RECK在食管鳞癌组织中的表达及其与浸润转移关系的研究
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摘要
食管癌是我国最常见的恶性肿瘤之一,我国特别是河南等省份是食管癌的高发地区,其死亡率居恶性肿瘤的第4位。由于浸润、转移是引起食管癌患者死亡的主要原因,因此对食管癌浸润转移机制的研究已成为当前研究的热点课题。
肿瘤的发生发展是多因素、多步骤、多阶段和多基因参与并发生改变的过程,原癌基因的激活和抑癌基因的功能失活是人类肿瘤形成和发展的物质基础,以往对抑癌基因的研究发现,p53、Rb和p16等的突变和缺失与多数恶性肿瘤的发生密切相关。
RECK(reversion inducing cysteine rich protein with Kazal motifs,RECK)是Takahashi新近发现的一种新型转移抑制基因,研究表明,该基因的表达与肿瘤的浸润转移及血管生成密切相关。近年来,围绕RECK与肿瘤侵袭转移这一主题,先后有肝癌、胰腺癌、乳腺癌和肺癌等相关文献报道。研究表明,RECK基因表达量与肿瘤的侵袭力呈负相关,且RECK基因表达较高的患者预后往往也明显好于表达量低的患者,该基因缺失或/和突变及mRNA或蛋白表达降低与肿瘤的发生、浸润、转移及组织学分级有关,其机制可能与RECK通过抑制MMP-2、MMP-9及MT1-MMP的分泌活性,从而对肿瘤生长、侵袭和转移具有负性调控作用有关。另外,RECK还可通过抑制肿瘤血管生成而抑制肿瘤生长、侵袭和转移。有关在食管癌中RECK的表达及其与食管鳞癌浸润转移关系的研究,除本课题组前期发表的相关文章外,迄今国内外未见其他文献报道。
为深入探讨RECK基因与食管鳞癌发生和浸润转移的关系,寻求早期检测食管鳞癌发生和浸润转移的指标及寻找抑制食管鳞癌发生和浸润转移的有效方法,本研究首先采用RT-PCR、原位杂交及免疫组化SP法联合检测了食管癌新鲜手术切除标本中RECK、MMP-9mRNA和蛋白的表达水平;采用免疫组化SP法检测了食管鳞癌、癌旁不典型增生组织及正常食管粘膜组织中血管内皮细胞生长因子(VEGF)的表达情况,同时用抗CD105抗体检测了62例食管鳞癌中微血管密度(MVD),探讨了RECK与VEGF、MVD的相关性,以揭示RECK与食管癌浸润、转移之间的关系。在此基础上,成功构建了RECK真核表达载体,然后将其转染入食管癌EC9706细胞中,分别应用RT-PCR、原位杂交联合检测了食管癌EC9706细胞中RECK、MMP-gmRNA表达水平;以免疫组化SP法和Western blot法联合检测了食管癌新鲜手术切除标本中RECK、MMP-9蛋白的表达水平;运用Boyden chamber侵袭实验技术观察转染细胞体外侵袭力的改变。最后通过裸鼠移植瘤实验,采取原位杂交、RT-PCR、免疫组织化学、联合检测了RECK、MMP-9等在裸鼠移植瘤中的表达情况,从基因、蛋白、细胞定位及肿瘤细胞生物学行为、体外细胞侵袭实验、体内裸鼠成瘤实验等多个方面深入探讨上调RECK的表达及活性对抑制食管癌侵袭的作用,试图阐明RECK的表达在食管鳞癌发生、发展及浸润、转移中的意义;并进一步阐明RECK基因抑制食管鳞癌发生、发展及浸润、转移的机制是否与下调VEGF表达、降低肿瘤中的MVD有关。为进一步探讨食管鳞癌发生、浸润、转移机制及寻找抑制食管鳞癌发生、发展的途径提供理论基础。本研究共分以下4个部分。
第一部分RECK在食管鳞癌组织中的表达及临床意义
方法
1.采用免疫组织化学的方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中RECK基因蛋白的表达情况,并进行形态学观察。
2.采用RT-PCR方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中RECK基因mRNA的表达情况,并进行形态学观察。
3.采用原位杂交方法检测62例食管鳞癌的癌组织、31例癌旁不典型增生组织、62例正常食管粘膜组织之中RECK基因mRNA的表达情况,并进行形态学观察。
4.统计学处理:应用SPSS11.0软件处理,采用χ2检验、t检验和方差分析。检验水准α=0.05。
结果
1.RECK mRNA及蛋白阳性表达主要位于正常食管粘膜鳞状上皮细胞的胞质内,在肿瘤细胞内无表达或者表达量较低。
2.RT-PCR和原位杂交联合检测结果显示:在正常食管粘膜组织、癌旁不典型增生组织和食管鳞癌组织中,RECK mRNA表达水平依次降低,三者组间比较差异有统计学意义(P＜0.05)。免疫组织化学方法检测结果显示:在正常食管粘膜组织、癌旁不典型增生组织和食管鳞癌组织中,阳性表达率依次降低,三者组间比较差异有统计学意义(P＜0.05)。
3.在浸达深肌层者和伴淋巴结转移的食管鳞癌组织中,RECK mRNA阳性表达率较侵达浅肌层者和无淋巴结转移者显著降低,组间比较差异有统计学意义(P＜0.05);在侵达深肌层者和伴淋巴结转移的食管癌组织中,RECK蛋白阳性表达率较侵达浅肌层者和伴无淋巴结转移者在显著降低,组间比较差异有统计学意义(P＜0.05)。
4.在食管高分化、中分化及低分化鳞癌组织中,其RECK蛋白和mRNA阳性表达率均依次降低,组间比较均有统计学意义(P＜0.05)。低分化食管鳞癌比高、中分化食管鳞癌RECK mRNA阳性表达率明显降低,差异有统计学意义(P＜0.05);低分化食管癌比高、中分化食管癌RECK蛋白阳性表达率明显降低,差异有统计学意义(P＜0.05)。
5.RECK mRNA表达与食管癌患者的性别、年龄、大体类型及肿瘤发生部位无关(P＜0.05)。
第二部分MMP-9、VEGF及CD105在食管鳞癌组织中的表达及其与RECK基因关系的研究
方法
1.采用RT-PCR方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中MMP-9 mRNA的表达情况及其相关性。
2.采用原位杂交方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中MMP-9 mRNA的表达情况及其相关性。
3.采用免疫组织化学的方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中MMP-9、VEGF的蛋白表达情况及起相关性,并进行形态学观察。
4.采用免疫组织化学的方法检测62例食管鳞癌组织之中CD105蛋白表达的情况及其相关性研究,并进行形态学观察。
5.统计学处理:应用SPSS11.0软件处理,采用χ~2检验、t检验和方差分析。检验水准α=0.05。
结果
1.MMP-9 mRNA及蛋白阳性表达主要位于肿瘤细胞的胞质内,在正常食管粘膜鳞状上皮细胞内无表达或者表达量较低。MMP-9蛋白及mRNA在三样品中表达与RECK的表达均呈负相关关系(P＜0.05)。
2.浸润深度达深层者和淋巴结转移的食管癌组织中,MMP-9蛋白及mRNA表达较浸润深度达浅层者和无淋巴结转移的显著升高,组间比较差异有统计学意义(P＜0.05)。
3.低分化食管癌比高、中分化食管鳞癌MMP-9蛋白及mRNA阳性表达显著降低,差异有统计学意义(P＜0.05)。
4.VEGF蛋白阳性表达主要位于肿瘤细胞的胞质内。正常食管粘膜组织、癌旁不典型增生组织和食管癌组织中VEGF蛋白表达水平依次升高,三者组间比较差异有统计学意义(P＜0.05)。食管鳞癌组织中VEGF蛋白表达与食管鳞癌组织的浸润深度及淋巴结转移相关。其蛋白表达与RECK蛋白表达呈负相关关系,与MMP-9蛋白表达呈正相关关系(P＜0.05)。
5.CD105蛋白阳性表达主要位于肿瘤间质的血管内皮细胞的胞浆中。CD105在浸润深度达深肌层和有淋巴结转移组的蛋白阳性表达率较浸润深度达浅层者和无淋巴结转移的显著降低,组间比较差异有统计学意义(P＜0.05)。其蛋白表达与RECK蛋白表达呈负相关关系,与MMP-9及VEGF蛋白表达呈正相关关系(P＜0.05)。
6.MMP-9 mRNA、蛋白表达及VEGF及CD105蛋白表达与食管癌患者的性别、年龄、等无关(P＞0.05)。
第三部分人RECK基因的分子克隆和真核表达载体的构建及其对人食管癌细胞株EC9706生物学行为的影响
方法
1.从食管组织中提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增出人RECK基因;限制性内切酶KpnⅠ和NotⅠ双酶切PCR产物和pcDNA3质粒,然后用T4 DNA Ligase连接,转化大肠杆菌JM109感受态细胞,PCR验证转化子,挑取阳性克隆扩大培养,提取其质粒,进行KpnⅠ和NotⅠ双酶切验证构建RECK基因的真核表达载体pcDNA3-RECK并经酶切鉴定。
2.采用脂质体Lipofectamine2000介导将真核表达载体pcDNA3-RECK导入体外培养的食管癌EC9706细胞。
3.采用RT-PCR、原位杂交方法检测转染前、后RECK及MMP-9在人食管癌细胞株EC9706 mRNA的表达情况。
4.采用免疫细胞化学、Western blot的方法检测转染前、后RECK及MMP-9在人食管癌细胞株EC9706蛋白的表达情况。
5.细胞计数法绘制各组细胞的生长曲线以观察RECK基因对细胞增殖能力的影响。
6.Boyden chamber实验计算各组穿膜的细胞数以比较转染RECK基因对细胞侵袭能力的变化。
7.统计学处理:应用SPSS11.0软件处理,采用χ2检验、方差分析。检验水准α=0.05。
结果
1.PCR扩增得到了特异性的4.4kb基因片段。基因片段的大小与预期一致。所获得的RECK基因测序及BLAST分析证明克隆的人RECK基因序列正确,pcDNA3-RECK经酶切后得到5.4kb和4.4kb两个片段。
2.PCR鉴定、酶切鉴定pcDNA3-RECK重组载体,结果显示:PCR扩增片段及KpnⅠ和NotⅠ双酶切片段大小与设计相符。
3.DNA测序结果证明:所构建真核表达载体上的目的基因核苷酸序列与GenBank上基因预计部分完全一致,且方向正确。
4.转染pcDNA3-RECK真核表达载体后,EC9706细胞均能稳定高表达RECKmRNA及蛋白。
5.Boyden chamber体外侵袭实验结果显示:与空白对照组相比,EC9706pcDNA3-RECK组穿越Matrigel胶的细胞数显著减少(P＜0.05),而pcDNA3空质粒组及空白对照组之间则无明显差异(P＞0.05)。
第四部分RECK基因对裸鼠移植瘤生长的抑制作用及机制研究
方法
1.分别将转染前、后及空质粒组的食管癌细胞株EC9706注入裸鼠皮下,比较各组裸鼠成瘤大小。
2.分别应用RT-PCR、原位杂交方法检测各组裸鼠肿瘤组织中RECK、MMP-9基因的mRNA表达情况。
3.应用免疫组织化学方法检测各组裸鼠肿瘤组织中RECK、MMP-9、VEGF基因的蛋白表达情况。
4.统计学处理:应用SPSS11.0软件处理,采用χ2检验、方差分析。检验水准α=0.05。
结果
1.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤体积显著大于EC9706/pcDNA3-RECK组裸鼠移植瘤体积,组间比较差异具有统计学意义(P＜0.05)。
2.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤RECK基因蛋白及mRNA表达显著低于EC9706/pcDNA3-RECK的表达,组间比较差异具有统计学意义(P＜0.05)。
3.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤MMP-9基因蛋白及mRNA表达显著高于EC9706/pcDNA3-RECK的表达,组间比较差异具有统计学意义(P＜0.05)。
4.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤VEGF基因蛋白表达显著高于EC9706/pcDNA3-RECK的表达,组间比较差异有统计学意义(P＜0.05)。
结论
1.食管鳞癌组织中RECK蛋白及mRNA呈低表达。
2.RECK可能是食管鳞癌侵袭、转移的重要生物学标记。
3.RECK在肿瘤的演进中具有重要的作用。
4.成功构建了RECK真核表达载体pcDNA3-RECK,并成功导入人食管癌EC9706细胞株中,筛选出了稳定细胞株,为进一步研究RECK的生物学功能和RECK的靶向治疗奠定了基础。
5.pcDNA3-RECK可上调EC9706细胞的蛋白及mRNA的表达。
6.RECK基因可通过抑制MMP-9来影响肿瘤的浸润、转移。
7.运用Boyden chamber技术检测了RECK基因转染前后及空质粒组EC9706细胞的侵袭能力的变化。
8.RECK基因有可能通过拮抗MMP-9来降低食管癌细胞的体外侵袭能力。
Esophagus cacinoma is one of the most common malignancy in our country,and Henan province also has higher morbility.It's mortality reside forth position in our country,whereas it's invasion and metastasis is the first cause of invoke esophagus cancer sufferer's obituary,so discussing the esophague cacinoma invasion and metastasis mechanism became one of the investgative hotspot.
Tumor's occurrence and development are the course of multifactor,multistep, multistage and polygene alterative.Oncogene activation and tumor suppressor gene inactivation are the material basis of human tumor formation and development.Previous research found that the mutations and deletions of some tumor suppressor genes,such as p53,Rb,p16 and so on,are relate to malignancy occurrence.
A new anti-oncogene named RECK(reversion inducing cysteine rich protein with Kazal motifs,RECK)was found in recent years.The expression of RECK closely relate to tumorous invasion、metastasis and angiogenesis.In recent years,there are several relevant studies about the relation between RECK and tumor invasion and metastasis in the fields of liver cancer,pancreatic cancer,breast cancer and lung cancer.Researches show that RECK gene's expression were negative correlate to tumorous invasiveness and the sufferers whose RECK gene higher expression have better prognoses than those with lower expression quantity.This gene's deletions or(and) mutation and depressing expression of mRNA or protein relate to tumorous occur,invasion,metastasis and histology grade.The mechanism may be that RECK regulated negatively tumour growth,invasion,metastasis through restraining the secretion and activity of MMP-2,MMP-9 and MT1-MMP.In addition, RECK can inhibit tumor growth,invasion and metastasis by inhibition of tumor angiogenesis.There have no other report both in China and abroad so far except the relevant articles pulished by our team before.
In order to discuss RECK gene's relation to esophagus squama carcinogenesis and invasion,metastasis for more,sought early detection esophagues squama carcinogenesis and soakage transitionary indices and found available approach to restrain esophagus squama carcinogenesis and soakage transitionary,this topic firstly used RT-PCR, hybridization in situ and immunohistochemistry SP combine detected the expression of esophagus cancer verdure excision specimen's RECK,MMP-9mRNA and protein.We use immunohistochemistry SP detected blood vssel endotheliocyte growth factor in esophague squama,adjacentatypical hyperplasia tissue and normal esophagus mucous membrane tissue.At the same time,use resist CD105 antibody detected microvascular density(MVD) in 62 cases' esophagus squama carcinoma,and discussed the relationship among RECK, VEGF and MVD,revealed the relation of RECK and esophagus cancer invasion and metastasis.On this foundation,we constructed RECK's eukaryon expression vector successfully,then transfected it into esophagus cancer cell EC9706,use RT-PCR, hybridization in situ combine detected esophagus cancer cell EC9706's RECK, MMP-9mRNA expression level,respectively;Immunohistochemistry SP combined Western blot detected esophagus cancer verdure excision specimen's RECK,MMP-9 protein's expression level;The transfected cell's invasion ability was determined by Boyden-chamber model in vitro.In the final,apply nude mice lotus tunour experiment, combined use hybridization in situ,RT-PCR,immunohistochemistry detected the expression of RECK,MMP-9 at nude mice transplantation tumor,we in depth explorate upper regulate RECK's expression and activity restrain esophagus cancer invade in multi-aspect,such as gene,protein,cellular localization and cellular biology of tumor behavior,vitro invade cell experiment,in vivo nude mice achieve tumour experiment and so on.We try to illuminate the meaning in RECK's expression at esophagus squama carcinogenesis and development,invasion and metastasis.For more illuminate RECK gene repress esophagus squama carcinogenesis and development,invasion and metastasis's mechanism whether related to down regulate the expression of VEGF,depress MVD in tumour.Providing fundamental basis for deeply discuss esophagus carcinogenesis,invasion and metastasis mechanism and soght restrain esophagus squama carcinogenesis, developmental way.This study includes the following four parts.
The first part:The clinicopathological significance of the expression of RECK in esophageal squamaous cell carcinoma
Methods:
1.Detected RECK gene protein expression in 62 cases esophageal squamous cell carcinoma tissue(ESCC),31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by immunohistochemical, then observed morphology.
2.Detected RECK gene's mRNA expression in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by RT-PCR,then observed morphology.
3.Detected RECK gene's mRNA expression in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by hybridization in situ,then observed morphology.
4.Statistical treatment:Statistics analysis was performed by SPSS 11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.RECK mRNA and protein masculine expression in esophagus cancer tissue mostly locate in normal esophagus mucous membrane squamous cells' cytoplast,no or lower expression in tumor cell.
2.Combine RT-PCR and hybridization in situ detect resulting:RECK mRNA expression level depress by turns in normal esophagus mucous membrane tissue,adjacent atypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).The result of immunohistochemical display:RECK mRNA expression level depress by turns in normal esophagus mucous membrane tissue, adjacentatypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).
3.RECK mRNA masculine expression rate in infiltrate to deep depth and lymphatic metastasis' esophagus cancer tissue signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).RECK protein masculine expression rate in infiltrate to deep depth and lymph node metastasis' esophagus cancer tissue signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).
4.RECK mRNA masculine expression rate in poorly differentiated esophagus cancer signifcant depress compare to well,moderately differentiated esophagus cancer,the difference make statistics sense(P＜0.05).RECK protein masculine expression rate in poorly differentiated esophagus cancer signifcant depress compare to well,moderately differentiated esophagus cancer,the difference make statistics sense(P＜0.05).
5.RECK mRNA's expression was not related esophagus cancer sufferer's sex,age, general style and tumorigenes is position.
The second part:The study on MMP-9,VEGF,CD105's expression in ESCC tissue and the relation to RECK gene
Methods:
1.Detected MMP-9 mRNA expression and relation in 62 eases ESCC tissue(ESCC), 31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by RT-PCR.
2.Detected MMP-9 mRNA expression and relation in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by hybridization in situ.
3.Detected MMP-9,VEGF's protein expression and relation 62 cases ESCC tissue(ESCC),31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by immunohistochemical,then observed morphology.
4.Detected CD105 protein expression and relation in 62 cases ESCC tissue by immunohistochemical,then observed morphology.
5.Statistical treatment:Statistics analysis was performed by SPSS11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.The expression of MMP-9 mRNA and protein mainly located in the cytoplasm of tumor cells and no or lower expression in the normal esophageal squamous mucosal epithelial cells.The expression of MMP-9 mRNA and protein in three groups was negatively correlated with the expression of RECK(P＜0.05).
2.MMP-9 mRNA masculine expression rate in infiltrate to deep depth and lymphatic metastasis' esophagus cancer tissue siguifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).
3.The expression MMP-9 protein and mRNA was significantly decreased in poorly differentiated esophageal cancer compare to well,moderately differentiated ones.The difference among groups make statistics sense(P＜0.05).
4.The expression of VEGF protein mainly located in the cytoplasm of tumor cells. The expression level step up by turns in normal esophagus mucous membrane tissue, adjacent atypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).Esophagus squama carcinoma tissue VEGF protein expression was related to esophagus squama carcinoma tissue soakage depth and lymphatic metastasis,VEGF protein masculine expression mostly locate in esophagus cancer cells' cytoplast.
5.CD105 protein masculine expression mostly locate in tumour interstitial blood vessel endotheliocyte's kytoplasm.The masculine expression rate in esophagus cancer tissue infiltrated to deep depth and with lymphatic metastasis signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis.The difference among groups make statistics sense(P<0.05).The protein expression is negatively correlated with the expression of RECK and positively correlated with the expression of MMP-9(P<0.05).
6.RECK mRNA's expression was not related esophagus cancer sufferer's sex,age, general style and tumorigenesis position(P>0.05).
The third part:The influence of biology behaviour on Human RECK gene molecular cloning and construction of eukaryotic expression bearer to human esophagus cancer strain EC9706
Methods:
1.Total RNA was extracted from from esophagus tissue,RECK cDNA was isolated by using reverse transcription-polymerase chain reaction(RT-PCR);use restrict enzyme Kpn I and Not I two enzyme cut PCR offspring and pcDNA3 plasmid,then use T4 DNA Ligase joint,translate colibacilus JM109 competent call,PCR demonstrat converter,pick masculine clone intermediate culture,abstract there plasmid,proceed Kpn I and Not I two enzyme cut demonstrat construct RECK genic eukaryon expression bearer pcDNA3-RECK and judged by enzyme cut.
2.Adopt liposome Lipofectamine2000 mediate eukaryon expression bearer pcDNA3 -RECK import esophagus cancer EC9706 cell in vitro culture.
3.Detect pro and post transfection RECK and MMP-9 in human esophagus cancer cell strain EC9706 mRNA's expression by RT-PCR,hybridization in situ.
4.Detect pro and post transfection RECK and MMP-9 in human esophagus cancer cell strain EC9706 protein's expression by immunocytochemistry,Western blot.
5.Cell count protract each group cellular growth curve and observe RECK gene's impact on cell multiplication capable.
6.Boyden chamber experiment count each group worn membranous cell count and compare transfect RECK gene varied to cell invade capable.
7.Statistical treatment:Statistics analysis was performed by SPSS 11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.Amplified idiosyncratic 4.4Kb gene fragment through PCR.Gene piecewise size accord with anticipate.The RECK gene sequencing and BLAST analysis proved the exactness of clone human RECK gene order,adopt enzyme cut pcDNA3-RECK got 5.4 kb and 4.4kb two fragment.
2.PCR judge,enzyme cut judge pcDNA3-RECK reform bearer,resulting display: PCR amplificate fragment and Kpn I and Not I two enzyme slab segment size match with design.
3.DNA sequencing result display:The construct eukaryon expression bearer's purpose gene nucleotidesequence perfect agreement with GeneBank's predict part of gene, and in the right direction.
4.After transfected pcDNA3-RECK eukarya expression bearer,EC9706 cells all can stable express RECK mRNA and protein.
5.Boyden chamber extraneous invade experimental result display:Compare with blank control group,EC9706pcDNA3-RECK group notable decrease in through Matrigel gluey's cell(P＜0.05),but pcDNA3 was no distinct difference in vacancy plasmid group and blank control group(P＞0.05)
The fourth part:The research of inhibitory action and mechanism on RECK gene to nude mice transplantation tumor's vegetal
Methods:
1.Separately inject pro,post transfection and vancancy plasmid group's esophagus cancer cell strain EC9706 into nude mice subsurface,compared each groups nude mice's tumor size and metastasis scale.
2.Detect each group's nude mice tissue's RECK,MMP-9 mRNA expression by RT-PCR,hybridization in situ.
3.Detect each group's nude mice turnout tissue's RECK,MMP-9,VEGF genie protein expression by immunohistochemical method.
4.Statistical treatment:Statistics analysis was performed by SPSS11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor volume significant larger than EC9706/pcDNA3-RECK group's,compare among groups had difference in statistic sence(P＜0.05).
2.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor RECK gene protein and mRNA expression significant lower than EC9706/pcDNA3 -RECK's expression,compare among groups had difference in statistic sence(P＜0.05).
3.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor MMP-9 gene protein and mRNA expression significant higher than EC9706/pcDNA3 -RECK's expression,compare among groups had difference in statistic sence(P＜0.05).
4.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor VEGF gene protein expression significant higher than EC9706/pcDNA3-RECK's expression,compare among groups had difference in statistic sence(P＜0.05).
Conclusion
1.The expression of RECK protein and mRNA is lower in esophagus squama carcinoma tissue.
2.RECK may be a important biological markers of invasion and metastasis of ESCC.
3.RECK play an important role in the tumor evolution.
4.Successfully constructing RECK genie eukaryon expression bearer peDNA3-RECK, and importing human esophageal cancer EC9706 cell lines.Selecting a stable cell line laid foundation for further research about the biological functions of RECK and target therapy on RECK.
5.pcDNA3-RECK can be increased RECK protein and mRNA expression of EC9706 cell.
6.RECK can influence tumor invasion and metastasis by inhibiting MMP-9.
7.Using Boyden chamber technology to detect the change of invasion in pro and post transfected plasmid groups and blank control group.
8.RECK can anti tumor invasion and metastasis by inhibiting MMP-9.
肿瘤的发生发展是多因素、多步骤、多阶段和多基因参与并发生改变的过程,原癌基因的激活和抑癌基因的功能失活是人类肿瘤形成和发展的物质基础,以往对抑癌基因的研究发现,p53、Rb和p16等的突变和缺失与多数恶性肿瘤的发生密切相关。
RECK(reversion inducing cysteine rich protein with Kazal motifs,RECK)是Takahashi新近发现的一种新型转移抑制基因,研究表明,该基因的表达与肿瘤的浸润转移及血管生成密切相关。近年来,围绕RECK与肿瘤侵袭转移这一主题,先后有肝癌、胰腺癌、乳腺癌和肺癌等相关文献报道。研究表明,RECK基因表达量与肿瘤的侵袭力呈负相关,且RECK基因表达较高的患者预后往往也明显好于表达量低的患者,该基因缺失或/和突变及mRNA或蛋白表达降低与肿瘤的发生、浸润、转移及组织学分级有关,其机制可能与RECK通过抑制MMP-2、MMP-9及MT1-MMP的分泌活性,从而对肿瘤生长、侵袭和转移具有负性调控作用有关。另外,RECK还可通过抑制肿瘤血管生成而抑制肿瘤生长、侵袭和转移。有关在食管癌中RECK的表达及其与食管鳞癌浸润转移关系的研究,除本课题组前期发表的相关文章外,迄今国内外未见其他文献报道。
为深入探讨RECK基因与食管鳞癌发生和浸润转移的关系,寻求早期检测食管鳞癌发生和浸润转移的指标及寻找抑制食管鳞癌发生和浸润转移的有效方法,本研究首先采用RT-PCR、原位杂交及免疫组化SP法联合检测了食管癌新鲜手术切除标本中RECK、MMP-9mRNA和蛋白的表达水平;采用免疫组化SP法检测了食管鳞癌、癌旁不典型增生组织及正常食管粘膜组织中血管内皮细胞生长因子(VEGF)的表达情况,同时用抗CD105抗体检测了62例食管鳞癌中微血管密度(MVD),探讨了RECK与VEGF、MVD的相关性,以揭示RECK与食管癌浸润、转移之间的关系。在此基础上,成功构建了RECK真核表达载体,然后将其转染入食管癌EC9706细胞中,分别应用RT-PCR、原位杂交联合检测了食管癌EC9706细胞中RECK、MMP-gmRNA表达水平;以免疫组化SP法和Western blot法联合检测了食管癌新鲜手术切除标本中RECK、MMP-9蛋白的表达水平;运用Boyden chamber侵袭实验技术观察转染细胞体外侵袭力的改变。最后通过裸鼠移植瘤实验,采取原位杂交、RT-PCR、免疫组织化学、联合检测了RECK、MMP-9等在裸鼠移植瘤中的表达情况,从基因、蛋白、细胞定位及肿瘤细胞生物学行为、体外细胞侵袭实验、体内裸鼠成瘤实验等多个方面深入探讨上调RECK的表达及活性对抑制食管癌侵袭的作用,试图阐明RECK的表达在食管鳞癌发生、发展及浸润、转移中的意义;并进一步阐明RECK基因抑制食管鳞癌发生、发展及浸润、转移的机制是否与下调VEGF表达、降低肿瘤中的MVD有关。为进一步探讨食管鳞癌发生、浸润、转移机制及寻找抑制食管鳞癌发生、发展的途径提供理论基础。本研究共分以下4个部分。
第一部分RECK在食管鳞癌组织中的表达及临床意义
方法
1.采用免疫组织化学的方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中RECK基因蛋白的表达情况,并进行形态学观察。
2.采用RT-PCR方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中RECK基因mRNA的表达情况,并进行形态学观察。
3.采用原位杂交方法检测62例食管鳞癌的癌组织、31例癌旁不典型增生组织、62例正常食管粘膜组织之中RECK基因mRNA的表达情况,并进行形态学观察。
4.统计学处理:应用SPSS11.0软件处理,采用χ2检验、t检验和方差分析。检验水准α=0.05。
结果
1.RECK mRNA及蛋白阳性表达主要位于正常食管粘膜鳞状上皮细胞的胞质内,在肿瘤细胞内无表达或者表达量较低。
2.RT-PCR和原位杂交联合检测结果显示:在正常食管粘膜组织、癌旁不典型增生组织和食管鳞癌组织中,RECK mRNA表达水平依次降低,三者组间比较差异有统计学意义(P＜0.05)。免疫组织化学方法检测结果显示:在正常食管粘膜组织、癌旁不典型增生组织和食管鳞癌组织中,阳性表达率依次降低,三者组间比较差异有统计学意义(P＜0.05)。
3.在浸达深肌层者和伴淋巴结转移的食管鳞癌组织中,RECK mRNA阳性表达率较侵达浅肌层者和无淋巴结转移者显著降低,组间比较差异有统计学意义(P＜0.05);在侵达深肌层者和伴淋巴结转移的食管癌组织中,RECK蛋白阳性表达率较侵达浅肌层者和伴无淋巴结转移者在显著降低,组间比较差异有统计学意义(P＜0.05)。
4.在食管高分化、中分化及低分化鳞癌组织中,其RECK蛋白和mRNA阳性表达率均依次降低,组间比较均有统计学意义(P＜0.05)。低分化食管鳞癌比高、中分化食管鳞癌RECK mRNA阳性表达率明显降低,差异有统计学意义(P＜0.05);低分化食管癌比高、中分化食管癌RECK蛋白阳性表达率明显降低,差异有统计学意义(P＜0.05)。
5.RECK mRNA表达与食管癌患者的性别、年龄、大体类型及肿瘤发生部位无关(P＜0.05)。
第二部分MMP-9、VEGF及CD105在食管鳞癌组织中的表达及其与RECK基因关系的研究
方法
1.采用RT-PCR方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中MMP-9 mRNA的表达情况及其相关性。
2.采用原位杂交方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中MMP-9 mRNA的表达情况及其相关性。
3.采用免疫组织化学的方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织之中MMP-9、VEGF的蛋白表达情况及起相关性,并进行形态学观察。
4.采用免疫组织化学的方法检测62例食管鳞癌组织之中CD105蛋白表达的情况及其相关性研究,并进行形态学观察。
5.统计学处理:应用SPSS11.0软件处理,采用χ~2检验、t检验和方差分析。检验水准α=0.05。
结果
1.MMP-9 mRNA及蛋白阳性表达主要位于肿瘤细胞的胞质内,在正常食管粘膜鳞状上皮细胞内无表达或者表达量较低。MMP-9蛋白及mRNA在三样品中表达与RECK的表达均呈负相关关系(P＜0.05)。
2.浸润深度达深层者和淋巴结转移的食管癌组织中,MMP-9蛋白及mRNA表达较浸润深度达浅层者和无淋巴结转移的显著升高,组间比较差异有统计学意义(P＜0.05)。
3.低分化食管癌比高、中分化食管鳞癌MMP-9蛋白及mRNA阳性表达显著降低,差异有统计学意义(P＜0.05)。
4.VEGF蛋白阳性表达主要位于肿瘤细胞的胞质内。正常食管粘膜组织、癌旁不典型增生组织和食管癌组织中VEGF蛋白表达水平依次升高,三者组间比较差异有统计学意义(P＜0.05)。食管鳞癌组织中VEGF蛋白表达与食管鳞癌组织的浸润深度及淋巴结转移相关。其蛋白表达与RECK蛋白表达呈负相关关系,与MMP-9蛋白表达呈正相关关系(P＜0.05)。
5.CD105蛋白阳性表达主要位于肿瘤间质的血管内皮细胞的胞浆中。CD105在浸润深度达深肌层和有淋巴结转移组的蛋白阳性表达率较浸润深度达浅层者和无淋巴结转移的显著降低,组间比较差异有统计学意义(P＜0.05)。其蛋白表达与RECK蛋白表达呈负相关关系,与MMP-9及VEGF蛋白表达呈正相关关系(P＜0.05)。
6.MMP-9 mRNA、蛋白表达及VEGF及CD105蛋白表达与食管癌患者的性别、年龄、等无关(P＞0.05)。
第三部分人RECK基因的分子克隆和真核表达载体的构建及其对人食管癌细胞株EC9706生物学行为的影响
方法
1.从食管组织中提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增出人RECK基因;限制性内切酶KpnⅠ和NotⅠ双酶切PCR产物和pcDNA3质粒,然后用T4 DNA Ligase连接,转化大肠杆菌JM109感受态细胞,PCR验证转化子,挑取阳性克隆扩大培养,提取其质粒,进行KpnⅠ和NotⅠ双酶切验证构建RECK基因的真核表达载体pcDNA3-RECK并经酶切鉴定。
2.采用脂质体Lipofectamine2000介导将真核表达载体pcDNA3-RECK导入体外培养的食管癌EC9706细胞。
3.采用RT-PCR、原位杂交方法检测转染前、后RECK及MMP-9在人食管癌细胞株EC9706 mRNA的表达情况。
4.采用免疫细胞化学、Western blot的方法检测转染前、后RECK及MMP-9在人食管癌细胞株EC9706蛋白的表达情况。
5.细胞计数法绘制各组细胞的生长曲线以观察RECK基因对细胞增殖能力的影响。
6.Boyden chamber实验计算各组穿膜的细胞数以比较转染RECK基因对细胞侵袭能力的变化。
7.统计学处理:应用SPSS11.0软件处理,采用χ2检验、方差分析。检验水准α=0.05。
结果
1.PCR扩增得到了特异性的4.4kb基因片段。基因片段的大小与预期一致。所获得的RECK基因测序及BLAST分析证明克隆的人RECK基因序列正确,pcDNA3-RECK经酶切后得到5.4kb和4.4kb两个片段。
2.PCR鉴定、酶切鉴定pcDNA3-RECK重组载体,结果显示:PCR扩增片段及KpnⅠ和NotⅠ双酶切片段大小与设计相符。
3.DNA测序结果证明:所构建真核表达载体上的目的基因核苷酸序列与GenBank上基因预计部分完全一致,且方向正确。
4.转染pcDNA3-RECK真核表达载体后,EC9706细胞均能稳定高表达RECKmRNA及蛋白。
5.Boyden chamber体外侵袭实验结果显示:与空白对照组相比,EC9706pcDNA3-RECK组穿越Matrigel胶的细胞数显著减少(P＜0.05),而pcDNA3空质粒组及空白对照组之间则无明显差异(P＞0.05)。
第四部分RECK基因对裸鼠移植瘤生长的抑制作用及机制研究
方法
1.分别将转染前、后及空质粒组的食管癌细胞株EC9706注入裸鼠皮下,比较各组裸鼠成瘤大小。
2.分别应用RT-PCR、原位杂交方法检测各组裸鼠肿瘤组织中RECK、MMP-9基因的mRNA表达情况。
3.应用免疫组织化学方法检测各组裸鼠肿瘤组织中RECK、MMP-9、VEGF基因的蛋白表达情况。
4.统计学处理:应用SPSS11.0软件处理,采用χ2检验、方差分析。检验水准α=0.05。
结果
1.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤体积显著大于EC9706/pcDNA3-RECK组裸鼠移植瘤体积,组间比较差异具有统计学意义(P＜0.05)。
2.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤RECK基因蛋白及mRNA表达显著低于EC9706/pcDNA3-RECK的表达,组间比较差异具有统计学意义(P＜0.05)。
3.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤MMP-9基因蛋白及mRNA表达显著高于EC9706/pcDNA3-RECK的表达,组间比较差异具有统计学意义(P＜0.05)。
4.EC9706细胞组、EC9706/pcDNA3细胞组裸鼠移植瘤VEGF基因蛋白表达显著高于EC9706/pcDNA3-RECK的表达,组间比较差异有统计学意义(P＜0.05)。
结论
1.食管鳞癌组织中RECK蛋白及mRNA呈低表达。
2.RECK可能是食管鳞癌侵袭、转移的重要生物学标记。
3.RECK在肿瘤的演进中具有重要的作用。
4.成功构建了RECK真核表达载体pcDNA3-RECK,并成功导入人食管癌EC9706细胞株中,筛选出了稳定细胞株,为进一步研究RECK的生物学功能和RECK的靶向治疗奠定了基础。
5.pcDNA3-RECK可上调EC9706细胞的蛋白及mRNA的表达。
6.RECK基因可通过抑制MMP-9来影响肿瘤的浸润、转移。
7.运用Boyden chamber技术检测了RECK基因转染前后及空质粒组EC9706细胞的侵袭能力的变化。
8.RECK基因有可能通过拮抗MMP-9来降低食管癌细胞的体外侵袭能力。
Esophagus cacinoma is one of the most common malignancy in our country,and Henan province also has higher morbility.It's mortality reside forth position in our country,whereas it's invasion and metastasis is the first cause of invoke esophagus cancer sufferer's obituary,so discussing the esophague cacinoma invasion and metastasis mechanism became one of the investgative hotspot.
Tumor's occurrence and development are the course of multifactor,multistep, multistage and polygene alterative.Oncogene activation and tumor suppressor gene inactivation are the material basis of human tumor formation and development.Previous research found that the mutations and deletions of some tumor suppressor genes,such as p53,Rb,p16 and so on,are relate to malignancy occurrence.
A new anti-oncogene named RECK(reversion inducing cysteine rich protein with Kazal motifs,RECK)was found in recent years.The expression of RECK closely relate to tumorous invasion、metastasis and angiogenesis.In recent years,there are several relevant studies about the relation between RECK and tumor invasion and metastasis in the fields of liver cancer,pancreatic cancer,breast cancer and lung cancer.Researches show that RECK gene's expression were negative correlate to tumorous invasiveness and the sufferers whose RECK gene higher expression have better prognoses than those with lower expression quantity.This gene's deletions or(and) mutation and depressing expression of mRNA or protein relate to tumorous occur,invasion,metastasis and histology grade.The mechanism may be that RECK regulated negatively tumour growth,invasion,metastasis through restraining the secretion and activity of MMP-2,MMP-9 and MT1-MMP.In addition, RECK can inhibit tumor growth,invasion and metastasis by inhibition of tumor angiogenesis.There have no other report both in China and abroad so far except the relevant articles pulished by our team before.
In order to discuss RECK gene's relation to esophagus squama carcinogenesis and invasion,metastasis for more,sought early detection esophagues squama carcinogenesis and soakage transitionary indices and found available approach to restrain esophagus squama carcinogenesis and soakage transitionary,this topic firstly used RT-PCR, hybridization in situ and immunohistochemistry SP combine detected the expression of esophagus cancer verdure excision specimen's RECK,MMP-9mRNA and protein.We use immunohistochemistry SP detected blood vssel endotheliocyte growth factor in esophague squama,adjacentatypical hyperplasia tissue and normal esophagus mucous membrane tissue.At the same time,use resist CD105 antibody detected microvascular density(MVD) in 62 cases' esophagus squama carcinoma,and discussed the relationship among RECK, VEGF and MVD,revealed the relation of RECK and esophagus cancer invasion and metastasis.On this foundation,we constructed RECK's eukaryon expression vector successfully,then transfected it into esophagus cancer cell EC9706,use RT-PCR, hybridization in situ combine detected esophagus cancer cell EC9706's RECK, MMP-9mRNA expression level,respectively;Immunohistochemistry SP combined Western blot detected esophagus cancer verdure excision specimen's RECK,MMP-9 protein's expression level;The transfected cell's invasion ability was determined by Boyden-chamber model in vitro.In the final,apply nude mice lotus tunour experiment, combined use hybridization in situ,RT-PCR,immunohistochemistry detected the expression of RECK,MMP-9 at nude mice transplantation tumor,we in depth explorate upper regulate RECK's expression and activity restrain esophagus cancer invade in multi-aspect,such as gene,protein,cellular localization and cellular biology of tumor behavior,vitro invade cell experiment,in vivo nude mice achieve tumour experiment and so on.We try to illuminate the meaning in RECK's expression at esophagus squama carcinogenesis and development,invasion and metastasis.For more illuminate RECK gene repress esophagus squama carcinogenesis and development,invasion and metastasis's mechanism whether related to down regulate the expression of VEGF,depress MVD in tumour.Providing fundamental basis for deeply discuss esophagus carcinogenesis,invasion and metastasis mechanism and soght restrain esophagus squama carcinogenesis, developmental way.This study includes the following four parts.
The first part:The clinicopathological significance of the expression of RECK in esophageal squamaous cell carcinoma
Methods:
1.Detected RECK gene protein expression in 62 cases esophageal squamous cell carcinoma tissue(ESCC),31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by immunohistochemical, then observed morphology.
2.Detected RECK gene's mRNA expression in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by RT-PCR,then observed morphology.
3.Detected RECK gene's mRNA expression in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by hybridization in situ,then observed morphology.
4.Statistical treatment:Statistics analysis was performed by SPSS 11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.RECK mRNA and protein masculine expression in esophagus cancer tissue mostly locate in normal esophagus mucous membrane squamous cells' cytoplast,no or lower expression in tumor cell.
2.Combine RT-PCR and hybridization in situ detect resulting:RECK mRNA expression level depress by turns in normal esophagus mucous membrane tissue,adjacent atypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).The result of immunohistochemical display:RECK mRNA expression level depress by turns in normal esophagus mucous membrane tissue, adjacentatypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).
3.RECK mRNA masculine expression rate in infiltrate to deep depth and lymphatic metastasis' esophagus cancer tissue signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).RECK protein masculine expression rate in infiltrate to deep depth and lymph node metastasis' esophagus cancer tissue signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).
4.RECK mRNA masculine expression rate in poorly differentiated esophagus cancer signifcant depress compare to well,moderately differentiated esophagus cancer,the difference make statistics sense(P＜0.05).RECK protein masculine expression rate in poorly differentiated esophagus cancer signifcant depress compare to well,moderately differentiated esophagus cancer,the difference make statistics sense(P＜0.05).
5.RECK mRNA's expression was not related esophagus cancer sufferer's sex,age, general style and tumorigenes is position.
The second part:The study on MMP-9,VEGF,CD105's expression in ESCC tissue and the relation to RECK gene
Methods:
1.Detected MMP-9 mRNA expression and relation in 62 eases ESCC tissue(ESCC), 31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by RT-PCR.
2.Detected MMP-9 mRNA expression and relation in 62 cases ESCC tissue,31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by hybridization in situ.
3.Detected MMP-9,VEGF's protein expression and relation 62 cases ESCC tissue(ESCC),31 cases adjacent atypical hyperplasia tissue,carcinoma in situ tissue and 62 cases normal esophagus mucous membrane tissue by immunohistochemical,then observed morphology.
4.Detected CD105 protein expression and relation in 62 cases ESCC tissue by immunohistochemical,then observed morphology.
5.Statistical treatment:Statistics analysis was performed by SPSS11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.The expression of MMP-9 mRNA and protein mainly located in the cytoplasm of tumor cells and no or lower expression in the normal esophageal squamous mucosal epithelial cells.The expression of MMP-9 mRNA and protein in three groups was negatively correlated with the expression of RECK(P＜0.05).
2.MMP-9 mRNA masculine expression rate in infiltrate to deep depth and lymphatic metastasis' esophagus cancer tissue siguifcant depress compare to infiltrate shallow layer and without lymphatic metastasis,the difference among groups make statistics sense(P＜0.05).
3.The expression MMP-9 protein and mRNA was significantly decreased in poorly differentiated esophageal cancer compare to well,moderately differentiated ones.The difference among groups make statistics sense(P＜0.05).
4.The expression of VEGF protein mainly located in the cytoplasm of tumor cells. The expression level step up by turns in normal esophagus mucous membrane tissue, adjacent atypical hyperplasia tissue and esophagus cancer tissue,the difference in three groups make statistics sense(P＜0.05).Esophagus squama carcinoma tissue VEGF protein expression was related to esophagus squama carcinoma tissue soakage depth and lymphatic metastasis,VEGF protein masculine expression mostly locate in esophagus cancer cells' cytoplast.
5.CD105 protein masculine expression mostly locate in tumour interstitial blood vessel endotheliocyte's kytoplasm.The masculine expression rate in esophagus cancer tissue infiltrated to deep depth and with lymphatic metastasis signifcant depress compare to infiltrate shallow layer and without lymphatic metastasis.The difference among groups make statistics sense(P<0.05).The protein expression is negatively correlated with the expression of RECK and positively correlated with the expression of MMP-9(P<0.05).
6.RECK mRNA's expression was not related esophagus cancer sufferer's sex,age, general style and tumorigenesis position(P>0.05).
The third part:The influence of biology behaviour on Human RECK gene molecular cloning and construction of eukaryotic expression bearer to human esophagus cancer strain EC9706
Methods:
1.Total RNA was extracted from from esophagus tissue,RECK cDNA was isolated by using reverse transcription-polymerase chain reaction(RT-PCR);use restrict enzyme Kpn I and Not I two enzyme cut PCR offspring and pcDNA3 plasmid,then use T4 DNA Ligase joint,translate colibacilus JM109 competent call,PCR demonstrat converter,pick masculine clone intermediate culture,abstract there plasmid,proceed Kpn I and Not I two enzyme cut demonstrat construct RECK genic eukaryon expression bearer pcDNA3-RECK and judged by enzyme cut.
2.Adopt liposome Lipofectamine2000 mediate eukaryon expression bearer pcDNA3 -RECK import esophagus cancer EC9706 cell in vitro culture.
3.Detect pro and post transfection RECK and MMP-9 in human esophagus cancer cell strain EC9706 mRNA's expression by RT-PCR,hybridization in situ.
4.Detect pro and post transfection RECK and MMP-9 in human esophagus cancer cell strain EC9706 protein's expression by immunocytochemistry,Western blot.
5.Cell count protract each group cellular growth curve and observe RECK gene's impact on cell multiplication capable.
6.Boyden chamber experiment count each group worn membranous cell count and compare transfect RECK gene varied to cell invade capable.
7.Statistical treatment:Statistics analysis was performed by SPSS 11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.Amplified idiosyncratic 4.4Kb gene fragment through PCR.Gene piecewise size accord with anticipate.The RECK gene sequencing and BLAST analysis proved the exactness of clone human RECK gene order,adopt enzyme cut pcDNA3-RECK got 5.4 kb and 4.4kb two fragment.
2.PCR judge,enzyme cut judge pcDNA3-RECK reform bearer,resulting display: PCR amplificate fragment and Kpn I and Not I two enzyme slab segment size match with design.
3.DNA sequencing result display:The construct eukaryon expression bearer's purpose gene nucleotidesequence perfect agreement with GeneBank's predict part of gene, and in the right direction.
4.After transfected pcDNA3-RECK eukarya expression bearer,EC9706 cells all can stable express RECK mRNA and protein.
5.Boyden chamber extraneous invade experimental result display:Compare with blank control group,EC9706pcDNA3-RECK group notable decrease in through Matrigel gluey's cell(P＜0.05),but pcDNA3 was no distinct difference in vacancy plasmid group and blank control group(P＞0.05)
The fourth part:The research of inhibitory action and mechanism on RECK gene to nude mice transplantation tumor's vegetal
Methods:
1.Separately inject pro,post transfection and vancancy plasmid group's esophagus cancer cell strain EC9706 into nude mice subsurface,compared each groups nude mice's tumor size and metastasis scale.
2.Detect each group's nude mice tissue's RECK,MMP-9 mRNA expression by RT-PCR,hybridization in situ.
3.Detect each group's nude mice turnout tissue's RECK,MMP-9,VEGF genie protein expression by immunohistochemical method.
4.Statistical treatment:Statistics analysis was performed by SPSS11.0 software,adopt chi-square test and ANOVA,test standardα=0.05.
Results:
1.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor volume significant larger than EC9706/pcDNA3-RECK group's,compare among groups had difference in statistic sence(P＜0.05).
2.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor RECK gene protein and mRNA expression significant lower than EC9706/pcDNA3 -RECK's expression,compare among groups had difference in statistic sence(P＜0.05).
3.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor MMP-9 gene protein and mRNA expression significant higher than EC9706/pcDNA3 -RECK's expression,compare among groups had difference in statistic sence(P＜0.05).
4.EC9706 cell group,EC9706/pcDNA3 cell group nude mice transplantation tumor VEGF gene protein expression significant higher than EC9706/pcDNA3-RECK's expression,compare among groups had difference in statistic sence(P＜0.05).
Conclusion
1.The expression of RECK protein and mRNA is lower in esophagus squama carcinoma tissue.
2.RECK may be a important biological markers of invasion and metastasis of ESCC.
3.RECK play an important role in the tumor evolution.
4.Successfully constructing RECK genie eukaryon expression bearer peDNA3-RECK, and importing human esophageal cancer EC9706 cell lines.Selecting a stable cell line laid foundation for further research about the biological functions of RECK and target therapy on RECK.
5.pcDNA3-RECK can be increased RECK protein and mRNA expression of EC9706 cell.
6.RECK can influence tumor invasion and metastasis by inhibiting MMP-9.
7.Using Boyden chamber technology to detect the change of invasion in pro and post transfected plasmid groups and blank control group.
8.RECK can anti tumor invasion and metastasis by inhibiting MMP-9.
引文
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