摘要
宫颈癌是女性生殖道最常见的妇科恶性肿瘤之一,在世界范围每年新增病例是50万。晚期宫颈癌和治疗后复发患者免疫功能的抑制,使得预后难以改善;虽然部分早期患者通过手术切除,能够治愈,手术造成生活质量的下降、生育功能的丧失或流产、早产的增加等,因此探索新的治疗策略势在必行。
作为恶性肿瘤第四种治疗模式的生物治疗研究蓬勃发展,如何改善宫颈癌治疗现状,在有效治疗的同时提高患者的生存质量甚至保留年轻患者的生育功能,免疫治疗是较为活跃的领域。细胞因子治疗是肿瘤免疫治疗的重要组成部分,它是基于对免疫反应具有调控作用,可以提高抗原呈递作用,并能加强细胞毒性抗肿瘤反应,目前临床上已应用IL-2、CSF、IFN、TNF-α等治疗肿瘤。IL-21是新近发现的细胞因子,属IL-2家族成员之一,主要是由CD4+T细胞分泌的,为Th1细胞因子,可以诱导被激活的T和NK细胞的增值,并提高了细胞毒性T细胞和NK细胞的溶解的功能,因此具有明显的抗肿瘤作用。
基因转染的载体多种多样,包括以腺病毒、脂质体、细胞电穿孔等。但是前两者具有生物不安全、免疫源性高,不稳定等因素,而脉冲电场作为载体能克服上面这些问题。脉冲电场能使细胞膜的通透性增加,脂质双膜结构发生改变,形成纳米级的亲水孔。一定场强范围内能产生可逆(reversible electroporation RE)和不可逆电穿孔(irreversible electroporation IRE)。IRE是一种新型的微创组织消融技术,不同于其它热消融技术,具有很多优势如:能量可控、能够监测、不受血流的影响,尤其是肿瘤组织的消融具有独特的治疗效果,利用正常组织和肿瘤组织的介电常数的不同,可以达到肿瘤细胞的消融。肿瘤细胞膜的持续穿孔及渗透作用,导致肿瘤细胞凋亡或死亡,而不影响间质细胞和大血管;局部创面很快愈合,对周围正常组织不造成损伤,死亡的细胞引流至淋巴结,在局部形成炎性细胞的浸润,不利于肿瘤细胞通过淋巴通道转移。由于宫颈的解剖位置利于电极的放置,宫颈癌是IRE治疗肿瘤的最佳器官之一。
但是随着电场的衰减,在周围可能残存有肿瘤细胞的区域形成不可逆穿孔,这些被可逆穿孔的肿瘤细胞势必再次增殖而复发,因此我们将IL-21基因配伍在IRE周围,在消融一定量的肿瘤实质的同时,使残存的肿瘤细胞能转染IL-21基因,在改变肿瘤的抗原的同时增强机体的抗肿瘤作用。为一种新型的肿瘤综合治疗方法提供理论和实验依据。
第一章IRE对宫颈癌细胞消融的电参数的筛选
目的:通过在活体外实验,筛查出IRE诱导宫颈癌细胞株完全消融电参数组合,同时找出不能完全消融脉冲参数对肿瘤细胞生物特性的影响。同时优化基因转染的电参数组合
方法:(1)将HeLa细胞悬液按场强从0.5kV/cm,梯度增加0.25kV/cm到2.5kV/cm,共9组,并设置对照组,固定脉宽100μs和频率1Hz,8个脉冲为一套。
(2)作用后的细胞在0h、6h和24h用苔盼蓝计数细胞的死亡率;细胞爬片6小时Giemsa染色证实电场作用于细胞的量效关系,以及免疫组化S-P法检测凋亡蛋白Caspase-3的表达;
(3)作用后的细胞24小时MTT检测细胞活力;流式细胞术检测细胞的周期改变和凋亡;
(4)用接近和达到半致死的场强间隙作用2-3次以找寻完全的宫颈癌细胞消融参数
(5)在半致死场强以下的电参数转染GFP质粒优化转染基因的电参数。
结果:0h苔盼蓝细胞计数0-1.5kV/cm之间没有差异,对照组与1.75-2.5kV/cm之间有明显的差异;6h、24h计数,对照组与1-2.5kV/cm之间出现了差异,Giemsa染色直观的显示了这个结果;6h细胞爬片免疫组化检测Caspase-3的表达,在1.5-2.5kV/cm的阳性信号明显的高于对照组,通过流式细胞术检测细胞凋亡,对照组与2-2.25kV/cm之间有明显的差异;0.5-1.75kV/cm组较对照组有增高的趋势;流式细胞术检测2-2.5kV/cm作用下细胞G2期的细胞数明显增高;24小时MTT结果0.5-1.75kV/cm之间增殖没有差异性,2-2.5kV/cm作用下细胞增殖受到明显的抑制。1.75-2.5kV/cm在电场间隙性作用3套时细胞能够完全消融,而没有热量的叠加。适时温度的检测,室温对流的情况下,各脉冲电场的温升,最高仅为4.9±1.17。
结论:随着电场的增加,细胞从RE到IRE,此过程中场强和脉冲个数同等重要,单独使用IRE是可以用于癌肿的治疗产生的热效应可以忽略,细胞死亡除了不可逆电穿孔机制外,可能还涉及了细胞凋亡途径。值得注意的是,用脉冲电场消融肿瘤时,务必要对肿瘤细胞实现完全杀伤,否则,细胞周期的改变可能会导致肿瘤的复发和加速生长。
第二章IL-21基因对HeLa细胞转染后的生物学影响
第一节人源性IL-21基因的构建和鉴定
目的:新近发现的Th1细胞因子IL-21具有广泛的免疫调节功能和抗肿瘤作用,是由被激活的CD4+ T细胞分泌,为了获得IL-21mRNA我们通过CD3和CD28抗体与外周血淋巴细胞共刺激,提取mRNA。
方法:1)分离人外周血淋巴细胞,在体外与CD3/CD28抗体共培养,提取总的mRNA,设计引物, RT-PCR法扩增片段,割胶纯化载体pcDNA4/HisMax-IL-21的连接反应
2)CaCl2法制备感受态菌,并将pcDNA4/HisMax-IL-21转化至感受态TOP10,蓝白筛选后震荡培养
3)用Qiagen-tipl00抽提试剂盒进行pcDNA4/HisMax-IL-21质粒的大量扩增
4)用XhoI/HindlII双酶切1%的琼脂糖凝胶电泳及送invitrogen公司测序鉴定
结果:从人外周血淋巴细胞中提取的总RNA,用紫外分光光度仪测定OD260/OD280=0.359/0.182=1.97表明RNA的质量完整。RT-PCR法扩增出一条带,与分子量Manker比较,约为489bp大小的特异性条带,与预期设计的全长IL-21cDNA的大小相符合。被酶切的质粒出现两条带,约5.3kb、489bp,分别与pcDNA4/HisMaxA和IL-21基因的片段大小一致。测序结果经NCBI提供的BLAST程序分析对比,显示本实验获得的IL-21序列与genebank公布的IL-21基因序列一致
结论:通过在外周血淋巴细胞内加入CD3/CD28抗体,进行共刺激后,提取的mRNA在引物的作用下,成功的扩增了IL-21cDNA ;并与质粒载体连接;产物经过双酶切和测序,与genebank公布的IL-21基因序列一致
第二节IL-21基因转导人宫颈癌HeLa细胞产生的生物学影响
目的:为了探索人源性IL-21转导至HeLa细胞对其有无生物学影响
方法:我们将成功构建并鉴定的的人源性IL-21质粒及质粒载体,用已经优化的电参数组合电转染至人宫颈癌HeLa细胞内;通过质粒Zeocin抗性筛选出具有IL-21和载体的HeLa细胞(HeLa-IL-21和HeLa-vector);细胞生长曲线的绘制,MTT检测比较HeLa、HeLa-vector和HeLa-IL-21生长活力,流式细胞术检测三者的周期和凋亡。
结果:HeLa、HeLa-vector和HeLa-IL-21生长活力没有明显的不同,流式细胞术检测的各细胞凋亡及周期也没有明显的不同
结论:转导人源性IL-21基因至HeLa细胞内,没有对HeLa细胞造成生物学的影响。
第三章IL-21基因转导人类宫颈癌细胞产生T和NK细胞依赖的抗肿瘤作用
目的:检测肿瘤细胞内表达白介素-21(IL-21)移植到小鼠体内能否产生抗肿瘤作用。
方法:用包含有人IL-21的质粒电转染至宫颈癌HeLa细胞,移植到用人外周淋巴细胞免疫重建和非免疫重建的SCID(severe combined immunodeficiency)小鼠体内,并用抗唾液酸神经节苷酯(anti-asialo GM1)删除小鼠的NK细胞,以及LDH法检测荷瘤鼠的脾细胞毒性和ELSAL双夹心法检测对靶细胞产生的IFN-γ。
结果: HeLa-IL-21细胞在免疫重建和非免疫重建小鼠中的生长完全被抑制,HeLa细胞在免疫重建鼠中的生长较非免疫重建小鼠滞后,肿瘤体积也明显小。删除NK细胞后暂时消除了HeLa-IL-21对肿瘤的生长抑制,同时非免疫重建组消除肿瘤生长抑制较免疫重建组明显;荷HeLa-IL-21细胞小鼠脾细胞毒性在免疫重建组高于非免疫重建组和荷HeLa细胞组,对靶细胞产生IFN-γ的量也大于非免疫重建和荷HeLa细胞组。
结论表达IL-21的HeLa细胞能产生依赖NK细胞和T淋巴细胞介导的抗肿瘤作用。脉冲电场;;IRE;;RE;;IL-21;;宫颈癌
Cervical cancer is one of the commonest malignant tumor in female reproductive system.Now epidemiological investigation shows that the cases will develop in about 500 000 women this year worldwide. The primary cause in development of cervical cancer is human papillomavirus (HPV)infection,smoking and lower immunological function and so on.The patients in advanced stage cervical cancer and recurrence were immunological deficient and poor outcome.Other patients in earlier stage ,even though hysterectomy and local treatment may be recovery.But their quality of life hard to development, abortion and even reproductive function lose. So explore new treatment strategy is imminent.
Biotherapy as a treatment modality of tumor has been extensively studied.To development outcome,survival quality and reproductive capability after effective surgerg, Immunotherapy has considerable potential for the treatment of cancer,cytokine therapy is one of the alternative. It can induction of tumour-specific cytotoxic T lymphocytes (CTLs) is thought to play a critical role in mediating the antitumour effects. Moreover, the ability of different cytokines to enhance NK-cell activity either alone or in the presence of tumour-specific antibodies. Cytokines, the biological response modifiers of the immune system, have had mixed success in cancer therapy. IFN-α,CSF,TNF-αand IL-2 are prototypical examples of successful cytokine-based immunotherapy for cancers. One such immune-stimulating substance is IL-21—a cytokine that belongs to the IL-2 superfamily of cytokines that all have a unique receptor but share a commonγc chain (γc) receptor—that has shown promising results in preclinical tumour models. we will discuss how the potential use of IL-21 in combination with other mothed may lead to better responses .
The vectors used for gene transfection had a wide variety types,such as adenovirus,liposomes,etc.These vectors have their own deficienc,such as biological unsafety, higher immunogenicity and unstable,while the pulsed electric field as a vector for gene transfection can overcome these adverse factors. Electroporation is the permeabilization of the cell membrane with electrical pulses applied across the cell. electrical field induces instabilities in the polarized cell membrane lipid bilayer then alters its shape forming aqueous pathways that possibly are nano-scale pores named electroporation.It can be divided into RE and IRE.RE is commonly used to electrochemtherapy and Electrogene-therapy. IRE ,nevertheless, was used primarily for ablation of microorganisms and cells in vitro and of undesirable tissues in vivo. IRE should facilitate real time control of electroporation during clinical applications. There appears to be complete ablation to the margin of blood vessels without compromising the functionality of the blood vessels. Specific inductive capacity of tumor cell is larger than that of normal cell due to karyoplasmic ratio of tumor cell larger than that of normal one. so identical field strength effect,tumor cell is easier sfuffer to lesion.IRE is a promising method for treatment of tumors near blood vessels. Cervical cancer could also benefit from this property of IRE.
With electric field gradually weaken,cells far away electrode will from IRE to beome RE. Tumor cells of RE within the tissue may be proliferation then tumor recurrence.So we will IRE combine with IL-21 electrotransfer for cervical cancer cell to therapy this disease, aim to IRE can ablate substantial volumes of tumor tissue and electrotransfect IL-21 to cells which were .reversible electroporated by pulse electric field. Tumor cells which were not ablated can transfection IL-21 gene for alter their immunogenicity and enforce immunity to tumor of patiens.
CHARPTTER ONE: CERVICAL CANCER CELL BIOLOGY ALTERATION WITH ELECTROPORATION AT DIFFERETION ELECTRIC STRENGTHS
OBJECTIVE: when in vivo tumor was treated by irreversible electroporation, no matter what shape of the electrode array, complete uniform pulsed electric field is difficult to achieve and there is always a gradient variation.In this study, cervical cancer HeLa cells were treated by pulsed electric field with different intensity, we aimed to explore the biological effects of changed electric field on tumor cells in vivo.
METHODS: 9 groups of HeLa cell suspension with field strength from 0.5kV/cm to 2.5kV/cm(the gradient was 0.25kV/cm) and the control group were included. Pulse width was 100μs and frequency was 1Hz.Every 8 pulses was a set. Trypan blue was used for calculating cell mortality after the cells were treated for 0h, 6h and 24h;Giemsa staining of 6h creep plates was used to prove dose-effect relationship between electric field and cells. The expression of apoptotic protein caspase-3 was detected by SP immunohistochemisty; MTT was used for detection of cell viability 24 hours after treating;flow cytometry was used for detection of cell cycle change and apoptosis;
RESULT: 0h trypan blue cell count showed that no significant difference was found between cell suspension with 0-1.5kV/cm field strength and there was significant difference between the control group and cell suspension with 1.75 - 2.5kV/cm field strength;6h and 24h cell count showed significant difference between the control group and cell suspension with 1-2.5kV/cm field strength,which was also showed directly by Giemsa staining;Caspase-3 expression after 6h creep plates was detected by immunohistochemisty and the results showed that positive signal in cell suspensions with 1.5-2.5kV/cm field strength was significantly higher than that of control group;flow cytometry showed significant difference of apoptosis between control group and cell suspension with 2-2.25kV/cm field strength,apoptosis in 0.5-1.75 kV/cm group showed increasing trend compared with control group;flow cytometry showed cells in G2 phase increased significantly when the field strength was 2-2.5kV/cm;24-hour MTT results showed no difference of proliferation between cell suspension with 0.5-1.75kV/cm field strength and cell proliferation was markedly inhibited when the field strength was 2-2.5kV/cm.The cells were ablated completely without heat superposition under the gap action of 3 sets of 1.75-2.5kV/cm field strength. The maximum of temperature rise of pulsed electric field is 4.9±1.17 under air convection and proper temperature;
CONCLUSIONS: With the increase of electric field,cells developed from reversible electroporation to Irreversible electroporation, in this process field strength is as important as the number of pulse.IRE can be used alone for the treatment of cancer and its thermal effect can be ignored.Cell death may involved irreversible electroporation and cell apoptosis pathway.It is noteworthy that complete destruction of tumor cells should be achieved when pulsed electric field was used for tumor ablation,otherwise the change of cell cycle may lead to tumor recurrence and accelerated growth.
CHARPTTER TWO: BIOLOGY EFFECTIVE TO HELA CELLS AFTER TRANSDUCTION IL-21 GENE FIRST ARTICLE :HOMO-IL-21 GENE CONSTRUCTION AND IDENTIFICATION
OBJECTIVE: IL-21,which Th1 cytokine is newly discovered possess extensive function of immunological regulation in antitumor. It is secreted by CD4+ T cell.To gain IL-21mRNA, gather mRNA was extracted by Trizol way from human peripheral blood lymphocyte that was costimulated with CD3/CD28 antibody.
METHODS: Human peripheral blood lymphocyte,which was draw off by gradient centrifugation ,was co-cultured with CD3/CD28 antibody in vitro. Gather mRNA ,which was extracted by Trizol way from human peripheral blood lymphocyte, was amplificated under primer existence by RT-PCR and purified. IL-21cDNA and pcDNA4/HisMax vehicle were cutted and then conjuncted by XhoI/HindlII and T4DNA ligase respectively.pcDNA4/HisMax-IL-21 was transformated to competence TOP10 cells(CaCl2).Plasmid pcDNA4/HisMax-IL-21 was bulk extracted by Qiagen-tipl00 kit. Plasmid was identificated by XhoI/HindlII dizymase cutted, 1% agarose gel electrophoresis and sequencing.
RESULT: The strap of amplification with RT-PCR was compared to Manke .It is show that length is 489bp and consistent with IL-21cDNA. There were two specificity straps of 5.3kb and 489bp respectively was appeared by dizymase cutted the plasmid. These are identical with fragment of pcDNA4/HisMaxA and IL-21 gene.Sequencing result show that gained IL-21 sequence is identical with that of genebank publication by BLAST programme to analyze in NCBI
CONCLUSIONS: Amplification of IL-21cDNA is succeed under primer effected on mRNA extracted after costimulated CD3/CD28 antibody and hPBL.
SECOND ARTICLE:BIOLOGY EFFECTIVE TO HELA CELLS AFTER TRANSDUCTION IL-21 GENE
OBJECTIVE: To search for biology effect of cervical cancer HeLa,which was transducted IL-21 by reversible electroporation.
METHODS: We apply electricity parameter that was optimized to electroblot HeLa cells with homo-IL-21 plasmid by reversible electroporation. The plasmid has resistivity to Zeocin. We have progressed elective culture to HeLa cells on the basis of its peculiarity and gained HeLa–IL-21 and HeLa-vector cell lines.HeLa-IL-21 cells was compared cell viability and with HeLa and HeLa-vector cell lines by growth curve, MTT. Flow cytometry was used comparison their apoptosis and cell cycle.
RESULT: Comparison viability of HeLa-IL-21 cells with HeLa and HeLa-vector cells showed not significant difference respectively. Their cycle and apoptosis showed yet not significant difference respectively
CONCLUSIONS: HeLa,which was transducted IL-21 has not produced biology peculiarity alteration
CHARPTTER THREE: TRANSDUCTION OF THE IL-21 GENES IN HUMAN CERVICAL CANCER CELLS PRODUCES T CELL-DEPENDENT AND IN-DEPENDENT ANTITUMOR EFFECTS
OBJECTIVE: To examined whether novel cytokines, interleukin (IL)-21 that were expressed in HeLa cells could produce antitumor effects in the inoculated mice.
METHODS: Human cervical cancer HeLa cells were electric transduced with human IL-21 and were injected into severe combined immunodeficiency (SCID) mice that were immune reconstituted by human peripheral blood lymphocytes (hPBL) and not reconstituted.Treatment of some of these SCID mices with anti-asialo GM1 antibody to depleted NK cells temporally in order to examine an involvement of T cells in the antitumor effects; We examined the production of IFN-γand Cytotoxic activities of spleen from SCID mice bearing parent and HeLa-IL-21 cells cells to HeLa and Caski.
RESULT: Growth of HeLa-IL-21 tumors developed in SCID mice was completely inhibited compared with that of parent tumors, no matter whether in immune reconstituted or not immune reconstituted group. Growth of HeLa tumors developed in SCID with hPBL was retarded compared with in that of haven’t hPBL. Afeter depleted NK cells temporally abrogated the growth retardation of HeLa-IL-21,the phenomenon in immune reconstituted group(IRG) were more significant than in nIRG. Cytotoxic activities of spleen cells to HeLa from spleen cells of SCID mice bearing HeLa-IL-21 cells with hPBL group was higher compare with in that of haven’t hPBL and bearing HeLa group,the production of IFN-γof splenocyts from IRG to target cells were higher than that from nIRG and bearing HeLa group.
CONCLUSIONS: Expression of IL-21 in HeLa cells can produce T cell dependent and -independent antitumor effects in an NK cell-defective condition.
CHARPTTER FOUR: IRE COMBINED IL-21 GENE THERAPY CERVICAL CANCER
OBJECTIVE: IRE can change to RE arounding tissues which electric field extinction is developed owing to its far away electrode. IL-21 may be transfected to relic tumor cells by RE then it produce antitumor effective.To explore effective and mechanism of IRE combination with RE as well as different electrode be used in treatment tumor.
METHODS: The experiment parameter is composed of field,strength 2500V/cm,pulse width100μs and 1Hz frequency and every 8 pulses was a set . Bearing tumor naked mice and SCID mice were treated by pliers shape parallel and needle-electrode respectively. Bearing tumor SCID mice were divided to two groups of pulse and pulse addition IL-21 gene treatment, control group as well. We observe to their tumor regression and survival time. Histopathology was observed by HE staining after IRE treatrment 5 minutes,24 and 72 hours respectively.
RESULT: No matter used pliers shape parallel or needle-electrode,they can not active suppression tumor growth to put 16 pulses into practice.In needle-electrode,however tumor accelerate growth after 16 pulses treatment short term. With increase number of pulses, IRE induce significant tumor regression especially in 64-80 pulses There are better results use of pliers shape parallel electrode than that of needle-electrode. Complete tumor regression occure in bearing tumor naked mice that were treated by 64 pulses There are not significant morphology alteration after 5 minutes with IRE treatment in tumour cell. However, the congestion of blood in the tumour vessels was evident. At 24 h, almost the entire tumor presented that the nuclei, extremely pycnotic, membranolysis.‘‘cytoplasm’’appeared to take delight in eosin staining. Under high power microscope,tumor cytomembrane were unintegrated, nuclei break into pieces and edge assemble. At 72 h, the nuclei were smaller even disappearance and tissue necrosis still more evident. Nevertheless,there are not morphology alteration of blood vessel and stroma cell to appear. There are significant different in tumor regression and survival time of bearing tumor SCID ,which were alone pulse treated and IRE combine with IL-21gene transfection At 12 day,tumor volume mean of IRE combine with IL-21 gene transfection and control group are 31.57±16.97 and 65.89±13.84 respectively.( p < 0.01) With increase number of pulses, Complete tumor regression in bearing tumor SCID mice of IRE combine with IL-21 gene transfection treatment was more evident
CONCLUSIONS:IRE can be used to effectively ablate tumors in vivo, Eelectric field used parallel electrode is uniformity over tumor area and effective to destroy tissue. Eelectric field reduction used needle-electrode is more evident and from IRE to RE. So,this property can transduce gene and impermeat anti-cancer to remaining tumor cell,which can supplementary to IRE treatment. IRE combine with IL-21 gene transfection is a effective tumor treatment mode
引文
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