【作者】 王旭；

【导师】 王洁；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2005， 博士

【摘要】 目的:涎腺多形性腺瘤(pleomorphic adenoma of salivery gland)是口腔颌面部最常见的肿瘤之一,约占全部涎腺肿瘤的50%以上。由于涎腺多形性腺瘤累及面部,影响面容,发生在腮腺的肿瘤常常危害面神经,容易造成面瘫。该肿瘤在生物学行为上虽属于良性,但多数肿瘤包膜不完整甚至无包膜。临床上涎腺多形性腺瘤具有好种植、易复发的特点,反复复发可造成恶性转变。目前手术治疗对于复发性肿瘤效果并不理想,因此寻求一种辅助治疗方法是很有必要的,尤其是防止肿瘤的复发及恶性变。随着肿瘤发生的分子机制研究的深入,人们已逐渐认识到肿瘤的发生是一个多因素、多基因参与的复杂过程。其中抑癌基因的失活和癌基因的激活是最基本的分子事件。研究表明,c-erbB-2、H-ras、p53、bcl-2 等多种癌基因和抑癌基因的异常与涎腺多形性腺瘤的发生有关。其中抑癌基因p53 的突变是涎腺多形性腺瘤中最常见的基因改变。采用多聚酶链式反应(PCR)、免疫组织化学及流式细胞等技术研究表明, p53 基因蛋白的过度表达率在涎腺多形性腺瘤中为83%,单股构象多态分析研究显示, p53基因的突变率在涎腺多形性腺瘤为41%。在涎腺多形性腺瘤p53 基因的突变中,尤其以点突变最为常见。核酸序列分析发现在该肿瘤发生中, p53基因的第5~8 外显子均有突变。同一肿瘤可出现多个密码子的点突变。此外,p53 基因的缺失突变和基因重排也在涎腺多形性腺瘤中被检测出来。基因治疗是近年来肿瘤学研究的热点,它是针对肿瘤发生的分子生物学特点,通过基因转移技术,将外源目的基因转入肿瘤细胞中以纠正或补偿肿瘤细胞内源性的基因缺陷,从而达到治疗的目的。野生型p53 (wt-p53) 基因具有细胞周期调控,细胞生长抑制,促进肿瘤细胞凋亡等功能。将外源性wt-p53 基因导入肿瘤细胞内,以恢复p53 基因功能,发挥其对肿瘤细胞的抑制作用。这项研究在目前肿瘤抑癌基因治疗中最引人瞩目,并且已经进入临床试验阶段。单纯疱疹病毒胸苷激酶(herpesl simplexvirus thymidinekinase HSV-tk) 丙氧鸟苷(gancyclovir,GCV)系统是通过GCV在细胞内被HSV-tk 的产物胸苷激酶磷酸化成GCV 磷酸,参与细胞DNA更多还原

【Abstract】 Objectives:Salivary pleomorphic adenoma （SPA） is one of the most common tumors of salivary gland, accounting for 50% of the total epithelial tumors. This tumor usually involves the oral maxillofacial regions and affects the facial features. SPA occurred in parotid could destroy the facial nerve and result in facial paralysis. Although benign and slow-growing painless masses, some tumors have no an entire or even no capsule. So, the tumor is easy to relapse and implant after surgery, furthermore it tends to malignant transformation. Up to now, the surgery is not reasonable for the recurrence. A new approach for the treatment of SPA has to be developed, especial for the recurrence. With the progress of research for oncogene, it has been realized that tumourigenesis is a complicated procedure involved multiple factor and polygene mutations. Current studies showed that some oncogenes such as H-ras, wt-p53, bcl-2 was nearly associated with development of SPA and mutation of anti-oncogene wt-p53 was most common occurred. The p53 protein was investigated by using polymerase chain reaction （PCR）, immunohistochemical staining and single strand conformational polymorphism （SSCP） analysis. The mutation rate of wt-p53 in SPA was 83% immunohistochemically and 41% by SSCP. The sequencing analysis revealed that the mutation of wt-p53 in SPA showed poly-codon mutation and poly-pattern mutations, chiefly showed point mutations in exon 5⁸. Gene therapy mainly introduced exogenous gene into tumors by using gene transfer technique to compensate deficiency of endogenous gene. The tumor suppressor activity of wt-p53 is mainly mediated through its ability to cause cell growth arrest or apoptosis, whereas a lack of functional wt-p53 usually leads to increased genomic instability, deregulated cell proliferation, accelerated tumor progression. Several reports have suggested that the p53 status in tumor cells is responsible for the biological behavior. The tumors with deficiency of functional wt-p53 transduced by adenovirus mediated human wt-p53 gene has been shown a significant suppressing effect in the pro-clinical studies and in clinical trials. Herpes simplex virus thymidine kinase/ganciclovir （HSV-TK/GCV） method is one of the most frequently utilized forms of gene therapy. GCV, a kind of antiviral drug, can be phosphorylated by HSV-tk protein, and finally converted into triphosphates, which are potent inhibitors of DNA polymerase, leading to the disruption of cellar DNA synthesis and ultimately cell death. Experiments showed that HSV-tk gene causes efficiently anti-tumor effect in transduced tumor cells. However, suicide gene therapy alone cannot completely eliminate tumor cells, so it is necessary to combine suicide gene therapy with other therapy to improve performance. In order to explore the feasibility of combing HSV-tk and wt-p53 gene in SPA therapy, we used adenoviral vector containing HSV-tk and wt-p53 gene to transfer SPA cells and to evaluate the possibility of such a strategy as combing gene therapy in vitro. Methods 1. Culture of SPA cells and identification:Specimens of 4 SPA were obtained from surgical resection, tumor tissues were placed in RPMI1640 supplemented with 15% fetal calf serum （FCS）, streptomycin （500ug/ml） and penicillin （1000U/ml）, for 30 minutes. They were rid of necrotic tissue, plasma clot and mucosa, then minced into fine pieces and washed with PBS. Fragmental tissues were planted in culture flasks and incubated in 5%CO2 and 37℃. The medium was added or changed according to the variation of pH. When the cells reached 80% confluency, they were harvested by trypsinization which was adopted to get rid of fibroblasts through 2³ times. The pure tumor cells were obtained and evaluated by Giemsa stain. Immunohistochemistry and transmission electronic micoscope （TEM） were used to identify the histological features of SPA cells. 2. Exogenous HSV-tk and wt-p53 expression analysis:SPA cells weretransduced with 102 MOI Ad-wt-p53 and HSV-tk gene at 37℃and 5%CO₂ incubation. After 24h incubation, cells were harvested and washed with PBS. Total RNA was extracted from 5×10⁶ using SK1351 （Gibco）. Then cDNA was generated by using the QuantiTectTM SYBR Green RT-PCR （Qiagen, germany）, PCR was performed by an initial heating step at 50°C for 30 min, followed by 40 cycles at 94°C for 15 second, at 55°C for 30 second and at 72°C for 30 second. The PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining and UV light. 3. Endogenous p53 gene status of SPA after wt-p53 infection:Cells were infected with Ad-wt-p53 at an MOI of 200 for 24h. After 24h incubation period, the cells were harvested and washed with cold PBS, then centrifuged for 10min at 8000r/min, DNA was extracted from 5×10⁶ cultured cells. Simultaneously, the corresponding tumor tissue DNA was extracted, the oligonucleotide primers of wt-p53 were designed. The condition for amplification was 94℃45 seconds,56℃45 seconds and 72℃45 seconds for 35 cycles. The PCR products were loaded onto 10% nondenaturing polyacrylamidedel gels and run at room temperature for 10¹2h. Staining of the single-and-double-stranded DNA was performed according to a silver staining protocol. Then DNA sequencing analysis was used to determined. 4. The Effects of HSV-tk suicide gene and wt-p53 gene on SPA:SPA cells were seeded in 96 well plates at a density of 4×10³cells per well, transfection were performed after the cells adhered, the cells were divided into 4 groups treated by wt-p53, HSV-tk /GCV, wt-p53+HSV-tk /GCV respectively. After 5 days, the plates were taken out and cells were added with 20μl of 5g/L MTT to each well, culturing was continued for 4h and the supernatant was removed. Add 150μl DMSO to react for 15 minutes. Absorbance was measured at 570nm by using a spectrophotometer. The growth inhibition ratio was calculated. Bystand effect was determined by mixing no infected cells and infected cells at varying rations. Thepercentage of infected cells was 0%, 25%, 50%, 75% and 100%. Cell mixtures were plated at 4×10³ cells per well. The survival ratios were measured. Results 1. The growth status and morphologic feature of SPA :Tumor tissue had the histologic features of SPA, they were characterized by a lobular architecture consisting of loose chondromyxoid stroma. The cellular component was made up of small rounded epithelial cells and slightly spindled myoepithelial cells. Both these cell types could either lie loose in the stroma or form cysts and ducts with a lining of epithelial cells surrounded by a more solid mass of myoepithelial cells. Under inverted microscope, tumor cells were large spindle-shaped or polygonal, nuclear was round or oval. In primary culture, cells overlapped growth and formed “cell clusters”. Growth curve of the cells was gentle and doubling time was longer than that of malignancy tumors. On the 5th, 6th days, the curve reached a peak. Histologic observation: Cells were stained by Giemsa stain, adenoepithelial cells were round or polygonal, the cells were arranged cluster, and formed gland lumen, there were much mucus in cytoplasm of the cells. Myoepithelial cells were spindle, arranged in mass with some mucoid substance around them. Immunohistochemical stain showed that cytokeratin was labelled in columnar-shaped cells, and spindle -shaped cells were positive for the smooth muscle actin. It suggested adenoepithelial and myoepithelial natures. 2. Detection of HSV-tk and gene expression:Amplification fragment 1134 bp （wt-p53） and 1150 bp （TK） could be seen in the experimental group, while none was found in the control group. The results suggested that the exogenous HSV-tk and wt-p53 gene had been transferred and expressed by SPA cells. 3. Endogenous wt-p53 gene status of SPA after wt-p53 infection:PCR-SSCP analysis showed that 2 out of 4 SPA with abnormal exon 8. Comparison of SSCP with DNA sequencing analysis, the results showed that all samples更多还原