EPO对下颌下腺细胞放射性损伤后细胞增殖和凋亡的影响

发布时间：2018-03-11 00:35

本文选题：EPO　切入点：下颌下腺　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:体外培养大鼠下颌下腺原代细胞,探索EPO对体外培养的大鼠下颌下腺细胞放射性损伤后细胞增殖和凋亡的影响,为临床预防和治疗涎腺放射性损伤提供实验基础。方法:无菌环境下取7d龄Wistar大鼠下颌下腺,利用组织块培养法提取大鼠下颌下腺原代细胞,并运用酶消化法和细胞差速贴壁法对所培养的细胞进行筛选提纯,采用免疫细胞化学染色(SP法)检测细胞角蛋白(CK-8)抗体、α淀粉酶抗体的表达,及PAS染色检验细胞糖原分泌功能,以此对所提取细胞进行鉴定。取第二代下颌下腺细胞,通过HE染色、Hoechst 33342染色及扫描电镜观察细胞形态学特征。对体外培养的正常下颌下腺细胞加入不同浓度EPO(5、50、500、1000IU/ml)后,CCK-8法检测细胞增殖率变化,倒置相差显微镜观察细胞形态学变化,观察EPO对正常下颌下腺细胞生长的影响。然后,分别给予第二代下颌下腺细胞0、2.5、5.0、7.5、10Gy剂量电离辐射,检测细胞存活率,筛选出引起下颌下腺细胞可复性放射性损伤的适宜放射剂量,并用于后续实验。实验共分为五组,分别为空白对照组,单放组(只放射,不加EPO),放射前加EPO组,放射后加EPO组,放射前后均加EPO组,24h后分别运用CCK-8检测细胞增殖,流式细胞仪Annexin-V/FITC荧光染色检测细胞凋亡,观察EPO对下颌下腺细胞放射性损伤的预防和恢复效果。结果:体外培养大鼠下颌下腺细胞生物学特性:原代培养第4天,倒置相差显微镜下观察可见大部分组织块周围有细胞爬出,从中间至周围,细胞密度由密变疏,形状以多边形和短梭形居多,并有长短不一的伪足伸出相互交联;原代培养第7-8天,大部分组织块之间细胞基本长满,密度较大,并开始出现复层生长现象。此时,加入胰蛋白酶消化分散细胞当做第一次传代,细胞进行纯化后继续培养48h后观察,见大部分细胞已贴壁,为多角形和短梭形,约6-7天长满,呈多边形排列成铺路石状。PAS染色可见,胞质为紫红色。免疫组化染色可见胞质为棕黄色,胞核为蓝紫色,即兔抗鼠Cytokeratin-8抗体和α-Amylase抗体阳性表达。HE染色可见细胞呈多边形或多角形,蓝染的细胞核较大,椭圆形,胞质呈粉红色;荧光显微镜下观察Hoechst 33342染色后的细胞,可见椭圆形蓝色亮染细胞核较大,淡染细胞质平铺开来,并相互连接;扫描电镜下观察可见细胞平铺在爬片上,位于中间的胞核颜色较深,细胞向周围伸出许多长短不一的伪足,有些细胞还可见分泌颗粒。EPO对正常下颌下腺细胞增殖的影响:EPO作用24h、48h、72h后细胞增殖率各组之间未见明显差异,细胞形态未见明显变化。适宜放射剂量筛选:放射线照射24h后观察,细胞存活率随着放射剂量的增加而呈现下降趋势,且当放射剂量为7.5Gy时,细胞存活率下降39%,此时P0.05,差异具有统计学意义。EPO对细胞增殖和凋亡的影响:一次性给予实验组7.5Gy剂量照射,24h后观察各实验组之间细胞增殖及凋亡率未见明显差异。结论:组织块培养法能成功获得大鼠下颌下腺原代细胞,并能成功进行传代培养。EPO对大鼠下颌下腺细胞放射性损伤后细胞增殖和凋亡无明显影响,其作用仍需进一步研究。
[Abstract]:Objective: the rat submandibular gland cells cultured in vitro, explore EPO on cultured rat submandibular gland cells after radiation induced cell proliferation and apoptosis, to provide experimental basis for clinical prevention and treatment of radiation injury of salivary gland. Methods: the sterile environment of 7D old Wistar rat submandibular gland by tissue cultured cells from rat submandibular gland and primary cells by enzymatic digestion and differential adhesion method for screening purification of the cultured cells, using immunocytochemical staining (SP method) to detect cytokeratin (CK-8) antibody, expression of alpha amylase antibodies, and PAS staining test cell glycogen secretion were identified based on the extracted cells. The second generation of submandibular gland cells and cell morphology were observed by HE staining, Hoechst 33342 staining and scanning electron microscopy. The submandibular gland cells cultured in vitro under normal entry Different concentrations of EPO (5,505001000IU/ml), CCK-8 method was used to detect cell proliferation rate, cell morphological changes were observed under inverted microscope, to observe the effect of EPO on the growth of normal submandibular gland cells. Then, were given to the second generation of submandibular gland cells 0,2.5,5.0,7.5,10Gy dose of ionizing radiation, cell viability was detected by screening of submandibular gland cells after appropriate doses of radiation injury of radioactive, and used for subsequent experiments. The experiments were divided into five groups, namely control group, radiotherapy group (radiotherapy only, no EPO), plus EPO group before radiotherapy, after radiotherapy plus EPO group, both before and after radiotherapy plus EPO group, 24h cell proliferation was detected by CCK-8 using flow cytometry, Annexin-V/FITC staining for detection of apoptosis, the effect of EPO on prevention of radiation injury of submandibular gland cells and the recovery effect. Results: the biology of the submandibular gland cells of SD rat in vitro Characteristics: primary culture for fourth days, observed under inverted phase contrast microscope visible around most tissues cells crawl, from the middle to around the cell density by dense thinning, in the shape of polygonal and spindle shaped and have different lengths are pseudopodium interconnected; the original Daipei raised 7-8 day, most organizations between the blocks the cells covered, high density, and the beginning of double layer growth phenomenon. At this time, adding trypsin dispersed cells as the first passage, cells were observed after purification to 48h after culture, most of the cells were adherent, polygonal and spindle shaped, about 6-7 days full, polygonal arranged paving stone.PAS staining, the cytoplasm was purple. Immunohistochemical staining of the cytoplasm was brown, the nucleus is blue purple, namely the Rabbit anti mouse Cytokeratin-8 antibody and alpha -Amylase antibody positive expression of.HE staining cells were visible polygon Blue or polygonal, oval, large nuclei, cytoplasm was pink; fluorescence microscope after Hoechst 33342 stain cells, visible oval bright blue staining large, pale staining cytoplasm spread, and connected with each other; were observed in tile slip under scanning electron microscope, the nucleus is located in the middle of the dark and many different lengths of cells extended to surrounding pseudopodia, some cells also secrete visible effects of particle.EPO on the proliferation of normal submandibular gland cells: EPO 24h, 48h 72h, the proliferation rate of cells without obvious differences between the groups, no obvious changes in cell morphology. The suitable radiation dose screening: To observe radiation after 24h cells the survival rate with the increase of radiation doses showed a declining trend, and when the radiation dose was 7.5Gy, the cell survival rate decreased by 39%, at P0.05, the difference was statistically significant in.EPO cells Effect of proliferation and apoptosis in experimental group: given a one-time dose of 7.5Gy irradiation, 24h were observed between the experimental group and the apoptosis rate of cell proliferation showed no significant difference. Conclusion: the tissue culture method can successfully get the rat submandibular gland cells were cultured successfully, and.EPO had no significant effect on the injury of rat submandibular gland cells after radiation of cell proliferation and apoptosis, the effect still need further study.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.7

【相似文献】