垂体生长激素腺瘤靶向性基因治疗的实验研究
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摘要
垂体腺瘤虽然是一种良性肿瘤，但其引起的视力视野障碍和内分泌功能紊乱极大地影响了患者的生活质量。传统的手术、药物和放射治疗方法可以治愈大部分垂体腺瘤，但仍有部分患者不能得到有效治疗，寻找新的治疗方法已成为当今神经外科追求的目标之一。基因治疗已经从遗传性疾病、恶性肿瘤发展到其他疾病的治疗，有望成为垂体腺瘤继手术、药物治疗和放疗之外的另一种有前途的治疗策略。目前，国际上已有多个研究中心应用腺病毒介导的单纯疱疹病毒胸苷激酶(HSV-TK)基因结合更昔洛韦(ganciclovir,GCV)进行了垂体腺瘤基因治疗的实验研究，但存在着可能影响正常垂体细胞功能的副作用。
我们对受体介导的GE7基因导入系统的靶向性转染及垂体生长激素启动子的靶向性转录进行了评价，并将二者结合应用于垂体腺瘤的基因治疗，进行了以下几方面的探索：
1．大鼠垂体生长激素腺瘤(GH_3)细胞、人骨髓瘤(U-2OS)细胞和人卵巢癌(HO8910PM)细胞表面表皮生长因子受体(EGFR)的表达
目的：判明实验细胞GH_3细胞、U-2OS细胞和HO8910PM细胞的EGFR表达情况。
方法：实验中应用免疫组织化学的方法，取细胞附壁良好的盖玻片，经丙酮固定，1％H_2O_2作用，非免疫羊血清覆盖，依次加一抗、二抗孵化，0.05％DAB+0.03％H_2O_2显色，苏木素衬染、分化，封片，镜检，棕黄色为阳性结果。
结果：GH_3细胞细胞膜上明显黄染，EGFR强表达。HO8910PM细胞细胞膜上亦明显黄染，EGFR强表达。U-2OS细胞细胞膜未见黄染，EGFR表达阴性。
结论：实验证明GH_3细胞、HO8910PM细胞表面EGFR强表达，U-2OS细胞表面无EGFR表达。本研究拟应用EGFR介导的GE7基因导入系统达到靶向性基因转染的目的。这一结果表明：(1)GH3细胞可以作为GE7基因导入系统基因治疗研究的实验细胞；(2)U-2OS细胞可以作为阴性对照细胞；
(3)HO89lopM细胞可以作为阳性对照细胞。
2.细胞特异性表达质粒的构建
目的:构建人生长激素启动子(human growth hormone promoter，hGHp)调
控的报告基因及杀伤基因表达质粒。
方法:根据GenBank中hGHp的核普酸序列和基因克隆的要求，用PCGENE
软件进行分析，选择最佳扩增引物，通过聚合酶链式反应(PCR)，得到hGHp
的克隆，经测序证实。将作为载体的质粒pGL3一Basic双酶切，并与hGHp粘端
连接，构建报告质粒pGL3一Basic一hGHp。通过相似的步骤完成杀伤基因质粒
peDNA3.1舰isA一hGHp一TK的构建。
结果:PCR反应得到hGHp片段，大小为399bP，DNA测序结果正确。成
功合成质粒pGL3一Basie一hGHp和PeDNA3.1/H isA一hGHp·TK。
结论:应用分子生物学实验技术，可以得到正确的hGHp片段，并通过重
组质粒的方法，成功构建hGHp调控的报告基因及杀伤基因质粒。
3.基因治疗系统转染及表达的研究
目的:评价基因治疗系统的有效性及靶向性。
方法:应用GE7基因导入系统，将报告质粒pGL3一Basic一hGHp分别转染
GH3、U一205及HO8910PM细胞，通过测定报告基因—荧光素酶(lueiferase，
Luc)基因表达活性，证明基因治疗系统的有效性和靶向性。同样应用GE7基
因导入系统，将杀伤基因质粒peDNA3.l舰isA一hoHp一TK转染oH3、u一205及
H08910PM细胞，经W七Stem blot检测，证明基因治疗系统的有效性和靶向性。
同时，研究转染GH3细胞的最佳剂量、最佳转染时间及表达时间。
结果:用GE7基因导入系统转染pGL3一Basic一hGHp后，GH3细胞的荧光素
酶活性(77732.25)明显高于U一205(2207.25，p<0.001)和HO8910pM细胞
(1958.25，p<0.001)。同样转染peDNA3.l胜isA一hGHp一TK的细胞进行Western
blot检测，结果显示GH3细胞在约46kD处有一明显条带，而对照细胞则无此
条带。实验表明GH3细胞最佳转染剂量为2.0卜留ml，最佳转染时间为24小时，
基因表达在7天达高峰，之后缓慢下降，但是直至21天仍有较高表达。
结论:(1)研究所采用的基因治疗系统是高效的，能够将足量的目的基因
导入细胞，并保证目的基因有较高的表达。(2)该基因治疗系统具有较强的靶
向性，其中GE7基因导入系统的靶向性转染，将目的基因导入EGFR高表达的
细胞;生长激素启动子的靶向性转录调控目的基因只在分泌生长激素的细胞中
转录并表达;二者结合的双重靶向性保证目的基因只在GH3细胞中发挥作用。
4.基因治疗的体外实验
目的:评价基因治疗系统进行体外细胞杀伤实验的效果和靶向性。
方法:应用GE7系统将pcnNA3 .1舰isA一hGHp一TK分别转染GH3、u一205
和HO8910PM三种细胞24小时，换完全培养液继续培养24小时，加入GCV
0.1林留孔，培养48小时，MTT法观察细胞杀伤靶向性。GH3细胞GE7转染
PeDNA3 .1倪isA一hGHp一TK后，按0，l，2，4，8，16林g/ml6个梯度加入GCv 0.lml/
孔，培养48小时，MTT法观察GCv的杀伤效果，选择最佳GCv浓度(最低的
有效浓度)。GH:细胞GE7转染peDNA3 .1/H isA一hGHp一TK，24小时后加入Gev，
培养24小时，每隔24小时更换l次GCV，MTT法在第1一7天每天观察杀伤
效果。
结果:在实验中MTT检测表明:(1)GE7转染的peDNA3.l舰
Pituitary rumors are typically benign adenomas that may cause severe clinical symptoms. Although many pituitary tumors are treated successfully, there are several tumors that do not respond to currently available therapies. Gene therapy is always used in the treatment of cancer. However, as the technology improves, gene therapy applications will increase and gain clinical acceptance for the treatment of non-life-threatening diseases, such as pituitary adenoma. Gene therapy is a very attractive alternative to classic therapeutic modalities - chemotherapy, radiotherapy and surgery. Recently, gene therapy using adenoviral vectors to deliver the herpes simplex virus-thymidine kinase (HSV-TK) followed by ganciclovir (GCV) administration has been developed as a strategy for the treatment of pituitary adenomas by several research groups. However, one of the limitations of this type of strategy is that it may affect normal pituitary cells.
Our group appraised the receptor-mediated GE7 gene delivery system, the cell -specific promoters-human growth hormone promoter (hGHp) and developed the strategy of gene therapy for pituitary adenoma using both of them at the same time. The main results of the in vitro and in vivo experimental studies were as follows: Expression of EGFR in cell lines
The GE7 system is a gene delivery system capable of delivering therapeutic gene to the target cells, organs or tissues that express epidermal growth hormone factor receptor (EGFR). So, we detected the expression of EGFR of the cultured GH3, U-OS and HO8910PM cell lines using immunohistochemical method initially. The result suggests that there is EGFR in the membrane of GH3 and HO8910PM cell line, and not in that of U-2OS cell line. So GH3 cell line can be act as the objective cells in the EGFR-mediated gene therapy, HO8910PM cell line as positive control cells, and U-2OS cell line as negative control cells.
Generation of recombinant plasmids expressing transgenes under the control of the human growth hormone promoter
The growth hormone promoter (hGHp) fragment was amplified by PCR using the primer pair and the sperm DNA as a template. The transgene, HSV-TK, was excised from the plasmids pcDNA3.1/His A-TK and cloned into the plasmid pcDNA3.1/His A, whose CMV promoter was cut, generating pcDNA3.1/His A-hGHp-TK. The plasmid pGL3-Basic-hGHp was constructed using the hGHp promoter driving the expression of the marker gene luciferase. Cell type-specific expression of luciferase or HSV-TK in the pituitary tumor cell line, GH3
When the GH3, U-2OS and HO8910PM cells were infected with GE7-deliveried pGL3-Basic-hGHp, only the somatotrophic GH3 cells expressed the luciferase enzyme. No expression of luciferase was observed in U-2OS or HO8910PM cells. These results show that the GE7 gene delivery system and hGHp
promoter can restrict expression of the marker gene luciferase to established somatotrophic tumor cell lines in vitro.
Simultaneous detection of HSV-TK protein within GH3 cells following GE7 infection with pcDNA3.1/His A-hGHp-TK was performed by Western blot techniques. Infection of these cells with pcDNA3.1/His A-hGHp-TK resulted in a 46KD staining line in the somatotrophic GH3 cells. In U-2OS and HO8910 cells, we observed no HSV-TK expression.
To quantitate promoter strength of the hGHp promoter within pGL3-Basic-hGHp, luciferase enzyme activity was assessed in the somatotrophic GHs cells following GE7 infection. The levels of enzyme activity increased with increasing plasmid used, and peaked in 2.0ng/ml plasmid.
Target cytotoxicity of HSV-TK driven by hGHp promoter in combination with GCV in vitro
The most widely used approach of gene therapy for tumor treatment is conditional cytotoxicity. Therapy with conditional cytotoxic genes, such as HSV-TK, has shown toxicity in the presence of GCV. Therefore, we assessed the effect of HSV-TK expression on GH3, U-2OS and HO8910PM cells after GE7 infection with pcDNA3.1/His A-hGHp-TK, in the absence or presence of 0-16ug/ml GCV. We detected cell exist rate using MTT methods. We
我们对受体介导的GE7基因导入系统的靶向性转染及垂体生长激素启动子的靶向性转录进行了评价，并将二者结合应用于垂体腺瘤的基因治疗，进行了以下几方面的探索：
1．大鼠垂体生长激素腺瘤(GH_3)细胞、人骨髓瘤(U-2OS)细胞和人卵巢癌(HO8910PM)细胞表面表皮生长因子受体(EGFR)的表达
目的：判明实验细胞GH_3细胞、U-2OS细胞和HO8910PM细胞的EGFR表达情况。
方法：实验中应用免疫组织化学的方法，取细胞附壁良好的盖玻片，经丙酮固定，1％H_2O_2作用，非免疫羊血清覆盖，依次加一抗、二抗孵化，0.05％DAB+0.03％H_2O_2显色，苏木素衬染、分化，封片，镜检，棕黄色为阳性结果。
结果：GH_3细胞细胞膜上明显黄染，EGFR强表达。HO8910PM细胞细胞膜上亦明显黄染，EGFR强表达。U-2OS细胞细胞膜未见黄染，EGFR表达阴性。
结论：实验证明GH_3细胞、HO8910PM细胞表面EGFR强表达，U-2OS细胞表面无EGFR表达。本研究拟应用EGFR介导的GE7基因导入系统达到靶向性基因转染的目的。这一结果表明：(1)GH3细胞可以作为GE7基因导入系统基因治疗研究的实验细胞；(2)U-2OS细胞可以作为阴性对照细胞；
(3)HO89lopM细胞可以作为阳性对照细胞。
2.细胞特异性表达质粒的构建
目的:构建人生长激素启动子(human growth hormone promoter，hGHp)调
控的报告基因及杀伤基因表达质粒。
方法:根据GenBank中hGHp的核普酸序列和基因克隆的要求，用PCGENE
软件进行分析，选择最佳扩增引物，通过聚合酶链式反应(PCR)，得到hGHp
的克隆，经测序证实。将作为载体的质粒pGL3一Basic双酶切，并与hGHp粘端
连接，构建报告质粒pGL3一Basic一hGHp。通过相似的步骤完成杀伤基因质粒
peDNA3.1舰isA一hGHp一TK的构建。
结果:PCR反应得到hGHp片段，大小为399bP，DNA测序结果正确。成
功合成质粒pGL3一Basie一hGHp和PeDNA3.1/H isA一hGHp·TK。
结论:应用分子生物学实验技术，可以得到正确的hGHp片段，并通过重
组质粒的方法，成功构建hGHp调控的报告基因及杀伤基因质粒。
3.基因治疗系统转染及表达的研究
目的:评价基因治疗系统的有效性及靶向性。
方法:应用GE7基因导入系统，将报告质粒pGL3一Basic一hGHp分别转染
GH3、U一205及HO8910PM细胞，通过测定报告基因—荧光素酶(lueiferase，
Luc)基因表达活性，证明基因治疗系统的有效性和靶向性。同样应用GE7基
因导入系统，将杀伤基因质粒peDNA3.l舰isA一hoHp一TK转染oH3、u一205及
H08910PM细胞，经W七Stem blot检测，证明基因治疗系统的有效性和靶向性。
同时，研究转染GH3细胞的最佳剂量、最佳转染时间及表达时间。
结果:用GE7基因导入系统转染pGL3一Basic一hGHp后，GH3细胞的荧光素
酶活性(77732.25)明显高于U一205(2207.25，p<0.001)和HO8910pM细胞
(1958.25，p<0.001)。同样转染peDNA3.l胜isA一hGHp一TK的细胞进行Western
blot检测，结果显示GH3细胞在约46kD处有一明显条带，而对照细胞则无此
条带。实验表明GH3细胞最佳转染剂量为2.0卜留ml，最佳转染时间为24小时，
基因表达在7天达高峰，之后缓慢下降，但是直至21天仍有较高表达。
结论:(1)研究所采用的基因治疗系统是高效的，能够将足量的目的基因
导入细胞，并保证目的基因有较高的表达。(2)该基因治疗系统具有较强的靶
向性，其中GE7基因导入系统的靶向性转染，将目的基因导入EGFR高表达的
细胞;生长激素启动子的靶向性转录调控目的基因只在分泌生长激素的细胞中
转录并表达;二者结合的双重靶向性保证目的基因只在GH3细胞中发挥作用。
4.基因治疗的体外实验
目的:评价基因治疗系统进行体外细胞杀伤实验的效果和靶向性。
方法:应用GE7系统将pcnNA3 .1舰isA一hGHp一TK分别转染GH3、u一205
和HO8910PM三种细胞24小时，换完全培养液继续培养24小时，加入GCV
0.1林留孔，培养48小时，MTT法观察细胞杀伤靶向性。GH3细胞GE7转染
PeDNA3 .1倪isA一hGHp一TK后，按0，l，2，4，8，16林g/ml6个梯度加入GCv 0.lml/
孔，培养48小时，MTT法观察GCv的杀伤效果，选择最佳GCv浓度(最低的
有效浓度)。GH:细胞GE7转染peDNA3 .1/H isA一hGHp一TK，24小时后加入Gev，
培养24小时，每隔24小时更换l次GCV，MTT法在第1一7天每天观察杀伤
效果。
结果:在实验中MTT检测表明:(1)GE7转染的peDNA3.l舰
Pituitary rumors are typically benign adenomas that may cause severe clinical symptoms. Although many pituitary tumors are treated successfully, there are several tumors that do not respond to currently available therapies. Gene therapy is always used in the treatment of cancer. However, as the technology improves, gene therapy applications will increase and gain clinical acceptance for the treatment of non-life-threatening diseases, such as pituitary adenoma. Gene therapy is a very attractive alternative to classic therapeutic modalities - chemotherapy, radiotherapy and surgery. Recently, gene therapy using adenoviral vectors to deliver the herpes simplex virus-thymidine kinase (HSV-TK) followed by ganciclovir (GCV) administration has been developed as a strategy for the treatment of pituitary adenomas by several research groups. However, one of the limitations of this type of strategy is that it may affect normal pituitary cells.
Our group appraised the receptor-mediated GE7 gene delivery system, the cell -specific promoters-human growth hormone promoter (hGHp) and developed the strategy of gene therapy for pituitary adenoma using both of them at the same time. The main results of the in vitro and in vivo experimental studies were as follows: Expression of EGFR in cell lines
The GE7 system is a gene delivery system capable of delivering therapeutic gene to the target cells, organs or tissues that express epidermal growth hormone factor receptor (EGFR). So, we detected the expression of EGFR of the cultured GH3, U-OS and HO8910PM cell lines using immunohistochemical method initially. The result suggests that there is EGFR in the membrane of GH3 and HO8910PM cell line, and not in that of U-2OS cell line. So GH3 cell line can be act as the objective cells in the EGFR-mediated gene therapy, HO8910PM cell line as positive control cells, and U-2OS cell line as negative control cells.
Generation of recombinant plasmids expressing transgenes under the control of the human growth hormone promoter
The growth hormone promoter (hGHp) fragment was amplified by PCR using the primer pair and the sperm DNA as a template. The transgene, HSV-TK, was excised from the plasmids pcDNA3.1/His A-TK and cloned into the plasmid pcDNA3.1/His A, whose CMV promoter was cut, generating pcDNA3.1/His A-hGHp-TK. The plasmid pGL3-Basic-hGHp was constructed using the hGHp promoter driving the expression of the marker gene luciferase. Cell type-specific expression of luciferase or HSV-TK in the pituitary tumor cell line, GH3
When the GH3, U-2OS and HO8910PM cells were infected with GE7-deliveried pGL3-Basic-hGHp, only the somatotrophic GH3 cells expressed the luciferase enzyme. No expression of luciferase was observed in U-2OS or HO8910PM cells. These results show that the GE7 gene delivery system and hGHp
promoter can restrict expression of the marker gene luciferase to established somatotrophic tumor cell lines in vitro.
Simultaneous detection of HSV-TK protein within GH3 cells following GE7 infection with pcDNA3.1/His A-hGHp-TK was performed by Western blot techniques. Infection of these cells with pcDNA3.1/His A-hGHp-TK resulted in a 46KD staining line in the somatotrophic GH3 cells. In U-2OS and HO8910 cells, we observed no HSV-TK expression.
To quantitate promoter strength of the hGHp promoter within pGL3-Basic-hGHp, luciferase enzyme activity was assessed in the somatotrophic GHs cells following GE7 infection. The levels of enzyme activity increased with increasing plasmid used, and peaked in 2.0ng/ml plasmid.
Target cytotoxicity of HSV-TK driven by hGHp promoter in combination with GCV in vitro
The most widely used approach of gene therapy for tumor treatment is conditional cytotoxicity. Therapy with conditional cytotoxic genes, such as HSV-TK, has shown toxicity in the presence of GCV. Therefore, we assessed the effect of HSV-TK expression on GH3, U-2OS and HO8910PM cells after GE7 infection with pcDNA3.1/His A-hGHp-TK, in the absence or presence of 0-16ug/ml GCV. We detected cell exist rate using MTT methods. We
引文
1． Klibanski A. Nonsecreting pituitary tumors. Endocrinol Metab Clin North Am, 1987,16:793-804．
2． LeRiche VK, Asa SL, Ezzat S. Epidemal growth factor and it's receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness. J Clin Endocrinol Metab, 1996; 81(2) : 656-662．
3． Chaidarun SS, Eggo MC, Sheppard MC, Stewart PM. Expression of epidermal growth factor (EGF), its receptor, and related oncoprotein (erbB-2) in human pituitary tumors and response to EGF in vitro. Endocrinology, 1994; 135(5) : 2012-21．
4． 张龙，雷霆，薛得麟，EGF、TGF-α及其受体在垂体瘤中的表达。
5． Sugawa N, Ekstrand AJ, James CD, et al. Proc Natl Acad Sci USA, 1990; 87: 8602．
6． Wong AJ, Ruppert JM, Biger SH, et al. Proc Natl Acad Sci USA, 1992; 89: 2965．
7． Finley EL, Ramsdell JS. A transforming growth factor-alpha pathway is expressed in GH4C1 rat pituitary tumors and appears necessary for tumor formation. Endocrinology, 1994; 135(1) : 416-22．
8． Andries M, Tilemans D, Denef C. Modulation of epidermal growth factor receptor binding and action by N-acetyl-TGF alpha (34-43) methyl ester. Peptides, 1994; 15(4) : 619-25．
9．王连刚，王喜青，武毅军，等．表皮生长因子、转化因子-α及其受体在人垂体腺瘤组织中的表达．细胞与分子免疫学杂志，2000；16(2)：156-158．
10. Johnson LK, Baxter JD, Vlodavsky I, Gospodarowicz D. Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response. Proc Natl Acad Sci U S A, 1980; 77(1): 394-8.
2． LeRiche VK, Asa SL, Ezzat S. Epidemal growth factor and it's receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness. J Clin Endocrinol Metab, 1996; 81(2) : 656-662．
3． Chaidarun SS, Eggo MC, Sheppard MC, Stewart PM. Expression of epidermal growth factor (EGF), its receptor, and related oncoprotein (erbB-2) in human pituitary tumors and response to EGF in vitro. Endocrinology, 1994; 135(5) : 2012-21．
4． 张龙，雷霆，薛得麟，EGF、TGF-α及其受体在垂体瘤中的表达。
5． Sugawa N, Ekstrand AJ, James CD, et al. Proc Natl Acad Sci USA, 1990; 87: 8602．
6． Wong AJ, Ruppert JM, Biger SH, et al. Proc Natl Acad Sci USA, 1992; 89: 2965．
7． Finley EL, Ramsdell JS. A transforming growth factor-alpha pathway is expressed in GH4C1 rat pituitary tumors and appears necessary for tumor formation. Endocrinology, 1994; 135(1) : 416-22．
8． Andries M, Tilemans D, Denef C. Modulation of epidermal growth factor receptor binding and action by N-acetyl-TGF alpha (34-43) methyl ester. Peptides, 1994; 15(4) : 619-25．
9．王连刚，王喜青，武毅军，等．表皮生长因子、转化因子-α及其受体在人垂体腺瘤组织中的表达．细胞与分子免疫学杂志，2000；16(2)：156-158．
10. Johnson LK, Baxter JD, Vlodavsky I, Gospodarowicz D. Epidermal growth factor and expression of specific genes: effects on cultured rat pituitary cells are dissociable from the mitogenic response. Proc Natl Acad Sci U S A, 1980; 77(1): 394-8.