摘要
子宫颈癌的发病率在女性生殖系统恶性肿瘤中居首位,是引起女性患者死亡最常见的妇科恶性肿瘤之一。子宫颈癌的发病年龄呈现出愈来愈年轻化趋势,这可能与性生活的过早发生、性伴侣多、性生活不注意清洁卫生等相关。高危型(又称致癌型)人乳头状瘤病毒的持续感染是宫颈癌发生、发展的必要因素。在确定的15种高危型HPV类型中,HPV18和HPV16是最常见的两种,最新的研究揭示,它们与将近70%的宫颈癌相关。细胞周期调控与宫颈恶性肿瘤发生、发展关系的研究近年来成为热点。
P19ARF是新发现的重要的抑癌基因,它来源于细胞周期性蛋白依赖酶抑制剂(cyclin dependent kinase inhibitor CDKI)家族中的INK4a (inhibitor of CDK4),在小鼠胚胎表达为P14ARF,在人类编码同源物是分子量为14KD的P19ARF蛋白,它是INK4a/ARF基因通过变更剪接编码出的与P16Ink4a的阅读框不同的蛋白质。P19ARF是重要的抑癌基因P53的上游基因,能够通过P19ARF-MDM2-P53途径,直接结合MDM2或者发挥核定位功能,减少MDM2对P53的降解,使P53积聚,稳定性升高。其通过非磷酸化的视网膜癌蛋白RB在细胞周期的重要作用使细胞周期于G1-S期发生停滞或直接诱导细胞凋亡,而达到抑制细胞的无限制增殖。
P18Ink4c是同P19ARF一样也属于CDKI。其作用方式主要是通过抑制周期蛋白D与周期蛋白激酶CDK4／6结合,以及P18Ink4c自身与CDK4／6特异性结合起作用,抑制该两种复合物磷酸化酶功能,从而达到抑制磷酸化蛋白RB的活性的作用。非磷酸化的RB能够抑制E2F在E2F-RB复合物中的释放,转录因子E2F不能启动遗传物质的转录,使相关的周期蛋白依赖性激酶失活,限制细胞周期通过检测点,细胞周期停滞。P18Ink4c恰好抑制RB的磷酸化,限制细胞通G1-S期限制点,抑制细胞无限制增殖。
目的
采用逆转录-聚合酶链反应(RT-PCR)方法,研究宫颈癌、宫颈上皮内瘤变(CIN)、正常宫颈组织中人类P19ARFmRNA、P18Ink4cmRNA的表达情况,应用免疫组织化学方法(S-P法)研究两种蛋白的阳性表达情况,探讨该两种因子在宫颈癌发病中的表达及临床意义,为宫颈癌的临床诊断和治疗提供新的思路。
材料和方法
1材料
选取郑州大学第三附属医院2011年1月至2012年1月在门诊行宫颈活检术或者住院手术治疗的子宫颈癌标本47例,CIN20例；同期因子宫腺肌症或子宫肌瘤行全子宫切除的正常子宫颈组织20例。宫颈癌标本按照国际FIGO分期I期21例,II期20例,III期3例,IV期3例；按照病理学分级G1级19例,G2级15例,G3级13例；17例患者有淋巴结转移,30例患者无淋巴结转移；所有标本经病理学证实,均无放疗、化疗及免疫学治疗病史。所有标本于切下后5min取材,严格遵守无菌原则,一部分取材后迅速置于-80℃液氮中保存,另一部分置于10%甲醛固定后进行石蜡包埋,4μm连续切片。
2实验方法
2.1实验方法和主要试剂
采用采用免疫组织化学方法(S-P法)、逆转录-聚合酶链式反应(RT-PCR)方法进行检测。免疫组化SP试剂盒,DAB染色剂均购自北京生物博奥森有限公司。一抗P18Ink4c多克隆抗体购自北京生物博奥森有限公司,P19ARF多克隆抗体购自santa cruz生物试剂有限公司。
2.2免疫组织化学方法
标本经10%甲醛固定,严格按照试剂盒说明书进行操作,用石蜡包埋,以均匀4μm厚度进行切片。P18Ink4c和P19ARF蛋白阳性结果判定标准：在400倍显微镜下随机计数5个视野,根据染色强度及阳性细胞占总细胞数的比例(阳性率)同时评分。根据染色强度进行评分：基本无着色为0分、弱阳性为1分、强阳性为3分,介于二者之间为2分；根据阳性率进行评分：≤5%为0分,6%-25%为1分,26%-50%为2分,>50%为3分。两种评分的乘积判定阳性结果。0-2分为阴性,3-9分为阳性。
2.3RT-PCR
设计引物,组织总RNA提取后,进行聚合酶链反应。PCR扩增产物采用琼脂凝胶电泳,在紫外线投射仪下观察电泳条带,用D-140图像记录分析系统进行分析,目的基因的表达量以目的DNA条带和ACTIN条带的灰度值比值计算。
3统计学方法
采用SPSS17.0统计软件包进行数据处理,采用K-W检验、X2检验、Fisher's确切概率法等。检验标准：a=0.05
结果
1P19ARF mRNA在不同宫颈组织中的表达
P19ARF mRNA在586bp处显示有清晰明亮的条带。在三组中均有表达,在宫颈癌组的表达量为0.098±0.005,明显低于CIN组和正常宫颈组(0.502±0.009,0.915±0.027),表达量依次升高,差异有统计学意义(P<0.05)。
2P18ARF蛋白在不同宫颈组织中的表达
P19ARF蛋白在正常宫颈组织、CIN、宫颈癌中主要表达于胞浆和胞膜,随病变程度的加重,染色强度逐渐减轻,阳性细胞比例依次降低,其阳性率分别为80%、45%、21.28%,差异有统计学意义(P<0.05)。
3P19ARF蛋白与宫颈癌病临床理特征的关系
P19ARF与淋巴结转移有关,在伴随淋巴结转移的宫颈癌组织中,P19ARF的表达率降低,差异有统计学意义(P<0.05),其在宫颈癌I期的阳性表达率高于其他分期,但差异无统计学意义(P>0.05)。与宫颈癌组织学分级、组织学类型无关(P>0.05)。
4P18Ink4cmRNA在不同宫颈组织中的表达
P18Ink4cmRNA在479bp处有清晰明亮的条带。条带亮度在正常宫颈组织、CIN、宫颈癌组织中呈现递增水平变化。P18Ink4cmRNA的表达量为依次升高,分别为0.187±0.002,0.331±0.006,0.616±0.009。差异具有统计学意义(P<0.05)。
5P18Ink4c蛋白在不同宫颈组织中的表达
18Ink4c蛋白在正常宫颈组织、CIN、宫颈癌中主要表达于胞浆和胞膜,随着病变程度的加重,染色强度逐渐加重,阳性细胞比例依次升高,阳性表达率依次升高,分别为10%、45%、65.96%,差异有统计学意义(P<0.05)。
6P18Ink4c蛋白与宫颈癌病临床理特征的关系
P18Ink4c与宫颈癌分期、淋巴结转移有关,伴随着淋巴结转移、分期的增加其表达率也增加,差异有统计学意义(P<0.05)。与宫颈癌组织学分级、组织学类型无关(P>0.05)。
7宫颈癌中P19ARF和P18Ink4c关联性分析
RT-PCR结果显示P18Ink4c和P19ARF不存在简单线性关系,免疫组化结果显示宫颈癌组织中P18Ink4c和P19ARF无明显相关性(P>0.05)。
结论
1.在宫颈癌组织中人类P19ARF和P18Ink4c的基因水平和蛋白水平均有异常表达。
2.人类P19ARF的低表达P18Ink4c的高表达可能参与了宫颈癌的发生、发展的过程。
Cervival cancer is one of the most common therioma of the female procreational system.The morbidity of cervical cancer is the first malignant tumor of female therioma.Though it is one of highest percentage in female pernicious neoplasm causa mortis. The morbidity of cervical cancer presents a growing trend among young women.This probably because younger women' sex happen early, they have more sex partner, they don't pay attention to clean sanitation when they have sex. Persistent infection of high risk human papilloma virus is a necessary factor among attacks and progression of uterine neck pernicious neoplasm.In the known15high risk HPV, HPV16and HPV18are two of the most common.The new research demonstrates that they have relation with70%of cervical cancer.
P19ARF is a newfound potential tumor suppressor gene.It roots in Ink4a family which is from cyclin dependent kinase inhibitor family. P19ARF gene coding P19ARF protein whose molecular weight is14KD. P19ARF and P16Ink4a have the same coding gene,but their open reading frame are different.Meantime, P19ARF is the upstream gene of P53gene,it can arrest the cell cycle through P53-MDM2pathway.In this way RB protein can be non-phosphorylation.Phosphorylated RB protein can not bind with E2F,as a result signal pathway is interrupted,then P19ARF-MDM2-P53signal pathway is interrupted.Cell cycle can be stoped in G1/S,in order to inhibit attacks and progression of uterine neck pernicious neoplasm.
P18Ink4c is a cell cycle control gene and it can inhibit attacks and progression of uterine neck pernicious neoplasm.The transcription product P18Ink4c protein is a cyclin dependent kinase inhibitor.cThe transcription factor E2F activated transcription of genetic material,and it ia able to escape the cell cycle checkpoint,activation of cyclin dependent kinase related.It comes into paly through binding with CDK4/6,this makes cell still in G1phase,preventing cancer cells continue to proliferation.
Objective
This study use RT-PCR methods to research the expression of P19ARF and P18Ink4c mRNA in three different condition uterine neck tissue,use immunohistochemistry technique S-P to study the positive expression of P19ARF and P18Ink4c, in order to disuss the expression and clinical ananlysis of P19ARF and P18Ink4c in uterine nack neoplasm.
Materials and Methods
1Materials
Choosing47cervical cancer and20CIN samples from third Affiliated Hospital of Zhengzhou University outpatient cervival biopsy and inpatient department patients during junary2011and junary2012,and20normal cervical tissue samples from resectional uterus because of ademomyosis of uterus or uterine myoma.According to FIGO standard,cervical cancer sample including21Ⅰ phase samples,20Ⅱ pahase samples,3Ⅲ phase samples,3Ⅳ phase samples.According pathology classification,19G1samples,15G2samples,13G3samples.Among them,17patients have lymphatic metastasis, and30patients have not lymphatic metastasis.All the patients do not have chemotherapy and radiational and immunology treatment history.All the specimen was drew within5min after downcut.Following close to aseptique principe.One part of materials were put in-80℃liquid nitrogen,another part were put in paraffin embedding,serial section per44um.
2Methods
2.1Experimental method and main reagent
Immunohistochemisty and RT-PCR,the SPim-sty kit and DAB colouring agent and primary antibodies P18Ink4c polyclonal antibody are bought from Beijing biological bo orson limited company. P19ARF polyclonal antibody is bought from santa cruz biological reagent limited company.
2.2Immunohistochemisty
The tissue is fabricated by10%specific drug solvent and solid chemical substances,serial section per4um.In strict accordance with the kit instructions for operation.The evalution standard of P18and P19protein positive results,to prefer5random field vision in400times microscope.Giving a mark according to the dyeing strength and positive cell percentae of total cell number.According to the dyeing strength rating:zero means no coloring,1means weak positive coloring,3means strong positive coloring,2is between1and3.According to the positive cell rating:≤5%defines0,6%-25%defines1,26%-50%defines2,>50%defines3.The two scores determine the product,0-2is devided into negative,3-9is devided into positive.
2.3RT-PCR
Designing primers,extracting tissue total RNA,PCR amplification product using AGAR gel electrophoresis,observing electrophoresis band under ultraviolet rays.Analysis by D-140picture record analysis system.Quqantity of purpose gene expression is computed by the ratio of objective DNA banding and grave value of ACTINA banding.
3Statistical methods
The data is dealed by SPSS17.0.Using K-W test, X2test,Fisher's exact propability method.Inspection atandard:a=0.05.
Results
1The expression of P19ARF mRNA in cervical tissue
In progression of uterine neck, lesioning to serious analysis tendency,P19ARF mRNA has gradually lower expression(P<0.05). Its expression are0.098±0.005,0.502±0.009,0.915±0.027respectively and is seated in568bp.
2The expression of P19ARF protein in cervival tissue
In progression of uterine neck morbidity,lesioning to serious analysis tendency,P19ARF has gradually lower expression. Its expression are80%、45%、21.28%respectively(P<0.05).
3. P19ARF and cervical cancer clinical and pathological characteristics
P19ARF is related with lymph node metastasis(P<0.05).The positive expression of P19ARF in cancer has not relationship with clinical stages and histological grading(P>0.05).
4. The expression of P18Ink4c mRNA protein in different cervival tissue
The rate of positive expression of P18Ink4c mRNAprotein in cervical cancer,CIN,normal cervical tissue is gradually died down(P<0.05). Its expression are0.616±0.009,0.331±0.006,0.187±0.002respectively and is seated in479bp.
5. The expression of P18Ink4c protein in cervical tissue
In progression of uterine neck morbidity,lesioning to serious analysis tendency, The gradually higher expression of P18Ink4c are10%、45%、65.96%(P<0.05).
6. P18Ink4c and cervical cancer clinical and pathological characteristics
It is related with cervival cancer stages.The expressional rate of P18Ink4c is gradually higher along with lymphatic metastasis and cervical cancer stages increased.And it has not relationship with histological grading.
7. Correlation analysis of P19ARF and P18Ink4c in cervical cancer
RT-PCR showed that they do not have linear ralationship.The results showed that there is no obvious correlationship of P19ARF and P18Ink4c in cervical cancer.(P>0.05).
Conclusion
1. The low expression of P19ARF and high expression of P18Ink4c may with the relevant to the attack and progression of uterine neck neoplasm.
2. In cervical cancer P19ARF and P18Ink4c has abnornal expression in gene level and protein level.
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