本文選題：EPO　切入點(diǎn)：下頜下腺　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:體外培養(yǎng)大鼠下頜下腺原代細(xì)胞,探索EPO對(duì)體外培養(yǎng)的大鼠下頜下腺細(xì)胞放射性損傷后細(xì)胞增殖和凋亡的影響,為臨床預(yù)防和治療涎腺放射性損傷提供實(shí)驗(yàn)基礎(chǔ)。方法:無菌環(huán)境下取7d齡Wistar大鼠下頜下腺,利用組織塊培養(yǎng)法提取大鼠下頜下腺原代細(xì)胞,并運(yùn)用酶消化法和細(xì)胞差速貼壁法對(duì)所培養(yǎng)的細(xì)胞進(jìn)行篩選提純,采用免疫細(xì)胞化學(xué)染色(SP法)檢測(cè)細(xì)胞角蛋白(CK-8)抗體、α淀粉酶抗體的表達(dá),及PAS染色檢驗(yàn)細(xì)胞糖原分泌功能,以此對(duì)所提取細(xì)胞進(jìn)行鑒定。取第二代下頜下腺細(xì)胞,通過HE染色、Hoechst 33342染色及掃描電鏡觀察細(xì)胞形態(tài)學(xué)特征。對(duì)體外培養(yǎng)的正常下頜下腺細(xì)胞加入不同濃度EPO(5、50、500、1000IU/ml)后,CCK-8法檢測(cè)細(xì)胞增殖率變化,倒置相差顯微鏡觀察細(xì)胞形態(tài)學(xué)變化,觀察EPO對(duì)正常下頜下腺細(xì)胞生長(zhǎng)的影響。然后,分別給予第二代下頜下腺細(xì)胞0、2.5、5.0、7.5、10Gy劑量電離輻射,檢測(cè)細(xì)胞存活率,篩選出引起下頜下腺細(xì)胞可復(fù)性放射性損傷的適宜放射劑量,并用于后續(xù)實(shí)驗(yàn)。實(shí)驗(yàn)共分為五組,分別為空白對(duì)照組,單放組(只放射,不加EPO),放射前加EPO組,放射后加EPO組,放射前后均加EPO組,24h后分別運(yùn)用CCK-8檢測(cè)細(xì)胞增殖,流式細(xì)胞儀Annexin-V/FITC熒光染色檢測(cè)細(xì)胞凋亡,觀察EPO對(duì)下頜下腺細(xì)胞放射性損傷的預(yù)防和恢復(fù)效果。結(jié)果:體外培養(yǎng)大鼠下頜下腺細(xì)胞生物學(xué)特性:原代培養(yǎng)第4天,倒置相差顯微鏡下觀察可見大部分組織塊周圍有細(xì)胞爬出,從中間至周圍,細(xì)胞密度由密變疏,形狀以多邊形和短梭形居多,并有長(zhǎng)短不一的偽足伸出相互交聯(lián);原代培養(yǎng)第7-8天,大部分組織塊之間細(xì)胞基本長(zhǎng)滿,密度較大,并開始出現(xiàn)復(fù)層生長(zhǎng)現(xiàn)象。此時(shí),加入胰蛋白酶消化分散細(xì)胞當(dāng)做第一次傳代,細(xì)胞進(jìn)行純化后繼續(xù)培養(yǎng)48h后觀察,見大部分細(xì)胞已貼壁,為多角形和短梭形,約6-7天長(zhǎng)滿,呈多邊形排列成鋪路石狀。PAS染色可見,胞質(zhì)為紫紅色。免疫組化染色可見胞質(zhì)為棕黃色,胞核為藍(lán)紫色,即兔抗鼠Cytokeratin-8抗體和α-Amylase抗體陽(yáng)性表達(dá)。HE染色可見細(xì)胞呈多邊形或多角形,藍(lán)染的細(xì)胞核較大,橢圓形,胞質(zhì)呈粉紅色;熒光顯微鏡下觀察Hoechst 33342染色后的細(xì)胞,可見橢圓形藍(lán)色亮染細(xì)胞核較大,淡染細(xì)胞質(zhì)平鋪開來,并相互連接;掃描電鏡下觀察可見細(xì)胞平鋪在爬片上,位于中間的胞核顏色較深,細(xì)胞向周圍伸出許多長(zhǎng)短不一的偽足,有些細(xì)胞還可見分泌顆粒。EPO對(duì)正常下頜下腺細(xì)胞增殖的影響:EPO作用24h、48h、72h后細(xì)胞增殖率各組之間未見明顯差異,細(xì)胞形態(tài)未見明顯變化。適宜放射劑量篩選:放射線照射24h后觀察,細(xì)胞存活率隨著放射劑量的增加而呈現(xiàn)下降趨勢(shì),且當(dāng)放射劑量為7.5Gy時(shí),細(xì)胞存活率下降39%,此時(shí)P0.05,差異具有統(tǒng)計(jì)學(xué)意義。EPO對(duì)細(xì)胞增殖和凋亡的影響:一次性給予實(shí)驗(yàn)組7.5Gy劑量照射,24h后觀察各實(shí)驗(yàn)組之間細(xì)胞增殖及凋亡率未見明顯差異。結(jié)論:組織塊培養(yǎng)法能成功獲得大鼠下頜下腺原代細(xì)胞,并能成功進(jìn)行傳代培養(yǎng)。EPO對(duì)大鼠下頜下腺細(xì)胞放射性損傷后細(xì)胞增殖和凋亡無明顯影響,其作用仍需進(jìn)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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R781.7

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