靶向H-ras基因的小干扰性RNA联合能量可控陡脉冲治疗卵巢癌的实验研究
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摘要
陡脉冲电场对细胞膜的不可逆性电击穿导致的细胞死亡成为了恶性肿瘤低创或微创物理治癌新方式,而对肿瘤边缘残存的有活性肿瘤细胞的补充杀伤则是影响陡脉冲治疗效果的关键。肿瘤是一种以癌基因激活和肿瘤抑制基因失活为特征的基因组功能性疾病,这些基因组功能的改变限制了传统治疗手段的有效性。随着分子生物学、免疫学和病毒学的发展,通过对致瘤基因的抑制或缺陷基因的修复等方法,基因治疗已成为继手术、化疗和放疗之后的可望根治癌症的新方法。ras基因是存在于正常细胞的原癌基因,与细胞的增殖、分化有密切的关系;ras 基因的持续的激活可导致细胞的增殖和永生化。野生型 H-ras基因是一种在卵巢浆液性囊腺癌细胞中高表达的基因,我们运用 RNAi技术沉默其表达后观察细胞增殖、凋亡和肿瘤生长情况,进一步明确H-ras 基因在卵巢癌发生发展过程的作用,并将 H-ras 基因的干扰性RNA 与陡脉冲联合应用解决陡脉冲治疗后瘤细胞的残余和复发问题,为癌症的陡脉冲联合基因治疗提供理论和实验依据。
目的:构建针对卵巢癌高表达的H-ras基因编码区的小发夹状RNA重组质粒载体 pshRNA/H-ras,并行序列分析,为进一步研究 H-ras 基因在卵巢癌发生发展中的作用打下基础。
方法:应用基因克隆技术,设计含 21bp H-ras 基因编码序列片段及中间以 4-5 个 bp 间隔的反向重复序列,经退火形成互补双链,克隆至转录载体 pTZU6+1 上,转化 JM109 菌株,提取重组质粒酶切鉴定并序列分析。
结果:将合成的 DNA 片段成功克隆至载体上,经酶切及 DNA 序列鉴定为正确的目的序列。
结论:靶向 H-ras 基因的发夹状 RNA 干扰重组质粒的成功构建,可进一步研究其对细胞内源 H-ras 基因 mRNA 转录和蛋白质表达的影响,为卵巢癌的基因治疗奠定基础。
Steep pulsed electric fields(SPEFs) is a new low-invasive or mini-invasive physical approach in treatment neoplasm, which causes irreversible electrical breakdown(IREB) on the tumor cell membrane and results in cell death. Killing the survival tumor cells suppletively in marginal tissue after ECSP is a key question that influences the effect of energy-controllable steep pulse(ECSP) therapy. Tumor is a genome functional disease with characteristics of activation of cancer gene and nonactivation of tumor suppressor gene, which limits the availability of traditional therapy. With the development of molecularbiology 、immunology and virology , gene therapy could become a new method to effect a radical cure for cancer following surgical operation、chemotherapy and radiotherapy. Ras gene is a kind of proto-oncogene in normal cells which has a close relation with proliferation and differentiation of cells. Continuing activation of ras gene will lead to cell proliferation and cause immortal cells. Wild H-ras gene is higher expression in ovarian serous cystadenocarcinoma. We have used RNAi technology to silence H-ras
expression and observe the cells proliferation, apoptosis and growth of tumor in order to ascertain the function of H-ras gene in development of ovarian carcinoma. ECSP combined with short hairpin RNA targeting H-ras gene were used to solve the remnants and relapse after ECSP. Our experiments provide a theoretical and experimental basis of combination ECSP and gene treatment for cancer.
PART ONE CONSTRUCTING AND IDENTIFICATION OF THE SHORT HAIRPIN RNA RECOMBINANT PLASMID TARGETING H-RAS GENE
OBJECTIVE: To construct the recombinant plasmid targeting H-ras gene coding sequence pshRNA/H-ras and analyzing nucleic acid sequence for further researching the role in the development of ovarian carcinoma.
METHODS: A 21bp reverse repeated motif of H-ras gene target sequence with 4-5bp spacer were designed and synthesized. Annealing and inserted into pTZU6+1. These recombinant plasmids were transformed into JM109 strain. Then the recombinant plasmids were identified by restriction enzyme and DNA sequence analyzing.
RESULTS: The recombinant plasmids targeting H-ras gene were constructed successfully.
CONCLUSION: Constructing the pshRNA/H-ras recombinant plasmid helps to further study the inhibitory role of H-rasmRNA and the protein expression.
PART TWO INHIBITION OF ENDOGENETIC H-RAS GENE EXPRESSION BY PSHRNA/H-RAS IN OVARIAN CANCER CELLS AND THE OVARIAN CANCER RESISTANT CELLS
OBJECTIVE: To investigate the inhibition of endogenetic H-ras gene expression applying siRNA directed against H-ras in SKOV3 and SKOV3/ADM strains.
METHODS: The expression level of H-ras gene in SKOV3 and SKOV3/ADM cells were measured by Immunocytochemistry. The recombinant plasmid targeting H-ras gene were transfected into SKOV3 and SKOV3/ADM with DOTAP lipofectmine and the expression of H-ras were detected by Reverse Transcripitional RCR、Western blot.
RESULTS: There are higher expression of H-ras in SKOV3 and SKOV3/ADM cells. The expression of H-ras are all suppressed after transfected pshRNA/H-ras1 and pshRNA/H-ras2 respectively by RT-PCR and Western blot.
CONCLUSION: These results indicated that the siRNA directed against H-ras could inhibit the expression of H-ras in SKOV3 and SKOV3/ADM cells and established a foundation to further study the
unusual proliferation in cancer cells.
PART THREE THE PROLIFERATIVE EFFECT ON SILENCING OF ENDOGENETIC H-RAS GENE EXPRESSION BY RNA INTERFERENCE IN SKOV3 AND SKOV3/ADM
OBJECTIVE: To investigate the cells proliferation effect after transfected pshRNA/H-ras and discuss the role of H-ras gene in the development of ovarian cancer.
METHODS: There are four groups in our study : normal control group, 24h , 48h and 72h experimental group after transfected pshRNA/H-ras. The influence on proliferation and apoptosis were investigated by MTT assay, cloning test, flow cytometry and AO/EB respectively.
RESULTS: It shows the inhibition of cell cycle after transfected pshRNA/H-ras 24h and 48h in
目的:构建针对卵巢癌高表达的H-ras基因编码区的小发夹状RNA重组质粒载体 pshRNA/H-ras,并行序列分析,为进一步研究 H-ras 基因在卵巢癌发生发展中的作用打下基础。
方法:应用基因克隆技术,设计含 21bp H-ras 基因编码序列片段及中间以 4-5 个 bp 间隔的反向重复序列,经退火形成互补双链,克隆至转录载体 pTZU6+1 上,转化 JM109 菌株,提取重组质粒酶切鉴定并序列分析。
结果:将合成的 DNA 片段成功克隆至载体上,经酶切及 DNA 序列鉴定为正确的目的序列。
结论:靶向 H-ras 基因的发夹状 RNA 干扰重组质粒的成功构建,可进一步研究其对细胞内源 H-ras 基因 mRNA 转录和蛋白质表达的影响,为卵巢癌的基因治疗奠定基础。
Steep pulsed electric fields(SPEFs) is a new low-invasive or mini-invasive physical approach in treatment neoplasm, which causes irreversible electrical breakdown(IREB) on the tumor cell membrane and results in cell death. Killing the survival tumor cells suppletively in marginal tissue after ECSP is a key question that influences the effect of energy-controllable steep pulse(ECSP) therapy. Tumor is a genome functional disease with characteristics of activation of cancer gene and nonactivation of tumor suppressor gene, which limits the availability of traditional therapy. With the development of molecularbiology 、immunology and virology , gene therapy could become a new method to effect a radical cure for cancer following surgical operation、chemotherapy and radiotherapy. Ras gene is a kind of proto-oncogene in normal cells which has a close relation with proliferation and differentiation of cells. Continuing activation of ras gene will lead to cell proliferation and cause immortal cells. Wild H-ras gene is higher expression in ovarian serous cystadenocarcinoma. We have used RNAi technology to silence H-ras
expression and observe the cells proliferation, apoptosis and growth of tumor in order to ascertain the function of H-ras gene in development of ovarian carcinoma. ECSP combined with short hairpin RNA targeting H-ras gene were used to solve the remnants and relapse after ECSP. Our experiments provide a theoretical and experimental basis of combination ECSP and gene treatment for cancer.
PART ONE CONSTRUCTING AND IDENTIFICATION OF THE SHORT HAIRPIN RNA RECOMBINANT PLASMID TARGETING H-RAS GENE
OBJECTIVE: To construct the recombinant plasmid targeting H-ras gene coding sequence pshRNA/H-ras and analyzing nucleic acid sequence for further researching the role in the development of ovarian carcinoma.
METHODS: A 21bp reverse repeated motif of H-ras gene target sequence with 4-5bp spacer were designed and synthesized. Annealing and inserted into pTZU6+1. These recombinant plasmids were transformed into JM109 strain. Then the recombinant plasmids were identified by restriction enzyme and DNA sequence analyzing.
RESULTS: The recombinant plasmids targeting H-ras gene were constructed successfully.
CONCLUSION: Constructing the pshRNA/H-ras recombinant plasmid helps to further study the inhibitory role of H-rasmRNA and the protein expression.
PART TWO INHIBITION OF ENDOGENETIC H-RAS GENE EXPRESSION BY PSHRNA/H-RAS IN OVARIAN CANCER CELLS AND THE OVARIAN CANCER RESISTANT CELLS
OBJECTIVE: To investigate the inhibition of endogenetic H-ras gene expression applying siRNA directed against H-ras in SKOV3 and SKOV3/ADM strains.
METHODS: The expression level of H-ras gene in SKOV3 and SKOV3/ADM cells were measured by Immunocytochemistry. The recombinant plasmid targeting H-ras gene were transfected into SKOV3 and SKOV3/ADM with DOTAP lipofectmine and the expression of H-ras were detected by Reverse Transcripitional RCR、Western blot.
RESULTS: There are higher expression of H-ras in SKOV3 and SKOV3/ADM cells. The expression of H-ras are all suppressed after transfected pshRNA/H-ras1 and pshRNA/H-ras2 respectively by RT-PCR and Western blot.
CONCLUSION: These results indicated that the siRNA directed against H-ras could inhibit the expression of H-ras in SKOV3 and SKOV3/ADM cells and established a foundation to further study the
unusual proliferation in cancer cells.
PART THREE THE PROLIFERATIVE EFFECT ON SILENCING OF ENDOGENETIC H-RAS GENE EXPRESSION BY RNA INTERFERENCE IN SKOV3 AND SKOV3/ADM
OBJECTIVE: To investigate the cells proliferation effect after transfected pshRNA/H-ras and discuss the role of H-ras gene in the development of ovarian cancer.
METHODS: There are four groups in our study : normal control group, 24h , 48h and 72h experimental group after transfected pshRNA/H-ras. The influence on proliferation and apoptosis were investigated by MTT assay, cloning test, flow cytometry and AO/EB respectively.
RESULTS: It shows the inhibition of cell cycle after transfected pshRNA/H-ras 24h and 48h in
引文
[1]. 曹泽毅. 妇科肿瘤学[M]. 北京:北京出版社, 814.
[2]. Wingo P, Tong T, Bolden S. Cancer statistics[J]. CA cancer J Clin, 1995;45:8-30.
[3]. G. A.Hofmann and G. Evans. Electronic genetic-physical and biological aspects of cellular electromanipulation[J]. IEEE Eng. Med.Biol.Mag, 1986, 5(4):6-25.
[4]. G. A.Hofmann, S.B.Dev, S.Dimmer, et al. Electroperation Therpy: A New Approach for the Treatment of Head and Neck Cancer. IEEE Trans[J]. On Biomedical Engineering, 1999, (46)6: 752-759.
[5]. 孙才新. 姚成果. 熊 兰. 陡脉冲电场对恶性肿瘤细胞杀伤效应的研究[J]. 生物物理学报. 2002.12, 18(4):474-477.
[6]. 姚成果.孙才新.熊 兰. 陡脉冲电场对荷瘤BALB/C小鼠生存期的影响[J]. 生物医学工程学杂志. 2004.21(3):433-435.
[7]. Xiong Lan. Sun Caixin. Yao Chen-guo. Vascular Effect and Immunity Effect of Steep Pulse Electric Fileds on Walker 256-bearing Wistar Mice[C]. 2004 IEEE BMES/EMBS Conference. San Francisco, CA, U.S.A., 2004.9.1-5.
[8]. 陈 竺 主编. 医学遗传学[M]. 北京: 人民卫生出版社, 2001, 198.
[9]. Stevenson JP, Kang-Shen Yao, Gallagher M, et al. Phase I clinical pharmacokinetic and pharmacdynamic trial of the antisense oligonucleotide ISIS 5132(CGP6984A).
J Clin Oncol, 1999, 17(7): 2227-2236.
[10]. Kogerman P, Sy MS, Gulp LA. Oncogene-dependent expression of CD44 in Balb/c 3T3 derivatives: correlation with metastatic competence[J]. Clin Exp Matastasis, 1996, 14(1): 73-82.
[11]. Chambers AF, Tuck AB. Ras-responsive genes and tumor metastasis[J]. Crit Rev Oncog, 1993;4(2):95-114.
[12]. Sanz L, Berra E, Municio MM, et al. Zeta PKC plays a critical role during stromelysin promoter activation by platelet-derived growth factor through a novel palindromic element[J]. J Biol Chem , 1994; 269(13):10044-10049.
[13]. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C.elegans embryos, encodes a putative Ser/Thr kinse that is asymmetrically distributed[J]. Cell, 1995; 81: 611-620.
[14]. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998:391:806-811.
[15].Salvi A, Arici B, De Petro G, et al. Small interfering RNA urokinase siliencing inhibits a invasion and migration of human hepatocellular carcinoma cells[J]. Mol Cancer Ther. 2004 Jun; 3(6):671-678.
[16]. Pardrige Pardridge WM. Intravenous, non-viral RNAi gene therapy of brain cancer[J]. Expert Opin Biol Ther, 2004; 4(7):1103-1113.
[17]. Li K, Lin SY, Brunicardi FC, et al. Use of RNA interferce to target cyclin E-overexpressing hepatocellular carcinoma[J]. Cancer Res. 2003 Jul 1; 63(13):3593-3597.
[18]. Hang L,Yang N,Mohamed-Hadley A,et al. Vector-based RNAi,a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer[J]. Biochem Biophys Res.Commun.2003 Apr 18; 303(4):1169-1178.
[19]. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J].Cancer cell, 2002,2(3):245-247.
[20]. Gong Yang, Jennifer AT, Bingliang F, et al. Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer[J]. Oncogene,2003,22:5694-5701.
[21]. Slamon DJ, deKernion JB, Verma IM, et al. Expression of cellular oncogenes in human malignancies[J]. Science. 1984 Apr 20;224(4646):256-262.
[22]. Kacinski BM, Carter D, Kohorn EI, et al. Oncogene expression in vivo by ovarian adenocarcinomas and mixed-mullerian tumors[J]. Yale J Biol Med. 1989 Jul-Aug;62(4):379-392.
[23]. Varras MN, Sourvinos G, Diakomanolis E, et al. Detection and clinical correlations of ras gene mutations in human ovarian[J]. Oncology. 1999;56(2):89-96.
[2]. Wingo P, Tong T, Bolden S. Cancer statistics[J]. CA cancer J Clin, 1995;45:8-30.
[3]. G. A.Hofmann and G. Evans. Electronic genetic-physical and biological aspects of cellular electromanipulation[J]. IEEE Eng. Med.Biol.Mag, 1986, 5(4):6-25.
[4]. G. A.Hofmann, S.B.Dev, S.Dimmer, et al. Electroperation Therpy: A New Approach for the Treatment of Head and Neck Cancer. IEEE Trans[J]. On Biomedical Engineering, 1999, (46)6: 752-759.
[5]. 孙才新. 姚成果. 熊 兰. 陡脉冲电场对恶性肿瘤细胞杀伤效应的研究[J]. 生物物理学报. 2002.12, 18(4):474-477.
[6]. 姚成果.孙才新.熊 兰. 陡脉冲电场对荷瘤BALB/C小鼠生存期的影响[J]. 生物医学工程学杂志. 2004.21(3):433-435.
[7]. Xiong Lan. Sun Caixin. Yao Chen-guo. Vascular Effect and Immunity Effect of Steep Pulse Electric Fileds on Walker 256-bearing Wistar Mice[C]. 2004 IEEE BMES/EMBS Conference. San Francisco, CA, U.S.A., 2004.9.1-5.
[8]. 陈 竺 主编. 医学遗传学[M]. 北京: 人民卫生出版社, 2001, 198.
[9]. Stevenson JP, Kang-Shen Yao, Gallagher M, et al. Phase I clinical pharmacokinetic and pharmacdynamic trial of the antisense oligonucleotide ISIS 5132(CGP6984A).
J Clin Oncol, 1999, 17(7): 2227-2236.
[10]. Kogerman P, Sy MS, Gulp LA. Oncogene-dependent expression of CD44 in Balb/c 3T3 derivatives: correlation with metastatic competence[J]. Clin Exp Matastasis, 1996, 14(1): 73-82.
[11]. Chambers AF, Tuck AB. Ras-responsive genes and tumor metastasis[J]. Crit Rev Oncog, 1993;4(2):95-114.
[12]. Sanz L, Berra E, Municio MM, et al. Zeta PKC plays a critical role during stromelysin promoter activation by platelet-derived growth factor through a novel palindromic element[J]. J Biol Chem , 1994; 269(13):10044-10049.
[13]. Guo S, Kemphues KJ. Par-1, a gene required for establishing polarity in C.elegans embryos, encodes a putative Ser/Thr kinse that is asymmetrically distributed[J]. Cell, 1995; 81: 611-620.
[14]. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998:391:806-811.
[15].Salvi A, Arici B, De Petro G, et al. Small interfering RNA urokinase siliencing inhibits a invasion and migration of human hepatocellular carcinoma cells[J]. Mol Cancer Ther. 2004 Jun; 3(6):671-678.
[16]. Pardrige Pardridge WM. Intravenous, non-viral RNAi gene therapy of brain cancer[J]. Expert Opin Biol Ther, 2004; 4(7):1103-1113.
[17]. Li K, Lin SY, Brunicardi FC, et al. Use of RNA interferce to target cyclin E-overexpressing hepatocellular carcinoma[J]. Cancer Res. 2003 Jul 1; 63(13):3593-3597.
[18]. Hang L,Yang N,Mohamed-Hadley A,et al. Vector-based RNAi,a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer[J]. Biochem Biophys Res.Commun.2003 Apr 18; 303(4):1169-1178.
[19]. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J].Cancer cell, 2002,2(3):245-247.
[20]. Gong Yang, Jennifer AT, Bingliang F, et al. Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumorgrowth in a model of human ovarian cancer[J]. Oncogene,2003,22:5694-5701.
[21]. Slamon DJ, deKernion JB, Verma IM, et al. Expression of cellular oncogenes in human malignancies[J]. Science. 1984 Apr 20;224(4646):256-262.
[22]. Kacinski BM, Carter D, Kohorn EI, et al. Oncogene expression in vivo by ovarian adenocarcinomas and mixed-mullerian tumors[J]. Yale J Biol Med. 1989 Jul-Aug;62(4):379-392.
[23]. Varras MN, Sourvinos G, Diakomanolis E, et al. Detection and clinical correlations of ras gene mutations in human ovarian[J]. Oncology. 1999;56(2):89-96.