海带多糖抗大鼠颌下腺辐射损伤以及鼻咽癌裸鼠移植瘤的实验研究

发布时间：2018-05-31 15:13

  本文选题：电离辐射 + 颌下腺　； 参考：《广西医科大学》2012年博士论文

【摘要】：目的：放射治疗是鼻咽癌等头颈部恶性肿瘤的主要治疗手段。鼻咽癌等头颈部恶性肿瘤患者放射治疗的常规照射野以两侧对穿外照射为主,涎腺特别是腮腺位于放疗靶区内,辐射造成涎腺组织损伤,引起腺体功能障碍,唾液分泌减少,放疗后患者发生口腔干燥症,严重影响生活质量。目前对辐射导致的口干症仍以综合对症治疗,缺乏有效防治的措施。研究涎腺辐射诱导的损伤机制,寻找既能对抗辐射又能抑制肿瘤的药物,对疾病的治疗有重要的临床意义。本研究通过脱氧核苷酸末端转移酶介导的dTP缺口末端标记(TdT-mediated dUTP nick end labeling, TUNEL)、电镜病理形态学、免疫组织化学染色、细胞培养、细胞毒性试验(MTT法)、裸鼠移植瘤模型、逆转录聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)等检测技术,旨在了解不同辐射剂量对大鼠颌下腺辐射诱导损伤的早期(1-3天)情况,在确定辐射诱导涎腺损伤的干预辐射剂量及探讨损伤机制后,予以LJP药物干预以及观察其对颌下腺辐射损伤是否具有防护作用,再观察LJP对人鼻咽癌HONE1和CNE2细胞增殖以及人鼻咽癌HONE1裸鼠移植瘤是否有影响,探讨LJP抗人鼻咽癌细胞的机制,为LJP的涎腺防辐射和抗鼻咽癌提供实验依据。 方法：本研究分三部分：第一部分,不同剂量60Co Y射线照射对大鼠颌下腺凋亡相关因子表达及形态学的影响；第二部分,海带多糖对颌下腺60Co Y射线诱导损伤的防护作用；第三部分,海带多糖对人鼻咽癌细胞株裸鼠移植瘤的抑制作用。第一部分实验方法：将48只Wistar大鼠随机分组原则分为：(1)正常对照组(n=12)；(2)放疗7.5Gy组(n=12)；(3)放疗15Gy组(n=12)；(4)放疗22.5Gy组(n=12)。放疗组予以每只一次性总剂量15Gy的γ-ray照射,而对照组未予照射。于放疗后1 d、3 d,取各组6只大鼠颌下腺组织固定后冷冻切片,免疫组织化学SP法检测P53,Caspase-3表达情况以及TUNEL法检测细胞凋亡。每组随机取1只颌下腺扫描电镜(Transmission electron microscope,SEM)检查,观察颌下腺的形态学病理变化。第二部分实验方法：24只Wistar大鼠随机分组原则分为：(1)正常对照组(n=6)；(2)LJP高剂量放疗组(300mg/kg/只/天的LJP)(n=6)；(3)LJP低剂量放疗组(30m/kg/只/天的LJP)(n=6)。(4)放疗对照组(n=6)。于放疗前3 d、后3 d之间予以上述药物腹腔注射；放疗组以每只一次性总剂量15Gy的γ-ray照射,而对照组未予照射。各组6只在放疗后第3天上午活体各取大鼠双颌下腺组织,固定后冷冻切片,免疫组织化学SP法检测P53, Caspase-3表达情况以及TUNEL法检测细胞凋亡。15只Wistar大鼠作电镜观察,分成7.5Gy、15Gy、22.5 Gy三个剂量组①正常对照组(n=1)；②LJP高剂量放疗组(n=1)；③LJP低剂量放疗组(n=1)；④放疗对照组(n=1)；⑤蔗糖阴性对照组(n=1),同上述方法给药,在放疗后第3天上午活体取大鼠颌下腺组织电镜检查,观察颌下腺的形态学病理变化。第三部分实验方法：应用MTT法检测LJP对人NPC细胞株(HONE1和CNE2)增殖的抑制作用。30只雄性裸鼠随机分为组：①NS(正常对照组：NSO. lml/lOg/d) (n=6);②LJP 12.5 (LJP12.5mg/kg/d) (n=6);③LJP 25(LJP25mg/kg/d) (n=6);④LJP50 (LJP50mg/kg/d) (n=6);⑤DDP(阳性对照组：DDP 2 mg/kg/d) (n=6)。以人NPC细胞株HONE1建立裸鼠皮下移植瘤模型,以分组的药物进行体内抑瘤实验,计算抑瘤率,RT-PCR检测移植瘤凋亡相关基因Bax、Bcl-2、Caspase-3,8,9信使核糖核酸(messenger RNA, mRNA)的表达。 结果：第一部分：①p53的表达为：7.5 Gvld、3d照射组与对照组差异无统计学意义(p0.05)；15 Gy1d、3d照射组与对照组比较差异有显著性统计学意义(p0.01),22.5Gy3d照射组与对照组比较差异有统计学意义(p0.05)；15 Gy照射组1d和3d间的比较差异有显著性统计学意义(p0.01),7.5 Gy、22.5 Gy照射组1 d与3d间的比较差异无统计学意义(p0.05)。②Caspase-3表达为：7.5 Gy、22.5 Gy照射组1 d和3d、15 Gy照射组3d与对照组比较差异有显著性统计学意义(p0.01)；15 Gy照射组1d与对照组比较差异有统计学意义(p0.05)。15 Gy照射组1d与3d间的比较差异有统计学意义(p0.05)；7.5 Gy、22.5 Gy照射组1 d与3d间的比较差异无统计学意义(p0.05)。③tunel染色表达为：7.5 Gy照射组3d和正常组比较差异有统计学意义(p0.05)；15 Gy照射组1d、22.5 Gy照射组1d、3d与对照组比较差异有统计学意义(p0.05)；15 Gy照射组3d与对照组比较差异有显著统计学意义(p0.01)；7.5 G、15 Gy、22.5 Gy照射组1 d与3d比较差异无统计学意义(p0.05)；7.5 G、15 Gy、22.5 Gy照射组组间比较差异无统计学意义(p0.05)。④电镜观察结果：7.5Gy放疗组浆液性腺泡细胞及导管细胞,核膜完整,核仁消失,核内异染色质凝集成块状并边集于核膜内侧,胞质内粗面内质网轻度扩张,线粒体稍肿胀,嵴模糊不清。导管细胞,可见细胞膜皱缩,胞质内线粒体肿胀,嵴断裂、减少或消失,胞核完整,核仁清晰。15Gy放疗组浆液性腺泡细胞及导管细胞,细胞皱缩,电子密度增高,核膜完整,核仁消失,核内异染色质明显增多、凝集成块状并边集于核膜内侧；粗面内质网明显减少并扩张,分泌颗粒亦减少明显,且颗粒内排列紊乱,线粒体结构模糊不清。22.5Gy放疗组浆液性腺泡细胞及导管细胞,细胞皱缩,电子密度增高,核膜不完整,核内异染色质凝集成块状并边集于核膜内侧,核周隙增宽；胞质内粗面内质网大量减少,并扩张明显,结构紊乱,分泌颗粒消失,残余少量线粒体,核内异染色质开始增多。第二部分：①p53的表达为：放疗组与对照组比较差异有统计学意义(p0.05)；放疗组组内比较差异有统计学意义(p0.05),高剂量LJP组的细胞凋亡值最低。②Caspase-3表达结果为：放疗组与对照组比较差异有统计学意义(p0.05)；LJP30组与LJP300组比较差异无统计学意义(p0.05),LJP30组、LJP300组与放疗组比较减少差异有统计学意义(p0.05)。③TUNEL染色结果为：LJP30、LJP300组与放疗组相比差异有统计学意义(p0.05)；LJP30组与LJP300组比较差异无统计学意义(p0.05)。④电镜观察结果：7.5 Gy、15 Gy、22.5 Gy三组的细胞核、线粒体、内质网以及分泌泡等结构随着剂量的增加,呈现损伤加重的特征,LJP30. LJP300组细胞与放疗对照、蔗糖对照组比较,细胞结构损伤较轻,对细胞有保护作用。第三部分：①MTT结果：LJP对HONE1细胞的IC10为76.85μg/ml, IC20为115.31μg/ml, IC30为153.77μg/ml; LJP对CNE2细胞的IC10为18.92μg/ml, IC20为57.38μg/ml, IC30为98.85μg/ml。LJP对HONE1细胞、CNE2细胞工C5。浓度分别为240μg/ml、180μg/ml。②裸鼠移植瘤的干预结果：DDP组的平均抑瘤率为63.5%(与NS组比较,p0.01),12.5mg/kg LJP组对移植瘤的平均抑瘤率仅为16.4%(与NS组比较,p0.05),抑制效果不明显,而25mg/kg和50mg/kg LJP组对移植瘤的平均抑瘤率分别为33.7%(与NS组比较,p0.05)和47.0%(与NS组比较,p0.01)。③RT-PCR:LJP12.5、LJP25、LJP50、DDP2组的Bax mRNA表达与对照组NS比较差异有统计学意义(p0.05); LJP12.5、LJP25、LJP50、DDP2组的Bcl-2 mRNA表达与对照组比较差异有统计学意义(p0.05); LJP 12.5、LJP 25、LJP 50、DDP2组的Bax/Bcl-2比值与对照组比较差异有统计学意义(p0.05)；Bax mRNA表达：LJP25 LJP50 LJP12.5DDP2,组间的比较差异有统计学意义(p0.05)；Bcl-2mRNA表达：LJP25/LJP12.5LJP 50DDP2,组间的比较差异有统计学意义(p0.05)。LJP 12.5、LJP 25、LJP 50、DDP2组Caspase-3、Caspase-9 mRNA表达与对照组比较差异有统计学意义(p0.05); LJP25、LJP50、DDP2组Caspase-8 mRNA表达与对照组比较差异有统计学意义(p0.05)。Caspase-3 mRNA表达：DDP2 LJP 50 LJP 25 LJP 12.5,各组之间表达比较差异有统计学意义(p0.05)；Caspase-8 mRNA表达：NS组和LJP12.5组无差异,LJP25、LJP 50表达无差异,LJP 25、DDP2表达无差异；Caspase-9mRNA表达：LJP 12.5、LJP 25、LJP 50表达均无差异,且均低于DDP2处理组的表达。 结论： 1.60Coγ射线照射可引起大鼠颌下腺早期细胞凋亡。 2.60Coγ射线(7.5Gy、15 Gy、22.5 Gy)照射诱导的颌下腺细胞凋亡有剂量-效应关系。 3.15 Gy照射剂量诱导的颌下腺细胞损伤在早期(1-3天)处于修复和凋亡的变动阶段。 4.海带多糖对大鼠颌下腺辐射诱导的细胞凋亡具有抑制作用。 5.海带多糖对辐射诱导的大鼠颌下腺细胞损伤具有保护作用。 6.海带多糖对人鼻咽癌HONE1和CNE2细胞增殖有抑制作用。 7.海带多糖对人鼻咽癌HONE1和CNE2细胞抑制具有浓度依赖效应。 8.海带多糖对人鼻咽癌HONE1移植瘤的抑瘤机制是促进癌细胞的凋亡。
[Abstract]:Objective: radiation therapy is the main treatment for nasopharyngeal carcinoma and other head and neck malignant tumors. The conventional radiation field of radiotherapy in patients with nasopharyngeal carcinoma, such as head and neck cancer, is mainly on both sides. The salivary gland, especially the parotid gland is located in the target area of the radiotherapy. The radiation causes the injury of the salivary gland tissue, the gland dysfunction and the decrease of saliva secretion. Oral xerostomia has a serious impact on the quality of life in patients after radiotherapy. At present, the radiation induced dry mouth disease is still treated with comprehensive symptomatic treatment and lack of effective prevention and treatment. It is important to study the mechanism of radiation induced injury of salivary glands and find drugs that can both antagonize radiation and inhibit tumor. This study is of important clinical significance. Deoxynucleotidyl terminal transferase mediated dTP nick end labeling (TdT-mediated dUTP nick end labeling, TUNEL), electron microscope Pathomorphology, immunohistochemical staining, cell culture, cytotoxicity test (MTT), nude mouse transplanted tumor model, reverse transcription polymerase chain reaction (reverse transcription polymerase chain) R) detection techniques are designed to understand the early (1-3 days) conditions of radiation induced damage to the submandibular gland of rats by different radiation doses. After determining the radiation dose of radiation induced salivary gland injury and exploring the mechanism of injury, the intervention of LJP drugs and the observation of its protective effect on the radiation injury of the submandibular gland and the observation of LJP for human nasopharyngeal carcinoma are observed. HONE1 and CNE2 cell proliferation and the effect of human nasopharyngeal carcinoma (nasopharyngeal carcinoma) nude mice transplanted tumor were affected, and the mechanism of LJP against human nasopharyngeal carcinoma cells was discussed, which provided experimental basis for the anti radiation of LJP salivary gland and anti nasopharyngeal carcinoma.
Methods: This study was divided into three parts: the first part, the effect of different doses of 60Co Y ray on the expression and morphology of the apoptosis related factors of submandibular gland in rats; the second part, the protective effect of Laminaria Polysaccharide on 60Co Y ray induced injury of submandibular gland; the third part, the inhibition of Laminaria Polysaccharide on human nasopharyngeal carcinoma cell lines in nude mice The first part of the experimental method: 48 Wistar rats were divided into two groups: (1) the normal control group (n=12); (2) radiotherapy 7.5Gy group (n=12); (3) radiotherapy 15Gy group (n=12); (4) radiotherapy 22.5Gy group (n=12). The radiotherapy group was irradiated with each one-time dose of 15Gy gamma -ray, while the control group was not irradiated. 1 D, 3 D, 6 rats in each group after radiotherapy. After the submandibular gland was fixed, the rats were frozen and frozen. The immunohistochemical SP method was used to detect the expression of P53 and Caspase-3 and the apoptosis of the cells by TUNEL method. 1 submandibular gland scanning electron microscopy (Transmission electron microscope, SEM) were taken in each group to observe the morphological and pathological changes of the submandibular gland. The second part of the experimental methods: 24 Wistar rats The random grouping principles were divided into: (1) the normal control group (n=6); (2) LJP high dose radiotherapy group (300mg/kg/ only / LJP) (n=6); (3) LJP low dose radiotherapy group (30m/kg/ only / day LJP) (n=6). (4) the radiotherapy control group (n=6). The 3 D before radiotherapy and the latter 3 D were given the above drugs. The control group was not irradiated. 6 rats in each group were taken from the submandibular gland in each group at third days after the radiotherapy. After the fixation, the frozen section was fixed, the expression of P53, Caspase-3 expression and the TUNEL method were used to detect the apoptosis of.15 only Wistar rats by electron microscopy, and divided into 7.5Gy, 15Gy, and 22.5 Gy three doses group (1 normal control). Group (n=1); (2) LJP high dose radiotherapy group (n=1); LJP low dose radiotherapy group (n=1); (n=1); (n=1); (5) sucrose negative control group (n=1), with the above method, in the morning after radiotherapy, the submandibular gland tissue of the rat was examined by electron microscopy, and the morphological and pathological changes of the submandibular gland were observed. The experimental method of the application of MTT: the application of MTT The inhibitory effects of LJP on the proliferation of human NPC cell lines (HONE1 and CNE2).30 were randomly divided into groups: (1) NS (normal control group: NSO. lml/lOg/d) (n=6); (2) LJP 12.5 (LJP12.5mg/kg/d) (n=6); (25); (positive control group: 2). The cell line HONE1 established a nude mouse model of subcutaneous transplantation tumor. The tumor suppressor rate was calculated by grouping drugs in vivo and the tumor suppressor rate was calculated. RT-PCR was used to detect the expression of Bax, Bcl-2, Caspase-3,8,9 messenger ribonucleic acid (mRNA), Bcl-2, Caspase-3,8,9.
Results: the first part: (1) the expression of p53 was as follows: 7.5 Gvld, there was no significant difference between the 3D irradiation group and the control group (P0.05), and there was a significant statistical difference between the 15 Gy1d and the control group (P0.01), and the difference between the 22.5Gy3d irradiation group and the control group was statistically significant (P0.05); the difference between 1D and between 3D and 15 Gy irradiated groups was different. There was significant statistical significance (P0.01). There was no significant difference between 1 D and 3D in 7.5 Gy and 22.5 Gy irradiated groups (P0.05). (2) Caspase-3 expression was 7.5 Gy, 1 D and 3D in 22.5 Gy irradiation group, and 15 Gy group was significantly different from the control group. There was a statistically significant difference between the 15 irradiated group and the control group. The difference between 1D and 3D in P0.05.15 Gy irradiation group was statistically significant (P0.05); 7.5 Gy, 22.5 Gy irradiation group had no significant difference between 1 D and 3D (P0.05). (3) TUNEL staining expression was statistically significant (7.5); 15 The difference was statistically significant (P0.05); the difference between 3D and control group in 15 Gy irradiation group was statistically significant (P0.01); 7.5 G, 15 Gy, 1 D in 22.5 Gy irradiation group had no statistical significance (P0.05), 7.5 G, 15 Gy, and 22.5 differences between groups. Group serous acinar cells and ductal cells, complete nuclear membrane and disappearance of nucleolus. Heterochromatin integrates lumps and sides in the inner part of the nuclear membrane. In the cytoplasm, the rough endoplasmic reticulum dilated slightly, mitochondria swollen slightly, and the crista blurred. Cell membrane crinkle, mitochondria swollen in cytoplasm, crista breaks, crest breaks, nuclei complete, and nuclei are intact. .15Gy radiotherapy group had serous acinar cells and ductal cells, cell shrinkage, increased electron density, complete nuclear membrane, nucleolus disappearing, heterochromatin in nucleus increased obviously, agglutinating lump and edge of the inner membrane; rough endoplasmic reticulum decreased and expanded obviously, and the granules were arranged in disorder, mitochondrial structure model It is not clear that the serous acinus cells and ductal cells in the.22.5Gy radiotherapy group are crinkled, the electron density is increased, the nuclear membrane is incomplete, the heterochromatin is integrated into the inner nucleus of the nuclear membrane and the nuclear perinuclear gap widened, and the rough endoplasmic reticulum in the cytoplasm is greatly reduced, and the structure is disorganized, the secretory granules disappear, the remnants of the remnants are a small amount of mitochondria, and the nucleus is in the nucleus. The heterochromatin began to increase. The second part: (1) the expression of p53 was: the difference between the radiotherapy group and the control group was statistically significant (P0.05); the difference of the group in the radiotherapy group was statistically significant (P0.05), and the number of apoptosis in the high dose LJP group was the lowest. (P0) the expression of Caspase-3 was statistically significant (P0 .05); there was no significant difference between group LJP30 and group LJP300 (P0.05). Group LJP30, group LJP300 and radiotherapy group had statistical significance (P0.05). (P0.05). (P0.05). (P0.05). (3) TUNEL staining results were statistically significant (P0.05) compared with radiotherapy group (P0.05), and there was no significant difference between the LJP30 group and the group. (4) electron microscopy The results of observation: 7.5 Gy, 15 Gy, 22.5 Gy three groups of nuclei, mitochondria, endoplasmic reticulum and secretory vesicles, with the increase of dosage, showed the characteristics of damage aggravation, LJP30. LJP300 group cells were compared with radiotherapy, and the sucrose control group was compared with light cell structure damage and protective effect on cells. Third parts: (1) MTT results: LJP to HONE1 The IC10 of the cells was 76.85 mu g/ml, IC20 was 115.31 mu g/ml, IC30 was 153.77 mu g/ml, IC10 of CNE2 cells was 18.92 mu g/ml, IC20 57.38 micron g/ml. Compared with P0.01), the average inhibitory rate of the 12.5mg/kg LJP group was only 16.4% (compared with the NS group, P0.05), and the inhibitory effect was not obvious, while the average inhibition rate of the 25mg/kg and 50mg/kg LJP groups was 33.7% (compared with the NS group, P0.05) and 47% (P0.01). There were statistical significance (P0.05) in the comparison of group NS, and the expression of Bcl-2 mRNA in group LJP12.5, LJP25, LJP50 and DDP2 was statistically significant (P0.05), LJP 12.5, LJP 25, LJP 50, compared with the control group, there were statistically significant differences. The difference was statistically significant (P0.05), and the expression of Bcl-2mRNA: LJP25/LJP12.5LJP 50DDP2, the difference between the groups was statistically significant (P0.05).LJP 12.5, LJP 25, LJP 50, DDP2 group Caspase-3, Caspase-9 mRNA expression was statistically significant compared with the control group. P0.05.Caspase-3 mRNA expression: DDP2 LJP 50 LJP 25 LJP 12.5, and there was a significant difference in expression between each group (P0.05), Caspase-8 mRNA expression: NS group and LJP12.5 group, there was no difference between 50 and 25. No difference was found, and the expression was lower than that of the DDP2 treatment group.
Conclusion:
1.60Co gamma irradiation can induce early apoptosis of rat submandibular gland.
2.60Co - ray (7.5Gy, 15 Gy, 22.5 Gy) irradiation induced apoptosis of submandibular gland cells in a dose-response relationship.
3.15 the injury of submandibular gland cells induced by Gy irradiation was in the early stage (1-3 days) at the stage of repair and apoptosis.
4. Laminaria japonica polysaccharide inhibited the apoptosis induced by radiation-induced apoptosis in the submandibular gland of rats.
5. Laminaria japonica polysaccharide has protective effect on radiation induced injury of rat submandibular gland cells.
6. Laminaria japonica polysaccharide inhibited the proliferation of HONE1 and CNE2 cells in NPC.
7. Laminaria japonica polysaccharide has a concentration dependent effect on HONE1 and CNE2 cell inhibition in NPC.
8. the inhibitory mechanism of Laminaria japonica polysaccharide on human nasopharyngeal carcinoma HONE1 xenografts is to promote the apoptosis of cancer cells.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63
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