡检测显示,PCNA 标记指数(PCNA Label Index, PCNA LI)随着肿瘤分级的升高而递增,而凋亡指数 (Apoptosis Index, AI)随着肿瘤分级的升高而递减, PCNA LI/AI之比随着肿瘤分级的升高而递增.结论 p53异常表达和突变是胶质瘤中最常见的基因改变,与胶质瘤的组织学分级呈显著正相关;p53突变更常位于5、7、8外显子,突变类型主要为错义突变,且多为单碱基的改变,碱基突变以G→A或A→G最多.p53基因突变可以通过p53蛋白的表达来间接反映,p53基因的异常表达及突变与胶质瘤的发生和进展密切相关.p16的缺失更多见于高级别的胶质瘤,与胶质瘤的发生和进展密切相关.胶质瘤发生、发展与PCNA的高表达和细胞凋亡的抑制密切相关,PCNA LI/AI比PCNA LI或AI单独检测更能反映胶质瘤的病理特征及恶性行为.p53基因突变、p16基因表达的缺失及PCNA LI的增高和细胞凋亡的抑制在胶质瘤发生、发展中可能起着重要协同作用并与肿瘤的分化和异常增生显著相关,而胶质瘤细胞凋亡的发生受p53、p16基因的调控,p53及p16抑癌基因的失活,是胶质瘤逃逸凋亡的重要原因之一.
    목적 탐토P53기인돌변、p16기인결실、종류세포증식활성(PCMNA)화조망재효질류발생발전중적작용、상호관계급기여림상병리특정적상관성.방법 응용LSAB면역조화법검측96례불동류형효질류중P53, P16단백표체급기종류세포증식활성(PCNA);응용비동위소PCR-SSCP기술검측p53기인5-8외현자돌변.응용PCR-DNA측서기술검측SSCP양성표본중p53기인5-8외현자감기돌변보화안기산순서개변.응용TUNEL기술검측종류세포중조망적개변.결과 결과현시96례불동류형효질류중P53단백양성표체솔위41.7%(40/96),기중Ⅱ급종류양성솔위27.3%(13/53),Ⅲ、Ⅳ급종류양성솔분별위62.5%(20/32)、63.6%(7/11),저분급여고분급효질류중P53단백양성표체솔유현저성차이 (χ2 = 4.88, P＜0.05).SSCP검측결과현시34/96(35.4%)례효질류출현p53기인이상이동적단련DNA전영대,주요분포재5,7,8외현자.재40례P53단백양성적병례중유32례정현해기인적단련구상다태성개변,량충방법검측적부합솔기본일치.DNA서렬분석현시,17례SSCP유이상전영대적표본중균존재p53기인돌변,이차주요발생재5-8외현자,돌변류형다수위점돌변혹감기결실,돌변위점다분포재제5외현자130-175호밀마자지간,제8외현자270-291밀마자지간,돌변류형주요위착의돌변,차다위단감기적개변,감기돌변이G→A혹A→G최다.p16단백적표체결실솔위60.4%(58/96),기중Ⅱ급종류위39.6%(21/53),Ⅲ、Ⅳ급종류위81.2%(26/32)、100%(11/11),재고급별효질류중P16표체결실솔균현저고우저급별적종류.복합표체연구현시,p53단백적표체여p16결실지간존재부상관성(P＜0.05).96례효질류세포증식활성급조망검측현시,PCNA 표기지수(PCNA Label Index, PCNA LI)수착종류분급적승고이체증,이조망지수 (Apoptosis Index, AI)수착종류분급적승고이체감, PCNA LI/AI지비수착종류분급적승고이체증.결론 p53이상표체화돌변시효질류중최상견적기인개변,여효질류적조직학분급정현저정상관;p53돌변경상위우5、7、8외현자,돌변류형주요위착의돌변,차다위단감기적개변,감기돌변이G→A혹A→G최다.p53기인돌변가이통과p53단백적표체래간접반영,p53기인적이상표체급돌변여효질류적발생화진전밀절상관.p16적결실경다견우고급별적효질류,여효질류적발생화진전밀절상관.효질류발생、발전여PCNA적고표체화세포조망적억제밀절상관,PCNA LI/AI비PCNA LI혹AI단독검측경능반영효질류적병리특정급악성행위.p53기인돌변、p16기인표체적결실급PCNA LI적증고화세포조망적억제재효질류발생、발전중가능기착중요협동작용병여종류적분화화이상증생현저상관,이효질류세포조망적발생수p53、p16기인적조공,p53급p16억암기인적실활,시효질류도일조망적중요원인지일.
    Objective To investigate P53 gene mutation, abnormal expression of p16 gene, proliferation (PCNA)and apoptosis of tumor cells, as well as their roles and their relationships with each other in tumorigenesis of gliomas. Thses results are contacted with clinicopathological data of gliomas (grade, tumor size, site, age, sex, prognosis).Methods LSAB immunohistochemical staining method were respectively used to detect expression of P53, P16 and the activities of cellular proliferation (PCNA)in 96 cases of gliomas. The base mutations in exon 5～8 of p53 gene were detected by using non-isotopic PCR-SSCP technique. PCR-sequencing was directly used to detect DNA mutations in exon 5～8 of cases with positive SSCP. Apopotosis in 96 cases of gliomas was investigated by using TUNEL technology.Results Results showed that the expression rate of P53 protein was 41.7%(40/96)in glioma samples. Immunoreactivity to the p53 gene product was observed 27.3%(13/53)in grade Ⅱ and 62.5%(20/32)in grade Ⅲ and 63.6% (7/11)grade Ⅳ gliomas respectively. There was a significant difference in expression rate of p53 protein between low grade and high grade gliomas (χ2=4.88, P＜0.05). p53 mutations (aberrant band)were detected in 34 out of 96 cases (35.42%), distributing mainly in exon 5,7,8. Significant electrophoretic mobility shifts were detected in 32 out of 40 gliomas of p53 protein positive. The correlative rate of two methods was 85.0% (32/40). Analysis of DNA sequences indicated that all of p53 mutations in 17 of positive SSCP cases occured in it's exon 5～8, and most of them were p53 gene point mutation or base pair (bp)deletion. The mutations of p53 gene mostly occured in 130～175 codons of exon 5 and 270～291 codons of exon 8. The type of mutation was mainly the missense mutation (mostly the alteration of single bp). G→A or A→G were at most observed in bp mutations.The absence rate of p16 expression was 60.4%(58/96).The absence rate of p16 expression showed 39.6%,81.2%,100% in grade Ⅱ,Ⅲ and Ⅳ gliomas respectively. P16 deletion was singnificantly higher in high grade gliomas than that in low grade gliomas. There was a significant negative correlation between p53 expression and p16 deletion (P＜0.05).The detections of PCNA and apoptosis of 96 cases indicated that PCNA Label Index(PCNA LI)increased progressively along with grade of gliomas steping up, but Apoptosis Index (AI)decreased,PCNA LI/AI also increased progressively.Conclusion The expression and mutation of p53 gene were most commonly genetic alteration in gliomas, which were significantly associated with the grade of tumor differentiation. P53 mutations are mainly located in exon 5, 7 and 8. The type of mutation was mainly the missense mutation ( mostly the alteration of single bp). G→A or A→G were at most observed in bp mutations. p53 protein accumulation would indirectly reflect p53 mutation. The abnormal expression and mutation of P53 gene were significantly related to the tumorigenesis and development of gliomas. p16 deletion were singnificantly higher in high grade than that in low grade gliomas and related to the tumorigenesis and development of gliomas. The high expression of PCNA and the inhibitation of apoptosis were significantly associated with the tumorigenesis and development of gliomas. Detecting PCNA LI/AI was more significant than doing PCNA LI or AI respectively in determing clinicopathlogic features and malignant behaviors. P53 mutation , p16 deletion, PCNA LI increasing and AI decreasing might play an important congenerous roles in the pathogenesis of gliomas and were significantly associated with the differentiation and abnormal proliferation of gliomas. The cellular apoptosis of glioma might be controled by p53 and p16 genes. The inactivation of p53 and p16 might be one of the important causes in escaping apoptosis of gliomas.
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