發(fā)布時(shí)間：2018-01-31 00:28

  本文關(guān)鍵詞： 羧甲基殼聚糖鋅多肽 牙周膜成纖維細(xì)胞 細(xì)胞毒性 動(dòng)物毒性　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究羧甲基殼聚糖鋅多肽復(fù)合材料(carboxymethyl chitosan zinc and peptide,CMC-Zn+-P)的生物安全性,為材料的安全使用提供依據(jù)。方法:1.細(xì)胞毒性檢測(cè):在體外培養(yǎng)人牙周膜成纖維細(xì)胞(human periodontal ligament cells,HPDLCs),對(duì)其形態(tài)學(xué)進(jìn)行觀察,并進(jìn)行免疫組織化學(xué)鑒定。制取材料浸提液,以不同的濃度與細(xì)胞培養(yǎng)液混合,用于培養(yǎng)細(xì)胞,并進(jìn)行分組,1~4組為實(shí)驗(yàn)組,材料浸提液的濃度分別為100%、75%、50%、25%,第5組為對(duì)照組,不含材料浸提液,采用CCK-8法測(cè)定不同濃度的材料浸提液對(duì)細(xì)胞增殖活性的影響,通過(guò)計(jì)算細(xì)胞的相對(duì)增殖率(RGR),對(duì)細(xì)胞毒性進(jìn)行評(píng)級(jí)。2.急性毒性檢測(cè):選取20只Wistar大鼠,隨機(jī)分為實(shí)驗(yàn)組與對(duì)照組,實(shí)驗(yàn)組單次灌胃5 g/kg復(fù)合材料,對(duì)照組單次灌胃相同劑量的生理鹽水。給藥以后,大鼠正常飲食飲水,連續(xù)觀察一周,記錄大鼠的行為活動(dòng)狀態(tài),7天后對(duì)其進(jìn)行稱重、取血,檢測(cè)大鼠血常規(guī)及生化指標(biāo),處死后進(jìn)行尸檢,完整取出大鼠各臟器,計(jì)算大鼠體重相對(duì)增長(zhǎng)率和臟器指數(shù),并進(jìn)行病理切片檢查。3.口腔黏膜刺激試驗(yàn):選取10只Wistar大鼠,雌雄各半,麻醉后,將復(fù)合材料涂抹于大鼠的左側(cè)口腔黏膜,將生理鹽水涂抹于大鼠的右側(cè)口腔黏膜,每側(cè)均涂5 min,觀察24 h后,將大鼠處死,并取用藥部位的口腔黏膜,用10%福爾馬林固定,進(jìn)行HE染色,并于光學(xué)顯微鏡下觀察。4.急性皮膚刺激試驗(yàn):選取10只Wistar大鼠,雌雄各半。試驗(yàn)前24 h剔除背部的被毛,約3 cm×5 cm大小區(qū)域,將5 g/kg體重劑量的復(fù)合材料均勻涂抹于大鼠的背部,將藥物用紗布蓋住,使用無(wú)刺激性的膠布將其固定。24 h后去除紗布及藥物,觀察試驗(yàn)區(qū)皮膚是否出現(xiàn)紅斑、水腫等情況,并觀察大鼠是否有中毒表現(xiàn)及死亡情況,連續(xù)觀察一周。5.長(zhǎng)期毒性檢測(cè):選取健康的Wistar大鼠40只,將它們隨機(jī)分為4組,高、中、低劑量組分別給予大鼠灌胃5 g/kg、2 g/kg、0.5 g/kg復(fù)合材料,對(duì)照組灌胃給予相同劑量的生理鹽水,每天1次,連續(xù)給藥3個(gè)月。3個(gè)月后,取血檢測(cè)大鼠血常規(guī)及生化指標(biāo),處死后進(jìn)行尸檢,完整取出大鼠各臟器,計(jì)算大鼠體重相對(duì)增長(zhǎng)率和臟器指數(shù),并進(jìn)行病理檢查。6.統(tǒng)計(jì)學(xué)處理:采用SPSS 17.0軟件包對(duì)數(shù)據(jù)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:1.細(xì)胞毒性檢測(cè):體外培養(yǎng)的HPDLCs呈成纖維細(xì)胞的形態(tài);免疫組化結(jié)果示細(xì)胞抗波形絲蛋白染色呈陽(yáng)性,抗角蛋白染色呈陰性,培養(yǎng)的細(xì)胞來(lái)源于中胚層組織;不同濃度的材料浸提液對(duì)體外培養(yǎng)的HPDLCs均有促進(jìn)增殖的作用,各組細(xì)胞的RGR均大于100%,細(xì)胞毒性分級(jí)為0級(jí)。2.急性毒性檢測(cè):單次灌胃給藥后,實(shí)驗(yàn)組與對(duì)照組的試驗(yàn)大鼠行為狀態(tài)良好,均未出現(xiàn)大鼠死亡的現(xiàn)象,兩組大鼠在體重增長(zhǎng)、臟器指數(shù)、血常規(guī)及血液生化指標(biāo)各方面的差異均無(wú)統(tǒng)計(jì)學(xué)意義,病理檢查各組大鼠的內(nèi)臟器官,未發(fā)現(xiàn)明顯異常。3.口腔黏膜刺激試驗(yàn):試驗(yàn)用鼠未出現(xiàn)死亡,雙側(cè)口腔黏膜均無(wú)充血、水腫、糜爛等情況,病理切片也未見(jiàn)明顯異常。4.急性皮膚刺激試驗(yàn):試驗(yàn)用鼠皮膚未出現(xiàn)紅斑、充血、水腫及糜爛等異常情況,也未出現(xiàn)實(shí)驗(yàn)動(dòng)物的死亡。5.長(zhǎng)期毒性檢測(cè):高、中、低劑量組與對(duì)照組相比,大鼠的體重變化、臟器指數(shù)無(wú)明顯差異,各臟器無(wú)肉眼可見(jiàn)的病損,病理切片檢查也未見(jiàn)明顯異常,血常規(guī)結(jié)果在四組之間也無(wú)統(tǒng)計(jì)學(xué)差異,在血液生化指標(biāo)的檢測(cè)中,谷丙轉(zhuǎn)氨酶(AST)、谷草轉(zhuǎn)氨酶(ALT)、尿素(BUN)、葡萄糖(GLU)及肌酐(CREA)在四組中的結(jié)果差異無(wú)統(tǒng)計(jì)學(xué)意義,但總膽固醇(CHOL)結(jié)果出現(xiàn)組間差異,進(jìn)一步進(jìn)行兩兩比較發(fā)現(xiàn),高劑量組與對(duì)照組之間差異顯著,P0.001,高劑量組的CHOL含量明顯高于對(duì)照組;高劑量組與中劑量、低劑量組相比,CHOL含量偏高,P=0.015,0.001;中劑量組與對(duì)照組相比,CHOL含量偏高,P=0.003;中劑量組與低劑量組以及低劑量組與對(duì)照組之間,差異無(wú)統(tǒng)計(jì)學(xué)意義結(jié)論:CMC-Zn+-P復(fù)合材料對(duì)體外培養(yǎng)的HPDLCs不僅無(wú)細(xì)胞毒性,而且還可促進(jìn)細(xì)胞的增殖,對(duì)大鼠無(wú)急性毒性、無(wú)急性皮膚刺激毒性、無(wú)口腔黏膜刺激毒性、無(wú)長(zhǎng)期毒性。
[Abstract]:Objective: To study the effect of carboxymethyl chitosan zinc composite (carboxymethyl chitosan zinc peptide and, peptide, CMC-Zn+-P) of the biological safety, provide the basis for the safe use of materials. Methods: to detect 1. cell toxicity of cultured human periodontal ligament fibroblasts in vitro (human periodontal ligament cells, HPDLCs), the morphology was observed, and were identified by immunohistochemistry. The preparation of material extract, with different concentration and cell culture fluid mixing for cultured cells, and were divided into two groups, 1~4 group for the experimental group, material extract concentration was 100%, respectively, 75%, 50%, 25%, fifth for the control group, without material extracts were determined by CCK-8 method, the material effects of extracts on cell proliferation, cell proliferation rate calculated by the relative rating (RGR),.2. acute toxicity test on the cell toxicity: 20 Wistar rats were randomly divided into As the experimental group and control group, experimental group, single dose 5 g/kg composite materials, the control group normal saline single oral administration of the same dose. After administrated rats with normal diet and water, continuous observation of the week, recording the activity status of rats, 7 days after the blood sampling, weighing, large blood routine and biochemical indexes of rats were sacrificed after detection, autopsy, complete removal of the organs of rats, the body weight of rats relative growth rate and organ index calculation, and pathological examination of.3. oral mucosa irritation test: a total of 10 Wistar rats, male and female, after anesthesia, the left buccal composite smear in rats the right of oral mucosal saline smear in rats, each side is coated with 5 min, 24 h was observed after rats were killed, and the oral mucosal administration area, fixed with 10% formalin, HE staining, and observed under optical microscope in acute skin.4. Stimulation test: a total of 10 Wistar rats, half male and half female. Before the test 24 h to eliminate the back coat, about 3 cm * 5 cm in size, the composite 5 g/kg body weight dose evenly on the back of a rat, the drug with gauze cover, use non irritating tape to fix the.24 h after the removal of gauze and drugs, to observe whether test area of skin erythema, edema, and rat are manifestations of poisoning and death, continuous observation of the long-term toxicity detection a week.5.: select 40 healthy Wistar rats, they were randomly divided into 4 groups, high, low dose group the rats were given intragastric administration of 5 g/kg, 2 g/kg, 0.5 g/kg composite, saline control group were lavaged with the same dose, 1 times a day, continuous administration of 3.3 months after the blood serum of rats and biochemical indicators were examined, the complete removal of various organs in in the calculation The body weight of rats relative growth rate and organ index, and pathological examination of.6. statistical packages for statistical analysis of data using SPSS 17 software. Results: the detection of 1. cell toxicity: HPDLCs fibroblast cell morphology in vitro; immunohistochemistry showed cell anti vimentin staining was positive, anti keratin staining was negative, cultured cells derived from the mesoderm tissue; different concentrations of material extract on cultured HPDLCs could promote the proliferation of the cells in each group, the RGR was more than 100%, cell toxicity was graded as acute toxicity test: single level 0.2. after intragastric behavior of experimental rats the experimental group and control group, there was no death in rats, the rats in the two groups in body weight, organ index, the differences of blood routine and blood biochemical index were not statistically significant, pathological examination of the Rats of the visceral organs, found no obvious abnormal.3. oral mucosa irritation test: test rats caused no death, no bilateral oral mucosal hyperemia, edema, erosion, pathological sections showed no obvious abnormal.4. acute skin irritation test with rat skin not appear erythema, hyperaemia, edema and erosion etc. abnormal situation, long-term toxicity of death.5. detection does not appear in the experimental animal: high, low dose group, compared with the control group, the changes of body weight of rats, no significant difference between the organ index of each organ, no visible lesions, pathological examination showed no obvious abnormalities, blood test results had no significant difference in the four between the groups, in the detection of blood biochemical index, alanine aminotransferase (AST), aspartate aminotransferase (ALT), urea (BUN), glucose (GLU) and creatinine (CREA) in the four groups showed no significant difference, but the total cholesterol (CHOL) results There differences between groups, further comparison showed that in 22, the difference between high dose group and the control group was P0.001, CHOL content of high dose group was significantly higher than the control group; high dose group and middle dose, low dose group, high levels of CHOL, P=0.015,0.001; compared with the control group, the content of CHOL is high. P=0.003; between middle dose group and low dose group, low dose group and control group, the difference was not statistically significant. Conclusion: CMC-Zn+-P composite material not only has no cytotoxicity of HPDLCs in vitro, but also can promote cell proliferation and no acute toxicity in rats, skin irritation and no acute toxicity, no oral mucosal irritation. No long-term toxicity.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.1

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