基质金属蛋白酶-2靶向siRNA抑制成釉细胞瘤侵袭性的实验研究
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摘要
成釉细胞瘤(ameloblastoma, AM)是口腔颌面部最常见的牙源性肿瘤,占牙源性肿瘤的59.3%。AM虽为良性肿瘤,但具有局部侵袭性,肿瘤细胞常向骨小梁间侵润,临床治疗容易复发,其复发率高。反复多次手术给患者造成严重的面部畸形,并有可能恶变,因而研究其侵袭机制具有重要的意义。
到目前为止,成釉细胞瘤局部侵袭的机制尚未明了。一般认为,肿瘤侵袭主要是指肿瘤细胞侵犯和破坏周围正常组织的过程。肿瘤细胞的周边为结缔组织,即基底膜和细胞外基质,这些结构的降解与破坏是肿瘤侵袭转移多阶段过程中的重要步骤。细胞外基质和基底膜的主要组织结构为胶原、层粘素和纤维结合素等,这些结构的破坏与降解需要相应的溶解酶参与。研究表明,基质金属蛋白酶(matrix metalloproteinase,MMP)和相应的金属酶组织抑制剂在肿瘤的侵袭和转移过程中担任了重要的角色。其中,基质金属蛋白酶-2(MMP-2)是目前研究发现的与肿瘤侵袭关系最为密切的一种基质金属蛋白酶。
已有的研究表明,MMP的调节具有三个水平,即基因转录水平、酶原的活化激活水平、抑制剂水平。现有的研究主要集中在后2个方面,而在MMP基因转录水平对MMP进行调节的研究很少。近年来,出现了一种新的基因研究手段-RNA干扰(RNA interference, RNAi),它是将与mRNA对应的正义RNA和反义RNA组成的双链RNA导入细胞,使mRNA发生特异性的降解,导致其相应的基因沉默。RNAi在研究基因功能、基因敲除、基因表达调控和基因治疗方面发挥着重要作用。
本研究旨在以MMP-2为靶基因,采用RNA干扰的方法沉默MMP-2基因,从而从MMP-2基因转录水平调控MMP-2的表达,探讨MMP-2基因沉默后AM细胞侵袭性生
物学行为的改变,进一步研究MMP-2与AM局部侵袭的关系。本研究分四部分,现摘要如下:
第一部分成釉细胞瘤原代细胞培养及生长特性
迄今为止,AM侵袭性生长的机制尚未完全明确。为了研究其侵袭机制,采用体外培养的AM细胞进行研究是一种重要的方法。由于AM细胞在常规培养基中生长缓慢,存活时间短,不容易纯化,不利于AM的细胞生物学研究。为了探索更为适合AM细胞生长的培养基,本实验拟采用DK-SFM无血清培养基和DMEM培养基培养AM细胞,研究AM细胞在无血清培养基中的生长特性,为进一步研究AM的细胞生物学行为奠定基础。
方法:采用DK-SFM无血清培养基和DMEM培养基培养AM细胞,CK14、CK16、CK18及Vimentin免疫组织化学染色鉴定细胞来源,观察细胞的生长特性、细胞形态和传代次数,流式细胞仪测定S期细胞比率(SPF)和增殖指数(PI)。结果:体外培养的AM细胞CK14、CK16免疫组化染色阳性,CK18及Vimentin免疫组化染色阴性。在DK-SFM培养基中,细胞形态清晰,仅见少许散在的成纤维细胞;细胞传代3-6次,平均传代4.8代;细胞存活48-97d,平均存活73.8 d; SPF平均值为6.8%,PI平均值为15.9%。在DMEM中,细胞形态不清晰,可见较多的成纤维细胞;细胞传代2-4次,平均传代3.2代;细胞存活27-61 d,平均存活46.7 d;SPF平均值为5.4%,PI平均值为11.0%。结论:在DK-SFM中培养的AM细胞存活时间长,传代次数较多,DK-SFM较DMEM更适合AM细胞的体外培养。
第二部分MMP-2靶向siRNA表达质粒的构建及序列优化
RNA干扰是生物进化过程中抵抗病毒和转座子的一种保守防御反应。它是由正义RNA和反义RNA组成的双链RNA在细胞内被Dicer酶切割成约19~22nt的siRNA,进而形成siRNA蛋白复合物(siRNP),siRNP通过识别、降解具有同源序列的mRNA,最终导致特异性的基因表达沉默,属于转录后基因沉默机制。当进行RNA干扰实验研究时,需要制备siRNA,目前,siRNA的制备的方法有体外制备法和体内表达法两种。前者是在体外合成siRNA,再导入siRNA于细胞内,该法存在基因沉默维持时间短、转染效果不稳定等问题。后者是在体外构建siRNA表达载体,如质粒载体或病毒载体,载体导入体内后自动合成siRNA,引发基因沉默,该法的基因沉默时间长、转染效果稳定。因此,本实验将构建MMP-2特异性siRNA表达质粒,并优化特异性序列,为MMP-2基因相关的后续实验研究奠定基础。
方法:采用定向克隆的方法构建P1、P2共2条MMP-2特异性siRNA表达质粒,将其转染Hela细胞,采用荧光显微镜观察其转染效果,流式细胞仪检测转染效率,RT-PCR检测MMP-2 mRNA的表达情况。结果:构建的2条MMP-2特异性siRNA表达质粒经基因测序显示克隆的序列完全正确,2条siRNA表达质粒成功转染Hela细胞,转染效率随着时间的延长而增加,72h时,P1的转染效率为41.8%, P2的转染效率为93.2%,P2的转染效率大于P1的转染效率,差异具有显著性,P<0.05;72h时,P1、P2对MMP-2 mRNA的沉默效率分别为38.6%、68.9%。结论:体外构建的MMP-2靶向siRNA表达质粒成功转染Hela细胞并有效沉默MMP-2基因,但有序列差异性,P2的转染效率和基因沉默效率优于P1。
第三部分MMP-2靶向siRNA对成釉细胞瘤侵袭性的抑制作用
MMP-2的表达与活性调节一般有三个水平,即基因转录水平、酶原的活化激活、抑制剂水平。在本课题组的前期研究中,应用MMP-2特异性抑制剂Ro31-9790明显抑制了MMP-2的活性,使裸鼠肾包膜下的AM移植瘤的生长被抑制,这种对MMP-2的调节是在抑制剂水平上的调节。本实验拟以第二部分构建的MMP-2靶向siRNA表达质粒转染AM细胞,以沉默MMP-2基因的表达。从而从MMP-2基因转录水平调控MMP-2的表达,探讨MMP-2基因沉默后AM细胞侵袭性生物学行为的改变,进一步研究AM局部侵袭性生长的机制。
方法:采用第一部分的无血清培养法培养AM细胞,将体外构建的MMP-2靶向siRNA表达质粒转染AM细胞,荧光显微镜检测siRNA表达质粒的转染效果,流式细胞仪检测转染效率及细胞周期和细胞凋亡情况,明胶酶谱分析法检测培养上清液中MMP-2的活性, RT-PCR检测MMP-2 mRNA表达的改变,Western blotting印迹法检测AM细胞MMP-2蛋白表达,三维培养检测AM细胞的侵袭性,Transwell微侵袭分析检测细胞的侵袭抑制率,细胞黏附分析法检测细胞在Fn基质上的黏附率。采用SPSS统计软件对数据进行统计学分析。结果:siRNA表达质粒成功转染AM细胞,实验组细胞的凋亡和细胞周期无明显改变;96h时,质粒转染率为63.6%,培养上清液中酶原性MMP-2和活性MMP-2的表达分别下降了39.6%和56.5%;转染96h后,AM细胞的MMP-2 mRNA表达水平下降了66.0%,MMP-2蛋白表达下降64.6%,细胞的侵袭抑制率为61.3%,黏附抑制率为48.3%,P<0.05。结论:MMP-2靶向siRNA成功转染AM细胞并沉默MMP-2基因,抑制了MMP-2基因的表达,AM的侵袭性被明显抑制,MMP-2与AM细胞的侵袭性密切相关。
第四部分MMP-2靶向siRNA对成釉细胞瘤裸鼠移植瘤的抑制作用
通过前面的实验研究,我们已经证明MMP-2靶向siRNA可以在体外沉默MMP-2基因从而抑制MMP-2基因的表达,进而抑制了AM细胞的侵袭性。为了进一步探讨RNA干扰在生物体内的效应,本实验旨在构建AM移植瘤模型,通过在移植瘤周围局部注射MMP-2靶向siRNA表达质粒,探讨体内RNA干扰对AM侵袭性的抑制作用。方法:取新鲜的AM组织标本,剪切成1-2mm3组织小块,接种于裸鼠双侧前肢根部背侧和后背部皮下。设立自身对照,即左侧前肢处的移植瘤为空白组,注射250μl Opti-MEMR培养基;后背部的移植瘤为脂质体组,在移植瘤周围注射脂质体混合物,空白组和脂质体组均为对照组,右侧前肢处的移植瘤为siRNA组,在移植瘤周围注射MMP-2靶向siRNA表达质粒混合物;接种后第2周末开始施加处理因素,记录移植瘤体积和实验终止时的瘤重,采用SPSS统计软件包中的t检验法对数据进行检验,P<0.05表示有显著性差异。RT-PCR检测移植瘤中MMP-2 mRNA的表达,Western blotting检测移植瘤中MMP-2蛋白的表达,并对移植瘤进行组织病理学分析。结果:所有移植瘤均成活,实验组移植瘤体积增加量少于对照组移植瘤体积的增加量,实验组移植瘤的瘤重少于对照组移植瘤的瘤重,差异有显著性,P<0.05。对照组移植瘤周边的疏松结缔组织中都出现了AM滤泡样结构,滤泡样结构呈现典型的V-G三联征,实验组移植瘤周边的疏松结缔组织中没有AM滤泡样结构或仅仅残留滤泡样结构的痕迹。对照组MMP-2 mRNA和MMP-2蛋白的表达水平均显著性地高于实验组的表达;实验组MMP-2 mRNA表达相对抑制率为42.35%;MMP-2蛋白抑制率为51%。结论:成功构建AM裸鼠皮下移植瘤动物模型; AM移植瘤的侵袭和生长被抑制可能是由于MMP-2靶向siRNA对MMP-2基因的沉默作用引起的。
Human ameloblastoma is the most frequently encounted odontogenic epithelium tumor in oral and maxillofacial region. About 59.3-63.2% of odontogenic epithelium tumors are ameloblastoma. Although it is usually considered as benign tumor, it has the potential of local invasion and bone destruction. The tumor cells of ameloblastoma often invade into bone trabecula. The recurrence rate of ameloblastoma is significantly high after surgical treatment, which might reach to 50%～90%. Repeated surgical operation is often harmful to patient and may cause severe facial deformity. For the sake of these, it is important to study the mechanisms of the invasion of ameloblastomas.
The invasion of tumor mainly means the process that tumor cells invade and destroy the normal tissue around tumor cells. Usually, tissue around tumor is connective tissue, such as basement membrane and extracellular matrix. It is an important step of tumor invasion that degradation and destruction of these structure. The main tissue structures of basement membrane and extracellular matrix including collagen, laminin and fibronectin. To decompose and deconstruct these structure need the effect of resolvase. Studies indicated that matrix metalloproteinase (MMP) and its corresponding tissue inhibitors had taken an important role in the process of tumor invasion and metastasis. Among all of the MMPs discovered recently, MMP-2 is one of MMP which has the closest relationship with tumor invasion. Many studies shown that many kinds of malignant tumor has high expression of MMP-2, and the expression level of MMP- 2 has close relationship with the degree of tumor invasion and poor prognosis. The previous study of our group shows that MMP-2 was positive stain in the cytoplasm of peripheral cylindrocellular around tumor. MMP-2 mRNA also expressed in ameloblastoma, and the expression level was significant higher than that in normal dental follicle tissue.
Usually, there are three levels of modulation for MMP-2 expression and activation, namely, the level of gene transcription, proenzyme activation and inhibitor. Recently, the highlight studies are mainly in latter two aspects. However, the study of the modulation for MMP-2 on transcription level is very few. In the previous study of our group, MMP-2 activity was significantly inhibited by MMP-2 specific inhibitor Ro31-9790, and so did the growth of xenograft of ameloblastoma in submembrane of kidney. This kind of modulation for MMP-2 was based on MMP-2 inhibitor level.
RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing that is thought to inhibit the replication and expression of selfish DNA elements and viruses. RNAi was mediated by the generation of 21-23nt dsRNA molecules, termed small interference RNA (siRNA). dsRNA molecules are processed by the RNaseIII-like enzyme Dicer to generate siRNA, and the siRNAs direct the recognition and subsequent degradation of homologous mRNAs by a multiprotein complex, and the expression of mRNA was silenced. Now, RNAi is a useful method for gene study. It has very important effect in the area of study gene function, gene knock out, adjustment of gene expression and gene therapy.
The main purpose of this study is to suppress MMP-2 expression using RNA interference, and regulate MMP-2 expression on the gene transcript level. The invasive behavior of ameloblastoma after MMP-2 gene silenced by siRNA was studied. Then we further to study the mechanisims of the invasive growth of ameloblastomas. Therefore, this study including five parts a following:
PartⅠPrimary Culture and Growth Characteristic of Ameloblastoma Cells
Up to now, the mechanisims of invasive growth behavior into bone of ameloblastoma are not very clear. So, it is an important method to study the molecular mechanisims of invasiveness and biological characteristic of ameloblastoma by culture ameloblastoma cells in vitro. However, due to the short life span and slow growth of ameloblastoma cells and difficulty to purification in routing culture medium such as DMEM, it is not easy to study the biological behavior of ameloblastoma. In order to find a better culture medium for ameloblastoma cells’growth, serum free culture medium was adopted to culture ameloblastoma cells in this experiment. The growth characteristic of ameloblastoma cells was studied to provide a basis for further study of biological behavior of ameloblastoma.
Methods: Defined Keratinocyte-serum free medium (DK-SFM) and DMEM medium were used for ameloblastoma cell culture in vitro. Cytokeratin 14, 16, 18 and Vimentin monoclonal antibodies were used for immunohistochemistric analysis to identify the origin of cultured cells. The growth rate in different culture medium was observed. The morphology and passages of ameloblastoma cell in different medium were studied. The SPF value and PI value of cells were detected by flow cytometry. Results: Cytokeratin 14 and 16 was positive stain in cultured cells, however, cytokeratin 18 and Vimentin was negative stain in culture cells. In DK-SFM medium, the morphology of cultured cells was distinct. Only some sporadic fibroblast was seen in it.
Ameloblastoma cells were taken through 3 to 6 passages and the average passages were 4.8. The survival time of the cells was maintained 48 days to 97 days, and the average survival time was 73.8 days. The average value of SPF and PI of cells cultured in DK-SFM was 6.8% and 15.9%, respectively. However, in DMEM medium, the morphology of the cells cultured was not clear. On the other hand, a lot of fibroblast was seen in it. The cells were only taken through 2 to 4 passages and the average passages of it were 3.2. The survival time of the cells was maintained 27 days to 61 days, and the average survival time was 46.7 days. The average value of SPF and PI of cells cultured in DMEM are 5.4% and 11.0%, respectively. Conclusion: Ameloblastoma cells have long survival time in DK-SFM medium, which is more suitable for the growth of ameloblastoma cell than that of DMEM medium.
PartⅡConstruction and Sequence Optimization of MMP-2 Gene Specific siRNA Expression Plasmid
RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing that is thought to inhibit the replication and expression of selfish DNA elements and viruses. RNAi was mediated by the generation of 21-23nt dsRNA molecules, termed small interference RNA (siRNA). dsRNA molecules are processed by the RNaseIII-like enzyme Dicer to generate siRNA, and the siRNAs direct the recognition and subsequent degradation of homologous mRNAs by a multiprotein complex, and finally, the expression of mRNA was silenced. It is belong to post transcription silence. In RNAi experiment, there are two methods of constructing siRNA, namely, construct siRNA in vitro and siRNA expression in vivo. The former is to synthesis siRNA in vitro, and then the siRNA was transfected into cells. There are some problems in this kind of method to construct siRNA, such as short gene silence time and unstable transfection effect. The latter is to construct siRNA expression vector such as plasmid vector or virus vector. After tansfecting this vector into cells, it will produce siRNA automatically. It has long gene silence time and stable transfection effect. So, in present experiment, MMP-2 specific siRNA expression plasmid will be constructed, and the specific sequence of siRNA will be optimized in order to provide basis for next experiment study related to MMP-2 gene.
Methods: Two items of siRNA expression plasmids, namely P1 and P2, were constructed by directed cloning and transfected into Hela cell by Lipofectamine 2000. The transfection effect was confirmed by fluorescence microscope. The transfection rate was checked by flow cytometry. Reverse transcript polymerase chain reaction (RT-PCR) was used to detect the expression of MMP-2 mRNA. Results: All of the two items of siRNA expression plasmids were successfully tranfected into Hela cells. The transfection rate at 72h of P1 and P2 was 41.8% and 93.2%, respectively. The transfection rate of P2 was significant higher than that of P1. The inhibition rate of MMP-2 mRNA expression of P1 and P2 was 38.6% and 68.9%, respectively. Conclusion: The MMP-2 specific siRNA expression plasmid constructed in vitro was successfully transfected into Hela cells, but it had sequence difference. The trasfection rate and gene silence rate of P2 is better than that of P1.
PartⅢInhibition of Invasiveness of Ameloblastoma by Suppression MMP-2 Gene Expression Using MMP-2 Gene Targeting siRNA
Usually, there are three levels of modulation for expression and activity of MMP-2, namely, the level of MMP-2 gene transcription, activation of proenzyme and inhibitor of MMP-2. In our previous study, the activity of MMP-2 was obviously inhibited by using MMP-2 specific inhibitor Ro31-990, and so did the growth of xenograft of ameloblastoma in submembrane of kidney. This adjustment of MMP-2 was based on MMP-2 inhibitor level. In present experiment, MMP-2 targeting siRNA expression plasmid constructed in Part III was transfected into ameloblastoma cells in order to silence MMP-2 gene and to regulate the expression of MMP-2 on the level of MMP-2 gene transcription. The invasive behavior of ameloblastoma after MMP-2 gene silencing by siRNA was studied.
Methods: Ameloblastoma cells were cultured in Defined Keratinocyte-SFM medium. MMP-2 specific siRNA expression plasmid constructed in vitro was transfected into ameloblastoma cells. Fluorescence microscope was used to detect the result of transfection. Flow cytometry was used to check the transfection rate, the cell cycle and apoptosis. Gelatinolytic zymography analysis was used to investigate the activity of MMP-2 in supernatant of culture medium. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to check the expression of MMP-2 mRNA. Western-blot analysis was performed to investigate the expression of MMP-2 protein. The invasiveness of ameloblastoma cells was detected by three dimension culture system. Transwell analysis was done to measure the invasion of ameloblastoma cells. Cell adhesion rate on fibronectin was detected by cell adhesive analysis. The analysis of students t test was used to analyze the data. Results: There is no significant difference of apoptosis between experimental study and control study. MMP-2 specific siRNA expression plasmid was successfully transfected into ameloblastoma cells. The expression of proferment MMP-2 and active MMP-2 in supernatant of experimental group were decreased by 39.6% and 56.5%, respectively. The transfection rate at 96h after transfection was 63.6%. At the same time, MMP-2 mRNA expression was reduced to 66.0%. MMP-2 protein expression was reduced to 64.6%. The inhibition rate of the invasiveness of ameloblastoma cells was 61.3%. The inhibition rate of adhesion of ameloblastoma cell was 48.3%. P<0.05. Conclusion: MMP-2 gene was successfully silenced by MMP-2 targeting siRNA. MMP-2 has close relation with the invasiveness of ameloblastoma.
PartⅣInhibition Against Xenografts of Ameloblastoma in Nude Mice Using MMP-2 Targeting siRNA
In previous experiment, it has been demonstrated that MMP-2 gene could be silenced by MMP-2 targeting siRNA in vitro, and the expression of MMP-2 was inhibited, and then, the invasive behavior of ameloblastoma cells had been inhibited. In order to study the effects of RNAi in vivo, in present experiment, subcutaneous xenograft of ameloblastoma in nude mice was established and MMP-2 targeting siRNA expression plasmid was injected around the xenograft. Methods: Fresh ameloblastoma tissue was minced into 1-2 mm3 size and subcutaneous transplanted into the back of the two anterior extremities root and the posterior back of nude mice. Self control was designed. Namely, left anterior limb side was taken as blank group and 250μl Opti-MEMR I culture medium was injected around the xenograft, posterior back side was taken as liposome group and compounds of liposome was injected around the xenograft, right anterior limb side was taken as experiment side and MMP-2 specific siRNA expression plasmid was injected around the xenografts. Two weeks after transplantation of the ameloblastoma tissue lump, the experimental factor was exerted on control side and experimental side according to the group design. The volume of the xenografts and the weight of xenografts in the termination time of the experiment were recorded. The students t test of SPSS11.0 software was taken as statistic analysis. If the P value is less than 0.05, the result has significance. The pathological analysis was performed to study the xenografts. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to check the expression of MMP-2 mRNA in xenografts. Western-blot analysis was performed to investigate MMP-2 protein in xenografts. Results: All of the xenografts were survival. The volume of xenografts in control side was significant larger than that in experimental side. So did the weight of xenografts. P<0.05. There was some follicular structure of ameloblastoma in the connective tissue around the xenografts of all of the control side. The follicular structure had the typical V-G standard. There was no follicular structure in connective tissue of the experimental side, and only some residual follicular structure scattered in connective tissue. The expression of MMP-2 mRNA and MMP-2 protein in control group was significant higher than that in experimental group, respectively. Conclusion: Nude mice model of xenograft of ameloblastoma was successfully established. The invasion and growth of xenograft of ameloblastoma was suppressed might due to the reason of genesilence effect of MMP-2 targeting siRNA.
到目前为止,成釉细胞瘤局部侵袭的机制尚未明了。一般认为,肿瘤侵袭主要是指肿瘤细胞侵犯和破坏周围正常组织的过程。肿瘤细胞的周边为结缔组织,即基底膜和细胞外基质,这些结构的降解与破坏是肿瘤侵袭转移多阶段过程中的重要步骤。细胞外基质和基底膜的主要组织结构为胶原、层粘素和纤维结合素等,这些结构的破坏与降解需要相应的溶解酶参与。研究表明,基质金属蛋白酶(matrix metalloproteinase,MMP)和相应的金属酶组织抑制剂在肿瘤的侵袭和转移过程中担任了重要的角色。其中,基质金属蛋白酶-2(MMP-2)是目前研究发现的与肿瘤侵袭关系最为密切的一种基质金属蛋白酶。
已有的研究表明,MMP的调节具有三个水平,即基因转录水平、酶原的活化激活水平、抑制剂水平。现有的研究主要集中在后2个方面,而在MMP基因转录水平对MMP进行调节的研究很少。近年来,出现了一种新的基因研究手段-RNA干扰(RNA interference, RNAi),它是将与mRNA对应的正义RNA和反义RNA组成的双链RNA导入细胞,使mRNA发生特异性的降解,导致其相应的基因沉默。RNAi在研究基因功能、基因敲除、基因表达调控和基因治疗方面发挥着重要作用。
本研究旨在以MMP-2为靶基因,采用RNA干扰的方法沉默MMP-2基因,从而从MMP-2基因转录水平调控MMP-2的表达,探讨MMP-2基因沉默后AM细胞侵袭性生
物学行为的改变,进一步研究MMP-2与AM局部侵袭的关系。本研究分四部分,现摘要如下:
第一部分成釉细胞瘤原代细胞培养及生长特性
迄今为止,AM侵袭性生长的机制尚未完全明确。为了研究其侵袭机制,采用体外培养的AM细胞进行研究是一种重要的方法。由于AM细胞在常规培养基中生长缓慢,存活时间短,不容易纯化,不利于AM的细胞生物学研究。为了探索更为适合AM细胞生长的培养基,本实验拟采用DK-SFM无血清培养基和DMEM培养基培养AM细胞,研究AM细胞在无血清培养基中的生长特性,为进一步研究AM的细胞生物学行为奠定基础。
方法:采用DK-SFM无血清培养基和DMEM培养基培养AM细胞,CK14、CK16、CK18及Vimentin免疫组织化学染色鉴定细胞来源,观察细胞的生长特性、细胞形态和传代次数,流式细胞仪测定S期细胞比率(SPF)和增殖指数(PI)。结果:体外培养的AM细胞CK14、CK16免疫组化染色阳性,CK18及Vimentin免疫组化染色阴性。在DK-SFM培养基中,细胞形态清晰,仅见少许散在的成纤维细胞;细胞传代3-6次,平均传代4.8代;细胞存活48-97d,平均存活73.8 d; SPF平均值为6.8%,PI平均值为15.9%。在DMEM中,细胞形态不清晰,可见较多的成纤维细胞;细胞传代2-4次,平均传代3.2代;细胞存活27-61 d,平均存活46.7 d;SPF平均值为5.4%,PI平均值为11.0%。结论:在DK-SFM中培养的AM细胞存活时间长,传代次数较多,DK-SFM较DMEM更适合AM细胞的体外培养。
第二部分MMP-2靶向siRNA表达质粒的构建及序列优化
RNA干扰是生物进化过程中抵抗病毒和转座子的一种保守防御反应。它是由正义RNA和反义RNA组成的双链RNA在细胞内被Dicer酶切割成约19~22nt的siRNA,进而形成siRNA蛋白复合物(siRNP),siRNP通过识别、降解具有同源序列的mRNA,最终导致特异性的基因表达沉默,属于转录后基因沉默机制。当进行RNA干扰实验研究时,需要制备siRNA,目前,siRNA的制备的方法有体外制备法和体内表达法两种。前者是在体外合成siRNA,再导入siRNA于细胞内,该法存在基因沉默维持时间短、转染效果不稳定等问题。后者是在体外构建siRNA表达载体,如质粒载体或病毒载体,载体导入体内后自动合成siRNA,引发基因沉默,该法的基因沉默时间长、转染效果稳定。因此,本实验将构建MMP-2特异性siRNA表达质粒,并优化特异性序列,为MMP-2基因相关的后续实验研究奠定基础。
方法:采用定向克隆的方法构建P1、P2共2条MMP-2特异性siRNA表达质粒,将其转染Hela细胞,采用荧光显微镜观察其转染效果,流式细胞仪检测转染效率,RT-PCR检测MMP-2 mRNA的表达情况。结果:构建的2条MMP-2特异性siRNA表达质粒经基因测序显示克隆的序列完全正确,2条siRNA表达质粒成功转染Hela细胞,转染效率随着时间的延长而增加,72h时,P1的转染效率为41.8%, P2的转染效率为93.2%,P2的转染效率大于P1的转染效率,差异具有显著性,P<0.05;72h时,P1、P2对MMP-2 mRNA的沉默效率分别为38.6%、68.9%。结论:体外构建的MMP-2靶向siRNA表达质粒成功转染Hela细胞并有效沉默MMP-2基因,但有序列差异性,P2的转染效率和基因沉默效率优于P1。
第三部分MMP-2靶向siRNA对成釉细胞瘤侵袭性的抑制作用
MMP-2的表达与活性调节一般有三个水平,即基因转录水平、酶原的活化激活、抑制剂水平。在本课题组的前期研究中,应用MMP-2特异性抑制剂Ro31-9790明显抑制了MMP-2的活性,使裸鼠肾包膜下的AM移植瘤的生长被抑制,这种对MMP-2的调节是在抑制剂水平上的调节。本实验拟以第二部分构建的MMP-2靶向siRNA表达质粒转染AM细胞,以沉默MMP-2基因的表达。从而从MMP-2基因转录水平调控MMP-2的表达,探讨MMP-2基因沉默后AM细胞侵袭性生物学行为的改变,进一步研究AM局部侵袭性生长的机制。
方法:采用第一部分的无血清培养法培养AM细胞,将体外构建的MMP-2靶向siRNA表达质粒转染AM细胞,荧光显微镜检测siRNA表达质粒的转染效果,流式细胞仪检测转染效率及细胞周期和细胞凋亡情况,明胶酶谱分析法检测培养上清液中MMP-2的活性, RT-PCR检测MMP-2 mRNA表达的改变,Western blotting印迹法检测AM细胞MMP-2蛋白表达,三维培养检测AM细胞的侵袭性,Transwell微侵袭分析检测细胞的侵袭抑制率,细胞黏附分析法检测细胞在Fn基质上的黏附率。采用SPSS统计软件对数据进行统计学分析。结果:siRNA表达质粒成功转染AM细胞,实验组细胞的凋亡和细胞周期无明显改变;96h时,质粒转染率为63.6%,培养上清液中酶原性MMP-2和活性MMP-2的表达分别下降了39.6%和56.5%;转染96h后,AM细胞的MMP-2 mRNA表达水平下降了66.0%,MMP-2蛋白表达下降64.6%,细胞的侵袭抑制率为61.3%,黏附抑制率为48.3%,P<0.05。结论:MMP-2靶向siRNA成功转染AM细胞并沉默MMP-2基因,抑制了MMP-2基因的表达,AM的侵袭性被明显抑制,MMP-2与AM细胞的侵袭性密切相关。
第四部分MMP-2靶向siRNA对成釉细胞瘤裸鼠移植瘤的抑制作用
通过前面的实验研究,我们已经证明MMP-2靶向siRNA可以在体外沉默MMP-2基因从而抑制MMP-2基因的表达,进而抑制了AM细胞的侵袭性。为了进一步探讨RNA干扰在生物体内的效应,本实验旨在构建AM移植瘤模型,通过在移植瘤周围局部注射MMP-2靶向siRNA表达质粒,探讨体内RNA干扰对AM侵袭性的抑制作用。方法:取新鲜的AM组织标本,剪切成1-2mm3组织小块,接种于裸鼠双侧前肢根部背侧和后背部皮下。设立自身对照,即左侧前肢处的移植瘤为空白组,注射250μl Opti-MEMR培养基;后背部的移植瘤为脂质体组,在移植瘤周围注射脂质体混合物,空白组和脂质体组均为对照组,右侧前肢处的移植瘤为siRNA组,在移植瘤周围注射MMP-2靶向siRNA表达质粒混合物;接种后第2周末开始施加处理因素,记录移植瘤体积和实验终止时的瘤重,采用SPSS统计软件包中的t检验法对数据进行检验,P<0.05表示有显著性差异。RT-PCR检测移植瘤中MMP-2 mRNA的表达,Western blotting检测移植瘤中MMP-2蛋白的表达,并对移植瘤进行组织病理学分析。结果:所有移植瘤均成活,实验组移植瘤体积增加量少于对照组移植瘤体积的增加量,实验组移植瘤的瘤重少于对照组移植瘤的瘤重,差异有显著性,P<0.05。对照组移植瘤周边的疏松结缔组织中都出现了AM滤泡样结构,滤泡样结构呈现典型的V-G三联征,实验组移植瘤周边的疏松结缔组织中没有AM滤泡样结构或仅仅残留滤泡样结构的痕迹。对照组MMP-2 mRNA和MMP-2蛋白的表达水平均显著性地高于实验组的表达;实验组MMP-2 mRNA表达相对抑制率为42.35%;MMP-2蛋白抑制率为51%。结论:成功构建AM裸鼠皮下移植瘤动物模型; AM移植瘤的侵袭和生长被抑制可能是由于MMP-2靶向siRNA对MMP-2基因的沉默作用引起的。
Human ameloblastoma is the most frequently encounted odontogenic epithelium tumor in oral and maxillofacial region. About 59.3-63.2% of odontogenic epithelium tumors are ameloblastoma. Although it is usually considered as benign tumor, it has the potential of local invasion and bone destruction. The tumor cells of ameloblastoma often invade into bone trabecula. The recurrence rate of ameloblastoma is significantly high after surgical treatment, which might reach to 50%～90%. Repeated surgical operation is often harmful to patient and may cause severe facial deformity. For the sake of these, it is important to study the mechanisms of the invasion of ameloblastomas.
The invasion of tumor mainly means the process that tumor cells invade and destroy the normal tissue around tumor cells. Usually, tissue around tumor is connective tissue, such as basement membrane and extracellular matrix. It is an important step of tumor invasion that degradation and destruction of these structure. The main tissue structures of basement membrane and extracellular matrix including collagen, laminin and fibronectin. To decompose and deconstruct these structure need the effect of resolvase. Studies indicated that matrix metalloproteinase (MMP) and its corresponding tissue inhibitors had taken an important role in the process of tumor invasion and metastasis. Among all of the MMPs discovered recently, MMP-2 is one of MMP which has the closest relationship with tumor invasion. Many studies shown that many kinds of malignant tumor has high expression of MMP-2, and the expression level of MMP- 2 has close relationship with the degree of tumor invasion and poor prognosis. The previous study of our group shows that MMP-2 was positive stain in the cytoplasm of peripheral cylindrocellular around tumor. MMP-2 mRNA also expressed in ameloblastoma, and the expression level was significant higher than that in normal dental follicle tissue.
Usually, there are three levels of modulation for MMP-2 expression and activation, namely, the level of gene transcription, proenzyme activation and inhibitor. Recently, the highlight studies are mainly in latter two aspects. However, the study of the modulation for MMP-2 on transcription level is very few. In the previous study of our group, MMP-2 activity was significantly inhibited by MMP-2 specific inhibitor Ro31-9790, and so did the growth of xenograft of ameloblastoma in submembrane of kidney. This kind of modulation for MMP-2 was based on MMP-2 inhibitor level.
RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing that is thought to inhibit the replication and expression of selfish DNA elements and viruses. RNAi was mediated by the generation of 21-23nt dsRNA molecules, termed small interference RNA (siRNA). dsRNA molecules are processed by the RNaseIII-like enzyme Dicer to generate siRNA, and the siRNAs direct the recognition and subsequent degradation of homologous mRNAs by a multiprotein complex, and the expression of mRNA was silenced. Now, RNAi is a useful method for gene study. It has very important effect in the area of study gene function, gene knock out, adjustment of gene expression and gene therapy.
The main purpose of this study is to suppress MMP-2 expression using RNA interference, and regulate MMP-2 expression on the gene transcript level. The invasive behavior of ameloblastoma after MMP-2 gene silenced by siRNA was studied. Then we further to study the mechanisims of the invasive growth of ameloblastomas. Therefore, this study including five parts a following:
PartⅠPrimary Culture and Growth Characteristic of Ameloblastoma Cells
Up to now, the mechanisims of invasive growth behavior into bone of ameloblastoma are not very clear. So, it is an important method to study the molecular mechanisims of invasiveness and biological characteristic of ameloblastoma by culture ameloblastoma cells in vitro. However, due to the short life span and slow growth of ameloblastoma cells and difficulty to purification in routing culture medium such as DMEM, it is not easy to study the biological behavior of ameloblastoma. In order to find a better culture medium for ameloblastoma cells’growth, serum free culture medium was adopted to culture ameloblastoma cells in this experiment. The growth characteristic of ameloblastoma cells was studied to provide a basis for further study of biological behavior of ameloblastoma.
Methods: Defined Keratinocyte-serum free medium (DK-SFM) and DMEM medium were used for ameloblastoma cell culture in vitro. Cytokeratin 14, 16, 18 and Vimentin monoclonal antibodies were used for immunohistochemistric analysis to identify the origin of cultured cells. The growth rate in different culture medium was observed. The morphology and passages of ameloblastoma cell in different medium were studied. The SPF value and PI value of cells were detected by flow cytometry. Results: Cytokeratin 14 and 16 was positive stain in cultured cells, however, cytokeratin 18 and Vimentin was negative stain in culture cells. In DK-SFM medium, the morphology of cultured cells was distinct. Only some sporadic fibroblast was seen in it.
Ameloblastoma cells were taken through 3 to 6 passages and the average passages were 4.8. The survival time of the cells was maintained 48 days to 97 days, and the average survival time was 73.8 days. The average value of SPF and PI of cells cultured in DK-SFM was 6.8% and 15.9%, respectively. However, in DMEM medium, the morphology of the cells cultured was not clear. On the other hand, a lot of fibroblast was seen in it. The cells were only taken through 2 to 4 passages and the average passages of it were 3.2. The survival time of the cells was maintained 27 days to 61 days, and the average survival time was 46.7 days. The average value of SPF and PI of cells cultured in DMEM are 5.4% and 11.0%, respectively. Conclusion: Ameloblastoma cells have long survival time in DK-SFM medium, which is more suitable for the growth of ameloblastoma cell than that of DMEM medium.
PartⅡConstruction and Sequence Optimization of MMP-2 Gene Specific siRNA Expression Plasmid
RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing that is thought to inhibit the replication and expression of selfish DNA elements and viruses. RNAi was mediated by the generation of 21-23nt dsRNA molecules, termed small interference RNA (siRNA). dsRNA molecules are processed by the RNaseIII-like enzyme Dicer to generate siRNA, and the siRNAs direct the recognition and subsequent degradation of homologous mRNAs by a multiprotein complex, and finally, the expression of mRNA was silenced. It is belong to post transcription silence. In RNAi experiment, there are two methods of constructing siRNA, namely, construct siRNA in vitro and siRNA expression in vivo. The former is to synthesis siRNA in vitro, and then the siRNA was transfected into cells. There are some problems in this kind of method to construct siRNA, such as short gene silence time and unstable transfection effect. The latter is to construct siRNA expression vector such as plasmid vector or virus vector. After tansfecting this vector into cells, it will produce siRNA automatically. It has long gene silence time and stable transfection effect. So, in present experiment, MMP-2 specific siRNA expression plasmid will be constructed, and the specific sequence of siRNA will be optimized in order to provide basis for next experiment study related to MMP-2 gene.
Methods: Two items of siRNA expression plasmids, namely P1 and P2, were constructed by directed cloning and transfected into Hela cell by Lipofectamine 2000. The transfection effect was confirmed by fluorescence microscope. The transfection rate was checked by flow cytometry. Reverse transcript polymerase chain reaction (RT-PCR) was used to detect the expression of MMP-2 mRNA. Results: All of the two items of siRNA expression plasmids were successfully tranfected into Hela cells. The transfection rate at 72h of P1 and P2 was 41.8% and 93.2%, respectively. The transfection rate of P2 was significant higher than that of P1. The inhibition rate of MMP-2 mRNA expression of P1 and P2 was 38.6% and 68.9%, respectively. Conclusion: The MMP-2 specific siRNA expression plasmid constructed in vitro was successfully transfected into Hela cells, but it had sequence difference. The trasfection rate and gene silence rate of P2 is better than that of P1.
PartⅢInhibition of Invasiveness of Ameloblastoma by Suppression MMP-2 Gene Expression Using MMP-2 Gene Targeting siRNA
Usually, there are three levels of modulation for expression and activity of MMP-2, namely, the level of MMP-2 gene transcription, activation of proenzyme and inhibitor of MMP-2. In our previous study, the activity of MMP-2 was obviously inhibited by using MMP-2 specific inhibitor Ro31-990, and so did the growth of xenograft of ameloblastoma in submembrane of kidney. This adjustment of MMP-2 was based on MMP-2 inhibitor level. In present experiment, MMP-2 targeting siRNA expression plasmid constructed in Part III was transfected into ameloblastoma cells in order to silence MMP-2 gene and to regulate the expression of MMP-2 on the level of MMP-2 gene transcription. The invasive behavior of ameloblastoma after MMP-2 gene silencing by siRNA was studied.
Methods: Ameloblastoma cells were cultured in Defined Keratinocyte-SFM medium. MMP-2 specific siRNA expression plasmid constructed in vitro was transfected into ameloblastoma cells. Fluorescence microscope was used to detect the result of transfection. Flow cytometry was used to check the transfection rate, the cell cycle and apoptosis. Gelatinolytic zymography analysis was used to investigate the activity of MMP-2 in supernatant of culture medium. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to check the expression of MMP-2 mRNA. Western-blot analysis was performed to investigate the expression of MMP-2 protein. The invasiveness of ameloblastoma cells was detected by three dimension culture system. Transwell analysis was done to measure the invasion of ameloblastoma cells. Cell adhesion rate on fibronectin was detected by cell adhesive analysis. The analysis of students t test was used to analyze the data. Results: There is no significant difference of apoptosis between experimental study and control study. MMP-2 specific siRNA expression plasmid was successfully transfected into ameloblastoma cells. The expression of proferment MMP-2 and active MMP-2 in supernatant of experimental group were decreased by 39.6% and 56.5%, respectively. The transfection rate at 96h after transfection was 63.6%. At the same time, MMP-2 mRNA expression was reduced to 66.0%. MMP-2 protein expression was reduced to 64.6%. The inhibition rate of the invasiveness of ameloblastoma cells was 61.3%. The inhibition rate of adhesion of ameloblastoma cell was 48.3%. P<0.05. Conclusion: MMP-2 gene was successfully silenced by MMP-2 targeting siRNA. MMP-2 has close relation with the invasiveness of ameloblastoma.
PartⅣInhibition Against Xenografts of Ameloblastoma in Nude Mice Using MMP-2 Targeting siRNA
In previous experiment, it has been demonstrated that MMP-2 gene could be silenced by MMP-2 targeting siRNA in vitro, and the expression of MMP-2 was inhibited, and then, the invasive behavior of ameloblastoma cells had been inhibited. In order to study the effects of RNAi in vivo, in present experiment, subcutaneous xenograft of ameloblastoma in nude mice was established and MMP-2 targeting siRNA expression plasmid was injected around the xenograft. Methods: Fresh ameloblastoma tissue was minced into 1-2 mm3 size and subcutaneous transplanted into the back of the two anterior extremities root and the posterior back of nude mice. Self control was designed. Namely, left anterior limb side was taken as blank group and 250μl Opti-MEMR I culture medium was injected around the xenograft, posterior back side was taken as liposome group and compounds of liposome was injected around the xenograft, right anterior limb side was taken as experiment side and MMP-2 specific siRNA expression plasmid was injected around the xenografts. Two weeks after transplantation of the ameloblastoma tissue lump, the experimental factor was exerted on control side and experimental side according to the group design. The volume of the xenografts and the weight of xenografts in the termination time of the experiment were recorded. The students t test of SPSS11.0 software was taken as statistic analysis. If the P value is less than 0.05, the result has significance. The pathological analysis was performed to study the xenografts. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to check the expression of MMP-2 mRNA in xenografts. Western-blot analysis was performed to investigate MMP-2 protein in xenografts. Results: All of the xenografts were survival. The volume of xenografts in control side was significant larger than that in experimental side. So did the weight of xenografts. P<0.05. There was some follicular structure of ameloblastoma in the connective tissue around the xenografts of all of the control side. The follicular structure had the typical V-G standard. There was no follicular structure in connective tissue of the experimental side, and only some residual follicular structure scattered in connective tissue. The expression of MMP-2 mRNA and MMP-2 protein in control group was significant higher than that in experimental group, respectively. Conclusion: Nude mice model of xenograft of ameloblastoma was successfully established. The invasion and growth of xenograft of ameloblastoma was suppressed might due to the reason of genesilence effect of MMP-2 targeting siRNA.
引文
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