SLPI启动子调控靶向EGFR的人工microRNA用于喉癌的基因治疗研究
发布时间：2018-09-19 15:37
【摘要】：研究背景： 喉癌是上呼吸道最常见的恶性肿瘤,占头颈部肿瘤的25%左右。传统的喉癌治疗手段为根治性手术或放射治疗,辅以或不辅以化学治疗,但是传统治疗手段导致的喉功能部分甚至全部丧失或严重的副反应极大地影响了患者的生存质量,且仍有部分患者出现治疗后复发。尽管手术术式、放疗方法及化疗方案在革新中不断进步,近30年来患者的生存率并无明显改善。而失去根治性治疗机会的晚期患者多行姑息性化疗,长期以来该期患者的5年生存率始终停留在30%左右。 近20年来,分子靶向治疗作为一种新型的治疗手段在肿瘤的治疗中取得了重大突破。对于头颈部鳞状细胞癌(Head and Neck Squamous Cell Carcinoma, HNSCC),最具代表性的分子靶向药物为特异性抑制表皮生长因子受体(Epidermal Growth Factor Receptor, EGFR)的单克隆抗体及小分子酪氨酸激酶抑制剂。然而以吉非替尼为代表的小分子抑制剂对HNSCC并无明确疗效,而以西妥西单抗为代表的单克隆抗体虽然对传统的放、化疗起到一定的辅助作用,然而由于有限的反应率,较高的耐药率及频发的皮肤毒性、消化道症状等副作用使得该类药物的应用空间仍然不大。 随着分子克隆技术的进步,以病毒为表达载体,通过RNA干扰技术特异性下调EGFR表达的基因治疗策略为肿瘤的治疗开辟了新的方向。而随着RNA干扰技术的不断改进,目前已出现人工microRNA (artificial microRNA, amiR),即第二代shRNA,与siRNA及第一代shRNA相比,人工microRNA在保留第一代shRNA发夹结构的基础上,加入了天然microRNA的框架,除了更高效之外,最大的优势在于可以被哺乳动物体内大多数的启动子所启动。而人工microRNA的这一优势使得采用肿瘤组织特异性启动子来调控靶向EGFR的RNA干扰成为可能。 基于以上研究背景,我们拟设计以EGFR为靶点的人工microRNA,并利用重组腺病毒为载体,通过喉癌特异性的SLPI启动子调控该人工microRNA的表达,并以Hep-2为研究对象,探讨该基因治疗策略抑制肿瘤生长的作用。 研究目的： 构建荷载有在SLPI启动子调控下的靶向EGFR的人工microRNA的重组腺病毒载体,并研究其安全性及对喉癌细胞的体内外抑制作用。 研究方法： 1、构建腺病毒穿梭质粒pDC312-SLPI-EGFRamiR-pA, Ad-SLPI-GFP-pA,分别与骨架质粒pBGHlox (delta) E1,3Cre共转染HEK293细胞进行腺病毒包装。以PCR, western blot等方法鉴定重组腺病毒Ad-SLPI-EGFRamiR, Ad-SLPI-GFP。扩增病毒,并以CsCl密度梯度离心法纯化扩增的腺病毒,TCID50法测定病毒滴度。 2、将重组腺病毒Ad-SLPI-EGFRamiR及对照病毒Ad-SLPI-GFP分别感染人喉鳞状上皮癌细胞株Hep-2和人正常脐静脉内皮细胞株HuVEC,通过显微镜观察,MTT,流式细胞仪细胞凋亡分析检测病毒对喉癌细胞Hep-2及正常细胞HuVEC增殖的影响。 3、建立裸鼠喉癌荷瘤模型,予瘤内注射重组腺病毒Ad-SLPI-EGFRamiR、 Ad-SLPI-GFP,或每日口服吉非替尼。比较治疗过程中各组瘤体体积变化及观察期结束后的瘤体重量。观察治疗过程中裸鼠的饮食、活动及精神状态,并比较裸鼠体重的变化情况。 研究结果： 1重组腺病毒Ad-SLPI-EGFRamiR, Ad-SLPI-GFP的包装,鉴定、扩增、纯化及滴度测定 腺病毒穿梭质粒pDC312-SLPI-EGFRamiR-pA、pDC312-SLPI-GFP-pA分别与骨架质粒pBGHlox (delta) E1,3Cre共转染HEK293细胞,转染第13天左右,倒置显微镜下观察,发现80%以上的细胞都发生细胞病变效应,呈葡萄状,贴壁不牢易脱落。收集感染细胞,反复冻融,病毒上清经蛋白酶K处理后,PCR检测示Ad-SLPI-EGFRamiR扩增出不同重复序列数的EGFRamiR片段,142bp、284bp、426bp片段,Ad-SLPI-GFP扩增出1482bp片段,示重组腺病毒Ad-SLPI-EGFRamiR及对照病毒Ad-SLPI-GFP构建成功。予扩增纯化后进行滴度测定。Ad-SLPI-EGFRamiR滴度为1×1010pfu/ml, Ad-SLPI-GFP滴度为6.3×109pfu/ml。 Western blot结果显示,Ad-SLPI-EGFRamiR病毒作用72小时的Hep-2细胞,170kdEGFR表达显著降低。而Ad-SLPI-GFP感染72小时后,荧光显微镜下可见大量Hep-2细胞呈现较强的绿色荧光信号,而HuVEC细胞仍无绿色荧光。以上结果表明,SLPI启动子能够在喉癌细胞内有效下调EGFR,并特异性调控绿荧光蛋白在人喉鳞状细胞癌细胞株Hep-2细胞中表达,而在正常人脐静脉内皮细胞HuVEC中无表达,72小时为合适的感染时间。 2重组腺病毒Ad-SLPI-EGFRamiR对喉癌细胞Hep-2的体外抑制作用 2.1重组腺病毒对细胞增殖抑制作用的试验 MTT结果显示,感染72小时后重组腺病毒Ad-SLPI-EGFRamiR对喉癌细胞Hep-2的增殖有较强的抑制作用,在MOI为50pfu/cell时就能有效地抑制喉癌细胞的增殖(抑制率为22.5%),但对正常细胞HuVEC的增殖却无明显抑制作用(抑制率为-4.2%)。 2.2重组腺病毒作用72小时后细胞形态学变化 重组腺病毒Ad-SLPI-EGFRamiR (MOI=50)作用72小时后的喉癌细胞Hep-2在形态学上发生了显著的变化：细胞变圆,皱缩,部分呈串珠状、浮起。而正常细胞HuVEC在病毒作用前后细胞形态差异不明显。而对照病毒Ad-SLPI-GFP (MOI=50)作用前后,Hep-2及HuVEC细胞形态均无明显差异。 2.3流式细胞仪定量分析细胞凋亡 流式细胞仪检测显示重组腺病毒Ad-SLPI-EGFRamiR在MOI=35及MOI=50下作用72小时后,Hep-2细胞的凋亡率(层Annexin V-R-PE和7-AAD均染色的细胞)分别为32.8%和31.8%,而Ad-SLPI-GFP作用72小时后相应的凋亡率分别为9.2%和10.0%,两种病毒差异显著,从定量角度说明重组腺病毒Ad-SLPI-EGFRamiR能通过有效诱导凋亡而对喉癌细胞的生长增殖起抑制作用。而对于正常脐静脉内皮细胞HuVEC,在MOI=35的Ad-SLPI-EGFRamiR, Ad-SLPI-GFP作用72小时后的凋亡率分别为11.1%,8.2%,在MOI=50的相应病毒作用下凋亡率分别为15.5%,4.8%,差异均不显著。 3重组腺病毒Ad-SLPI-EGFRamiR对喉癌荷瘤模型肿瘤组织生长的抑制作用 3.1重组腺病毒Ad-SLPI-EGFRamiR的体内抑制肿瘤生长作用 在首次治疗后第13天,Ad-SLPI-EGFRamiR治疗组的瘤体体积小于Ad-SLPI-GFP组及吉非替尼组,但是这一差别无统计学上的显著意义(P0.05)。与首次治疗日的瘤体体积相比,Ad-SLPI-EGFRamiR组的瘤体体积增长速度小于Ad-SLPI-GFP组及吉非替尼组,但是仍无统计学上的显著意义(P0.05)。在观察结束时(首次给药后第20天)Ad-SLPI-EGFRamiR组瘤重小于Ad-SLPI-GFP组及吉非替尼组,但这一差别无统计学上的显著意义(P0.05)。 3.2重组腺病毒Ad-SLPI-EGFRamiR的体内不良反应观察 在治疗过程中,Ad-SLPI-EGFRamiR组及Ad-SLPI-GFP组裸鼠的身体状况良好,精神状态、活动及饮食情况均无明显异常。但是吉非替尼组的部分小鼠出现精神萎靡、腹泻、食欲不振情况。Ad-SLPI-EGFRamiR组裸鼠的体重增长率与Ad-SLPI-GFP组裸鼠相比无明显差异(P0.05),但是却明显高于吉非替尼组(P0.05)。 研究结论： 1、重组腺病毒Ad-SLPI-EGFRamiR和Ad-SLPI-GFP均能有效感染Hep-2细胞,并表达相应的基因,具有良好的喉癌组织特异性。 2、重组腺病毒Ad-SLPI-EGFRamiR在体外能显著地特异性抑制喉癌细胞的生长,而对正常细胞抑制作用较低。 3、在荷瘤裸鼠模型中,重组腺病毒Ad-SLPI-EGFRamiR有抑制肿瘤组织生长的趋势。 4、在荷瘤裸鼠模型中,重组腺病毒与吉非替尼相比具有较高的安全性。
[Abstract]:Research background:
Laryngeal cancer is the most common malignant tumor of the upper respiratory tract, accounting for about 25% of the head and neck tumors. The traditional treatment of laryngeal cancer is radical surgery or radiotherapy, supplemented by or not supplemented by chemotherapy. However, the loss of laryngeal function or serious side effects caused by traditional treatment greatly affect the quality of life of patients. Although surgical procedures, radiotherapy and chemotherapy regimens have progressed steadily, the survival rate of patients has not improved significantly in the past 30 years.
In the past 20 years, molecular targeted therapy has made great breakthroughs in the treatment of tumors. For head and neck squamous cell carcinoma (HNSCC), the most representative molecular targeted drug is the specific inhibitor of epidermal growth factor receptor (EGF). R) Monoclonal antibodies and small molecule tyrosine kinase inhibitors. However, the small molecule inhibitors represented by gefitinib have no definite effect on HNSCC. While the monoclonal antibodies represented by cetuximab play an auxiliary role in traditional radiotherapy and chemotherapy, however, due to the limited response rate, high drug resistance rate and frequent occurrence. Skin toxicity, gastrointestinal symptoms and other side effects make the application space of these drugs still small.
With the advancement of molecular cloning technology, gene therapy strategy with virus as expression vector and down-regulation of EGFR expression by RNA interference technology has opened up a new direction for tumor treatment. Compared with shRNA, artificial microRNAs incorporate a natural microRNA framework based on retaining the hairpin structure of the first generation of shRNA. In addition to being more efficient, the greatest advantage is that they can be activated by most of the promoters in mammals. This advantage of artificial microRNAs enables the use of tumor tissue-specific promoters to regulate targets. RNA interference with EGFR becomes possible.
Based on the above research background, we intend to design an artificial microRNA targeting EGFR, and use recombinant adenovirus as vector to regulate the expression of the artificial microRNA through the specific SLPI promoter of laryngeal cancer. We also take Hep-2 as the research object to explore the effect of this gene therapy strategy on tumor growth.
Research purposes:
To construct recombinant adenovirus vector loaded with artificial microRNA targeting EGFR under the control of SLPI promoter, and to study its safety and inhibitory effect on laryngeal cancer cells in vitro and in vivo.
Research methods:
1. Adenovirus shuttle plasmids pDC312-SLPI-EGFRamiR-pA and Ad-SLPI-GFP-pA were constructed and co-transfected into HEK293 cells with cytoskeleton plasmids pBGHlox (delta) E1 and 3Cre respectively for adenovirus packaging. Virus titer was determined by TCID50.
2. Human laryngeal squamous cell carcinoma cell line Hep-2 and human normal umbilical vein endothelial cell line HuVEC were infected with recombinant adenovirus Ad-SLPI-EGFRamiR and control virus Ad-SLPI-GFP respectively. The effects of the recombinant adenovirus on the proliferation of laryngeal carcinoma cell line Hep-2 and normal cell HuVEC were examined by microscopy, MTT and flow cytometry.
3. To establish a tumor-bearing model of laryngeal carcinoma in nude mice, we injected recombinant adenovirus Ad-SLPI-EGFRamiR, Ad-SLPI-GFP into the tumor, or orally administered gefitinib daily.
Research findings:
1 packaging, identification, amplification, purification and titer determination of recombinant adenovirus Ad-SLPI-EGFRamiR, Ad-SLPI-GFP
Adenovirus shuttle plasmids pDC312-SLPI-EGFRamiR-pA and pDC312-SLPI-GFP-pA were co-transfected into HEK293 cells with cytoskeleton plasmids pBGHlox (delta) E1 and 3Cre, respectively. About 13 days after transfection, the cells were observed under inverted microscope. More than 80% of the cells showed grape-like cytopathic effect and were not easy to fall off. After the supernatant was treated with protease K, PCR showed that the EGFRamiR fragments with different repeat numbers, 142 bp, 284 bp, 426 bp, were amplified by Ad-SLPI-EGFRamiR, and 1482 BP fragments were amplified by Ad-SLPI-EGFRamiR. The recombinant adenovirus Ad-SLPI-EGFRamiR and the control virus Ad-SLPI-GFP were successfully constructed. For 1 x 1010pfu/ml, the titer of Ad-SLPI-GFP is 6.3 * 109pfu/ml..
Western blot showed that the expression of 170kdEGFR was significantly decreased in Hep-2 cells treated with Ad-SLPI-EGFRamiR virus for 72 hours. However, after 72 hours of infection with Ad-SLPI-GFP, a large number of Hep-2 cells showed strong green fluorescence signal under fluorescence microscope, while HuVEC cells still showed no green fluorescence. EGFR was effectively down-regulated internally and the expression of green fluorescent protein was specifically regulated in human laryngeal squamous cell carcinoma cell line Hep-2, but not in normal human umbilical vein endothelial cell HuVEC. 72 hours was the appropriate infection time.
Inhibitory effect of recombinant adenovirus Ad-SLPI-EGFRamiR 2 on laryngeal carcinoma cell Hep-2 in vitro
2.1 inhibitory effect of recombinant adenovirus on cell proliferation
MTT results showed that the recombinant adenovirus Ad-SLPI-EGFRamiR had a strong inhibitory effect on the proliferation of laryngeal carcinoma Hep-2 cells 72 hours after infection, and could effectively inhibit the proliferation of laryngeal carcinoma cells at MOI 50 pfu/cell (inhibition rate was 22.5%), but had no significant inhibitory effect on the proliferation of normal HuVEC cells (inhibition rate was - 4.2%).
2.2 the morphological changes of the recombinant adenovirus after 72 hours.
After 72 hours of treatment with recombinant adenovirus Ad-SLPI-EGFRamiR (MOI=50), the morphological changes of Hep-2 cells were marked: the cells became round, shrunk, part beaded and floated. The morphological differences of HuVEC cells in normal cells were not obvious before and after treatment with adenovirus Ad-SLPI-EGFRamiR (MOI=50). There was no significant difference in cell morphology.
2.3 quantitative analysis of apoptosis by flow cytometry
Flow cytometry showed that the apoptosis rates of Hep-2 cells (cells stained by Annexin V-R-PE and 7-AAD) were 32.8% and 31.8% respectively 72 hours after the recombinant adenovirus Ad-SLPI-EGFRamiR was treated with MOI=35 and MOI=50, while the corresponding apoptosis rates of Hep-2 cells treated with Ad-SLPI-GFP for 72 hours were 9.2% and 10.0% respectively. The difference between the two viruses was significant quantitatively. These results suggest that recombinant adenovirus Ad-SLPI-EGFRamiR can effectively induce apoptosis and inhibit the growth and proliferation of laryngeal cancer cells, while for normal umbilical vein endothelial cells HuVEC, the apoptosis rates of AD-SLPI-EGFRamiR and Ad-SLPI-GFP after 72 hours treatment are 11.1% and 8.2% respectively, and the apoptosis rates of HuVEC treated with the corresponding virus MOI=50 are 15. 5%, 4.8%, the difference is not significant.
Inhibitory effect of recombinant adenovirus Ad-SLPI-EGFRamiR 3 on growth of tumor tissue in laryngeal carcinoma model
3.1 inhibitory effect of recombinant adenovirus Ad-SLPI-EGFRamiR on tumor growth in vivo
On the 13th day after the first treatment, the tumor volume of the Ad-SLPI-EGFRamiR group was smaller than that of the Ad-SLPI-GFP group and the gefitinib group, but the difference was not statistically significant (P 0.05). Compared with the tumor volume of the first treatment day, the growth rate of the tumor volume of the Ad-SLPI-EGFRamiR group was smaller than that of the Ad-SLPI-GFP group and the gefitinib group, but there was no significant difference. Significant statistical significance (P 0.05). At the end of the observation (20 days after the first administration), the tumor weight of the Ad-SLPI-EGFRamiR group was less than that of the Ad-SLPI-GFP group and the gefitinib group, but there was no significant difference (P 0.05).
3.2 adverse effects of recombinant adenovirus Ad-SLPI-EGFRamiR in vivo
During the course of treatment, the nude mice in the Ad-SLPI-EGFRamiR group and the Ad-SLPI-GFP group were in good physical condition, mental state, activity and dietary condition without obvious abnormalities. The difference (P0.05) was significantly higher than that in gefitinib group (P0.05).
Research conclusions:
1. The recombinant adenovirus Ad-SLPI-EGFRamiR and AD-SLPI-GFP can effectively infect Hep-2 cells and express the corresponding genes, which has good tissue specificity for laryngeal carcinoma.
2. The recombinant adenovirus Ad-SLPI-EGFRamiR can inhibit the growth of laryngeal carcinoma cells in vitro, but has a low inhibitory effect on normal cells.
3, in nude mice bearing tumor, recombinant adenovirus Ad-SLPI-EGFRamiR has a tendency to inhibit the growth of tumor tissue.
4, in nude mice bearing tumor, the recombinant adenovirus has high safety compared with gefitinib.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.65;R450
本文编号：2250550
[Abstract]:Research background:
Laryngeal cancer is the most common malignant tumor of the upper respiratory tract, accounting for about 25% of the head and neck tumors. The traditional treatment of laryngeal cancer is radical surgery or radiotherapy, supplemented by or not supplemented by chemotherapy. However, the loss of laryngeal function or serious side effects caused by traditional treatment greatly affect the quality of life of patients. Although surgical procedures, radiotherapy and chemotherapy regimens have progressed steadily, the survival rate of patients has not improved significantly in the past 30 years.
In the past 20 years, molecular targeted therapy has made great breakthroughs in the treatment of tumors. For head and neck squamous cell carcinoma (HNSCC), the most representative molecular targeted drug is the specific inhibitor of epidermal growth factor receptor (EGF). R) Monoclonal antibodies and small molecule tyrosine kinase inhibitors. However, the small molecule inhibitors represented by gefitinib have no definite effect on HNSCC. While the monoclonal antibodies represented by cetuximab play an auxiliary role in traditional radiotherapy and chemotherapy, however, due to the limited response rate, high drug resistance rate and frequent occurrence. Skin toxicity, gastrointestinal symptoms and other side effects make the application space of these drugs still small.
With the advancement of molecular cloning technology, gene therapy strategy with virus as expression vector and down-regulation of EGFR expression by RNA interference technology has opened up a new direction for tumor treatment. Compared with shRNA, artificial microRNAs incorporate a natural microRNA framework based on retaining the hairpin structure of the first generation of shRNA. In addition to being more efficient, the greatest advantage is that they can be activated by most of the promoters in mammals. This advantage of artificial microRNAs enables the use of tumor tissue-specific promoters to regulate targets. RNA interference with EGFR becomes possible.
Based on the above research background, we intend to design an artificial microRNA targeting EGFR, and use recombinant adenovirus as vector to regulate the expression of the artificial microRNA through the specific SLPI promoter of laryngeal cancer. We also take Hep-2 as the research object to explore the effect of this gene therapy strategy on tumor growth.
Research purposes:
To construct recombinant adenovirus vector loaded with artificial microRNA targeting EGFR under the control of SLPI promoter, and to study its safety and inhibitory effect on laryngeal cancer cells in vitro and in vivo.
Research methods:
1. Adenovirus shuttle plasmids pDC312-SLPI-EGFRamiR-pA and Ad-SLPI-GFP-pA were constructed and co-transfected into HEK293 cells with cytoskeleton plasmids pBGHlox (delta) E1 and 3Cre respectively for adenovirus packaging. Virus titer was determined by TCID50.
2. Human laryngeal squamous cell carcinoma cell line Hep-2 and human normal umbilical vein endothelial cell line HuVEC were infected with recombinant adenovirus Ad-SLPI-EGFRamiR and control virus Ad-SLPI-GFP respectively. The effects of the recombinant adenovirus on the proliferation of laryngeal carcinoma cell line Hep-2 and normal cell HuVEC were examined by microscopy, MTT and flow cytometry.
3. To establish a tumor-bearing model of laryngeal carcinoma in nude mice, we injected recombinant adenovirus Ad-SLPI-EGFRamiR, Ad-SLPI-GFP into the tumor, or orally administered gefitinib daily.
Research findings:
1 packaging, identification, amplification, purification and titer determination of recombinant adenovirus Ad-SLPI-EGFRamiR, Ad-SLPI-GFP
Adenovirus shuttle plasmids pDC312-SLPI-EGFRamiR-pA and pDC312-SLPI-GFP-pA were co-transfected into HEK293 cells with cytoskeleton plasmids pBGHlox (delta) E1 and 3Cre, respectively. About 13 days after transfection, the cells were observed under inverted microscope. More than 80% of the cells showed grape-like cytopathic effect and were not easy to fall off. After the supernatant was treated with protease K, PCR showed that the EGFRamiR fragments with different repeat numbers, 142 bp, 284 bp, 426 bp, were amplified by Ad-SLPI-EGFRamiR, and 1482 BP fragments were amplified by Ad-SLPI-EGFRamiR. The recombinant adenovirus Ad-SLPI-EGFRamiR and the control virus Ad-SLPI-GFP were successfully constructed. For 1 x 1010pfu/ml, the titer of Ad-SLPI-GFP is 6.3 * 109pfu/ml..
Western blot showed that the expression of 170kdEGFR was significantly decreased in Hep-2 cells treated with Ad-SLPI-EGFRamiR virus for 72 hours. However, after 72 hours of infection with Ad-SLPI-GFP, a large number of Hep-2 cells showed strong green fluorescence signal under fluorescence microscope, while HuVEC cells still showed no green fluorescence. EGFR was effectively down-regulated internally and the expression of green fluorescent protein was specifically regulated in human laryngeal squamous cell carcinoma cell line Hep-2, but not in normal human umbilical vein endothelial cell HuVEC. 72 hours was the appropriate infection time.
Inhibitory effect of recombinant adenovirus Ad-SLPI-EGFRamiR 2 on laryngeal carcinoma cell Hep-2 in vitro
2.1 inhibitory effect of recombinant adenovirus on cell proliferation
MTT results showed that the recombinant adenovirus Ad-SLPI-EGFRamiR had a strong inhibitory effect on the proliferation of laryngeal carcinoma Hep-2 cells 72 hours after infection, and could effectively inhibit the proliferation of laryngeal carcinoma cells at MOI 50 pfu/cell (inhibition rate was 22.5%), but had no significant inhibitory effect on the proliferation of normal HuVEC cells (inhibition rate was - 4.2%).
2.2 the morphological changes of the recombinant adenovirus after 72 hours.
After 72 hours of treatment with recombinant adenovirus Ad-SLPI-EGFRamiR (MOI=50), the morphological changes of Hep-2 cells were marked: the cells became round, shrunk, part beaded and floated. The morphological differences of HuVEC cells in normal cells were not obvious before and after treatment with adenovirus Ad-SLPI-EGFRamiR (MOI=50). There was no significant difference in cell morphology.
2.3 quantitative analysis of apoptosis by flow cytometry
Flow cytometry showed that the apoptosis rates of Hep-2 cells (cells stained by Annexin V-R-PE and 7-AAD) were 32.8% and 31.8% respectively 72 hours after the recombinant adenovirus Ad-SLPI-EGFRamiR was treated with MOI=35 and MOI=50, while the corresponding apoptosis rates of Hep-2 cells treated with Ad-SLPI-GFP for 72 hours were 9.2% and 10.0% respectively. The difference between the two viruses was significant quantitatively. These results suggest that recombinant adenovirus Ad-SLPI-EGFRamiR can effectively induce apoptosis and inhibit the growth and proliferation of laryngeal cancer cells, while for normal umbilical vein endothelial cells HuVEC, the apoptosis rates of AD-SLPI-EGFRamiR and Ad-SLPI-GFP after 72 hours treatment are 11.1% and 8.2% respectively, and the apoptosis rates of HuVEC treated with the corresponding virus MOI=50 are 15. 5%, 4.8%, the difference is not significant.
Inhibitory effect of recombinant adenovirus Ad-SLPI-EGFRamiR 3 on growth of tumor tissue in laryngeal carcinoma model
3.1 inhibitory effect of recombinant adenovirus Ad-SLPI-EGFRamiR on tumor growth in vivo
On the 13th day after the first treatment, the tumor volume of the Ad-SLPI-EGFRamiR group was smaller than that of the Ad-SLPI-GFP group and the gefitinib group, but the difference was not statistically significant (P 0.05). Compared with the tumor volume of the first treatment day, the growth rate of the tumor volume of the Ad-SLPI-EGFRamiR group was smaller than that of the Ad-SLPI-GFP group and the gefitinib group, but there was no significant difference. Significant statistical significance (P 0.05). At the end of the observation (20 days after the first administration), the tumor weight of the Ad-SLPI-EGFRamiR group was less than that of the Ad-SLPI-GFP group and the gefitinib group, but there was no significant difference (P 0.05).
3.2 adverse effects of recombinant adenovirus Ad-SLPI-EGFRamiR in vivo
During the course of treatment, the nude mice in the Ad-SLPI-EGFRamiR group and the Ad-SLPI-GFP group were in good physical condition, mental state, activity and dietary condition without obvious abnormalities. The difference (P0.05) was significantly higher than that in gefitinib group (P0.05).
Research conclusions:
1. The recombinant adenovirus Ad-SLPI-EGFRamiR and AD-SLPI-GFP can effectively infect Hep-2 cells and express the corresponding genes, which has good tissue specificity for laryngeal carcinoma.
2. The recombinant adenovirus Ad-SLPI-EGFRamiR can inhibit the growth of laryngeal carcinoma cells in vitro, but has a low inhibitory effect on normal cells.
3, in nude mice bearing tumor, recombinant adenovirus Ad-SLPI-EGFRamiR has a tendency to inhibit the growth of tumor tissue.
4, in nude mice bearing tumor, the recombinant adenovirus has high safety compared with gefitinib.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.65;R450
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