沉默Id-1基因对腺样囊性癌生物学行为影响的实验研究
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摘要
研究目的：
1.建立能够实时监测、连续动态观察肿瘤生长、转移等生物学行为的裸鼠在体绿色荧光腺样囊性癌ACCM肿瘤模型。
2.探讨裸鼠舌体移植腺样囊性癌瘤体内注射小干扰RNA慢病毒载体进行RNA干扰的有效性和可行性以及应用慢病毒载体的可能性。
3.探讨RNA干扰技术沉默Id-1基因在体内和体外实验中对腺样囊性癌ACCM的抑制作用。
4.探讨Id-1基因的表达在腺样囊性癌ACCM的细胞增殖、侵袭、凋亡等生物学行为中的作用,探讨其作为ACCM新的靶向治疗的重要分子的可能性。
研究方法：
1. ACCM细胞用含-10%胎牛血清、青霉素100mg/ml和链霉素100mg/ml的DMEM培养基,在37℃,5%CO2培养箱培养。逆转录病毒颗粒pLEGFP-N1(病毒滴度为108 Cfu／L)用0.45μm的滤膜过滤,再加入终浓度为8μg/ml的Polybrene组成感染液。用0.25%胰蛋白酶消化收集ACCM细胞,按1×105个／孔接种于6孔板中,常规培养12-16h后,加入上述感染液,培养24 h后更换新鲜培养基,继续培养48h,经G418 (800μg/ml)筛选出阳性克隆。应用有限稀释法进行单细胞克隆筛选,并进行扩大培养,最终获得单细胞克隆细胞株(ACCM-GFP)。
2.胰蛋白酶消化收集处于对数生长期的逆转录病毒载体pLEGFP-N1感染的ACCM细胞(ACCM-GFP),用含有胎牛血清的4℃预冷的DMEM清洗3遍,最终稀释成密度为2×106个／ml,收集后40min内完成细胞注射,注射前可置于冰上暂时保存。注射部位为裸鼠舌背中线处前2／3与后1／3交界处粘膜下,注射剂量为0.1ml／只。裸鼠为6周龄,体重20-25g。
3. ACCM-GFP肿瘤细胞接种7天后,在不同的时间点,用99.9%的乙醚进行吸入麻醉,并用实体荧光成像系统进行摄像分析。以465nm蓝光激发,观察裸鼠原发灶及淋巴结等部位GFP表达情况。同时,对获得的ACCM-GFP细胞进行绿色荧光观测,并与ACCM细胞在细胞形态学、细胞增殖能力、侵袭能力以及组织学等方面进行分析比较。体外进行细胞培养,提取ACCM-GFP细胞和ACCM细胞的总RNA和总蛋白,进行RT-PCR和Western Blot检测细胞内Id-1 mRNA表达水平和蛋白表达水平,并进行分析比较。
4. ACCM-GFP细胞注射2周后,以瘤体内多角度注射慢病毒载体对ACCM-GFP肿瘤进行RNAi,隔日注射1次,连续注射3次。Id-1-siRNA-Lentivirus和NC-siRNA-Lentivirus注射量为100μl (Id-1病毒滴度为5×108TU/ml; NC病毒滴度为1×109TU/ml,应用时稀释成5×108TU/ml);复感染指数(MOI)为1：50。
5.自注射慢病毒载体后隔日测量肿瘤体积大小以观测RNAi后肿瘤生长的变化；在肿瘤RNAi后的第3、4、5、6、7、10、14天分别处死实验组(感染Id-1-siRNA-Lentivirus组)、阴性组(感染NC-siRNA-Lentivirus组)和对照组(空白对照组：未感染慢病毒载体组)裸鼠并切取组织标本,同时观测RNAi后的肿瘤在实体荧光成像系统下的图像；对切取标本进行组织总RNA提取和组织总蛋白提取,分别进行实时定量RT-PCR和Western Blot检测肿瘤组织内Id-1 mRNA表达水平和蛋白表达水平；对肿瘤RNAi后第7天的组织切取标本制备肿瘤标本蜡块,进行增殖细胞核抗原标记物Ki-67的表达检测。
6.体外以Id-1-siRNA-Lentivirus感染ACCM细胞(MOI为1：50),感染24h、48h、72h后以流式细胞仪检测慢病毒感染效率(激发波长488nm,发射波长675nm),同时在荧光倒置相差显微镜下观测被Id-1-siRNA-Lentivirus感染的ACCM-GFP细胞,判断慢病毒感染效率；6孔板内每孔种入2×104ACCM细胞,过夜,使汇合率达到50%-80%,按照MOI为1：50加入Id-1-siRNA-Lentivirus和NC-siRNA-Lentivirus;感染4天后进行Hoechst法凋亡检测,研究RNAi Id-1基因对肿瘤细胞凋亡的影响；将ACCM细胞96孔板种板,每孔200μl,103个／孔,置37℃; 5%CO2温箱培养,待细胞贴壁,培养24小时后按照MOI为1：50加入Id-1-siRNA-Lentivirus和NC-siRNA-Lentivirus,感染1、2、3、4、5、6、7、8天分别以MTT法进行细胞活力检测,研究RNAi沉默Id-1基因对肿瘤细胞增殖活性的影响；在6孔板中用Id-1-siRNA-Lentivirus感染ACCM细胞后第5天,应用Transwell侵袭小室检测肿瘤细胞侵袭能力的变化(瑞氏染色法)。
研究结果：
1.经高浓度G418筛选培养并通过有限稀释法后获得的稳定表达GFP的单克隆ACCM细胞株(ACCM-GFP),在去除G418筛选压力后继续培养2个月以及冻存6个月复苏后,细胞仍能稳定表达GFP,仍可观测到强烈的绿色荧光信号,GFP分布于整个ACCM-GFP细胞质中。
2.组织形态学及组织病理学观测ACCM细胞与ACCM-GFP细胞无显著差异,倒置相差显微镜下观察为圆形、椭圆形或多角形上皮样细胞,大小较为一致,细胞单层贴壁生长,界限清楚。胞浆少,可见透亮度深浅不等的颗粒,核大,类圆形或不规则形,核浆比例高,核仁2-3个,清晰可见,并可见巨大融合细胞。细胞分裂相多见,并有多级核分裂,排列紧密时细胞互相镶嵌成铺路石样,密度再增高时可重叠生长,表现为接触抑制消失；ACCM-GFP细胞与ACCM细胞相比,其增殖能力无显著差异；体外培养发现其侵袭能力无显著差异；其Id-1基因的mRNA和蛋白水平表达无显著差异。
3.裸鼠舌体种植ACCM-GFP细胞成瘤率为100%；体外检测Id-1-siRNA-Lentivirus慢病毒感染效率,感染24h时,病毒的感染效率为24.89%；48h时感染效率为68.45%；72h时可高达83.5%；同时分别以以绿色和红色荧光观察被Id-1-siRNA-Lentivirus感染的ACCM-GFP细胞,通过细胞计数判断慢病毒的感染效率可高达80%。
4.活体荧光成像系统下观察ACCM-GFP裸鼠肿瘤模型,瘤体的绿色荧光信号强烈,肿瘤实性质地,呈分叶或结节状,边界呈波浪状,瘤体内可见囊腔；3周时,可以观测到颌下淋巴结转移灶,转移波及区域范围较小,但是荧光信号强烈；3周时未见肺转移及其它远处转移病灶。进行RNAi的ACCM-GFP肿瘤在465nm波长光线下激发出较弱强度的绿色荧光,在560nm波长光线下激发出高强度的红色荧光。
5.RNAi后阴性对照组与空白对照组的肿瘤生长趋势及生长速度较为一致,肿瘤体积大小无显著差异,实验组在RNAi后12d之前与阴性对照组及空白对照组比较无显著差异,第14天后肿瘤的体积虽然仍为增长上升趋势,但与阴性对照组及空白对照组比较差异非常显著(P<0.01), RNAi对肿瘤生长的抑制率可达26.14%-39.17%。RNAi后增殖细胞核抗原标记物Ki-67在实验组的表达明显降低,与空白对照组与阴性对照组两两比较P<0.01,差异有非常显著的统计学意义；而阴性对照组与空白对照组相比则无显著差异(P>0.05)。
6.进行Id-1 RNAi的ACCM-GFP肿瘤组织Id-1 mRNA自RNAi第4天起出现表达水平下调,与阴性对照组及空白对照组比较差异有非常显著的统计学意义(P<0.01), Id-1-siRNA对Id-1 mRNA的表达抑制率可达42.76%; RNAi后第4天至第14天之间的裸鼠肿瘤mRNA表达水平下调后保持相对稳定水平；RNAi第4天开始,Id-1蛋白表达水平与阴性对照组和空白对照组比较显著下降(P<0.01),并且到达第6天时,蛋白表达下降至相对稳定水平。
7.相关体外实验研究中,感染Id-1-siRNA-Lentivirus的ACCM细胞体积缩小,膜皱缩,细胞核皱缩呈致密浓染、核凝聚变小,染色较深；染色质破裂成大小不等的碎片,或呈块状,并有边集现象,核染色强度变淡,呈现典型的细胞凋亡表现。计数相同视野内发生细胞凋亡的细胞数目,实验组与空白对照组及阴性对照组之间的差异有显著统计学意义(p<0.01). Transwell侵袭小室检测细胞侵袭能力,实验组中感染Id-1-siRNA-Lentivirus的ACCM细胞穿过微孔膜的细胞数与空白对照组和阴性对照组比较显著降低(P<0.01)。MTT法检测细胞增殖能力,接受Id-1-siRNAi的ACCM细胞第5天起生长速度明显减慢,实验组对细胞抑制率大约为40%-50%。
结论：
1.同一ACCM细胞可以相继接受2次以上的逆转录病毒感染,其基因组可以多次嵌入外源病毒基因而本身的基因表达和生物学特性不受影响。Id-1基因的mRNA表达和蛋白表达在ACCM细胞和ACCM-GFP细胞中基本一致。
2. ACCM-GFP荧光肿瘤模型可以长期稳定地表达GFP,有利于长期无创、实时和动态地观测肿瘤的生长变化、浸润、侵袭、远处转移以及治疗效果等,同时也为早期肿瘤变化的观测提供了可能,可以及时捕捉到早于临床症状出现的变化。是研究腺样囊性癌的最佳肿瘤模型之一。
3.本实验应用的慢病毒载体具有高感染效率、表达稳定和较高的生物安全性等特点。
4.特异性Id-1-siRNA可以显著降低Id-1基因在腺样囊性癌ACCM肿瘤组织中的mRNA和蛋白表达；对肿瘤的生长和细胞的增殖有明显的抑制作用；可以显著抑制肿瘤细胞的侵袭能力；并且可以促进肿瘤细胞的凋亡。
5.Id-1基因在腺样囊性癌肿瘤中与细胞的增殖能力、侵袭能力等生物学行为呈正相关；Id-1基因是治疗腺样囊性癌的理想的靶基因。
6.慢病毒载体介导的RNAi具有稳定、高效、给药方式接近临床等优势,是一种有效的肿瘤基因治疗技术,在腺样囊性癌的基因治疗中具有非常好的应用前景。
Objective:
1. To establish a novel green fluorescent protein (GFP)-expressing nude mice orthotopic model which can be visualized in real time with fluorescence imaging and detected in a noninvasive and continuous way for tumor's development, invasion and metastasis.
2. To demonstrate the effectiveness and feasibility of RNAi in the tongue of orthotopic mice model which is mediated by lentivirus injection into the tumor.
3. To investigate the inhibition of silencing Id-1 gene by RNAi on adenoid cystic carcinoma (ACCM) in vivo and in vitro.
4. To research the effect of Id-1 expression on cell proliferation, invasion, apoptosis, and so on in ACCM and to explore the possibility of Id-1 being a new therapeutic target for ACCM treatment.
Methods:
1. ACCM cells were cultured in DMEM supplemented with 10%fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin,100 U/ml streptomycin) at 37℃, 5%CO2. After collecting viral supernatant through a 0.45μm celluslose acetate filter, viral titer was determined as 108 cfu/L finally.1×105 ACCM cells in a 6-well plate, cultured for about 12-16h, were exposed to viral supernatant at a multiplicity of infection, in the presence of 8μg/ml polybrene for 24h. Subsequently, the viral supernatant was replaced with fresh medium. ACCM-GFP cells were acquired with 800μg/ml G418 selection for 2 weeks and then were collected, counted. Split one cell per well in 96-well plate. Colonies were grown in DMEM containing FBS and antibiotics at 37℃,5%CO2.
2. ACCM-GFP cells were harvested with trypsin and washed with prechilling DMEM for 3 times.2×105 ACCM-GFP cells in 0.1 ml medium were injected submucosally into lingual central part at the cross-point of forward 2/3 and backward 1/3 in 60 BALB/c nu/nu male mice. Injection should be finished in 40 minutes and the medium containing cells can be placed on the ice temporarily. The mice were six weeks old, with a body weight of 20-25g.
3.7 days after ACCM-GFP cells injection, mice were imaged at various time points using a cooled CCD camera. To acquire better images, mice were anesthetized, and the tongue was pulled out of the oral cavity. Next, anesthetized mice were pictured by the whole-body optical imaging system. Green fluorescence images were also collected using 60s exposure time with 465nm excitation/535nm emission filters but no binning. The comparison between ACCM-GFP and ACCM cells in morphology, capability of cell growth and invasion, and histology was analyzed. RNA and protein were extracted from ACCM-GFP and ACCM cells in vitro. mRNA and protein expression of Id-1 were evaluated with RT-PCR and Western Blot.
4.2 weeks after ACCM-GFP injection,5×107TU in 100μl of Id-1-siRNA-Lentivirus and NC-siRNA-Lentivirus were injected in a multiple intratumoral direction respectively every other day and for 3 times continuously. The multiplicity of infection (MOI) is 50.
5. Each tumor was measured every other day by using a caliper. Graphs were plotted to illustrate tumor growth in each group. Mice were photographed for fluorescent images by KODAK Image Station. The mice were scanned with fluorescence with excitation maximum 465nm and 560nm for GFP and RFP respectively. Mice were sacrificed at day 3,4,5,6,7,10 and 14 after RNAi. Total RNA and protein were isolated from the tissue and Id-1 expression in mRNA and protein level were detected by qRT-PCR and Western Blot. Immunohistochemistry analysis of Ki-67 expression was conducted 7 days after RNAi.
6. Infection of ACCM cells by Id-1-siRNA-Lentivirus in vitro was applied to analyze the transduction efficiency. At different time points (24h,48h, and 72h), cells were harvested for FACS analysis to confirm the transduction efficiency (465nm excitation/535nm emission). Meanwhile, ACCM-GFP cells infected with Id-1-siRNA-Lentivirus were observed under the green/red fluorescence to determine the transduction efficiency.2 X 104 ACCM cells were seeded in each well of a 6-well plate. The cells were infected in DMEM containing 10%FBS and 5μg/ml polybrene when the confluence was about 50%-80%and Id-1-siRNA-Lentivirus was introduced at a multiplicity of infection (MOI) of 50 for 24h.4 days later, apoptosis was measured using Hoechst 33258 staining. Cells at a concentration of 103 per well were seeded in the 96-well plate and incubated for 24h and then infected with lentivirus particles at a MOI of 50. MTT assay was performed for each day after RNAi to study the effect of Id-1 siRNA on cell proliferation. Cell invasion was assayed using a Boyden chamber cell with culture-chamber-insert system. On the 5th day of RNAi, ACCM cells infected by Id-1-siRNA-Lentivirus in 6-well plate were collected for cell invasion analysis (Wright's stain).
Results:
1. ACCM-GFP cells isolated by G418 screening were able to express GFP stably after 2 months'culture and thawed after 6 months'freezing-storage when G418 was already removed. The GFP sign was completely strong. GFP was distributed in the cytoplasm.
2. From the viewpoint of histology, there was no significant difference between ACCM and ACCM-GFP cells. Histopathologically, most ACCM-GFP tumors presented solid form. It was composed of solid epithelial islands with central areas of necrosis; the cells were small, basophilic and hyperchromatic with a densely granulated nucleus and easily visualized mitotic figures. The epithelial island was composed of cubic cells with scarce cytoplasm, oval nuclei with eosinophilic content, surrounded by a band of dense muscular fibrous tissue, well vascularized, and moderate diffuse chronic inflammatory infiltration. ACCM-GFP cells showed no significant difference from ACCM cells in morphology, Id-1 protein expression, cell proliferation, and invasion ability.
3. ACCM-GFP tumor incidence measured by GFP expression and histological analysis was 100%for all mice in the control and test groups. Transduction efficiency in vitro was evaluated by flow cytometry. With the stable expression of RFP, the high transduction efficiency of 83.5%,68.45%and 24.89%could be detected 72h,48h and 24h after lentiviral infection respectively. Meanwhile, ACCM-GFP cells infected with Id-1-siRNA-Lentivirus were observed with green and red fluorescence respectively and the transduction efficiency of lentivirus was as high as about 80%.
4. Detected with green fluorescence, some features of original ACCM tumor, such as notches along the tumor border, cystic cavities inside the tumor, etc. can present in ACCM-GFP tumors. Meanwhile, a metastic lymphatic node invaded by ACCM-GFP cells can be detected by GFP 3 weeks after ACCM-GFP cell injection, although it is not palpable. The fluorescence sign was strong in spite of the limited areas involved by metastasis. However, we did not find metastasis in lung and other tissues. ACCM-GFP tumors treated with RNAi presented weak green fluorescence with excitation of 465nm, while strong red fluorescence with excitation of 560nm.
5.12 days after the infection with RNAi-containing lentivirus particles, tumor growth began to decrease. Although the tumor size was still increasing after 12 days of RNAi, the difference of test and controls group was significant. However, the negative controls showed the same tumor growth speed as the blank controls. Tumor growth inhibition efficiency could be 26.14%-39.17%. Immunohisto-chemical staining showed Ki-67 positive cells in tumors treated with NC-siRNA-Lentivirus were similar in quantity to ACCM-GFP tumors, however, cellular proliferation indices of tumors derived from Id-1-siRNA-Lentivirus-treated mice differed significantly from others.
6. Until the 3rd day after injection, Id-1-siRNA-Lentivirus-treated tumors, NC- siRNA-Lentivirus-treated tumors and ACCM-GFP tumors showed no reduction in the levels of Id-1; whereas those infected with Id-1-siRNA-Lentivirus for more than 3 days showed significantly reduced levels of mRNA. mRNA level decreased to a relative stable state from the 4th to the 14th day. The expression was markedly downregulated in tumors from the 4th day of infection with Id-1-siRNA-Lentivirus (p<0.01), while it remained similar in blank control, NC-siRNA and Id-1-siRNA group infected for no more than 3 days. Protein level decreased to a relative stable state from the 6th day.
7. Cells in culture after 4 days of infection with Id-1-siRNA exhibited lots of apoptosis, which was characterized by pyknotic and fragmented nuclei. Condensed bright apoptotic nuclei were readily observed amidst the transducted cells. In control cultures, round and large nuclei appeared with regular contours and cells with smaller nuclei and condensed chromatin were rarely seen. Repression of Id-1 expression in ACCM-GFP was found to inhibit invasion in vitro. Cells passed PET membrane in test group were significantly less than control groups (p<0.01). MTT assays showed that Id-1-siRNA-Lentivirus-treated tumor cells grew significantly more slowly than others (p<0.01) frome the 5th day after RNAi. Inhibition efficiency in test group was around 40%-50%. No significant difference was detected in cells treated with NC-siRNA-Lentivirus compared with untreated cells.
Conclusion:
1. ACCM cells can be infected 2 times successively by retrovirus. The insertion of retroviral genome into ACCM's had no influence on the physiological behavior in our study. ACCM-GFP cells displayed the same characteristics as ACCM cells in cell proliferation, invasion ability, Id-1 mRNA and protein expression.
2. Our established GFP-expressing ACCM tumor model should be useful for the evaluation of novel treatment strategies for ACCs including neoadjuvant chemotherapy or gene therapy. It is also an important new tool to help us to understand the key events of cancer development and metastasis especially interaction of the metastatic tumor with a wide array of normal organs and tissues. It is one of the best tumor models of adenoid cystic carcinoma.
3. Lentiviral vector shows high transfection efficiency, stable expression and reliable safety in this research.
4. Specific Id-1-siRNA can downregulate significantly Id-1 gene expression in mRNA and protein level. It can also inhibit tumor development, cell proliferation and cell invasiveness as well. Cell apoptosis can be induced at the same time.
5. Id-1 has a positive relationship with cell proliferation, invasiveness and so on in adenoid cystic carcinoma. It is an ideal target gene for adenoid cystic carcinoma treatment.
6. Lentivirus-mediated RNAi presents advantage of stability, high efficiency and similar administration to clinic. This effective technique proves to be promising for genetic therapy of adenoid cystic carcinoma.
1.建立能够实时监测、连续动态观察肿瘤生长、转移等生物学行为的裸鼠在体绿色荧光腺样囊性癌ACCM肿瘤模型。
2.探讨裸鼠舌体移植腺样囊性癌瘤体内注射小干扰RNA慢病毒载体进行RNA干扰的有效性和可行性以及应用慢病毒载体的可能性。
3.探讨RNA干扰技术沉默Id-1基因在体内和体外实验中对腺样囊性癌ACCM的抑制作用。
4.探讨Id-1基因的表达在腺样囊性癌ACCM的细胞增殖、侵袭、凋亡等生物学行为中的作用,探讨其作为ACCM新的靶向治疗的重要分子的可能性。
研究方法：
1. ACCM细胞用含-10%胎牛血清、青霉素100mg/ml和链霉素100mg/ml的DMEM培养基,在37℃,5%CO2培养箱培养。逆转录病毒颗粒pLEGFP-N1(病毒滴度为108 Cfu／L)用0.45μm的滤膜过滤,再加入终浓度为8μg/ml的Polybrene组成感染液。用0.25%胰蛋白酶消化收集ACCM细胞,按1×105个／孔接种于6孔板中,常规培养12-16h后,加入上述感染液,培养24 h后更换新鲜培养基,继续培养48h,经G418 (800μg/ml)筛选出阳性克隆。应用有限稀释法进行单细胞克隆筛选,并进行扩大培养,最终获得单细胞克隆细胞株(ACCM-GFP)。
2.胰蛋白酶消化收集处于对数生长期的逆转录病毒载体pLEGFP-N1感染的ACCM细胞(ACCM-GFP),用含有胎牛血清的4℃预冷的DMEM清洗3遍,最终稀释成密度为2×106个／ml,收集后40min内完成细胞注射,注射前可置于冰上暂时保存。注射部位为裸鼠舌背中线处前2／3与后1／3交界处粘膜下,注射剂量为0.1ml／只。裸鼠为6周龄,体重20-25g。
3. ACCM-GFP肿瘤细胞接种7天后,在不同的时间点,用99.9%的乙醚进行吸入麻醉,并用实体荧光成像系统进行摄像分析。以465nm蓝光激发,观察裸鼠原发灶及淋巴结等部位GFP表达情况。同时,对获得的ACCM-GFP细胞进行绿色荧光观测,并与ACCM细胞在细胞形态学、细胞增殖能力、侵袭能力以及组织学等方面进行分析比较。体外进行细胞培养,提取ACCM-GFP细胞和ACCM细胞的总RNA和总蛋白,进行RT-PCR和Western Blot检测细胞内Id-1 mRNA表达水平和蛋白表达水平,并进行分析比较。
4. ACCM-GFP细胞注射2周后,以瘤体内多角度注射慢病毒载体对ACCM-GFP肿瘤进行RNAi,隔日注射1次,连续注射3次。Id-1-siRNA-Lentivirus和NC-siRNA-Lentivirus注射量为100μl (Id-1病毒滴度为5×108TU/ml; NC病毒滴度为1×109TU/ml,应用时稀释成5×108TU/ml);复感染指数(MOI)为1：50。
5.自注射慢病毒载体后隔日测量肿瘤体积大小以观测RNAi后肿瘤生长的变化；在肿瘤RNAi后的第3、4、5、6、7、10、14天分别处死实验组(感染Id-1-siRNA-Lentivirus组)、阴性组(感染NC-siRNA-Lentivirus组)和对照组(空白对照组：未感染慢病毒载体组)裸鼠并切取组织标本,同时观测RNAi后的肿瘤在实体荧光成像系统下的图像；对切取标本进行组织总RNA提取和组织总蛋白提取,分别进行实时定量RT-PCR和Western Blot检测肿瘤组织内Id-1 mRNA表达水平和蛋白表达水平；对肿瘤RNAi后第7天的组织切取标本制备肿瘤标本蜡块,进行增殖细胞核抗原标记物Ki-67的表达检测。
6.体外以Id-1-siRNA-Lentivirus感染ACCM细胞(MOI为1：50),感染24h、48h、72h后以流式细胞仪检测慢病毒感染效率(激发波长488nm,发射波长675nm),同时在荧光倒置相差显微镜下观测被Id-1-siRNA-Lentivirus感染的ACCM-GFP细胞,判断慢病毒感染效率；6孔板内每孔种入2×104ACCM细胞,过夜,使汇合率达到50%-80%,按照MOI为1：50加入Id-1-siRNA-Lentivirus和NC-siRNA-Lentivirus;感染4天后进行Hoechst法凋亡检测,研究RNAi Id-1基因对肿瘤细胞凋亡的影响；将ACCM细胞96孔板种板,每孔200μl,103个／孔,置37℃; 5%CO2温箱培养,待细胞贴壁,培养24小时后按照MOI为1：50加入Id-1-siRNA-Lentivirus和NC-siRNA-Lentivirus,感染1、2、3、4、5、6、7、8天分别以MTT法进行细胞活力检测,研究RNAi沉默Id-1基因对肿瘤细胞增殖活性的影响；在6孔板中用Id-1-siRNA-Lentivirus感染ACCM细胞后第5天,应用Transwell侵袭小室检测肿瘤细胞侵袭能力的变化(瑞氏染色法)。
研究结果：
1.经高浓度G418筛选培养并通过有限稀释法后获得的稳定表达GFP的单克隆ACCM细胞株(ACCM-GFP),在去除G418筛选压力后继续培养2个月以及冻存6个月复苏后,细胞仍能稳定表达GFP,仍可观测到强烈的绿色荧光信号,GFP分布于整个ACCM-GFP细胞质中。
2.组织形态学及组织病理学观测ACCM细胞与ACCM-GFP细胞无显著差异,倒置相差显微镜下观察为圆形、椭圆形或多角形上皮样细胞,大小较为一致,细胞单层贴壁生长,界限清楚。胞浆少,可见透亮度深浅不等的颗粒,核大,类圆形或不规则形,核浆比例高,核仁2-3个,清晰可见,并可见巨大融合细胞。细胞分裂相多见,并有多级核分裂,排列紧密时细胞互相镶嵌成铺路石样,密度再增高时可重叠生长,表现为接触抑制消失；ACCM-GFP细胞与ACCM细胞相比,其增殖能力无显著差异；体外培养发现其侵袭能力无显著差异；其Id-1基因的mRNA和蛋白水平表达无显著差异。
3.裸鼠舌体种植ACCM-GFP细胞成瘤率为100%；体外检测Id-1-siRNA-Lentivirus慢病毒感染效率,感染24h时,病毒的感染效率为24.89%；48h时感染效率为68.45%；72h时可高达83.5%；同时分别以以绿色和红色荧光观察被Id-1-siRNA-Lentivirus感染的ACCM-GFP细胞,通过细胞计数判断慢病毒的感染效率可高达80%。
4.活体荧光成像系统下观察ACCM-GFP裸鼠肿瘤模型,瘤体的绿色荧光信号强烈,肿瘤实性质地,呈分叶或结节状,边界呈波浪状,瘤体内可见囊腔；3周时,可以观测到颌下淋巴结转移灶,转移波及区域范围较小,但是荧光信号强烈；3周时未见肺转移及其它远处转移病灶。进行RNAi的ACCM-GFP肿瘤在465nm波长光线下激发出较弱强度的绿色荧光,在560nm波长光线下激发出高强度的红色荧光。
5.RNAi后阴性对照组与空白对照组的肿瘤生长趋势及生长速度较为一致,肿瘤体积大小无显著差异,实验组在RNAi后12d之前与阴性对照组及空白对照组比较无显著差异,第14天后肿瘤的体积虽然仍为增长上升趋势,但与阴性对照组及空白对照组比较差异非常显著(P<0.01), RNAi对肿瘤生长的抑制率可达26.14%-39.17%。RNAi后增殖细胞核抗原标记物Ki-67在实验组的表达明显降低,与空白对照组与阴性对照组两两比较P<0.01,差异有非常显著的统计学意义；而阴性对照组与空白对照组相比则无显著差异(P>0.05)。
6.进行Id-1 RNAi的ACCM-GFP肿瘤组织Id-1 mRNA自RNAi第4天起出现表达水平下调,与阴性对照组及空白对照组比较差异有非常显著的统计学意义(P<0.01), Id-1-siRNA对Id-1 mRNA的表达抑制率可达42.76%; RNAi后第4天至第14天之间的裸鼠肿瘤mRNA表达水平下调后保持相对稳定水平；RNAi第4天开始,Id-1蛋白表达水平与阴性对照组和空白对照组比较显著下降(P<0.01),并且到达第6天时,蛋白表达下降至相对稳定水平。
7.相关体外实验研究中,感染Id-1-siRNA-Lentivirus的ACCM细胞体积缩小,膜皱缩,细胞核皱缩呈致密浓染、核凝聚变小,染色较深；染色质破裂成大小不等的碎片,或呈块状,并有边集现象,核染色强度变淡,呈现典型的细胞凋亡表现。计数相同视野内发生细胞凋亡的细胞数目,实验组与空白对照组及阴性对照组之间的差异有显著统计学意义(p<0.01). Transwell侵袭小室检测细胞侵袭能力,实验组中感染Id-1-siRNA-Lentivirus的ACCM细胞穿过微孔膜的细胞数与空白对照组和阴性对照组比较显著降低(P<0.01)。MTT法检测细胞增殖能力,接受Id-1-siRNAi的ACCM细胞第5天起生长速度明显减慢,实验组对细胞抑制率大约为40%-50%。
结论：
1.同一ACCM细胞可以相继接受2次以上的逆转录病毒感染,其基因组可以多次嵌入外源病毒基因而本身的基因表达和生物学特性不受影响。Id-1基因的mRNA表达和蛋白表达在ACCM细胞和ACCM-GFP细胞中基本一致。
2. ACCM-GFP荧光肿瘤模型可以长期稳定地表达GFP,有利于长期无创、实时和动态地观测肿瘤的生长变化、浸润、侵袭、远处转移以及治疗效果等,同时也为早期肿瘤变化的观测提供了可能,可以及时捕捉到早于临床症状出现的变化。是研究腺样囊性癌的最佳肿瘤模型之一。
3.本实验应用的慢病毒载体具有高感染效率、表达稳定和较高的生物安全性等特点。
4.特异性Id-1-siRNA可以显著降低Id-1基因在腺样囊性癌ACCM肿瘤组织中的mRNA和蛋白表达；对肿瘤的生长和细胞的增殖有明显的抑制作用；可以显著抑制肿瘤细胞的侵袭能力；并且可以促进肿瘤细胞的凋亡。
5.Id-1基因在腺样囊性癌肿瘤中与细胞的增殖能力、侵袭能力等生物学行为呈正相关；Id-1基因是治疗腺样囊性癌的理想的靶基因。
6.慢病毒载体介导的RNAi具有稳定、高效、给药方式接近临床等优势,是一种有效的肿瘤基因治疗技术,在腺样囊性癌的基因治疗中具有非常好的应用前景。
Objective:
1. To establish a novel green fluorescent protein (GFP)-expressing nude mice orthotopic model which can be visualized in real time with fluorescence imaging and detected in a noninvasive and continuous way for tumor's development, invasion and metastasis.
2. To demonstrate the effectiveness and feasibility of RNAi in the tongue of orthotopic mice model which is mediated by lentivirus injection into the tumor.
3. To investigate the inhibition of silencing Id-1 gene by RNAi on adenoid cystic carcinoma (ACCM) in vivo and in vitro.
4. To research the effect of Id-1 expression on cell proliferation, invasion, apoptosis, and so on in ACCM and to explore the possibility of Id-1 being a new therapeutic target for ACCM treatment.
Methods:
1. ACCM cells were cultured in DMEM supplemented with 10%fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin,100 U/ml streptomycin) at 37℃, 5%CO2. After collecting viral supernatant through a 0.45μm celluslose acetate filter, viral titer was determined as 108 cfu/L finally.1×105 ACCM cells in a 6-well plate, cultured for about 12-16h, were exposed to viral supernatant at a multiplicity of infection, in the presence of 8μg/ml polybrene for 24h. Subsequently, the viral supernatant was replaced with fresh medium. ACCM-GFP cells were acquired with 800μg/ml G418 selection for 2 weeks and then were collected, counted. Split one cell per well in 96-well plate. Colonies were grown in DMEM containing FBS and antibiotics at 37℃,5%CO2.
2. ACCM-GFP cells were harvested with trypsin and washed with prechilling DMEM for 3 times.2×105 ACCM-GFP cells in 0.1 ml medium were injected submucosally into lingual central part at the cross-point of forward 2/3 and backward 1/3 in 60 BALB/c nu/nu male mice. Injection should be finished in 40 minutes and the medium containing cells can be placed on the ice temporarily. The mice were six weeks old, with a body weight of 20-25g.
3.7 days after ACCM-GFP cells injection, mice were imaged at various time points using a cooled CCD camera. To acquire better images, mice were anesthetized, and the tongue was pulled out of the oral cavity. Next, anesthetized mice were pictured by the whole-body optical imaging system. Green fluorescence images were also collected using 60s exposure time with 465nm excitation/535nm emission filters but no binning. The comparison between ACCM-GFP and ACCM cells in morphology, capability of cell growth and invasion, and histology was analyzed. RNA and protein were extracted from ACCM-GFP and ACCM cells in vitro. mRNA and protein expression of Id-1 were evaluated with RT-PCR and Western Blot.
4.2 weeks after ACCM-GFP injection,5×107TU in 100μl of Id-1-siRNA-Lentivirus and NC-siRNA-Lentivirus were injected in a multiple intratumoral direction respectively every other day and for 3 times continuously. The multiplicity of infection (MOI) is 50.
5. Each tumor was measured every other day by using a caliper. Graphs were plotted to illustrate tumor growth in each group. Mice were photographed for fluorescent images by KODAK Image Station. The mice were scanned with fluorescence with excitation maximum 465nm and 560nm for GFP and RFP respectively. Mice were sacrificed at day 3,4,5,6,7,10 and 14 after RNAi. Total RNA and protein were isolated from the tissue and Id-1 expression in mRNA and protein level were detected by qRT-PCR and Western Blot. Immunohistochemistry analysis of Ki-67 expression was conducted 7 days after RNAi.
6. Infection of ACCM cells by Id-1-siRNA-Lentivirus in vitro was applied to analyze the transduction efficiency. At different time points (24h,48h, and 72h), cells were harvested for FACS analysis to confirm the transduction efficiency (465nm excitation/535nm emission). Meanwhile, ACCM-GFP cells infected with Id-1-siRNA-Lentivirus were observed under the green/red fluorescence to determine the transduction efficiency.2 X 104 ACCM cells were seeded in each well of a 6-well plate. The cells were infected in DMEM containing 10%FBS and 5μg/ml polybrene when the confluence was about 50%-80%and Id-1-siRNA-Lentivirus was introduced at a multiplicity of infection (MOI) of 50 for 24h.4 days later, apoptosis was measured using Hoechst 33258 staining. Cells at a concentration of 103 per well were seeded in the 96-well plate and incubated for 24h and then infected with lentivirus particles at a MOI of 50. MTT assay was performed for each day after RNAi to study the effect of Id-1 siRNA on cell proliferation. Cell invasion was assayed using a Boyden chamber cell with culture-chamber-insert system. On the 5th day of RNAi, ACCM cells infected by Id-1-siRNA-Lentivirus in 6-well plate were collected for cell invasion analysis (Wright's stain).
Results:
1. ACCM-GFP cells isolated by G418 screening were able to express GFP stably after 2 months'culture and thawed after 6 months'freezing-storage when G418 was already removed. The GFP sign was completely strong. GFP was distributed in the cytoplasm.
2. From the viewpoint of histology, there was no significant difference between ACCM and ACCM-GFP cells. Histopathologically, most ACCM-GFP tumors presented solid form. It was composed of solid epithelial islands with central areas of necrosis; the cells were small, basophilic and hyperchromatic with a densely granulated nucleus and easily visualized mitotic figures. The epithelial island was composed of cubic cells with scarce cytoplasm, oval nuclei with eosinophilic content, surrounded by a band of dense muscular fibrous tissue, well vascularized, and moderate diffuse chronic inflammatory infiltration. ACCM-GFP cells showed no significant difference from ACCM cells in morphology, Id-1 protein expression, cell proliferation, and invasion ability.
3. ACCM-GFP tumor incidence measured by GFP expression and histological analysis was 100%for all mice in the control and test groups. Transduction efficiency in vitro was evaluated by flow cytometry. With the stable expression of RFP, the high transduction efficiency of 83.5%,68.45%and 24.89%could be detected 72h,48h and 24h after lentiviral infection respectively. Meanwhile, ACCM-GFP cells infected with Id-1-siRNA-Lentivirus were observed with green and red fluorescence respectively and the transduction efficiency of lentivirus was as high as about 80%.
4. Detected with green fluorescence, some features of original ACCM tumor, such as notches along the tumor border, cystic cavities inside the tumor, etc. can present in ACCM-GFP tumors. Meanwhile, a metastic lymphatic node invaded by ACCM-GFP cells can be detected by GFP 3 weeks after ACCM-GFP cell injection, although it is not palpable. The fluorescence sign was strong in spite of the limited areas involved by metastasis. However, we did not find metastasis in lung and other tissues. ACCM-GFP tumors treated with RNAi presented weak green fluorescence with excitation of 465nm, while strong red fluorescence with excitation of 560nm.
5.12 days after the infection with RNAi-containing lentivirus particles, tumor growth began to decrease. Although the tumor size was still increasing after 12 days of RNAi, the difference of test and controls group was significant. However, the negative controls showed the same tumor growth speed as the blank controls. Tumor growth inhibition efficiency could be 26.14%-39.17%. Immunohisto-chemical staining showed Ki-67 positive cells in tumors treated with NC-siRNA-Lentivirus were similar in quantity to ACCM-GFP tumors, however, cellular proliferation indices of tumors derived from Id-1-siRNA-Lentivirus-treated mice differed significantly from others.
6. Until the 3rd day after injection, Id-1-siRNA-Lentivirus-treated tumors, NC- siRNA-Lentivirus-treated tumors and ACCM-GFP tumors showed no reduction in the levels of Id-1; whereas those infected with Id-1-siRNA-Lentivirus for more than 3 days showed significantly reduced levels of mRNA. mRNA level decreased to a relative stable state from the 4th to the 14th day. The expression was markedly downregulated in tumors from the 4th day of infection with Id-1-siRNA-Lentivirus (p<0.01), while it remained similar in blank control, NC-siRNA and Id-1-siRNA group infected for no more than 3 days. Protein level decreased to a relative stable state from the 6th day.
7. Cells in culture after 4 days of infection with Id-1-siRNA exhibited lots of apoptosis, which was characterized by pyknotic and fragmented nuclei. Condensed bright apoptotic nuclei were readily observed amidst the transducted cells. In control cultures, round and large nuclei appeared with regular contours and cells with smaller nuclei and condensed chromatin were rarely seen. Repression of Id-1 expression in ACCM-GFP was found to inhibit invasion in vitro. Cells passed PET membrane in test group were significantly less than control groups (p<0.01). MTT assays showed that Id-1-siRNA-Lentivirus-treated tumor cells grew significantly more slowly than others (p<0.01) frome the 5th day after RNAi. Inhibition efficiency in test group was around 40%-50%. No significant difference was detected in cells treated with NC-siRNA-Lentivirus compared with untreated cells.
Conclusion:
1. ACCM cells can be infected 2 times successively by retrovirus. The insertion of retroviral genome into ACCM's had no influence on the physiological behavior in our study. ACCM-GFP cells displayed the same characteristics as ACCM cells in cell proliferation, invasion ability, Id-1 mRNA and protein expression.
2. Our established GFP-expressing ACCM tumor model should be useful for the evaluation of novel treatment strategies for ACCs including neoadjuvant chemotherapy or gene therapy. It is also an important new tool to help us to understand the key events of cancer development and metastasis especially interaction of the metastatic tumor with a wide array of normal organs and tissues. It is one of the best tumor models of adenoid cystic carcinoma.
3. Lentiviral vector shows high transfection efficiency, stable expression and reliable safety in this research.
4. Specific Id-1-siRNA can downregulate significantly Id-1 gene expression in mRNA and protein level. It can also inhibit tumor development, cell proliferation and cell invasiveness as well. Cell apoptosis can be induced at the same time.
5. Id-1 has a positive relationship with cell proliferation, invasiveness and so on in adenoid cystic carcinoma. It is an ideal target gene for adenoid cystic carcinoma treatment.
6. Lentivirus-mediated RNAi presents advantage of stability, high efficiency and similar administration to clinic. This effective technique proves to be promising for genetic therapy of adenoid cystic carcinoma.
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