摘要
背景：肺癌是一种对人类健康和生命威胁极大的恶性肿瘤，其发生与多种因素有关。大量的遗传学和分子生物学研究表明p53基因与肺癌的发生密切相关，在非小细胞肺癌中其突变率为47.8％，小细胞肺癌中其突变率为80％左右。p73基因是p53基因家族的新成员，蛋白序列与p53蛋白高度同源，也定位于细胞核内，现已发现有p73α、p73β、p73γ、p73δ、p73ε、p73Φ六种变异体。△Np73基因是一种缩短了的p73基因，它缺乏转录激活区。血管生成是实体瘤生长和转移的基础。当肿瘤体积增至2mm~3左右时，已不能依靠弥散获取氧气和营养物质，如无新生血管长入，肿瘤组织将保持休眠状态或发生退化。一旦血管长入肿瘤，这些血管将为肿瘤提供养料和排泄代谢产物，肿瘤在生长过程中也分泌多种物质来促进新生血管的进一步形成，这些丰富的毛细血管也为肿瘤细胞侵袭进入循环系统提供了良好的机会。血管生成过程包括：血管内皮基底膜溶解，内皮细胞向肿瘤内迁移，内皮细胞的增殖，内皮细胞形成管道，小分支形成血管网络，形成新的血管基底膜。这一复杂过程是肿瘤细胞、血管内皮细胞以及细胞外基质微环境相互作用的结果，受多种促血管生成因子和血管生成抑制因子的调节。已有研究表明，p73基因与某些肿瘤的血管生成密切相关。目前的研究表明p73基因在非小细胞肺癌中表达增高，而且很少发生突变。但是这种高表达对肺癌的发展，尤其是在肺癌血管生成方面起什么作用，目前尚不清楚。
目的：了解高表达p73基因在人类肺腺癌血管生成方面的作用，以及p73基因不同变异体之间功能的差异，并且为非小细胞肺癌的基因治疗提供一种新的方法。
方法：A549细胞株(wtp53)、H1299细胞株(p53-null)是两种人类肺腺癌细胞株。p73α、p73β、△Np73基因构建于真核表达质粒pcDNA3，通过脂质体基因转染的方法将外源性p73基因转染以上两种细胞，用G418筛选过表达p73基因的细胞克隆，RT-PCR和western blot对所筛选的细胞克隆进行鉴定。将筛选得到的过表达p73基因细胞株放大培养，观察p73基因不同的异构体对两种细胞株生长曲线的影响。应用逆转录多聚酶链反应(RT-PCR)技术，检测转染前后以上两种肿瘤细胞中VEGF、bFGF、TSP-1的mRNA表达水平的变化，western-blot法检测转染前后肿瘤细胞中VEGF、bFGF蛋白表达水平的变化，观察过表达的p73基因对肿瘤细胞产生血管生成相关因子的作
第三军医大学博士学位论文
用。将过表达p73基因的A549细胞以及对照组的A549细胞接种于裸鼠，观察种植瘤
的大小、生长速度，免疫组化检测种植瘤中vEGF、bFGF、iNos的表达程度，Fvln一RAg
免疫组化染色计数种植瘤微血管密度(M vD)，观察过表达p73基因对裸鼠种植瘤血管
生成的影响。应用SPSS 10.0统计软件进行统计分析。
结果:将p73a、p73p、△Np73基因以脂质体法转染A549细胞、H1299细胞后，
经G418成功的筛选出过表达p73基因细胞株。细胞生长曲线的测定发现过表达的p73
Q、p73p基因能够抑制以上两种细胞的生长，而过表达的△Np73基因可以促进以上
两种细胞生长。RT一PCR半定量法检测以上两种肺腺癌细胞中VEGF、bFGF、TSP一l
mRNA的表达，发现过表达的p73a基因能够降低vEGF、bFGF mRN^的表达(p<0.05)，
增加TsP一1 mRN^的表达(p<0.05);westem blot半定量法检测vEGF、bFGF蛋白的表
达，发现过表达的p73“基因能够降低以上两种肺腺癌细胞中vEGF、bFGF蛋白的表
达水平(P<0.05)。过表达的p73p作用与p73。相似，但是其对vEGF mRNA和蛋白表
达的抑制作用更明显(P<0.01)。而过表达△Np73基因的作用与p73。、p73p相反，其
功能更像一个癌基因。免疫组织化学方法检测裸鼠种植瘤VEGF、bFGF、iNOS蛋白
的表达，发现过表达p73。基因能够降低种植瘤中vEGF、bFGF、iNos蛋白表达水平
(P<0 .05)，减少裸鼠种植瘤MVD(p<0.01)，从而抑制裸鼠种植瘤生长。过表达△Np73
基因在裸鼠种植瘤中的作用与过表达p73Q相反，能够促进裸鼠种植瘤的生长。
结论:过表达的p73“、p73p基因在肺腺癌中可以起到抑制肿瘤血管生成的作用，
其中p73p基因的作用更为明显。而△Np73基因的作用与p73。、p73p基因相反，能
够促进肿瘤血管生成，它的表现象是一个癌基因。利用p73基因在非小细胞肺癌很少
突变的这一现象，可以人工诱导p73基因的高表达，尤其是p73p的高表达，通过其
能抑制肿瘤血管生成这一作用，来抑制肺癌的发展，为非小细胞肺癌的基因治疗提供
了一种新的选择。
Background: Lung cancer is extremely harmful to the life and health of human beings. Its genesis is related to multiple factors. It has been proved that there is approximately 47.8% in NSCLC and 80% in SCLC with p53 gene mutation. p73 gene is a homologue of p53. p73 protein has the similary structure to p53 protein and it is located in the nucleus too. p73 gene has some kinds of variants, such as p73 a , p73 P , p73 Y , p73 5 , p73 and p73 4. A Np73 gene is a kind of shortening p73 which lack the transcriptional activation domain. p73 gene is located in human chromosome Ip36 where was found delete frequently in many human tumors. All these imply that p73 gene may be an anti-oncogene and some researches have proved it can inhibit cell growth and induce cell apoptosis.
Angiogenesis is a process that form the new blood vessels from preexisting micro-vascular. The tumor can't grow beyond 2~3mm3 for lack of oxygen and nutrient without angiogenesis. With angiogenesis the tumor can obtain nutrition and the capillaries can provide a chance for the tumor metastasis. The process includes the following steps: dissolve the vascular endothelial basal membrane, endothelium cell migrate to tumor tissue, endothelium cell proliferate, form new capillary, form new basal membrane. This complex process involves the interaction of tumor cells, endothelial cell and extracellular matrix. Its regulation depends on many cytokines. Some cytokines have been separated and purified, such as VEGF, bFGF, angiogenin, TSP-1, iNOS and so on. The effect of p73 gene on angiogenesis in some human tumors has been reported. It has been proved that p73 gene was overexpression in NSCLC and nearly hasn't mutation. But the effect of p73 gene on angiogenesis in NSCLC is still unknown.
Objective: To study the effect of p73 gene overexpression on angiogenesis in human lung adenocarcinoma and the difference function between the p73 variants. Then find a new way for NSCLC gene therapy.
Methods: This study use A549 cell, HI299 cell, 2 kinds of lung adenocarcinoma cell
lines as a study object. p73oc, p73 0 and A Np73 gene were transfered into these 2 kinds of cells by liposome and the overexpresion of p73 gene cell clones were chosen by G418. The overexpression of different p73 varients cells were cultured and their effects on the cell growth curve were observed. The mRNA expression of VEGF^ bFGF and TSP-1 were detected by RT-PCR, the protein expression of VEGF^ bFGF were detected by western blot in vitro. A549 cell, A549-p73oc cell and A549-ANp73 cell were transplanted into nude mice. The effect of p73 gene on angiogenesis in transplanted tumor was studied. The volume and growth speed of transplanted tumors were observed. VEGF> bFGF and iNOS protein expression in transplanted tumors were detected by immunohistochemistry staining. MVD of transplanted tumors were counted by FVTII-RAg immunohistochemistry staining.
Results: Overexpression of p73 gene cell clones were chosen successfully. The cell growth curve demonstrated that overexpression of p73cc and p73 P could inhibit the cell growth but the A Np73 could promote cell growth. Overexpression of p73a could inhibit VEGF, bFGF mRNA and protein expression and promote TSP-1 mRNA expression(p<0.05). The overexpression of p73 3 has similar function with p73a but has more obviously effect on inhibiting VEGF expression(p<0.01). Overexpression of p73ot can inhibit nude mice transplanted tumor growth, inhibit VEGF, bFGF, iNOS protein expression(p<0.05), reduced the tumor MVD(p<0.01). A Np73 gene has the contrary function of p73a in vitro and in vivo, its behavior is more like an oncogene.
Conclusion: Overexpression of p73cc and p73 P can inhibit the angiogenesis in human lung adenocarcinoma. The p73 P may has more obviously effect on inhibiting angiogenesis. A Np73 gene has the contrary effect of p73cc, p73 P on angiogenesis, it seems like an oncogene. We can apply the characteristic that p73 gene nearly hasn't mutation in NSCLC to induce p73 gene overexpression in lung adenocarcinoma, especially the p73 P overexpress
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