CpG ODN对非小细胞肺癌放射增敏作用的实验研究
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摘要
据世界卫生组织统计全球每年肺癌新发病例超过120万,每年因肺癌而死亡的人高达约110万,已经成为第一癌症杀手。非小细胞肺癌(NSCLC)占全部肺癌的85%以上。放疗在NSCLC综合治疗中占据了重要地位,但癌细胞自身对放射线的抗拒性往往导致放疗失败,寻找行之有效的放射增敏剂是多年来肺癌研究领域倍受关注的热点。既往研究证实,含有非甲基化CpG二核苷酸的寡聚脱氧核苷酸(CpG ODN oligodeoxynucleotide, CpG ODN)可激活机体免疫系统,从而增强多种抗癌措施的疗效；但近年的文献报道,CpG ODN可直接诱导癌细胞凋亡,抑制癌细胞生长,并对NSCLC和前列腺癌有化疗增敏作用。诱导细胞凋亡是肿瘤治疗的关键,对放疗敏感的癌细胞较不敏感细胞更易进入放射线诱导的细胞凋亡过程。而目前CPG ODN是否能增强人NSCLC细胞放射敏感性尚未见报道。
在本研究的第一部分,为了探索CpG ODN对人NSCLC放射增敏作用及其可能作用机理,我们以人肺腺癌A549细胞株为观察对象,首先用MTS法检测不同浓度CpG ODN7909 (5,10,30,60μg/ml)分别作用24 h、48 h后,对A549细胞活力的影响。结果表明,CpG ODN7909可直接抑制A549细胞活力,并呈剂量和时间的依赖性。随后将A549细胞随机分为四组：空白对照组、CpGODN7909组、照射组、CpG ODN7909+照射组。并用克隆集落形成法检测各组细胞增殖情况；流式细胞仪检测细胞凋亡情况及细胞周期分布；ELISA法检测细胞培养液中肿瘤坏死因子(TNF-a)的浓度。结果表明,CpG ODN7909(10μg/ml)联合照射组与空白对照组相比,细胞克隆存活分数降低、细胞凋亡率明显增高、细胞周期G2/M期阻滞、TNF-a因子分泌增多。随后我们用RT-PCR法检测A549细胞TLR9的mRNA的表达。结果表明,A549细胞表达TLR9,但CpG ODN7909是否通过A549细胞内TLR9信号途径起放射增敏作用,还有待于进一步的研究。因此,我们认为CpG ODN7909 (10μg/ml)能有效提高A549细胞放射敏感性,为今后临床上使用CpG ODN7909联合放疗治疗非小细胞肺癌提供了实验依据。
本课题组在之前体内试验中发现,CPG ODN1826(每只小鼠腹腔注射0.3mg)可通过增强小鼠免疫功能来提高C57BL/6小鼠Lewis肺癌移植瘤放射敏感性。为进一步探索CPG ODN1826对C57BL/6小鼠Lewis肺癌移植瘤放射增敏作用的剂量效应,本研究第二部分,我们以Lewis肺癌移植瘤模型为对象,首先将成功建立Lewis肺癌移植瘤模型随机分为6组,每组8只：对照组(腹腔注射PBS液)、X线照射组(单纯给予分次照射：250 cGY/fx,共1250 cGY/5fx)、CpG ODN组(每只小鼠腹腔注射CpG ODN 1826,注射时间为第1,2,9天,共0.3 mg)及低、中、高剂量CpG ODN联合X线照射组(每只小鼠腹腔注射CpG ODN1826联合分次照射,CpG ODN总剂量分别为0.15、0.3、0.6 mmg)(注射时间及照射方法同上)；其次随移植瘤的生长,定期观察荷瘤小鼠一般生存状况、移植瘤生长情况、小鼠免疫功能(脾指数)、TUNEL法检测移植瘤细胞凋亡情况等。研究结果表明,CpG ODN有利于改善荷瘤小鼠一般生存状况,并呈剂量依赖性；且各处理组与对照组相比,肿瘤生长速度减慢,肿瘤绝对生长延迟时间(AGD)延长,其中高剂量CPG ODN联合照射组AGD最长,为9.0天,放射增敏比(SER)为17.9; CPG ODN组和CPG ODN联合照射组与对照组、单纯照射组相比,瘤重明显减轻(p<0.05),且抑瘤率随着CPG ODN浓度增高而逐渐增大；单纯CPGODN组和中、高剂量CPG ODN联合照射组脾指数分别为(19.13±2.19)mg/g、(17.41±3.25) mg/g、(22.45±0.73) mg/g,与对照组(11.97±3.73) mg/g、单纯照射组(12.71±3.33) mg/g相比增大,有统计学差异(p<0.05)；单纯X线照射组、CpG ODN组及低、中、高剂量CPG ODN联合X线组移植瘤细胞凋亡率分别为(5.78±1.32)%、(14.12±2.89)%、(12.42±2.41)%、(20.89±4.87)%、(28.63±2.69)%,均高于对照组(2.67±2.14)%(p<0.05)。因此,CpG ODN1826可通过改善小鼠一般生存状况、抑制肿瘤生长、增强小鼠的免疫功能、促进移植瘤细胞凋亡等途径,明显提高Lewis肺癌放射敏感性,且呈剂量依赖性。
Lung cancer worldwide each year, according to World Health Organization (WH O) more than 1.2 million new cases each year die from it up to about 1.1 million people, has become the first cancer killer. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer. Comprehensive treatment of radiotherapy in NSCLC occupies an important position, but the cancer cells themselves on the radiation resistance to radiation therapy often lead to failure. To find an effective radiosensitizer of lung cancer research over the years is a hot issue. Previous studies confirmed that contain unmethylated CpG oligodeoxynucleotides (CpG ODN) can activate the immune system, thereby enhancing the efficacy of various anti-cancer measures; but recent reports showed that CpG ODN may direc tly induce apoptosis, inhibit cancer cell growth and enhance the chemotherapy sensitivity of NSCLC or prostate cancer. Induction of apoptosis is a key mechanism of cytotoxicity for most antitumor agents, including radiation, and radiation-sensitive tumors undergo radiation-induced apoptosis more readily than do radiation-resistant tumors. Whether the CPG ODN can enhance the radio-sensitivity of NSCLC cells has not been reported.
In the first part of this study, to explore the CpG ODN radiosensitization of human NSCLC and its possible mechanism, human lung adenocarcinoma A549 cell line was used. Firstly, MTS assay was used to determine the cell viability at 24 h,48 h after different concentrations of CpG ODN7909 (5,10,30,60μg/ml) treated. The results showed that CpG ODN7909 directly inhibited cell viability in a dose-and time-dependent manner. Sencondly, A549 cell randomly divided into four groups: control group, CpG ODN7909 group, irradiated group, CpG ODN7909+ irradiation group. Colony formation was tested in each group to further determine clonogenic cell survival; flow cytometry was used to detect cell apoptosis and cell cycle distribution; ELISA assay was used to measure tumor necrosis factorα(TNF-α) concentrations in cell culture medium. Decreased cell clonogenic survival, enhanced cell apoptotic index, accumulated pertentage of cells in G2/M phase and increased TNF-a secretion were showed in the combined treatments of CpG ODN7909 and irradiation as compared to either treatment alone (p<0.05). Thirdly, TLR9 mRNA was found express in A549 cells by RT-PCR. Therefore, we believe that CpG ODN7909 (10μg/ml) can effectively improve the radiosensitivity of A549 cells.
Our group found in the previous studies in vivo, CPG ODN1826 (each mouse intraperitoneally 0.30 mg) can enhance the radiosensitivity of Lewis lung cancer by activating the immune system of C57BL/6 mice. To further explore the dose effect of CPG ODN1826 combined with irradiation, in the second part of this study, firstly, we successfully established model of Lewis lung cancer in C57BL/6J mice, which were randomly divided into 6 groups (8 mice in each group):control group (intraperitoneal injection of PBS solution), X-ray irradiation group (only for fractionated radiation, 250 cGY/fx, a total of 1250 cGY/5fx), CpG ODN group (intraperitoneal injection of each mouse CpG ODN1826, a total dose of 0.3 mg) and low, medium and high doses of CpG ODN (a total dose of 0.15,0.3,0.6 mg, respectively) combined with X-ray irradiation group (as above). Followed with tumor growth, mice were regularly viewed, like the general living conditions, the tumor growth, the immune function (spleen index), the apoptosis of tumor cells and so on. The results show that, CpG will help to improve the general living conditions of tumor-bearing mice in a dose-dependent manner; And mice in the treatment group compared with the control group, tumor growth delayed, the absolute tumor growth delay (AGD) extended; In high doses of CpG ODN combined with X-ray irradiation group radiosensitization ratio (SER) was 17.9; Compared with control group or irradiation alone group, tumor weight significantly reduced in CpG ODN and CpG ODN combined with X-ray irradiation group (p<0.05), and the inhibition rate increased gradually with the increasing concentration of CpG ODN; In CpG ODN group, medium and high doses of CpG ODN combined with X-ray irradiation group spleen index were (19.13±2.19) mg/g, (17.41±3.25) mg/g, (22.45±0.73) mg/g, increased compared with that in control group (11.97±3.73) mg/g or X-ray irradiation group (12.71±3.33) mg/g (p<0.05); Tumor cell apoptosis rates were (5.78±1.32)%, (14.12±2.89)%, (12.42±2.41)%, (20.89±4.87)%, (28.63±2.69)% in X-ray irradiation group, CpG ODN group and the low, medium and high doses of CpG ODN group combined with X-ray irradiation group, higher than that in the control group (2.67±2.14)%, respecitively (p<0.05). Therefore, CpG ODN1826 can significantly enhance radiosensitivity of Lewis lung cancer in a dose-dependent manner, by improving the general living conditions of mice, delaying tumor growth, increasing immune function and promoting tumor cell apoptosis.
在本研究的第一部分,为了探索CpG ODN对人NSCLC放射增敏作用及其可能作用机理,我们以人肺腺癌A549细胞株为观察对象,首先用MTS法检测不同浓度CpG ODN7909 (5,10,30,60μg/ml)分别作用24 h、48 h后,对A549细胞活力的影响。结果表明,CpG ODN7909可直接抑制A549细胞活力,并呈剂量和时间的依赖性。随后将A549细胞随机分为四组：空白对照组、CpGODN7909组、照射组、CpG ODN7909+照射组。并用克隆集落形成法检测各组细胞增殖情况；流式细胞仪检测细胞凋亡情况及细胞周期分布；ELISA法检测细胞培养液中肿瘤坏死因子(TNF-a)的浓度。结果表明,CpG ODN7909(10μg/ml)联合照射组与空白对照组相比,细胞克隆存活分数降低、细胞凋亡率明显增高、细胞周期G2/M期阻滞、TNF-a因子分泌增多。随后我们用RT-PCR法检测A549细胞TLR9的mRNA的表达。结果表明,A549细胞表达TLR9,但CpG ODN7909是否通过A549细胞内TLR9信号途径起放射增敏作用,还有待于进一步的研究。因此,我们认为CpG ODN7909 (10μg/ml)能有效提高A549细胞放射敏感性,为今后临床上使用CpG ODN7909联合放疗治疗非小细胞肺癌提供了实验依据。
本课题组在之前体内试验中发现,CPG ODN1826(每只小鼠腹腔注射0.3mg)可通过增强小鼠免疫功能来提高C57BL/6小鼠Lewis肺癌移植瘤放射敏感性。为进一步探索CPG ODN1826对C57BL/6小鼠Lewis肺癌移植瘤放射增敏作用的剂量效应,本研究第二部分,我们以Lewis肺癌移植瘤模型为对象,首先将成功建立Lewis肺癌移植瘤模型随机分为6组,每组8只：对照组(腹腔注射PBS液)、X线照射组(单纯给予分次照射：250 cGY/fx,共1250 cGY/5fx)、CpG ODN组(每只小鼠腹腔注射CpG ODN 1826,注射时间为第1,2,9天,共0.3 mg)及低、中、高剂量CpG ODN联合X线照射组(每只小鼠腹腔注射CpG ODN1826联合分次照射,CpG ODN总剂量分别为0.15、0.3、0.6 mmg)(注射时间及照射方法同上)；其次随移植瘤的生长,定期观察荷瘤小鼠一般生存状况、移植瘤生长情况、小鼠免疫功能(脾指数)、TUNEL法检测移植瘤细胞凋亡情况等。研究结果表明,CpG ODN有利于改善荷瘤小鼠一般生存状况,并呈剂量依赖性；且各处理组与对照组相比,肿瘤生长速度减慢,肿瘤绝对生长延迟时间(AGD)延长,其中高剂量CPG ODN联合照射组AGD最长,为9.0天,放射增敏比(SER)为17.9; CPG ODN组和CPG ODN联合照射组与对照组、单纯照射组相比,瘤重明显减轻(p<0.05),且抑瘤率随着CPG ODN浓度增高而逐渐增大；单纯CPGODN组和中、高剂量CPG ODN联合照射组脾指数分别为(19.13±2.19)mg/g、(17.41±3.25) mg/g、(22.45±0.73) mg/g,与对照组(11.97±3.73) mg/g、单纯照射组(12.71±3.33) mg/g相比增大,有统计学差异(p<0.05)；单纯X线照射组、CpG ODN组及低、中、高剂量CPG ODN联合X线组移植瘤细胞凋亡率分别为(5.78±1.32)%、(14.12±2.89)%、(12.42±2.41)%、(20.89±4.87)%、(28.63±2.69)%,均高于对照组(2.67±2.14)%(p<0.05)。因此,CpG ODN1826可通过改善小鼠一般生存状况、抑制肿瘤生长、增强小鼠的免疫功能、促进移植瘤细胞凋亡等途径,明显提高Lewis肺癌放射敏感性,且呈剂量依赖性。
Lung cancer worldwide each year, according to World Health Organization (WH O) more than 1.2 million new cases each year die from it up to about 1.1 million people, has become the first cancer killer. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer. Comprehensive treatment of radiotherapy in NSCLC occupies an important position, but the cancer cells themselves on the radiation resistance to radiation therapy often lead to failure. To find an effective radiosensitizer of lung cancer research over the years is a hot issue. Previous studies confirmed that contain unmethylated CpG oligodeoxynucleotides (CpG ODN) can activate the immune system, thereby enhancing the efficacy of various anti-cancer measures; but recent reports showed that CpG ODN may direc tly induce apoptosis, inhibit cancer cell growth and enhance the chemotherapy sensitivity of NSCLC or prostate cancer. Induction of apoptosis is a key mechanism of cytotoxicity for most antitumor agents, including radiation, and radiation-sensitive tumors undergo radiation-induced apoptosis more readily than do radiation-resistant tumors. Whether the CPG ODN can enhance the radio-sensitivity of NSCLC cells has not been reported.
In the first part of this study, to explore the CpG ODN radiosensitization of human NSCLC and its possible mechanism, human lung adenocarcinoma A549 cell line was used. Firstly, MTS assay was used to determine the cell viability at 24 h,48 h after different concentrations of CpG ODN7909 (5,10,30,60μg/ml) treated. The results showed that CpG ODN7909 directly inhibited cell viability in a dose-and time-dependent manner. Sencondly, A549 cell randomly divided into four groups: control group, CpG ODN7909 group, irradiated group, CpG ODN7909+ irradiation group. Colony formation was tested in each group to further determine clonogenic cell survival; flow cytometry was used to detect cell apoptosis and cell cycle distribution; ELISA assay was used to measure tumor necrosis factorα(TNF-α) concentrations in cell culture medium. Decreased cell clonogenic survival, enhanced cell apoptotic index, accumulated pertentage of cells in G2/M phase and increased TNF-a secretion were showed in the combined treatments of CpG ODN7909 and irradiation as compared to either treatment alone (p<0.05). Thirdly, TLR9 mRNA was found express in A549 cells by RT-PCR. Therefore, we believe that CpG ODN7909 (10μg/ml) can effectively improve the radiosensitivity of A549 cells.
Our group found in the previous studies in vivo, CPG ODN1826 (each mouse intraperitoneally 0.30 mg) can enhance the radiosensitivity of Lewis lung cancer by activating the immune system of C57BL/6 mice. To further explore the dose effect of CPG ODN1826 combined with irradiation, in the second part of this study, firstly, we successfully established model of Lewis lung cancer in C57BL/6J mice, which were randomly divided into 6 groups (8 mice in each group):control group (intraperitoneal injection of PBS solution), X-ray irradiation group (only for fractionated radiation, 250 cGY/fx, a total of 1250 cGY/5fx), CpG ODN group (intraperitoneal injection of each mouse CpG ODN1826, a total dose of 0.3 mg) and low, medium and high doses of CpG ODN (a total dose of 0.15,0.3,0.6 mg, respectively) combined with X-ray irradiation group (as above). Followed with tumor growth, mice were regularly viewed, like the general living conditions, the tumor growth, the immune function (spleen index), the apoptosis of tumor cells and so on. The results show that, CpG will help to improve the general living conditions of tumor-bearing mice in a dose-dependent manner; And mice in the treatment group compared with the control group, tumor growth delayed, the absolute tumor growth delay (AGD) extended; In high doses of CpG ODN combined with X-ray irradiation group radiosensitization ratio (SER) was 17.9; Compared with control group or irradiation alone group, tumor weight significantly reduced in CpG ODN and CpG ODN combined with X-ray irradiation group (p<0.05), and the inhibition rate increased gradually with the increasing concentration of CpG ODN; In CpG ODN group, medium and high doses of CpG ODN combined with X-ray irradiation group spleen index were (19.13±2.19) mg/g, (17.41±3.25) mg/g, (22.45±0.73) mg/g, increased compared with that in control group (11.97±3.73) mg/g or X-ray irradiation group (12.71±3.33) mg/g (p<0.05); Tumor cell apoptosis rates were (5.78±1.32)%, (14.12±2.89)%, (12.42±2.41)%, (20.89±4.87)%, (28.63±2.69)% in X-ray irradiation group, CpG ODN group and the low, medium and high doses of CpG ODN group combined with X-ray irradiation group, higher than that in the control group (2.67±2.14)%, respecitively (p<0.05). Therefore, CpG ODN1826 can significantly enhance radiosensitivity of Lewis lung cancer in a dose-dependent manner, by improving the general living conditions of mice, delaying tumor growth, increasing immune function and promoting tumor cell apoptosis.
引文
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