重组腺相关Ⅱ型病毒介导人TIMP1基因抑制肝癌侵袭和生长的实验研究
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摘要
目的全球每年死于肝细胞癌(hepatocellular carcinoma，HCC)的病人超过100万人。在我国，肝癌已成为恶性肿瘤的第二大病因，每年死于HCC的病人数列全球第一位，对人类健康威胁极大。目前虽已有许多办法和措施用于HCC的治疗，包括：手术切除、放疗、化疗、介入治疗、免疫治疗、中药治疗和肝移植等，但获得根治的仅为10％左右。影响HCC疗效的最重要原因是肝癌转移。
在恶性肿瘤侵袭转移的进程中，完善的ECM(extracellular matrix，ECM)和BM(basement membrane，BM)是一个重要的屏障，可以限制肿瘤细胞的浸润和转移，在组织学上，BM完整性的破坏被认为是恶性肿瘤侵袭开始的一个标志。有许多酶类参与降解ECM和BM中成分。肿瘤细胞或肿瘤间质组织能产生降解ECM和BM的蛋白水解酶系列，其中，基质金属蛋白酶(Matrixmetalloproteinases，MMPs)在肿瘤的转移过程中起着关键作用。
金属蛋白酶组织抑制因子(tissue inhibitor of metalloproteinase，TIMPs)亦可为肿瘤细胞、肿瘤间质组织或正常组织产生。但却具有拮抗MMP的功效。目前已有提取的天然MMPs抑制剂(MMPI)和人工合成的MMPIs用于抗癌研究。但除从鲨鱼软骨中提取的Neovastat(癌立消)进入Ⅲ期临床试验外，其它尚皆在实验室研究阶段。以分子生物学方法促进TIMPs表达而抑制MMPs表达，重建MMPs-TIMPs平衡，达到抑制肝癌的侵袭和转移已成为目前TIMP研究的一大亮点。
基因治疗的理想结果与载体系统的选择具有非常密切的关系，载体需兼具精确、低毒、高表达、大容量等特点，常用的病毒载体包括腺病毒、腺相关病毒、逆转录病毒、乙肝病毒、单纯疱疹病毒等。其中，腺相关病毒是目前公认的较理想载体。rAAV2是近年来出现的新型基因治疗载体，具有长时间有效表达外源性基因、转染后对机体无免疫反应以及对治疗者无致病性和基因定点整合等多项优点，已成为基因治疗研究的热点。
因此，本研究选择腺相关病毒rAAV2为载体，重组携带全长TIMP1基因cDNA的rAAV2-TIMP1，以探讨其可能的抗癌效能及机制。
研究目标
1构建高滴度的携带人全长TIMP1基因的重组腺相关病毒rAAV2-TIMP1，为进一步探讨其抑制HCC生长、侵袭和转移的实验奠定基础。
2观察重组腺病毒介导TIMP1基因对HCC细胞侵袭性的影响，探讨其抑制HCC细胞侵袭性的作用机理。
3观察重组腺病毒介导TIMP1基因对活体HCC生长的影响，探讨rAAV2-TIMP1抑制活体HCC的效能及rAAV2-TIMP1对HCC的治疗潜力。
研究内容
1携带人全长TIMP1基因cDNA的重组腺相关病毒载体的构建
1．1人TIMP1基因全长cDNA的选择与提取
从Genbank查获人TIMP1基因全序列，进行特异性引物设计；从HCC组织中提取总mRNA，用逆转录聚合酶链反应(reverse-transcriptionpolymerase chain reaction，RT-PCR)扩增TIMP1基因全长cDNA。
1．2携带全长TIMP1基因cDNA的重组腺相关病毒rAAV2-TIMP1的构建：
①TIMP-1基因cDNA片段定向克隆到pMD18-T质粒，得重组质粒命名为pMD18-T-TIMP1。将pMD18-T—TIMP1转入Top10中。筛选和鉴定重组子，用EcoRⅠ／BamHⅠ双酶切鉴定，并送上海申能博彩有限公司进行基因测序鉴定。
②rAAV2载体pSNAV-TIMP1质粒的构建与鉴定：将测序正确的pMD18-T-TIMP1重组子大规模扩增，提取质粒DNA，用EcoRⅠ／BamHⅠ双酶切切下TIMP-1全长510bp cDNA，正向插入pSNAV载体质粒的多克隆位点中，得到的重组质粒命名为pSNVA-TIMP1。
③rAAV2-TIMP1病毒细胞株的建立用脂质体转染法将pSNVA-TIMP1重组质粒转染BHK-21细胞，将细胞株命名为BHK-21／rAAV2-TIMP1。
④rAAV2-TIMP1重组病毒的制备与纯化，先进行重组AAV2病毒rAAV2-TIMP1的包装：用具有rAAV2包装功能的1型单纯疱疹病毒(HSV1-rc／△UL2)感染BHK-TIMP1。首先大量扩增BHK-21／rAAV2-TIMP1细胞及辅助病毒rHSV1-rc／△UL2，用rHSV1-rc／△UL2(MOI为1.0)感染BHK-21／rAAV2-TIMP1细胞，待细胞完全病变后收集细胞及上清液。初步纯化，获得rAAV2-TIMP1，再用亲和层析、离子交换层析和分子筛层析进一步精纯化。
⑤rAAV2-TIMP1的纯度的检测：用SDS-聚丙烯酰胺电泳(SDS-PAGE)法检测rAAV2-TIMP1的纯度，rAAV2-TIMP1纯度采用凝胶扫描图像分析系统进行。再对纯化的rAAV2-TIMP1病毒样品行高压液相(HPLC)分析。
⑥rAAV2-TIMP1重组病毒的滴度测定：用点杂交方法检测纯化rAAV2-TIMP1的滴度。同时，构建空白腺相关病毒(rMV2-luc，无TIMP1)作为对照。
2 rMV2-TIMP1抑制HCC细胞侵袭的细胞实验
2．1 rAAV2-TIMP1抑制HCC细胞株侵袭的实验
分别用感染复数(multiplicities of infection，MOI)为100的rAAV2-TIMP1和rAAV2-luc感染人肝癌细胞(Bel-7402)。用侵袭小室(Boydenchamber)检测Bel-7402细胞侵袭人工基底膜(Matrigel)的能力。
2．2 rAAV2-TIHP1抑制HCC细胞生长曲线
用MTT法检测HCC细胞Bel-7402细胞生长曲线，确定rAAV2-TIMP1对Bel-7402细胞生长的影响
2．3 rAAV2-TIMP1抑制HCC细胞的生产和促进凋亡
用流式细胞仪(FCM)检测感染rAAV2-TIMP1的Bel-7402细胞生长指数和凋亡指数
2．4 RT-PCR检测感染rAAV2-TIHP1后Bel-7402细胞中TIHP1 mRNA的变化
2．5 Western blot法检测感染rAAV2-TIMP1对HCC细胞分泌TIHP1蛋白表达的影响
用Western blot检测Bel-7402细胞培养液中TIMP1蛋白的表达，以确定感染rAAV2-TIMP1的HCC细胞株(Bel-7402)分泌TIMP1蛋白的变化。
3动物实验
3．1成瘤实验
用100 MOI的rAAV2-TIMP1感染Bel-7402细胞，然后注射入裸鼠皮下组织成瘤，观察rAAV2-TIMP1对HCC成瘤的影响。以rAAV2-luc为对照。
3．2治疗实验
用rAAV2-TIMP1对已成瘤的HCC行瘤内注射，观察rAAV2-TIMP1对HCC的治疗作用，以瘤内注射PBS为对照。
3．3组织学检测
所有实验HCC行病理切片，HE(hematoxylin eosin)染色，观察肿瘤组织结构；免疫组化染色检测TIMP-1基因在荷瘤鼠组织中的表达。结果
1成功地从HCC组织中提取并扩增出全长TIMP1基因cDNA，经DNA测序证实该基因片段序列与Genbank中人类TIMP1基因序列完全一致
2成功地构建出携带人类TIMP1基因的重组腺相关病毒rAAV2-TIMP1，病毒滴度达1×10~(12)v．g／ml
3受rAAV2-TIMP1感染的Bel-7402细胞穿过Matrigel的细胞数下降52％
4受rAAV2-TIMP1感染的Bel-7402细胞分泌TIMP1的能力明显增强且持续表达5受rAAV2-TIMP1感染的Bel-7402细胞的成瘤能力下降，瘤内注射rAAV2-TIMP1使瘤体生长缓慢
结论携带人TIMP1全长cDNA的重组腺相关病毒rAAV2-TIMP1，体内、体外实验均证实能够稳定高效地表达TIMP-1。rAAV2-TIMP1重组体显示出降低HCC的侵袭力、抑制HCC生长和增殖的能力。rAAV2-TIMP1的成功构建及其稳定表达，以及对人肝癌侵袭、生长的抑制，为进一步开展HCC的基因治疗建立了实验基础和技术平台。
Objective This study was design to conduct (a) to construct a recombinant adeno-associated virus-2 carrying tissue inhibitor metalloproteinase-l(TIMP1) gene (rAAV2-TIMP 1): (b) to study the effects that the rAAV2-TIMP 1 inhibit the invasiveness of hepatocellular carcinoma(HCC) cells in vitro model and the mchamism of rAAV2-TIMP1 in inhibiting the invasiveness ofHCC. (c) to study the effects on the growth and angiogenesis of HCC in vivo models by treatment of rAAV2-TIMP1. So as to investigate the possible potency of rAAV2-TIMP1 in treatment ofhepatocellular carcinoma
Methods: Total RNA was extracted from human hepatocellular carcinoma tissue, and a 510-bp of TIMP 1 cDNA was amplified by RT-PCR and extracted, the TIMP 1 gene was then cloned into the adeno-assciated virus-2 vector pSNAV to form the recombinant pSNAV-TIMP1, Which was transfected into BHK-21 cells by means of lipofectamine. Using G418 Selection from mixed cells, BHK-TIMP1 was isolated, which was capable of TIMP1 expression and was subsequently infected with recombinant herpes simplex virus 1(HSVI-rc/AUL2) that was able to package the rAAV2-TIMP1 to form a functional and infectious virus. After purification, the packaged rAAV2-TIMP 1 was obtained. The human HCC cell line (Bel-7402) were transfected in vitro and treated in vivo with rAAV2-TIMP1, and a series of experiments was performed to observe the possoble effects of rAAV2-TIMP1 on the invasion and growth of Bel-7402 cells. Compared with the conventional transfection method, our modified method using "one provirus cell line, one helper virus" has an advantage to increase the yield of rAAV2 by two orders of magnitude. (a) The mRNA expression of TIMP 1 were detected by RT-PCR. (b) The protein production of TIMP 1 in the Bel-7402 cells was detected with Western Blot analysis. (c) The invasiveness of the Bel-7402 cells was assayed in Matrigel. (d) The index of proliferation and apoptosis of Bel-7402 cells were detected with MTT and FCM respectively. (e) The in vitro experiments was done by subcutaneously inoculation with rAAV2-TIMP1 infected BHK-21 cells in nude mice, and injected intratumorally into pre-existing tumors. The tumors were removed, sectioned, stained with H&E and immunohistochemical method for inspection.
Results: The recombinant adeno-associated virus-2 vector carrying TIMP1 was constructed successfully, Using RT-PCR and Western Blot, the destination gene TIMP1 was detected. The recombinat viral titer was 1×10~(12)v.g/ml. Compared with PBS and rAAV2-1uc(empty control) infected cells, the invasiveness of Bel-7402 cells in Matrigel assays with transfected rAAV2-TIMP1 resulted in a significantly reduction by 52%, and the tumor mass was also obviously reduced in nude mice with subcutaneously inoculation by rAAV2-TIMP1. Direct intratumoral injection of rAAV2-TIMP 1 into pre-existing tumors significantly inhibited the further growth of the HCC tumor, the tumor cells show necrosis and shrink, and the apoptosis rate also increased in Bel-7402 cells infected with rAAV2-TIMP 1
Conclusion: (1)The constructed recombinant adeno-associated virus-2 with TIMP 1 (rAAV2-TIMP1) can express TIMP 1 and effectively inhibit the invasiveness of Bel-7402 cells of HCC in vitro. (2) rAAV2-TIMP1 can effectively promote the apoptosis and inhibit the growth of Bel-7402 cells in vivo; (3) rAAV2-TIMP1 can effectively attenuate the growth of HCC in vivo models, and demonstrat a therapeutic potential for HCC.
在恶性肿瘤侵袭转移的进程中，完善的ECM(extracellular matrix，ECM)和BM(basement membrane，BM)是一个重要的屏障，可以限制肿瘤细胞的浸润和转移，在组织学上，BM完整性的破坏被认为是恶性肿瘤侵袭开始的一个标志。有许多酶类参与降解ECM和BM中成分。肿瘤细胞或肿瘤间质组织能产生降解ECM和BM的蛋白水解酶系列，其中，基质金属蛋白酶(Matrixmetalloproteinases，MMPs)在肿瘤的转移过程中起着关键作用。
金属蛋白酶组织抑制因子(tissue inhibitor of metalloproteinase，TIMPs)亦可为肿瘤细胞、肿瘤间质组织或正常组织产生。但却具有拮抗MMP的功效。目前已有提取的天然MMPs抑制剂(MMPI)和人工合成的MMPIs用于抗癌研究。但除从鲨鱼软骨中提取的Neovastat(癌立消)进入Ⅲ期临床试验外，其它尚皆在实验室研究阶段。以分子生物学方法促进TIMPs表达而抑制MMPs表达，重建MMPs-TIMPs平衡，达到抑制肝癌的侵袭和转移已成为目前TIMP研究的一大亮点。
基因治疗的理想结果与载体系统的选择具有非常密切的关系，载体需兼具精确、低毒、高表达、大容量等特点，常用的病毒载体包括腺病毒、腺相关病毒、逆转录病毒、乙肝病毒、单纯疱疹病毒等。其中，腺相关病毒是目前公认的较理想载体。rAAV2是近年来出现的新型基因治疗载体，具有长时间有效表达外源性基因、转染后对机体无免疫反应以及对治疗者无致病性和基因定点整合等多项优点，已成为基因治疗研究的热点。
因此，本研究选择腺相关病毒rAAV2为载体，重组携带全长TIMP1基因cDNA的rAAV2-TIMP1，以探讨其可能的抗癌效能及机制。
研究目标
1构建高滴度的携带人全长TIMP1基因的重组腺相关病毒rAAV2-TIMP1，为进一步探讨其抑制HCC生长、侵袭和转移的实验奠定基础。
2观察重组腺病毒介导TIMP1基因对HCC细胞侵袭性的影响，探讨其抑制HCC细胞侵袭性的作用机理。
3观察重组腺病毒介导TIMP1基因对活体HCC生长的影响，探讨rAAV2-TIMP1抑制活体HCC的效能及rAAV2-TIMP1对HCC的治疗潜力。
研究内容
1携带人全长TIMP1基因cDNA的重组腺相关病毒载体的构建
1．1人TIMP1基因全长cDNA的选择与提取
从Genbank查获人TIMP1基因全序列，进行特异性引物设计；从HCC组织中提取总mRNA，用逆转录聚合酶链反应(reverse-transcriptionpolymerase chain reaction，RT-PCR)扩增TIMP1基因全长cDNA。
1．2携带全长TIMP1基因cDNA的重组腺相关病毒rAAV2-TIMP1的构建：
①TIMP-1基因cDNA片段定向克隆到pMD18-T质粒，得重组质粒命名为pMD18-T-TIMP1。将pMD18-T—TIMP1转入Top10中。筛选和鉴定重组子，用EcoRⅠ／BamHⅠ双酶切鉴定，并送上海申能博彩有限公司进行基因测序鉴定。
②rAAV2载体pSNAV-TIMP1质粒的构建与鉴定：将测序正确的pMD18-T-TIMP1重组子大规模扩增，提取质粒DNA，用EcoRⅠ／BamHⅠ双酶切切下TIMP-1全长510bp cDNA，正向插入pSNAV载体质粒的多克隆位点中，得到的重组质粒命名为pSNVA-TIMP1。
③rAAV2-TIMP1病毒细胞株的建立用脂质体转染法将pSNVA-TIMP1重组质粒转染BHK-21细胞，将细胞株命名为BHK-21／rAAV2-TIMP1。
④rAAV2-TIMP1重组病毒的制备与纯化，先进行重组AAV2病毒rAAV2-TIMP1的包装：用具有rAAV2包装功能的1型单纯疱疹病毒(HSV1-rc／△UL2)感染BHK-TIMP1。首先大量扩增BHK-21／rAAV2-TIMP1细胞及辅助病毒rHSV1-rc／△UL2，用rHSV1-rc／△UL2(MOI为1.0)感染BHK-21／rAAV2-TIMP1细胞，待细胞完全病变后收集细胞及上清液。初步纯化，获得rAAV2-TIMP1，再用亲和层析、离子交换层析和分子筛层析进一步精纯化。
⑤rAAV2-TIMP1的纯度的检测：用SDS-聚丙烯酰胺电泳(SDS-PAGE)法检测rAAV2-TIMP1的纯度，rAAV2-TIMP1纯度采用凝胶扫描图像分析系统进行。再对纯化的rAAV2-TIMP1病毒样品行高压液相(HPLC)分析。
⑥rAAV2-TIMP1重组病毒的滴度测定：用点杂交方法检测纯化rAAV2-TIMP1的滴度。同时，构建空白腺相关病毒(rMV2-luc，无TIMP1)作为对照。
2 rMV2-TIMP1抑制HCC细胞侵袭的细胞实验
2．1 rAAV2-TIMP1抑制HCC细胞株侵袭的实验
分别用感染复数(multiplicities of infection，MOI)为100的rAAV2-TIMP1和rAAV2-luc感染人肝癌细胞(Bel-7402)。用侵袭小室(Boydenchamber)检测Bel-7402细胞侵袭人工基底膜(Matrigel)的能力。
2．2 rAAV2-TIHP1抑制HCC细胞生长曲线
用MTT法检测HCC细胞Bel-7402细胞生长曲线，确定rAAV2-TIMP1对Bel-7402细胞生长的影响
2．3 rAAV2-TIMP1抑制HCC细胞的生产和促进凋亡
用流式细胞仪(FCM)检测感染rAAV2-TIMP1的Bel-7402细胞生长指数和凋亡指数
2．4 RT-PCR检测感染rAAV2-TIHP1后Bel-7402细胞中TIHP1 mRNA的变化
2．5 Western blot法检测感染rAAV2-TIMP1对HCC细胞分泌TIHP1蛋白表达的影响
用Western blot检测Bel-7402细胞培养液中TIMP1蛋白的表达，以确定感染rAAV2-TIMP1的HCC细胞株(Bel-7402)分泌TIMP1蛋白的变化。
3动物实验
3．1成瘤实验
用100 MOI的rAAV2-TIMP1感染Bel-7402细胞，然后注射入裸鼠皮下组织成瘤，观察rAAV2-TIMP1对HCC成瘤的影响。以rAAV2-luc为对照。
3．2治疗实验
用rAAV2-TIMP1对已成瘤的HCC行瘤内注射，观察rAAV2-TIMP1对HCC的治疗作用，以瘤内注射PBS为对照。
3．3组织学检测
所有实验HCC行病理切片，HE(hematoxylin eosin)染色，观察肿瘤组织结构；免疫组化染色检测TIMP-1基因在荷瘤鼠组织中的表达。结果
1成功地从HCC组织中提取并扩增出全长TIMP1基因cDNA，经DNA测序证实该基因片段序列与Genbank中人类TIMP1基因序列完全一致
2成功地构建出携带人类TIMP1基因的重组腺相关病毒rAAV2-TIMP1，病毒滴度达1×10~(12)v．g／ml
3受rAAV2-TIMP1感染的Bel-7402细胞穿过Matrigel的细胞数下降52％
4受rAAV2-TIMP1感染的Bel-7402细胞分泌TIMP1的能力明显增强且持续表达5受rAAV2-TIMP1感染的Bel-7402细胞的成瘤能力下降，瘤内注射rAAV2-TIMP1使瘤体生长缓慢
结论携带人TIMP1全长cDNA的重组腺相关病毒rAAV2-TIMP1，体内、体外实验均证实能够稳定高效地表达TIMP-1。rAAV2-TIMP1重组体显示出降低HCC的侵袭力、抑制HCC生长和增殖的能力。rAAV2-TIMP1的成功构建及其稳定表达，以及对人肝癌侵袭、生长的抑制，为进一步开展HCC的基因治疗建立了实验基础和技术平台。
Objective This study was design to conduct (a) to construct a recombinant adeno-associated virus-2 carrying tissue inhibitor metalloproteinase-l(TIMP1) gene (rAAV2-TIMP 1): (b) to study the effects that the rAAV2-TIMP 1 inhibit the invasiveness of hepatocellular carcinoma(HCC) cells in vitro model and the mchamism of rAAV2-TIMP1 in inhibiting the invasiveness ofHCC. (c) to study the effects on the growth and angiogenesis of HCC in vivo models by treatment of rAAV2-TIMP1. So as to investigate the possible potency of rAAV2-TIMP1 in treatment ofhepatocellular carcinoma
Methods: Total RNA was extracted from human hepatocellular carcinoma tissue, and a 510-bp of TIMP 1 cDNA was amplified by RT-PCR and extracted, the TIMP 1 gene was then cloned into the adeno-assciated virus-2 vector pSNAV to form the recombinant pSNAV-TIMP1, Which was transfected into BHK-21 cells by means of lipofectamine. Using G418 Selection from mixed cells, BHK-TIMP1 was isolated, which was capable of TIMP1 expression and was subsequently infected with recombinant herpes simplex virus 1(HSVI-rc/AUL2) that was able to package the rAAV2-TIMP1 to form a functional and infectious virus. After purification, the packaged rAAV2-TIMP 1 was obtained. The human HCC cell line (Bel-7402) were transfected in vitro and treated in vivo with rAAV2-TIMP1, and a series of experiments was performed to observe the possoble effects of rAAV2-TIMP1 on the invasion and growth of Bel-7402 cells. Compared with the conventional transfection method, our modified method using "one provirus cell line, one helper virus" has an advantage to increase the yield of rAAV2 by two orders of magnitude. (a) The mRNA expression of TIMP 1 were detected by RT-PCR. (b) The protein production of TIMP 1 in the Bel-7402 cells was detected with Western Blot analysis. (c) The invasiveness of the Bel-7402 cells was assayed in Matrigel. (d) The index of proliferation and apoptosis of Bel-7402 cells were detected with MTT and FCM respectively. (e) The in vitro experiments was done by subcutaneously inoculation with rAAV2-TIMP1 infected BHK-21 cells in nude mice, and injected intratumorally into pre-existing tumors. The tumors were removed, sectioned, stained with H&E and immunohistochemical method for inspection.
Results: The recombinant adeno-associated virus-2 vector carrying TIMP1 was constructed successfully, Using RT-PCR and Western Blot, the destination gene TIMP1 was detected. The recombinat viral titer was 1×10~(12)v.g/ml. Compared with PBS and rAAV2-1uc(empty control) infected cells, the invasiveness of Bel-7402 cells in Matrigel assays with transfected rAAV2-TIMP1 resulted in a significantly reduction by 52%, and the tumor mass was also obviously reduced in nude mice with subcutaneously inoculation by rAAV2-TIMP1. Direct intratumoral injection of rAAV2-TIMP 1 into pre-existing tumors significantly inhibited the further growth of the HCC tumor, the tumor cells show necrosis and shrink, and the apoptosis rate also increased in Bel-7402 cells infected with rAAV2-TIMP 1
Conclusion: (1)The constructed recombinant adeno-associated virus-2 with TIMP 1 (rAAV2-TIMP1) can express TIMP 1 and effectively inhibit the invasiveness of Bel-7402 cells of HCC in vitro. (2) rAAV2-TIMP1 can effectively promote the apoptosis and inhibit the growth of Bel-7402 cells in vivo; (3) rAAV2-TIMP1 can effectively attenuate the growth of HCC in vivo models, and demonstrat a therapeutic potential for HCC.
引文
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