腺病毒介导IL-24基因对U251胶质瘤细胞的杀伤作用及相关机制研究
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摘要
神经胶质瘤是人类最常见的脑部肿瘤,已成为15岁以下儿童肿瘤致死的第二大病因,是导致35~54岁成年男性和15~34岁女性肿瘤死亡的第四大病因。目前,恶性胶质瘤的常规治疗手段(手术加放化疗)治疗困难、远期效果差,死亡率高。对于恶性程度高的胶质瘤患者,生存期短、5年生存率小于1%。怎样改善胶质瘤的治疗效果,是临床面对的一个难题。近年来,随着分子生物学、分子遗传学的研究进展,对于肿瘤的基因治疗方面取得了长足进步,为提高恶性胶质瘤的治疗效果,增加患者的生存率开拓了新的思路。
白细胞介素24(interleukin-24,IL-24)是细胞因子白介素10家族的新成员,由于最初在人类黑色素瘤中发现,因此也被称为黑色素瘤分化相关因子-7(melanoma differentiation-associated gene-7,mda-7)基因。研究发现,IL-24对多种肿瘤细胞(肺癌、前列腺癌、乳腺癌、卵巢癌、胰腺癌、肝癌等)均有选择性的抑制作用。此外,IL-24与化疗、放疗相结合的研究表明,将IL-24基因转染入人非小细胞肺癌的细胞后,可增强此肿瘤细胞对放射治疗的敏感性,而同时接受照射的人正常成纤维细胞则未受此放射线照射的影响。由于IL-24特有的杀伤肿瘤细胞,而对正常细胞没有损伤作用的生物学特点,现已成为基因治疗的候选基因,并成功用于1期临床研究。目前,关于IL-24对胶质瘤作用的研究报道尚少,尤其对于IL-24诱导胶质瘤细胞凋亡的分子机制尚缺乏足够的了解。
本研究应用新型重组腺病毒载体(Ad5F35-IL24),将IL-24基因导入人胶质瘤U251细胞,应用MTT、流式细胞术、双荧光染色、Hoechst 33258荧光染色、Annexin V-FITC凋亡试剂盒和单细胞凝胶电泳(Single cell gel electrophoresis; SCGE)等技术,体外观察Ad5F35-IL24对U251细胞的杀伤作用。同时用transwell细胞侵袭实验、细胞划痕实验(Cell scratch assay)检测药物作用后肿瘤细胞侵袭、迁移能力的改变。应用Western blot印迹检测外源性IL-24基因转染U251细胞后, ERK、p-ERK、p38 MAPK和p-p38 MAPK等细胞信号转导通路蛋白的表达水平变化。同时应用MTT、流式细胞术、双荧光染色观察ERK、p38 MAPK蛋白抑制剂PD98059、SB203580作用后,对IL-24基因介导U251细胞杀伤中作用的影响。应用Western blot印迹、RT-PCR、流式细胞术、免疫细胞化学的方法检测bcl-2、caspase-3和TopoⅡα蛋白的表达变化情况。以期更深入的了解IL-24基因可能的效应途径和作用靶点,为进一步提高临床神经胶质瘤治疗效果提供研究基础。
第一部分Ad5F35-IL24重组腺病毒载体的构建
目的:构建重组腺病毒载体Ad5F35-IL24。
方法:先用HindⅢ和SalⅠ双酶切ATCC质粒,得到目的条带,然后再用HindⅢ和SalⅠ双酶切pDC316质粒,得到线性化的载体条带,进行片断连接,转化,筛选得到重组的pDC316-IL24质粒。随后用PCR和酶切的方法对重组pDC316-IL24质粒进行鉴定;最后经全自动核酸分析仪上进行序列测定证实连接正确。制备感受态细胞,进行转化,大量制备质粒DNA;之后用双质粒共转染293细胞,产生重组腺病毒Ad5F35-IL24。进行病毒扩增、纯化,计算病毒滴度,按照公式:病毒滴度(PFU/mL)=GFP阳性细胞数×病毒上清稀释倍数/0.5 mL。
结果:成功构建载有IL-24基因的重组pDC316-IL24质粒,并用PCR、酶切、序列测定和BLAST的方法进行了鉴定,证明连接正确;获得了携带IL-24基因的大肠杆菌菌株;成功构建成载有IL-24基因的增殖能力缺陷的新型重组腺病毒Ad5F35-IL24,并进行了扩增和纯化。测得病毒滴度为2.8×108 PFU/mL。
结论:成功构建携带IL-24基因,具有增殖能力缺陷的新型重组腺病毒Ad5F35-IL24载体。
第二部分腺病毒介导IL-24基因对U251胶质瘤细胞的侵袭、迁移及杀伤作用
目的:观察IL-24基因对U251人胶质瘤细胞的杀伤作用,及其对肿瘤细胞侵袭、迁移能力的影响。
方法:用MTT、流式细胞术、双荧光染色、Hoechst 33258荧光染色、Annexin V-FITC凋亡试剂盒和单细胞凝胶电泳( Single cell gel electrophoresis; SCGE)等技术,观察Ad5F35-IL24对U251细胞的杀伤作用。同时用transwell细胞侵袭实验和细胞划痕实验(Cell scratch assay),检测药物作用后对肿瘤细胞侵袭和迁移能力的影响。
结果:与空白对照组和空载体组相比,Ad5F35-IL24能够抑制人U251细胞的增殖,诱导细胞凋亡,且随着Ad5F35-IL24药物浓度的增加,细胞杀伤率增加明显。结果表明,Ad5F35-IL24对U251细胞细胞周期进程有抑制作用,可使细胞周期被阻滞于G2/M期。同时,研究表明Ad5F35-IL24组可显著降低U251细胞的侵袭和迁移能力。
结论:Ad5F35-IL24能够诱导人U251细胞凋亡,诱导G2/M期阻滞,降低肿瘤细胞的侵袭和迁移能力。
第三部分腺病毒介导IL-24基因对U251胶质瘤细胞ERK和p38 MAPK信号转导通路的影响
目的:探讨外源性IL-24基因对U251胶质瘤细胞ERK和p38 MAPK信号转导通路的影响。
方法:IL-24基因转染U251细胞24小时后,应用Western blot印迹检测细胞信号转导通路蛋白ERK、p-ERK、p38 MAPK和p-p38 MAPK的表达水平变化。同时,应用MTT、流式细胞术、双荧光染色观察ERK、p38 MAP蛋白抑制剂PD98059、SB203580作用后,对IL-24基因介导U251细胞杀伤作用的影响,以及细胞内相关蛋白表达水平的变化。
结果:Western Blot结果表明:外源性IL-24基因作用于胶质瘤细胞U251后,p38 MAPK和p-p38 MAPK蛋白表达(0.46±0.07, 0.60±0.07)明显增强(P<0.05)。将p38 MAPK抑制剂SB203580与Ad5F35-IL24合用后(20.83±1.34),较Ad5F35-IL24单用组(36.15±2.61)杀伤作用减弱(P<0.05)。ERK和p-ERK蛋白的与对照组相比没有显著改变(P>0.05)。ERK蛋白抑制剂PD98059与Ad5F35-IL24合用,能够提高IL-24基因对U251的杀伤作用(P<0.05)。
结论:外源性IL-24基因对U251的选择性诱导凋亡作用,可能与其上调并激活p38 MAPK表达有关。而阻断ERK途径可进一步提高IL-24基因对肿瘤细胞的杀伤。
第四部分腺病毒介导IL-24基因对U251胶质瘤细胞拓扑异构酶Ⅱα、bcl-2及caspase-3表达的影响
目的:观察外源性IL-24基因对bcl-2、caspase-3以及拓扑异构酶Ⅱα表达(TopoⅡα)的影响。
方法:应用Western blot印迹、RT-PCR、流式细胞术、免疫细胞化学的方法检测bcl-2、caspase-3和TopoⅡα蛋白的表达变化情况。
结果:Western blot印迹结果表明,Ad5F35-IL24组TopoⅡα蛋白表达(0.48±0.03, 0.31±0.02)较对照组(1.35±0.07)和空载体VEC组(1.39±0.11, 1.26±0.06)表达减低(P<0.05)。且随IL-24基因表达浓度增高TopoⅡα表达随之减低。Ad5F35-IL24组bcl-2的表达(0.02±0.01)也较对照组(0.17±0.02)和空载体VEC组(0.18±0.01)表达减低(P<0.05)。Ad5F35-IL24组caspase-3蛋白的表达(0.71±0.09)较对照组(0.43±0.04)和空载体VEC组(0.47±0.02)表达增加(P<0.05)。
流式细胞术检测表明,caspase-3表达增加,bcl-2表达降低(P<0.05)。Western blot印迹、RT-PCR、流式细胞术的方法观察到TopoⅡα表达降低,且与药物浓度增加成反比。免疫细胞化学检测发现TopoⅡα表达降低。
结论:外源性IL-24基因杀伤U251细胞,TopoⅡα、bcl-2、caspase-3为其重要的作用靶点。研究结果为今后IL-24基因用于胶质瘤的临床治疗提供了实验资料。
Malignant gliomas are the most common brain tumors in humans. It has become the second death inducing factor in the group under 15 among people suffered from the diease. The results are fourth for men in 35-54 ages’group and women in 15-34 ages’group. Current treatments fail to provide long-term management of these tumors. The prognosis for patients with high-grade glioma remains poor: survival for less than 1 year, even following surgery and adjuvant therapies such as chemotherapy and radiation therapy. It clearly requires development of more effective therapeutic strategies that will improve long-term control and survival. To date, gene therapy has provided a new way to effectively therapy of glioma along with the development of molecular biology and molecular genetics in the past few years.
Interleukin-24(IL-24) is a new member of interleukin-10 family. It is also called melanoma differentiation-associated gene-7(mda-7), since it was discovered overexpression in human melanoma during terminal differentiation. Now, several groups have demonstrated that Ad-mda7 (using Ad-mda7) induces apoptosis in a wide range of cancer cells (lung, prostate, breast, ovary, pancreas, hepatoma). In addition, there are some researches about combination of IL-24 and radiotherapy or chemotherapy. For example, it was found that IL-24 can enhance the radiosensitizing effect of non-small cell lung cancer (NSCLC) cell lines. Because of its characteristic killing cancer cell without harm normal cell, IL-24 has become candidate gene and successful carried out in Phase 1 clinic trail. But, the effects of IL-24 gene on glioma cell and its molecular events have been not clearly understood.
In the present study, we construct a new type recombine adenovirus included IL-24 gene (Ad5F35-IL24). After transfected human glioma cell line U251 cells with this new type recombined adenovirus, we use MTT, flow cytometry, double fluorochrome stain, Hoechst 33258 fluorochrome stain, Annexin V-FITC apoptosis kit, Single cell gel electrophoresis(SCGE) assays to observe the effects of Ad5F35-IL24 on U251 cells. The transwell assay and Cell scratch assay were used to detect the invasion and migration ability of the cells after transfected by Ad5F35-IL24. And then, we use Western blot and flow cytometry to detect cell signal transduction protein ERK, p-ERK, p38 MAPK and p-p38 expression induced by Ad5F35-IL24. And also, the MTT, flow cytometry, double fluorochrome stain methods were used to detect the effect of PD98059 (inhibitor of ERK) and SB203580 (inhibitor of p38 MAPK) combined with Ad5F35-IL24 on killing U251 cells. The expression of bcl-2, caspase-3 and TopoⅡα. proteins were examined with Western blot, RT-PCR, flow cytometry, immuocytochemistry assays.
Part 1 Construction of recombinant adenovirus Ad5F35-IL24 vector
Objective: to construct recombinant adenovirus Ad5F35-IL24 vector.
Methods: When plasmids of ATCC were cut by both HindⅢand SalⅠenzymes, the goal bands were obtained. Plasmids of pDC316 were cut by both HindⅢand SalⅠenzymes. Subsequently, after some fragments were connected, transformed and filtered, reorganized plasmids of pDC316 were obtained. Thereafter,it was identified with PCR and enzymes cut analysis. Then the plasmids were carried out sequence determination in whole automatic nucleic acid analyzer. Subsequently competent cells were prepared and transformed. After large-scale preparation of plasmid DNA, recombined adenoviruses Ad5F35-IL24 were generated with homologous recombination by cotransfection of 293 cells with a couple plasmids. After identified with PCR, Ad5F35-IL24 recombinant adenovirus species was amplified and purified. Then the titre was determined according to formula: PFU/mL =GFP post cell number×deliquation of virus supernatant/0.5 mL.
Results: Reconstructed plasmids of pDC316-IL24 containing IL-24 gene was successfully constructed and identified by PCR, enzymes cut and sequence determination. Escherichia coli strains containing IL-24 gene was obtained. Reconstructed adenovirus Ad5F35-IL24 containing IL-24 gene with functional defect of proliferation was successfully constructed, amplified, purified and identified with PCR. Its titre was assayed according to formula: PFU/mL =GFP post cell number×deliquation of virus supernatant/0.5 mL, and the titer was 2.8×108 PFU/mL.
Conclusions: Reconstructed adenovirus vector Ad5F35-IL24 was successfully constructed.
Part 2 Ad5F35-IL24 induces apoptosis and its influence on cell invasion and migration of glioma U251 cells
Objective: Observe the effects of Ad5F35-IL24 on the apoptosis, invasion and migration of U251 cells. Methods: MTT, flow cytometry, double fluorochrome stain, Hoechst 33258 fluorochrome stain, Annexin V-FITC apoptosis kit were used to detect Ad5F35-IL24 inducing U251 cell apoptosis, and observed its influnces on cell invasion and migration with the transwell assay and Cell scratch assay.
Results: Ad5F35-IL24 induces growth suppression and apoptosis in human glioma cells U251 cells. The apoptosis was increased obviously along with Ad5F35-IL24 drug concertration increase. Ad5F35-IL24 could suppress U251 cell cycle; the cells were blocked at G2/M phase. Ad5F35-IL24 could effective decrese the invasion and migration of U251 cells.
Conclusion: Ad5F35-IL24 could inhibit the growth, invasion and migration of human glioma U251 cell, and induces apoptosis.
Part 3 The effects of Ad5F35-IL24 on expression of ERK and p38 MAPK cell signel transduction pathways
Objective: to investigate mechanism of Ad5F35-IL24 effect on ERK and p38 MAPK protein expression.
Methods: After administration of Ad5F35-IL24 for 24 hour. We used Western blot and flow cytometry to detect cell signal transduction protein ERK, p-ERK, p38 MAPK and p-p38 expressions. And the MTT, flow cytometry, double fluorochrome stain were also used to detect the effect of PD98059 (inhibitor of ERK) and SB203580 (inhibitor of p38 MAPK) combined with Ad5F35-IL24 on killing U251 cells.
Results: Western Blot analyses found that IL-24 selectively induced U251 cell apoptosis by up-regulating and activating p38 MAPK and p-p38 MAPK protein expression(0.46±0.07, 0.60±0.07)(P<0.05). After combined with p38 MAPK inhibitor SB203580,it was showed that the inhibition rate was decreased(20.83±1.34)contrasted with the single Ad5F35-IL24 group(36.15±2.61)(P<0.05). On the other hand, ERK and p-ERK protein expression have not significant changed in contrast with control group(P>0.05). After blocked ERK signal transduction pathway with dministration of ERK inhibitor PD98059, the results showed that it could increase apoptosis of U251 cells(P<0.05).
Conclusions: Ad5F35-IL24 selectively induced U251 cell apoptosis by up-regulating and activating p38 MAPK pathway. Blocking ERK pathway could increase Ad5F35-IL24 to induce cell apoptosis.
Part 4 Ad5F35-IL24 influence of TopoⅡα、bcl-2 and caspase-3 protein expression
Objective: To observe Ad5F35-IL24 influence of bcl-2、caspase-3 and TopoⅡαexpression
Methods: After transfected U251 cell with Ad5F35-IL24 for 24 hours, the bcl-2, caspase-3 and TopoⅡαprotein expression were detected with Western blot, RT-PCR, immuocytochemistry assays.
Results: After Ad5F35-IL24 transfection, the related proteins were detacted by Western blot. We found that caspase-3 significantly raised up (0.71±0.09)contrasted with that of control group(0.43±0.04), and bcl-2 expression was significantly cut down(0.02±0.01)compared to the control group(0.17±0.02)(P<0.05). TopoⅡαexpression were found significantly decreased ( 0.48±0.03, 0.31±0.02 ) in contrast with the control group(1.35±0.07)(P<0.05). Immuocytochemistry method also showed that TopoⅡαexpression was decreased down after Ad5F35-IL24 transfected(P<0.05).
Conclusions: IL-24 could inhibit TopoⅡαand bcl-2 expression, and increase caspase-3 expression in U251 glioma cell, which provided refrences for effectively therapy of glioma in future.
白细胞介素24(interleukin-24,IL-24)是细胞因子白介素10家族的新成员,由于最初在人类黑色素瘤中发现,因此也被称为黑色素瘤分化相关因子-7(melanoma differentiation-associated gene-7,mda-7)基因。研究发现,IL-24对多种肿瘤细胞(肺癌、前列腺癌、乳腺癌、卵巢癌、胰腺癌、肝癌等)均有选择性的抑制作用。此外,IL-24与化疗、放疗相结合的研究表明,将IL-24基因转染入人非小细胞肺癌的细胞后,可增强此肿瘤细胞对放射治疗的敏感性,而同时接受照射的人正常成纤维细胞则未受此放射线照射的影响。由于IL-24特有的杀伤肿瘤细胞,而对正常细胞没有损伤作用的生物学特点,现已成为基因治疗的候选基因,并成功用于1期临床研究。目前,关于IL-24对胶质瘤作用的研究报道尚少,尤其对于IL-24诱导胶质瘤细胞凋亡的分子机制尚缺乏足够的了解。
本研究应用新型重组腺病毒载体(Ad5F35-IL24),将IL-24基因导入人胶质瘤U251细胞,应用MTT、流式细胞术、双荧光染色、Hoechst 33258荧光染色、Annexin V-FITC凋亡试剂盒和单细胞凝胶电泳(Single cell gel electrophoresis; SCGE)等技术,体外观察Ad5F35-IL24对U251细胞的杀伤作用。同时用transwell细胞侵袭实验、细胞划痕实验(Cell scratch assay)检测药物作用后肿瘤细胞侵袭、迁移能力的改变。应用Western blot印迹检测外源性IL-24基因转染U251细胞后, ERK、p-ERK、p38 MAPK和p-p38 MAPK等细胞信号转导通路蛋白的表达水平变化。同时应用MTT、流式细胞术、双荧光染色观察ERK、p38 MAPK蛋白抑制剂PD98059、SB203580作用后,对IL-24基因介导U251细胞杀伤中作用的影响。应用Western blot印迹、RT-PCR、流式细胞术、免疫细胞化学的方法检测bcl-2、caspase-3和TopoⅡα蛋白的表达变化情况。以期更深入的了解IL-24基因可能的效应途径和作用靶点,为进一步提高临床神经胶质瘤治疗效果提供研究基础。
第一部分Ad5F35-IL24重组腺病毒载体的构建
目的:构建重组腺病毒载体Ad5F35-IL24。
方法:先用HindⅢ和SalⅠ双酶切ATCC质粒,得到目的条带,然后再用HindⅢ和SalⅠ双酶切pDC316质粒,得到线性化的载体条带,进行片断连接,转化,筛选得到重组的pDC316-IL24质粒。随后用PCR和酶切的方法对重组pDC316-IL24质粒进行鉴定;最后经全自动核酸分析仪上进行序列测定证实连接正确。制备感受态细胞,进行转化,大量制备质粒DNA;之后用双质粒共转染293细胞,产生重组腺病毒Ad5F35-IL24。进行病毒扩增、纯化,计算病毒滴度,按照公式:病毒滴度(PFU/mL)=GFP阳性细胞数×病毒上清稀释倍数/0.5 mL。
结果:成功构建载有IL-24基因的重组pDC316-IL24质粒,并用PCR、酶切、序列测定和BLAST的方法进行了鉴定,证明连接正确;获得了携带IL-24基因的大肠杆菌菌株;成功构建成载有IL-24基因的增殖能力缺陷的新型重组腺病毒Ad5F35-IL24,并进行了扩增和纯化。测得病毒滴度为2.8×108 PFU/mL。
结论:成功构建携带IL-24基因,具有增殖能力缺陷的新型重组腺病毒Ad5F35-IL24载体。
第二部分腺病毒介导IL-24基因对U251胶质瘤细胞的侵袭、迁移及杀伤作用
目的:观察IL-24基因对U251人胶质瘤细胞的杀伤作用,及其对肿瘤细胞侵袭、迁移能力的影响。
方法:用MTT、流式细胞术、双荧光染色、Hoechst 33258荧光染色、Annexin V-FITC凋亡试剂盒和单细胞凝胶电泳( Single cell gel electrophoresis; SCGE)等技术,观察Ad5F35-IL24对U251细胞的杀伤作用。同时用transwell细胞侵袭实验和细胞划痕实验(Cell scratch assay),检测药物作用后对肿瘤细胞侵袭和迁移能力的影响。
结果:与空白对照组和空载体组相比,Ad5F35-IL24能够抑制人U251细胞的增殖,诱导细胞凋亡,且随着Ad5F35-IL24药物浓度的增加,细胞杀伤率增加明显。结果表明,Ad5F35-IL24对U251细胞细胞周期进程有抑制作用,可使细胞周期被阻滞于G2/M期。同时,研究表明Ad5F35-IL24组可显著降低U251细胞的侵袭和迁移能力。
结论:Ad5F35-IL24能够诱导人U251细胞凋亡,诱导G2/M期阻滞,降低肿瘤细胞的侵袭和迁移能力。
第三部分腺病毒介导IL-24基因对U251胶质瘤细胞ERK和p38 MAPK信号转导通路的影响
目的:探讨外源性IL-24基因对U251胶质瘤细胞ERK和p38 MAPK信号转导通路的影响。
方法:IL-24基因转染U251细胞24小时后,应用Western blot印迹检测细胞信号转导通路蛋白ERK、p-ERK、p38 MAPK和p-p38 MAPK的表达水平变化。同时,应用MTT、流式细胞术、双荧光染色观察ERK、p38 MAP蛋白抑制剂PD98059、SB203580作用后,对IL-24基因介导U251细胞杀伤作用的影响,以及细胞内相关蛋白表达水平的变化。
结果:Western Blot结果表明:外源性IL-24基因作用于胶质瘤细胞U251后,p38 MAPK和p-p38 MAPK蛋白表达(0.46±0.07, 0.60±0.07)明显增强(P<0.05)。将p38 MAPK抑制剂SB203580与Ad5F35-IL24合用后(20.83±1.34),较Ad5F35-IL24单用组(36.15±2.61)杀伤作用减弱(P<0.05)。ERK和p-ERK蛋白的与对照组相比没有显著改变(P>0.05)。ERK蛋白抑制剂PD98059与Ad5F35-IL24合用,能够提高IL-24基因对U251的杀伤作用(P<0.05)。
结论:外源性IL-24基因对U251的选择性诱导凋亡作用,可能与其上调并激活p38 MAPK表达有关。而阻断ERK途径可进一步提高IL-24基因对肿瘤细胞的杀伤。
第四部分腺病毒介导IL-24基因对U251胶质瘤细胞拓扑异构酶Ⅱα、bcl-2及caspase-3表达的影响
目的:观察外源性IL-24基因对bcl-2、caspase-3以及拓扑异构酶Ⅱα表达(TopoⅡα)的影响。
方法:应用Western blot印迹、RT-PCR、流式细胞术、免疫细胞化学的方法检测bcl-2、caspase-3和TopoⅡα蛋白的表达变化情况。
结果:Western blot印迹结果表明,Ad5F35-IL24组TopoⅡα蛋白表达(0.48±0.03, 0.31±0.02)较对照组(1.35±0.07)和空载体VEC组(1.39±0.11, 1.26±0.06)表达减低(P<0.05)。且随IL-24基因表达浓度增高TopoⅡα表达随之减低。Ad5F35-IL24组bcl-2的表达(0.02±0.01)也较对照组(0.17±0.02)和空载体VEC组(0.18±0.01)表达减低(P<0.05)。Ad5F35-IL24组caspase-3蛋白的表达(0.71±0.09)较对照组(0.43±0.04)和空载体VEC组(0.47±0.02)表达增加(P<0.05)。
流式细胞术检测表明,caspase-3表达增加,bcl-2表达降低(P<0.05)。Western blot印迹、RT-PCR、流式细胞术的方法观察到TopoⅡα表达降低,且与药物浓度增加成反比。免疫细胞化学检测发现TopoⅡα表达降低。
结论:外源性IL-24基因杀伤U251细胞,TopoⅡα、bcl-2、caspase-3为其重要的作用靶点。研究结果为今后IL-24基因用于胶质瘤的临床治疗提供了实验资料。
Malignant gliomas are the most common brain tumors in humans. It has become the second death inducing factor in the group under 15 among people suffered from the diease. The results are fourth for men in 35-54 ages’group and women in 15-34 ages’group. Current treatments fail to provide long-term management of these tumors. The prognosis for patients with high-grade glioma remains poor: survival for less than 1 year, even following surgery and adjuvant therapies such as chemotherapy and radiation therapy. It clearly requires development of more effective therapeutic strategies that will improve long-term control and survival. To date, gene therapy has provided a new way to effectively therapy of glioma along with the development of molecular biology and molecular genetics in the past few years.
Interleukin-24(IL-24) is a new member of interleukin-10 family. It is also called melanoma differentiation-associated gene-7(mda-7), since it was discovered overexpression in human melanoma during terminal differentiation. Now, several groups have demonstrated that Ad-mda7 (using Ad-mda7) induces apoptosis in a wide range of cancer cells (lung, prostate, breast, ovary, pancreas, hepatoma). In addition, there are some researches about combination of IL-24 and radiotherapy or chemotherapy. For example, it was found that IL-24 can enhance the radiosensitizing effect of non-small cell lung cancer (NSCLC) cell lines. Because of its characteristic killing cancer cell without harm normal cell, IL-24 has become candidate gene and successful carried out in Phase 1 clinic trail. But, the effects of IL-24 gene on glioma cell and its molecular events have been not clearly understood.
In the present study, we construct a new type recombine adenovirus included IL-24 gene (Ad5F35-IL24). After transfected human glioma cell line U251 cells with this new type recombined adenovirus, we use MTT, flow cytometry, double fluorochrome stain, Hoechst 33258 fluorochrome stain, Annexin V-FITC apoptosis kit, Single cell gel electrophoresis(SCGE) assays to observe the effects of Ad5F35-IL24 on U251 cells. The transwell assay and Cell scratch assay were used to detect the invasion and migration ability of the cells after transfected by Ad5F35-IL24. And then, we use Western blot and flow cytometry to detect cell signal transduction protein ERK, p-ERK, p38 MAPK and p-p38 expression induced by Ad5F35-IL24. And also, the MTT, flow cytometry, double fluorochrome stain methods were used to detect the effect of PD98059 (inhibitor of ERK) and SB203580 (inhibitor of p38 MAPK) combined with Ad5F35-IL24 on killing U251 cells. The expression of bcl-2, caspase-3 and TopoⅡα. proteins were examined with Western blot, RT-PCR, flow cytometry, immuocytochemistry assays.
Part 1 Construction of recombinant adenovirus Ad5F35-IL24 vector
Objective: to construct recombinant adenovirus Ad5F35-IL24 vector.
Methods: When plasmids of ATCC were cut by both HindⅢand SalⅠenzymes, the goal bands were obtained. Plasmids of pDC316 were cut by both HindⅢand SalⅠenzymes. Subsequently, after some fragments were connected, transformed and filtered, reorganized plasmids of pDC316 were obtained. Thereafter,it was identified with PCR and enzymes cut analysis. Then the plasmids were carried out sequence determination in whole automatic nucleic acid analyzer. Subsequently competent cells were prepared and transformed. After large-scale preparation of plasmid DNA, recombined adenoviruses Ad5F35-IL24 were generated with homologous recombination by cotransfection of 293 cells with a couple plasmids. After identified with PCR, Ad5F35-IL24 recombinant adenovirus species was amplified and purified. Then the titre was determined according to formula: PFU/mL =GFP post cell number×deliquation of virus supernatant/0.5 mL.
Results: Reconstructed plasmids of pDC316-IL24 containing IL-24 gene was successfully constructed and identified by PCR, enzymes cut and sequence determination. Escherichia coli strains containing IL-24 gene was obtained. Reconstructed adenovirus Ad5F35-IL24 containing IL-24 gene with functional defect of proliferation was successfully constructed, amplified, purified and identified with PCR. Its titre was assayed according to formula: PFU/mL =GFP post cell number×deliquation of virus supernatant/0.5 mL, and the titer was 2.8×108 PFU/mL.
Conclusions: Reconstructed adenovirus vector Ad5F35-IL24 was successfully constructed.
Part 2 Ad5F35-IL24 induces apoptosis and its influence on cell invasion and migration of glioma U251 cells
Objective: Observe the effects of Ad5F35-IL24 on the apoptosis, invasion and migration of U251 cells. Methods: MTT, flow cytometry, double fluorochrome stain, Hoechst 33258 fluorochrome stain, Annexin V-FITC apoptosis kit were used to detect Ad5F35-IL24 inducing U251 cell apoptosis, and observed its influnces on cell invasion and migration with the transwell assay and Cell scratch assay.
Results: Ad5F35-IL24 induces growth suppression and apoptosis in human glioma cells U251 cells. The apoptosis was increased obviously along with Ad5F35-IL24 drug concertration increase. Ad5F35-IL24 could suppress U251 cell cycle; the cells were blocked at G2/M phase. Ad5F35-IL24 could effective decrese the invasion and migration of U251 cells.
Conclusion: Ad5F35-IL24 could inhibit the growth, invasion and migration of human glioma U251 cell, and induces apoptosis.
Part 3 The effects of Ad5F35-IL24 on expression of ERK and p38 MAPK cell signel transduction pathways
Objective: to investigate mechanism of Ad5F35-IL24 effect on ERK and p38 MAPK protein expression.
Methods: After administration of Ad5F35-IL24 for 24 hour. We used Western blot and flow cytometry to detect cell signal transduction protein ERK, p-ERK, p38 MAPK and p-p38 expressions. And the MTT, flow cytometry, double fluorochrome stain were also used to detect the effect of PD98059 (inhibitor of ERK) and SB203580 (inhibitor of p38 MAPK) combined with Ad5F35-IL24 on killing U251 cells.
Results: Western Blot analyses found that IL-24 selectively induced U251 cell apoptosis by up-regulating and activating p38 MAPK and p-p38 MAPK protein expression(0.46±0.07, 0.60±0.07)(P<0.05). After combined with p38 MAPK inhibitor SB203580,it was showed that the inhibition rate was decreased(20.83±1.34)contrasted with the single Ad5F35-IL24 group(36.15±2.61)(P<0.05). On the other hand, ERK and p-ERK protein expression have not significant changed in contrast with control group(P>0.05). After blocked ERK signal transduction pathway with dministration of ERK inhibitor PD98059, the results showed that it could increase apoptosis of U251 cells(P<0.05).
Conclusions: Ad5F35-IL24 selectively induced U251 cell apoptosis by up-regulating and activating p38 MAPK pathway. Blocking ERK pathway could increase Ad5F35-IL24 to induce cell apoptosis.
Part 4 Ad5F35-IL24 influence of TopoⅡα、bcl-2 and caspase-3 protein expression
Objective: To observe Ad5F35-IL24 influence of bcl-2、caspase-3 and TopoⅡαexpression
Methods: After transfected U251 cell with Ad5F35-IL24 for 24 hours, the bcl-2, caspase-3 and TopoⅡαprotein expression were detected with Western blot, RT-PCR, immuocytochemistry assays.
Results: After Ad5F35-IL24 transfection, the related proteins were detacted by Western blot. We found that caspase-3 significantly raised up (0.71±0.09)contrasted with that of control group(0.43±0.04), and bcl-2 expression was significantly cut down(0.02±0.01)compared to the control group(0.17±0.02)(P<0.05). TopoⅡαexpression were found significantly decreased ( 0.48±0.03, 0.31±0.02 ) in contrast with the control group(1.35±0.07)(P<0.05). Immuocytochemistry method also showed that TopoⅡαexpression was decreased down after Ad5F35-IL24 transfected(P<0.05).
Conclusions: IL-24 could inhibit TopoⅡαand bcl-2 expression, and increase caspase-3 expression in U251 glioma cell, which provided refrences for effectively therapy of glioma in future.
引文
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