本文關(guān)鍵詞： 三氧化二砷 子宮肉瘤 凋亡　出處：《青島大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:采用體內(nèi)實(shí)驗(yàn)研究的方法,選用人子宮肉瘤MES-SA細(xì)胞系,建立人子宮肉瘤裸鼠皮下移植瘤模型,研究三氧化二砷(Arsenic Trioxide,As_2O_3)對(duì)人子宮肉瘤細(xì)胞系(MES-SA)是否具有抗腫瘤作用并初步研究其可能機(jī)制。方法:人子宮肉瘤細(xì)胞株MES-SA在含體積分?jǐn)?shù)0.10標(biāo)準(zhǔn)胎牛血清的RPM I-1640培養(yǎng)液中進(jìn)行細(xì)胞培養(yǎng),并獲取單細(xì)胞懸液;實(shí)驗(yàn)動(dòng)物為40只4-6周齡的裸鼠,MES-SA單細(xì)胞懸液皮下注入裸鼠體內(nèi),產(chǎn)生瘤結(jié)節(jié),建立人子宮肉瘤細(xì)胞裸鼠皮下移植瘤模型。隨機(jī)分為5組:As_2O_3低、中、高劑量組(劑量分別為1.0、2.5、5.0mg/kg)、生理鹽水(NS)空白對(duì)照組、異環(huán)磷酰胺(IFO)陽(yáng)性對(duì)照組,每組8只,連續(xù)10天腹腔注射相應(yīng)藥物,觀察裸鼠一般情況及瘤結(jié)節(jié)生長(zhǎng)情況。停藥后處死裸鼠剝離瘤組織,檢測(cè)以下指標(biāo):稱(chēng)瘤質(zhì)量并計(jì)算抑瘤率;流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率;提取瘤組織中RNA,采用熒光標(biāo)記的特異性Taq探針?lè)?進(jìn)行PCR反應(yīng),測(cè)定CT值,進(jìn)行半胱天冬氨酸蛋白酶(Caspase3)基因表達(dá)的相對(duì)定量分析,檢測(cè)并比較caspase-3基因表達(dá);制備腫瘤組織石蠟切片,進(jìn)行p-ERK抗體免疫組化染色,觀察著色情況,測(cè)定p-ERK表達(dá)(免疫組化SP法)。進(jìn)行數(shù)據(jù)處理及統(tǒng)計(jì)學(xué)分析。結(jié)果:1.一般情況:所有裸鼠均有瘤結(jié)節(jié)長(zhǎng)出,成功建立移植瘤模型。用藥后各As_2O_3組裸鼠一般狀態(tài)良好,精神飲食可、大小便正常、正�；顒�(dòng)、生長(zhǎng)良好、無(wú)明顯消瘦,與NS組差別不大;IFO陽(yáng)性對(duì)照組裸鼠用藥后一般狀態(tài)不佳,出現(xiàn)坐立不安等疑似腹膜刺激癥狀,且精神差,飲食少,反應(yīng)遲鈍,活動(dòng)少,并有腹瀉,消瘦等。2.移植瘤質(zhì)量及抑瘤率:三組As_2O_3組及IFO組的瘤質(zhì)量均小于NS組瘤質(zhì)量,差異均有統(tǒng)計(jì)學(xué)意義,(P0.05);As_2O_3高劑量組及IFO組瘤質(zhì)量最低,較其他組有顯著差異(P0.05);As_2O_3高劑量組與IFO組瘤質(zhì)量差別不大,As_2O_3中、低劑量組差別也不明顯。結(jié)果提示As_2O_3有抑制腫瘤作用,且高劑量As_2O_3與IFO有相似的抑瘤作用,抑瘤作用最強(qiáng)。3.凋亡率:三組As_2O_3組及IFO組均有典型凋亡峰出現(xiàn);與NS組相比,As_2O_3組及IFO組凋亡率均升高;且As_2O_3組凋亡率高于IFO組(P0.05)。As_2O_3組凋亡率隨劑量增加而升高,中、低劑量組差別不明顯(P0.05),高劑量組凋亡率高于中、低劑量組�？梢�(jiàn)As_2O_3能促進(jìn)細(xì)胞凋亡,且高劑量As_2O_3組促凋亡作用最明顯。4.caspase-3基因表達(dá):各As_2O_3組caspase-3基因表達(dá)高于NS組及IFO組(P0.05),提示As_2O_3可上調(diào)caspase-3基因表達(dá);高劑量As_2O_3組表達(dá)高于中、低劑量組,但差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.192)。5.p-ERK表達(dá):各劑量As_2O_3組及IFO組p-ERK的表達(dá)均低于NS組,但無(wú)顯著差異;高劑量As_2O_3組p-ERK表達(dá)率最低,NS組最高,高劑量As_2O_3組顯著低于NS組(Z=-2.063,P=0.0390.05)。提示As_2O_3可下調(diào)p-ERK的表達(dá),且高劑量組作用最明顯。結(jié)論:1.As_2O_3對(duì)人子宮肉瘤裸鼠皮下移植瘤增殖發(fā)揮出較好的抑制生長(zhǎng)作用,能誘導(dǎo)腫瘤細(xì)胞凋亡;As_2O_3可上調(diào)Caspase-3、下調(diào)p-ERK基因的表達(dá);促凋亡可能與上調(diào)Caspase-3基因表達(dá)有關(guān),抑瘤作用還可能與下調(diào)p-ERK表達(dá)影響ERK-MAPK信號(hào)傳導(dǎo)通路有關(guān)。2.與IFO相比,As_2O_3不良反應(yīng)相對(duì)較小,耐受可,相對(duì)安全有效,有望應(yīng)用于除白血病外的多種實(shí)體瘤的治療,為腫瘤的治療帶來(lái)新的希望。
[Abstract]:Objective: To study the in vivo, the human uterine sarcoma cell line MES-SA, establish the model of subcutaneous sarcoma of uterus in nude mice, to study the effect of arsenic trioxide (Arsenic Trioxide, As_2O_3) on human uterine sarcoma cell lines (MES-SA) have antitumor effect and study its possible mechanism. Methods: human uterine sarcoma cell line MES-SA the volume fraction of 0.10 standard fetal bovine serum RPM I-1640 culture medium for cell culture, and to obtain single cell suspension; experimental animal for 40 4-6 week old mice, MES-SA single cell suspension into nude mice subcutaneous nodules, produce, establish the model of subcutaneous human uterine sarcoma xenografts were randomly. Divided into 5 groups: As_2O_3, low and high dose group (the dose was 1.0,2.5,5.0mg/kg), normal saline (NS) control group, ifosfamide (IFO) positive control group, 8 rats in each group, intraperitoneal injection for 10 days The corresponding drugs, observe the growth of nude mice in general and tumor nodules were sacrificed. Peel the tumor tissue after discontinuation, detection of the following indicators: quality of tumor and the inhibition rate was calculated; cell apoptosis was detected by flow cytometry; extraction of tumor tissue RNA, using specific Taq probe fluorescence labeling, PCR reaction determination of CT value of caspase (Caspase3) gene expression relative quantitative analysis, comparison of Caspase-3 gene expression and detection; preparation of paraffin sections of tumor tissues, p-ERK immunohistochemical staining, observe the staining, the expression of p-ERK was determined (SP immunohistochemical method). Analysis of data processing and statistics results: 1.. General: all nude mice have long nodules, xenografts were established successfully. After the treatment of As_2O_3 mice were generally in good condition, the spirit of diet, urine normal, normal activity, good growth, ignorance Significant weight loss, and NS group had no significant difference; IFO positive control group of nude mice after administration of general condition, appeared on tenterhooks suspected peritoneal irritation symptoms, and poor spirit, eating less, unresponsive, less activity, and diarrhea, weight loss and quality.2. tumor inhibition rate: three group As_2O_3 group and IFO group. Tumor mass was less than that of NS group in quality, the differences were statistically significant (P0.05); high dose of As_2O_3 group and IFO group of minimum quality than other groups have significant difference (P0.05); high dose of As_2O_3 group and IFO group, tumor mass difference, As_2O_3 in low dose group, the difference is not obvious. The results suggest that As_2O_3 has inhibitory effect on tumor, and high doses of As_2O_3 and IFO have similar anti-tumor effect, the antitumor effect of.3. was the strongest apoptosis rate: three group As_2O_3 group and IFO group showed typical apoptosis peak; compared with NS group, As_2O_3 group and IFO group. The apoptosis rate was increased and the apoptosis rate of As_2O_3 group is high; In group IFO (P0.05).As_2O_3 group apoptosis rate increased along with the increase of the dose, low dose group, the difference was not significant (P0.05), the apoptosis rate in high dose group than in low dose group. The As_2O_3 can promote cell apoptosis, and apoptosis of high dose of As_2O_3 group the most obvious expression of.4.caspase-3 gene by As_2O_3 group the expression of Caspase-3 gene was higher than that of NS group and IFO group (P0.05), suggesting that As_2O_3 can upregulate the expression of Caspase-3 gene; high dose of As_2O_3 group was higher than that in the low dose group, but the difference was not statistically significant (P=0.192).5.p-ERK expression: the expression of As_2O_3 in each dose group IFO and group p-ERK were lower than NS group, but no significant difference; high dose group As_2O_3 p-ERK expression was the lowest and the highest in NS group, high dose of As_2O_3 group was significantly lower than that of group NS (Z=-2.063, P=0.0390.05). It is suggested that As_2O_3 can downregulate the expression of p-ERK, and the most obvious effect in the high dose group. Conclusion: 1.As_2O_3 of uterine sarcoma nude 榧犵毊涓嬬Щ妞嶇槫澧炴畺鍙戞尌鍑?guó)櫨冨ソ鐨勬姂鍒剁敓闀夸綔鐢?鑳借瀵艱偪鐦ょ粏鑳?yōu)鍑嬩海?

本文編號(hào)：1523583