肿瘤的分子生物学检验省名师优质课赛课获奖课件市赛课一等奖课件.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,本资料仅供参考，不能作为科学依据。谢谢。本资料仅供参考，不能作为科学依据。谢谢,1,肿瘤分子诊疗,Molecular diagnosis of tumor,李江滨 副教授,第1页,2,第一节 肿瘤诊疗生物标志物,染色体异常、基因异常、单核苷酸多态性、表观遗传异常、,miRNA,第二节 肿瘤分子生物学检验技术,第三节 肿瘤分子生物学检验临床应用,乳腺癌,肿瘤分子诊疗与个体化医疗,第2页,3,什么是肿瘤？,肿瘤（,Tumor,）是机体在各种致癌原因作用下，局部组织某一个细胞在基因水平上失去对其生长正常调控，造成其,克隆性异常增生,而形成异常病变。,学界普通将肿瘤分为良性和恶性两大类。,第3页,4,Cancer,vs,Tumor,Tumors are sometimes cancerous but this does not mean that tumors and cancers are synonyms(as most people think).,第4页,5,Abnormal cellular growth,Tumors and cancers are different.,A tumor,develops when a lesion or lump,病变或肿块,is formed in your body due to,abnormal cellular growth,.In the case of cancer,this cellular growth is uncontrollable and it spreads in the body,.,第5页,6,Treatment,Cancer,Surgery,chemotherapy and radiotherapy.,Tumor,Removing a benign tumor is relatively easy through surgery,and the condition does not recur.,第6页,7,第一节 肿瘤诊疗生物标志物,最早肿瘤标志物：本,-,周蛋白，,1846,年,当前已发觉一百各种肿瘤标志物,蛋白与,核酸,两大类,肿瘤相关染色体异常、基因异常、单核苷酸多态性、表观遗传异常、,miRNA,第7页,8,一、肿瘤相关染色体异常,多数肿瘤细胞存在染色体异常。,1.,染色体数目异常,例：某个癌细胞染色体共,104,条，包含许多异常染色体,第8页,9,2.,染色体结构异常,包含易位、缺失、重复、环状染色体和双着丝粒染色体等。,例：,Ph,染色体（费城,1,号染色体），慢性粒细胞性白血病（,CML,），,9,号和,22,号染色体长臂易位,9,号原癌基因,abl,和,22,号,bcr,基因组合成融合基因增高酪氨酸激酶活性。,Ph,临床意义在于：,95,CML,都是,Ph,阳性，能够作为诊疗依据。有时,Ph,先于临床症状出现，故又可用于早期诊疗。,第9页,10,二、肿瘤相关基因表示异常,原癌基因,:,sis,、,VEGF,、,EGFR,、,c-myc,抑癌基因,:APC,、,BRCA,、,p53,、,Rb,细胞周期调整基因,:,cyclins,、,CDKs,、,CKIs,细胞凋亡相关基因,:,Bcl-1,、,p53,、,bcr-abl,基因组稳定相关基因,(DNA,修复基因,):,APE1,肿瘤转移相关基因,:,nm23,、,WDNM,、,sis,、,p53,肿瘤血管生成相关基因,:,VEGF,、,EGFR,、,p53,第10页,11,三、肿瘤相关单核苷酸多态性,What are single nucleotide polymorphisms(SNPs)?,SNPs(pronounced“snips”),are the most common type of genetic variation among people.Each SNP represents a difference in a single DNA building block,called a nucleotide.For example,a SNP may replace the nucleotide cytosine(C)with the nucleotide thymine(T)in a certain stretch of DNA.,第11页,12,第12页,13,SNPs occur normally throughout a persons DNA.They occur once in every 300 nucleotides on average,which means there are roughly 10 million SNPs in the human genome.They can act as biological markers,helping scientists locate genes that are associated with disease.When SNPs occur within a gene or in a regulatory region near a gene,they may play a more direct role in disease by affecting the genes function.,第13页,14,Most SNPs have no effect on health.Researchers have found SNPs that may help predict an individuals response to certain drugs,susceptibility to environmental factors such as toxins,and risk of developing particular diseases.SNPs can also be used to track the inheritance of disease genes within families.Future studies will work to identify SNPs associated with complex diseases such as heart disease,diabetes,and cancer.,第14页,15,三、肿瘤相关单核苷酸多态性,SNP:Single nucleotide polymorphisms,1.SNP,与肿瘤,肿瘤易感基因、肿瘤药品治疗相关基因,肿瘤个体化诊疗,2.SNP,研究思绪,研究对象差异性,研究技术：,PCR,、芯片,研究难点：样本采集,第15页,16,四、肿瘤相关表观遗传异常,表观遗传：,表观遗传（,epigenetics,）是指,DNA,序列不发生改变,，但,基因表示却发生了可遗传改变,。这种改变是细胞内除了遗传信息以外其它可遗传物质发生改变，且这种改变在发育和细胞增殖过程中能稳定传递。,几乎全部类型人类肿瘤都存,在表观遗传异常,抑癌基因高甲基化,和,癌基因去甲基化,。,异常甲基化常发生在肿瘤细胞形成,早期,。,第16页,17,五、肿瘤相关,miRNA,MicroRNA formation and function.flv,第17页,18,第18页,19,五、肿瘤相关,miRNA,1.miRNA,与肿瘤,miRNA,与肿瘤发生、发展、诊疗、治疗、预后,2.miRNA,类肿瘤标志物研究思绪,研究对象：实体瘤、细胞、循环血、其它,研究技术：定量,PCR,、,western blot,、芯片、免疫组化、细胞培养（增殖、迁移、浸润）等等,第19页,肿瘤相关长链非编码RNA,长链非编码RNA(long noncoding RNA,lncRNA),长度在200-100000 nt之间RNA分子,不编码蛋白,lncRNA参加细胞内各种过程调控,种类、数量、功效都不明确,长链非编码RNA 在肿瘤发生发展中位置,肿瘤分子生物学检验技术,肿瘤分子诊疗惯用检测标本,外周血细胞,病灶局部受损组织,提取,DNA,或,mRNA,检测,第21页,22,肿瘤早期诊疗标本选择及处理,（,1,）样本选择,体液、分泌物、排泄物作为早期分子诊疗检测样本，如：血、痰、大便等。,（,2,）样本处理：获取血浆,DNA,首先获取,无血细胞血浆,（,Cell free plasma,）：,重复离心法,：,EDTA,抗凝血,4 1500rpm 20min,吸收上层血浆,3000rpm 10min,。,磁珠法和,QIAamp DNA mini kit,在内各种方法提取循环,DNA,。,第22页,23,循环,DNA,定量检测方法,早期检测方法：二苯胺法、溴化乙锭法、对流免疫电泳法、,RNA-DNA,杂交法,新检测技术：放射免疫法，,实时荧光定量,PCR,法,等,第23页,24,新型检测标志物：血浆,DNA,何谓血浆,DNA(plasma DNA),？,又称循环,DNA(circulating DNA),，是一个无细胞状态细胞外,DNA(extra cellular DNA),，由长度不等,单链或双链,DNA,及其混合物组成，,主要以,DNA-,蛋白质混合物形式存在，但也存在部分游离,DNA,。,第24页,25,血浆,DNA,作为检测对象优势,循环,DNA,来自外周血浆或血清，较组织标本,易于取得,，可实现无创检验。,DNA,比,RNA,和蛋白质含有更高稳定性，,不宜降解,，更适合作为检测靶分子。,能够对其进行,扩增,，检测敏感性高。,越来越多证据表明，疾病（尤其是肿瘤）患者循环,DNA,存在量和质改变,。,第25页,26,Cell-free DNA resuscitated for tumor testing,Nature Medicine,14,914-915(),in situ hybridization,FISH,）,第28页,29,致病基因结构异常检测,（,1,）斑点杂交（,dot blot hybridization,）,（,2,）等位基因特异寡核苷酸探针杂交（,allele-specific oligonucleotide,，,ASO,）,（,3,）单链构象多态性（,single strand conformation polymorphism,SSCP,）,（,4,）限制性内切酶图谱（,restriction map,）分析,（,5,）限制性片段长度多态性（,restriction fragment length polymorphism,RFLP,）,（,6,）,DNA,分子杂交（,Southern blot,）,第29页,30,致病基因表示异常检测,（,1,）mRNA检测,定量PCR,（2）蛋白检测,免疫组化：定性、半定量、定位,Western blot：定量,第30页,31,第三节 肿瘤分子生物学检验临床应用,乳腺癌,第31页,32,一、乳腺癌,女性发病率高，,5%-10%,为家族性。,90%,家族性乳腺癌包括,BRCA1,和,BRCA2,基因突变。,BRCA1,突变可发生在全部细胞，若发生在精子或卵子则能够传给下一代。,其它包括基因：,p53,、,PTEN,、,c-myc,、端粒酶等。,论文导读,:,A strong candidate for the breast and ovarian,卵巢,cancer susceptibility gene BRCA1,www.sciencemag.org/cgi/rapidpdf/266/5182/66.pdf,第32页,33,BRCA1,BRCA1(breast cancer 1,early onset,早发,)is a human tumor suppressor gene,肿瘤抑制基因,which produces a protein,called breast cancer type 1 susceptibility,易感性,protein.It is found in the cells of breast and other tissue,where it helps repair damaged DNA,and destroy the cell when DNA cant be repaired.If BRCA1 itself is damaged,the damaged DNA can let the cell duplicate,复制,without control,and turn into a cancer.,第33页,34,Mutations and cancer risk,Certain variations of the BRCA1 gene lead to an increased risk for breast cancer.Researchers have identified hundreds of mutations in the BRCA1 gene,many of which are associated with an increased risk of cancer.Women who have an abnormal BRCA1 or BRCA2 gene have up to an 60%risk of developing breast cancer;increased risk of developing ovarian cancer is about 55%for women with BRCA1 mutations and about 25%for women with BRCA2 mutations.,第34页,35,These mutations can be changes in one or a small number of DNA base pairs.Those mutations can be identified with PCR and DNA sequencing.Other methods are proposed:Q-PCR,定量,and Quantitative Multiplex PCR,多重定量,of Shorts Fluorescents Fragments(QMPSF).New methods have been recently proposed:heteroduplex analysis,异源双链分析,(HDA)by multi-capillary electrophoresis,多毛细管电泳,or also oligonucleotides array based on comparative genomic hybridization,比较基因组杂交,(array-CGH).,第35页,36,Researchers believe that the defective BRCA1 protein is unable to help fix mutations that occur in other genes.These defects accumulate and may allow cells to grow and divide uncontrollably to form a tumor.,第36页,37,女性排名第一常见恶性肿瘤。,美国每,8,个人就有,1,个人一生中会得乳腺癌。美国患乳腺癌女性占新发恶性肿瘤,30%,，而其中大约,10%,乳腺癌是遗传性。,乳癌“株连”，一旦家里有些人患此疾病，一级亲属（母亲、姐妹或孩子）女性都应该做检验。,第37页,38,乳腺癌高危人群 有,乳腺癌家族史、基因检测到,BRAC1/2,基因突变,、曾有过胸部放疗史、乳腺活检为高危良性病变、乳腺密度高、初潮年纪早于,12,岁,以及绝经年纪推迟等人群，普通患乳腺癌危险性会比较高。,第38页,39,第39页,40,安吉丽娜,朱莉在,年,5,月,纽约时报,上刊文“,My Medical Choice”,，讲述了决定手术原因及经过，最终呼吁全部女性注意预防乳腺癌。,My doctors estimated that I had an 87 percent risk of breast cancer and a 50 percent risk of ovarian cancer,although the risk is different in the case of each woman,。,Only a fraction of breast cancers result from an inherited gene mutation.Those with a defect in BRCA1 have a 65 percent risk of getting it,on average,。,第40页,41,Once I knew that this was my reality,I decided to be proactive and to minimize the risk as much I could.I made a decision to have a preventive double mastectomy,预防性双乳切除手术,.I started with the breasts,as my risk of breast cancer is higher than my risk of ovarian cancer,。,第41页,42,年,3,月,纽约时报,上刊文“,diary of a surgery”,I wanted other women at risk to know about the options.I promised to follow up with any information that could be useful,including about my next preventive surgery,the removal of my ovaries and fallopian tubes,卵巢和输卵管,.,I had been planning this for some time.It is a less complex surgery than the mastectomy,but its effects are more severe.,It puts a woman into forced menopause,更年期,.,第42页,43,Then two weeks ago I got a call from my doctor with,blood-test results.“Your CA-125 is normal,”,he said.I breathed a sigh of relief.That test measures the amount of the,protein CA-125 in the blood,and is used to monitor ovarian cancer,.I have it every year because of my family history.,But that wasnt all.He went on.“,There are a number of inflammatory markers that are elevated,and taken together they could be a sign of early cancer,.”I took a pause.“,CA-125 has a 50 to 75 percent chance of missing ovarian cancer at early stages,”he said.He wanted me to see the surgeon immediately to check my ovaries.,第43页,44,The day of the results came.,The PET/CT scan looked clear,and the tumor test was negative.,I was full of happiness,although the radioactive tracer meant I couldnt hug my children.,There was still a chance of early stage cancer,but that was minor compared with a full-blown tumor.,To my relief,I still had the option of removing my ovaries and fallopian tubes and I chose to do it.,第44页,45,In my case,the Eastern and Western,doctors I met agreed that surgery to remove my tubes and ovaries was the best option,because on top of the BRCA gene,three women in my family have died from cancer,.My doctors indicated I should have preventive surgery about a decade before the earliest onset of cancer in my female relatives.My mothers ovarian cancer was diagnosed when she was 49.Im 39.,第45页,46,Last week,I had the procedure:a laparoscopic bilateral salpingo-oophorectomy,腹腔镜双侧输卵管卵巢切除术,.There was a small benign tumor on one ovary,but no signs of cancer in any of the tissues.,在一侧卵巢有一个小良性肿瘤，任何组织都没有癌症迹象。,第46页,47,I have a little clear patch,透明贴,that contains bio-identical estrogen,雌激素,.A progesterone IUD,黄体酮节育器,was inserted in my uterus,子宫,.It will help me maintain a hormonal balance,but more important it will help prevent uterine cancer,子宫癌,.I chose to keep my uterus because cancer in that location is not part of my family history.,第47页,48,Regardless of the hormone replacements Im taking,I am now in menopause,更年期,.I will not be able to have any more children,and I expect some physical changes.But I feel at ease with whatever will come,not because I am strong but because this is a part of life.It is nothing to be feared.,第48页,49,I feel deeply for women for whom this moment comes very early in life,before they have had their children.Their situation is far harder than mine.I inquired and found out that there are options for women to remove their fallopian tubes but keep their ovaries,and so retain the ability to bear children and not go into menopause.I hope they can be aware of that.,第49页,50,预防性切乳术能确保今后百分百不患乳癌吗？,国外研究表明，预防性乳房乳腺切除手术，是当前对乳腺癌高危妇女最有效预防办法。,预防性切乳术不能确保今后百分百不患乳癌,预防性双侧乳房切除术，分为两种。一个是乳房全切，即为整个乳房全部切掉。还有一个是保留乳头乳晕皮下乳腺全切。,朱莉选择是后者。癌变几率从手术前,87%,，降到了,5%,。,切除输卵管保留卵巢还能怀孕吗？,切除卵巢及输卵管还能怀孕吗？,第50页,51,肿瘤分子筛查,经过特异性，敏感性分子生物学技术伎俩，能够有效筛查易患癌症体质人群，对他们进行早期检测和治疗。,易感,=,患病？,第51页,52,临床检测实例：,华大基因,birth.bgi-research program of anewplasmanucleic aciddiagnostic markerof tumor,2.,How to carry outthedetectionof cancer susceptibility,第53页,