Ad-p16、Ad-p53联合紫杉醇治疗乳腺癌的实验研究
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摘要
第一部分乳腺癌p16，p53基因蛋白及其mRNA表达的研究
目的乳腺癌位居女性恶性肿瘤之首，全球每年大约有40万人死于乳腺癌，同时发病率呈逐年上升、发病呈现年轻化之势。大量研究表明，癌基因、肿瘤抑制基因(抑癌基因)的异常与乳腺癌的发生、发展密切相关。如p16、p53、C-cerbB-2及p21基因等。p16抑癌基因突变和／或缺失广泛存在于各种肿瘤中，参与肿瘤细胞增殖的调控，在肿瘤的发生、发展过程中起着重要作用。通过免疫组织化学方法检测p16基因表达蛋白，发现阳性率在24％～60％不等。p53抑癌基因突变在人类肿瘤中最为常见，是目前发现与人类肿瘤关系最为密切的基因，已知乳腺癌组织p53基因的突变率为15％～60％。我们通过检测乳腺癌组织内p16、p53基因蛋白以及mRNA的表达，分析其表达与乳腺癌临床病理特征的相互关系，探讨其表达对乳腺癌分子生物学特性的影响，为临床和临床基础研究乳腺癌的治疗和预后评估提供理论基础和依据。
方法我们利用免疫组织化学SP法和逆转录-聚合酶链反应(reverse transcription polymerase chain reaction，RT-PCR)技术，检测乳腺癌组织内p16、p53基因蛋白和mRNA的表达。运用统计学方法分析各种表达与乳腺癌临床病理学特征之间的关系。
结果免疫组织化学检测癌旁正常乳腺组织中p16基因蛋白的表达率为90％(27／30)，其中48.1％(13／27)为(+++)；29.6％(8／27)为(++)；22.2％(6／27)为(+)；p53基因蛋白的表达率为6.67％(2／30)，全部为(+)。乳腺癌组织中p16基因蛋白阳性表达率为38.3％(23／60)，其中60.9％(14／23)为(+++)，26.1％(6／23)为(++)，13.0％(3／23)为(+)；p53基因蛋白阳性表达率为48.3％(29／60)，其中79.3％(23／29)为(+++)，13.8％(4／29)为(++)，6.9％(2／29)为(+)。RT-PCR检测癌旁正常乳腺组织的p16mRNA显著高于乳腺癌组织(P=0.023)，而p53mRNA在乳腺癌组织的水平显著高于癌旁正常乳腺组织(P=0.001)。p16、p53蛋白表达与乳腺癌组织学分级、淋巴结和／或器官转移相关。乳腺癌细胞分化程度越高p16蛋白阳性表达率越高，而p53表达蛋白正相反；有淋巴结和／或器官转移的病例p16蛋白的表达率明显低于无淋巴结和／或器官转移者，而p53蛋白的表达率显著增加。p16mRNA在组织学Ⅰ级的水平显著高于Ⅱ、Ⅲ级，而Ⅱ、Ⅲ级之间无明显差异；p53mRNA与组织学分型无明显关系。有淋巴结和／或器官转移的p16mRNA、p53mRNA表达均明显高于无淋巴结和／或器官转移。
结论(1)p16、p53基因蛋白的表达与乳腺癌组织学分级、淋巴结和／或器官转移有密切关系。(2)p16mRNA的表达与乳腺癌组织学分级有关。p16mRNA、p53mRNA表达与淋巴结和／或器官转移有关。(3)p16、p53基因的缺失、突变或甲基化等改变与乳腺癌的发生发展密切相关，可以用于评估乳腺癌的预后。(4)通过特殊载体将外源性野生型p16和p53基因进行转导，以纠正p16、p53基因的异常改变，将有帮助于乳腺癌的治疗。第二部分重组p16和p53腺病毒的构建、鉴定、纯化和测定
目的研究显示恶性肿瘤的发生、发展与基因突变、缺失、磷酸化和甲基化等密切相关，其中基因突变在恶性肿瘤细胞表型中起重要作用。表明通过转导外源正常的目的基因的方法，进行修饰突变的基因可以治疗肿瘤。设计肿瘤基因治疗方案的关键在于构建有效的目的基因载体系统，以达到特异性地将目的基因转送至靶细胞。非病毒载体在体内的转染效率远低于病毒载体。腺病毒载体可以有效地感染和传递目的基因，广泛地适用于分裂和静息的组织和细胞，腺病毒DNA不与宿主细胞DNA整合。腺病毒载体的缺点是如要保持转基因的持续表达可导致机体强烈的炎性和免疫反应。但是这种免疫特性可增加目的基因对肿瘤的杀伤作用。腺病毒载体易于构建，经辅助细胞培养可产生大量腺病毒载体。该研究应用基因重组腺病毒的方法，构建携带抑癌基因p16、p53的C亚群5型重组腺病毒(Ad5)，并对其进行鉴定、筛选和纯化；应用人胚胎肾293细胞进行扩增，并确定重组腺病毒p53(Ad-p53)、p16(Ad-p16)和Lac Z(Ad-Lac Z)的纯化程度、病毒滴度、感染率、转导率和扩增情况，以及细胞感染后p53、p16蛋白的表达状况。
方法应用HindⅢ和SpeⅠ酶对含野生型p16cDNA质粒pBluescript-p16进行双酶切；应用KpnⅠ和XaⅠ酶对野生型p53cDNA质粒pGEM-p53进行双酶切。酶切后进行电泳，回收p16cDNA和p53cDNA。用HindⅢ／KpnⅠ和SpeⅠ酶对pAdCMV穿梭质粒进行双酶切，酶切后将回收的p16cDNA和p53cDNA插入定向pAdCMV中，并得到pAdCMV-p16和pAdCMV-p53。pAdCMV-p16／p53重组质粒与腺病毒质粒pJM17在293细胞内进行同源重组，p16／p53基因被转移到腺病毒基因中，生成的E1基因缺陷的重组腺病毒在293细胞中进行繁殖，因E1基因产物的反式作用下大量增殖。以腺病毒、p16、p53特异引物进行扩增，琼脂糖凝胶电泳分析PCR扩增产物，根据PCR扩增产物特有的片段长短筛选p53cDNA、p16cDNA阳性的重组腺病毒空斑，通过传代293细胞扩增阳性空斑内的病毒，以氯化铯密度梯度超速离心机进行病毒的纯化。再次在293细胞中进行繁殖以增加滴度，最多繁殖5代。采用293细胞空斑实验(plaque formation test)测定重组腺病毒滴度。应用报告基因Lac Z以测定重组腺病毒感染率。免疫组织化学技术(SP方法)检测重组腺病毒介导的基因转移效率。Western blot分析p53和p16蛋白表达。
结果p16cDNA、p53cDNA插入pAdCMV得到pAdCMV-p16、pAdCMV-p53。经293细胞中进行繁殖，10-14d即可出现细胞病变(cytopathic effect，CPE)，即典型的病毒空斑，此时显微镜下可见293细胞圆缩脱落，表明pAdCMV-p16、pAdCMV-p53重组质粒与腺病毒质粒pJM17已在293细胞内发生了同源重组，p16、p53基因被转移到腺病毒基因中，培养液的上清液中含有重组腺病毒颗粒，所制备的重组腺病毒分别命各为Ad-p16、Ad-p53。PCR扩增出860bp的腺病毒DNA片段和570 bp的p16cDNA片段、642 bp的p53cDNA片段即为重组p16、p53腺病毒。空斑形成试验计算出的滴度为：Ad-p53 2.1×10~(10)；Ad-p16 6.7×10~(10)；Ad-Lac Z1.0×10~(10)。当Ad-Lac Z的MOI为25时，293细胞的感染率为32％：MOI为50时，感染率达到78.9％；而MOI为100时，感染率达到98.6％，因此可以应用重组腺病毒MOI≥100感染人乳腺癌MDA-MB-231细胞株，以观察细胞的基因表达和凋亡情况。免疫组织化学染色结果表明重组腺病毒介导的基因转导率(％)p16为93.8±2.5，p53为96.3±3.8。Ad-Lac Z感染的肿瘤细胞无p53和p16蛋白表达。但是，p53和p16联合感染的肿瘤细胞有明显的p53和p16蛋白表达，说明重组腺病毒可有效地介导外源基因的高效转移。
结论(1)重组腺病毒能够正确携带目的基因进入靶细胞，并准确表达目的基因蛋白。(2)生产重组腺病毒载体较为经济、滴度高。(3)治疗量病毒的体积小，有助于在体内使用。第三部分Ad-p16、Ad-p53联合紫杉醇治疗乳腺癌的研究
目的正常细胞通过关卡作用以调节细胞周期的进程，保持细胞增殖和凋亡地平衡。发现癌基因和抑癌基因均是通过调控细胞周期而发挥作用。肿瘤的发生常涉及多个抑癌基因的突变、磷酸化或／和失活。将正常肿瘤抑制基因导入肿瘤细胞以替代失去功能的基因，而达到逆转其恶性表型、抑制肿瘤生长，甚至消灭肿瘤成为肿瘤基因治疗的热点。p16和p53是人体内重要的肿瘤抑制基因，直接或间接调控细胞周期，使细胞周期停滞于G1期。p16和p53基因发生缺失、突变、重排、磷酸化或不表达可存在于多种肿瘤的发生、发展过程中。恢复野生型p16和p53基因的功能成为抗肿瘤基因治疗研究的重要途径。紫杉醇可诱导多种肿瘤的凋亡，尤其对转移性乳腺癌的作用尤为引人注目。该研究对Tax、Ad-p16、Ad-p53及其联合应用对人乳腺癌细胞株MDA-MB-231的生长抑制和细胞凋亡进行深入研究。然后，应用Tax、Ad-p16、Ad-p53及其联合对乳腺癌细胞MDA-MB-231成瘤裸鼠模型进行动物治疗实验。观察治疗效果，为研究肿瘤治疗以及治疗途径提供依据。
方法显微镜下观察Ad-p16、Ad-p53及其联合对MDA-MB-231细胞形态学以及生长抑制的影响。MTT法监测Tax、Ad-p16、Ad-p53及联合应用对MDA-MB-231细胞生长的抑制率。应用流式细胞仪Annexin V-FITC／PI法检测分析Tax、Ad-p16、Ad-p53及其联合应用后MDA-MB-231细胞的凋亡率。应用流式细胞仪分析Tax、Ad-p16、Ad-p53及其联合对MDA-MB-231细胞周期和凋亡影响。裸鼠乳房脂肪垫内注射MDA-MB-231细胞悬浮液，使裸鼠形成乳腺癌模型。应用Ad-Lac Z、Tax、Ad-p16、Ad-p53、Tax+Ad-p16、Tax+Ad-p53、Ad-p16+Ad-p53和Tax+Ad-p16+Ad-p53对成瘤裸鼠进行治疗，观察肿瘤体积和抑瘤率的变化治疗，分析治疗效果。并对治疗后的肿瘤标本进行常规病理检查。应用脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测分析肿瘤组织细胞凋亡的状况。实验数据采用SPSS11.0统计软件包进行分析。
结果Ad-p16、Ad-p53和二者联合感染可导致人乳腺癌MDA-MB-231细胞生长抑制。Ad-p16对细胞生长的抑制作用比Ad-p53强，但导致的细胞形态学发生改变时间晚。Ad-p16和Ad-p53联合感染可导致细胞生长近乎停止，在细胞生长抑制和形态学改变均较单一感染明显且发生时间早。而Ad-Lac Z感染后细胞生长良好，细胞形态学无明显变化。Tax对MDA-MB-231细胞的抑制随着剂量的增加，细胞生长抑制率明显增加，细胞抑制率与时间呈正相关(r=0.923，P=0.000)。与Ad-Lac Z相比较，Ad-p16、Ad-p53以及二者联合感染MDA-MB-231细胞后，对肿瘤细胞均有明显的抑制作用(P值均为0.000)。Ad-p16的抑制率在各个时相均高于Ad-p53(P_(24h)=0.038，P_(48h)=0.004，P_(72h)=0.003，P_(96h)=0.001，P_(120h)=0000)。Ad-p16的抑制作用呈现逐渐上升趋势，而Ad-p53在72h时达到高峰后有下降趋势，但是均无统计学意义。Ad-p16与Ad-p53联合感染MDA-MB-231细胞后，在各个时相的细胞抑制率均显著大于单一感染的抑制率(vs Ad-p16，P值在0.001-0.004之间；vs Ad-p53，P值均为0.000)；并且随着时间的延长，细胞抑制率明显增加(120h vs72h，P=0.006)，与感染时间有明显的正相关(r=0.89，P=0.001)。Tax与Ad-p16联合作用于MDA-MB-231细胞后，在各个时相上细胞生长抑制率均显著大于单一应用，于96h时达到高峰，抑制率达到70.96％(70.96±3.30)(vs Ad-p16，P=0.001；vs Tax，P=0.000)；并且细胞抑制率随着时间的延长明显增加(120h vs 72h，P=0.028)。Tax与Ad-p53联合有同样效果，但与Tax与Ad-p16联合的抑制率在各个时相上相比较无显著性差异(P值在0.069-0.86之间)。Tax、Ad-p16和Ad-p53三者联合应用，对MDA-MB-231细胞的抑制作用更加明显，在各个时相上均有明显增加(P值均为0.000)，最高抑制率达到90％以上。Tax、Ad-p16或Ad-p53单一作用于MDA-MB-231细胞可诱导其发生凋亡，并且随着时间的延长细胞凋亡率明显增加，72h时分别达到15.31％、19.34％和29.05％。但是，感染Ad-p16的MDA-MB-231细胞发生凋亡较Tax和Ad-p53晚，凋亡率明显低于Ad-p53。Tax与Ad-p16或Ad-p53联合可明显增加MDA-MB-231细胞的凋亡率。Ad-p16与Ad-p53联合也可明显增加MDA-MB-231细胞的凋亡。Tax、Ad-p16和Ad-p53三者联合诱导MDA-MB-231细胞的能力明显提高，效果更加显著。Tax可导致MDA-MB-231细胞停滞于G2／M期，Ad-p16、Ad-p53或二者联合感染使MDA-MB-231细胞停滞于G0／G1期。Tax、Ad-p16和Ad-p53联合时，大多数细胞停滞于G0／G1和G2／M期。Tax、Ad-p16、Ad-p53及其联合作用于MDA-MB-231细胞72h后琼脂糖凝胶电泳呈现典型的凋亡梯形电泳带，而对照组仅仅呈现基因组DNA。Tax、Ad-p16和Ad-p53组裸鼠体内肿瘤明显受到抑制，第20天时与Ad-Lac Z组比较的P值均为0.000；各组较治疗前肿瘤体积虽有所减小，但是均无统计学意义(P值分别为0.216、0.895和0.138)。Ad-p16与Ad-p53联合应用不仅可以抑制肿瘤的增长，尚可以使肿瘤体积明显减小(0d vs 16d，P=0.034；0d vs 20d，P=0.007)。Tax、Ad-p16和Ad-p53三者联合，裸鼠肿瘤减小更加明显(0d vs 12d，P=0.041；0d vs 16d，P=0.003；0d vs 20d，P=0.000)。治疗组的肿瘤组织间质明显增加，肿瘤细胞以及细胞核分裂明显减少，细胞核呈现浅蓝色，细胞核与细胞浆比例减小，可见散在的新月形核染色质和细胞核碎裂的碎片。Ad-Lac Z、Tax、Ad-p16、Ad-p53、Ad-p16+Ad-p53和Tax+Ad-p16+Ad-p53组肿瘤组织细胞凋亡率(％)分别为1.4±0.9、14.7±2.6、19.5±2.5、21.3±3.1、30.5±3.2和42.3±4.3。
结论(1)Ad-p16、Ad-p53和二者联合感染可导致人乳腺癌MDA-MB-231细胞形态学改变和生长抑制。而此现象并非是腺病毒本身所致，而是外源性p16和p53基因所发挥的作用。(2)Tax、Ad-p16和Ad-p53在诱导MDA-MB-231细胞凋亡上有明显的协同作用，p16和p53基因的表达可以增加Tax对MDA-MB-231细胞的敏感性，p16基因可以促进p53基因所诱导的细胞凋亡。(3)Tax可导致MDA-MB-231细胞停滞于G2／M期，Ad-p16、Ad-p53或二者联合感染使MDA-MB-231细胞停滞于G0／G1期。Tax、Ad-p16和Ad-p53联合时，大多数细胞停滞于G0／G1和G2／M期。(4)Tax、Ad-p16和Ad-p53三者之间有协同抑制裸鼠体内肿瘤生长的作用，尤其是三者联合更加明显。在抑制肿瘤生长同时，可导致肿瘤组织细胞凋亡。
Part 1
Study on breast cancer p16 and p53 gene protein and mRNA expression
Objectives Breast cancer ranks the top among the malignant tumors of women. About 400,000 people die of breast cancer all over the world every year. Meanwhile, the incidence of breast cancer has been increasing annually and breast cancer patients tend to be younger. A great deal of research shows that abnormality of oncogene and tumor suppressor gene (antioncogene) is closely related to the occurrence and development of breast cancer, such as p16, p53, C-cerbB-2 and p21 genes, p16 antioncogene mutation and /or deficiency is widely available in various tumors, is involved in regulation of tumor cell proliferation and plays an important role in the occurrence and development process of tumor. Through immunohistochemical method, we found out that the positive rate of p16 gene protein expression varied between 24% and 60%. p53 antioncogene mutation is the most popular in the human tumors, and p53 is the gene with the closest relation with the human tumors according to the existing researches. It is known that the p53 gene mutation rate in breast cancer tissue is 15%～60%. Through testing the breast cancer tissue p16 and p53 gene protein and mRNA expression, we analyzed the correlation between the expression and the clinical pathological characteristics of breast cancer and explored the effects of the expression on the molecular biological characteristics of breast cancer to provide a theoretical foundation and basis for treatment and prognosis assessment of breast cancer in clinical and basic researches.
Methods We tested p16 and p53 gene protein and mRNA expression within the breast cancer tissue through immunohistochemical SP method and reverse transcription polymerase chain reaction (RT-PCR) technique and analyzed the relationship between the expressions and the clinical pathological characteristics of breast cancer through statistical methods.
Results Through immunohistochemical method we found out that the expression rate of p16 gene protein within the tumor-adjacent normal breast tissue was 90%(27/30), including 48.1%(13/27)(+++); 29.6% (8/27)(++); 22.2%(6/27)
(+) ; the expression rate of p53 gene protein was 6.67% (2/30) , all as (+) ; while the positive rate of p16 gene protein expression within the breast cancer tissue was 38.3% (23/60) , including 60.9% (14/23) (+++), 26.1% (6/23) (++) and 13.0%
(3/23) (+) ; the positive rate of p53 gene protein expression was 48.3% (29/60) , including 79.3% (23/29) (+++) , 13.8% (4/29) (++) and 6.9% (2/29) (+) .The results of RT-PCR test showed that pl6mRNA of tumor-adjacent normal breast tissue was obvious higher than that of breast cancer tissue (P=0.023), while p53mRNA of the breast cancer tissue was significantly higher than that of tumor-adjacent normal breast tissue (P=0.001) . Expression of pl6 and p53 proteins is closely related to histological grade of breast cancer tissue, and lymph node and/or organ metastasis. The higher the cell differentiation of breast cancer is, the higher the positive rate of p16 protein expression. The p53 expression rate was on the contrary. The cases with lymph node and/or organ metastasis showed significantly lower expression rate of p16 protein than that of those without lymph node and / or organ metastasis, while the expression rate of p53 protein in metastasis cases was significantly higher. p16mRNA at histological grade I was obviously higher than that at grade II and III, but no obvious difference was observed between the p16mRNA level at grade II and grade III. No obvious relation was found between p53mRNA and histological classification. The p16mRNA and p53mRNA expression rates for cases with lymph node and /or organ metastasis were obviously higher than those for cases without lymph node and / or organ metastasis.
Conclusion (1) Expression of p16 and p53 gene proteins is closely related to histological grade of breast cancer, and lymph node and/or organ metastasis. (2) Expression of pl6mRNA is related to histological grade of breast cancer. Expression of pl6mRNA and p53mRNA is related to lymph node and/or organ metastasis. (3) Deficiency, mutation or methylation, etc of p16 and p53 genes is closely related to the occurrence and development of breast cancer and can be used in prognosis assessment of breast cancer. (4) Exogenous wild-type p16 and p53 genes can be transfected through specific vectors to correct the abnormal changes of p16 and p53 genes and contribute to the treatment of breast cancer.
Part 2
Construction, identification, purification and determination of recombinant pl6 and p53 adenovirus
Objectives The study shows that the occurrence and development of malignant tumor is closely related to gene mutation, deficiency, phosphorylation, methylation, etc, and gene mutation plays an important role in malignant tumor cell phenotype. It is shown that transfection of exogenous normal target genes can be used to modify the mutant genes and serves as a tumor therapy. The key to designing a gene therapy is to construct an effective target gene vector system for specific transfer of target genes to target cells. The transfection efficiency of non-virus vectors within the body is far lower than that of virus vectors. Adenovirus vectors can effectively infect and transfer the target genes, and are widely applicable to both dividing and static tissues and cells, and the adenovirus DNA does not integrate with DNA of host cells. The disadvantages of adenovirus vectors include severe inflammation and immunoreaction if continuous expression of transgenes is maintained. But such immune characteristics may enhance the effects of target genes on killing tumors. Adenovirus vectors are easily constructed and a large number of adenovirus vectors may be cultured through helper cells. In this study, gene recombinant adenovirus method was adopted to construct recombinant adenovirus type 5 (Ad5) of sub-group C of antioncogene p16 and p53, which were identified, sifted and purified. Human embryonic kidney 293 cells were used for amplification, and the purification rate, virus titer, infection rate, transfection rate, and amplification of recombinant adenovirus p53 (Ad-p53) , p16 (Ad-p16) , as well as expression of p53 and p16 protein after cell infection was determined to provide sound recombinant adenovirus vectors for future experiments, namely Ad-p16 and Ad-p53.
Methods HindIII and SpeI enzymes were used for double enzyme digestion of pBluescript-p16 plasmids containing wild type p16cDNA; KpnI and XaI enzymes were used for double enzyme digestion of pGEM-p53 plasmid containing wild type p53cDNA, and then p16cDNA and p53cDNA were recovered through electrophoresis. HindIII/KpnI and SpeI enzymes were used for double enzyme digestion of pAdCMV shuttle plasmid and the recovered pl6cDNA and p53cDNA were then inserted into directional pAdCMV to get pAdCMV-p16 and pAdCMV-p53. Homologous recombination between pAdCMV-p53 recombinant plasmid and adenovirus plasmid pJM17 was made within 293 cells and p53 gene was transferred into the adenovirus gene. The generated El gene-defective recombinant adenovirus multiplied within the 293 cells and proliferated vigorously under transacting effects of El gene products. Adenovirus-, p16-, p53-specific primers were adopted for amplification and PCR amplification products were analyzed through agarose gel electrophoresis. Based on the length of specific fragments of PCR amplification products, the recombinant adenovirus plaques with positive p16cDNA, p53cDNA were selected. The virus in the positive plaques proliferated through subculture 293 cells. The viruses were purified through ultracentrifuge with cesium chloride density gradient. The viruses re-multiplied within 293 cells up to 5 generations to increase titer. 293 cell plaque formation testing was adopted to measure the recombinant adenovirus titer. Reporter gene Lac Z was used to measure the recombinant adenovirus infection rate. Immunohistochemical method (SP method) was used to test the efficiency of recombinant adenovirus-mediated gene transfer, and Western blot method was used to analyze expression of p53 and p16 protein.
Results pl6cDNA, p53cDNA were inserted into pAdCMV to get pAdCMV- p16, pAdCMV- p53. Through multiplication within 293 cells, cytopathic effect (CPE) , namely, typical virus plaque occurred after 10-14 days. Then we saw through a microscope that 293 cells rounded up, shrank and fell off, indicating homologous recombination between pAdCMV-pl6/p53 recombinant plasmids and adenovirus plasmid pJM17within 293 cells, p16/p53 gene transferred into adenovirus gene and the upper clean solution of culture media containing the recombinant adenovirus plasmids, with the prepared recombinant adenovirus respectively named Ad-p16, Ad-p53. From PCR amplification, there were 860bp adenovirus DNA segment, 570 bp p16cDNA segment, 642 bp p53cDNA segment, namely, the recombinant p53, pl6 adenovirus. The titer calculated after the plaque formation test was Ad-p53 2.1×10~(10); Ad-p16 6.7×10~(10); Ad-LacZ1.0×10~(10). When MOI of Ad-Lac Z was 25, 50 and 100, the infection rate of 293 cells was 32%, approached 78.9% and reached 98.6% respectively. Therefore, it is feasible to use recombinant adenovirus with MOI≥100 to infect human breast cancer MDA-MB-231 cell strain to observe the gene expression and apoptosis. Immunohistochemical staining results showed the recombinant adenovirus mediated gene transfection rate of pl6 being 93.8±2.5% and of p53 being 96.3±3.8%. No p53 or p16 protein expression was available in Ad-Lac Z infected tumor cells. However, tumor cells infected by both p53 and p16 showed obvious expression of p53 and p16 proteins, indicating that recombinant adenovirus could effectively mediate highly efficient transfer of exogenous genes.
Conclusion (1) The recombinant adenovirus can properly carry target genes into target cells and accurately express target gene protein. (2) It is economical to produce recombinant adenovirus vector with high titer. (3) The therapeutic dosage volume of virus is small and will be good for applications within the human body.
Part 3 Study on breast cancer therapy combining Ad-p16 and Ad-p53 with Tax
Objectives Normal cells rely on checkpoint function to regulate cell cycle progression and maintain balance between cell proliferation and apoptosis. We found that both oncogene and antioncogene act through regulation of cell cycle. Occurrence of tumor often involves mutation, phosphorylation and/or inactivation of a number of antioncogenes. It has become a popular tumor gene therapy to transduce normal tumor suppressor genes into tumor cells to replace dysfunctional genes to reverse malignant phenotype and inhibit tumor growth and even eliminate tumor, p16 and p53 are important tumor suppressor genes in human body and directly or indirectly regulate cell cycle to cause stasis in G1 stage. Deficiency, mutation, rearrangement, phosphorylation or non-expression of p16 and p53 genes may be present in the occurrence and development of various tumors. It has become an important way of anti-tumor gene therapy study to restore the functions of wild type p16 and p53 genes. Tax may induce apoptosis of various tumors, and is especially effective in treatment of metastatic breast cancer. In this study, in-depth research was conducted over the effects of Tax, Ad-p16, Ad-p53 and their combination on growth inhibition and cell apoptosis of human breast cancer cell strain MDA-MB-231. Then Tax, Ad-p16, Ad-p53 and their combination was applied to breast cancer cell MDA-MB-231 in the tumorigenesis model of nude mice for an animal therapy experiment. The therapeutic effects were observed to provide basis for tumor therapy and therapeutic approaches.
Methods The effects of Ad-p16, Ad-p53 and their combination on morphology and growth inhibition of MDA-MB-231 cells were observed through a microscope. MTT method was used to monitor and measure the growth inhibition rate of MDA-MB-231 cells applied with Tax, Ad-p16, Ad-p53 or their combination. The flow cytometer Annexin V-FITC/PI method was applied to test and analyze the apoptosis rate of MDA-MB-231 cells after application of Tax, Ad-p16, Ad-p53 and their combination. The flow cytometer was used to analyze the effects of Tax, Ad-p16, Ad-p53 and their combination on cell cycle and apoptosis of MDA-MB-231. MDA-MB-231 cell suspension was injected into the mammary fat pad of nude mice to form a breast cancer model in the nude mice. Ad-Lac Z, Tax, Ad-p16, Ad-p53, Tax+Ad-pl6, Tax+Ad-p53, Ad-pl6+ Ad-p53 and Tax+Ad-p16+Ad-p53 was used as a therapy for tumorigenesis in nude mice, and the changes in tumor size and tumor inhibition rate were observed and then the therapeutic effects were analyzed. In addition, routine pathological examination was made over the tumor specimens after treatment. Terminal deoxynucleotide transferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) method was used to test and analyze the apoptosis of tumor tissue cells. SPSS11.0 statistical software package was used to analyze the experiment data.
Results Ad-p16, .Ad-p53 or their combined infection can inhibit growth of human breast cancer cell MDA-MB-23. Ad-p16 was more effective in inhibiting cell growth than Ad-p53, but with morphologic changes occurring later. Combined Ad-p16 and Ad-p53 infection may almost stop cell growth, with obviously more effective cell growth inhibition and earlier morphologic changes compared with Ad-pl6 or Ad-p53 infection. In case of Ad-Lac Z infection, the cell grew well without obvious morphologic changes. With increasing dose of Tax, the inhibition effects on MDA-MB-231 cells increased with obviously higher inhibition rate and positive correlation between cell inhibition rate and time (r=0.923, P=0.000) . Compared with Ad-Lac Z, Ad-p16, Ad-p53 or their combined infection was obviously effective in inhibiting the tumor cells (P=0.000 in all cases) . The inhibition rate of Ad-p16 ]was higher than that of Ad-p53 in all the time phases (P_(24h)=0.038, P_(48h)=0.004, P_(72h)=0.003, P_(96h)=0.001, P_(120h)=0.000) . The inhibition effects of Ad-p16 tended to go up gradually, while the inhibition effects of Ad-p53 tended to go down after reaching the peak value at 72h, but without statistical significance in both cases. In case of combined Ad-p16 and Ad-p53 infection, the inhibition rate of MDA-MB-231 cells was higher than that of Ad-p16 or Ad-p53 infection in all the time phases (vs Ad-p16, P = 0.001-0.004; vs Ad-p53,P=0.000); and obviously increased with time( 120h vs72h, P=0.006) , indicating obvious positive correlation with infection time (r=0.89, P=0.001) . After application of combined Tax and Ad-p16 into MDA-MB-231 cells, the cell growth inhibition rate was notably higher than that of Tax or Ad-p16application in all the time phases and reached the peak value at 96h up to 70.96% (70.96±3.30) (vsAd-p16,P=0.001;vsTax,P=0.000) ; and notably increased with time (120h vs 72h,P=0.028) . Same effects were observed in case of combined application of Tax and Ad-p53 but without obvious difference of inhibition rate in various time phases (P = 0.069-0.86) compared with the inhibition rate in case of application of Tax and Ad-p16. Inhibition of MDA-MB-231 cells was more obvious in case of combined application of Tax, Ad-pl6 and Ad-p53, and increased in all the time phases (P = 0.000 in all phases) with the highest inhibition rate up to over 90%. Application of Tax, Ad-p16 or Ad-p53 to MDA-MB-231 cells may induce cell apoptosis and the apoptosis rate obviously increased with time and reached 15.31%, 19.34% and 29.05% respectively at 72h. However, Ad-p16-infected MDA-MB-231 cell apoptosis occurred later than apoptosis of Tax- or Ad-p53-infected cells with apoptosis rate for Ad-p16 obviously lower than that for Ad-p53. Application of combined Tax and Ad-p16 or Ad-p53 can obviously increase the apoptosis rate of MDA-MB-231 cells, and combined application of Ad-p16 and Ad-p53 can also increase apoptosis of MDA-MB-231 cells. Application of combined Tax, Ad-p16 and Ad-p53 obviously improved the ability to induce MDA-MB-231 cell apoptosis with more notable effects. Tax may cause MDA-MB-231 cell stasis in G2/M stage, while application of Ad-p16, Ad-p53 or their combined infection may cause MDA-MB-231 cell stasis in G0/G1 stage. Application of combined Tax, Ad-p16 and Ad-p53 caused stasis of most cells in G0/G1 and G2/M stage. In case of application of Tax, Ad-p16, Ad-p53 or their combination into MDA-MB-231 cells, 72h agarose gel electrophoresis showed typical apoptosis trapezoidal belt, while the control group only showed gene group DNA. The tumors in the nude mice were obviously inhibited for Tax, Ad-p16 and Ad-p53 group, and P = 0.000 for all cases at the 20~(th) day compared with Ad-Lac Z group. Although the tumor size decreased for all groups compared with pre-treatment but without statistical significance (P = 0.216, 0.895, 0.138 respectively) . Application of combined Ad-p16 and Ad-p53 can both inhibit tumor growth and obviously reduce tumor size (0d vs 20d,P=0.034; 0d vs 20d,P=0.007). Application of combined Tax, Ad-pl6 and Ad-p53 more obviously reduced tumor size (0d vs 12d, P=0.041; 0d vs 16d, P=0.003; 0d vs 20d, P=0.000) . For the therapy group, tumor tissue stroma increased obviously and the tumor cells and nuclear division decreased notably with light blue nucleus, decreasing ratio between nucleus and cell plasma and scattered crescent chromatin and nucleus fragments visible. The tumor tissue cell apoptosis rate was 1.4±0.9%, 14.7±2.6%, 19.5±2.5%, 21.3±3.1%, 30.5±3.2% and 42.3±4.3 respectively in case of application of Ad-Lac Z, Tax, Ad-p16, Ad-p53, Ad-p16+Ad-p53 and Tax+Ad-p16+Ad-p53.
Conclusion (1) Ad-p16, Ad-p53 or combined application of both can contribute to morphologic change and growth inhibition of oncogenic MDA-MB-231 cells, while this is due to the functions of exogenous p16 and p53 genes, instead of adenovirus virus. (2) There is obvious synergetic effect of Tax, Ad-p16 and Ad-p53 in inducing apoptosis of MDA-MB-231 cells, expression of p16 and p53 genes can enhance sensitivity of Tax to MDA-MB-231 cells, and p16 genes can contribute to apoptosis induced by p53 genes. (3) Tax can cause MDA-MB-231 cell stasis in G2/M stage, while Ad-p16 infection, Ad-p53 infection or combined Ad-pl6 and Ad-p53 infection can cause MDA-MB-231 cell stasis in G0/G1 stage. Action of combined Tax, Ad-p16 and Ad-p53 causes stasis in G0/G1 stage and G2/M stage of most cells. (4) Tax, Ad-p16 and Ad-p53 have synergetic effects of inhibiting tumor growth in Balb/c nu/nu, and the synergetic effects of combined Tax, Ad-p16 and Ad-p53 are more obvious. Besides inhibiting the tumor growth, they can also cause apoptosis of tumor cells.
目的乳腺癌位居女性恶性肿瘤之首，全球每年大约有40万人死于乳腺癌，同时发病率呈逐年上升、发病呈现年轻化之势。大量研究表明，癌基因、肿瘤抑制基因(抑癌基因)的异常与乳腺癌的发生、发展密切相关。如p16、p53、C-cerbB-2及p21基因等。p16抑癌基因突变和／或缺失广泛存在于各种肿瘤中，参与肿瘤细胞增殖的调控，在肿瘤的发生、发展过程中起着重要作用。通过免疫组织化学方法检测p16基因表达蛋白，发现阳性率在24％～60％不等。p53抑癌基因突变在人类肿瘤中最为常见，是目前发现与人类肿瘤关系最为密切的基因，已知乳腺癌组织p53基因的突变率为15％～60％。我们通过检测乳腺癌组织内p16、p53基因蛋白以及mRNA的表达，分析其表达与乳腺癌临床病理特征的相互关系，探讨其表达对乳腺癌分子生物学特性的影响，为临床和临床基础研究乳腺癌的治疗和预后评估提供理论基础和依据。
方法我们利用免疫组织化学SP法和逆转录-聚合酶链反应(reverse transcription polymerase chain reaction，RT-PCR)技术，检测乳腺癌组织内p16、p53基因蛋白和mRNA的表达。运用统计学方法分析各种表达与乳腺癌临床病理学特征之间的关系。
结果免疫组织化学检测癌旁正常乳腺组织中p16基因蛋白的表达率为90％(27／30)，其中48.1％(13／27)为(+++)；29.6％(8／27)为(++)；22.2％(6／27)为(+)；p53基因蛋白的表达率为6.67％(2／30)，全部为(+)。乳腺癌组织中p16基因蛋白阳性表达率为38.3％(23／60)，其中60.9％(14／23)为(+++)，26.1％(6／23)为(++)，13.0％(3／23)为(+)；p53基因蛋白阳性表达率为48.3％(29／60)，其中79.3％(23／29)为(+++)，13.8％(4／29)为(++)，6.9％(2／29)为(+)。RT-PCR检测癌旁正常乳腺组织的p16mRNA显著高于乳腺癌组织(P=0.023)，而p53mRNA在乳腺癌组织的水平显著高于癌旁正常乳腺组织(P=0.001)。p16、p53蛋白表达与乳腺癌组织学分级、淋巴结和／或器官转移相关。乳腺癌细胞分化程度越高p16蛋白阳性表达率越高，而p53表达蛋白正相反；有淋巴结和／或器官转移的病例p16蛋白的表达率明显低于无淋巴结和／或器官转移者，而p53蛋白的表达率显著增加。p16mRNA在组织学Ⅰ级的水平显著高于Ⅱ、Ⅲ级，而Ⅱ、Ⅲ级之间无明显差异；p53mRNA与组织学分型无明显关系。有淋巴结和／或器官转移的p16mRNA、p53mRNA表达均明显高于无淋巴结和／或器官转移。
结论(1)p16、p53基因蛋白的表达与乳腺癌组织学分级、淋巴结和／或器官转移有密切关系。(2)p16mRNA的表达与乳腺癌组织学分级有关。p16mRNA、p53mRNA表达与淋巴结和／或器官转移有关。(3)p16、p53基因的缺失、突变或甲基化等改变与乳腺癌的发生发展密切相关，可以用于评估乳腺癌的预后。(4)通过特殊载体将外源性野生型p16和p53基因进行转导，以纠正p16、p53基因的异常改变，将有帮助于乳腺癌的治疗。第二部分重组p16和p53腺病毒的构建、鉴定、纯化和测定
目的研究显示恶性肿瘤的发生、发展与基因突变、缺失、磷酸化和甲基化等密切相关，其中基因突变在恶性肿瘤细胞表型中起重要作用。表明通过转导外源正常的目的基因的方法，进行修饰突变的基因可以治疗肿瘤。设计肿瘤基因治疗方案的关键在于构建有效的目的基因载体系统，以达到特异性地将目的基因转送至靶细胞。非病毒载体在体内的转染效率远低于病毒载体。腺病毒载体可以有效地感染和传递目的基因，广泛地适用于分裂和静息的组织和细胞，腺病毒DNA不与宿主细胞DNA整合。腺病毒载体的缺点是如要保持转基因的持续表达可导致机体强烈的炎性和免疫反应。但是这种免疫特性可增加目的基因对肿瘤的杀伤作用。腺病毒载体易于构建，经辅助细胞培养可产生大量腺病毒载体。该研究应用基因重组腺病毒的方法，构建携带抑癌基因p16、p53的C亚群5型重组腺病毒(Ad5)，并对其进行鉴定、筛选和纯化；应用人胚胎肾293细胞进行扩增，并确定重组腺病毒p53(Ad-p53)、p16(Ad-p16)和Lac Z(Ad-Lac Z)的纯化程度、病毒滴度、感染率、转导率和扩增情况，以及细胞感染后p53、p16蛋白的表达状况。
方法应用HindⅢ和SpeⅠ酶对含野生型p16cDNA质粒pBluescript-p16进行双酶切；应用KpnⅠ和XaⅠ酶对野生型p53cDNA质粒pGEM-p53进行双酶切。酶切后进行电泳，回收p16cDNA和p53cDNA。用HindⅢ／KpnⅠ和SpeⅠ酶对pAdCMV穿梭质粒进行双酶切，酶切后将回收的p16cDNA和p53cDNA插入定向pAdCMV中，并得到pAdCMV-p16和pAdCMV-p53。pAdCMV-p16／p53重组质粒与腺病毒质粒pJM17在293细胞内进行同源重组，p16／p53基因被转移到腺病毒基因中，生成的E1基因缺陷的重组腺病毒在293细胞中进行繁殖，因E1基因产物的反式作用下大量增殖。以腺病毒、p16、p53特异引物进行扩增，琼脂糖凝胶电泳分析PCR扩增产物，根据PCR扩增产物特有的片段长短筛选p53cDNA、p16cDNA阳性的重组腺病毒空斑，通过传代293细胞扩增阳性空斑内的病毒，以氯化铯密度梯度超速离心机进行病毒的纯化。再次在293细胞中进行繁殖以增加滴度，最多繁殖5代。采用293细胞空斑实验(plaque formation test)测定重组腺病毒滴度。应用报告基因Lac Z以测定重组腺病毒感染率。免疫组织化学技术(SP方法)检测重组腺病毒介导的基因转移效率。Western blot分析p53和p16蛋白表达。
结果p16cDNA、p53cDNA插入pAdCMV得到pAdCMV-p16、pAdCMV-p53。经293细胞中进行繁殖，10-14d即可出现细胞病变(cytopathic effect，CPE)，即典型的病毒空斑，此时显微镜下可见293细胞圆缩脱落，表明pAdCMV-p16、pAdCMV-p53重组质粒与腺病毒质粒pJM17已在293细胞内发生了同源重组，p16、p53基因被转移到腺病毒基因中，培养液的上清液中含有重组腺病毒颗粒，所制备的重组腺病毒分别命各为Ad-p16、Ad-p53。PCR扩增出860bp的腺病毒DNA片段和570 bp的p16cDNA片段、642 bp的p53cDNA片段即为重组p16、p53腺病毒。空斑形成试验计算出的滴度为：Ad-p53 2.1×10~(10)；Ad-p16 6.7×10~(10)；Ad-Lac Z1.0×10~(10)。当Ad-Lac Z的MOI为25时，293细胞的感染率为32％：MOI为50时，感染率达到78.9％；而MOI为100时，感染率达到98.6％，因此可以应用重组腺病毒MOI≥100感染人乳腺癌MDA-MB-231细胞株，以观察细胞的基因表达和凋亡情况。免疫组织化学染色结果表明重组腺病毒介导的基因转导率(％)p16为93.8±2.5，p53为96.3±3.8。Ad-Lac Z感染的肿瘤细胞无p53和p16蛋白表达。但是，p53和p16联合感染的肿瘤细胞有明显的p53和p16蛋白表达，说明重组腺病毒可有效地介导外源基因的高效转移。
结论(1)重组腺病毒能够正确携带目的基因进入靶细胞，并准确表达目的基因蛋白。(2)生产重组腺病毒载体较为经济、滴度高。(3)治疗量病毒的体积小，有助于在体内使用。第三部分Ad-p16、Ad-p53联合紫杉醇治疗乳腺癌的研究
目的正常细胞通过关卡作用以调节细胞周期的进程，保持细胞增殖和凋亡地平衡。发现癌基因和抑癌基因均是通过调控细胞周期而发挥作用。肿瘤的发生常涉及多个抑癌基因的突变、磷酸化或／和失活。将正常肿瘤抑制基因导入肿瘤细胞以替代失去功能的基因，而达到逆转其恶性表型、抑制肿瘤生长，甚至消灭肿瘤成为肿瘤基因治疗的热点。p16和p53是人体内重要的肿瘤抑制基因，直接或间接调控细胞周期，使细胞周期停滞于G1期。p16和p53基因发生缺失、突变、重排、磷酸化或不表达可存在于多种肿瘤的发生、发展过程中。恢复野生型p16和p53基因的功能成为抗肿瘤基因治疗研究的重要途径。紫杉醇可诱导多种肿瘤的凋亡，尤其对转移性乳腺癌的作用尤为引人注目。该研究对Tax、Ad-p16、Ad-p53及其联合应用对人乳腺癌细胞株MDA-MB-231的生长抑制和细胞凋亡进行深入研究。然后，应用Tax、Ad-p16、Ad-p53及其联合对乳腺癌细胞MDA-MB-231成瘤裸鼠模型进行动物治疗实验。观察治疗效果，为研究肿瘤治疗以及治疗途径提供依据。
方法显微镜下观察Ad-p16、Ad-p53及其联合对MDA-MB-231细胞形态学以及生长抑制的影响。MTT法监测Tax、Ad-p16、Ad-p53及联合应用对MDA-MB-231细胞生长的抑制率。应用流式细胞仪Annexin V-FITC／PI法检测分析Tax、Ad-p16、Ad-p53及其联合应用后MDA-MB-231细胞的凋亡率。应用流式细胞仪分析Tax、Ad-p16、Ad-p53及其联合对MDA-MB-231细胞周期和凋亡影响。裸鼠乳房脂肪垫内注射MDA-MB-231细胞悬浮液，使裸鼠形成乳腺癌模型。应用Ad-Lac Z、Tax、Ad-p16、Ad-p53、Tax+Ad-p16、Tax+Ad-p53、Ad-p16+Ad-p53和Tax+Ad-p16+Ad-p53对成瘤裸鼠进行治疗，观察肿瘤体积和抑瘤率的变化治疗，分析治疗效果。并对治疗后的肿瘤标本进行常规病理检查。应用脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测分析肿瘤组织细胞凋亡的状况。实验数据采用SPSS11.0统计软件包进行分析。
结果Ad-p16、Ad-p53和二者联合感染可导致人乳腺癌MDA-MB-231细胞生长抑制。Ad-p16对细胞生长的抑制作用比Ad-p53强，但导致的细胞形态学发生改变时间晚。Ad-p16和Ad-p53联合感染可导致细胞生长近乎停止，在细胞生长抑制和形态学改变均较单一感染明显且发生时间早。而Ad-Lac Z感染后细胞生长良好，细胞形态学无明显变化。Tax对MDA-MB-231细胞的抑制随着剂量的增加，细胞生长抑制率明显增加，细胞抑制率与时间呈正相关(r=0.923，P=0.000)。与Ad-Lac Z相比较，Ad-p16、Ad-p53以及二者联合感染MDA-MB-231细胞后，对肿瘤细胞均有明显的抑制作用(P值均为0.000)。Ad-p16的抑制率在各个时相均高于Ad-p53(P_(24h)=0.038，P_(48h)=0.004，P_(72h)=0.003，P_(96h)=0.001，P_(120h)=0000)。Ad-p16的抑制作用呈现逐渐上升趋势，而Ad-p53在72h时达到高峰后有下降趋势，但是均无统计学意义。Ad-p16与Ad-p53联合感染MDA-MB-231细胞后，在各个时相的细胞抑制率均显著大于单一感染的抑制率(vs Ad-p16，P值在0.001-0.004之间；vs Ad-p53，P值均为0.000)；并且随着时间的延长，细胞抑制率明显增加(120h vs72h，P=0.006)，与感染时间有明显的正相关(r=0.89，P=0.001)。Tax与Ad-p16联合作用于MDA-MB-231细胞后，在各个时相上细胞生长抑制率均显著大于单一应用，于96h时达到高峰，抑制率达到70.96％(70.96±3.30)(vs Ad-p16，P=0.001；vs Tax，P=0.000)；并且细胞抑制率随着时间的延长明显增加(120h vs 72h，P=0.028)。Tax与Ad-p53联合有同样效果，但与Tax与Ad-p16联合的抑制率在各个时相上相比较无显著性差异(P值在0.069-0.86之间)。Tax、Ad-p16和Ad-p53三者联合应用，对MDA-MB-231细胞的抑制作用更加明显，在各个时相上均有明显增加(P值均为0.000)，最高抑制率达到90％以上。Tax、Ad-p16或Ad-p53单一作用于MDA-MB-231细胞可诱导其发生凋亡，并且随着时间的延长细胞凋亡率明显增加，72h时分别达到15.31％、19.34％和29.05％。但是，感染Ad-p16的MDA-MB-231细胞发生凋亡较Tax和Ad-p53晚，凋亡率明显低于Ad-p53。Tax与Ad-p16或Ad-p53联合可明显增加MDA-MB-231细胞的凋亡率。Ad-p16与Ad-p53联合也可明显增加MDA-MB-231细胞的凋亡。Tax、Ad-p16和Ad-p53三者联合诱导MDA-MB-231细胞的能力明显提高，效果更加显著。Tax可导致MDA-MB-231细胞停滞于G2／M期，Ad-p16、Ad-p53或二者联合感染使MDA-MB-231细胞停滞于G0／G1期。Tax、Ad-p16和Ad-p53联合时，大多数细胞停滞于G0／G1和G2／M期。Tax、Ad-p16、Ad-p53及其联合作用于MDA-MB-231细胞72h后琼脂糖凝胶电泳呈现典型的凋亡梯形电泳带，而对照组仅仅呈现基因组DNA。Tax、Ad-p16和Ad-p53组裸鼠体内肿瘤明显受到抑制，第20天时与Ad-Lac Z组比较的P值均为0.000；各组较治疗前肿瘤体积虽有所减小，但是均无统计学意义(P值分别为0.216、0.895和0.138)。Ad-p16与Ad-p53联合应用不仅可以抑制肿瘤的增长，尚可以使肿瘤体积明显减小(0d vs 16d，P=0.034；0d vs 20d，P=0.007)。Tax、Ad-p16和Ad-p53三者联合，裸鼠肿瘤减小更加明显(0d vs 12d，P=0.041；0d vs 16d，P=0.003；0d vs 20d，P=0.000)。治疗组的肿瘤组织间质明显增加，肿瘤细胞以及细胞核分裂明显减少，细胞核呈现浅蓝色，细胞核与细胞浆比例减小，可见散在的新月形核染色质和细胞核碎裂的碎片。Ad-Lac Z、Tax、Ad-p16、Ad-p53、Ad-p16+Ad-p53和Tax+Ad-p16+Ad-p53组肿瘤组织细胞凋亡率(％)分别为1.4±0.9、14.7±2.6、19.5±2.5、21.3±3.1、30.5±3.2和42.3±4.3。
结论(1)Ad-p16、Ad-p53和二者联合感染可导致人乳腺癌MDA-MB-231细胞形态学改变和生长抑制。而此现象并非是腺病毒本身所致，而是外源性p16和p53基因所发挥的作用。(2)Tax、Ad-p16和Ad-p53在诱导MDA-MB-231细胞凋亡上有明显的协同作用，p16和p53基因的表达可以增加Tax对MDA-MB-231细胞的敏感性，p16基因可以促进p53基因所诱导的细胞凋亡。(3)Tax可导致MDA-MB-231细胞停滞于G2／M期，Ad-p16、Ad-p53或二者联合感染使MDA-MB-231细胞停滞于G0／G1期。Tax、Ad-p16和Ad-p53联合时，大多数细胞停滞于G0／G1和G2／M期。(4)Tax、Ad-p16和Ad-p53三者之间有协同抑制裸鼠体内肿瘤生长的作用，尤其是三者联合更加明显。在抑制肿瘤生长同时，可导致肿瘤组织细胞凋亡。
Part 1
Study on breast cancer p16 and p53 gene protein and mRNA expression
Objectives Breast cancer ranks the top among the malignant tumors of women. About 400,000 people die of breast cancer all over the world every year. Meanwhile, the incidence of breast cancer has been increasing annually and breast cancer patients tend to be younger. A great deal of research shows that abnormality of oncogene and tumor suppressor gene (antioncogene) is closely related to the occurrence and development of breast cancer, such as p16, p53, C-cerbB-2 and p21 genes, p16 antioncogene mutation and /or deficiency is widely available in various tumors, is involved in regulation of tumor cell proliferation and plays an important role in the occurrence and development process of tumor. Through immunohistochemical method, we found out that the positive rate of p16 gene protein expression varied between 24% and 60%. p53 antioncogene mutation is the most popular in the human tumors, and p53 is the gene with the closest relation with the human tumors according to the existing researches. It is known that the p53 gene mutation rate in breast cancer tissue is 15%～60%. Through testing the breast cancer tissue p16 and p53 gene protein and mRNA expression, we analyzed the correlation between the expression and the clinical pathological characteristics of breast cancer and explored the effects of the expression on the molecular biological characteristics of breast cancer to provide a theoretical foundation and basis for treatment and prognosis assessment of breast cancer in clinical and basic researches.
Methods We tested p16 and p53 gene protein and mRNA expression within the breast cancer tissue through immunohistochemical SP method and reverse transcription polymerase chain reaction (RT-PCR) technique and analyzed the relationship between the expressions and the clinical pathological characteristics of breast cancer through statistical methods.
Results Through immunohistochemical method we found out that the expression rate of p16 gene protein within the tumor-adjacent normal breast tissue was 90%(27/30), including 48.1%(13/27)(+++); 29.6% (8/27)(++); 22.2%(6/27)
(+) ; the expression rate of p53 gene protein was 6.67% (2/30) , all as (+) ; while the positive rate of p16 gene protein expression within the breast cancer tissue was 38.3% (23/60) , including 60.9% (14/23) (+++), 26.1% (6/23) (++) and 13.0%
(3/23) (+) ; the positive rate of p53 gene protein expression was 48.3% (29/60) , including 79.3% (23/29) (+++) , 13.8% (4/29) (++) and 6.9% (2/29) (+) .The results of RT-PCR test showed that pl6mRNA of tumor-adjacent normal breast tissue was obvious higher than that of breast cancer tissue (P=0.023), while p53mRNA of the breast cancer tissue was significantly higher than that of tumor-adjacent normal breast tissue (P=0.001) . Expression of pl6 and p53 proteins is closely related to histological grade of breast cancer tissue, and lymph node and/or organ metastasis. The higher the cell differentiation of breast cancer is, the higher the positive rate of p16 protein expression. The p53 expression rate was on the contrary. The cases with lymph node and/or organ metastasis showed significantly lower expression rate of p16 protein than that of those without lymph node and / or organ metastasis, while the expression rate of p53 protein in metastasis cases was significantly higher. p16mRNA at histological grade I was obviously higher than that at grade II and III, but no obvious difference was observed between the p16mRNA level at grade II and grade III. No obvious relation was found between p53mRNA and histological classification. The p16mRNA and p53mRNA expression rates for cases with lymph node and /or organ metastasis were obviously higher than those for cases without lymph node and / or organ metastasis.
Conclusion (1) Expression of p16 and p53 gene proteins is closely related to histological grade of breast cancer, and lymph node and/or organ metastasis. (2) Expression of pl6mRNA is related to histological grade of breast cancer. Expression of pl6mRNA and p53mRNA is related to lymph node and/or organ metastasis. (3) Deficiency, mutation or methylation, etc of p16 and p53 genes is closely related to the occurrence and development of breast cancer and can be used in prognosis assessment of breast cancer. (4) Exogenous wild-type p16 and p53 genes can be transfected through specific vectors to correct the abnormal changes of p16 and p53 genes and contribute to the treatment of breast cancer.
Part 2
Construction, identification, purification and determination of recombinant pl6 and p53 adenovirus
Objectives The study shows that the occurrence and development of malignant tumor is closely related to gene mutation, deficiency, phosphorylation, methylation, etc, and gene mutation plays an important role in malignant tumor cell phenotype. It is shown that transfection of exogenous normal target genes can be used to modify the mutant genes and serves as a tumor therapy. The key to designing a gene therapy is to construct an effective target gene vector system for specific transfer of target genes to target cells. The transfection efficiency of non-virus vectors within the body is far lower than that of virus vectors. Adenovirus vectors can effectively infect and transfer the target genes, and are widely applicable to both dividing and static tissues and cells, and the adenovirus DNA does not integrate with DNA of host cells. The disadvantages of adenovirus vectors include severe inflammation and immunoreaction if continuous expression of transgenes is maintained. But such immune characteristics may enhance the effects of target genes on killing tumors. Adenovirus vectors are easily constructed and a large number of adenovirus vectors may be cultured through helper cells. In this study, gene recombinant adenovirus method was adopted to construct recombinant adenovirus type 5 (Ad5) of sub-group C of antioncogene p16 and p53, which were identified, sifted and purified. Human embryonic kidney 293 cells were used for amplification, and the purification rate, virus titer, infection rate, transfection rate, and amplification of recombinant adenovirus p53 (Ad-p53) , p16 (Ad-p16) , as well as expression of p53 and p16 protein after cell infection was determined to provide sound recombinant adenovirus vectors for future experiments, namely Ad-p16 and Ad-p53.
Methods HindIII and SpeI enzymes were used for double enzyme digestion of pBluescript-p16 plasmids containing wild type p16cDNA; KpnI and XaI enzymes were used for double enzyme digestion of pGEM-p53 plasmid containing wild type p53cDNA, and then p16cDNA and p53cDNA were recovered through electrophoresis. HindIII/KpnI and SpeI enzymes were used for double enzyme digestion of pAdCMV shuttle plasmid and the recovered pl6cDNA and p53cDNA were then inserted into directional pAdCMV to get pAdCMV-p16 and pAdCMV-p53. Homologous recombination between pAdCMV-p53 recombinant plasmid and adenovirus plasmid pJM17 was made within 293 cells and p53 gene was transferred into the adenovirus gene. The generated El gene-defective recombinant adenovirus multiplied within the 293 cells and proliferated vigorously under transacting effects of El gene products. Adenovirus-, p16-, p53-specific primers were adopted for amplification and PCR amplification products were analyzed through agarose gel electrophoresis. Based on the length of specific fragments of PCR amplification products, the recombinant adenovirus plaques with positive p16cDNA, p53cDNA were selected. The virus in the positive plaques proliferated through subculture 293 cells. The viruses were purified through ultracentrifuge with cesium chloride density gradient. The viruses re-multiplied within 293 cells up to 5 generations to increase titer. 293 cell plaque formation testing was adopted to measure the recombinant adenovirus titer. Reporter gene Lac Z was used to measure the recombinant adenovirus infection rate. Immunohistochemical method (SP method) was used to test the efficiency of recombinant adenovirus-mediated gene transfer, and Western blot method was used to analyze expression of p53 and p16 protein.
Results pl6cDNA, p53cDNA were inserted into pAdCMV to get pAdCMV- p16, pAdCMV- p53. Through multiplication within 293 cells, cytopathic effect (CPE) , namely, typical virus plaque occurred after 10-14 days. Then we saw through a microscope that 293 cells rounded up, shrank and fell off, indicating homologous recombination between pAdCMV-pl6/p53 recombinant plasmids and adenovirus plasmid pJM17within 293 cells, p16/p53 gene transferred into adenovirus gene and the upper clean solution of culture media containing the recombinant adenovirus plasmids, with the prepared recombinant adenovirus respectively named Ad-p16, Ad-p53. From PCR amplification, there were 860bp adenovirus DNA segment, 570 bp p16cDNA segment, 642 bp p53cDNA segment, namely, the recombinant p53, pl6 adenovirus. The titer calculated after the plaque formation test was Ad-p53 2.1×10~(10); Ad-p16 6.7×10~(10); Ad-LacZ1.0×10~(10). When MOI of Ad-Lac Z was 25, 50 and 100, the infection rate of 293 cells was 32%, approached 78.9% and reached 98.6% respectively. Therefore, it is feasible to use recombinant adenovirus with MOI≥100 to infect human breast cancer MDA-MB-231 cell strain to observe the gene expression and apoptosis. Immunohistochemical staining results showed the recombinant adenovirus mediated gene transfection rate of pl6 being 93.8±2.5% and of p53 being 96.3±3.8%. No p53 or p16 protein expression was available in Ad-Lac Z infected tumor cells. However, tumor cells infected by both p53 and p16 showed obvious expression of p53 and p16 proteins, indicating that recombinant adenovirus could effectively mediate highly efficient transfer of exogenous genes.
Conclusion (1) The recombinant adenovirus can properly carry target genes into target cells and accurately express target gene protein. (2) It is economical to produce recombinant adenovirus vector with high titer. (3) The therapeutic dosage volume of virus is small and will be good for applications within the human body.
Part 3 Study on breast cancer therapy combining Ad-p16 and Ad-p53 with Tax
Objectives Normal cells rely on checkpoint function to regulate cell cycle progression and maintain balance between cell proliferation and apoptosis. We found that both oncogene and antioncogene act through regulation of cell cycle. Occurrence of tumor often involves mutation, phosphorylation and/or inactivation of a number of antioncogenes. It has become a popular tumor gene therapy to transduce normal tumor suppressor genes into tumor cells to replace dysfunctional genes to reverse malignant phenotype and inhibit tumor growth and even eliminate tumor, p16 and p53 are important tumor suppressor genes in human body and directly or indirectly regulate cell cycle to cause stasis in G1 stage. Deficiency, mutation, rearrangement, phosphorylation or non-expression of p16 and p53 genes may be present in the occurrence and development of various tumors. It has become an important way of anti-tumor gene therapy study to restore the functions of wild type p16 and p53 genes. Tax may induce apoptosis of various tumors, and is especially effective in treatment of metastatic breast cancer. In this study, in-depth research was conducted over the effects of Tax, Ad-p16, Ad-p53 and their combination on growth inhibition and cell apoptosis of human breast cancer cell strain MDA-MB-231. Then Tax, Ad-p16, Ad-p53 and their combination was applied to breast cancer cell MDA-MB-231 in the tumorigenesis model of nude mice for an animal therapy experiment. The therapeutic effects were observed to provide basis for tumor therapy and therapeutic approaches.
Methods The effects of Ad-p16, Ad-p53 and their combination on morphology and growth inhibition of MDA-MB-231 cells were observed through a microscope. MTT method was used to monitor and measure the growth inhibition rate of MDA-MB-231 cells applied with Tax, Ad-p16, Ad-p53 or their combination. The flow cytometer Annexin V-FITC/PI method was applied to test and analyze the apoptosis rate of MDA-MB-231 cells after application of Tax, Ad-p16, Ad-p53 and their combination. The flow cytometer was used to analyze the effects of Tax, Ad-p16, Ad-p53 and their combination on cell cycle and apoptosis of MDA-MB-231. MDA-MB-231 cell suspension was injected into the mammary fat pad of nude mice to form a breast cancer model in the nude mice. Ad-Lac Z, Tax, Ad-p16, Ad-p53, Tax+Ad-pl6, Tax+Ad-p53, Ad-pl6+ Ad-p53 and Tax+Ad-p16+Ad-p53 was used as a therapy for tumorigenesis in nude mice, and the changes in tumor size and tumor inhibition rate were observed and then the therapeutic effects were analyzed. In addition, routine pathological examination was made over the tumor specimens after treatment. Terminal deoxynucleotide transferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) method was used to test and analyze the apoptosis of tumor tissue cells. SPSS11.0 statistical software package was used to analyze the experiment data.
Results Ad-p16, .Ad-p53 or their combined infection can inhibit growth of human breast cancer cell MDA-MB-23. Ad-p16 was more effective in inhibiting cell growth than Ad-p53, but with morphologic changes occurring later. Combined Ad-p16 and Ad-p53 infection may almost stop cell growth, with obviously more effective cell growth inhibition and earlier morphologic changes compared with Ad-pl6 or Ad-p53 infection. In case of Ad-Lac Z infection, the cell grew well without obvious morphologic changes. With increasing dose of Tax, the inhibition effects on MDA-MB-231 cells increased with obviously higher inhibition rate and positive correlation between cell inhibition rate and time (r=0.923, P=0.000) . Compared with Ad-Lac Z, Ad-p16, Ad-p53 or their combined infection was obviously effective in inhibiting the tumor cells (P=0.000 in all cases) . The inhibition rate of Ad-p16 ]was higher than that of Ad-p53 in all the time phases (P_(24h)=0.038, P_(48h)=0.004, P_(72h)=0.003, P_(96h)=0.001, P_(120h)=0.000) . The inhibition effects of Ad-p16 tended to go up gradually, while the inhibition effects of Ad-p53 tended to go down after reaching the peak value at 72h, but without statistical significance in both cases. In case of combined Ad-p16 and Ad-p53 infection, the inhibition rate of MDA-MB-231 cells was higher than that of Ad-p16 or Ad-p53 infection in all the time phases (vs Ad-p16, P = 0.001-0.004; vs Ad-p53,P=0.000); and obviously increased with time( 120h vs72h, P=0.006) , indicating obvious positive correlation with infection time (r=0.89, P=0.001) . After application of combined Tax and Ad-p16 into MDA-MB-231 cells, the cell growth inhibition rate was notably higher than that of Tax or Ad-p16application in all the time phases and reached the peak value at 96h up to 70.96% (70.96±3.30) (vsAd-p16,P=0.001;vsTax,P=0.000) ; and notably increased with time (120h vs 72h,P=0.028) . Same effects were observed in case of combined application of Tax and Ad-p53 but without obvious difference of inhibition rate in various time phases (P = 0.069-0.86) compared with the inhibition rate in case of application of Tax and Ad-p16. Inhibition of MDA-MB-231 cells was more obvious in case of combined application of Tax, Ad-pl6 and Ad-p53, and increased in all the time phases (P = 0.000 in all phases) with the highest inhibition rate up to over 90%. Application of Tax, Ad-p16 or Ad-p53 to MDA-MB-231 cells may induce cell apoptosis and the apoptosis rate obviously increased with time and reached 15.31%, 19.34% and 29.05% respectively at 72h. However, Ad-p16-infected MDA-MB-231 cell apoptosis occurred later than apoptosis of Tax- or Ad-p53-infected cells with apoptosis rate for Ad-p16 obviously lower than that for Ad-p53. Application of combined Tax and Ad-p16 or Ad-p53 can obviously increase the apoptosis rate of MDA-MB-231 cells, and combined application of Ad-p16 and Ad-p53 can also increase apoptosis of MDA-MB-231 cells. Application of combined Tax, Ad-p16 and Ad-p53 obviously improved the ability to induce MDA-MB-231 cell apoptosis with more notable effects. Tax may cause MDA-MB-231 cell stasis in G2/M stage, while application of Ad-p16, Ad-p53 or their combined infection may cause MDA-MB-231 cell stasis in G0/G1 stage. Application of combined Tax, Ad-p16 and Ad-p53 caused stasis of most cells in G0/G1 and G2/M stage. In case of application of Tax, Ad-p16, Ad-p53 or their combination into MDA-MB-231 cells, 72h agarose gel electrophoresis showed typical apoptosis trapezoidal belt, while the control group only showed gene group DNA. The tumors in the nude mice were obviously inhibited for Tax, Ad-p16 and Ad-p53 group, and P = 0.000 for all cases at the 20~(th) day compared with Ad-Lac Z group. Although the tumor size decreased for all groups compared with pre-treatment but without statistical significance (P = 0.216, 0.895, 0.138 respectively) . Application of combined Ad-p16 and Ad-p53 can both inhibit tumor growth and obviously reduce tumor size (0d vs 20d,P=0.034; 0d vs 20d,P=0.007). Application of combined Tax, Ad-pl6 and Ad-p53 more obviously reduced tumor size (0d vs 12d, P=0.041; 0d vs 16d, P=0.003; 0d vs 20d, P=0.000) . For the therapy group, tumor tissue stroma increased obviously and the tumor cells and nuclear division decreased notably with light blue nucleus, decreasing ratio between nucleus and cell plasma and scattered crescent chromatin and nucleus fragments visible. The tumor tissue cell apoptosis rate was 1.4±0.9%, 14.7±2.6%, 19.5±2.5%, 21.3±3.1%, 30.5±3.2% and 42.3±4.3 respectively in case of application of Ad-Lac Z, Tax, Ad-p16, Ad-p53, Ad-p16+Ad-p53 and Tax+Ad-p16+Ad-p53.
Conclusion (1) Ad-p16, Ad-p53 or combined application of both can contribute to morphologic change and growth inhibition of oncogenic MDA-MB-231 cells, while this is due to the functions of exogenous p16 and p53 genes, instead of adenovirus virus. (2) There is obvious synergetic effect of Tax, Ad-p16 and Ad-p53 in inducing apoptosis of MDA-MB-231 cells, expression of p16 and p53 genes can enhance sensitivity of Tax to MDA-MB-231 cells, and p16 genes can contribute to apoptosis induced by p53 genes. (3) Tax can cause MDA-MB-231 cell stasis in G2/M stage, while Ad-p16 infection, Ad-p53 infection or combined Ad-pl6 and Ad-p53 infection can cause MDA-MB-231 cell stasis in G0/G1 stage. Action of combined Tax, Ad-p16 and Ad-p53 causes stasis in G0/G1 stage and G2/M stage of most cells. (4) Tax, Ad-p16 and Ad-p53 have synergetic effects of inhibiting tumor growth in Balb/c nu/nu, and the synergetic effects of combined Tax, Ad-p16 and Ad-p53 are more obvious. Besides inhibiting the tumor growth, they can also cause apoptosis of tumor cells.
引文
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