以抗体为基础的肿瘤基因治疗及导向药物治疗实验研究
摘要
侵袭与转移是恶性肿瘤最显著的生物学特征，是导致癌症患者临床死亡的主要原因。研究恶性肿瘤侵袭转移的机制及其阻断途径是当前癌症研究的重大课题。恶性肿瘤细胞的转移是一个多步骤的复杂过程。在此过程中，癌细胞必须经过的三个共同步骤是细胞粘附、基质降解和运动移行，其中癌细胞对基底膜(BM)和细胞外基质(ECM)的降解是癌细胞侵袭始发和转移的必经过程。
研究发现，恶性肿瘤细胞的侵袭转移与肿瘤细胞及其基质诱导产生的基质金属蛋白酶(Matrix Metalloproteinase，MMPs)的水平呈正相关，MMPs与许多恶性肿瘤细胞的恶性表型有关。由于IV型胶原是BM的主要成分，Ⅳ型胶原酶(包括MMP-2和MMP-9)与肿瘤转移的关系成为金属蛋白酶研究的热点。众多的研究表明，在人类前列腺癌、结直肠癌、乳腺癌、鳞癌、恶性星形细胞瘤、转移瘤细胞株中，均有Ⅳ型胶原酶的高表达，而且与肿瘤的侵袭转移和恶性进展密切相关。
胞内抗体(intracellular antibody，Intrabody)技术是指应用基因重组技术在非淋巴细胞内表达具有生物活性的抗体，并通过对抗体分子进行适当修饰，使之定向分布于细胞核、细胞浆或某些细胞器中，从而特异性干扰或阻断分布于该部位的某些生物大分子的活性或加工、分泌过程，引起细胞的一系列生物过程发生改变。它是继反义RNA技术、特异性核酶(ribozyme)、显性负突变(dorminant-negative mutation)、“自杀”基因等技术之后又一新兴基因治疗途径。已有许多研究证明，运用胞内抗体技术，可以在分泌途径上对癌蛋白进行阻断、并可在细胞质和细胞核内阻断癌蛋白的活性。基于Ⅳ型胶原酶(包括MMP-2和MMP-9)在恶性肿瘤转移扩散中的重要作用，本研究拟应用胞内抗体技术，探讨通过内质网滞留型胞内抗体在内质网与Ⅳ型胶原酶结合，从而阻断其向细胞表面的分泌，达到对恶性肿瘤细胞侵袭转移能力的逆转的可能性。人高转移性肺巨细胞癌系PG亚克隆，是在裸鼠体内建立的人类高转移性肿瘤细胞系。Zymography和Western Blot研究表明该细胞有着高水平表达和活性的MMP-2和MMP-9。本研究通过构建可在真核细胞内质网表达抗Ⅳ型胶原酶scFv M97抗体基因的真核表达载体，对人高转移性肺巨细胞癌系PG进行基因治疗研究。结果表明，以质粒PCANTAB
Tumor cell invasion and metastasis to secondary sites are complex, multistep processes including cell detachment from the primary site, migration through lymph or blood vessels, adhesion to cellular and extracellular matrix elements of the organs involved, proteolytic degradation of matrix proteins and cell proliferation. Several above processes involved proteolysis of the extracellular matrix and tissue remodeling. Matrix metalloproteinases (MMPs) comprise a family of enzymes whose main function is the degradation of extracellular matrix, and have been implicated in tumor progression. It was documented that type IV collagenase with two subtypes of 72kDa/MMP-2 and 92kDa/MMP-9 plays an important role in tumor invasion and metastasis, based on the mechanism of proteolytic degradation of collagen IV in basement membrane by which malignant tumor initiates its invasion and spread.
It has been demonstrated that natural and synthetic inhibitors of type IV collagenase such as TIMPs, BB94 have reduced the metastasis of murine Lewis lung carcinoma and tumor neovascularization . These finding suggested that blocking the function of type IV collagenase represents a useful strategy in cancer therapy and prevention of metastasis.
One possible method to inhibit specifically the function of a protein is to express intracellular antibody (intrabody) combining site that can block the function or prevent the expression of the targeted molecule. Intracellular antibodies are novel agents that can be used to study or to alter the function of specific cellular proteins. Intracellular scFv has been used as a novel approach to gene therapy to inhibit a variety of HIV proteins, resulting in ablating the replication of HIV virus in T cells. Many groups have used scFv to abrogate the expression of oncoproteins. Thus, scFv can achieve effectively functional knock-out of target oncoproteins. Based on this concept, intracellular antibodies against type IV collagenase may have therapeutic use against tumor
研究发现，恶性肿瘤细胞的侵袭转移与肿瘤细胞及其基质诱导产生的基质金属蛋白酶(Matrix Metalloproteinase，MMPs)的水平呈正相关，MMPs与许多恶性肿瘤细胞的恶性表型有关。由于IV型胶原是BM的主要成分，Ⅳ型胶原酶(包括MMP-2和MMP-9)与肿瘤转移的关系成为金属蛋白酶研究的热点。众多的研究表明，在人类前列腺癌、结直肠癌、乳腺癌、鳞癌、恶性星形细胞瘤、转移瘤细胞株中，均有Ⅳ型胶原酶的高表达，而且与肿瘤的侵袭转移和恶性进展密切相关。
胞内抗体(intracellular antibody，Intrabody)技术是指应用基因重组技术在非淋巴细胞内表达具有生物活性的抗体，并通过对抗体分子进行适当修饰，使之定向分布于细胞核、细胞浆或某些细胞器中，从而特异性干扰或阻断分布于该部位的某些生物大分子的活性或加工、分泌过程，引起细胞的一系列生物过程发生改变。它是继反义RNA技术、特异性核酶(ribozyme)、显性负突变(dorminant-negative mutation)、“自杀”基因等技术之后又一新兴基因治疗途径。已有许多研究证明，运用胞内抗体技术，可以在分泌途径上对癌蛋白进行阻断、并可在细胞质和细胞核内阻断癌蛋白的活性。基于Ⅳ型胶原酶(包括MMP-2和MMP-9)在恶性肿瘤转移扩散中的重要作用，本研究拟应用胞内抗体技术，探讨通过内质网滞留型胞内抗体在内质网与Ⅳ型胶原酶结合，从而阻断其向细胞表面的分泌，达到对恶性肿瘤细胞侵袭转移能力的逆转的可能性。人高转移性肺巨细胞癌系PG亚克隆，是在裸鼠体内建立的人类高转移性肿瘤细胞系。Zymography和Western Blot研究表明该细胞有着高水平表达和活性的MMP-2和MMP-9。本研究通过构建可在真核细胞内质网表达抗Ⅳ型胶原酶scFv M97抗体基因的真核表达载体，对人高转移性肺巨细胞癌系PG进行基因治疗研究。结果表明，以质粒PCANTAB
Tumor cell invasion and metastasis to secondary sites are complex, multistep processes including cell detachment from the primary site, migration through lymph or blood vessels, adhesion to cellular and extracellular matrix elements of the organs involved, proteolytic degradation of matrix proteins and cell proliferation. Several above processes involved proteolysis of the extracellular matrix and tissue remodeling. Matrix metalloproteinases (MMPs) comprise a family of enzymes whose main function is the degradation of extracellular matrix, and have been implicated in tumor progression. It was documented that type IV collagenase with two subtypes of 72kDa/MMP-2 and 92kDa/MMP-9 plays an important role in tumor invasion and metastasis, based on the mechanism of proteolytic degradation of collagen IV in basement membrane by which malignant tumor initiates its invasion and spread.
It has been demonstrated that natural and synthetic inhibitors of type IV collagenase such as TIMPs, BB94 have reduced the metastasis of murine Lewis lung carcinoma and tumor neovascularization . These finding suggested that blocking the function of type IV collagenase represents a useful strategy in cancer therapy and prevention of metastasis.
One possible method to inhibit specifically the function of a protein is to express intracellular antibody (intrabody) combining site that can block the function or prevent the expression of the targeted molecule. Intracellular antibodies are novel agents that can be used to study or to alter the function of specific cellular proteins. Intracellular scFv has been used as a novel approach to gene therapy to inhibit a variety of HIV proteins, resulting in ablating the replication of HIV virus in T cells. Many groups have used scFv to abrogate the expression of oncoproteins. Thus, scFv can achieve effectively functional knock-out of target oncoproteins. Based on this concept, intracellular antibodies against type IV collagenase may have therapeutic use against tumor
引文
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