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
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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(p<0.05)；15 Gy1d、3d照射组与对照组比较差异有显著性统计学意义(p<0.01),22.5Gy3d照射组与对照组比较差异有统计学意义(p<0.05)；15 Gy照射组1d和3d间的比较差异有显著性统计学意义(p<0.01),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(p<0.05)。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(p<0.01)；15 Gy照射组1d与对照组比较差异有统计学意义(p<0.05)。15 Gy照射组1d与3d间的比较差异有统计学意义(p<0.05)；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(p>0.05)。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(p<0.05)；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(p<0.05)；15 Gy照射组3d与对照组比较差异有显著统计学意义(p<0.01)；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(p<0.05)；7.5 G、15 Gy、22.5 Gy照射组组间比较差异无统计学意义(p<0.05)。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(p<0.05)；放疗组组内比较差异有统计学意义(p<0.05),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(p<0.05)；LJP30组与LJP300组比较差异无统计学意义(p>0.05),LJP30组、LJP300组与放疗组比较减少差异有统计学意义(p<0.05)。③TUNEL染色结果为：LJP30、LJP300组与放疗组相比差异有统计学意义(p<0.05)；LJP30组与LJP300组比较差异无统计学意义(p>0.05)。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,p<0.01),12.5mg/kg LJP组对移植瘤的平均抑瘤率仅为16.4%(与NS组比较,p>0.05),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,p<0.05)和47.0%(与NS组比较,p<0.01)。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(p<0.05); 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(p<0.05); 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(p<0.05)；Bax mRNA表达：LJP25> LJP50> LJP12.5>DDP2,组间的比较差异有统计学意义(p<0.05)；Bcl-2mRNA表达：LJP25/LJP12.5>LJP 50>DDP2,组间的比较差异有统计学意义(p<0.05)。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(p<0.05); LJP25、LJP50、DDP2组Caspase-8 mRNA表达与对照组比较差异有统计学意义(p<0.05)。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(p<0.05)；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。
Objective:Radiationtherapy is the main treatment for Nasopharyngeal carcinoma and head and neck cancer. With conventional radiation way to nasopharyngeal carcinoma, the patients must bear external irradiation to both sides of the main salivary gland in radiation therapy and head and neck cancer, especially the parotidgland is located in the irrdiation target area. Radiation-induced salivary gland injury cause the gland dysfunction and reduce saliva secretion leading to xerostomia after radiotherapy in patients with serious impact on the quality of life.Lacking of effective prevention and control measures, must comprehensive symptomatic treatment is still used to radiation-induced xerostomia. Study of mechanisms against the radiation for salivary gland radiation-induced damage may find drugs to inhibit tumor and treat diseases, which has important clinical significance. To explore mechanism and effective doses of radiation-induced damage in this study, we observed firstly rat submandibular gland damage by different radiation-induced dose at early time(1-3 day), and observed secondly protective effect to the submandibular gland radiation damage with LJP drug intervention.To explore mechanism of LJP anti-human nasopharyngeal carcinoma cell, we observed lastly inhibition to human nasopharyngeal HONE1 and CNE2 cell proliferation and established up xenografts of human nasopharyngeal carcinoma HONE1 in nude mice model by detection technologies includining terminal deoxynuclotidyl transferase-mediated dTP nick end labeling(TUNEL), electronmicroscopy pathomorphology, immunohistochemistry, cell culture cytotoxicity test(MTT assay) and reverse transcriptase polymerase chain reaction(RT-PCR), which can provide experimental basis for salivary gland radiation and anti-nasopharyngeal carcinoma with LJP.
Methods:The study is divided into three parts:Part one, the gene-related expression of apoptosis and morphology to different doses of 60Co y-ray irradiationon in rat submandibular gland. Part two,protective effect of LJP to destruction induced by 60Co y-rays in rat Submandibular gland.Parts three, inhibition to xenografts of human carcinoma Cell line in nude mice. The methods of part one,48 Wistarrats were randomly divided into:(1) normal control group(n=12); (2) radiotherapy 7.5Gy group(n=12); (3)radiotherapy 15Gy group (n=12); (4) radiation therapy 22.5Gy group (n=12). Radiotherapy group were irradiated by one-time total dose of y-ray irradiation according to group dose, while the control group were not irradiated.6 of each group in rat were sacrificed in 1 day and 3 days after irradiation All specimens were dehydrated, embeded in paraffin and serially cutted into 5μm slicesion.The expression of P53, Caspase-3 were detected by immunohistochemistry SP method and TUNEL assay cell apoptosis. All specimens taken randomly from a submandibular gland in each group were observed pathological changes of morphology by Transmission of electron microscope (TEM). The methods of part two,24 Wistar rats were randomly divided into:(1)normal control group(n=6); (2)LJP high-dose radiotherapy group(300mg/kg/one/day)(n=6); (3)the LJP low-dose radiotherapy group (30m/kg/one/day)(n=6); (4)radiotherapy control group(n=6). From 3 days before irradiation to 3 day after irradiation, each one was treated by intraperitoneal injection of LJP according to group dose.With one-time total dose of 15Gy of y-ray irradiation,each one was irradiated in radiotherapy group, while the control group were not irradiated.6 of each group in rat were sacrificed in 3 days after irradiation. All specimens were dehydrated, embeded in paraffin and serially cutted into 5μm slicesion. The expression of p53, Caspase-3 were detected by immunohistochemistry SP method and TUNEL assay cell apoptosis.15 Wistar rats were divided into three dose groups by 7.5Gy,15Gy,22.5 Gy for electron microscopy, and each group was divided into:(1)control group (n=1); (2)LJP high-dose radiotherapy group (n=1); (3) LJP low-dose radiotherapy group(n=1); (4) radiotherapy control group(n=1); (5)sucrose-negative control group (n=1), with above method of administration of radiotherapy, all rat were sacrificed in 3 days after irradiation and the morphology of rat submandibular gland were observed to understand pathological changes with drug intervention. The methods of part three, MTT assay was detected effect of LJP on inhibition to the proliferation of the human NPC cell line (HONE1 and of CNE2).30 male mice were randomly divided into groups:(1) normal control group(NS0.1ml/10g/d) (n=6); (2)LJP12.5 group (LJP12.5mg/kg/d) (n=6); (3)LJP25 group (LJP25mg/kg/d) (n=6); (4)LJP50 group (LJP50mg/kg/d)(n=6); (5) positive control group(DDP2mg/kg/d)(n=6). The drug intervention of LJP were used to detected effect on inhibition to human nasopharyngeal HONE1 and CNE2 cell proliferation, and the model of xenografts of human nasopharyngeal carcinoma HONE1 were established in nude mice, which were observed to detect the expression of Bax, Bcl-2,Caspase-3,8,9 messenger RNA (mRNA) with LJP drug intervention by RT-PCR.
Results:Part I:①Gross specimen observation, there were differences on mental state, reaction of the outside and diet in rat after irradiation, the greater the radiation dose, the worse the mental status, ability to respond to outside and appetite.②p53 expression as follows:7.5 Gy Id,3d irradiation group compared with control group showed no statistically significant(p>0.05); 15 Gy 1d,3d irradiation group compared with control group increased statistically (p<0.01),22.5Gy 3d group compared with control group was statistically significant(p<0.05); 15 Gy group difference between Id and 3d was statistical significance(p<0.01), difference between 1d and 3d in 7.5Gy,22.5Gy groups were no significant differences(p>0.05).③Caspase-3 expression as follows: 1d,3d in 7.5Gy and 22.5Gy group,3d in 15Gy group compared with the control group was significant statistically significance(p<0.01); Id in 15Gy group compared with the control group was statistically significant (p<0.05). Difference between Id and 3d in 15Gy group was statistically significant (p<0.05); difference between 1d and 3d in 7.5Gy,22.5Gy group was not statistically significant(p>0.05).④TUNEL staining as follows:3d in 7.5Gy group compared with the normal group was statistically significant(p<0.05); Id in 15 Gy and 1d,3d in 22.5 Gy groups compared with the control group was statistically significant(p<0.05); 3d in 15 Gy group compared with the control group was statistically significant(p<0.01); difference between 1d and 3d in 7.5Gy,15Gy,22.5Gy groups was not statistically significant(p>0.05). Difference in 7.5Gy,15Gy,22.5Gy groups was not statistically significant(p>0.05).⑤Electron microscopy results:in 7.5Gy group, serous acinar cells showing as follows:the nuclear membrane integrity and nucleolus disappear, nuclear chromatin condensate integrated block and the set of edges in the nuclear membrane, the inside of the rough endoplasmic reticulum is slight expansion in the cytoplasm and the mitochondria is a little swelling, cristae blurred. Ductal cells showing that membrane shrinkage, the cytoplasm of mitochondrial swelling, cristae,reduced or disappear, the complete nucleus, nucleolus clear.15Gy group,serous acinar cells and ductal cells showing with cell shrinkage, increased electron density, the nuclear membrane integrity, the nucleolus disappears, the nuclear heterochromatin increased significantly, the condensate integrated block and the set of edges in the inside nuclear membrane; rough endoplasmic reticulum was significantly reduced and the expansion of the secretory granules also decreased significantly, and the particles inside the disorganized mitochondrial structure was mbiguous. 22.5Gy group, serous acinar cells and ductal cells showing that cell shrinkage, increased electron density, incomplete nuclear membrane, nucleus different chromatin coagulation integrated block and the edge set of the inside at the nuclear membrane, perinuclear gap widened; quality within the rough endoplasmic reticulum were significant reduction and dilated, the structural disorder, secretory granules disappeared, remnants of a small amount of mitochondria, nuclear heterochromatin began to increase. PartⅡ,①p53 expression as follows:radiotherapy group compared with control group was statistically significant(p<0.05); the difference in radiotherapy group was statistically significant(p<0.05), the apoptos of high-dose LJP group was the lowest value.②Caspase-3 expression results as follows:radiotherapy group compared with control group was statistically significant(p<0.05); difference between LJP30 and LJP300 group was not statistically significant(p>0.05), LJP30 and LJP300 group compared with radiotherapy group reduced statistically significant(p<0.05).③TUNEL staining results:LJP30 and LJP300 group compared with radiotherapy group was statistically significant (p<0.05); there were no statistically significant difference between the LJP30 and LJP300 group.④Electron microscopy results:the nucleus, mitochondria, endoplasmic reticulum and secretory vesicles structure of cells in 7.5 Gy,15Gy,22.5 Gy groups showing the injuries characteristics aggravated with increasing dose. Compared with radiation and sucrose control group, damage of cell structure in high and low LJP concentrations became lighter, which mean a protective effect on cell structure with LJP.PartⅢ:①MTT results, LJP IC10 of HONE 1 cells 76.85μg/ml, IC20 115.31μg/ml, IC30153.77μg/ml, IC50 240μg/ml; LJP CNE2 cells IC10 18.92μg/ml, IC20 57.38μg/ml, IC30 98.85μg/ml, IC50 180μg/ml.②Intervention results of the xenografts in nude mice:DDP group average tumor inhibition rate was 63.5%(compared with NS group, p<0.01), the LJP 12.5 group of transplanted tumors average tumor inhibition rate was 16.4% (with NS group, p>0.05) and inhibitory effect was not obvious, and the LJP 25 and LJP 50 group of transplanted tumor inhibition rates were 33.7%(compared with the NS group, p<0.05) and 47.0%(compared with NS group, p<0.01).③RT-PCR, Bax mRNA expression of LJP12.5, LJP25, LJP50, DDP2 group compared with the control group(NS) was statistically significant(p<0.05); difference of Bcl-2 mRNA expression in LJP12.5, LJP25, LJP 50, DDP2 group was statistically significant (p<0.05); the ratio of Bax/Bcl-2 in LJP50, LJP12.5, LJP25, DDP2 group compared with the control group was statistically significant (p<0.05); expression of Bax mRNA as follows:LJP25/LJP50> LJP12.5>DDP2, which difference was statistically significant(P<0.05); expression of Bcl-2 mRNA as follows:LJP25/LJP12.5>LJP50>DDP2, the difference was statistically significant (P<0.05). Expression of Caspase-3,9 mRNA in LJP12.5, LJP25, LJP50, DDP2 group compared with control group difference was statistically significant (p<0.05); LJP25, LJP50, DDP2 group of Caspase-8 mRNA expression and the control group the difference was statistically significance (p<0.05). Difference of Caspase-3 mRNA expression were statistically significant as follows:DDP2>LJP50>LJP25>LJP12.5; Caspase-8 mRNA expression between the NS and LJP12.5 group was not different (p>0.05) and no differences in LJP25 and LJP50 as well as LJP25 and DDP2; expression of Caspase-9 mRNA in LJP12.5, LJP25, LJP50 group was no difference, and were lower than expression of DDP2 treatment group.
Conclusions:
1.60Co y-ray irradiation could lead to apoptosis of rat submandibular gland in the early stage (1-3 days).
2.60Co y-ray irradiation induced apoptosis of rat submandibular gland cells in a dose-effect relationship.
3. Damage of submandibular gland cells could be induced by 15 Gy dose irradiation in the early stage (1-3 days),which has changes in the repair and apoptosis.
4. Radiation-induced apoptosis of rat submandibular gland is inhibited by LJP.
5. LJP can protect cell injury of rat submandibular gland induced by radiation.
6. LJP inhibit human nasopharyngeal HONE1 and CNE2 cells proliferation.
7.LJP inhibit nasopharyngeal HONE1 and CNE2 cells of human in a concentration-dependent effect.
8. One of the inhibition mechanism to xenografts of human nasopharyngeal carcinoma HONE1 is to promote apoptosis of cancer cells.
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(p<0.05)；15 Gy1d、3d照射组与对照组比较差异有显著性统计学意义(p<0.01),22.5Gy3d照射组与对照组比较差异有统计学意义(p<0.05)；15 Gy照射组1d和3d间的比较差异有显著性统计学意义(p<0.01),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(p<0.05)。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(p<0.01)；15 Gy照射组1d与对照组比较差异有统计学意义(p<0.05)。15 Gy照射组1d与3d间的比较差异有统计学意义(p<0.05)；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(p>0.05)。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(p<0.05)；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(p<0.05)；15 Gy照射组3d与对照组比较差异有显著统计学意义(p<0.01)；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(p<0.05)；7.5 G、15 Gy、22.5 Gy照射组组间比较差异无统计学意义(p<0.05)。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(p<0.05)；放疗组组内比较差异有统计学意义(p<0.05),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(p<0.05)；LJP30组与LJP300组比较差异无统计学意义(p>0.05),LJP30组、LJP300组与放疗组比较减少差异有统计学意义(p<0.05)。③TUNEL染色结果为：LJP30、LJP300组与放疗组相比差异有统计学意义(p<0.05)；LJP30组与LJP300组比较差异无统计学意义(p>0.05)。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,p<0.01),12.5mg/kg LJP组对移植瘤的平均抑瘤率仅为16.4%(与NS组比较,p>0.05),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,p<0.05)和47.0%(与NS组比较,p<0.01)。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(p<0.05); 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(p<0.05); 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(p<0.05)；Bax mRNA表达：LJP25> LJP50> LJP12.5>DDP2,组间的比较差异有统计学意义(p<0.05)；Bcl-2mRNA表达：LJP25/LJP12.5>LJP 50>DDP2,组间的比较差异有统计学意义(p<0.05)。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(p<0.05); LJP25、LJP50、DDP2组Caspase-8 mRNA表达与对照组比较差异有统计学意义(p<0.05)。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(p<0.05)；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。
Objective:Radiationtherapy is the main treatment for Nasopharyngeal carcinoma and head and neck cancer. With conventional radiation way to nasopharyngeal carcinoma, the patients must bear external irradiation to both sides of the main salivary gland in radiation therapy and head and neck cancer, especially the parotidgland is located in the irrdiation target area. Radiation-induced salivary gland injury cause the gland dysfunction and reduce saliva secretion leading to xerostomia after radiotherapy in patients with serious impact on the quality of life.Lacking of effective prevention and control measures, must comprehensive symptomatic treatment is still used to radiation-induced xerostomia. Study of mechanisms against the radiation for salivary gland radiation-induced damage may find drugs to inhibit tumor and treat diseases, which has important clinical significance. To explore mechanism and effective doses of radiation-induced damage in this study, we observed firstly rat submandibular gland damage by different radiation-induced dose at early time(1-3 day), and observed secondly protective effect to the submandibular gland radiation damage with LJP drug intervention.To explore mechanism of LJP anti-human nasopharyngeal carcinoma cell, we observed lastly inhibition to human nasopharyngeal HONE1 and CNE2 cell proliferation and established up xenografts of human nasopharyngeal carcinoma HONE1 in nude mice model by detection technologies includining terminal deoxynuclotidyl transferase-mediated dTP nick end labeling(TUNEL), electronmicroscopy pathomorphology, immunohistochemistry, cell culture cytotoxicity test(MTT assay) and reverse transcriptase polymerase chain reaction(RT-PCR), which can provide experimental basis for salivary gland radiation and anti-nasopharyngeal carcinoma with LJP.
Methods:The study is divided into three parts:Part one, the gene-related expression of apoptosis and morphology to different doses of 60Co y-ray irradiationon in rat submandibular gland. Part two,protective effect of LJP to destruction induced by 60Co y-rays in rat Submandibular gland.Parts three, inhibition to xenografts of human carcinoma Cell line in nude mice. The methods of part one,48 Wistarrats were randomly divided into:(1) normal control group(n=12); (2) radiotherapy 7.5Gy group(n=12); (3)radiotherapy 15Gy group (n=12); (4) radiation therapy 22.5Gy group (n=12). Radiotherapy group were irradiated by one-time total dose of y-ray irradiation according to group dose, while the control group were not irradiated.6 of each group in rat were sacrificed in 1 day and 3 days after irradiation All specimens were dehydrated, embeded in paraffin and serially cutted into 5μm slicesion.The expression of P53, Caspase-3 were detected by immunohistochemistry SP method and TUNEL assay cell apoptosis. All specimens taken randomly from a submandibular gland in each group were observed pathological changes of morphology by Transmission of electron microscope (TEM). The methods of part two,24 Wistar rats were randomly divided into:(1)normal control group(n=6); (2)LJP high-dose radiotherapy group(300mg/kg/one/day)(n=6); (3)the LJP low-dose radiotherapy group (30m/kg/one/day)(n=6); (4)radiotherapy control group(n=6). From 3 days before irradiation to 3 day after irradiation, each one was treated by intraperitoneal injection of LJP according to group dose.With one-time total dose of 15Gy of y-ray irradiation,each one was irradiated in radiotherapy group, while the control group were not irradiated.6 of each group in rat were sacrificed in 3 days after irradiation. All specimens were dehydrated, embeded in paraffin and serially cutted into 5μm slicesion. The expression of p53, Caspase-3 were detected by immunohistochemistry SP method and TUNEL assay cell apoptosis.15 Wistar rats were divided into three dose groups by 7.5Gy,15Gy,22.5 Gy for electron microscopy, and each group was divided into:(1)control group (n=1); (2)LJP high-dose radiotherapy group (n=1); (3) LJP low-dose radiotherapy group(n=1); (4) radiotherapy control group(n=1); (5)sucrose-negative control group (n=1), with above method of administration of radiotherapy, all rat were sacrificed in 3 days after irradiation and the morphology of rat submandibular gland were observed to understand pathological changes with drug intervention. The methods of part three, MTT assay was detected effect of LJP on inhibition to the proliferation of the human NPC cell line (HONE1 and of CNE2).30 male mice were randomly divided into groups:(1) normal control group(NS0.1ml/10g/d) (n=6); (2)LJP12.5 group (LJP12.5mg/kg/d) (n=6); (3)LJP25 group (LJP25mg/kg/d) (n=6); (4)LJP50 group (LJP50mg/kg/d)(n=6); (5) positive control group(DDP2mg/kg/d)(n=6). The drug intervention of LJP were used to detected effect on inhibition to human nasopharyngeal HONE1 and CNE2 cell proliferation, and the model of xenografts of human nasopharyngeal carcinoma HONE1 were established in nude mice, which were observed to detect the expression of Bax, Bcl-2,Caspase-3,8,9 messenger RNA (mRNA) with LJP drug intervention by RT-PCR.
Results:Part I:①Gross specimen observation, there were differences on mental state, reaction of the outside and diet in rat after irradiation, the greater the radiation dose, the worse the mental status, ability to respond to outside and appetite.②p53 expression as follows:7.5 Gy Id,3d irradiation group compared with control group showed no statistically significant(p>0.05); 15 Gy 1d,3d irradiation group compared with control group increased statistically (p<0.01),22.5Gy 3d group compared with control group was statistically significant(p<0.05); 15 Gy group difference between Id and 3d was statistical significance(p<0.01), difference between 1d and 3d in 7.5Gy,22.5Gy groups were no significant differences(p>0.05).③Caspase-3 expression as follows: 1d,3d in 7.5Gy and 22.5Gy group,3d in 15Gy group compared with the control group was significant statistically significance(p<0.01); Id in 15Gy group compared with the control group was statistically significant (p<0.05). Difference between Id and 3d in 15Gy group was statistically significant (p<0.05); difference between 1d and 3d in 7.5Gy,22.5Gy group was not statistically significant(p>0.05).④TUNEL staining as follows:3d in 7.5Gy group compared with the normal group was statistically significant(p<0.05); Id in 15 Gy and 1d,3d in 22.5 Gy groups compared with the control group was statistically significant(p<0.05); 3d in 15 Gy group compared with the control group was statistically significant(p<0.01); difference between 1d and 3d in 7.5Gy,15Gy,22.5Gy groups was not statistically significant(p>0.05). Difference in 7.5Gy,15Gy,22.5Gy groups was not statistically significant(p>0.05).⑤Electron microscopy results:in 7.5Gy group, serous acinar cells showing as follows:the nuclear membrane integrity and nucleolus disappear, nuclear chromatin condensate integrated block and the set of edges in the nuclear membrane, the inside of the rough endoplasmic reticulum is slight expansion in the cytoplasm and the mitochondria is a little swelling, cristae blurred. Ductal cells showing that membrane shrinkage, the cytoplasm of mitochondrial swelling, cristae,reduced or disappear, the complete nucleus, nucleolus clear.15Gy group,serous acinar cells and ductal cells showing with cell shrinkage, increased electron density, the nuclear membrane integrity, the nucleolus disappears, the nuclear heterochromatin increased significantly, the condensate integrated block and the set of edges in the inside nuclear membrane; rough endoplasmic reticulum was significantly reduced and the expansion of the secretory granules also decreased significantly, and the particles inside the disorganized mitochondrial structure was mbiguous. 22.5Gy group, serous acinar cells and ductal cells showing that cell shrinkage, increased electron density, incomplete nuclear membrane, nucleus different chromatin coagulation integrated block and the edge set of the inside at the nuclear membrane, perinuclear gap widened; quality within the rough endoplasmic reticulum were significant reduction and dilated, the structural disorder, secretory granules disappeared, remnants of a small amount of mitochondria, nuclear heterochromatin began to increase. PartⅡ,①p53 expression as follows:radiotherapy group compared with control group was statistically significant(p<0.05); the difference in radiotherapy group was statistically significant(p<0.05), the apoptos of high-dose LJP group was the lowest value.②Caspase-3 expression results as follows:radiotherapy group compared with control group was statistically significant(p<0.05); difference between LJP30 and LJP300 group was not statistically significant(p>0.05), LJP30 and LJP300 group compared with radiotherapy group reduced statistically significant(p<0.05).③TUNEL staining results:LJP30 and LJP300 group compared with radiotherapy group was statistically significant (p<0.05); there were no statistically significant difference between the LJP30 and LJP300 group.④Electron microscopy results:the nucleus, mitochondria, endoplasmic reticulum and secretory vesicles structure of cells in 7.5 Gy,15Gy,22.5 Gy groups showing the injuries characteristics aggravated with increasing dose. Compared with radiation and sucrose control group, damage of cell structure in high and low LJP concentrations became lighter, which mean a protective effect on cell structure with LJP.PartⅢ:①MTT results, LJP IC10 of HONE 1 cells 76.85μg/ml, IC20 115.31μg/ml, IC30153.77μg/ml, IC50 240μg/ml; LJP CNE2 cells IC10 18.92μg/ml, IC20 57.38μg/ml, IC30 98.85μg/ml, IC50 180μg/ml.②Intervention results of the xenografts in nude mice:DDP group average tumor inhibition rate was 63.5%(compared with NS group, p<0.01), the LJP 12.5 group of transplanted tumors average tumor inhibition rate was 16.4% (with NS group, p>0.05) and inhibitory effect was not obvious, and the LJP 25 and LJP 50 group of transplanted tumor inhibition rates were 33.7%(compared with the NS group, p<0.05) and 47.0%(compared with NS group, p<0.01).③RT-PCR, Bax mRNA expression of LJP12.5, LJP25, LJP50, DDP2 group compared with the control group(NS) was statistically significant(p<0.05); difference of Bcl-2 mRNA expression in LJP12.5, LJP25, LJP 50, DDP2 group was statistically significant (p<0.05); the ratio of Bax/Bcl-2 in LJP50, LJP12.5, LJP25, DDP2 group compared with the control group was statistically significant (p<0.05); expression of Bax mRNA as follows:LJP25/LJP50> LJP12.5>DDP2, which difference was statistically significant(P<0.05); expression of Bcl-2 mRNA as follows:LJP25/LJP12.5>LJP50>DDP2, the difference was statistically significant (P<0.05). Expression of Caspase-3,9 mRNA in LJP12.5, LJP25, LJP50, DDP2 group compared with control group difference was statistically significant (p<0.05); LJP25, LJP50, DDP2 group of Caspase-8 mRNA expression and the control group the difference was statistically significance (p<0.05). Difference of Caspase-3 mRNA expression were statistically significant as follows:DDP2>LJP50>LJP25>LJP12.5; Caspase-8 mRNA expression between the NS and LJP12.5 group was not different (p>0.05) and no differences in LJP25 and LJP50 as well as LJP25 and DDP2; expression of Caspase-9 mRNA in LJP12.5, LJP25, LJP50 group was no difference, and were lower than expression of DDP2 treatment group.
Conclusions:
1.60Co y-ray irradiation could lead to apoptosis of rat submandibular gland in the early stage (1-3 days).
2.60Co y-ray irradiation induced apoptosis of rat submandibular gland cells in a dose-effect relationship.
3. Damage of submandibular gland cells could be induced by 15 Gy dose irradiation in the early stage (1-3 days),which has changes in the repair and apoptosis.
4. Radiation-induced apoptosis of rat submandibular gland is inhibited by LJP.
5. LJP can protect cell injury of rat submandibular gland induced by radiation.
6. LJP inhibit human nasopharyngeal HONE1 and CNE2 cells proliferation.
7.LJP inhibit nasopharyngeal HONE1 and CNE2 cells of human in a concentration-dependent effect.
8. One of the inhibition mechanism to xenografts of human nasopharyngeal carcinoma HONE1 is to promote apoptosis of cancer cells.
引文
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[13]王汉渝,冼超贵,钟睿,等.黄芩中酚性苷类对腮腺放射损伤的防护作用的研究.肿瘤防治杂志,2003,10(6)：637-639.
[14]李厚勇,王蕊,高晓奇,等.海带提取物对脂质过氧化和血液流变学的影响.中国公共卫生[J],2002,18(3)：263-264.
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